Perivascular neurons instruct 3D vascular lattice formation via neurovascular contact.

The vasculature of the central nervous system is a 3D lattice composed of laminar vascular beds interconnected by penetrating vessels. The mechanisms controlling 3D lattice network formation remain largely unknown. Combining viral labeling, genetic marking, and single-cell profiling in the mouse retina, we discovered a perivascular neuronal subset, annotated as Fam19a4/Nts-positive retinal ganglion cells (Fam19a4/Nts-RGCs), directly contacting the vasculature with perisomatic endfeet. Developmental ablation of Fam19a4/Nts-RGCs led to disoriented growth of penetrating vessels near the ganglion cell layer (GCL), leading to a disorganized 3D vascular lattice. We identified enriched PIEZO2 expression in Fam19a4/Nts-RGCs. Piezo2 loss from all retinal neurons or Fam19a4/Nts-RGCs abolished the direct neurovascular contacts and phenocopied the Fam19a4/Nts-RGC ablation deficits. The defective vascular structure led to reduced capillary perfusion and sensitized the retina to ischemic insults. Furthermore, we uncovered a Piezo2-dependent perivascular granule cell subset for cerebellar vascular patterning, indicating neuronal Piezo2-dependent 3D vascular patterning in the brain.

Diverse CMT2 neuropathies are linked to aberrant G3BP interactions in stress granules.

Complex diseases often involve the interplay between genetic and environmental factors. Charcot-Marie-Tooth type 2 neuropathies (CMT2) are a group of genetically heterogeneous disorders, in which similar peripheral neuropathology is inexplicably caused by various mutated genes. Their possible molecular links remain elusive. Here, we found that upon environmental stress, many CMT2-causing mutant proteins adopt similar properties by entering stress granules (SGs), where they aberrantly interact with G3BP and integrate into SG pathways. For example, glycyl-tRNA synthetase (GlyRS) is translocated from the cytoplasm into SGs upon stress, where the mutant GlyRS perturbs the G3BP-centric SG network by aberrantly binding to G3BP. This disrupts SG-mediated stress responses, leading to increased stress vulnerability in motoneurons. Disrupting this aberrant interaction rescues SG abnormalities and alleviates motor deficits in CMT2D mice. These findings reveal a stress-dependent molecular link across diverse CMT2 mutants and provide a conceptual framework for understanding genetic heterogeneity in light of environmental stress.

Natural Killer Cells Degenerate Intact Sensory Afferents following Nerve Injury.

Sensory axons degenerate following separation from their cell body, but partial injury to peripheral nerves may leave the integrity of damaged axons preserved. We show that an endogenous ligand for the natural killer (NK) cell receptor NKG2D, Retinoic Acid Early 1 (RAE1), is re-expressed in adult dorsal root ganglion neurons following peripheral nerve injury, triggering selective degeneration of injured axons. Infiltration of cytotoxic NK cells into the sciatic nerve by extravasation occurs within 3 days following crush injury. Using a combination of genetic cell ablation and cytokine-antibody complex stimulation, we show that NK cell function correlates with loss of sensation due to degeneration of injured afferents and reduced incidence of post-injury hypersensitivity. This neuro-immune mechanism of selective NK cell-mediated degeneration of damaged but intact sensory axons complements Wallerian degeneration and suggests the therapeutic potential of modulating NK cell function to resolve painful neuropathy through the clearance of partially damaged nerves.

Sodium Channels in Human Pain Disorders: Genetics and Pharmacogenomics.

Acute pain is adaptive, but chronic pain is a global challenge. Many chronic pain syndromes are peripheral in origin and reflect hyperactivity of peripheral pain-signaling neurons. Current treatments are ineffective or only partially effective and in some cases can be addictive, underscoring the need for better therapies. Molecular genetic studies have now linked multiple human pain disorders to voltage-gated sodium channels, including disorders characterized by insensitivity or reduced sensitivity to pain and others characterized by exaggerated pain in response to normally innocuous stimuli. Here, we review recent developments that have enhanced our understanding of pathophysiological mechanisms in human pain and advances in targeting sodium channels in peripheral neurons for the treatment of pain using novel and existing sodium channel blockers.

AIF translocation into nucleus caused by Aifm1 R450Q mutation: generation and characterization of a mouse model for AUNX1.

