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Tang K, Zhou L, Tian X, Fang SY, Vandenbulcke E, Du A, Shen J, Cao H, Zhou J, Chen K, Kim HR, Luo Z, Xin S, Lin SH, Park D, Yang L, Zhang Y, Suzuki K, Majety M, Ling X, Lam SZ, Chow RD, Ren P, Tao B, Li K, Codina A, Dai X, Shang X, Bai S, Nottoli T, Levchenko A, Booth CJ, Liu C, Fan R, Dong MB, Zhou X, Chen S. Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering. Nat Biomed Eng 2025:10.1038/s41551-025-01371-2. [PMID: 40114032 DOI: 10.1038/s41551-025-01371-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2024] [Accepted: 02/13/2025] [Indexed: 03/22/2025]
Abstract
The pleiotropic effects of human disease and the complex nature of gene-interaction networks require knock-in mice allowing for multiplexed gene perturbations. Here we describe a series of knock-in mice with a C57BL/6 background and with the conditional or constitutive expression of LbCas12a or of high-fidelity enhanced AsCas12a, which were inserted at the Rosa26 locus. The constitutive expression of Cas12a in the mice did not lead to discernible pathology and enabled efficient multiplexed genome engineering. We used the mice for the retrovirus-based immune-cell engineering of CD4+ and CD8+ T cells, B cells and bone-marrow-derived dendritic cells, for autochthonous cancer modelling through the delivery of multiple CRISPR RNAs as a single array using adeno-associated viruses, and for the targeted genome editing of liver tissue using lipid nanoparticles. We also describe a system for simultaneous dual-gene activation and knockout (DAKO). The Cas12a-knock-in mice and the viral and non-viral delivery vehicles provide a versatile toolkit for ex vivo and in vivo applications in genome editing, disease modelling and immune-cell engineering, and for the deconvolution of complex gene interactions.
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Affiliation(s)
- Kaiyuan Tang
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA
- Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA
| | - Liqun Zhou
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA
- Immunobiology Program, Yale University, New Haven, CT, USA
| | - Xiaolong Tian
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Department of Biomedical Engineering, Yale University, New Haven, CT, USA
| | - Shao-Yu Fang
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
| | - Erica Vandenbulcke
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Yale College, Yale University, New Haven, CT, USA
| | - Andrew Du
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Yale College, Yale University, New Haven, CT, USA
| | - Johanna Shen
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Yale College, Yale University, New Haven, CT, USA
| | - Hanbing Cao
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
| | - Jerry Zhou
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Yale College, Yale University, New Haven, CT, USA
| | - Krista Chen
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Yale College, Yale University, New Haven, CT, USA
| | - Hyunu R Kim
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
| | - Zhicheng Luo
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA
| | - Shan Xin
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
| | - Shawn H Lin
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA
- Immunobiology Program, Yale University, New Haven, CT, USA
| | - Daniel Park
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Yale College, Yale University, New Haven, CT, USA
| | - Luojia Yang
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA
- Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA
| | - Yueqi Zhang
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
| | - Kazushi Suzuki
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
| | - Medha Majety
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Yale College, Yale University, New Haven, CT, USA
| | - Xinyu Ling
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
| | - Stanley Z Lam
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Yale College, Yale University, New Haven, CT, USA
| | - Ryan D Chow
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- M.D.-Ph.D. Program, Yale University, West Haven, CT, USA
| | - Ping Ren
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
| | - Bo Tao
- System Biology Institute, Yale University, West Haven, CT, USA
- Department of Biomedical Engineering, Yale University, New Haven, CT, USA
| | - Keyi Li
- Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA
| | - Adan Codina
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA
- Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA
| | - Xiaoyun Dai
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
- System Biology Institute, Yale University, West Haven, CT, USA
- Center for Genome Editing, Westlake Laboratory of Life Sciences and Biomedicine, School of Medicine, Westlake University, Hangzhou, China
| | - Xingbo Shang
- System Biology Institute, Yale University, West Haven, CT, USA
- Department of Biomedical Engineering, Yale University, New Haven, CT, USA
| | - Suxia Bai
- Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA
| | - Timothy Nottoli
- Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA
| | - Andre Levchenko
- System Biology Institute, Yale University, West Haven, CT, USA
- Immunobiology Program, Yale University, New Haven, CT, USA
- Department of Biomedical Engineering, Yale University, New Haven, CT, USA
- Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA
| | - Carmen J Booth
- Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA
| | - Chen Liu
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
| | - Rong Fan
- System Biology Institute, Yale University, West Haven, CT, USA
- Department of Biomedical Engineering, Yale University, New Haven, CT, USA
- Department of Pathology, Yale University School of Medicine, New Haven, CT, USA
- Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA
- Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA
- Yale Center for RNA Science and Medicine, Yale University School of Medicine, New Haven, CT, USA
| | - Matthew B Dong
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
- System Biology Institute, Yale University, West Haven, CT, USA.
- M.D.-Ph.D. Program, Yale University, West Haven, CT, USA.
- Department of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA.
| | - Xiaoyu Zhou
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
- System Biology Institute, Yale University, West Haven, CT, USA.
| | - Sidi Chen
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
- System Biology Institute, Yale University, West Haven, CT, USA.
- Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA.
- Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA.
- Immunobiology Program, Yale University, New Haven, CT, USA.
- Yale College, Yale University, New Haven, CT, USA.
- M.D.-Ph.D. Program, Yale University, West Haven, CT, USA.
- Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA.
- Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA.
- Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA.
- Yale Center for RNA Science and Medicine, Yale University School of Medicine, New Haven, CT, USA.
- Yale Liver Center, Yale University School of Medicine, New Haven, CT, USA.
- Yale Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA.
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Osanai Y, Xing YL, Mochizuki S, Kobayashi K, Homman-Ludiye J, Cooray A, Poh J, Inutsuka A, Ohno N, Merson TD. 5' Transgenes drive leaky expression of 3' transgenes in Cre-inducible bi-cistronic vectors. Mol Ther Methods Clin Dev 2024; 32:101288. [PMID: 39104576 PMCID: PMC11298883 DOI: 10.1016/j.omtm.2024.101288] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2023] [Accepted: 06/21/2024] [Indexed: 08/07/2024]
Abstract
Molecular cloning techniques enabling contemporaneous expression of two or more protein-coding sequences provide an invaluable tool for understanding the molecular regulation of cellular functions. The Cre-lox system is used for inducing the expression of recombinant proteins encoded within a bi-/poly-cistronic cassette. However, leak expression of transgenes is often observed in the absence of Cre recombinase activity, compromising the utility of this approach. To investigate the mechanism of leak expression, we generated Cre-inducible bi-cistronic vectors to monitor the expression of transgenes positioned either 5' or 3' of a 2A peptide or internal ribosomal entry site (IRES) sequence. Cells transfected with these bi-cistronic vectors exhibited Cre-independent leak expression specifically of transgenes positioned 3' of the 2A peptide or IRES sequence. Similarly, AAV-FLEX vectors encoding bi-cistronic cassettes or fusion proteins revealed the selective Cre-independent leak expression of transgenes positioned at the 3' end of the open reading frame. Our data demonstrate that 5' transgenes confer promoter-like activity that drives the expression of 3' transgenes. An additional lox-STOP-lox cassette between the 2A sequence and 3' transgene dramatically decreased Cre-independent transgene expression. Our findings highlight the need for appropriate experimental controls when using Cre-inducible bi-/poly-cistronic constructs and inform improved design of vectors for more tightly regulated inducible transgene expression.
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Affiliation(s)
- Yasuyuki Osanai
- Australian Regenerative Medicine Institute, Monash University, 15 Innovation Walk, Clayton, VIC 3800, Australia
- Department of Anatomy, Division of Histology and Cell Biology, School of Medicine, Jichi Medical University, Shimotsuke, Tochigi 329-0431, Japan
| | - Yao Lulu Xing
- Australian Regenerative Medicine Institute, Monash University, 15 Innovation Walk, Clayton, VIC 3800, Australia
| | - Shinya Mochizuki
- Department of Anatomy, Bioimaging and Neuro-cell Science, Jichi Medical University, Shimotsuke, Tochigi 329-0431, Japan
| | - Kenta Kobayashi
- Section of Viral Vector Development, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan
- The Graduate University for Advanced Studies (SOKENDAI), Shonan Village, Hayama, Kanagawa 240-0193, Japan
| | - Jihane Homman-Ludiye
- Australian Regenerative Medicine Institute, Monash University, 15 Innovation Walk, Clayton, VIC 3800, Australia
| | - Amali Cooray
- Australian Regenerative Medicine Institute, Monash University, 15 Innovation Walk, Clayton, VIC 3800, Australia
| | - Jasmine Poh
- Australian Regenerative Medicine Institute, Monash University, 15 Innovation Walk, Clayton, VIC 3800, Australia
| | - Ayumu Inutsuka
- Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University, Shimotsuke, Tochigi 329-0431, Japan
| | - Nobuhiko Ohno
- Department of Anatomy, Division of Histology and Cell Biology, School of Medicine, Jichi Medical University, Shimotsuke, Tochigi 329-0431, Japan
- Division of Ultrastructure Research, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan
| | - Tobias D. Merson
- Australian Regenerative Medicine Institute, Monash University, 15 Innovation Walk, Clayton, VIC 3800, Australia
- Oligodendroglial Interactions Group, Systems Neurodevelopment Laboratory, National Institute of Mental Health, Bethesda, MD 20892, USA
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Murphy KC, Ruscetti M. Advances in Making Cancer Mouse Models More Accessible and Informative through Non-Germline Genetic Engineering. Cold Spring Harb Perspect Med 2024; 14:a041348. [PMID: 37277206 PMCID: PMC10982712 DOI: 10.1101/cshperspect.a041348] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Genetically engineered mouse models (GEMMs) allow for modeling of spontaneous tumorigenesis within its native microenvironment in mice and have provided invaluable insights into mechanisms of tumorigenesis and therapeutic strategies to treat human disease. However, as their generation requires germline manipulation and extensive animal breeding that is time-, labor-, and cost-intensive, traditional GEMMs are not accessible to most researchers, and fail to model the full breadth of cancer-associated genetic alterations and therapeutic targets. Recent advances in genome-editing technologies and their implementation in somatic tissues of mice have ushered in a new class of mouse models: non-germline GEMMs (nGEMMs). nGEMM approaches can be leveraged to generate somatic tumors de novo harboring virtually any individual or group of genetic alterations found in human cancer in a mouse through simple procedures that do not require breeding, greatly increasing the accessibility and speed and scale on which GEMMs can be produced. Here we describe the technologies and delivery systems used to create nGEMMs and highlight new biological insights derived from these models that have rapidly informed functional cancer genomics, precision medicine, and immune oncology.
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Affiliation(s)
- Katherine C Murphy
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts 01605, USA
| | - Marcus Ruscetti
- Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, Massachusetts 01605, USA;
- Immunology and Microbiology Program, University of Massachusetts Chan Medical School, Worcester, Massachusetts 01605, USA
- Cancer Center, University of Massachusetts Chan Medical School, Worcester, Massachusetts 01605, USA
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Tong Y, Cho S, Coenen VA, Döbrössy MD. Input-output relation of midbrain connectomics in a rodent model of depression. J Affect Disord 2024; 345:443-454. [PMID: 37890539 DOI: 10.1016/j.jad.2023.10.124] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/06/2023] [Revised: 09/13/2023] [Accepted: 10/18/2023] [Indexed: 10/29/2023]
Abstract
BACKGROUND The symptoms associated with depression are believed to arise from disruptions in information processing across brain networks. The ventral tegmental area (VTA) influences reward-based behavior, motivation, addiction, and psychiatric disorders, including depression. Deep brain stimulation (DBS) of the medial forebrain bundle (MFB), is an emerging therapy for treatment-resistant depression. Understanding the depression associated anatomical networks crucial for comprehending its antidepressant effects. METHODS Flinders Sensitive Line (FSL), a rodent model of depression and Sprague-Dawley rats (n = 10 each) were used in this study. We used monosynaptic tracing to map inputs of VTA efferent neurons: VTA-to-NAc nucleus accumbens (NAc) (both core and shell) and VTA-to-prefrontal cortex (PFC). Quantitative analysis explored afferent diversity and strengths. RESULTS VTA efferent neurons receive a variety of afferents with varying input weights and predominant neuromodulatory representation. Notably, NAc-core projecting VTA neurons showed stronger afferents from dorsal raphe, while NAc shell-projecting VTA neurons displayed lower input strengths from cortex, thalamus, zona incerta and pretectal area in FSL rats. NAc shell-projecting VTA neurons showed the most difference in connectivity across the experimental groups. LIMITATIONS Lack of functional properties of the anatomical connections is the major limitation of this study. Incomplete labeling and the cytotoxicity of the rabies virus should be made aware of. CONCLUSIONS These findings provide the first characterization of inputs to different VTA ascending projection neurons, shedding light on critical differences in the connectome of the midbrain-forebrain system. Moreover, these differences support potential network effects of these circuits in the context of MFB DBS neuromodulation for depression.
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Affiliation(s)
- Y Tong
- Laboratory of Stereotaxy and Interventional Neurosciences, Department of Stereotactic and Functional Neurosurgery, University Hospital Freiburg, 79106 Freiburg, Germany; Department of Stereotactic and Functional Neurosurgery, University Hospital Freiburg, 79106 Freiburg, Germany
| | - S Cho
- Laboratory of Stereotaxy and Interventional Neurosciences, Department of Stereotactic and Functional Neurosurgery, University Hospital Freiburg, 79106 Freiburg, Germany; Department of Stereotactic and Functional Neurosurgery, University Hospital Freiburg, 79106 Freiburg, Germany; Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
| | - V A Coenen
- Laboratory of Stereotaxy and Interventional Neurosciences, Department of Stereotactic and Functional Neurosurgery, University Hospital Freiburg, 79106 Freiburg, Germany; Department of Stereotactic and Functional Neurosurgery, University Hospital Freiburg, 79106 Freiburg, Germany; Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany; Center for Basics in Neuromodulation, University of Freiburg, 79106 Freiburg, Germany; IMBIT (Institute for Machine-Brain Interfacing Technology), University of Freiburg, 79110 Freiburg, Germany
| | - M D Döbrössy
- Laboratory of Stereotaxy and Interventional Neurosciences, Department of Stereotactic and Functional Neurosurgery, University Hospital Freiburg, 79106 Freiburg, Germany; Department of Stereotactic and Functional Neurosurgery, University Hospital Freiburg, 79106 Freiburg, Germany; Center for Basics in Neuromodulation, University of Freiburg, 79106 Freiburg, Germany.
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Bai T, Zhan L, Zhang N, Lin F, Saur D, Xu C. Learning-prolonged maintenance of stimulus information in CA1 and subiculum during trace fear conditioning. Cell Rep 2023; 42:112853. [PMID: 37481720 DOI: 10.1016/j.celrep.2023.112853] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2022] [Revised: 04/12/2023] [Accepted: 07/08/2023] [Indexed: 07/25/2023] Open
Abstract
Temporal associative learning binds discontiguous conditional stimuli (CSs) and unconditional stimuli (USs), possibly by maintaining CS information in the hippocampus after its offset. Yet, how learning regulates such maintenance of CS information in hippocampal circuits remains largely unclear. Using the auditory trace fear conditioning (TFC) paradigm, we identify a projection from the CA1 to the subiculum critical for TFC. Deep-brain calcium imaging shows that the peak of trace activity in the CA1 and subiculum is extended toward the US and that the CS representation during the trace period is enhanced during learning. Interestingly, such plasticity is consolidated only in the CA1, not the subiculum, after training. Moreover, CA1 neurons, but not subiculum neurons, increasingly become active during CS-and-trace and shock periods, respectively, and correlate with CS-evoked fear retrieval afterward. These results indicate that learning dynamically enhances stimulus information maintenance in the CA1-subiculum circuit during learning while storing CS and US memories primarily in the CA1 area.
