Selective neuronal vulnerability is a hallmark of many neurodegenerative diseases, yet how ubiquitous genetic insults cause highly selective neuronal loss remains poorly understood. In spinal muscular atrophy (SMA), reduced SMN levels trigger degeneration of specific motor neuron pools. Although non-apoptotic, p53-mediated death pathways have been implicated, p53 is expressed in both vulnerable and resistant neurons, leaving the downstream determinants of selective vulnerability unresolved. Here, we identify a p53-ΔNp73 signaling axis as a previously unrecognized execution pathway driving motor neuron degeneration. Using differential transcriptional profiling of SMA motor neurons following pharmacological modulation of p53 activity, we uncover p73 as a critical downstream mediator of neuronal death. Notably, SMN deficiency induces cell-autonomous, p53-dependent expression of the ΔNp73 isoform selectively in vulnerable, but not resistant, motor neurons. ΔNp73 induction precisely parallels the spatial and temporal pattern of degeneration in mouse models and is also detected in motor neurons from SMA patients. Strikingly, despite its established role as a pro-survival antagonist of p53, depletion of ΔNp73 improves motor neuron survival and partially preserves neuromuscular junction integrity in SMA mice. These findings reveal a context-dependent, isoform-specific functional switch in p53 family signaling that redirects a canonical survival factor into a driver of neurodegeneration, identifying a novel molecular mechanism underlying selective neuronal vulnerability in SMA and a potential therapeutic target for neuroprotection.

Motor neuron (MN) loss is a hallmark of neurodegenerative disorders, yet its assessment remains variable, confounding mechanistic and therapeutic interpretation. To address this, we conducted a systematic review and meta-analysis of spinal muscular atrophy (SMA) mouse studies, revealing 60

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by ubiquitous survival motor neuron (SMN) deficiency and loss of motor neurons. The persistence of motor and communication impairments, together with emerging cognitive and social deficits in severe type I SMA patients treated early with SMN-restoring therapies, suggests a broader dysfunction involving neural circuits of the brain. To explore the potential supraspinal contributions to these emerging phenotypes, we investigated the cerebellum, a brain region crucial for both motor and cognitive behaviours. Here, we identify cerebellar pathology in both post-mortem tissue from type I SMA patients and a severe mouse model, which is characterized by lobule-specific Purkinje cell death driven by cell-autonomous, non-apoptotic p53-dependent mechanisms. Loss and dysfunction of excitatory parallel fibre synapses onto Purkinje cells contribute further to cerebellar circuit disruption and altered Purkinje cell firing. Furthermore, we identified impaired ultrasonic vocalization (a proxy for early-developing social communication skills that depend on cerebellar function) in a severe SMA mouse model. Cell-specific rescue experiments demonstrate that intrinsic cerebellar pathology contributes to motor and social communication impairments independently of spinal motor circuit abnormalities. Together, these findings establish cerebellar dysfunction as a pathogenic driver of neurodevelopmental motor and social defects, providing mechanistic insight into the persisting and emerging phenotypes of SMA.

Charcot-Marie-Tooth disease type 1E (CMT1E) is a rare, autosomal dominant peripheral neuropathy caused by missense variants, deletions, and truncations within the peripheral myelin protein-22 (PMP22) gene. CMT1E phenotypes vary depending on the specific variant, ranging from mild to severe, and there is little natural history and phenotypic progression data on individuals with CMT1E. Patients with CMT1E were evaluated during initial and follow-up visits at sites within the Inherited Neuropathy Consortium. Clinical characteristics were obtained from history, neurological exams, and nerve conduction studies. Clinical outcome measures were used to quantify baseline and longitudinal changes, including the Rasch-modified CMT Examination Score version 2 (CMTESv2-R) and the CMT Pediatric Scale (CMTPedS). The trafficking of PMP22 variants in transfected cells was correlated to disease severity. Twenty-four, presumed disease-causing PMP22 variants were identified in 50 individuals from 35 families, including 19 missense variants, three in-frame deletions, and two truncations. Twenty-nine patients presented with delayed walking during childhood. At their baseline evaluation, the mean CMTESv2-R in 46 patients was 16 ± 7.72 (out of 32), and the mean CMTPedS from 17 patients was 28 ± 6.35 (out of 44). Six individuals presented with hearing loss, eleven with scoliosis, three with hip dysplasia, and one with both scoliosis and hip dysplasia. Twenty variants were localized within in transmembrane domains; 31 of 35 individuals with these variants had moderate to severe phenotypes. Three variants were found in the extracellular domain and were associated with milder phenotypes. Reduced expression of PMP22 at the cell surface, and the location of missense variants within in the transmembrane domain correlated with disease severity. Pathogenic PMP22 variants located within the transmembrane regions usually cause a moderate to severe clinical phenotype, beginning in early childhood, and have impaired trafficking to the plasma membrane.