Mutations in AIFM1, encoding for apoptosis-inducing factor (AIF), cause AUNX1, an X-linked neurologic disorder with late-onset auditory neuropathy (AN) and peripheral neuropathy. Despite significant research on AIF, there are limited animal models with the disrupted AIFM1 representing the corresponding phenotype of human AUNX1, characterized by late-onset hearing loss and impaired auditory pathways. Here, we generated an Aifm1 p.R450Q knock-in mouse model (KI) based on the human AIFM1 p.R451Q mutation. Hemizygote KI male mice exhibited progressive hearing loss from P30 onward, with greater severity at P60 and stabilization until P210. Additionally, muscle atrophy was observed at P210. These phenotypic changes were accompanied by a gradual reduction in the number of spiral ganglion neuron cells (SGNs) at P30 and ribbons at P60, which coincided with the translocation of AIF into the nucleus starting from P21 and P30, respectively. The SGNs of KI mice at P210 displayed loss of cytomembrane integrity, abnormal nuclear morphology, and dendritic and axonal demyelination. Furthermore, the inner hair cells and myelin sheath displayed abnormal mitochondrial morphology, while fibroblasts from KI mice showed impaired mitochondrial function. In conclusion, we successfully generated a mouse model recapitulating AUNX1. Our findings indicate that disruption of Aifm1 induced the nuclear translocation of AIF, resulting in the impairment in the auditory pathway.

Spectrum of Diabetic Neuropathy: New Insights in Diagnosis and Treatment.

Diabetic neuropathy is a highly prevalent complication of diabetes. It consists of a broad range of neuropathic conditions, such as distal symmetric polyneuropathy and various forms of autonomic neuropathies involving the cardiovascular, gastrointestinal, and urogenital systems. Prevention or diagnosis in early stages of disease is crucial to prevent symptomatic onset and progression, particularly in the absence of current disease-modifying therapies. In this review, we describe the four main types of diabetic neuropathy. We review current understanding with respect to diagnosis and treatment while highlighting knowledge gaps and future directions.

A neuropathy-associated kinesin KIF1A mutation hyper-stabilizes the motor-neck interaction during the ATPase cycle.

The mechanochemical coupling of ATPase hydrolysis and conformational dynamics in kinesin motors facilitates intramolecular interaction cycles between the kinesin motor and neck domains, which are essential for microtubule-based motility. Here, we characterized a charge-inverting KIF1A-E239K mutant that we identified in a family with axonal-type Charcot-Marie-Tooth disease and also in 24 cases in human neuropathies including spastic paraplegia and hereditary sensory and autonomic neuropathy. We show that Glu239 in the ß7 strand is a key residue of the motor domain that regulates the motor-neck interaction. Expression of the KIF1A-E239K mutation has decreased ability to complement Kif1a+/- neurons, and significantly decreases ATPase activity and microtubule gliding velocity. X-ray crystallography shows that this mutation causes an excess positive charge on ß7, which may electrostatically interact with a negative charge on the neck. Quantitative mass spectrometric analysis supports that the mutation hyper-stabilizes the motor-neck interaction at the late ATP hydrolysis stage. Thus, the negative charge of Glu239 dynamically regulates the kinesin motor-neck interaction, promoting release of the neck from the motor domain upon ATP hydrolysis.

Low disease risk and penetrance in Leber hereditary optic neuropathy.

The risk of Leber hereditary optic neuropathy (LHON) has largely been extrapolated from disease cohorts, which underestimate the population prevalence of pathogenic primary LHON variants as a result of incomplete disease penetrance. Understanding the true population prevalence of primary LHON variants, alongside the rate of clinical disease, provides a better understanding of disease risk and variant penetrance. We identified pathogenic primary LHON variants in whole-genome sequencing data of a well-characterized population-based control cohort and found that the prevalence is far greater than previously estimated, as it occurs in approximately 1 in 800 individuals. Accordingly, we were able to more accurately estimate population risk and disease penetrance in LHON variant carriers, validating our findings by using other large control datasets. These findings will inform accurate counseling in relation to the risk of vision loss in LHON variant carriers and disease manifestation in their family. This Matters Arising paper is in response to Lopez Sanchez et al. (2021), published in The American Journal of Human Genetics. See also the response by Mackey et al. (2022), published in this issue.

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

NT-3 contributes to chemotherapy-induced neuropathic pain through TrkC-mediated CCL2 elevation in DRG neurons.