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Affiliation(s)
- Tao Bai
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China; University of the Chinese Academy of Sciences, Beijing 100049, China
| | - Lijie Zhan
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China
| | - Na Zhang
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China
| | - Feikai Lin
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China
| | - Dieter Saur
- Department of Internal Medicine 2, Technische Universität München, Ismaningerstrasse 22, 81675 Munich, Germany
| | - Chun Xu
- Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China; University of the Chinese Academy of Sciences, Beijing 100049, China; Shanghai Center for Brain Science and Brain-Inspired Technology, Shanghai 201210, China.
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Andrianova L, Yanakieva S, Margetts-Smith G, Kohli S, Brady ES, Aggleton JP, Craig MT. No evidence from complementary data sources of a direct glutamatergic projection from the mouse anterior cingulate area to the hippocampal formation. eLife 2023; 12:e77364. [PMID: 37545394 PMCID: PMC10425170 DOI: 10.7554/elife.77364] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2022] [Accepted: 08/03/2023] [Indexed: 08/08/2023] Open
Abstract
The connectivity and interplay between the prefrontal cortex and hippocampus underpin various key cognitive processes, with changes in these interactions being implicated in both neurodevelopmental and neurodegenerative conditions. Understanding the precise cellular connections through which this circuit is organised is, therefore, vital for understanding these same processes. Overturning earlier findings, a recent study described a novel excitatory projection from anterior cingulate area to dorsal hippocampus. We sought to validate this unexpected finding using multiple, complementary methods: anterograde and retrograde anatomical tracing, using anterograde and retrograde adeno-associated viral vectors, monosynaptic rabies tracing, and the Fast Blue classical tracer. Additionally, an extensive data search of the Allen Projection Brain Atlas database was conducted to find the stated projection within any of the deposited anatomical studies as an independent verification of our own results. However, we failed to find any evidence of a direct, monosynaptic glutamatergic projection from mouse anterior cingulate cortex to the hippocampus proper.
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Affiliation(s)
- Lilya Andrianova
- Institute of Biomedical and Clinical Science, University of Exeter Medical SchoolExeterUnited Kingdom
- School of Psychology & Neuroscience, College of Medical, Veterinary and Life Sciences, University of GlasgowGlasgowUnited Kingdom
- School of Infection & Immunity, College of Medical, Veterinary and Life Sciences, University of GlasgowGlasgowUnited Kingdom
| | - Steliana Yanakieva
- School of Infection & Immunity, College of Medical, Veterinary and Life Sciences, University of GlasgowGlasgowUnited Kingdom
| | - Gabriella Margetts-Smith
- Institute of Biomedical and Clinical Science, University of Exeter Medical SchoolExeterUnited Kingdom
| | - Shivali Kohli
- Institute of Biomedical and Clinical Science, University of Exeter Medical SchoolExeterUnited Kingdom
| | - Erica S Brady
- Institute of Biomedical and Clinical Science, University of Exeter Medical SchoolExeterUnited Kingdom
| | - John P Aggleton
- School of Psychology, Cardiff UniversityCardiffUnited Kingdom
| | - Michael T Craig
- Institute of Biomedical and Clinical Science, University of Exeter Medical SchoolExeterUnited Kingdom
- School of Psychology & Neuroscience, College of Medical, Veterinary and Life Sciences, University of GlasgowGlasgowUnited Kingdom
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7
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Lee C, Côté SL, Raman N, Chaudhary H, Mercado BC, Chen SX. Whole-brain mapping of long-range inputs to the VIP-expressing inhibitory neurons in the primary motor cortex. Front Neural Circuits 2023; 17:1093066. [PMID: 37275468 PMCID: PMC10237295 DOI: 10.3389/fncir.2023.1093066] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2022] [Accepted: 05/05/2023] [Indexed: 06/07/2023] Open
Abstract
The primary motor cortex (MOp) is an important site for motor skill learning. Interestingly, neurons in MOp possess reward-related activity, presumably to facilitate reward-based motor learning. While pyramidal neurons (PNs) and different subtypes of GABAergic inhibitory interneurons (INs) in MOp all undergo cell-type specific plastic changes during motor learning, the vasoactive intestinal peptide-expressing inhibitory interneurons (VIP-INs) in MOp have been shown to preferentially respond to reward and play a critical role in the early phases of motor learning by triggering local circuit plasticity. To understand how VIP-INs might integrate various streams of information, such as sensory, pre-motor, and reward-related inputs, to regulate local plasticity in MOp, we performed monosynaptic rabies tracing experiments and employed an automated cell counting pipeline to generate a comprehensive map of brain-wide inputs to VIP-INs in MOp. We then compared this input profile to the brain-wide inputs to somatostatin-expressing inhibitory interneurons (SST-INs) and parvalbumin-expressing inhibitory interneurons (PV-INs) in MOp. We found that while all cell types received major inputs from sensory, motor, and prefrontal cortical regions, as well as from various thalamic nuclei, VIP-INs received more inputs from the orbital frontal cortex (ORB) - a region associated with reinforcement learning and value predictions. Our findings provide insight on how the brain leverages microcircuit motifs by both integrating and partitioning different streams of long-range input to modulate local circuit activity and plasticity.
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Affiliation(s)
- Candice Lee
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Sandrine L. Côté
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Nima Raman
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Hritvic Chaudhary
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Bryan C. Mercado
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada
| | - Simon X. Chen
- Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada
- Brain and Mind Research Institute, University of Ottawa, Ottawa, ON, Canada
- Center for Neural Dynamics, University of Ottawa, Ottawa, ON, Canada
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Frezel N, Ranucci M, Foster E, Wende H, Pelczar P, Mendes R, Ganley RP, Werynska K, d'Aquin S, Beccarini C, Birchmeier C, Zeilhofer HU, Wildner H. c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy. Cell Rep 2023; 42:112295. [PMID: 36947543 PMCID: PMC10157139 DOI: 10.1016/j.celrep.2023.112295] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Revised: 02/02/2023] [Accepted: 03/06/2023] [Indexed: 03/23/2023] Open
Abstract
Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-MafEX) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-MafEX neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-MafEX neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-MafEX neuron activation. Our study identifies c-MafEX neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control.
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Affiliation(s)
- Noémie Frezel
- Institute of Pharmacology and Toxicology, University of Zürich, 8057 Zürich, Switzerland
| | - Matteo Ranucci
- Institute of Pharmacology and Toxicology, University of Zürich, 8057 Zürich, Switzerland
| | - Edmund Foster
- Institute of Pharmacology and Toxicology, University of Zürich, 8057 Zürich, Switzerland
| | | | - Pawel Pelczar
- Center for Transgenic Models (CTM), University of Basel, 4001 Basel, Switzerland
| | - Raquel Mendes
- Institute of Pharmacology and Toxicology, University of Zürich, 8057 Zürich, Switzerland
| | - Robert P Ganley
- Institute of Pharmacology and Toxicology, University of Zürich, 8057 Zürich, Switzerland
| | - Karolina Werynska
- Institute of Pharmacology and Toxicology, University of Zürich, 8057 Zürich, Switzerland
| | - Simon d'Aquin
- Institute of Pharmacology and Toxicology, University of Zürich, 8057 Zürich, Switzerland
| | - Camilla Beccarini
- Institute of Pharmacology and Toxicology, University of Zürich, 8057 Zürich, Switzerland
| | | | - Hanns Ulrich Zeilhofer
- Institute of Pharmacology and Toxicology, University of Zürich, 8057 Zürich, Switzerland; Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zürich, 8092 Zürich, Switzerland.
| | - Hendrik Wildner
- Institute of Pharmacology and Toxicology, University of Zürich, 8057 Zürich, Switzerland.
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9
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Paul MC, Schneeweis C, Falcomatà C, Shan C, Rossmeisl D, Koutsouli S, Klement C, Zukowska M, Widholz SA, Jesinghaus M, Heuermann KK, Engleitner T, Seidler B, Sleiman K, Steiger K, Tschurtschenthaler M, Walter B, Weidemann SA, Pietsch R, Schnieke A, Schmid RM, Robles MS, Andrieux G, Boerries M, Rad R, Schneider G, Saur D. Non-canonical functions of SNAIL drive context-specific cancer progression. Nat Commun 2023; 14:1201. [PMID: 36882420 PMCID: PMC9992512 DOI: 10.1038/s41467-023-36505-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Accepted: 02/03/2023] [Indexed: 03/09/2023] Open
Abstract
SNAIL is a key transcriptional regulator in embryonic development and cancer. Its effects in physiology and disease are believed to be linked to its role as a master regulator of epithelial-to-mesenchymal transition (EMT). Here, we report EMT-independent oncogenic SNAIL functions in cancer. Using genetic models, we systematically interrogated SNAIL effects in various oncogenic backgrounds and tissue types. SNAIL-related phenotypes displayed remarkable tissue- and genetic context-dependencies, ranging from protective effects as observed in KRAS- or WNT-driven intestinal cancers, to dramatic acceleration of tumorigenesis, as shown in KRAS-induced pancreatic cancer. Unexpectedly, SNAIL-driven oncogenesis was not associated with E-cadherin downregulation or induction of an overt EMT program. Instead, we show that SNAIL induces bypass of senescence and cell cycle progression through p16INK4A-independent inactivation of the Retinoblastoma (RB)-restriction checkpoint. Collectively, our work identifies non-canonical EMT-independent functions of SNAIL and unravel its complex context-dependent role in cancer.
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Affiliation(s)
- Mariel C Paul
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany
| | - Christian Schneeweis
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany
| | - Chiara Falcomatà
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany
| | - Chuan Shan
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany
| | - Daniel Rossmeisl
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany
| | - Stella Koutsouli
- Institute of Medical Psychology, Faculty of Medicine, LMU Munich, Goethe Str. 31, 80336, Munich, Germany
| | - Christine Klement
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany.,Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675, Munich, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Munich, Munich, Germany
| | - Magdalena Zukowska
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany
| | - Sebastian A Widholz
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany.,Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675, Munich, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Munich, Munich, Germany
| | - Moritz Jesinghaus
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany.,Institute of Pathology, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Institute of Pathology, University Hospital Marburg, Baldingerstraße, 35043, Marburg, Germany
| | - Konstanze K Heuermann
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany
| | - Thomas Engleitner
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany.,Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675, Munich, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Munich, Munich, Germany
| | - Barbara Seidler
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany
| | - Katia Sleiman
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany
| | - Katja Steiger
- Institute of Pathology, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany
| | - Markus Tschurtschenthaler
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany
| | - Benjamin Walter
- Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany
| | - Sören A Weidemann
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany
| | - Regina Pietsch
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany
| | - Angelika Schnieke
- Livestock Biotechnology, School of Life Sciences, Technische Universität München, Liesel-Beckmann Str. 1, 85354, Freising, Germany
| | - Roland M Schmid
- Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany
| | - Maria S Robles
- Institute of Medical Psychology, Faculty of Medicine, LMU Munich, Goethe Str. 31, 80336, Munich, Germany
| | - Geoffroy Andrieux
- Institute of Medical Bioinformatics and Systems Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 79110, Freiburg, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Freiburg, 79106, Freiburg, Germany
| | - Melanie Boerries
- Institute of Medical Bioinformatics and Systems Medicine, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 79110, Freiburg, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Freiburg, 79106, Freiburg, Germany
| | - Roland Rad
- Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany.,Institute of Molecular Oncology and Functional Genomics, School of Medicine, Technische Universität München, 81675, Munich, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Partner Site Munich, Munich, Germany
| | - Günter Schneider
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany.,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany.,University Medical Center Göttingen, Department of General, Visceral and Pediatric Surgery, 37075, Göttingen, Germany
| | - Dieter Saur
- Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. .,Chair of Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany. .,Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81675, Munich, Germany. .,Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675, Munich, Germany.
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10
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Ganley RP, de Sousa MM, Werder K, Öztürk T, Mendes R, Ranucci M, Wildner H, Zeilhofer HU. Targeted anatomical and functional identification of antinociceptive and pronociceptive serotonergic neurons that project to the spinal dorsal horn. eLife 2023; 12:78689. [PMID: 36752606 PMCID: PMC9943064 DOI: 10.7554/elife.78689] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2022] [Accepted: 02/06/2023] [Indexed: 02/09/2023] Open
Abstract
Spinally projecting serotonergic neurons play a key role in controlling pain sensitivity and can either increase or decrease nociception depending on physiological context. It is currently unknown how serotonergic neurons mediate these opposing effects. Utilizing virus-based strategies and Tph2-Cre transgenic mice, we identified two anatomically separated populations of serotonergic hindbrain neurons located in the lateral paragigantocellularis (LPGi) and the medial hindbrain, which respectively innervate the superficial and deep spinal dorsal horn and have contrasting effects on sensory perception. Our tracing experiments revealed that serotonergic neurons of the LPGi were much more susceptible to transduction with spinally injected AAV2retro vectors than medial hindbrain serotonergic neurons. Taking advantage of this difference, we employed intersectional chemogenetic approaches to demonstrate that activation of the LPGi serotonergic projections decreases thermal sensitivity, whereas activation of medial serotonergic neurons increases sensitivity to mechanical von Frey stimulation. Together these results suggest that there are functionally distinct classes of serotonergic hindbrain neurons that differ in their anatomical location in the hindbrain, their postsynaptic targets in the spinal cord, and their impact on nociceptive sensitivity. The LPGi neurons that give rise to rather global and bilateral projections throughout the rostrocaudal extent of the spinal cord appear to be ideally poised to contribute to widespread systemic pain control.
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Affiliation(s)
- Robert Philip Ganley
- Institute for Pharmacology and Toxicology, University of ZurichZurichSwitzerland
| | | | - Kira Werder
- Institute for Pharmacology and Toxicology, University of ZurichZurichSwitzerland
| | - Tugce Öztürk
- Institute for Pharmacology and Toxicology, University of ZurichZurichSwitzerland
| | - Raquel Mendes
- Institute for Pharmacology and Toxicology, University of ZurichZurichSwitzerland
| | - Matteo Ranucci
- Institute for Pharmacology and Toxicology, University of ZurichZurichSwitzerland
| | - Hendrik Wildner
- Institute for Pharmacology and Toxicology, University of ZurichZurichSwitzerland
| | - Hanns Ulrich Zeilhofer
- Institute for Pharmacology and Toxicology, University of ZurichZurichSwitzerland,Institute of Pharmaceutical Sciences, Swiss Federal Institute of TechnologyZurichSwitzerland
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11
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Ozawa M, Taguchi J, Katsuma K, Ishikawa-Yamauchi Y, Kikuchi M, Sakamoto R, Yamada Y, Ikawa M. Efficient simultaneous double DNA knock-in in murine embryonic stem cells by CRISPR/Cas9 ribonucleoprotein-mediated circular plasmid targeting for generating gene-manipulated mice. Sci Rep 2022; 12:21558. [PMID: 36513736 PMCID: PMC9748034 DOI: 10.1038/s41598-022-26107-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Accepted: 12/09/2022] [Indexed: 12/15/2022] Open
Abstract
Gene targeting of embryonic stem (ES) cells followed by chimera production has been conventionally used for developing gene-manipulated mice. Although direct knock-in (KI) using murine zygote via CRISPR/Cas9-mediated genome editing has been reported, ES cell targeting still has merits, e.g., high throughput work can be performed in vitro. In this study, we first compared the KI efficiency of mouse ES cells with CRISPR/Cas9 expression vector and ribonucleoprotein (RNP), and confirmed that KI efficiency was significantly increased by using RNP. Using CRISPR/Cas9 RNP and circular plasmid with homologous arms as a targeting vector, knock-in within ES cell clones could be obtained efficiently without drug selection, thus potentially shortening the vector construction or cell culture period. Moreover, by incorporating a drug-resistant cassette into the targeting vectors, double DNA KI can be simultaneously achieved at high efficiency by a single electroporation. This technique will help to facilitate the production of genetically modified mouse models that are fundamental for exploring topics related to human and mammalian biology.
