Supervised and unsupervised methods have emerged to address the complexity of single cell data analysis in the context of large pools of independent studies. Here, we present ClusterFoldSimilarity (CFS), a novel statistical method design to quantify the similarity between cell groups across any number of independent datasets, without the need for data correction or integration. By bypassing these processes, CFS avoids the introduction of artifacts and loss of information, offering a simple, efficient, and scalable solution. This method match groups of cells that exhibit conserved phenotypes across datasets, including different tissues and species, and in a multimodal scenario, including single-cell RNA-Seq, ATAC-Seq, single-cell proteomics, or, more broadly, data exhibiting differential abundance effects among groups of cells. Additionally, CFS performs feature selection, obtaining cross-dataset markers of the similar phenotypes observed, providing an inherent interpretability of relationships between cell populations. To showcase the effectiveness of our methodology, we generated single-nuclei RNA-Seq data from the motor cortex and spinal cord of adult mice. By using CFS, we identified three distinct sub-populations of astrocytes conserved on both tissues. CFS includes various visualization methods for the interpretation of the similarity scores and similar cell populations.

Cell plasticity enables the dynamic changes in cell identities necessary for normal development and tissue repair. Induced cell reprogramming, which leverages this plasticity, holds great promise for regenerative medicine and personalized therapies. However, the success of cell reprogramming is often impeded by various molecular barriers, such as epigenetic marks, cell senescence, and the activation of alternative or refractory routes. In this review, we examine the cell reprogramming events that occur within or between germ layers and adult stem cell lineages and propose that the overall similarity in the pre-existing chromatin accessibility landscape is a major determinant of reprogramming efficiency from one cell type to another. A better understanding of the regulation and control of chromatin accessibility should facilitate the development of new methods and strategies to improve cell reprogramming efficiency and advance translational research.

JOURNAL/nrgr/04.03/01300535-202506000-00030/figure1/v/2024-08-05T133530Z/r/image-tiff Direct in vivo conversion of astrocytes into functional new neurons induced by neural transcription factors has been recognized as a potential new therapeutic intervention for neural injury and degenerative disorders. However, a few recent studies have claimed that neural transcription factors cannot convert astrocytes into neurons, attributing the converted neurons to pre-existing neurons mis-expressing transgenes. In this study, we overexpressed three distinct neural transcription factors--NeuroD1, Ascl1, and Dlx2--in reactive astrocytes in mouse cortices subjected to stab injury, resulting in a series of significant changes in astrocyte properties. Initially, the three neural transcription factors were exclusively expressed in the nuclei of astrocytes. Over time, however, these astrocytes gradually adopted neuronal morphology, and the neural transcription factors was gradually observed in the nuclei of neuron-like cells instead of astrocytes. Furthermore, we noted that transcription factor-infected astrocytes showed a progressive decrease in the expression of astrocytic markers AQP4 (astrocyte endfeet signal), CX43 (gap junction signal), and S100β. Importantly, none of these changes could be attributed to transgene leakage into pre-existing neurons. Therefore, our findings suggest that neural transcription factors such as NeuroD1, Ascl1, and Dlx2 can effectively convert reactive astrocytes into neurons in the adult mammalian brain.

Important advances have been made in reperfusion therapies for acute ischemic stroke. However, a majority of patients are either ineligible for or do not respond to treatments and continue to have considerable functional deficits. Stroke results in a pathological disruption of the neurovascular unit (NVU) that involves blood-brain barrier leakage, glial activation, neuronal damage and chronic inflammation, all of which create a microenvironment that hinders recovery. Therefore, finding ways to promote central nervous system recovery remains the holy grail of stroke research. Here we propose a conceptual framework to synthesize recent progress in the field, which is currently dispersed and disconnected in the literature. We suggest that stroke recovery requires an integrated reprogramming process throughout the brain that occurs at multiple levels, including changes in gene expression, endogenous cellular transdifferentiation within the NVU, and reorganization of larger-scale neural and social networks.

Reactive gliosis is a hallmark of neuropathology and offers a potential target for addressing numerous neurological diseases. Here, we show that growth arrest and DNA damage inducible gamma (GADD45G), a stress sensor in astrocytes, is a nodal orchestrator of reactive gliosis and neurodegeneration. GADD45G expression in astrocytes is sufficient to incite astrogliosis, microgliosis, synapse loss, compromised animal behavior, and the aggravation of Alzheimer's disease (AD). Conversely, silencing GADD45G specifically in astrocytes preserves synapses and rescues the histological and behavioral phenotypes of AD. Mechanistically, GADD45G controls the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) and neuroimmune signaling pathways, including nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF3), leading to profound molecular changes and the secretion of various factors that regulate both cell-autonomous and cell-nonautonomous reactive gliosis and glia-neuron interactions. These results uncover GADD45G signaling as a promising therapeutic target for AD and potentially for numerous other neurological disorders.