Despite the remarkable benefits of nusinersen and other disease-modifying therapies in spinal muscular atrophy (SMA), patients may still experience clinical manifestations of the disease. Here we assessed the potential for high-dose nusinersen to rapidly slow neurodegeneration and lead to improved outcomes for patients. The global, three-part, phase 2/3 DEVOTE trial evaluated the efficacy and safety of high-dose nusinersen (50-mg loading dose; 28-mg maintenance dose) in individuals with SMA. In Part B, treatment-naive individuals (n = 75) were randomized 2:1 to 50/28 mg or 12/12 mg nusinersen. In a supportive open-label cohort (Part C), nusinersen-experienced individuals (12/12 mg for more than 1 year) were enrolled. The primary endpoint (Part B infantile-onset participants) was a 6-month change in the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND) total score comparing 50/28 mg with matched ENDEAR participants (n = 20) who received sham. DEVOTE met its primary endpoint: at day 183, the CHOP-INTEND total score significantly improved (+15.1 points) in those who received 50/28 mg nusinersen and worsened (-11.1 points) in matched ENDEAR participants who received sham (difference, 26.19 (95% confidence interval = 20.7 to 31.74); statistical testing was performed using the joint-rank test where the difference in ranks was 26.06 (95% confidence interval = 17.9 to 34.2; P < 0.0001). The safety profile of 50/28 mg nusinersen was similar to the 12/12 mg regimen. The data support that high-dose nusinersen provides benefit in patients with SMA, with a generally well-tolerated safety profile. ClinicalTrials.gov registration: https://clinicaltrials.gov/study/NCT04089566 . EudraCT no: 2019-002663-10.

Severe spinal muscular atrophy (SMA) is a life-limiting neurodegenerative disease of infancy and early childhood, caused by reduced expression of the ubiquitous survival motor neuron protein (SMN). While current therapies aim to increase SMN levels and preserve motor neurons, significant deficits remain in treated patients and non-neuronal manifestations of SMN deficiency are underexplored. Vascular abnormalities including intrinsic endothelial cell dysfunction, altered vessel morphology, and altered vascular distribution have been reported in preclinical SMA models. Here, we characterised vascular architecture and blood-spinal cord barrier (BSCB) morphology and integrity in post-mortem spinal cord samples from severe SMA patients compared with unaffected controls. Von Willebrand Factor (vWF), a marker of endothelial cell health, was reduced within individual vascular endothelial cells, and associated with ultrastructural endothelial cell oedema, vacuolisation and compromised endothelial integrity. Ultrastructural damage extended to other components of the BSCB as evidenced by extravascular leakage of fibrinogen into the neural parenchyma and microglial activation consistent with a neuroinflammatory environment. Together, these findings suggest that vascular defects with associated dysfunction of the BSCB are present in the spinal cord of infants with severe SMA. This work adds to a growing body of evidence linking microvascular dysfunction to neurodegeneration in human neurodegenerative diseases. Further studies are warranted to define the contribution of vascular dysfunction to SMA pathogenesis and to assess whether current therapies adequately address this aspect of the disease.

Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder caused by mutations in the survival motor neuron 1 (SMN1) gene leading to decreased SMN protein levels and motor neuron dysfunction. SMN-restoring therapies offer clinical benefit, but the downstream molecular consequences of SMN reduction remain incompletely understood. SMN deficiency resulted in downregulation of kinesin heavy chain isoform 5A (KIF5A) in human neurons and in a mouse model of SMA. SMN associated with KIF5A mRNA and contributed to its stability. Reduced SMN levels impaired axon regeneration, which was rescued by KIF5A overexpression. Because KIF5A has also been connected to ALS, these findings provide evidence of a molecular link between SMA and ALS pathophysiology, highlighting KIF5A as an SMN-regulated factor. Our findings suggest that SMNindependent interventions targeting KIF5A could represent a complementary therapeutic approach for SMA and other motor neuron diseases.

