Brain-body mechanisms contribute to sexual dimorphism in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common form of human motor neuron disease. It is characterized by the progressive degeneration of upper and lower motor neurons, leading to generalized motor weakness and, ultimately, respiratory paralysis and death within 3-5 years. The disease is shaped by genetics, age, sex and environmental stressors, but no cure or routine biomarkers exist for the disease. Male individuals have a higher propensity to develop ALS, and a different manifestation of the disease phenotype, than female individuals. However, the mechanisms underlying these sex differences remain a mystery. In this Review, we summarize the epidemiology of ALS, examine the sexually dimorphic presentation of the disease and highlight the genetic variants and molecular pathways that might contribute to sex differences in humans and animal models of ALS. We advance the idea that sexual dimorphism in ALS arises from the interactions between the CNS and peripheral organs, involving vascular, metabolic, endocrine, musculoskeletal and immune systems, which are strikingly different between male and female individuals. Finally, we review the response to treatments in ALS and discuss the potential to implement future personalized therapeutic strategies for the disease.

Pharmacologic Activation of Integrated Stress Response Kinases Inhibits Pathologic Mitochondrial Fragmentation.

Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically-diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic, stress-independent activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic, stress-independent activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that stress-independent activation of these ISR kinases reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic, stress-independent activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

Valosin-Containing Protein (VCP): A Review of Its Diverse Molecular Functions and Clinical Phenotypes.

In this review we examine the functionally diverse ATPase associated with various cellular activities (AAA-ATPase), valosin-containing protein (VCP/p97), its molecular functions, the mutational landscape of VCP and the phenotypic manifestation of VCP disease. VCP is crucial to a multitude of cellular functions including protein quality control, endoplasmic reticulum-associated degradation (ERAD), autophagy, mitophagy, lysophagy, stress granule formation and clearance, DNA replication and mitosis, DNA damage response including nucleotide excision repair, ATM- and ATR-mediated damage response, homologous repair and non-homologous end joining. VCP variants cause multisystem proteinopathy, and pathology can arise in several tissue types such as skeletal muscle, bone, brain, motor neurons, sensory neurons and possibly cardiac muscle, with the disease course being challenging to predict.

Identification of a mosaic MTOR variant in purified neuronal DNA in a patient with focal cortical dysplasia using a novel depth electrode harvesting technique.

OBJECTIVE: Recent studies have identified brain somatic variants as a cause of focal epilepsy. These studies relied on resected tissue from epilepsy surgery, which is not available in most patients. The use of trace tissue adherent to depth electrodes used for stereo electroencephalography (EEG) has been proposed as an alternative but is hampered by the low cell quality and contamination by nonbrain cells. Here, we use our improved depth electrode harvesting technique that purifies neuronal nuclei to achieve molecular diagnosis in a patient with focal cortical dysplasia (FCD). METHODS: Depth electrode tips were collected, pooled by brain region and seizure onset zone, and nuclei were isolated and sorted using fluorescence-activated nuclei sorting (FANS). Somatic DNA was amplified from neuronal and astrocyte nuclei using primary template amplification followed by exome sequencing of neuronal DNA from the affected pool, unaffected pool, and saliva. The identified variant was validated using droplet digital polymerase chain reaction (PCR). RESULTS: An 11-year-old male with drug-resistant genetic-structural epilepsy due to left anterior insula FCD had seizures from age 3 years. Stereo EEG confirmed seizure onset in the left anterior insula. The two anterior insula electrodes were combined as the affected pool and three frontal electrodes as the unaffected pool. FANS isolated 140 neuronal nuclei from the affected and 245 neuronal nuclei from the unaffected pool. A novel somatic missense MTOR variant (p.Leu489Met, CADD score 23.7) was identified in the affected neuronal sample. Droplet digital PCR confirmed a mosaic gradient (variant allele frequency = .78% in affected neuronal sample; variant was absent in all other samples). SIGNIFICANCE: Our findings confirm that harvesting neuronal DNA from depth electrodes followed by molecular analysis to identify brain somatic variants is feasible. Our novel method represents a significant improvement compared to the previous method by focusing the analysis on high-quality cells of the cell type of interest.

Life-Saving Treatments for Spinal Muscular Atrophy: Global Access and Availability.