Retinal Müller glia in cold-blooded vertebrates can reprogram into neurogenic progenitors to replace neurons lost to injury, but mammals lack this ability. While recent studies have shown that transgenic overexpression of neurogenic bHLH factors and glial-specific disruption of NFI family transcription factors and Notch signaling induce neurogenic competence in mammalian Müller glia, induction of neurogenesis in wildtype glia has thus far proven elusive. Here, we report that viral-mediated overexpression of the pluripotency factor Pou5f1 (Oct4) induces transdifferentiation of mouse Müller glia into bipolar neurons, and synergistically stimulates glial-derived neurogenesis in parallel with Notch loss of function. Single-cell multiomic analysis shows that Pou5f1 overexpression leads to widespread changes in gene expression and chromatin accessibility, inducing activity of both the neurogenic transcription factor Rfx4 and the Yamanaka factors Sox2 and Klf4. This study demonstrates that viral-mediated overexpression of Pou5f1 induces neurogenic competence in adult mouse Müller glia, identifying mechanisms that could be used in cell-based therapies for treating retinal dystrophies.

Neuronal direct cell reprogramming approach allows direct conversion of somatic cells into neurons via forced expression of neuronal cell-lineage transcription factors (TFs). These so-called induced neuronal cells have significant potential as research tools and for therapeutic applications, such as in cell replacement therapy. However, the optimization of TF delivery strategies is crucial to reach clinical practice. In this review, we outlined the currently explored delivery technologies in neuronal direct cell reprogramming and their limitations and advantages. The first employed delivery strategies were mainly integrating viral systems, such as lentiviruses that exert consistently high transgene expression in most cell types. On the other hand, viral systems cause major safety concerns, including the risk for insertional mutagenesis and inflammation. More recently, several safer nonviral delivery systems have been investigated as well; however, these systems generally exert inferior reprogramming efficiency compared with viral systems. Emerging delivery technologies could provide new opportunities in the achievement of safe and effective delivery for neuronal direct cell reprogramming.

In vivo transcription factor (TF) -mediated gene therapy through astrocyte-to-neuron (AtN) conversion has shown therapeutic effects on rodent and non-human primate cortical ischemic injury in the subacute phase. However, in the clinic, subcortical regions including striatum as well as white matter are vulnerable regions of stroke, with millions of patients beyond subacute phase. In this study, we investigate whether TF-mediated AtN conversion therapy can be extended to treat chronic-phase ischemic stroke involving subcortical regions (e.g., striatum) and white matter, beyond cortical injuries.

Rat middle cerebral artery occlusion (MCAO)-like models were established to induce broad ischemic injuries including cortical and striatal regions. Then multiple rounds of TF-mediated gene therapy treatments through adeno-associated virus (AAV) system to cover the large-scaled infarct areas were conducted in the chronic phase of the stroke models. Magnetic resonance imaging (MRI), [18F] FDG-PET/CT, behavioral tests, immunohistochemistry and bulk-RNA seq were applied to evaluate the AtN conversion, tissue repair and functional recovery.

Our results revealed that administrated in the chronic phase of ischemic stroke, TF-mediated gene therapy can efficiently regenerate new neurons in both cortical and striatal regions, and promote tissue repair in both grey and white matter. Compared with single round of AAV administration, multiple rounds of treatment regenerated more neurons and led to a significant functional recovery.

Our study demonstrates that TF-mediated gene therapy has a broad therapeutic time window and can be applied multiple rounds to treat severe ischemic stroke, making it an attractive therapeutic intervention in the chronic phase after stroke, when current approaches are largely ineffective.

NEUROD1 (ND1)-induced astrocyte-to-neuron (AtN) conversion shows promise for treating neurological disorders. To gain insight into the molecular mechanisms of neuronal reprogramming, we established an in vitro system using primary cortical astrocyte cultures from postnatal rats and employed single-cell and multiomics sequencing. Our findings indicate that the initial cultures primarily consisted of immature astrocytes (ImAs), with potentially a minor presence of radial glial cells. The ImAs initially went through an intermediate state, activating both astrocyte and neural progenitor genes. Subsequently, they mimic in vivo neurogenesis to acquire mature neuronal characteristics. We show that ND1 acted as a pioneer factor that reshapes the chromatin landscape of astrocytes to that of neurons. This restructuring promotes the expression of neurogenic genes via inducing H3K27ac modification. Through integrative analysis of various ND1-induced neuronal specification systems, we identified 25 ND1 targets, including Hes6, as key regulators. Thus, our work highlights the key role of ND1 and its downstream regulators in neuronal reprogramming.

Direct conversion of reactive glial cells to neurons is a promising avenue for neuronal replacement therapies after brain injury or neurodegeneration. The overexpression of neurogenic fate determinants in glial cells results in conversion to neurons. For repair purposes, the conversion should ideally be induced in the pathology-induced neuroinflammatory environment. However, very little is known regarding the influence of the injury-induced neuroinflammatory environment and released growth factors on the direct conversion process.

We establish a new in vitro culture system of postnatal astrocytes without epidermal growth factor that reflects the direct conversion rate in the injured, neuroinflammatory environment in vivo. We demonstrate that the growth factor combination corresponding to the injured environment defines the ability of glia to be directly converted to neurons. Using this culture system, we show that chromatin structural protein high mobility group box 2 (HMGB2) regulates the direct conversion rate downstream of the growth factor combination. We further demonstrate that Hmgb2 cooperates with neurogenic fate determinants, such as Neurog2, in opening chromatin at the loci of genes regulating neuronal maturation and synapse formation. Consequently, early chromatin rearrangements occur during direct fate conversion and are necessary for full fate conversion.

Our data demonstrate novel growth factor-controlled regulation of gene expression during direct fate conversion. This regulation is crucial for proper maturation of induced neurons and could be targeted to improve the repair process.