Cancer patients undergoing treatment with antineoplastic drugs often experience chemotherapy-induced neuropathic pain (CINP), and the therapeutic options for managing CINP are limited. Here, we show that systemic paclitaxel administration upregulates the expression of neurotrophin-3 (Nt3) mRNA and NT3 protein in the neurons of dorsal root ganglia (DRG), but not in the spinal cord. Blocking NT3 upregulation attenuates paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities and spontaneous pain without altering acute pain and locomotor activity in male and female mice. Conversely, mimicking this increase produces enhanced responses to mechanical, heat, and cold stimuli and spontaneous pain in naive male and female mice. Mechanistically, NT3 triggers tropomyosin receptor kinase C (TrkC) activation and participates in the paclitaxel-induced increases of C-C chemokine ligand 2 (Ccl2) mRNA and CCL2 protein in the DRG. Given that CCL2 is an endogenous initiator of CINP and that Nt3 mRNA co-expresses with TrkC and Ccl2 mRNAs in DRG neurons, NT3 likely contributes to CINP through TrkC-mediated activation of the Ccl2 gene in DRG neurons. NT3 may be thus a potential target for CINP treatment.

Misregulation of mitochondria-lysosome contact dynamics in Charcot-Marie-Tooth Type 2B disease Rab7 mutant sensory peripheral neurons.

Inter-organelle contact sites between mitochondria and lysosomes mediate the crosstalk and bidirectional regulation of their dynamics in health and disease. However, mitochondria-lysosome contact sites and their misregulation have not been investigated in peripheral sensory neurons. Charcot-Marie-Tooth type 2B disease is an autosomal dominant axonal neuropathy affecting peripheral sensory neurons caused by mutations in the GTPase Rab7. Using live super-resolution and confocal time-lapse microscopy, we showed that mitochondria-lysosome contact sites dynamically form in the soma and axons of peripheral sensory neurons. Interestingly, Charcot-Marie-Tooth type 2B mutant Rab7 led to prolonged mitochondria-lysosome contact site tethering preferentially in the axons of peripheral sensory neurons, due to impaired Rab7 GTP hydrolysis-mediated contact site untethering. We further generated a Charcot-Marie-Tooth type 2B mutant Rab7 knock-in mouse model which exhibited prolonged axonal mitochondria-lysosome contact site tethering and defective downstream axonal mitochondrial dynamics due to impaired Rab7 GTP hydrolysis as well as fragmented mitochondria in the axon of the sciatic nerve. Importantly, mutant Rab7 mice further demonstrated preferential sensory behavioral abnormalities and neuropathy, highlighting an important role for mutant Rab7 in driving degeneration of peripheral sensory neurons. Together, this study identifies an important role for mitochondria-lysosome contact sites in the pathogenesis of peripheral neuropathy.

ELP1, the Gene Mutated in Familial Dysautonomia, Is Required for Normal Enteric Nervous System Development and Maintenance and for Gut Epithelium Homeostasis.

Familial dysautonomia (FD) is a rare sensory and autonomic neuropathy that results from a mutation in the ELP1 gene. Virtually all patients report gastrointestinal (GI) dysfunction and we have recently shown that FD patients have a dysbiotic gut microbiome and altered metabolome. These findings were recapitulated in an FD mouse model and moreover, the FD mice had reduced intestinal motility, as did patients. To understand the cellular basis for impaired GI function in FD, the enteric nervous system (ENS; both female and male mice) from FD mouse models was analyzed during embryonic development and adulthood. We show here that not only is Elp1 required for the normal formation of the ENS, but it is also required in adulthood for the regulation of both neuronal and non-neuronal cells and for target innervation in both the mucosa and in intestinal smooth muscle. In particular, CGRP innervation was significantly reduced as was the number of dopaminergic neurons. Examination of an FD patient's gastric biopsy also revealed reduced and disoriented axons in the mucosa. Finally, using an FD mouse model in which Elp1 was deleted exclusively from neurons, we found significant changes to the colon epithelium including reduced E-cadherin expression, perturbed mucus layer organization, and infiltration of bacteria into the mucosa. The fact that deletion of Elp1 exclusively in neurons is sufficient to alter the intestinal epithelium and perturb the intestinal epithelial barrier highlights a critical role for neurons in regulating GI epithelium homeostasis.

Single-Cell Analysis of Rohon-Beard Neurons Implicates Fgf Signaling in Axon Maintenance and Cell Survival.

Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon-Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular makeup of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely nonoverlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug.

Defective post-transcriptional modification of tRNA disrupts mitochondrial homeostasis in Leber's hereditary optic neuropathy.

