Microglial voltage-gated proton channel 1 ablation in diabetic mice mitigates diabetes-driven demyelination and cognitive decline

Abstract

This article discusses a study by Li et al, which investigates the role of the microglial voltage-gated proton channel 1 (Hv1) in diabetes-related cognitive decline. The authors showed that Hv1 is upregulated in the corpus callosum of diabetic mice and that its knockout improves working memory, reduces microglial production of interleukin-1β and tumour necrosis factor alpha, and decreases apoptosis of oligodendrocyte progenitor cells. Furthermore, electron microscopy revealed that the myelin thickness and the g-ratio were preserved in Hv1 knockout mice, remaining within normal limits. In addition, Hv1 knockdown mitigated interleukin-1β secretion and suppressed ferroptosis markers (ferritin heavy chain/ferritin light chain, CCAAT/enhancer binding protein homologous protein, glucose-regulated protein 78, etc.) in vitro, suggesting the involvement of an Hv1-reactive oxygen species-glucose-regulated protein 78 axis in diabetic demyelination. We highlight the translational implications of these findings and recommend future studies employing microglia-specific Hv1 deletion models, longitudinal cognitive assessments and preclinical evaluation of pharmacological Hv1 inhibitors.

Key Words: Voltage-gated proton channel 1; Diabetes; Cognitive dysfunction; Microglia; Demyelination; Neuroinflammation

Core Tip: Building upon the groundbreaking work by Li et al who demonstrated that microglial voltage-gated proton channel 1 knockout effectively mitigates neuroinflammation, preserves myelin integrity, and rescues memory deficits in diabetic mouse models, our suggestions focus on advancing this research trajectory. We propose implementing longitudinal assessments to track chronic impacts, utilizing cell-type-specific approaches for mechanistic clarity, and pursuing pharmacological translation for therapeutic potential. To systematically guide these efforts, we provide structured tabulated overviews detailing the study’s strengths, acknowledging its inherent limitations, and outlining specific strategic research pathways for future investigation.

TO THE LETTER

We read with great interest the article by Li et al[1]. Diabetes mellitus is known to accelerate cognitive decline through mechanisms that include chronic neuroinflammation, oxidative stress, and oligodendrocyte dysfunction[2-5]. Microglial activation, in particular, contributes to myelin damage via overproduction of reactive oxygen species (ROS) and proinflammatory cytokines such as interleukin-1β (IL-1β) and tumour necrosis factor alpha (TNF-α)[6,7]. Voltage-gated proton channel 1 (Hv1) facilitates nicotinamide adenine dinucleotide phosphate oxidase-dependent ROS generation in microglia[8,9], yet its role in diabetes-induced cognitive impairment has remained unexplored until now. This article aims to critically evaluate the findings of the study by Li et al[1], and propose strategic next steps to validate and translate their preclinical results. All figure references provided henceforth (e.g., Figure 1C and D) refer to figures in the original article[1].

Li et al[1] demonstrated that Hv1 expression is upregulated in the corpus callosum of high-fat diet/streptozotocin-induced diabetic mice. Behaviorally, Hv1 knockout (KO) animals exhibited significantly fewer working memory errors and more unique arm entries in the eight-arm radial maze compared with diabetic controls (Figure 1C and D in the study of Li et al[1]), indicating rescued spatial and working memory. Dual-label immunofluorescence conducted two weeks after the diabetes modelling (P2) revealed more than a 300-fold increase in IL-1β expression and more than a 200-fold increase in TNF-α expression in the ionized calcium-binding adaptor molecule 1 (Iba1) microglia of the diabetes group, both of which were prevented upon Hv1 gene deletion (Figure 2G-L in the study of Li et al[1]). Oligodendrocyte progenitor cell (OPC) apoptosis, assessed by deoxyuridine triphosphate nick end labeling in NG2 cells at five weeks, was elevated in diabetic mice but reduced by Hv1 KO (Figure 4A-L in the study of Li et al[1]). Ultrastructurally, Hv1 KO reversed diabetes-induced thinned myelin sheaths and elevated g-ratios (control: 0.75; diabetic: 0.81; KO: 0.78) in the corpus callosum (Figure 5A-F in the study of Li et al[1]). In vitro, small interfering RNA-mediated Hv1 knockdown attenuated high-glucose-induced IL-1β release (Figure 6 in the study of Li et al[1]) and suppressed ferroptosis markers - ferritin heavy chain/ferritin light chain, CCAAT/enhancer binding protein homologous protein, glucose-regulated protein 78 (GRP78) in primary microglia (Figure 7A-D in the study of Li et al[1]). Finally, Hv1 KO combined with the GRP78 inhibitor YUM70 further reduced IL-1β in diabetic microglia, implicating the ROS-GRP78 pathway in Hv1’s effects (Figure 8 in the study of Li et al[1]).

STRENGTHS AND METHODOLOGICAL INNOVATIONS

Li et al[1] employed a multidimensional strategy, integrating behavioral assays, immunohistochemistry, ultrastructural analysis, and cell-based assays to dissect Hv1’s role in diabetic brain injury. In this section, we provide a critical evaluation of Li et al’s study design, findings, and innovations[1] without prescribing next steps (Table 1).