Rationale: As a highly plastic population, parenchymal astrocytes have demonstrated the capacity to become activated and recapitulate neurogenic potential in response to traumatic central nervous system (CNS) injuries, representing a latent reservoir for neuronal regeneration in non-neurogenic brain regions. However, the extrinsic and intrinsic factors regulating this process remain poorly characterized. Elucidating these molecular mechanisms is crucial to harnessing the regenerative potential of reactive astrocytes in CNS repair. Methods: A multidisciplinary approach combining immunostaining, western blotting, RNA interference (RNAi), gene knock out and fate-mapping was used to investigate the role of topoisomerase IIα (TOP2a) in regulation of the stemness response in reactive astrocytes to traumatic brain injury (TBI). Results: Both in vitro and in vivo analyses demonstrated that TBI induces a stem cell-like response in reactive astrocytes concomitant with TOP2a upregulation. Pharmacological inhibition or genetic deletion of TOP2a significantly attenuated this stemness response. Neurosphere culture assay indicates that TOP2a might act as a downstream factor of Sonic Hedgehog (SHH) signaling to mediate the acquisition of stem cell potential. Conclusions: This study identifies TOP2a as a pivotal intrinsic regulator of astrocytic stem cell potential in the injured brain, which will advance our understanding of the molecular underpinnings of the stem cell response and its therapeutic application in neural regeneration.

Degeneration of the cochlear spiral ganglion neurons (SGNs) is one of the major causes of sensorineural hearing loss and significantly impacts the outcomes of cochlear implantation. Functional regeneration of SGNs holds great promise for treating sensorineural hearing loss. In this study, we systematically screened 33 transcriptional regulators implicated in neuronal and SGN fate. Using gene expression array and principal component analyses, we identified a sequential combination of Ascl1, Pou4f1 and Myt1l (APM) in promoting functional reprogramming of SGNs. The neurons induced by APM expressed mature neuronal and SGN lineage-specific markers, displayed mature SGN-like electrophysiological characteristics and exhibited single-cell transcriptomes resembling the endogenous SGNs. Thus, transcription factors APM may serve as novel candidates for direct reprogramming of SGNs and hearing recovery due to SGN damages.

Alzheimer's disease (AD) is a progressive and irreversible brain disorder with extensive neuronal loss in the neocortex and hippocampus. Current therapeutic interventions focus on the early stage of AD but lack effective treatment for the late stage of AD, largely due to the inability to replenish the lost neurons and repair the broken neural circuits. In this study, by using engineered adeno-associated virus vectors that efficiently cross the blood-brain-barrier in the mouse brain, a brain-wide neuroregenerative gene therapy is developed to directly convert endogenous astrocytes into functional neurons in a mouse model of AD. It is found that ≈500 000 new neurons are regenerated and widely distributed in the cerebral cortex and hippocampus. Importantly, it is demonstrated that the converted neurons can integrate into pre-existing neural networks and improve various cognitive performances in AD mice. Chemogenetic inhibition of the converted neurons abolishes memory enhancement in AD mice, suggesting a pivotal role for the newly converted neurons in cognitive restoration. Together, brain-wide neuroregenerative gene therapy may provide a viable strategy for the treatment of AD and other brain disorders associated with massive neuronal loss.

Retinal Muller glia in cold-blooded vertebrates can reprogram into neurogenic progenitors to replace neurons lost to injury, but mammals lack this ability. While recent studies have shown that transgenic overexpression of neurogenic bHLH factors and glial-specific disruption of NFI family transcription factors and Notch signaling induce neurogenic competence in mammalian Muller glia, induction of neurogenesis in wild-type glia has thus far proven elusive. Here, we report that viral-mediated overexpression of the pluripotency factor Oct4 (Pou5f1) induces transdifferentiation of mouse Muller glia into bipolar neurons, and synergistically stimulates glial-derived neurogenesis in parallel with Notch loss of function. Single cell multiomic analysis shows that Oct4 overexpression leads to widespread changes in gene expression and chromatin accessibility, inducing activity of both the neurogenic transcription factor Rfx4 and the Yamanaka factors Sox2 and Klf4. This study demonstrates that viral-mediated overexpression of Oct4 induces neurogenic competence in retinal Muller glia, identifying mechanisms that could be used in cell-based therapies for treating retinal dystrophies.

The successful generation of induced pluripotent stem cells (iPSCs) has significantly impacted many scientific fields. In the field of regenerative medicine, iPSC-derived somatic cells are expected to recover impaired organ functions through cell transplantation therapy. Subsequent studies using genetically engineered mouse models showed that somatic cells are also reprogrammable in vivo. Notably, cyclic expression of reprogramming factors, so-called partial reprogramming in vivo ameliorates cellular and physiological hallmarks of aging without inducing teratoma formation or premature death of animals. Subsequent studies provided evidence supporting the beneficial effects of partial reprogramming in various organs. Although in vivo reprogramming appears to be a promising strategy for tissue regeneration and rejuvenation, there remain unsolved issues that hinder its clinical application, including concerns regarding its safety, controllability, and unexpected detrimental effects. Here, we review the pathway that research of in vivo reprogramming has followed and discuss the future perspective as we look toward its clinical application in regenerative medicine.