Background and Objectives: Spinal muscular atrophy (SMA) is a neurodegenerative disorder manifesting with progressive muscle weakness and atrophy. SMA type 1 used to be fatal within the first 2 years of life, but is now treatable with therapies targeting splicing modification and gene replacement. Nusinersen, risdiplam, and onasemnogene abeparvovec-xioi improve survival, motor strength, endurance, and ability to thrive, allowing many patients to potentially attain a normal life; all have been recently approved by major regulatory agencies. Although these therapies have revolutionized the world of SMA, they are associated with a high economic burden, and access to these therapies is limited in some countries. The primary objective of this study was to compare the availability and implementation of treatment of SMA from different regions of the world. Methods: In this qualitative study, we surveyed health care providers from 21 countries regarding their experiences caring for patients with SMA. The main outcome measures were provider survey responses on newborn screening, drug availability/access, barriers to treatment, and related questions. Results: Twenty-four providers from 21 countries with decades of experience (mean 26 years) in treating patients with SMA responded to the survey. Nusinersen was the most available therapy for SMA. Our survey showed that while genetic testing is usually available, newborn screening is still unavailable in many countries. The provider-reported treatment cost also varied between countries, and economic burden was a major barrier in treating patients with SMA. Discussion: Overall, this survey highlights the global inequality in managing patients with SMA. The spread of newborn screening is essential in ensuring improved access to care for patients with SMA. With the advancement of neurotherapeutics, more genetic diseases will soon be treatable, and addressing the global inequality in clinical care will require novel approaches to mitigate such inequality in the future.

Prognostic Utility of Cardiovascular Magnetic Resonance-Based Phenotyping in Patients With Muscular Dystrophy.

Background The prognostic utility of cardiovascular magnetic resonance imaging, including strain analysis and tissue characterization, has not been comprehensively investigated in adult patients with muscular dystrophy. Methods and Results We prospectively enrolled 148 patients with dystrophinopathies (including heterozygotes), limb-girdle muscular dystrophy, and type 1 myotonic dystrophy (median age, 36.0 [interquartile range, 23.0-50.0] years; 51 [34.5%] women) over 7.7 years in addition to an age- and sex-matched healthy control cohort (n=50). Cardiovascular magnetic resonance markers, including 3-dimensional strain and fibrosis, were assessed for their respective association with major adverse cardiac events. Our results showed that markers of contractile performance were reduced across all muscular dystrophy groups. In particular, the dystrophinopathies cohort experienced reduced left ventricular (LV) ejection fraction and high burden of replacement fibrosis. Patients with type 1 myotonic dystrophy showed a 26.8% relative reduction in LV mass with corresponding reduction in chamber volumes. Eighty-two major adverse cardiac events occurred over a median follow-up of 5.2 years. Although LV ejection fraction was significantly associated with major adverse cardiac events (adjusted hazard ratio [aHR], 3.0 [95% CI, 1.4-6.4]) after adjusting for covariates, peak 3-dimensional strain amplitude demonstrated greater predictive value (minimum principal amplitude: aHR, 5.5 [95% CI, 2.5-11.9]; maximum principal amplitude: aHR, 3.3 [95% CI, 1.6-6.8]; circumferential amplitude: aHR, 3.4 [95% CI, 1.6-7.2]; longitudinal amplitude: aHR, 3.4 [95% CI, 1.7-6.9]; and radial strain amplitude: aHR, 3.0 [95% CI, 1.4-6.1]). Minimum principal strain yielded incremental prognostic value beyond LV ejection fraction for association with major adverse cardiac events (change in χ2=13.8; P<0.001). Conclusions Cardiac dysfunction is observed across all muscular dystrophy subtypes; however, the subtypes demonstrate distinct phenotypic profiles. Myocardial deformation analysis highlights unique markers of principal strain that improve risk assessment over other strain markers, LV ejection fraction, and late gadolinium enhancement in this vulnerable patient population.

Novel RAB39B variant associated intellectual disability and levodopa-responsive young-onset parkinsonism.

We report a 37-year-old Caucasian male with history of developmental delay, childhood onset Intellectual Disability (ID) and attention deficit hyperactivity disorder (ADHD) who presented at the age of 34 with tremor-dominant parkinsonism. Next Generation Sequencing (NGS) revealed pathogenic hemizygous sequence variant, c.200G > T, in the RAB39B gene. This report expands the number of described individuals with young onset PD associated with RAB39B mutation.

Therapeutic developments for valosin-containing protein mediated multisystem proteinopathy.

PURPOSE OF REVIEW: Missense mutations in valosin-containing protein (VCP) can lead to a multisystem proteinopathy 1 (MSP1) with any combination of limb-girdle distribution inclusion body myopathy (IBM) (present in about 90% of cases), Paget's disease of bone, and frontotemporal dementia (IBMPFD). VCP mutations lead to gain of function activity with widespread disarray in cellular function, with enhanced ATPase activity, increased binding with its cofactors, and reduced mitofusin levels. RECENT FINDINGS: This review highlights novel therapeutic approaches in VCP-MSP in in-vitro and in-vivo models. Furthermore, we also discuss therapies targeting mitochondrial dysfunction, autophagy, TDP-43 pathways, and gene therapies in other diseases with similar pathway involvement which can also be applicable in VCP-MSP. SUMMARY: Being a rare disease, it is challenging to perform large-scale randomized control trials (RCTs) in VCP-MSP. However, it is important to recognize potential therapeutic targets, and assess their safety and efficacy in preclinical models, to initiate RCTs for potential therapies in this debilitating disease.