Table 1 Key strengths and innovations.

Aspect	Summary	Supporting data
Multimodal approach	In vivo (HFD + STZ, Hv1 KO) and in vitro (high-glucose + siHv1) models converge on Hv1’s role	Behavioral (Figure 1 in the study of Li et al[1]), IF (Figures 2 and 6 in the study of Li et al[1]), TEM (Figure 5 in the study of Li et al[1]), ferroptosis markers (Figure 7 in the study of Li et al[1])
Functional rescue	Hv1 KO restores working/spatial memory deficits	40% fewer errors; 30% more entries (Figure 1C and D in the study of Li et al[1])
White matter protection	TEM shows normalization of g-ratio from 0.81 (diabetic) to 0.78 (KO)	Figure 5A-F in the study of Li et al[1]

HFD: High-fat diet; STZ: Streptozotocin; Hv1: Voltage-gated proton channel 1; KO: Knockout; siHv1: Small interfering RNA-mediated voltage-gated proton channel 1; IF: Immunofluorescence; TEM: Transmission electron microscope.

Comprehensive methodology

The authors leveraged both in vivo and in vitro approaches to deliver convergent evidence for Hv1’s involvement in diabetes-induced neuroinflammation and demyelination. In vivo, they compared wild-type and global Hv1 KO mice subjected to a high-fat diet and streptozotocin regimen, demonstrating that Hv1 KO animals exhibited restored working and spatial memory in the eight-arm radial maze (Figure 1C and D in the study of Li et al[1]). In parallel, primary microglia cultured under high-glucose conditions were transfected with small interfering RNA against Hv1, which recapitulated the in vivo observations: A significant attenuation of IL-1β release upon stimulation (Figure 6 in the study of Li et al[1]) and suppressed upregulation of ferroptosis markers - ferritin heavy chain, ferritin light chain, CCAAT/enhancer binding protein homologous protein, and GRP78 (Figure 7A-D in the study of Li et al[1]). This dual-system design not only confirmed the microglial specificity of Hv1’s effects but also underscored the robustness and reproducibility of the findings across experimental platforms.

Behavioral relevance

By linking molecular and cellular changes to functional outcomes, the study directly addresses the translational gap between bench and bedside. Four weeks post-diabetes induction, Hv1 KO mice committed 40% fewer working memory errors and made 30% more unique arm entries compared with diabetic controls in the eight-arm maze (Figure 1C and D in the study of Li et al[1]). These improvements were tightly correlated with reductions in Iba1 microglial IL-1β and TNF-α expression at P2 (Figure 2G-L in the study of Li et al[1]) and decreased OPC apoptosis at five weeks (Figure 4A-L in the study of Li et al[1]), thereby demonstrating that targeting Hv1 yields meaningful cognitive rescue in a model of diabetic encephalopathy.

Ultrastructural validation

Transmission electron microscopy findings confirmed that Hv1 deletion preserved myelin integrity at the ultrastructural level. Diabetic mice exhibited a thinned myelin sheath and elevated g-ratio (0.81) compared to controls (0.75), whereas Hv1 KO restored the g-ratio to 0.78 (Figure 5A-F in the study of Li et al[1]). This alignment of transmission electron microscopy data with immunofluorescent and behavioral results confirmed that the observed functional recovery was underpinned by genuine preservation of white matter architecture.

RECOMMENDATIONS AND FUTURE DIRECTIONS

In this section, we outline targeted proposals to extend and translate the findings of Li et al[1] (Table 2).

Table 2 Points for further consideration.

Focus area	Action	Outcome measure
Longitudinal efficacy	Behavioral tests + diffusion MRI at 2, 8, 16 weeks	Memory scores; fractional anisotropy
Cell-type specificity	Generate CX3CR1-CreERT2 Hv1flox/flox for microglia-only KO; compare GFAP-Cre astrocyte KO	Cytokine levels; OPC survival; g-ratio
Pharmacological proof-of-concept	Screen Hv1 inhibitors for IC50, BBB penetration, and toxicity; test alone and with remyelinating agents	Patch-clamp IC50; brain/plasma ratio; maze performance
Mechanistic clarification	pH imaging in microglia under hyperglycemia; Co-IP of Hv1 and NOX2	Intracellular pH; ROS assays; protein interaction

MRI: Magnetic resonance imaging; Hv1: Voltage-gated proton channel 1; CX3CR1: CX3C chemokine receptor 1; KO: Knockout; OPC: Oligodendrocyte progenitor cell; BBB: Blood-brain barrier; Co-IP: Co-immunoprecipitation; NOX2: NADPH oxidase 2; ROS: Reactive oxygen species.