Abstract Spinal muscular atrophy (SMA) is caused by a deficiency in the survival motor neuron (SMN) protein, resulting in degeneration of spinal motor neurons (MNs). However, persistent neurological deficits despite postnatal SMN-restoring therapies suggest that recovery of sensorimotor and supraspinal circuits may be incomplete. The cerebellum has recently emerged as a supraspinal contributor to motor deficits in the severe SMNΔ7 mouse model, yet it remains unclear whether cerebellar pathology is a conserved and therapeutically reversible feature across severe SMA mouse models and clinical subtypes. Here, we identify cerebellar pathology in Taiwanese SMA mice, characterized by hypoplasia, disrupted organization and loss of Purkinje cells (PCs), altered synaptic circuitry, and impaired cerebellar cortical output. Unlike the previously described p53-dependent PC degeneration in SMNΔ7 mice, cerebellar pathology in Taiwanese SMA mice was associated with developmental disorganization and external granule layer (EGL)-restricted p53 activation. Human cerebellar tissue mirrored this distinction, with p53 activation found in PCs from SMA Type I and in the EGL from SMA Type 0 individuals, indicating that cerebellar pathology arises through distinct mechanisms across severe forms of SMA. Importantly, two SMN-restoring strategies produced divergent therapeutic outcomes. In SMNΔ7 mice, AAV9-SMN prevented PC degeneration yet incompletely restored cerebellar circuitry. AAV9-SMN-treated Taiwanese mice developed severe ataxia-like deficits, retained profound cerebellar pathology, and survived to approximately one month of age. In contrast, systemic risdiplam rescued cerebellar pathology, motor behavior, and survival in both models. Together, these findings identify cerebellar pathology as a conserved yet distinct feature across severe forms of SMA and reveal cell type-specific tropism as a critical determinant of therapeutic outcome. More broadly, these findings suggest that successful recovery requires restoration of distributed supraspinal circuit integrity in addition to rescue of spinal motor pathways.

BACKGROUND:Neurofilament light chain (Nf-L) has been identified as a biomarker of neurodegeneration in many neuromuscular conditions, including several subtypes of polyneuropathies. The purpose of this research was to investigate whether Nf-L is also a promising biomarker for idiopathic peripheral neuropathy (IPN), the second most common subtype of axonal polyneuropathy. METHODS:Nf-L levels were quantified using an ultrasensitive digital immunoassay SiMoA in plasma samples from 294 subjects. Participant inclusion required a diagnosis of IPN confirmed by electrodiagnostic testing, intraepidermal nerve fiber density (IENFD), and/or neuromuscular examination. Laboratory testing recommended by the American Academy of Neurology for the evaluation of polyneuropathy was normal in all subjects. RESULTS:In our cohort, the majority of participants (78.1%, N = 228) had Nf-L levels in the age-adjusted normal range. Those with elevated Nf-L levels had higher scores on two different neuropathy severity scores and were more likely to have abnormal electrodiagnostic testing, including reduced action potential amplitude in peroneal motor and sural sensory nerves. No differences in blood Nf-L levels were observed in those participants with a short duration (≤ 1.5 years) versus long duration (≥ 5 years) of disease. Nf-L levels were also not correlated with the presence of neuropathic pain, nor the location of paresthesia. Nf-L expression had the strongest correlation with age. CONCLUSIONS:In this cohort with IPN, Nf-L levels correlated with disease severity as assessed by clinical examination and electrophysiology. However, given that Nf-L was in the normal range for the majority of subjects in our cohort, its use as a biomarker for clinical trials evaluating new treatments for IPN will be limited.

The ankyrin repeat is one of the most abundant protein-protein interaction motifs in eukaryotes yet occurs in only a small number of ion channels. These channels are all members of the transient receptor potential (TRP) superfamily and contain prominent ankyrin repeat domains (ARDs) in their cytoplasmic N termini. In transient receptor potential vanilloid 4 (TRPV4), the importance of this domain has been highlighted by the finding that gain-of-function neuromuscular disease-causing missense mutations cluster on the ARD surface. Little is known currently about the extent of the TRPV4-ARD interactome, nor how it may be altered by disease-causing mutations. Here, we utilized a human proteome microarray to profile the ARD interactomes of WT and mutant TRPV4. Probing of the microarray with TRPV4WT-ARD revealed 78 interactors, including proteins related to ubiquitination and small GTPase signaling, such as the ubiquitin ligase NEDD4L and the RhoGEF ARHGEF10. In parallel experiments, we also identified the deubiquitinase OTUB2 as an interactor of the proximal N terminus. Comparison of the ARD interactomes of WT and mutant TRPV4 revealed 21 interactions affected by disease-causing mutations. Strikingly, one of these interactors, ARHGEF10, is also mutated in neuromuscular disease. Cell-based studies confirmed that ARHGEF10 exhibits a reduced capacity to coimmunoprecipitate with mutant TRPV4. Furthermore, calcium imaging studies demonstrated that ARHGEF10 overexpression suppressed TRPV4WT channel activity, but that this inhibition is abrogated by disease-causing mutations. Together, these findings provide insights into the functional roles of an ion channel ARD, as well as their disruption in disease, and offer a resource for future cell-based studies.