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Affiliation(s)
- Manabu Ozawa
- grid.26999.3d0000 0001 2151 536XLaboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Jumpei Taguchi
- grid.26999.3d0000 0001 2151 536XDivision of Stem Cell Pathology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Kento Katsuma
- grid.26999.3d0000 0001 2151 536XLaboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Yu Ishikawa-Yamauchi
- grid.26999.3d0000 0001 2151 536XLaboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Mio Kikuchi
- grid.26999.3d0000 0001 2151 536XDivision of Stem Cell Pathology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Reiko Sakamoto
- grid.26999.3d0000 0001 2151 536XDivision of Stem Cell Pathology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Yasuhiro Yamada
- grid.26999.3d0000 0001 2151 536XDivision of Stem Cell Pathology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan
| | - Masahito Ikawa
- grid.26999.3d0000 0001 2151 536XLaboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639 Japan ,grid.136593.b0000 0004 0373 3971Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871 Japan
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12
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Abstract
Cortical circuit tracing using modified rabies virus can identify input neurons making direct monosynaptic connections onto neurons of interest. However, challenges remain in our ability to establish the cell type identity of rabies-labeled input neurons. While transcriptomics may offer an avenue to characterize inputs, the extent of rabies-induced transcriptional changes in distinct neuronal cell types remains unclear, and whether these changes preclude characterization of rabies-infected neurons according to established transcriptomic cell types is unknown. We used single-nucleus RNA sequencing to survey the gene expression profiles of rabies-infected neurons and assessed their correspondence with established transcriptomic cell types. We demonstrated that when using transcriptome-wide RNA profiles, rabies-infected cortical neurons can be transcriptomically characterized despite global and cell-type-specific rabies-induced transcriptional changes. Notably, we found differential modulation of neuronal marker gene expression, suggesting that caution should be taken when attempting to characterize rabies-infected cells with single genes or small gene sets.
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13
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Jacobsen RI, Nair RR, Obenhaus HA, Donato F, Slettmoen T, Moser MB, Moser EI. All-viral tracing of monosynaptic inputs to single birthdate-defined neurons in the intact brain. CELL REPORTS METHODS 2022; 2:100221. [PMID: 35637903 PMCID: PMC9142754 DOI: 10.1016/j.crmeth.2022.100221] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Revised: 02/14/2022] [Accepted: 04/26/2022] [Indexed: 11/25/2022]
Abstract
Neuronal firing patterns are the result of inputs converging onto single cells. Identifying these inputs, anatomically and functionally, is essential to understand how neurons integrate information. Single-cell electroporation of helper genes and subsequent local injection of recombinant rabies viruses enable precise mapping of inputs to individual cells in superficial layers of the intact cortex. However, access to neurons in deeper structures requires more invasive procedures, including removal of overlying tissue. We developed a method that, through a combination of virus injections, allows us to target 4 or fewer hippocampal cells 48% of the time and a single cell 16% of the time in wild-type mice without use of electroporation or tissue aspiration. We identify local and distant monosynaptic inputs that can be functionally characterized in vivo. By expanding the toolbox for monosynaptic circuit tracing, this method will help further our understanding of neuronal integration at the level of single cells.
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Affiliation(s)
- R. Irene Jacobsen
- Kavli Institute for Systems Neuroscience and Centre for Neural Computation, NTNU, Trondheim 7030, Norway
| | - Rajeevkumar R. Nair
- Kavli Institute for Systems Neuroscience and Centre for Neural Computation, NTNU, Trondheim 7030, Norway
| | - Horst A. Obenhaus
- Kavli Institute for Systems Neuroscience and Centre for Neural Computation, NTNU, Trondheim 7030, Norway
| | - Flavio Donato
- Kavli Institute for Systems Neuroscience and Centre for Neural Computation, NTNU, Trondheim 7030, Norway
| | - Torstein Slettmoen
- Kavli Institute for Systems Neuroscience and Centre for Neural Computation, NTNU, Trondheim 7030, Norway
| | - May-Britt Moser
- Kavli Institute for Systems Neuroscience and Centre for Neural Computation, NTNU, Trondheim 7030, Norway
| | - Edvard I. Moser
- Kavli Institute for Systems Neuroscience and Centre for Neural Computation, NTNU, Trondheim 7030, Norway
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14
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Han Z, Luo N, Kou J, Li L, Xu Z, Wei S, Wu Y, Wang J, Ye C, Lin K, Xu F. Brain-wide TVA compensation allows rabies virus to retrograde target cell-type-specific projection neurons. Mol Brain 2022; 15:13. [PMID: 35093138 PMCID: PMC8800268 DOI: 10.1186/s13041-022-00898-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2021] [Accepted: 01/16/2022] [Indexed: 11/21/2022] Open
Abstract
Retrograde tracers based on viral vectors are powerful tools for the imaging and manipulation of upstream neural networks projecting to a specific brain region, and they play important roles in structural and functional studies of neural circuits. However, currently reported retrograde viral tracers have many limitations, such as brain area selectivity or the inability to retrograde label genetically defined brain-wide projection neurons. To overcome these limitations, a new retrograde tracing method, AAV-PHP.eB assisted retrograde tracing systems (PARTS) based on rabies virus, was established through brain-wide TVA-dependent targeting using an AAV-PHP.eB that efficiently crosses the blood-brain barrier in C57BL/6 J mice, and complementation of EnvA-pseudotyped defective rabies virus that specifically recognizes the TVA receptor. Furthermore, combined with Cre transgenic mice, cell-type-specific PARTS (cPARTS) was developed, which can retrograde label genetically defined brain-wide projection neurons. Our research provides new tools and technical support for the analysis of neural circuits.
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Affiliation(s)
- Zengpeng Han
- Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, Wuhan, 430071, People's Republic of China
- University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China
- The Brain Cognition and Brain Disease Institute (BCBDI), Shenzhen Key Laboratory of Viral Vectors for Biomedicine, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, People's Republic of China
- Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, NMPA Key Laboratory for Research and Evaluation of Viral Vector Technology in Cell and Gene Therapy Medicinal Products, Shenzhen, Key Laboratory of Quality Control Technology for Virus-Based Therapeutics, Guangdong Provincial Medical Products Administration, Shenzhen, 518055, People's Republic of China
| | - Nengsong Luo
- Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, Wuhan, 430071, People's Republic of China
- The Brain Cognition and Brain Disease Institute (BCBDI), Shenzhen Key Laboratory of Viral Vectors for Biomedicine, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, People's Republic of China
- Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, NMPA Key Laboratory for Research and Evaluation of Viral Vector Technology in Cell and Gene Therapy Medicinal Products, Shenzhen, Key Laboratory of Quality Control Technology for Virus-Based Therapeutics, Guangdong Provincial Medical Products Administration, Shenzhen, 518055, People's Republic of China
- Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, 430074, People's Republic of China
| | - Jiaxin Kou
- Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, Wuhan, 430071, People's Republic of China
- Department of Pathophysiology, Key Lab of Neurological Disorder of Education Ministry, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China
| | - Lei Li
- Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, Wuhan, 430071, People's Republic of China
| | - Zihong Xu
- College of Life Sciences, Wuhan University, Wuhan, People's Republic of China
| | - Siyuan Wei
- HongYi Honor College, Wuhan University, Wuhan, People's Republic of China
| | - Yang Wu
- Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, Wuhan, 430071, People's Republic of China
| | - Jie Wang
- Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, Wuhan, 430071, People's Republic of China
- University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China
| | - Chaohui Ye
- Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, Wuhan, 430071, People's Republic of China
- University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China
| | - Kunzhang Lin
- The Brain Cognition and Brain Disease Institute (BCBDI), Shenzhen Key Laboratory of Viral Vectors for Biomedicine, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, People's Republic of China.
- Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, NMPA Key Laboratory for Research and Evaluation of Viral Vector Technology in Cell and Gene Therapy Medicinal Products, Shenzhen, Key Laboratory of Quality Control Technology for Virus-Based Therapeutics, Guangdong Provincial Medical Products Administration, Shenzhen, 518055, People's Republic of China.
| | - Fuqiang Xu
- Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, Wuhan, 430071, People's Republic of China.
- University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China.
- The Brain Cognition and Brain Disease Institute (BCBDI), Shenzhen Key Laboratory of Viral Vectors for Biomedicine, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, People's Republic of China.
- Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, NMPA Key Laboratory for Research and Evaluation of Viral Vector Technology in Cell and Gene Therapy Medicinal Products, Shenzhen, Key Laboratory of Quality Control Technology for Virus-Based Therapeutics, Guangdong Provincial Medical Products Administration, Shenzhen, 518055, People's Republic of China.
- Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, 430074, People's Republic of China.
- Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, 518055, People's Republic of China.
- Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, 200031, People's Republic of China.
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15
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Mouse Lines with Cre-Mediated Recombination in Retinal Amacrine Cells. eNeuro 2022; 9:ENEURO.0255-21.2021. [PMID: 35045975 PMCID: PMC8856716 DOI: 10.1523/eneuro.0255-21.2021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2021] [Revised: 12/02/2021] [Accepted: 12/02/2021] [Indexed: 11/21/2022] Open
Abstract
Amacrine cells (ACs) are the most diverse neuronal cell type in the vertebrate retina. Yet little is known about the contribution of ACs to visual processing and retinal disease. A major challenge in evaluating AC function is genetic accessibility. A classic tool of mouse genetics, Cre-mediated recombination, can provide such access. We have screened existing genetically-modified mouse strains and identified multiple candidates that express Cre-recombinase in subsets of retinal ACs. The Cre-expressing mice were crossed to fluorescent-reporter mice to assay Cre expression. In addition, a Cre-dependent fluorescent reporter plasmid was electroporated into the subretinal space of Cre strains. Herein, we report three mouse lines (Tac1::IRES-cre, Camk2a-cre, and Scx-cre) that express Cre recombinase in sub-populations of ACs. In two of these lines, recombination occurred in multiple AC types and a small number of other retinal cell types, while recombination in the Camk2a-cre line appears specific to a morphologically distinct AC. We anticipate that these characterized mouse lines will be valuable tools to the community of researchers who study retinal biology and disease.
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16
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Grimes WN, Aytürk DG, Hoon M, Yoshimatsu T, Gamlin C, Carrera D, Nath A, Nadal-Nicolás FM, Ahlquist RM, Sabnis A, Berson DM, Diamond JS, Wong RO, Cepko C, Rieke F. A High-Density Narrow-Field Inhibitory Retinal Interneuron with Direct Coupling to Müller Glia. J Neurosci 2021; 41:6018-6037. [PMID: 34083252 PMCID: PMC8276741 DOI: 10.1523/jneurosci.0199-20.2021] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Revised: 05/16/2021] [Accepted: 05/17/2021] [Indexed: 11/21/2022] Open
Abstract
Amacrine cells are interneurons composing the most diverse cell class in the mammalian retina. They help encode visual features, such as edges or directed motion, by mediating excitatory and inhibitory interactions between input (i.e., bipolar) and output (i.e., ganglion) neurons in the inner plexiform layer (IPL). Like other brain regions, the retina also contains glial cells that contribute to neurotransmitter uptake, metabolic regulation, and neurovascular control. Here, we report that, in mouse retina (of either sex), an abundant, though previously unstudied inhibitory amacrine cell is coupled directly to Müller glia. Electron microscopic reconstructions of this amacrine type revealed chemical synapses with known retinal cell types and extensive associations with Müller glia, the processes of which often completely ensheathe the neurites of this amacrine cell. Microinjecting small tracer molecules into the somas of these amacrine cells led to selective labeling of nearby Müller glia, leading us to suggest the name "Müller glia-coupled amacrine cell," or MAC. Our data also indicate that MACs release glycine at conventional chemical synapses, and viral retrograde transsynaptic tracing from the dorsal lateral geniculate nucleus showed selective connections between MACs and a subpopulation of retinal ganglion cell types. Visually evoked responses revealed a strong preference for light increments; these "ON" responses were primarily mediated by excitatory chemical synaptic input and direct electrical coupling with other cells. This initial characterization of the MAC provides the first evidence for neuron-glia coupling in the mammalian retina and identifies the MAC as a potential link between inhibitory processing and glial function.SIGNIFICANCE STATEMENT Gap junctions between pairs of neurons or glial cells are commonly found throughout the nervous system and play multiple roles, including electrical coupling and metabolic exchange. In contrast, gap junctions between neurons and glia cells have rarely been reported and are poorly understood. Here we report the first evidence for neuron-glia coupling in the mammalian retina, specifically between an abundant (but previously unstudied) inhibitory interneuron and Müller glia. Moreover, viral tracing, optogenetics, and serial electron microscopy provide new information about the neuron's synaptic partners and physiological responses.
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Affiliation(s)
- William N Grimes
- Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195
- National Institute of Neurological Disease and Stroke, National Institutes of Health, Bethesda, Maryland 20892
| | - Didem Göz Aytürk
- Harvard Medical School, Blavatnik Institute, Howard Hughes Medical Institute, Boston, Massachusetts 02115
| | - Mrinalini Hoon
- Department of Biological Structure, University of Washington, Seattle, Washington 98195
| | - Takeshi Yoshimatsu
- Department of Biological Structure, University of Washington, Seattle, Washington 98195
| | - Clare Gamlin
- Department of Biological Structure, University of Washington, Seattle, Washington 98195
| | - Daniel Carrera
- National Institute of Neurological Disease and Stroke, National Institutes of Health, Bethesda, Maryland 20892
| | - Amurta Nath
- National Institute of Neurological Disease and Stroke, National Institutes of Health, Bethesda, Maryland 20892
| | - Francisco M Nadal-Nicolás
- Retinal Neurophysiology Section, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892
| | - Richard M Ahlquist
- Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195
| | - Adit Sabnis
- National Institute of Neurological Disease and Stroke, National Institutes of Health, Bethesda, Maryland 20892
| | - David M Berson
- Department of Neuroscience, Brown University, Providence, Rhode Island 02912
| | - Jeffrey S Diamond
- National Institute of Neurological Disease and Stroke, National Institutes of Health, Bethesda, Maryland 20892
| | - Rachel O Wong
- Department of Biological Structure, University of Washington, Seattle, Washington 98195
| | - Connie Cepko
- Harvard Medical School, Blavatnik Institute, Howard Hughes Medical Institute, Boston, Massachusetts 02115
| | - Fred Rieke
- Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195
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17
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Visani M, Acquaviva G, De Leo A, Sanza V, Merlo L, Maloberti T, Brandes AA, Franceschi E, Di Battista M, Masetti M, Jovine E, Fiorino S, Pession A, Tallini G, de Biase D. Molecular alterations in pancreatic tumors. World J Gastroenterol 2021; 27:2710-2726. [PMID: 34135550 PMCID: PMC8173386 DOI: 10.3748/wjg.v27.i21.2710] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/17/2021] [Revised: 02/25/2021] [Accepted: 04/14/2021] [Indexed: 02/06/2023] Open
Abstract
Genetic alterations in pancreatic tumors can usually be classified in: (1) Mutational activation of oncogenes; (2) Inactivation of tumor suppressor genes; and (3) Inactivation of genome maintenance genes controlling the repair of DNA damage. Endoscopic ultrasound-guided fine-needle aspiration has improved pre-operative diagnosis, but the management of patients with a pancreatic lesion is still challenging. Molecular testing could help mainly in solving these "inconclusive" specimens. The introduction of multi-gene analysis approaches, such as next-generation sequencing, has provided a lot of useful information on the molecular characterization of pancreatic tumors. Different types of pancreatic tumors (e.g., pancreatic ductal adenocarcinomas, intraductal papillary mucinous neoplasms, solid pseudopapillary tumors) are characterized by specific molecular alterations. The aim of this review is to summarize the main molecular alterations found in pancreatic tumors.