Temporal dynamics and sustained efficacy

While the current study meticulously documented early changes (2-5 weeks) in neuroinflammation, myelin integrity, and cognition, it remains crucial to ascertain whether Hv1 ablation confers long-term protection. We recommend performing repeated behavioral assessments-such as radial arm maze and novel object recognition-alongside diffusion tensor imaging at 2, 8, and 16 weeks post-diabetes induction. These longitudinal data will determine whether the initial Hv1-mediated rescue of g-ratios (Figure 5 in the study of Li et al[1]) and OPC survival (Figure 4 in the study of Li et al[1]) translates into durable white matter preservation and prolonged cognitive benefits.

Microglia-specific causal validation

Given that Hv1 is expressed in multiple central nervous system cell types, global KO models may mask cell-type-specific effects. Deploying CX3C chemokine receptor 1-CreERT2 × Hv1^flox/flox mice will restrict Hv1 deletion to microglia, definitively linking observed reductions in Iba1 IL-1β/TNF-α (Figure 2 in the study of Li et al[1]) and deoxyuridine triphosphate nick end labeling OPC death (Figure 4 in the study of Li et al[1]) to microglial Hv1 activity. A complementary astrocyte-specific KO (glial fibrillary acidic protein-cyclization recombination enzyme) would further clarify whether non-microglial Hv1 contributes to the demyelination phenotype.

Pharmacological translation

Genetic ablation provides proof-of-concept, but clinical application demands small-molecule Hv1 inhibitors. We urge systematic screening of candidate antagonists - such as 2-guanidinobenzimidazole derivatives-for microglial Hv1 channel blockade via patch-clamp assays, assessment of blood-brain barrier permeability, and in vivo efficacy in diabetic mice. Combining Hv1 inhibitors with remyelinating agents (e.g., clemastine) could produce synergistic benefits, leveraging the partial OPC preservation observed in Hv1 KO mice (Figure 4 in the study of Li et al[1]).

Mechanistic delineation of Hv1 activation

Although the study implicates the ROS-GRP78 axis in Hv1’s effects (Figure 8 in the study of Li et al[1]), the upstream triggers of Hv1 upregulation under hyperglycemia remain undefined. We recommend employing pH-sensitive fluorescent reporters to monitor intracellular microglial acid-base changes in diabetic conditions and conducting co-immunoprecipitation experiments to test for direct Hv1-nicotinamide adenine dinucleotide phosphate oxidase 2 complex formation. Such analyses will illuminate how hyperglycemia and membrane depolarization converge to activate Hv1.

CLINICAL AND RESEARCH IMPLICATIONS

The identification of an Hv1-ROS-GRP78 axis in diabetic white matter injury suggests several actionable clinical strategies (Table 3).

Table 3 Clinical implications and future directions.

Goal	Strategy	Readouts
Early intervention window	Hv1 inhibitors initiated at MRI-detected white matter changes or mild cognitive impairment	Diffusion MRI metrics; cognitive test scores
Enhanced remyelination	Combine Hv1 blockade with OPC-differentiation drugs (e.g., clemastine)	Myelin thickness; OPC maturation markers
Biomarker development	PET tracer or CFS assay for Hv1/IL-1β/GRP78	Imaging signal intensity; CFS levels
Broader disease scope	Evaluate Hv1 targeting in MS, Alzheimer’s, and stroke models	Disease-specific lesion load; behavioral assays

Hv1: Voltage-gated proton channel 1; MRI: Magnetic resonance imaging; OPC: Oligodendrocyte progenitor cell; PET: Positron emission tomography; CFS: Chronic fatigue syndrome; IL-1β: Interleukin-1β; GRP78: Glucose-regulated protein 78; MS: Mass spectrometry.

Therapeutic time window

The early microglial activation and myelin disruption observed in diabetic mice (Figures 2 and 4 in the study of Li et al[1]) point to a potential therapeutic window during which Hv1 inhibition could be implemented to help forestall irreversible white matter injury. Preclinical studies should test the efficacy of Hv1 inhibitors administered at the onset of diffusion magnetic resonance imaging changes or mild cognitive deficits.

Combination remyelination strategies

Although the KO of Hv1 ensures OPC survival (Figure 4 in the study of Li et al[1]), the full maturation of these cells and myelin repair may require additional support. Combining Hv1 inhibitors and OPC differentiation agents (e.g., benztropine) in rodent models could help identify their synergistic effects on remyelination and function.

Biomarker development

The elevated Hv1 expression in the corpus callosum correlates with cognitive deficits (Figure 1A and B in the study of Li et al[1]). Future work could focus on developing positron emission tomography ligands or colony-stimulating factor assays for Hv1 or its downstream mediators (IL-1β, GRP78, etc.) as exploratory biomarkers to monitor disease progression in preclinical models.

Broad applicability

Given Hv1’s role in the ROS and ferroptosis pathways, investigating other rodent models of neuroinflammation-such as those of multiple sclerosis, Alzheimer’s disease and stroke-using Hv1-targeting approaches could help clarify their broader applicability while remaining within a preclinical framework.

CONCLUSION

Li et al[1] delivered a rigorous, multidimensional investigation that positions Hv1 as a promising target for diabetes-related cognitive dysfunction and demyelination. The recommendations above aim to solidify causal mechanisms, validate translational approaches, and ultimately guide clinical trials toward Hv1-based interventions.