Astrocyte-to-neuron reprogramming presents a viable approach for regenerative medicine. The reprogramming factor NeuroD1 has demonstrated capability of neuronal reprogramming with high efficiency both in culture and in the injured central nervous system. High level of NeuroD1 expression is required to break down the cellular identity barrier for a successful reprogramming, and yet persistence of this high level drives the reprogrammed neurons primarily to glutamatergic subtype. This is consistent with the critical role of NeuroD1 in determination of glutamatergic neuronal lineage during development. However, diversified neuronal subtypes are needed to establish appropriate neuronal connectivity in disease/injury conditions. We reason that continuously high level of NeuroD1 expression forces the reprogrammed neurons into glutamatergic subtype, and that reducing NeuroD1 level after reprogramming may allow generation of neurons with diversified subtypes. For this purpose, we engineered a novel viral expression vector by which NeuroD1 expression can be dynamically regulated during the reprogramming process. Specifically, the target site of a neuron-specific microRNA (miR-124) is incorporated in the expression system. Therefore, this novel construct would still achieve a high NeuroD1 expression level in astrocytes for reprogramming to occur and yet reduce its level in the reprogrammed neurons by suppression of endogenous miR-124. In this study, we demonstrated that this construct elicits a dynamic gene expression pattern with much reduced level of NeuroD1 at later stages of neuronal reprogramming. We also showed that this construct still retains relatively high reprogramming efficiency and can generate mature neurons with an enhanced GABAergic neuronal phenotype.

Ischemic stroke, the second leading cause of death worldwide and the leading cause of long-term disabilities, presents a significant global health challenge, particularly in aging populations where the risk and severity of cerebrovascular events are significantly increased. The aftermath of stroke involves neuronal loss in the infarct core and reactive astrocyte proliferation, disrupting the neurovascular unit, especially in aged brains. Restoring the balance between neurons and non-neuronal cells within the perilesional area is crucial for post-stroke recovery. The aged post-stroke brain mounts a fulminant proliferative astroglial response, leading to gliotic scarring that prevents neural regeneration. While countless therapeutic techniques have been attempted for decades with limited success, alternative strategies aim to transform inhibitory gliotic tissue into an environment conducive to neuronal regeneration and axonal growth through genetic conversion of astrocytes into neurons. This concept gained momentum following discoveries that in vivo direct lineage reprogramming in the adult mammalian brain is a feasible strategy for reprogramming non-neuronal cells into neurons, circumventing the need for cell transplantation. Recent advancements in glial cell reprogramming, including transcription factor-based methods with factors like NeuroD1, Ascl1, and Neurogenin2, as well as small molecule-induced reprogramming and chemical induction, show promise in converting glial cells into functional neurons. These approaches leverage the brain's intrinsic plasticity for neuronal replacement and circuit restoration. However, applying these genetic conversion therapies in the aged, post-stroke brain faces significant challenges, such as the hostile inflammatory environment and compromised regenerative capacity. There is a critical need for safe and efficient delivery methods, including viral and non-viral vectors, to ensure targeted and sustained expression of reprogramming factors. Moreover, addressing the translational gap between preclinical successes and clinical applications is essential, emphasizing the necessity for robust stroke models that replicate human pathophysiology. Ethical considerations and biosafety concerns are critically evaluated, particularly regarding the long-term effects and potential risks of genetic reprogramming. By integrating recent research findings, this comprehensive review provides an in-depth understanding of the current landscape and future prospects of genetic conversion therapy for ischemic stroke rehabilitation, highlighting the potential to enhance personalized stroke management and regenerative strategies through innovative approaches.

Neurodegenerative diseases are characterized by progressive loss of neurons and persistent inflammation. Neurons are terminally differentiated cells, and lost neurons cannot be replaced since neurogenesis is restricted to only two neurogenic niches in the adult brain, whose neurogenic potential decreases with age. In this regard, the astrocytes reprogramming into neurons may represent a promising strategy for restoring the lost neurons and rebuilding neural circuits. To date, many anti-inflammatory agents have been shown to reduce neuroinflammation; however, their potential to restore neuronal loss was poorly investigated. This study investigates the anti-inflammatory effects of lactoferrin on DI-TNC1 astrocyte cell line and its ability to induce astrocyte reprogramming in a context of sustained inflammation. For this purpose, astrocytes were pre-treated with lactoferrin (4 μg/mL) for 24 h, then with lipopolysaccharide (LPS) (400 ng/mL), and examined 2, 9 and 16 days from treatment. The results demonstrate that lactoferrin attenuates astrocyte reactivity by reducing Toll-like receptor 4 (TLR4), Glial fibrillary acidic protein (GFAP) and IL-6 expression, as well as by upregulating Interleukin-10 (IL-10) cytokine and NRF2 expression. Moreover, lactoferrin promotes the reprogramming of reactive astrocytes into proliferative neuroblasts by inducing the overexpression of the Sex determining region Y/SRY-box 2 (SOX2) reprogramming transcription factor. Overall, this study highlights the potential effects of lactoferrin to attenuate neuroinflammation and improve neurogenesis, suggesting a future strategy for the treatment of neurodegenerative disorders.

Neural stem cells from the subventricular zone (SVZ) neurogenic niche provide neurons that integrate in the olfactory bulb circuitry. However, in response to cortical injuries, the neurogenic activity of the SVZ is significantly altered, leading to increased number of neuroblasts with a modified migration pattern that leads cells towards the site of injury. Despite the increased neurogenesis and migration, many newly generated neurons fail to survive or functionally integrate into the cortical circuitry. Providing the injured area with the adequate signaling molecules may improve both migration and functional integration of newly generated neurons.