Spinal muscular atrophy (SMA), a leading genetic cause of infant mortality worldwide, is caused by reduced levels of the ubiquitous survival motor neuron (SMN) protein in SMA patients. Despite significant advancement in recent research and clinical treatments, the cellular pathologies that underlie SMA disease manifestations are not well characterized beyond those of spinal motor neurons (MNs). We previously reported cerebellar abnormalities in an SMA mouse model at the late stage of the disease, including volumetric deficits and lobule‐selective structural changes with Purkinje cell degeneration, with colocalized astrocytic reactivity. However, when these cerebellar defects arise and whether they are a consequence of MN degeneration remain unknown. We used magnetic resonance imaging, immunohistochemistry, and electrophysiology to characterize cerebellar pathology in early‐stage symptomatic SMNΔ7 mice and late‐stage SMA mice with transgenic rescue of SMN in MNs. We found disproportionate structural and lobule‐specific surface area deficits, as well as abnormal functional properties in the cerebella of early symptomatic SMA mice, suggesting that cerebellar pathologies may be a primary contributor to murine SMA phenotypes. Moreover, cerebellar pathologies were not ameliorated in SMA mice with MN rescue, suggesting that cerebellar neurons are independently vulnerable to reduced SMN expression. Overall, our study shows that cerebellar defects are a primary pathology in SMA mouse models and that therapies targeting cerebellar neurons in SMA patients may be needed for optimal treatment outcomes.

Neurological disorders with onset before or at birth are a leading cause of morbidity and mortality in infants and children. Prenatal treatment has the potential to reduce or prevent irreversible neuronal loss and facilitate normal neurodevelopment. We hypothesized that antisense oligonucleotides (ASOs) delivered to the amniotic fluid by intra-amniotic (IA) injection could safely distribute to the fetal central nervous system (CNS) and provide therapeutic benefit in the motor neuron disease spinal muscular atrophy (SMA), caused by mutations of the survival of motor neuron 1 gene ( SMN1 ), leading to deficiency of SMN protein. Although the splice-switching ASO nusinersen ameliorates SMA when delivered postnatally, substantial deficits can remain in severely affected infants. Here, IA injection of ASOs into two mouse models of severe SMA increased SMN expression in the CNS. In SMAΔ7 mice, which manifest pathology in utero, prenatal treatment improved motor neuron numbers, motor axon development, motor behavioral tests, and survival when compared with those in mice treated postnatally (between P1 and P3). To assess the feasibility of prenatal treatment in a large-animal model, ASOs were delivered midgestation to fetal sheep by IA or intracranial injection. ASOs delivered by IA injection distributed to the spinal cord at therapeutic concentrations and to multiple peripheral tissues without evidence of substantial toxicity to the fetus or mother. These data demonstrated that IA delivery of ASOs holds potential as a minimally invasive approach for prenatal treatment of SMA and possibly other severe, early-onset neurological disorders.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by a varying degree of severity that is correlated with the reduction of SMN protein levels. Motor neuron degeneration and skeletal muscle atrophy are hallmarks of SMA, but it is unknown whether other mechanisms contribute to the spectrum of clinical phenotypes. Here, through a combination of physiological and morphological studies in mouse models and SMA patients, we identify dysfunction and loss of proprioceptive sensory synapses as key signatures of SMA pathology. We demonstrate that type 3 SMA patients exhibit impaired proprioception and that their proprioceptive synapses are dysfunctional as measured by the neurophysiological test of the Hoffmann reflex. We also show moderate loss of spinal motor neurons along with reduced excitatory afferent synapses and altered potassium channel expression in motor neurons from type 1 SMA patients. These are conserved pathogenic events found in both severely affected patients and mouse models. Lastly, we report that improved motor function and fatigability in ambulatory type 3 SMA patients and mouse models treated with SMN-inducing drugs are correlated with increased function of sensory-motor circuits that can be captured accurately by the Hoffmann reflex assay. Thus, sensory synaptic dysfunction is a clinically relevant event in SMA, and the Hoffmann reflex is a suitable assay to monitor disease progression and treatment efficacy of motor circuit pathology.

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 CMT type 1A (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.