Alternative Splicing Events Are a Late Feature of Pathology in a Mouse Model of Spinal Muscular Atrophy

Spinal muscular atrophy is a severe motor neuron disease caused by inactivating mutations in the SMN1 gene leading to reduced levels of full-length functional SMN protein. SMN is a critical mediator of spliceosomal protein assembly, and complete loss or drastic reduction in protein leads to loss of cell viability. However, the reason for selective motor neuron degeneration when SMN is reduced to levels which are tolerated by all other cell types is not currently understood. Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death. However, it remains unclear whether splicing abnormalities are present during early stages of the disease, which would be a requirement for a direct role in disease pathogenesis. We performed exon-array analysis of RNA from SMN deficient mouse spinal cord at 3 time points, pre-symptomatic (P1), early symptomatic (P7), and late-symptomatic (P13). Compared to littermate control mice, SMA mice showed a time-dependent increase in the number of exons showing differential expression, with minimal differences between genotypes at P1 and P7, but substantial variation in late-symptomatic (P13) mice. Gene ontology analysis revealed differences in pathways associated with neuronal development as well as cellular injury. Validation of selected targets by RT–PCR confirmed the array findings and was in keeping with a shift between physiologically occurring mRNA isoforms. We conclude that the majority of splicing changes occur late in SMA and may represent a secondary effect of cell injury, though we cannot rule out significant early changes in a small number of transcripts crucial to motor neuron survival

Incidence rates of progressive childhood encephalopathy in Oslo, Norway: a population based study

<p>Abstract</p> <p>Background</p> <p>Progressive encephalopathy (PE) in children is a heterogeneous group of diseases mainly composed of metabolic diseases, but it consists also of neurodegenerative disorders where neither metabolic nor other causes are found. We wanted to estimate the incidence rate and aetiology of PE, as well as the age of onset of the disease.</p> <p>Methods</p> <p>We included PE cases born between 1985 and 2003, living in Oslo, and registered the number presenting annually between 1985 and 2004. Person-years at risk between 0 and 15 years were based on the number of live births during the observation period which was divided into four 5-year intervals. We calculated incidence rates according to age at onset which was classified as neonatal (0–4 weeks), infantile (1–12 months), late infantile (1–5 years), and juvenile (6–12 years).</p> <p>Results</p> <p>We found 84 PE cases representing 28 diagnoses among 1,305,997 person years, giving an incidence rate of 6.43 per 100,000 person years. The age-specific incidence rates per 100,000 were: 79.89 (<1 year), 8.64 (1–2 years), 1.90 (2–5 years), and 0.65 (>5 years). 66% (55/84) of the cases were metabolic, 32% (27/54) were neurodegenerative, and 2% (2/84) had HIV encephalopathy. 71% (60/84) of the cases presented at < 1 year, 24% (20/84) were late infantile presentations, and 5% (4/84) were juvenile presentations. Neonatal onset was more common in the metabolic (46%) (25/55) compared to the neurodegenerative group (7%) (2/27). 20% (17/84) of all cases were classified as unspecified neurodegenerative disease.</p> <p>Conclusion</p> <p>The overall incidence rate of PE was 6.43 per 100,000 person years. There was a strong reduction in incidence rates with increasing age. Two-thirds of the cases were metabolic, of which almost half presented in the neonatal period.</p

Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

Genetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA.A sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed.The assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA.This SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts

Phosphatase and tensin homologue: a therapeutic target for SMA

Spinal muscular atrophy (SMA) is one of the most common juvenile neurodegenerative diseases, which can be associated with child mortality. SMA is caused by a mutation of ubiquitously expressed gene, Survival Motor Neuron1 (SMN1), leading to reduced SMN protein and the motor neuron death. The disease is incurable and the only therapeutic strategy to follow is to improve the expression of SMN protein levels in motor neurons. Significant numbers of motor neurons in SMA mice and SMA cultures are caspase positive with condensed nuclei, suggesting that these cells are prone to a process of cell death called apoptosis. Searching for other potential molecules or signaling pathways that are neuroprotective for central nervous system (CNS) insults is essential for widening the scope of developmental medicine. PTEN, a Phosphatase and Tensin homologue, is a tumor suppressor, which is widely expressed in CNS. PTEN depletion activates anti-apoptotic factors and it is evident that the pathway plays an important protective role in many neurodegenerative disorders. It functions as a negative regulator of PIP3/AKT pathway and thereby modulates its downstream cellular functions through lipid phosphatase activity. Moreover, previous reports from our group demonstrated that, PTEN depletion using viral vector delivery system in SMN delta7 mice reduces disease pathology, with significant rescue on survival rate and the body weight of the SMA mice. Thus knockdown/depletion/mutation of PTEN and manipulation of PTEN medicated Akt/PKB signaling pathway may represent an important therapeutic strategy to promote motor neuron survival in SMA