In here, we have studied the effect of a diterpene with the capacity to induce neuregulin release at promoting neurogenesis in a murine model of cortical brain injury. Using green fluorescent protein expressing vectors we have labeled SVZ cells and have studied the migration of newly generated neuroblasts toward the injury in response the treatment. In addition, using electrophysiological recordings we have studied the differentiation of these neuroblasts into mature neurons and their functional integration into the cortical circuitry. We have studied their electrical properties, their morphology and cortical location.

We have found that EOF2 treatment of adult mice with mechanical cortical injuries facilitates the delivery of neuroblasts into these injuries. The newly generated neurons develop features of fully functional neurons. Our results show that the newly generated neurons receive electrical inputs, fire action potentials, and undergo complete differentiation into neurons recapitulating the stages that distinguish ontogenic differentiation. These neurons develop features representative of neurons belonging the cortical layer in which they are situated. We have also studied that EOF2 facilitates neuregulin release in SVZ cells, a signaling factor that promotes neuronal differentiation. Neuregulin is expressed in microglial cells that reach the injury in response to the damage and its release is increased by EOF2 treatment.

Promoting neuregulin release via diterpene treatment facilitates migration of SVZ-derived neuroblasts to cortical injuries stimulating their differentiation into mature functional neurons, which receive electrical inputs and develop features of cortical neurons. These findings highlight the role of diterpenoids as a potential therapy to repair cortical brain injuries.

In the mammalian central nervous system (CNS), astrocytes are the ubiquitous glial cells that have complex morphological and molecular characteristics. These fascinating cells play essential neurosupportive and homeostatic roles in the healthy CNS and undergo morphological, molecular, and functional changes to adopt so-called 'reactive' states in response to CNS injury or disease. In recent years, interest in astrocyte research has increased dramatically and some new biological features and roles of astrocytes in physiological and pathological conditions have been discovered thanks to technological advances. Here, we will review and discuss the well-established and emerging astroglial biology and functions, with emphasis on their potential as therapeutic targets for CNS injury, including traumatic and ischemic injury. This review article will highlight the importance of astrocytes in the neuropathological process and repair of CNS injury.

Lineage reprogramming of glial cells into induced neurons (iNs) has emerged as an innovative strategy to replace neurons lost due to injury or neurological diseases. Here, we describe a step-by-step protocol to induce in vivo conversion of reactive glial cells, proliferating within the injured hippocampus, into mature and functional GABAergic iNs through retrovirus-mediated expression of two neurogenic fate determinants (Ascl1 and Dlx2). We have previously applied this method to study the integration and functional impact of GABAergic iNs in epileptic mice (Lentini et al., Cell Stem Cell 28:2104-2121.e10, 2021). We successfully generated GABAergic iNs that exhibited substantial integration within pathological circuits, leading to a significant reduction in epileptic seizures.

Stem cell pluripotency gene Sox2 stimulates expression of proneural basic-helix-loop-helix transcription factor Atoh1. Sox2 is necessary for the development of cochlear hair cells and binds to the Atoh1 3' enhancer to stimulate Atoh1 expression. We show here that Sox2 deletion in late embryogenesis results in the formation of extra hair cells, in contrast to the absence of hair cell development obtained after Sox2 knockout early in gestation. Sox2 overexpression decreased the level of Atoh1 protein despite an increase in Atoh1 mRNA. Sox2 upregulated E3 ubiquitin ligase, Huwe1, by direct binding to the Huwe1 gene. By upregulating its cognate E3 ligase, Sox2 disrupts the positive feedback loop through which Atoh1 protein increases the expression of Atoh1. We conclude that Sox2 initiates expression, while also limiting continued activity of bHLH transcription factor, Atoh1, and this inhibition represents a new mechanism for regulating the activity of this powerful initiator of hair cell development.

Parkinson's disease (PD) is a multifactorial disease caused by irreversible progressive loss of dopaminergic neurons (DANs). Recent studies have reported the successful conversion of astrocytes into DANs by repressing polypyrimidine tract binding protein 1 (PTBP1), which led to the rescue of motor symptoms in a chemically-induced mouse model of PD. However, follow-up studies have questioned the validity of this astrocyte-to-DAN conversion model. Here, we devised an adenine base editing strategy to downregulate PTBP1 in astrocytes and neurons in a chemically-induced PD mouse model. While PTBP1 downregulation in astrocytes had no effect, PTBP1 downregulation in neurons of the striatum resulted in the expression of the DAN marker tyrosine hydroxylase (TH) in non-dividing neurons, which was associated with an increase in striatal dopamine concentrations and a rescue of forelimb akinesia and spontaneous rotations. Phenotypic analysis using multiplexed iterative immunofluorescence imaging further revealed that most of these TH-positive cells co-expressed the dopaminergic marker DAT and the pan-neuronal marker NEUN, with the majority of these triple-positive cells being classified as mature GABAergic neurons. Additional research is needed to fully elucidate the molecular mechanisms underlying the expression of the observed markers and understand how the formation of these cells contributes to the rescue of spontaneous motor behaviors. Nevertheless, our findings support a model where downregulation of neuronal, but not astrocytic, PTBP1 can mitigate symptoms in PD mice.