Leber's Hereditary Optic Neuropathy (LHON) is a rare, maternally inherited eye disease, predominantly due to the degeneration of retinal ganglion cells (RGCs). It is associated with a mitochondrial DNA (mtDNA) point mutation. Our previous study identified that the m.15927G>A homoplasmic mutation damaged the highly conserved basepairing (28C-42G) in anticodon stem of tRNAThr, caused deficient t6A modification and significantly decreased efficiency in aminoacylation and steady-state levels of tRNAThr, and led to mitochondrial dysfunction. Meanwhile, mechanisms underlying mtDNA mutations regulate intracellular signaling related to the mitochondrial and cellular integrity are less explored. Here, we manifested that defective nucleotide modification induced by the m.15927G>A mutation interfered with the expression of nuclear genes involved in cytoplasmic proteins essential for oxidative phosphorylation system (OXPHOS), thereby impacting the assemble and integrity of OXPHOS complexes. As a result of these mitochondrial dysfunctions, there was an imbalance in mitochondrial dynamics, particularly distinguished by an increased occurrence of mitochondrial fission. Excessive fission compromised the autophagy process, including initiation phase, formation and maturation of autophagosome. Both Parkin-mediated mitophagy and receptor-dependent mitophagy were significantly impaired in cybrids haboring the m.15927G>A mutation. These changes facilitated intrinsic apoptosis, as indicated by increased cytochrome c release and elevated levels of apoptosis-associated proteins (e.g., BAK, BAX, cleaved caspase 9, cleaved caspase 3, and cleaved PARP) in the mutant cybrids. This study demonstrates that the m.15927G>A mutation contributes to LHON by dysregulating OXPHOS biogenesis, aberrant quality control, increased autophagy, inhibited mitophagy, and abnormal apoptosis.

Bi-allelic variants in neuronal cell adhesion molecule cause a neurodevelopmental disorder characterized by developmental delay, hypotonia, neuropathy/spasticity.

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcamaΔ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcamaΔ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system.

SCG10 is required for peripheral axon maintenance and regeneration in mice.

Proper microtubule dynamics are critical for neuronal morphogenesis and functions, and their dysregulation results in neurological disorders and regeneration failure. Superior cervical ganglion-10 (SCG10, also known as stathmin-2 or STMN2) is a well-known regulator of microtubule dynamics in neurons, but its functions in the peripheral nervous system remain largely unknown. Here, we show that Scg10 knockout mice exhibit severely progressive motor and sensory dysfunctions with significant sciatic nerve myelination deficits and neuromuscular degeneration. Additionally, increased microtubule stability, shown by a significant increase in tubulin acetylation and decrease in tubulin tyrosination, and decreased axonal transport were observed in Scg10 knockout dorsal root ganglion (DRG) neurons. Furthermore, SCG10 depletion impaired axon regeneration in both injured mouse sciatic nerve and cultured DRG neurons following replating, and the impaired axon regeneration was found to be induced by a lack of SCG10-mediated microtubule dynamics in the neurons. Thus, our results highlight the importance of SCG10 in peripheral axon maintenance and regeneration.

Skin keratinocyte-derived SIRT1 and BDNF modulate mechanical allodynia in mouse models of diabetic neuropathy.

Diabetic neuropathy is a debilitating disorder characterized by spontaneous and mechanical allodynia. The role of skin mechanoreceptors in the development of mechanical allodynia is unclear. We discovered that mice with diabetic neuropathy had decreased sirtuin 1 (SIRT1) deacetylase activity in foot skin, leading to reduced expression of brain-derived neurotrophic factor (BDNF) and subsequent loss of innervation in Meissner corpuscles, a mechanoreceptor expressing the BDNF receptor TrkB. When SIRT1 was depleted from skin, the mechanical allodynia worsened in diabetic neuropathy mice, likely due to retrograde degeneration of the Meissner-corpuscle innervating Aß axons and aberrant formation of Meissner corpuscles which may have increased the mechanosensitivity. The same phenomenon was also noted in skin-keratinocyte specific BDNF knockout mice. Furthermore, overexpression of SIRT1 in skin induced Meissner corpuscle reinnervation and regeneration, resulting in significant improvement of diabetic mechanical allodynia. Overall, the findings suggested that skin-derived SIRT1 and BDNF function in the same pathway in skin sensory apparatus regeneration and highlighted the potential of developing topical SIRT1-activating compounds as a novel treatment for diabetic mechanical allodynia.