Synaptic Defects in the Spinal and Neuromuscular Circuitry in a Mouse Model of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a major genetic cause of death in childhood characterized by marked muscle weakness. To investigate mechanisms underlying motor impairment in SMA, we examined the spinal and neuromuscular circuitry governing hindlimb ambulatory behavior in SMA model mice (SMNΔ7). In the neuromuscular circuitry, we found that nearly all neuromuscular junctions (NMJs) in hindlimb muscles of SMNΔ7 mice remained fully innervated at the disease end stage and were capable of eliciting muscle contraction, despite a modest reduction in quantal content. In the spinal circuitry, we observed a ∼28% loss of synapses onto spinal motoneurons in the lateral column of lumbar segments 3–5, and a significant reduction in proprioceptive sensory neurons, which may contribute to the 50% reduction in vesicular glutamate transporter 1(VGLUT1)-positive synapses onto SMNΔ7 motoneurons. In addition, there was an increase in the association of activated microglia with SMNΔ7 motoneurons. Together, our results present a novel concept that synaptic defects occur at multiple levels of the spinal and neuromuscular circuitry in SMNΔ7 mice, and that proprioceptive spinal synapses could be a potential target for SMA therapy

Bi-allelic <em>ACBD6</em> variants lead to a neurodevelopmental syndrome with progressive and complex movement disorders

\ua9 The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain. The acyl-CoA-binding domain-containing protein 6 (ACBD6) is ubiquitously expressed, plays a role in the acylation of lipids and proteins and regulates the N-myristoylation of proteins via N-myristoyltransferase enzymes (NMTs). However, its precise function in cells is still unclear, as is the consequence of ACBD6 defects on human pathophysiology. Using exome sequencing and extensive international data sharing efforts, we identified 45 affected individuals from 28 unrelated families (consanguinity 93%) with bi-allelic pathogenic, predominantly loss-of-function (18/20) variants in ACBD6. We generated zebrafish and Xenopus tropicalis acbd6 knockouts by CRISPR/Cas9 and characterized the role of ACBD6 on protein N-myristoylation with myristic acid alkyne (YnMyr) chemical proteomics in the model organisms and human cells, with the latter also being subjected further to ACBD6 peroxisomal localization studies. The affected individuals (23 males and 22 females), aged 1-50 years, typically present with a complex and progressive disease involving moderate-to-severe global developmental delay/intellectual disability (100%) with significant expressive language impairment (98%), movement disorders (97%), facial dysmorphism (95%) and mild cerebellar ataxia (85%) associated with gait impairment (94%), limb spasticity/hypertonia (76%), oculomotor (71%) and behavioural abnormalities (65%), overweight (59%), microcephaly (39%) and epilepsy (33%). The most conspicuous and common movement disorder was dystonia (94%), frequently leading to early-onset progressive postural deformities (97%), limb dystonia (55%) and cervical dystonia (31%). A jerky tremor in the upper limbs (63%), a mild head tremor (59%), parkinsonism/hypokinesia developing with advancing age (32%) and simple motor and vocal tics were among other frequent movement disorders. Midline brain malformations including corpus callosum abnormalities (70%), hypoplasia/agenesis of the anterior commissure (66%), short midbrain and small inferior cerebellar vermis (38% each) as well as hypertrophy of the clava (24%) were common neuroimaging findings. Acbd6-deficient zebrafish and Xenopus models effectively recapitulated many clinical phenotypes reported in patients including movement disorders, progressive neuromotor impairment, seizures, microcephaly, craniofacial dysmorphism and midbrain defects accompanied by developmental delay with increased mortality over time. Unlike ACBD5, ACBD6 did not show a peroxisomal localization and ACBD6-deficiency was not associated with altered peroxisomal parameters in patient fibroblasts. Significant differences in YnMyr-labelling were observed for 68 co- and 18 post-translationally N-myristoylated proteins in patient-derived fibroblasts. N-myristoylation was similarly affected in acbd6-deficient zebrafish and X. tropicalis models, including Fus, Marcks and Chchd-related proteins implicated in neurological diseases. The present study provides evidence that bi-allelic pathogenic variants in ACBD6 lead to a distinct neurodevelopmental syndrome accompanied by complex and progressive cognitive and movement disorders