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Affiliation(s)
- Michela Visani
- Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna–Molecular Diagnostic Unit, Azienda USL di Bologna, Bologna 40138, Italy
| | - Giorgia Acquaviva
- Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna–Molecular Diagnostic Unit, Azienda USL di Bologna, Bologna 40138, Italy
| | - Antonio De Leo
- Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna–Molecular Diagnostic Unit, Azienda USL di Bologna, Bologna 40138, Italy
- Division of Molecular Pathology Laboratory, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy
| | - Viviana Sanza
- Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna–Molecular Diagnostic Unit, Azienda USL di Bologna, Bologna 40138, Italy
| | - Lidia Merlo
- Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna–Molecular Diagnostic Unit, Azienda USL di Bologna, Bologna 40138, Italy
| | - Thais Maloberti
- Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna–Molecular Diagnostic Unit, Azienda USL di Bologna, Bologna 40138, Italy
| | - Alba A Brandes
- Medical Oncology Department, Azienda USL/IRCCS Istituto Delle Scienze Neurologiche di Bologna, Bologna 40139, Italy
| | - Enrico Franceschi
- Medical Oncology Department, Azienda USL/IRCCS Istituto Delle Scienze Neurologiche di Bologna, Bologna 40139, Italy
| | - Monica Di Battista
- Medical Oncology Department, Azienda USL/IRCCS Istituto Delle Scienze Neurologiche di Bologna, Bologna 40139, Italy
| | - Michele Masetti
- Division of Surgery, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40133, Italy
| | - Elio Jovine
- Division of Surgery, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40133, Italy
| | - Sirio Fiorino
- Internal Medicine Unit, Budrio Hospital Azienda USL, Bologna 40133, Italy
| | - Annalisa Pession
- Division of Molecular Pathology Laboratory, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy
- Department of Pharmacy and Biotechnology, University of Bologna, Bologna 40138, Italy
| | - Giovanni Tallini
- Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna–Molecular Diagnostic Unit, Azienda USL di Bologna, Bologna 40138, Italy
- Division of Molecular Pathology Laboratory, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy
| | - Dario de Biase
- Division of Molecular Pathology Laboratory, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna 40138, Italy
- Department of Pharmacy and Biotechnology, University of Bologna, Bologna 40138, Italy
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18
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Maheshwari U, Kraus D, Vilain N, Holwerda SJB, Cankovic V, Maiorano NA, Kohler H, Satoh D, Sigrist M, Arber S, Kratochwil CF, Di Meglio T, Ducret S, Rijli FM. Postmitotic Hoxa5 Expression Specifies Pontine Neuron Positional Identity and Input Connectivity of Cortical Afferent Subsets. Cell Rep 2021; 31:107767. [PMID: 32553152 DOI: 10.1016/j.celrep.2020.107767] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2019] [Revised: 03/18/2020] [Accepted: 05/21/2020] [Indexed: 12/20/2022] Open
Abstract
The mammalian precerebellar pontine nucleus (PN) has a main role in relaying cortical information to the cerebellum. The molecular determinants establishing ordered connectivity patterns between cortical afferents and precerebellar neurons are largely unknown. We show that expression of Hox5 transcription factors is induced in specific subsets of postmitotic PN neurons at migration onset. Hox5 induction is achieved by response to retinoic acid signaling, resulting in Jmjd3-dependent derepression of Polycomb chromatin and 3D conformational changes. Hoxa5 drives neurons to settle posteriorly in the PN, where they are monosynaptically targeted by cortical neuron subsets mainly carrying limb somatosensation. Furthermore, Hoxa5 postmigratory ectopic expression in PN neurons is sufficient to attract cortical somatosensory inputs regardless of position and avoid visual afferents. Transcriptome analysis further suggests that Hoxa5 is involved in circuit formation. Thus, Hoxa5 coordinates postmitotic specification, migration, settling position, and sub-circuit assembly of PN neuron subsets in the cortico-cerebellar pathway.
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Affiliation(s)
- Upasana Maheshwari
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, 4051 Basel, Switzerland
| | - Dominik Kraus
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, 4051 Basel, Switzerland
| | - Nathalie Vilain
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Sjoerd J B Holwerda
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Vanja Cankovic
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Nicola A Maiorano
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Hubertus Kohler
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Daisuke Satoh
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; Biozentrum, University of Basel, Kingelbergstrasse 70, 4056 Basel, Switzerland
| | - Markus Sigrist
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; Biozentrum, University of Basel, Kingelbergstrasse 70, 4056 Basel, Switzerland
| | - Silvia Arber
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; Biozentrum, University of Basel, Kingelbergstrasse 70, 4056 Basel, Switzerland
| | - Claudius F Kratochwil
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Thomas Di Meglio
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Sebastien Ducret
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Filippo M Rijli
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, 4051 Basel, Switzerland.
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19
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Chang CH, Pauklin S. ROS and TGFβ: from pancreatic tumour growth to metastasis. J Exp Clin Cancer Res 2021; 40:152. [PMID: 33941245 PMCID: PMC8091747 DOI: 10.1186/s13046-021-01960-4] [Citation(s) in RCA: 50] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2021] [Accepted: 04/22/2021] [Indexed: 02/06/2023] Open
Abstract
Transforming growth factor β (TGFβ) signalling pathway switches between anti-tumorigenic function at early stages of cancer formation and pro-tumorigenic effects at later stages promoting cancer metastasis. A similar contrasting role has been uncovered for reactive oxygen species (ROS) in pancreatic tumorigenesis. Down-regulation of ROS favours premalignant tumour development, while increasing ROS level in pancreatic ductal adenocarcinoma (PDAC) enhances metastasis. Given the functional resemblance, we propose that ROS-mediated processes converge with the spatial and temporal activation of TGFβ signalling and thereby differentially impact early tumour growth versus metastatic dissemination. TGFβ signalling and ROS could extensively orchestrate cellular processes and this concerted function can be utilized by cancer cells to facilitate their malignancy. In this article, we revisit the interplay of canonical and non-canonical TGFβ signalling with ROS throughout pancreatic tumorigenesis and metastasis. We also discuss recent insight that helps to understand their conflicting effects on different stages of tumour development. These considerations open new strategies in cancer therapeutics.
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Affiliation(s)
- Chao-Hui Chang
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Windmill Road, OX3 7LD, Oxford, UK
| | - Siim Pauklin
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Windmill Road, OX3 7LD, Oxford, UK.
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20
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Besnard A, Miller SM, Sahay A. Distinct Dorsal and Ventral Hippocampal CA3 Outputs Govern Contextual Fear Discrimination. Cell Rep 2021; 30:2360-2373.e5. [PMID: 32075769 PMCID: PMC7050277 DOI: 10.1016/j.celrep.2020.01.055] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2019] [Revised: 11/05/2019] [Accepted: 01/17/2020] [Indexed: 01/12/2023] Open
Abstract
Considerable work emphasizes a role for hippocampal circuits in governing contextual fear discrimination. However· the intra- and extrahippocampal pathways that route contextual information to cortical and subcortical circuits to guide adaptive behavioral responses are poorly understood. Using terminal-specific optogenetic silencing in a contextual fear discrimination learning paradigm· we identify opposing roles for dorsal CA3-CA1 (dCA3-dCA1) projections and dorsal CA3-dorsolateral septum (dCA3-DLS) projections in calibrating fear responses to certain and ambiguous contextual threats· respectively. Ventral CA3-DLS (vCA3-DLS) projections suppress fear responses in both certain and ambiguous contexts· whereas ventral CA3-CA1 (vCA3-vCA1) projections promote fear responses in both these contexts. Lastly· using retrograde monosynaptic tracing· ex vivo electrophysiological recordings· and optogenetics,· we identify a sparse population of DLS parvalbumin (PV) neurons as putative relays of dCA3-DLS projections to diverse subcortical circuits. Taken together· these studies illuminate how distinct dCA3 and vCA3 outputs calibrate contextual fear discrimination. Besnard et al. show that dorsal and ventral hippocampal CA3 projections to CA1 and dorsolateral septum (DLS) play distinct roles in calibration of contextual fear discrimination. DLS parvalbumin inhibitory neurons receive monosynaptic dorsal CA3 inputs and modulate fear responses in a context-specific manner.
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Affiliation(s)
- Antoine Besnard
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Department of Psychiatry, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Samara M Miller
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Department of Psychiatry, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Amar Sahay
- Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Department of Psychiatry, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA; BROAD Institute of Harvard and MIT, Cambridge, MA 02142, USA.
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21
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Kitazawa T, Machlab D, Joshi O, Maiorano N, Kohler H, Ducret S, Kessler S, Gezelius H, Soneson C, Papasaikas P, López-Bendito G, Stadler MB, Rijli FM. A unique bipartite Polycomb signature regulates stimulus-response transcription during development. Nat Genet 2021; 53:379-391. [PMID: 33603234 PMCID: PMC7610396 DOI: 10.1038/s41588-021-00789-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2020] [Accepted: 01/19/2021] [Indexed: 01/31/2023]
Abstract
Rapid cellular responses to environmental stimuli are fundamental for development and maturation. Immediate early genes can be transcriptionally induced within minutes in response to a variety of signals. How their induction levels are regulated and their untimely activation by spurious signals prevented during development is poorly understood. We found that in developing sensory neurons, before perinatal sensory-activity-dependent induction, immediate early genes are embedded into a unique bipartite Polycomb chromatin signature, carrying active H3K27ac on promoters but repressive Ezh2-dependent H3K27me3 on gene bodies. This bipartite signature is widely present in developing cell types, including embryonic stem cells. Polycomb marking of gene bodies inhibits mRNA elongation, dampening productive transcription, while still allowing for fast stimulus-dependent mark removal and bipartite gene induction. We reveal a developmental epigenetic mechanism regulating the rapidity and amplitude of the transcriptional response to relevant stimuli, while preventing inappropriate activation of stimulus-response genes.
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Affiliation(s)
- Taro Kitazawa
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
| | - Dania Machlab
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland,Swiss Institute of Bioinformatics, Basel, Switzerland,University of Basel, Basel, Switzerland
| | - Onkar Joshi
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
| | - Nicola Maiorano
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
| | - Hubertus Kohler
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
| | - Sebastien Ducret
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
| | - Sandra Kessler
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
| | - Henrik Gezelius
- Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas (UMH-CSIC), Sant Joan d’Alacant, Spain
| | - Charlotte Soneson
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland,Swiss Institute of Bioinformatics, Basel, Switzerland
| | - Panagiotis Papasaikas
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland,Swiss Institute of Bioinformatics, Basel, Switzerland
| | - Guillermina López-Bendito
- Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas (UMH-CSIC), Sant Joan d’Alacant, Spain
| | - Michael B. Stadler
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland,Swiss Institute of Bioinformatics, Basel, Switzerland
| | - Filippo M. Rijli
- Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland,University of Basel, Basel, Switzerland,Correspondence to:
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22
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Kanvinde PP, Malla AP, Connolly NP, Szulzewsky F, Anastasiadis P, Ames HM, Kim AJ, Winkles JA, Holland EC, Woodworth GF. Leveraging the replication-competent avian-like sarcoma virus/tumor virus receptor-A system for modeling human gliomas. Glia 2021; 69:2059-2076. [PMID: 33638562 PMCID: PMC8591561 DOI: 10.1002/glia.23984] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2021] [Revised: 02/15/2021] [Accepted: 02/16/2021] [Indexed: 12/20/2022]
Abstract
Gliomas are the most common primary intrinsic brain tumors occurring in adults. Of all malignant gliomas, glioblastoma (GBM) is considered the deadliest tumor type due to diffuse brain invasion, immune evasion, cellular, and molecular heterogeneity, and resistance to treatments resulting in high rates of recurrence. An extensive understanding of the genomic and microenvironmental landscape of gliomas gathered over the past decade has renewed interest in pursuing novel therapeutics, including immune checkpoint inhibitors, glioma-associated macrophage/microglia (GAMs) modulators, and others. In light of this, predictive animal models that closely recreate the conditions and findings found in human gliomas will serve an increasingly important role in identifying new, effective therapeutic strategies. Although numerous syngeneic, xenograft, and transgenic rodent models have been developed, few include the full complement of pathobiological features found in human tumors, and therefore few accurately predict bench-to-bedside success. This review provides an update on how genetically engineered rodent models based on the replication-competent avian-like sarcoma (RCAS) virus/tumor virus receptor-A (tv-a) system have been used to recapitulate key elements of human gliomas in an immunologically intact host microenvironment and highlights new approaches using this model system as a predictive tool for advancing translational glioma research.
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Affiliation(s)
- Pranjali P Kanvinde
- Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, USA.,Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Adarsha P Malla
- Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, USA.,Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Nina P Connolly
- Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, USA.,Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Frank Szulzewsky
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
| | - Pavlos Anastasiadis
- Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, USA.,Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Heather M Ames
- Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA.,Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Anthony J Kim
- Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, USA.,Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Jeffrey A Winkles
- Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, USA.,Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA.,Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, USA.,Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Eric C Holland
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.,Seattle Tumor Translational Research Center, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
| | - Graeme F Woodworth
- Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, Maryland, USA.,Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA
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23
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Zheng X, Salinas KJ, Velez DXF, Nakayama T, Lin X, Banerjee D, Xu X, Gandhi SP. Host interneurons mediate plasticity reactivated by embryonic inhibitory cell transplantation in mouse visual cortex. Nat Commun 2021; 12:862. [PMID: 33558487 PMCID: PMC7870960 DOI: 10.1038/s41467-021-21097-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2020] [Accepted: 01/07/2021] [Indexed: 11/12/2022] Open
Abstract
The adult brain lacks sensitivity to changes in the sensory environment found in the juvenile brain. The transplantation of embryonic interneurons has been shown to restore juvenile plasticity to the adult host visual cortex. It is unclear whether transplanted interneurons directly mediate the renewed cortical plasticity or whether these cells act indirectly by modifying the host interneuron circuitry. Here we find that the transplant-induced reorganization of mouse host circuits is specifically mediated by Neuregulin (NRG1)/ErbB4 signaling in host parvalbumin (PV) interneurons. Brief visual deprivation reduces the visual activity of host PV interneurons but has negligible effects on the responses of transplanted PV interneurons. Exogenous NRG1 both prevents the deprivation-induced reduction in the visual responses of host PV interneurons and blocks the transplant-induced reorganization of the host circuit. While deletion of ErbB4 receptors from host PV interneurons blocks cortical plasticity in the transplant recipients, deletion of the receptors from the donor PV interneurons does not. Altogether, our results indicate that transplanted embryonic interneurons reactivate cortical plasticity by rejuvenating the function of host PV interneurons.