Role of the repeat expansion size in predicting age of onset and severity in RFC1 disease.

RFC1 disease, caused by biallelic repeat expansion in RFC1, is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I-V ß = -1.06, P < 0.001; lobules VI-VII ß = -0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion.

Combined clinical, structural, and cellular studies discriminate pathogenic and benign TRPV4 variants.

Dominant mutations in the calcium-permeable ion channel TRPV4 (transient receptor potential vanilloid 4) cause diverse and largely distinct channelopathies, including inherited forms of neuromuscular disease, skeletal dysplasias, and arthropathy. Pathogenic TRPV4 mutations cause gain of ion channel function and toxicity that can be rescued by small molecule TRPV4 antagonists in cellular and animal models, suggesting that TRPV4 antagonism could be therapeutic for patients. Numerous variants in TRPV4 have been detected with targeted and whole exome/genome sequencing, but for the vast majority, their pathogenicity remains unclear. Here, we used a combination of clinical information and experimental structure-function analyses to evaluate 30 TRPV4 variants across various functional protein domains. We report clinical features of seven patients with TRPV4 variants of unknown significance and provide extensive functional characterization of these and an additional 17 variants, including structural position, ion channel function, subcellular localization, expression level, cytotoxicity, and protein-protein interactions. We find that gain-of-function mutations within the TRPV4 intracellular ankyrin repeat domain target charged amino acid residues important for RhoA interaction, whereas ankyrin repeat domain residues outside of the RhoA interface have normal or reduced ion channel activity. We further identify a cluster of gain-of-function variants within the intracellular intrinsically disordered region that may cause toxicity via altered interactions with membrane lipids. In contrast, assessed variants in the transmembrane domain and other regions of the intrinsically disordered region do not cause gain of function and are likely benign. Clinical features associated with gain of function and cytotoxicity include congenital onset of disease, vocal cord weakness, and motor predominant disease, whereas patients with likely benign variants often demonstrated late-onset and sensory-predominant disease. These results provide a framework for assessing additional TRPV4 variants with respect to likely pathogenicity, which will yield critical information to inform patient selection for future clinical trials for TRPV4 channelopathies.

TRPV4 neuromuscular disease registry highlights bulbar, skeletal and proximal limb manifestations.

Dominant missense mutations of the calcium-permeable cation channel TRPV4 cause Charcot-Marie-Tooth disease (CMT) type 2C and two forms of distal spinal muscular atrophy. These conditions are collectively referred to as TRPV4-related neuromuscular disease and share features of motor greater than sensory dysfunction and frequent vocal fold weakness. Pathogenic variants lead to gain of ion channel function that can be rescued by TRPV4 antagonists in cellular and animal models. As small molecule TRPV4 antagonists have proven safe in trials for other disease indications, channel inhibition is a promising therapeutic strategy for TRPV4 patients. However, the current knowledge of the clinical features and natural history of TRPV4-related neuromuscular disease is insufficient to enable rational clinical trial design. To address these issues, we developed a TRPV4 patient database and administered a TRPV4-specific patient questionnaire. Here, we report demographic and clinical information, including CMT examination scores (CMTES), from 68 patients with known pathogenic TRPV4 variants, 40 of whom also completed the TRPV4 patient questionnaire. TRPV4 patients showed a bimodal age of onset, with the largest peak occurring in the first 2 years of life. Compared to CMT1A patients, TRPV4 patients showed distinct symptoms and signs, manifesting more ambulatory difficulties and more frequent involvement of proximal arm and leg muscles. Although patients reported fewer sensory symptoms, sensory dysfunction was often detected clinically. Many patients were affected by vocal fold weakness (55%) and shortness of breath (55%), and 11% required ventilatory support. Skeletal abnormalities were common, including scoliosis (64%), arthrogryposis (33%), and foot deformities. Strikingly, patients with infantile onset of disease showed less sensory involvement and less progression of symptoms. These results highlight distinctive clinical features in TRPV4 patients, including motor-predominant disease, proximal arm and leg weakness, severe ambulatory difficulties, vocal fold weakness, respiratory dysfunction, and skeletal involvement. In addition, patients with infantile onset of disease appeared to have a distinct phenotype with less apparent disease progression based on CMTES. These collective observations indicate that clinical trial design for TRPV4-related neuromuscular disease should include outcome measures that reliably capture non-length dependent motor dysfunction, vocal fold weakness, and respiratory disease.