Heart disease, including myocardial infarction (MI), remains a leading cause of morbidity and mortality worldwide, necessitating the development of more effective regenerative therapies. Direct reprogramming of cardiomyocyte-like cells from resident fibroblasts offers a promising avenue for myocardial regeneration, but its efficiency and consistency in generating functional cardiomyocytes remain limited. Alternatively, reprogramming induced cardiac progenitor cells (iCPCs) could generate essential cardiac lineages, but existing methods often involve complex procedures. These limitations underscore the need for advanced mechanistic insights and refined reprogramming strategies to improve reparative outcomes in the heart. Partial cellular fate transitions, while still a relatively less well-defined area and primarily explored in longevity and neurobiology, hold remarkable promise for cardiac repair. It enables the reprogramming or rejuvenation of resident cardiac cells into a stem or progenitor-like state with enhanced cardiogenic potential, generating the reparative lineages necessary for comprehensive myocardial recovery while reducing safety risks. As an emerging strategy, partial cellular fate transitions play a pivotal role in reversing myocardial infarction damage and offer substantial potential for therapeutic innovation. This review will summarize current advances in these areas, including recent findings involving two transcription factors that critically regulate stemness and cardiogenesis. It will also explore considerations for further refining these approaches to enhance their therapeutic potential and safety.

Parkinson's disease is typically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Many studies have been performed based on the supplementation of lost dopaminergic neurons to treat Parkinson's disease. The initial strategy for cell replacement therapy used human fetal ventral midbrain and human embryonic stem cells to treat Parkinson's disease, which could substantially alleviate the symptoms of Parkinson's disease in clinical practice. However, ethical issues and tumor formation were limitations of its clinical application. Induced pluripotent stem cells can be acquired without sacrificing human embryos, which eliminates the huge ethical barriers of human stem cell therapy. Another widely considered neuronal regeneration strategy is to directly reprogram fibroblasts and astrocytes into neurons, without the need for intermediate proliferation states, thus avoiding issues of immune rejection and tumor formation. Both induced pluripotent stem cells and direct reprogramming of lineage cells have shown promising results in the treatment of Parkinson's disease. However, there are also ethical concerns and the risk of tumor formation that need to be addressed. This review highlights the current application status of cell reprogramming in the treatment of Parkinson's disease, focusing on the use of induced pluripotent stem cells in cell replacement therapy, including preclinical animal models and progress in clinical research. The review also discusses the advancements in direct reprogramming of lineage cells in the treatment of Parkinson's disease, as well as the controversy surrounding in vivo reprogramming. These findings suggest that cell reprogramming may hold great promise as a potential strategy for treating Parkinson's disease.

Cellular reprogramming of mammalian glia to an induced neuronal fate holds the potential for restoring diseased brain circuits. While the proneural factor achaete-scute complex-like 1 (Ascl1) is widely used for neuronal reprogramming, in the early postnatal mouse cortex, Ascl1 fails to induce the glia-to-neuron conversion, instead promoting the proliferation of oligodendrocyte progenitor cells (OPC). Since Ascl1 activity is posttranslationally regulated, here, we investigated the consequences of mutating six serine phospho-acceptor sites to alanine (Ascl1SA6) on lineage reprogramming in vivo. Ascl1SA6 exhibited increased neurogenic activity in the glia of the early postnatal mouse cortex, an effect enhanced by coexpression of B cell lymphoma 2 (Bcl2). Genetic fate-mapping revealed that most induced neurons originated from astrocytes, while only a few derived from OPCs. Many Ascl1SA6/Bcl2-induced neurons expressed parvalbumin and were capable of high-frequency action potential firing. Our study demonstrates the authentic conversion of astroglia into neurons featuring subclass hallmarks of cortical interneurons, advancing our scope of engineering neuronal fates in the brain.

In vivo astrocyte-to-neuron (AtN) conversion induced by overexpression of neural transcriptional factors has great potential for neural regeneration and repair. Here, we demonstrate that a single neural transcriptional factor, Dlx2, converts mouse striatal astrocytes into neurons in a dose-dependent manner. Lineage-tracing studies in Aldh1l1-CreERT2 mice confirm that Dlx2 can convert striatal astrocytes into DARPP32+ and Ctip2+ medium spiny neurons (MSNs). Time-course studies reveal a gradual conversion from astrocytes to neurons in 1 month, with a distinct intermediate state in between astrocytes and neurons. Interestingly, when Dlx2-infected astrocytes start to lose astrocytic markers, the other local astrocytes proliferate to maintain astrocytic levels in the converted areas. Unexpectedly, although Dlx2 efficiently reprograms astrocytes into neurons in the gray matter striatum, it also induces partial reprogramming of astrocytes in the white matter corpus callosum. Such partial reprogramming of white matter astrocytes is associated with neuroinflammation, which can be suppressed by the addition of NeuroD1. Our results highlight the importance of investigating AtN conversion in both the gray matter and white matter to thoroughly evaluate therapeutic potentials. This study also unveils the critical role of anti-inflammation by NeuroD1 during AtN conversion.