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Affiliation(s)
- XiaoTing Zheng
- Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA, 92697-2715, USA
| | - Kirstie J Salinas
- Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA, 92697-2715, USA
| | - Dario X Figueroa Velez
- Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA, 92697-2715, USA
| | - Taylor Nakayama
- Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA, 92697-2715, USA
| | - Xiaoxiao Lin
- Department of Anatomy and Neurobiology, University of California, Irvine, Irvine, CA, 92697-2715, USA
| | - Dhruba Banerjee
- Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA, 92697-2715, USA
| | - Xiangmin Xu
- Department of Anatomy and Neurobiology, University of California, Irvine, Irvine, CA, 92697-2715, USA
- Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, 92697-2715, USA
- Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA, 92697-2715, USA
- Center for the Neurobiology of Learning and Memory, University of California, Irvine, Irvine, CA, 92697-2715, USA
| | - Sunil P Gandhi
- Department of Neurobiology and Behavior, University of California, Irvine, Irvine, CA, 92697-2715, USA.
- Center for the Neurobiology of Learning and Memory, University of California, Irvine, Irvine, CA, 92697-2715, USA.
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24
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Frezel N, Platonova E, Voigt FF, Mateos JM, Kastli R, Ziegler U, Karayannis T, Helmchen F, Wildner H, Zeilhofer HU. In-Depth Characterization of Layer 5 Output Neurons of the Primary Somatosensory Cortex Innervating the Mouse Dorsal Spinal Cord. Cereb Cortex Commun 2020; 1:tgaa052. [PMID: 34296117 PMCID: PMC8152836 DOI: 10.1093/texcom/tgaa052] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2020] [Revised: 07/07/2020] [Accepted: 08/09/2020] [Indexed: 12/26/2022] Open
Abstract
Neuronal circuits of the spinal dorsal horn integrate sensory information from the periphery with inhibitory and facilitating input from higher central nervous system areas. Most previous work focused on projections descending from the hindbrain. Less is known about inputs descending from the cerebral cortex. Here, we identified cholecystokinin (CCK) positive layer 5 pyramidal neurons of the primary somatosensory cortex (CCK + S1-corticospinal tract [CST] neurons) as a major source of input to the spinal dorsal horn. We combined intersectional genetics and virus-mediated gene transfer to characterize CCK+ S1-CST neurons and to define their presynaptic input and postsynaptic target neurons. We found that S1-CST neurons constitute a heterogeneous population that can be subdivided into distinct molecular subgroups. Rabies-based retrograde tracing revealed monosynaptic input from layer 2/3 pyramidal neurons, from parvalbumin positive cortical interneurons, and from thalamic relay neurons in the ventral posterolateral nucleus. Wheat germ agglutinin-based anterograde tracing identified postsynaptic target neurons in dorsal horn laminae III and IV. About 60% of these neurons were inhibitory and about 60% of all spinal target neurons expressed the transcription factor c-Maf. The heterogeneous nature of both S1-CST neurons and their spinal targets suggest complex roles in the fine-tuning of sensory processing.
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Affiliation(s)
- N Frezel
- Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zürich, Switzerland
| | - E Platonova
- Center for Microscopy and Image Analysis, University of Zurich, CH-8057 Zürich CH-8057, Switzerland
| | - F F Voigt
- Brain Research Institute, University of Zurich, CHJ-8057 Zurich CH-8057 , Switzerland.,Neuroscience Center Zurich, University of Zurich and ETH Zurich, CH-8057 Zurich CH-8057, Switzerland
| | - J M Mateos
- Center for Microscopy and Image Analysis, University of Zurich, CH-8057 Zürich CH-8057, Switzerland
| | - R Kastli
- Brain Research Institute, University of Zurich, CHJ-8057 Zurich CH-8057 , Switzerland.,Neuroscience Center Zurich, University of Zurich and ETH Zurich, CH-8057 Zurich CH-8057, Switzerland
| | - U Ziegler
- Center for Microscopy and Image Analysis, University of Zurich, CH-8057 Zürich CH-8057, Switzerland
| | - T Karayannis
- Brain Research Institute, University of Zurich, CHJ-8057 Zurich CH-8057 , Switzerland.,Neuroscience Center Zurich, University of Zurich and ETH Zurich, CH-8057 Zurich CH-8057, Switzerland
| | - F Helmchen
- Brain Research Institute, University of Zurich, CHJ-8057 Zurich CH-8057 , Switzerland.,Neuroscience Center Zurich, University of Zurich and ETH Zurich, CH-8057 Zurich CH-8057, Switzerland
| | - H Wildner
- Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zürich, Switzerland
| | - H U Zeilhofer
- Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zürich, Switzerland.,Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH Zürich), CH-8090 Zürich, Switzerland
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25
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Salaymeh Y, Farago M, Sebban S, Shalom B, Pikarsky E, Katzav S. Vav1 and mutant K-Ras synergize in the early development of pancreatic ductal adenocarcinoma in mice. Life Sci Alliance 2020; 3:e202000661. [PMID: 32277014 PMCID: PMC7156281 DOI: 10.26508/lsa.202000661] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2020] [Revised: 03/20/2020] [Accepted: 03/23/2020] [Indexed: 02/06/2023] Open
Abstract
To explore the contribution of Vav1, a hematopoietic signal transducer, to pancreatic ductal adenocarcinoma (PDAC) development, we generated transgenic mouse lines expressing, Vav1, K-RasG12D, or both K-RasG12D and Vav1 in pancreatic acinar cells. Co-expression of Vav1 and K-RasG12D synergistically enhanced acinar-to-ductal metaplasia (ADM) formation, far exceeding the number of lesions developed in K-RasG12D mice. Mice expressing only Vav1 did not develop ADM. Moreover, the incidence of PDAC in K-RasG12D/Vav1 was significantly higher than in K-RasG12D mice. Discontinuing Vav1 expression in K-RasG12D/Vav1 mice elicited a marked regression of malignant lesions in the pancreas, demonstrating Vav1 is required for generation and maintenance of ADM. Rac1-GTP levels in the K-RasG12D/Vav1 mice pancreas clearly demonstrated an increase in Rac1 activity. Treatment of K-RasG12D and K-RasG12D/Vav1 mice with azathioprine, an immune-suppressor drug which inhibits Vav1's activity as a GDP/GTP exchange factor, dramatically reduced the number of malignant lesions. These results suggest that Vav1 plays a role in the development of PDAC when co-expressed with K-RasG12D via its activity as a GEF for Rac1GTPase.
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Affiliation(s)
- Yaser Salaymeh
- Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem, Israel
| | - Marganit Farago
- Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem, Israel
| | - Shulamit Sebban
- Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem, Israel
| | - Batel Shalom
- Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem, Israel
| | - Eli Pikarsky
- The Lautenberg Center for Immunology and Cancer Research and Department of Pathology, Institute for Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem, Israel
| | - Shulamit Katzav
- Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem, Israel
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26
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Lavin TK, Jin L, Lea NE, Wickersham IR. Monosynaptic Tracing Success Depends Critically on Helper Virus Concentrations. Front Synaptic Neurosci 2020; 12:6. [PMID: 32116642 PMCID: PMC7033752 DOI: 10.3389/fnsyn.2020.00006] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2019] [Accepted: 01/23/2020] [Indexed: 11/13/2022] Open
Abstract
Monosynaptically-restricted transsynaptic tracing using deletion-mutant rabies virus (RV) has become a widely used technique in neuroscience, allowing identification, imaging, and manipulation of neurons directly presynaptic to a starting neuronal population. Its most common implementation is to use Cre mouse lines in combination with Cre-dependent "helper" adeno-associated viral vectors (AAVs) to supply the required genes to the targeted population before subsequent injection of a first-generation (ΔG) rabies viral vector. Here we show that the efficiency of transsynaptic spread and the degree of nonspecific labeling in wild-type control animals depend strongly on the concentrations of these helper AAVs. Our results suggest practical guidelines for achieving good results.
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Affiliation(s)
| | | | | | - Ian R. Wickersham
- McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, United States
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27
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Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors. Proc Natl Acad Sci U S A 2019; 116:27001-27010. [PMID: 31843925 DOI: 10.1073/pnas.1915974116] [Citation(s) in RCA: 46] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
In combination with transgenic mouse lines expressing Cre or Flp recombinases in defined cell types, recombinase-dependent adeno-associated viruses (AAVs) have become the tool of choice for localized cell-type-targeted gene expression. Unfortunately, applications of this technique when expressing highly sensitive transgenes are impeded by off-target, or "leak" expression, from recombinase-dependent AAVs. We investigated this phenomenon and find that leak expression is mediated by both infrequent transcription from the inverted transgene in recombinant-dependent AAV designs and recombination events during bacterial AAV plasmid production. Recombination in bacteria is mediated by homology across the antiparallel recombinase-specific recognition sites present in recombinase-dependent designs. To address both of these issues we designed an AAV vector that uses mutant "cross-over insensitive" recognition sites combined with an "ATG-out" design. We show that these CIAO (cross-over insensitive ATG-out) vectors virtually eliminate leak expression. CIAO vectors provide reliable and targeted transgene expression and are extremely useful for recombinase-dependent expression of highly sensitive transgenes.
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28
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Li N, Yang G, Luo L, Ling L, Wang X, Shi L, Lan J, Jia X, Zhang Q, Long Z, Liu J, Hu W, He Z, Liu H, Liu W, Zheng G. lncRNA THAP9-AS1 Promotes Pancreatic Ductal Adenocarcinoma Growth and Leads to a Poor Clinical Outcome via Sponging miR-484 and Interacting with YAP. Clin Cancer Res 2019; 26:1736-1748. [PMID: 31831555 DOI: 10.1158/1078-0432.ccr-19-0674] [Citation(s) in RCA: 66] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2019] [Revised: 07/21/2019] [Accepted: 12/09/2019] [Indexed: 01/05/2023]
Abstract
PURPOSE Long noncoding RNAs (lncRNA) have been observed in various cancer types. Our bioinformatic analysis of existing databases demonstrated overexpression of lncRNA THAP9-AS1 in pancreatic ductal adenocarcinoma (PDAC). We aimed to investigate the roles and mechanisms of THAP9-AS1 in PDAC. EXPERIMENTAL DESIGN The overexpression of THAP9-AS1 in samples of patients with pancreatic cancer was characterized and was associated with clinical outcomes. The nonprotein coding property of the THAP9-AS1 was verified. Various in vitro and in vivo experiments were performed to investigate the interaction between THAP9-AS1 and YAP signaling. RESULTS We demonstrated that lncRNA THAP9-AS1 is overexpressed in PDAC in multiple patient sample sets, which is significantly associated with poor outcome of patients with PDAC. THAP9-AS1 promotes PDAC cells growth both in vitro and in vivo. THAP9-AS1 exerts its effects via enhancing YAP signaling. Ectopic YAP expression overcame the effects of THAP9-AS1 knockdown. Inversely, YAP knockdown diminished the effects of THAP9-AS1 overexpression. THAP9-AS1 acts as a competing endogenous RNA for miR-484, leading to YAP upregulation. Moreover, THAP9-AS1 binds to YAP protein and inhibits the phosphorylation-mediated inactivation of YAP by LATS1. Reciprocally, YAP/TEAD1 complex promotes THAP9-AS1 transcription to form a feed-forward circuit. Importantly, THAP9-AS1 level positively correlates with YAP expression in PDAC tissues. YAP overexpression also predicts a poor outcome in patients with PDAC. CONCLUSIONS Our findings indicate that THAP9-AS1 plays an important role in PDAC growth via enhancing YAP signaling, which in turn also modulates THAP9-AS1 transcription. THAP9-AS1/YAP axis may serve as a potential biomarker and therapeutic target for PDAC treatment.
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Affiliation(s)
- Nan Li
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Guohua Yang
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Liyun Luo
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Li Ling
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Xiaorong Wang
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Lejuan Shi
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Junsong Lan
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Xiaoting Jia
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Qiong Zhang
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Ze Long
- Department of Orthopedics, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Jinbao Liu
- Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, Guangdong, China
| | - Weimin Hu
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Zhimin He
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China
| | - Haiying Liu
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China.
| | - Wanqing Liu
- Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, Michigan. .,Department of Pharmacology, School of Medicine, Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, Michigan
| | - Guopei Zheng
- Affiliated Cancer Hospital & Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The State Key Laboratory of Respiratory, Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor Treatment," Guangzhou, Guangdong, China.
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29
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Lavin TK, Jin L, Wickersham IR. Monosynaptic tracing: a step-by-step protocol. J Chem Neuroanat 2019; 102:101661. [PMID: 31408693 PMCID: PMC11526806 DOI: 10.1016/j.jchemneu.2019.101661] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2019] [Revised: 07/01/2019] [Accepted: 07/14/2019] [Indexed: 01/25/2023]
Abstract
Monosynaptic tracing using deletion-mutant rabies virus allows whole-brain mapping of neurons that are directly presynaptic to a targeted population of neurons. The most common and robust way of implementing it is to use Cre mouse lines in combination with Cre-dependent adeno-associated viral vectors for expression of the required genes in the targeted neurons before subsequent injection of the rabies virus. Here we present a step-by-step protocol for performing such experiments using first-generation (ΔG) rabies viral vectors.
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Affiliation(s)
- Thomas K Lavin
- McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, United States
| | - Lei Jin
- McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, United States
| | - Ian R Wickersham
- McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, United States.
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30
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Sun Y, Jin S, Lin X, Chen L, Qiao X, Jiang L, Zhou P, Johnston KG, Golshani P, Nie Q, Holmes TC, Nitz DA, Xu X. CA1-projecting subiculum neurons facilitate object-place learning. Nat Neurosci 2019; 22:1857-1870. [PMID: 31548723 PMCID: PMC6819262 DOI: 10.1038/s41593-019-0496-y] [Citation(s) in RCA: 66] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2018] [Accepted: 08/09/2019] [Indexed: 11/09/2022]
Abstract
Recent anatomical evidence suggests a functionally significant back-projection pathway from the subiculum to the CA1. Here we show that the afferent circuitry of CA1-projecting subicular neurons is biased by inputs from CA1 inhibitory neurons and the visual cortex, but lacks input from the entorhinal cortex. Efferents of the CA1-projecting subiculum neurons also target the perirhinal cortex, an area strongly implicated in object-place learning. We identify a critical role for CA1-projecting subicular neurons in object-location learning and memory, and show that this projection modulates place-specific activity of CA1 neurons and their responses to displaced objects. Together, these experiments reveal a novel pathway by which cortical inputs, particularly those from the visual cortex, reach the hippocampal output region CA1. Our findings also implicate this circuitry in the formation of complex spatial representations and learning of object-place associations.
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Affiliation(s)
- Yanjun Sun
- Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, Irvine, CA, USA
- Department of Neurobiology, Stanford University School of Medicine, Stanford, CA, USA
| | - Suoqin Jin
- Department of Mathematics and Department of Developmental & Cell Biology, University of California, Irvine, Irvine, CA, USA
| | - Xiaoxiao Lin
- Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, Irvine, CA, USA
| | - Lujia Chen
- Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, Irvine, CA, USA
| | - Xin Qiao
- Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, Irvine, CA, USA
| | - Li Jiang
- Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, Irvine, CA, USA
| | - Pengcheng Zhou
- Department of Statistics and Center for Theoretical Neuroscience, Columbia University, New York, NY, USA
| | - Kevin G Johnston
- Department of Mathematics and Department of Developmental & Cell Biology, University of California, Irvine, Irvine, CA, USA
| | - Peyman Golshani
- Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
- West Los Angeles VA Medical Center, Los Angeles, CA, USA
| | - Qing Nie
- Department of Mathematics and Department of Developmental & Cell Biology, University of California, Irvine, Irvine, CA, USA
- Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA
| | - Todd C Holmes
- Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, Irvine, CA, USA
| | - Douglas A Nitz
- Department of Cognitive Science, University of California, San Diego, La Jolla, CA, USA.
| | - Xiangmin Xu
- Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, Irvine, CA, USA.
- Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA.
- Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA, USA.
- Department of Computer Science, University of California, Irvine, Irvine, CA, USA.
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31
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Acton D, Ren X, Di Costanzo S, Dalet A, Bourane S, Bertocchi I, Eva C, Goulding M. Spinal Neuropeptide Y1 Receptor-Expressing Neurons Form an Essential Excitatory Pathway for Mechanical Itch. Cell Rep 2019; 28:625-639.e6. [PMID: 31315043 PMCID: PMC6709688 DOI: 10.1016/j.celrep.2019.06.033] [Citation(s) in RCA: 65] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2018] [Revised: 05/20/2019] [Accepted: 06/06/2019] [Indexed: 01/08/2023] Open
Abstract
Acute itch can be generated by either chemical or mechanical stimuli, which activate separate pathways in the periphery and spinal cord. While substantial progress has been made in mapping the transmission pathway for chemical itch, the central pathway for mechanical itch remains obscure. Using complementary genetic and pharmacological manipulations, we show that excitatory neurons marked by the expression of the neuropeptide Y1 receptor (Y1Cre neurons) form an essential pathway in the dorsal spinal cord for the transmission of mechanical but not chemical itch. Ablating or silencing the Y1Cre neurons abrogates mechanical itch, while chemogenetic activation induces scratching. Moreover, using Y1 conditional knockout mice, we demonstrate that endogenous neuropeptide Y (NPY) acts via dorsal-horn Y1-expressing neurons to suppress light punctate touch and mechanical itch stimuli. NPY-Y1 signaling thus regulates the transmission of innocuous tactile information by establishing biologically relevant thresholds for touch discrimination and mechanical itch reflexes.
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Affiliation(s)
- David Acton
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Xiangyu Ren
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA; Biology Graduate Program, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
| | - Stefania Di Costanzo
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA; Biology Graduate Program, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
| | - Antoine Dalet
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Steeve Bourane
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Ilaria Bertocchi
- Department of Neuroscience, University of Torino, Neuroscience Institute of the Cavalieri-Ottolenghi Foundation, Regione Gonzole 1, 10043 Orbassano, Italy
| | - Carola Eva
- Department of Neuroscience, University of Torino, Neuroscience Institute of the Cavalieri-Ottolenghi Foundation, Regione Gonzole 1, 10043 Orbassano, Italy
| | - Martyn Goulding
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
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Albagli O, Maugein A, Huijbregts L, Bredel D, Carlier G, Martin P, Scharfmann R. New α- and SIN γ-retrovectors for safe transduction and specific transgene expression in pancreatic β cell lines. BMC Biotechnol 2019; 19:35. [PMID: 31208395 PMCID: PMC6580483 DOI: 10.1186/s12896-019-0531-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2018] [Accepted: 06/06/2019] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Viral vectors are invaluable tools to transfer genes and/or regulatory sequences into differentiated cells such as pancreatic cells. To date, several kinds of viral vectors have been used to transduce different pancreatic cell types, including insulin-producing β cells. However, few studies have used vectors derived from « simple » retroviruses, such as avian α- or mouse γ-retroviruses, despite their high experimental convenience. Moreover, such vectors were never designed to specifically target transgene expression into β cells. RESULTS We here describe two novel α- or SIN (Self-Inactivating) γ-retrovectors containing the RIP (Rat Insulin Promoter) as internal promoter. These two retrovectors are easily produced in standard BSL2 conditions, rapidly concentrated if needed, and harbor a large multiple cloning site. For the SIN γ-retrovector, either the VSV-G (pantropic) or the retroviral ecotropic (rodent specific) envelope was used. For the α-retrovector, we used the A type envelope, as its receptor, termed TVA, is only naturally present in avian cells and can efficiently be provided to mammalian β cells through either exogenous expression upon cDNA transfer or gesicle-mediated delivery of the protein. As expected, the transgenes cloned into the two RIP-containing retrovectors displayed a strong preferential expression in β over non-β cells compared to transgenes cloned in their non-RIP (CMV- or LTR-) regulated counterparts. We further show that RIP activity of both retrovectors mirrored fluctuations affecting endogenous INSULIN gene expression in human β cells. Finally, both α- and SIN γ-retrovectors were extremely poorly mobilized by the BXV1 xenotropic retrovirus, a common invader of human cells grown in immunodeficient mice, and, most notably, of human β cell lines. CONCLUSION Our novel α- and SIN γ-retrovectors are safe and convenient tools to stably and specifically express transgene(s) in mammalian β cells. Moreover, they both reproduce some regulatory patterns affecting INSULIN gene expression. Thus, they provide a helpful tool to both study the genetic control of β cell function and monitor changes in their differentiation status.
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Affiliation(s)
- Olivier Albagli
- INSERM U1016, CNRS UMR8104, Institut Cochin, Université Paris Descartes, 123 Boulevard de Port-Royal, 75014, Paris, France.
| | - Alicia Maugein
- INSERM U1016, CNRS UMR8104, Institut Cochin, Université Paris Descartes, 123 Boulevard de Port-Royal, 75014, Paris, France
| | - Lukas Huijbregts
- INSERM U1016, CNRS UMR8104, Institut Cochin, Université Paris Descartes, 123 Boulevard de Port-Royal, 75014, Paris, France
| | - Delphine Bredel
- INSERM U1016, CNRS UMR8104, Institut Cochin, Université Paris Descartes, 123 Boulevard de Port-Royal, 75014, Paris, France.,Present Address: Laboratoire de Recherche Translationnelle en Immunothérapie, Institut Gustave Roussy, 114 Rue Edouard Vaillant, 94800, Villejuif, France
| | - Géraldine Carlier
- INSERM U1016, CNRS UMR8104, Institut Cochin, Université Paris Descartes, 123 Boulevard de Port-Royal, 75014, Paris, France
| | - Patrick Martin
- Université Côte d'Azur, CNRS UMR7277 INSERM U1099, iBV (Institut de Biologie Valrose), Université Nice Sophia Antipolis, Bâtiment Sciences Naturelles; UFR Sciences, Parc Valrose, 28, avenue Valrose, 06108, Nice Cedex 2, France
| | - Raphaël Scharfmann
- INSERM U1016, CNRS UMR8104, Institut Cochin, Université Paris Descartes, 123 Boulevard de Port-Royal, 75014, Paris, France
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Prabhakar A, Vujovic D, Cui L, Olson W, Luo W. Leaky expression of channelrhodopsin-2 (ChR2) in Ai32 mouse lines. PLoS One 2019; 14:e0213326. [PMID: 30913225 PMCID: PMC6435231 DOI: 10.1371/journal.pone.0213326] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2019] [Accepted: 02/18/2019] [Indexed: 01/03/2023] Open
Abstract
Optogenetics enables the selective activation of genetically-targeted neuronal populations using light-sensitive ion channels. Genetic strategies using Cre-dependent mouse strains, especially the Ai32 line expressing Channelrhodopsin (ChR2)-EYFP fusion protein, have been a popular means to drive opsin expression in a cell-type specific manner. Here we report a low level of leaky ‘off-target’ (Cre-independent) ChR2-EYFP expression in Ai32/Ai32 homozygous mice throughout the nervous system. This leaky off-target expression was characterized in multiple prevalent nervous system regions using anti-EYFP immunostaining. Expression of full-length ChR2-EYFP protein was confirmed using immunoprecipitation followed by Western blotting. Notably, light stimulation of these ChR2-EYFP expressing neurons in the spinal cord dorsal horn did not induce detectable photocurrents in juvenile 4-week old mice. Given the wide use of the Ai32 line by many labs, our results suggest researchers should be vigilant of possible off-target ChR2-EYFP expression in their region of interest, especially when generating Ai32/Ai32 homozygotes to drive high levels of ChR2-EYFP expression in adult mice.
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Affiliation(s)
- Arthi Prabhakar
- University of Texas at Dallas, Dallas, TX, United States of America
| | - Dragan Vujovic
- Williams College, Williamstown, MA, United States of America
| | - Lian Cui
- Department of Neuroscience, University of Pennsylvania, Philadelphia, PA, United States of America
| | - William Olson
- Department of Neuroscience, University of Pennsylvania, Philadelphia, PA, United States of America
- * E-mail: (WO); (WL)
| | - Wenqin Luo
- Department of Neuroscience, University of Pennsylvania, Philadelphia, PA, United States of America
- * E-mail: (WO); (WL)
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Dorsolateral septum somatostatin interneurons gate mobility to calibrate context-specific behavioral fear responses. Nat Neurosci 2019; 22:436-446. [PMID: 30718902 PMCID: PMC6387640 DOI: 10.1038/s41593-018-0330-y] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2017] [Accepted: 12/17/2018] [Indexed: 12/29/2022]
Abstract
Adaptive fear responses to external threats rely upon efficient relay of computations underlying contextual encoding to subcortical circuits. Brain-wide analysis of highly coactivated ensembles following contextual fear discrimination identified the dorsolateral septum (DLS) as a relay of the dentate gyrus-CA3 circuit. Retrograde monosynaptic tracing and electrophysiological whole-cell recordings demonstrated that DLS somatostatin-expressing interneurons (SST-INs) receive direct CA3 inputs. Longitudinal in vivo calcium imaging of DLS SST-INs in awake, behaving mice identified a stable population of footshock-responsive SST-INs during contextual conditioning whose activity tracked and predicted non-freezing epochs during subsequent recall in the training context but not in a similar, neutral context or open field. Optogenetic attenuation or stimulation of DLS SST-INs bidirectionally modulated conditioned fear responses and recruited proximal and distal subcortical targets. Together, these observations suggest a role for a potentially hard-wired DLS SST-IN subpopulation as arbiters of mobility that calibrate context-appropriate behavioral fear responses.
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Abstract
Encoding and predicting aversive events are critical functions of circuits that support survival and emotional well-being. Maladaptive circuit changes in emotional valence processing can underlie the pathophysiology of affective disorders. The lateral habenula (LHb) has been linked to aversion and mood regulation through modulation of the dopamine and serotonin systems. We have defined the identity and function of glutamatergic (Vglut2) control of the LHb, comparing the role of inputs originating in the globus pallidus internal segment (GPi), and lateral hypothalamic area (LHA), respectively. We found that LHb-projecting LHA neurons, and not the proposed GABA/glutamate co-releasing GPi neurons, are responsible for encoding negative value. Monosynaptic rabies tracing of the presynaptic organization revealed a predominantly limbic input onto LHA Vglut2 neurons, while sensorimotor inputs were more prominent onto GABA/glutamate co-releasing GPi neurons. We further recorded the activity of LHA Vglut2 neurons, by imaging calcium dynamics in response to appetitive versus aversive events in conditioning paradigms. LHA Vglut2 neurons formed activity clusters representing distinct reward or aversion signals, including a population that responded to mild foot shocks and predicted aversive events. We found that the LHb-projecting LHA Vglut2 neurons encode negative valence and rapidly develop a prediction signal for negative events. These findings establish the glutamatergic LHA-LHb circuit as a critical node in value processing.
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Progressive divisions of multipotent neural progenitors generate late-born chandelier cells in the neocortex. Nat Commun 2018; 9:4595. [PMID: 30389944 PMCID: PMC6214958 DOI: 10.1038/s41467-018-07055-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2018] [Accepted: 10/02/2018] [Indexed: 01/12/2023] Open
Abstract
Diverse γ-aminobutyric acid (GABA)-ergic interneurons provide different modes of inhibition to support circuit operation in the neocortex. However, the cellular and molecular mechanisms underlying the systematic generation of assorted neocortical interneurons remain largely unclear. Here we show that NKX2.1-expressing radial glial progenitors (RGPs) in the mouse embryonic ventral telencephalon divide progressively to generate distinct groups of interneurons, which occupy the neocortex in a time-dependent, early inside-out and late outside-in, manner. Notably, the late-born chandelier cells, one of the morphologically and physiologically highly distinguishable GABAergic interneurons, arise reliably from continuously dividing RGPs that produce non-chandelier cells initially. Selective removal of Partition defective 3, an evolutionarily conserved cell polarity protein, impairs RGP asymmetric cell division, resulting in premature depletion of RGPs towards the late embryonic stages and a consequent loss of chandelier cells. These results suggest that consecutive asymmetric divisions of multipotent RGPs generate diverse neocortical interneurons in a progressive manner. Diverse GABAergic neurons arise from progenitors in the medial ganglionic eminence. Here, the authors show these progenitors are progressively fate-restricted, with early-born interneurons occupying cortex in an “inside-out” pattern and later-born types like chandelier cells generated “outside-in”.
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Sikdar N, Saha G, Dutta A, Ghosh S, Shrikhande SV, Banerjee S. Genetic Alterations of Periampullary and Pancreatic Ductal Adenocarcinoma: An Overview. Curr Genomics 2018; 19:444-463. [PMID: 30258276 PMCID: PMC6128383 DOI: 10.2174/1389202919666180221160753] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2017] [Revised: 01/31/2018] [Accepted: 02/01/2018] [Indexed: 02/06/2023] Open
Abstract
Pancreatic Ductal AdenoCarcinoma (PDAC) is one of the most lethal malignancies of all solid cancers. Precancerous lesions for PDAC include PanIN, IPMNs and MCNs. PDAC has a poor prognosis with a 5-year survival of approximately 6%. Whereas Periampulary AdenoCarcinoma (PAC) having four anatomic subtypes, pancreatic, Common Bile Duct (CBD), ampullary and duodenum shows relative better prognosis. The highest incidence of PDAC has been reported with black with respect to white population. Similarly, incidence rate of PAC also differs with different ethnic populations. Several lifestyle, environmental and occupational exposures including long-term diabetes, obesity, and smoking, have been linked to PDAC, however, for PAC the causal risk factors were poorly described. It is now clear that PDAC and PAC are a multi-stage process resulting from the accumulation of genomic alterations in the somatic DNA of normal cells as well as inherited mutations. Approximately 10% of PDAC have a familial inheritance. Germline mutations in CDKN2A, BRCA2, STK11, PALB2, PRSS1, etc., as well as certain syndromes have been well associated with predisposition to PDAC. KRAS, CDKN2A, TP53 and SMAD4 are the 4 "mountains" (high-frequency driver genes) which have been known to earliest somatic alterations for PDAC while relatively less frequent in PAC. Our understanding of the molecular carcinogenesis has improved in the last few years due to extensive research on PDAC which was not well explored in case of PAC. The genetic alterations that have been identified in PDAC and different subgroups of PAC are important implications for the development of genetic screening test, early diagnosis, and prognostic genetic markers. The present review will provide a brief overview of the incidence and prevalence of PDAC and PAC, mainly, increased risk in India, the several kinds of risk factors associated with the diseases as well as required genetic alterations for disease initiation and progression.