Microglia, as the immune cells of the central nervous system (CNS), play dynamic roles in both healthy and diseased conditions. The ability to genetically target microglia using viruses is crucial for understanding their functions and advancing microglia-based treatments. We here show that resident microglia can be simply and specifically targeted using adeno-associated virus (AAV) vectors containing a 466-bp DNA fragment from the human IBA1 (hIBA1) promoter. This targeting approach is applicable to both resting and reactive microglia. When combining the short hIBA1 promoter with the target sequence of miR124, up to 98% of transduced cells are identified as microglia. Such a simple and highly specific microglia-targeting strategy may be further optimized for research and therapeutics.

The therapeutic potential of suppressing polypyrimidine tract-binding protein 1 (Ptbp1) messenger RNA by viral transduction in a post-stroke dementia mouse model has not yet been examined. In this study, 3 days after cerebral ischemia, we injected a viral vector cocktail containing adeno-associated virus (AAV)-pGFAP-mCherry and AAV-pGFAP-CasRx (control vector) or a cocktail of AAV-pGFAP-mCherry and AAV-pGFAP-CasRx-SgRNA-(Ptbp1) (1:5, 1.0 × 1011 viral genomes) into post-stroke mice via the tail vein. We observed new mCherry/NeuN double-positive neuron-like cells in the hippocampus 56 days after cerebral ischemia. A portion of mCherry/GFAP double-positive astrocyte-like glia could have been converted into new mCherry/NeuN double-positive neuron-like cells with morphological changes. The new neuronal cells integrated into the dentate gyrus and recognition memory was significantly ameliorated. These results demonstrated that the in vivo conversion of hippocampal astrocyte-like glia into functional new neurons by the suppression of Ptbp1 might be a therapeutic strategy for post-stroke dementia.

JOURNAL/nrgr/04.03/01300535-202408000-00032/figure1/v/2023-12-16T180322Z/r/image-tiff Over the past decade, a growing number of studies have reported transcription factor-based in situ reprogramming that can directly convert endogenous glial cells into functional neurons as an alternative approach for neuroregeneration in the adult mammalian central nervous system. However, many questions remain regarding how a terminally differentiated glial cell can transform into a delicate neuron that forms part of the intricate brain circuitry. In addition, concerns have recently been raised around the absence of astrocyte-to-neuron conversion in astrocytic lineage-tracing mice. In this study, we employed repetitive two-photon imaging to continuously capture the in situ astrocyte-to-neuron conversion process following ectopic expression of the neural transcription factor NeuroD1 in both proliferating reactive astrocytes and lineage-traced astrocytes in the mouse cortex. Time-lapse imaging over several weeks revealed the step-by-step transition from a typical astrocyte with numerous short, tapered branches to a typical neuron with a few long neurites and dynamic growth cones that actively explored the local environment. In addition, these lineage-converting cells were able to migrate radially or tangentially to relocate to suitable positions. Furthermore, two-photon Ca2+ imaging and patch-clamp recordings confirmed that the newly generated neurons exhibited synchronous calcium signals, repetitive action potentials, and spontaneous synaptic responses, suggesting that they had made functional synaptic connections within local neural circuits. In conclusion, we directly visualized the step-by-step lineage conversion process from astrocytes to functional neurons in vivo and unambiguously demonstrated that adult mammalian brains are highly plastic with respect to their potential for neuroregeneration and neural circuit reconstruction.

Neuronal reprogramming is a promising approach for making major advancement in regenerative medicine. Distinct from the approach of induced pluripotent stem cells, neuronal reprogramming converts non-neuronal cells to neurons without going through a primitive stem cell stage. In vivo neuronal reprogramming brings this approach to a higher level by changing the cell fate of glial cells to neurons in neural tissue through overexpressing reprogramming factors. Despite the ongoing debate over the validation and interpretation of newly generated neurons, in vivo neuronal reprogramming is still a feasible approach and has the potential to become clinical treatment with further optimization and refinement. Here, we discuss the major neuronal reprogramming factors (mostly pro-neurogenic transcription factors during development), especially the significance of their expression levels during neurogenesis and the reprogramming process focusing on NeuroD1. In the developing central nervous system, these pro-neurogenic transcription factors usually elicit distinct spatiotemporal expression patterns that are critical to their function in generating mature neurons. We argue that these dynamic expression patterns may be similarly needed in the process of reprogramming adult cells into neurons and further into mature neurons with subtype identities. We also summarize the existing approaches and propose new ones that control gene expression levels for a successful reprogramming outcome.

Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for treating neurodegenerative diseases. In this study, we present an efficient method for reprogramming retinal glial cells into neurons. By suppressing Notch signaling by disrupting either Rbpj or Notch1/2, we induced mature Müller glial cells to reprogram into bipolar- and amacrine-like neurons. We demonstrate that Rbpj directly activates both Notch effector genes and genes specific to mature Müller glia while indirectly repressing expression of neurogenic basic helix-loop-helix (bHLH) factors. Combined loss of function of Rbpj and Nfia/b/x resulted in conversion of nearly all Müller glia to neurons. Last, inducing Müller glial proliferation by overexpression of dominant-active Yap promotes neurogenesis in both Rbpj- and Nfia/b/x/Rbpj-deficient Müller glia. These findings demonstrate that Notch signaling and NFI factors act in parallel to inhibit neurogenic competence in mammalian Müller glia and help clarify potential strategies for regenerative therapies aimed at treating retinal dystrophies.

Astrocytes are increasingly recognised as partaking in complex homeostatic mechanisms critical for regulating neuronal plasticity following central nervous system (CNS) insults. Ischaemic stroke and traumatic brain injury are associated with high rates of disability and mortality. Depending on the context and type of injury, reactive astrocytes respond with diverse morphological, proliferative and functional changes collectively known as astrogliosis, which results in both pathogenic and protective effects. There is a large body of research on the negative consequences of astrogliosis following brain injuries. There is also growing interest in how astrogliosis might in some contexts be protective and help to limit the spread of the injury. However, little is known about how astrocytes contribute to the chronic functional recovery phase following traumatic and ischaemic brain insults. In this review, we explore the protective functions of astrocytes in various aspects of secondary brain injury such as oedema, inflammation and blood-brain barrier dysfunction. We also discuss the current knowledge on astrocyte contribution to tissue regeneration, including angiogenesis, neurogenesis, synaptogenesis, dendrogenesis and axogenesis. Finally, we discuss diverse astrocyte-related factors that, if selectively targeted, could form the basis of astrocyte-targeted therapeutic strategies to better address currently untreatable CNS disorders.

Neuronal injury, aging, and cerebrovascular and neurodegenerative diseases such as cerebral infarction, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and Huntington's disease are characterized by significant neuronal loss. Unfortunately, the neurons of most mammals including humans do not possess the ability to self-regenerate. Replenishment of lost neurons becomes an appealing therapeutic strategy to reverse the disease phenotype. Transplantation of pluripotent neural stem cells can supplement the missing neurons in the brain, but it carries the risk of causing gene mutation, tumorigenesis, severe inflammation, and obstructive hydrocephalus induced by brain edema. Conversion of neural or non-neural lineage cells into functional neurons is a promising strategy for the diseases involving neuron loss, which may overcome the above-mentioned disadvantages of neural stem cell therapy. Thus far, many strategies to transform astrocytes, fibroblasts, microglia, Müller glia, NG2 cells, and other glial cells to mature and functional neurons, or for the conversion between neuronal subtypes have been developed through the regulation of transcription factors, polypyrimidine tract binding protein 1 (PTBP1), and small chemical molecules or are based on a combination of several factors and the location in the central nervous system. However, some recent papers did not obtain expected results, and discrepancies exist. Therefore, in this review, we discuss the history of neuronal transdifferentiation, summarize the strategies for neuronal replenishment and conversion from glia, especially astrocytes, and point out that biosafety, new strategies, and the accurate origin of the truly converted neurons in vivo should be focused upon in future studies. It also arises the attention of replenishing the lost neurons from glia by gene therapies such as up-regulation of some transcription factors or down-regulation of PTBP1 or drug interference therapies.

Central nervous system (CNS) damage is usually irreversible owing to the limited regenerative capability of neurons. Following CNS injury, astrocytes are reactively activated and are the key cells involved in post-injury repair mechanisms. Consequently, research on the reprogramming of reactive astrocytes into neurons could provide new directions for the restoration of neural function after CNS injury and in the promotion of recovery in various neurodegenerative diseases. This review aims to provide an overview of the means through which reactive astrocytes around lesions can be reprogrammed into neurons, to elucidate the intrinsic connection between the two cell types from a neurogenesis perspective, and to summarize what is known about the neurotranscription factors, small-molecule compounds and MicroRNA that play major roles in astrocyte reprogramming. As the malignant proliferation of astrocytes promotes the development of glioblastoma multiforme (GBM), this review also examines the research advances on and the theoretical basis for the reprogramming of GBM cells into neurons and discusses the advantages of such approaches over traditional treatment modalities. This comprehensive review provides new insights into the field of GBM therapy and theoretical insights into the mechanisms of neurological recovery following neurological injury and in GBM treatment.

Ectopic expression of transcription factor-mediated in vivo neuronal reprogramming provides promising strategy to compensate for neuronal loss, while its further clinical application may be hindered by delivery and safety concerns. As a novel and attractive alternative, small molecules may offer a non-viral and non-integrative chemical approach for reprogramming cell fates. Recent definitive evidences have shown that small molecules can convert non-neuronal cells into neurons in vitro. However, whether small molecules alone can induce neuronal reprogramming in vivo remains largely unknown.

To identify chemical compounds that can induce in vivo neuronal reprogramming in the adult spinal cord.

Immunocytochemistry, immunohistochemistry, qRT-PCR and fate-mapping are performed to analyze the role of small molecules in reprogramming astrocytes into neuronal cells in vitro and in vivo.

By screening, we identify a chemical cocktail with only two chemical compounds that can directly and rapidly reprogram cultured astrocytes into neuronal cells. Importantly, this chemical cocktail can also successfully trigger neuronal reprogramming in the injured adult spinal cord without introducing exogenous genetic factors. These chemically induced cells showed typical neuronal morphologies and neuron-specific marker expression and could become mature and survive for more than 12 months. Lineage tracing indicated that the chemical compound-converted neuronal cells mainly originated from post-injury spinal reactive astrocytes.

Our proof-of-principle study demonstrates that in vivo glia-to-neuron conversion can be manipulated in a chemical compound-based manner. Albeit our current chemical cocktail has a lowreprogramming efficiency, it will bring in vivo cell fate reprogramming closer to clinical application in brain and spinal cord repair. Future studies should focus on further refining our chemical cocktail and reprogramming approach to boost the reprogramming efficiency.