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Affiliation(s)
- Nilabja Sikdar
- Address correspondence to this author at the Human Genetics Unit, Indian Statistical Institute, 203, B.T. Road Kolkata 700108, India; Tel (1): +91-33
-25773240 (L); (2): +91-9830780397 (M); Fax: +91 33 35773049;, E-mail:
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Ralvenius WT, Neumann E, Pagani M, Acuña MA, Wildner H, Benke D, Fischer N, Rostaher A, Schwager S, Detmar M, Frauenknecht K, Aguzzi A, Hubbs JL, Rudolph U, Favrot C, Zeilhofer HU. Itch suppression in mice and dogs by modulation of spinal α2 and α3GABA A receptors. Nat Commun 2018; 9:3230. [PMID: 30104684 PMCID: PMC6089996 DOI: 10.1038/s41467-018-05709-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2018] [Accepted: 07/20/2018] [Indexed: 11/10/2022] Open
Abstract
Chronic itch is a highly debilitating condition affecting about 10% of the general population. The relay of itch signals is under tight control by inhibitory circuits of the spinal dorsal horn, which may offer a hitherto unexploited therapeutic opportunity. Here, we found that specific pharmacological targeting of inhibitory α2 and α3GABAA receptors reduces acute histaminergic and non-histaminergic itch in mice. Systemic treatment with an α2/α3GABAA receptor selective modulator alleviates also chronic itch in a mouse model of atopic dermatitis and in dogs sensitized to house dust mites, without inducing sedation, motor dysfunction, or loss of antipruritic activity after prolonged treatment. Transsynaptic circuit tracing, immunofluorescence, and electrophysiological experiments identify spinal α2 and α3GABAA receptors as likely molecular targets underlying the antipruritic effect. Our results indicate that drugs targeting α2 and α3GABAA receptors are well-suited to alleviate itch, including non-histaminergic chronic itch for which currently no approved treatment exists.
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Affiliation(s)
- William T Ralvenius
- Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
| | - Elena Neumann
- Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
| | - Martina Pagani
- Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland.,Neuroscience Center Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
| | - Mario A Acuña
- Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
| | - Hendrik Wildner
- Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
| | - Dietmar Benke
- Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland.,Neuroscience Center Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland.,Drug Discovery Network Zürich (DDNZ), Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
| | - Nina Fischer
- Dermatology Department, Clinic for Small Animal Internal Medicine, Vetsuisse Faculty, Winterthurerstrasse 260, CH-8057, Zürich, Switzerland
| | - Ana Rostaher
- Dermatology Department, Clinic for Small Animal Internal Medicine, Vetsuisse Faculty, Winterthurerstrasse 260, CH-8057, Zürich, Switzerland
| | - Simon Schwager
- Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zürich, Vladimir-Prelog-Weg 1-5/10, CH-8093, Zürich, Switzerland
| | - Michael Detmar
- Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zürich, Vladimir-Prelog-Weg 1-5/10, CH-8093, Zürich, Switzerland
| | - Katrin Frauenknecht
- Institute of Neuropathology, University of Zürich and University Hospital Zürich, Schmelzbergstrasse 12, CH-8091, Zürich, Switzerland
| | - Adriano Aguzzi
- Institute of Neuropathology, University of Zürich and University Hospital Zürich, Schmelzbergstrasse 12, CH-8091, Zürich, Switzerland
| | - Jed Lee Hubbs
- Laboratory of Organic Chemistry, Swiss Federal Institute of Technology (ETH) Zürich, Vladimir-Prelog-Weg 1-5/10, CH-8093, Zürich, Switzerland
| | - Uwe Rudolph
- Laboratory of Genetic Neuropharmacology, McLean Hospital, 115 Mill Street, Belmont, MA, 02478, USA.,Department of Psychiatry, Harvard Medical School, 401 Park Drive, Boston, MA, 02215, USA
| | - Claude Favrot
- Dermatology Department, Clinic for Small Animal Internal Medicine, Vetsuisse Faculty, Winterthurerstrasse 260, CH-8057, Zürich, Switzerland
| | - Hanns Ulrich Zeilhofer
- Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland. .,Neuroscience Center Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland. .,Drug Discovery Network Zürich (DDNZ), Winterthurerstrasse 190, CH-8057, Zürich, Switzerland. .,Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zürich, Vladimir-Prelog-Weg 1-5/10, CH-8093, Zürich, Switzerland.
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Inducible transgenic expression of tripeptidyl peptidase 1 in a mouse model of late-infantile neuronal ceroid lipofuscinosis. PLoS One 2018; 13:e0192286. [PMID: 29408933 PMCID: PMC5800698 DOI: 10.1371/journal.pone.0192286] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2017] [Accepted: 01/22/2018] [Indexed: 12/02/2022] Open
Abstract
Late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease of children caused by mutations resulting in loss of activity of the lysosomal protease, tripeptidyl peptidase 1 (TPP1). While Tpp1-targeted mouse models of LINCL exist, the goal of this study was to create a transgenic mouse with inducible TPP1 to benchmark treatment approaches, evaluate efficacy of treatment at different stages of disease, and to provide insights into the pathobiology of disease. A construct containing a loxP-flanked stop cassette inserted between the chicken-actin promoter and a sequence encoding murine TPP1 (TgLSL-TPP1) was integrated into the ROSA26 locus in mice by homologous recombination. Tested in both transfected CHO cells and in transgenic mice, the TgLSL-TPP1 did not express TPP1 until cre-mediated removal of the LSL cassette, which resulted in supraphysiological levels of TPP1 activity. We tested four cre/ERT2 transgenes to allow tamoxifen-inducible removal of the LSL cassette and subsequent TPP1 expression at any stage of disease. However, two of the cre/ERT2 driver transgenes had significant cre activity in the absence of tamoxifen, while cre-mediated recombination could not be induced by tamoxifen by two others. These results highlight potential problems with the use of cre/ERT2 transgenes in applications that are sensitive to low levels of basal cre expression. However, the germline-recombined mouse transgenic that constitutively overexpresses TPP1 will allow long-term evaluation of overexposure to the enzyme and in cell culture, the inducible transgene may be a useful tool in biomarker discovery projects.
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Opposing and Complementary Topographic Connectivity Gradients Revealed by Quantitative Analysis of Canonical and Noncanonical Hippocampal CA1 Inputs. eNeuro 2018; 5:eN-NWR-0322-17. [PMID: 29387780 PMCID: PMC5790753 DOI: 10.1523/eneuro.0322-17.2018] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2017] [Revised: 01/08/2018] [Accepted: 01/09/2018] [Indexed: 01/07/2023] Open
Abstract
Physiological studies suggest spatial representation gradients along the CA1 proximodistal axis. To determine the underlying anatomical basis, we quantitatively mapped canonical and noncanonical inputs to excitatory neurons in dorsal hippocampal CA1 along the proximal-distal axis in mice of both sexes using monosynaptic rabies tracing. Our quantitative analyses show comparable strength of subiculum complex and entorhinal cortex (EC) inputs to CA1, significant inputs from presubiculum and parasubiculum to CA1, and a threefold stronger input to proximal versus distal CA1 from CA3. Noncanonical subicular complex inputs exhibit opposing topographic connectivity gradients whereby the subiculum-CA1 input strength systematically increases but the presubiculum-CA1 input strength decreases along the proximal-distal axis. The subiculum input strength cotracks that of the lateral EC, known to be less spatially selective than the medial EC. The functional significance of this organization is verified physiologically for subiculum-to-CA1 inputs. These results reveal a novel anatomical framework by which to determine the circuit bases for CA1 representations.
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Gadek KE, Wang H, Hall MN, Sungello M, Libby A, MacLaskey D, Eckel RH, Olwin BB. Striated muscle gene therapy for the treatment of lipoprotein lipase deficiency. PLoS One 2018; 13:e0190963. [PMID: 29304082 PMCID: PMC5755938 DOI: 10.1371/journal.pone.0190963] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2017] [Accepted: 12/22/2017] [Indexed: 11/19/2022] Open
Abstract
Excessive circulating triglycerides due to reduction or loss of lipoprotein lipase activity contribute to hypertriglyceridemia and increased risk for pancreatitis. The only gene therapy treatment for lipoprotein lipase deficiency decreases pancreatitis but minimally reduces hypertriglyceridemia. Synthesized in multiple tissues including striated muscle and adipose tissue, lipoprotein lipase is trafficked to blood vessel endothelial cells where it is anchored at the plasma membrane and hydrolyzes triglycerides into free fatty acids. We conditionally knocked out lipoprotein lipase in differentiated striated muscle tissue lowering striated muscle lipoprotein lipase activity causing hypertriglyceridemia. We then crossed lipoprotein lipase striated muscle knockout mice with mice possessing a conditional avian retroviral receptor gene and injected mice with either a human lipoprotein lipase retrovirus or an mCherry control retrovirus. Post-heparin plasma lipoprotein lipase activity increased for three weeks following human lipoprotein lipase retroviral infection compared to mCherry infected mice. Human lipoprotein lipase infected mice had significantly lower blood triglycerides compared to mCherry controls and were comparable to wild-type blood triglyceride levels. Thus, targeted delivery of human lipoprotein lipase into striated muscle tissue identifies a potential therapeutic target for lipoprotein lipase deficiency.
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Affiliation(s)
- Katherine E. Gadek
- Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado United States of America
| | - Hong Wang
- Division of Endocrinology, Metabolism, and Diabetes, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado United States of America
| | - Monica N. Hall
- Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado United States of America
| | - Mitchell Sungello
- Division of Endocrinology, Metabolism, and Diabetes, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado United States of America
| | - Andrew Libby
- Division of Endocrinology, Metabolism, and Diabetes, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado United States of America
| | - Drew MacLaskey
- Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado United States of America
| | - Robert H. Eckel
- Division of Endocrinology, Metabolism, and Diabetes, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado United States of America
- * E-mail: (BBO); (RHE)
| | - Bradley B. Olwin
- Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado United States of America
- * E-mail: (BBO); (RHE)
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Koch SC, Del Barrio MG, Dalet A, Gatto G, Günther T, Zhang J, Seidler B, Saur D, Schüle R, Goulding M. RORβ Spinal Interneurons Gate Sensory Transmission during Locomotion to Secure a Fluid Walking Gait. Neuron 2017; 96:1419-1431.e5. [PMID: 29224725 PMCID: PMC5828033 DOI: 10.1016/j.neuron.2017.11.011] [Citation(s) in RCA: 75] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2017] [Revised: 09/27/2017] [Accepted: 11/08/2017] [Indexed: 01/17/2023]
Abstract
Animals depend on sensory feedback from mechanosensory afferents for the dynamic control of movement. This sensory feedback needs to be selectively modulated in a task- and context-dependent manner. Here, we show that inhibitory interneurons (INs) expressing the RORβ orphan nuclear receptor gate sensory feedback to the spinal motor system during walking and are required for the production of a fluid locomotor rhythm. Genetic manipulations that abrogate inhibitory RORβ IN function result in an ataxic gait characterized by exaggerated flexion movements and marked alterations to the step cycle. Inactivation of RORβ in inhibitory neurons leads to reduced presynaptic inhibition and changes to sensory-evoked reflexes, arguing that the RORβ inhibitory INs function to suppress the sensory transmission pathways that activate flexor motor reflexes and interfere with the ongoing locomotor program. VIDEO ABSTRACT.
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Affiliation(s)
- Stephanie C Koch
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Marta Garcia Del Barrio
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Antoine Dalet
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Graziana Gatto
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Thomas Günther
- Urologische Klinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, Breisacherstrasse 66, 79106 Freiburg, Germany
| | - Jingming Zhang
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Barbara Seidler
- Department of Medicine II, School of Medicine, Klinikum rechts der Isar, Technische Universität München, Ismaningerstr. 22, 81675 München, Germany
| | - Dieter Saur
- Department of Medicine II, School of Medicine, Klinikum rechts der Isar, Technische Universität München, Ismaningerstr. 22, 81675 München, Germany; Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
| | - Roland Schüle
- Urologische Klinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, Breisacherstrasse 66, 79106 Freiburg, Germany
| | - Martyn Goulding
- Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
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Garz AK, Wolf S, Grath S, Gaidzik V, Habringer S, Vick B, Rudelius M, Ziegenhain C, Herold S, Weickert MT, Smets M, Peschel C, Oostendorp RAJ, Bultmann S, Jeremias I, Thiede C, Döhner K, Keller U, Götze KS. Azacitidine combined with the selective FLT3 kinase inhibitor crenolanib disrupts stromal protection and inhibits expansion of residual leukemia-initiating cells in FLT3-ITD AML with concurrent epigenetic mutations. Oncotarget 2017; 8:108738-108759. [PMID: 29312564 PMCID: PMC5752477 DOI: 10.18632/oncotarget.21877] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2017] [Accepted: 09/20/2017] [Indexed: 12/18/2022] Open
Abstract
Effectively targeting leukemia-initiating cells (LIC) in FLT3-ITD-mutated acute myeloid leukemia (AML) is crucial for cure. Tyrosine kinase inhibitors (TKI) have limited impact as single agents, failing to eradicate LIC in the bone marrow. Using primary AML samples and a patient-derived xenograft model, we investigated whether combining the FLT3-selective TKI crenolanib with the hypomethylating agent azacitidine (AZA) eliminates FLT3-ITD LIC and whether efficacy of this combination depends on co-existing mutations. Using multiparameter flow cytometry, we show FLT3-ITD occurs within the most primitive Lin-/CD33(+)/CD45dim/CD34+CD38- LIC compartment. Crenolanib alone could not target FLT3-ITD LIC in contact with niche cells while addition of AZA overcame stromal protection resulting in dramatically reduced clonogenic capacity of LIC in vitro and severely impaired engraftment in NSG mice. Strikingly, FLT3-mutated samples harboring TET2 mutations were completely resistant to crenolanib whereas neither NPM1 nor DNMT3A mutations influenced response. Conversely, primary AML LIC harboring either TET2, DNMT3A or NPM1 mutations did not show increased sensitivity to AZA. In summary, resistance of FLT3-ITD LIC to TKI depends on co-existing epigenetic mutations. However, AZA + crenolanib effectively abrogates stromal protection and inhibits survival of FLT3-ITD LIC irrespective of mutations, providing evidence for this combination as a means to suppress residual LIC.
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Affiliation(s)
- Anne-Kathrin Garz
- Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Saskia Wolf
- Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany
| | - Sonja Grath
- Department of Biology II, Ludwig-Maximilians-University (LMU), Munich, Germany
| | - Verena Gaidzik
- Department of Internal Medicine III, University of Ulm, Ulm, Germany
| | - Stefan Habringer
- Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Binje Vick
- German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.,Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany
| | - Martina Rudelius
- Department of Pathology, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany
| | | | - Sylvia Herold
- German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.,Department of Internal Medicine I, Gustav Carus University Dresden, Dresden, Germany
| | - Marie-Theresa Weickert
- Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany
| | - Martha Smets
- Department of Biology II, Ludwig-Maximilians-University (LMU), Munich, Germany
| | - Christian Peschel
- Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Robert A J Oostendorp
- Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany
| | - Sebastian Bultmann
- Department of Biology II, Ludwig-Maximilians-University (LMU), Munich, Germany
| | - Irmela Jeremias
- German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.,Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany
| | - Christian Thiede
- German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany.,Department of Internal Medicine I, Gustav Carus University Dresden, Dresden, Germany
| | - Konstanze Döhner
- Department of Internal Medicine III, University of Ulm, Ulm, Germany
| | - Ulrich Keller
- Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Katharina S Götze
- Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany.,German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany
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44
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Sultan KT, Han Z, Zhang XJ, Xianyu A, Li Z, Huang K, Shi SH. Clonally Related GABAergic Interneurons Do Not Randomly Disperse but Frequently Form Local Clusters in the Forebrain. Neuron 2017; 92:31-44. [PMID: 27710787 DOI: 10.1016/j.neuron.2016.09.033] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Revised: 08/30/2016] [Accepted: 09/07/2016] [Indexed: 11/16/2022]
Abstract
Progenitor cells in the medial ganglionic eminence (MGE) and preoptic area (PoA) give rise to GABAergic inhibitory interneurons that are distributed in the forebrain, largely in the cortex, hippocampus, and striatum. Two previous studies suggest that clonally related interneurons originating from individual MGE/PoA progenitors frequently form local clusters in the cortex. However, Mayer et al. and Harwell et al. recently argued that MGE/PoA-derived interneuron clones disperse widely and populate different forebrain structures. Here, we report further analysis of the spatial distribution of clonally related interneurons and demonstrate that interneuron clones do not non-specifically disperse in the forebrain. Around 70% of clones are restricted to one brain structure, predominantly the cortex. Moreover, the regional distribution of clonally related interneurons exhibits a clear clustering feature, which cannot occur by chance from a random diffusion. These results confirm that lineage relationship influences the spatial distribution of inhibitory interneurons in the forebrain. This Matters Arising paper is in response to Harwell et al. (2015) and Mayer et al. (2015), published in Neuron. See also the response by Turrero García et al. (2016) and Mayer et al. (2016), published in this issue.
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Affiliation(s)
- Khadeejah T Sultan
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA; Neuroscience Graduate Program, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
| | - Zhi Han
- Department of Biomedical Informatics, Ohio State University, Columbus, OH 43210, USA; College of Software, Nankai University, Tianjin 300071, PR China
| | - Xin-Jun Zhang
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
| | - Anjin Xianyu
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA; Physiology, Biophysics, and Systems Biology Graduate Program, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
| | - Zhizhong Li
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
| | - Kun Huang
- Department of Biomedical Informatics, Ohio State University, Columbus, OH 43210, USA.
| | - Song-Hai Shi
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA; Neuroscience Graduate Program, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA.
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45
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Identification of Two Classes of Somatosensory Neurons That Display Resistance to Retrograde Infection by Rabies Virus. J Neurosci 2017; 37:10358-10371. [PMID: 28951448 PMCID: PMC5656993 DOI: 10.1523/jneurosci.1277-17.2017] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2017] [Revised: 08/29/2017] [Accepted: 09/11/2017] [Indexed: 12/25/2022] Open
Abstract
Glycoprotein-deleted rabies virus-mediated monosynaptic tracing has become a standard method for neuronal circuit mapping, and is applied to virtually all parts of the rodent nervous system, including the spinal cord and primary sensory neurons. Here we identified two classes of unmyelinated sensory neurons (nonpeptidergic and C-fiber low-threshold mechanoreceptor neurons) resistant to direct and trans-synaptic infection from the spinal cord with rabies viruses that carry glycoproteins in their envelopes and that are routinely used for infection of CNS neurons (SAD-G and N2C-G). However, the same neurons were susceptible to infection with EnvA-pseudotyped rabies virus in tumor virus A receptor transgenic mice, indicating that resistance to retrograde infection was due to impaired virus adsorption rather than to deficits in subsequent steps of infection. These results demonstrate an important limitation of rabies virus-based retrograde tracing of sensory neurons in adult mice, and may help to better understand the molecular machinery required for rabies virus spread in the nervous system. In this study, mice of both sexes were used. SIGNIFICANCE STATEMENT To understand the neuronal bases of behavior, it is important to identify the underlying neural circuitry. Rabies virus-based monosynaptic tracing has been used to identify neuronal circuits in various parts of the nervous system. This has included connections between peripheral sensory neurons and their spinal targets. These connections form the first synapse in the somatosensory pathway. Here we demonstrate that two classes of unmyelinated sensory neurons, which account for >40% of dorsal root ganglia neurons, display resistance to rabies infection. Our results are therefore critical for interpreting monosynaptic rabies-based tracing in the sensory system. In addition, identification of rabies-resistant neurons might provide a means for future studies addressing rabies pathobiology.
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Fabbri C, Gibiino G, Fornelli A, Cennamo V, Grifoni D, Visani M, Acquaviva G, Fassan M, Fiorino S, Giovanelli S, Bassi M, Ghersi S, Tallini G, Jovine E, Gasbarrini A, de Biase D. Team work and cytopathology molecular diagnosis of solid pancreatic lesions. Dig Endosc 2017; 29:657-666. [PMID: 28190274 DOI: 10.1111/den.12845] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/30/2016] [Accepted: 02/08/2017] [Indexed: 02/05/2023]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer-associated death in the next decade or so. It is widely accepted that tumorigenesis is linked to specific alterations in key genes and pancreatic neoplasms are some of the best characterized at the genomic level. Recent whole-exome and whole-genome sequencing analyses confirmed that PDAC is frequently characterized by mutations in a set of four genes among others: KRAS, TP53, CDKN2A/p16, and SMAD4. Sequencing, for example, is the preferable technique available for detecting KRAS mutations, whereas in situ immunochemistry is the main approach for detecting TP53 gene alteration. Nevertheless, the diagnosis of PDAC is still a clinical challenge, involving adequate acquisition of endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) and specific pathological assessment from tissue architecture to specific biomolecular tests. The aim of the present review is to provide a complete overview of the current knowledge of the biology of pancreatic cancer as detected by the latest biomolecular techniques and, moreover, to propose a paradigm for strict teamwork collaboration in order to improve the correct use of diagnostic sources.
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Affiliation(s)
- Carlo Fabbri
- Unit of Gastroenterology and Digestive Endoscopy, AUSL Bologna Bellaria-Maggiore Hospital, Italy
| | - Giulia Gibiino
- Medical Pathology, Department of Internal Medicine, Gastroenterology Division, Policlinico Universitario A. Gemelli, Catholic University of Sacred Heart, Rome, Italy
| | - Adele Fornelli
- Anatomic Pathology Unit, AUSL of Bologna, Maggiore Hospital, Italy
| | - Vincenzo Cennamo
- Unit of Gastroenterology and Digestive Endoscopy, AUSL Bologna Bellaria-Maggiore Hospital, Italy
| | - Daniela Grifoni
- Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Italy
| | - Michela Visani
- Department of Medicine (DIMES), Molecular Diagnostic Unit AUSL of Bologna, University of Bologna School of Medicine, Italy
| | - Giorgia Acquaviva
- Department of Medicine (DIMES), Molecular Diagnostic Unit AUSL of Bologna, University of Bologna School of Medicine, Italy
| | - Matteo Fassan
- Department of Medicine, Anatomic Pathology, University of Padua, Padova, Italy
| | - Sirio Fiorino
- Internal Medicine Unit, Maggiore Hospital, Bologna, Italy
| | - Silvia Giovanelli
- Unit of Gastroenterology and Digestive Endoscopy, AUSL Bologna Bellaria-Maggiore Hospital, Italy
| | - Marco Bassi
- Unit of Gastroenterology and Digestive Endoscopy, AUSL Bologna Bellaria-Maggiore Hospital, Italy
| | - Stefania Ghersi
- Unit of Gastroenterology and Digestive Endoscopy, AUSL Bologna Bellaria-Maggiore Hospital, Italy
| | - Giovanni Tallini
- Department of Medicine (DIMES), Molecular Diagnostic Unit AUSL of Bologna, University of Bologna School of Medicine, Italy
| | - Elio Jovine
- Unit of Gastroenterology and Digestive Endoscopy, AUSL Bologna Bellaria-Maggiore Hospital, Italy
| | - Antonio Gasbarrini
- Medical Pathology, Department of Internal Medicine, Gastroenterology Division, Policlinico Universitario A. Gemelli, Catholic University of Sacred Heart, Rome, Italy
| | - Dario de Biase
- Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Italy
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47
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Zhang XJ, Li Z, Han Z, Sultan KT, Huang K, Shi SH. Precise inhibitory microcircuit assembly of developmentally related neocortical interneurons in clusters. Nat Commun 2017; 8:16091. [PMID: 28703129 PMCID: PMC5511369 DOI: 10.1038/ncomms16091] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2017] [Accepted: 05/26/2017] [Indexed: 12/11/2022] Open
Abstract
GABA-ergic interneurons provide diverse inhibitions that are essential for the operation of neuronal circuits in the neocortex. However, the mechanisms that control the functional organization of neocortical interneurons remain largely unknown. Here we show that developmental origins influence fine-scale synapse formation and microcircuit assembly of neocortical interneurons. Spatially clustered neocortical interneurons originating from low-titre retrovirus-infected radial glial progenitors in the embryonic medial ganglionic eminence and preoptic area preferentially develop electrical, but not chemical, synapses with each other. This lineage-related electrical coupling forms predominantly between the same interneuron subtype over an extended postnatal period and across a range of distances, and promotes action potential generation and synchronous firing. Interestingly, this selective electrical coupling relates to a coordinated inhibitory chemical synapse formation between sparsely labelled interneurons in clusters and the same nearby excitatory neurons. These results suggest a link between the lineage relationship of neocortical interneurons and their precise functional organization. Developmental neuroscientists have long asked if clonally-related neurons retain functional relationships after maturation. The authors show that sparsely labelled neocortical interneurons in clusters with high possibility of clonal relation preferentially form electrical, but not chemical, synapses.
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Affiliation(s)
- Xin-Jun Zhang
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA
| | - Zhizhong Li
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA
| | - Zhi Han
- Department of Biomedical Informatics, The Ohio State University, Columbus, Ohio 43210, USA.,College of Software, Nankai University, Tianjin 300071, China
| | - Khadeejah T Sultan
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA.,Graduate Program in Neuroscience, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, USA
| | - Kun Huang
- Department of Biomedical Informatics, The Ohio State University, Columbus, Ohio 43210, USA
| | - Song-Hai Shi
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, USA.,Graduate Program in Neuroscience, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065, USA
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48
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Molecular, morphological and survival analysis of 177 resected pancreatic ductal adenocarcinomas (PDACs): Identification of prognostic subtypes. Sci Rep 2017; 7:41064. [PMID: 28145465 PMCID: PMC5286512 DOI: 10.1038/srep41064] [Citation(s) in RCA: 80] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2016] [Accepted: 12/14/2016] [Indexed: 02/07/2023] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) has generally a poor prognosis, but recent data suggest that there are molecular subtypes differing in clinical outcome. This study examines the association between histopathologic heterogeneity, genetic profile, and survival. Tumor histology from 177 resected PDAC patients with follow-up data was subclassified according to predominant growth pattern, and four key genes were analyzed. PDACs were classified as conventional (51%), combined with a predominant component (41%), variants and special carcinomas (8%). Patients with combined PDACs and a dominant cribriform component survived longer than patients with conventional or other combined PDACs. Genetic alterations in at least two out of four genes were found in 95% of the patients (KRAS 93%, TP53 79%, CDKN2A/p16 75%, SMAD4 37%). Patients with less than four mutations survived significantly longer (p = 0.04) than those with alterations in all four genes. Patients with either wildtype KRAS or CDKN2A/p16 lived significantly longer than those with alterations in these genes (p = 0.018 and p = 0.006, respectively). Our data suggest that the number of altered genes, the mutational status of KRAS and certain morphological subtypes correlate with the outcome of patients with PDAC. Future pathology reporting of PDAC should therefore include the KRAS status and a detailed morphological description.
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49
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Fadok JP, Krabbe S, Markovic M, Courtin J, Xu C, Massi L, Botta P, Bylund K, Müller C, Kovacevic A, Tovote P, Lüthi A. A competitive inhibitory circuit for selection of active and passive fear responses. Nature 2017; 542:96-100. [PMID: 28117439 DOI: 10.1038/nature21047] [Citation(s) in RCA: 303] [Impact Index Per Article: 37.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Accepted: 12/05/2016] [Indexed: 12/11/2022]
Abstract
When faced with threat, the survival of an organism is contingent upon the selection of appropriate active or passive behavioural responses. Freezing is an evolutionarily conserved passive fear response that has been used extensively to study the neuronal mechanisms of fear and fear conditioning in rodents. However, rodents also exhibit active responses such as flight under natural conditions. The central amygdala (CEA) is a forebrain structure vital for the acquisition and expression of conditioned fear responses, and the role of specific neuronal sub-populations of the CEA in freezing behaviour is well-established. Whether the CEA is also involved in flight behaviour, and how neuronal circuits for active and passive fear behaviour interact within the CEA, are not yet understood. Here, using in vivo optogenetics and extracellular recordings of identified cell types in a behavioural model in which mice switch between conditioned freezing and flight, we show that active and passive fear responses are mediated by distinct and mutually inhibitory CEA neurons. Cells expressing corticotropin-releasing factor (CRF+) mediate conditioned flight, and activation of somatostatin-positive (SOM+) neurons initiates passive freezing behaviour. Moreover, we find that the balance between conditioned flight and freezing behaviour is regulated by means of local inhibitory connections between CRF+ and SOM+ neurons, indicating that the selection of appropriate behavioural responses to threat is based on competitive interactions between two defined populations of inhibitory neurons, a circuit motif allowing for rapid and flexible action selection.
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Affiliation(s)
- Jonathan P Fadok
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Sabine Krabbe
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Milica Markovic
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.,University of Basel, 4000 Basel, Switzerland
| | - Julien Courtin
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Chun Xu
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Lema Massi
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Paolo Botta
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.,University of Basel, 4000 Basel, Switzerland
| | - Kristine Bylund
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Christian Müller
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Aleksandar Kovacevic
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Philip Tovote
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
| | - Andreas Lüthi
- Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.,University of Basel, 4000 Basel, Switzerland
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50
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Wang H, Zhai K, Xue Y, Yang J, Yang Q, Fu Y, Hu Y, Liu F, Wang W, Cui L, Chen H, Zhang J, He W. Global Deletion of TSPO Does Not Affect the Viability and Gene Expression Profile. PLoS One 2016; 11:e0167307. [PMID: 27907096 PMCID: PMC5131929 DOI: 10.1371/journal.pone.0167307] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2016] [Accepted: 11/12/2016] [Indexed: 01/30/2023] Open
Abstract
Translocator Protein (18kDa, TSPO) is a mitochondrial outer membrane transmembrane protein. Its expression is elevated during inflammation and injury. However, the function of TSPO in vivo is still controversial. Here, we constructed a TSPO global knockout (KO) mouse with a Cre-LoxP system that abolished TSPO protein expression in all tissues and showed normal phenotypes in the physiological condition. The birth rates of TSPO heterozygote (Het) x Het or KO x KO breeding were consistent with Mendel’s Law, suggesting a normal viability of TSPO KO mice at birth. RNA-seq analysis showed no significant difference in the gene expression profile of lung tissues from TSPO KO mice compared with wild type mice, including the genes associated with bronchial alveoli immune homeostasis. The alveolar macrophage population was not affected by TSPO deletion in the physiological condition. Our findings contradict the results of Papadopoulos, but confirmed Selvaraj’s findings. This study confirms TSPO deficiency does not affect viability and bronchial alveolar immune homeostasis.
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Affiliation(s)
- Huaishan Wang
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
| | - Kangle Zhai
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
| | - Yingchao Xue
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
| | - Jia Yang
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
| | - Qi Yang
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
| | - Yi Fu
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
| | - Yu Hu
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
| | - Fang Liu
- Beijing Thorgene Medical Laboratory, Yizhuang Biomedical Park, Beijing, China
| | - Weiqing Wang
- Department of Otolaryngology, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China
| | - Lianxian Cui
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
| | - Hui Chen
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
| | - Jianmin Zhang
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
- * E-mail: (WH); (JZ)
| | - Wei He
- Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology, Beijing, China
- * E-mail: (WH); (JZ)
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