Primary lateral sclerosis.

Like motor neuron diseases (MNDs) refer to a constellation of primarily sporadic neurodegenerative diseases characterized by a progressive loss of upper and/or lower motor neurons. Primary lateral sclerosis (PLS) is considered a neurodegenerative disorder that is characterized by a gradually progressive course affecting the central motor systems, designated by the phrase "upper motor neurons." Despite significant development in neuroimaging, neurophysiology, and molecular biology, there is a growing consensus that PLS is of unknown etiology. Currently there is no disease-modifying treatment for PLS, or prospective randomized trials being carried out, partly due to the rarity of the disease and lack of significant understanding of the underlying pathophysiology. Consequently, the approach to treatment remains largely symptomatic. In this chapter we provide an overview of primary lateral sclerosis including clinical and electrodiagnostic considerations, differential diagnosis, updates in genetics and pathophysiology, and future directions for research.

The Role of Vitamin D in Neuroprotection in Multiple Sclerosis: An Update.

Multiple sclerosis (MS) is a complex neurological condition that involves both inflammatory demyelinating and neurodegenerative components. MS research and treatments have traditionally focused on immunomodulation, with less investigation of neuroprotection, and this holds true for the role of vitamin D in MS. Researchers have already established that vitamin D plays an anti-inflammatory role in modulating the immune system in MS. More recently, researchers have begun investigating the potential neuroprotective role of vitamin D in MS. The active form of vitamin D, 1,25(OH)2D3, has a range of neuroprotective properties, which may be important in remyelination and/or the prevention of demyelination. The most notable finding relevant to MS is that 1,25(OH)2D3 promotes stem cell proliferation and drives the differentiation of neural stem cells into oligodendrocytes, which carry out remyelination. In addition, 1,25(OH)2D3 counteracts neurodegeneration and oxidative stress by suppressing the activation of reactive astrocytes and M1 microglia. 1,25(OH)2D3 also promotes the expression of various neuroprotective factors, including neurotrophins and antioxidant enzymes. 1,25(OH)2D3 decreases blood-brain barrier permeability, reducing leukocyte recruitment into the central nervous system. These neuroprotective effects, stimulated by 1,25(OH)2D3, all enhance neuronal survival. This review summarizes and connects the current evidence supporting the vitamin D-mediated mechanisms of action for neuroprotection in MS.

Provisional practice recommendation for the management of myopathy in VCP-associated multisystem proteinopathy.

Valosin-containing protein (VCP)-associated multisystem proteinopathy (MSP) is a rare genetic disorder with abnormalities in the autophagy pathway leading to various combinations of myopathy, bone diseases, and neurodegeneration. Ninety percent of patients with VCP-associated MSP have myopathy, but there is no consensus-based guideline. The goal of this working group was to develop a best practice set of provisional recommendations for VCP myopathy which can be easily implemented across the globe. As an initiative by Cure VCP Disease Inc., a patient advocacy organization, an online survey was initially conducted to identify the practice gaps in VCP myopathy. All prior published literature on VCP myopathy was reviewed to better understand the different aspects of management of VCP myopathy, and several working group sessions were conducted involving international experts to develop this provisional recommendation. VCP myopathy has a heterogeneous clinical phenotype and should be considered in patients with limb-girdle muscular dystrophy phenotype, or any myopathy with an autosomal dominant pattern of inheritance. Genetic testing is the only definitive way to diagnose VCP myopathy, and single-variant testing in the case of a known familial VCP variant, or multi-gene panel sequencing in undifferentiated cases can be considered. Muscle biopsy is important in cases of diagnostic uncertainty or lack of a definitive pathogenic genetic variant since rimmed vacuoles (present in ~40% cases) are considered a hallmark of VCP myopathy. Electrodiagnostic studies and magnetic resonance imaging can also help rule out disease mimics. Standardized management of VCP myopathy will optimize patient care and help future research initiatives.

A protocol for single nucleus RNA-seq from frozen skeletal muscle.

Single-cell technologies are a method of choice to obtain vast amounts of cell-specific transcriptional information under physiological and diseased states. Myogenic cells are resistant to single-cell RNA sequencing because of their large, multinucleated nature. Here, we report a novel, reliable, and cost-effective method to analyze frozen human skeletal muscle by single-nucleus RNA sequencing. This method yields all expected cell types for human skeletal muscle and works on tissue frozen for long periods of time and with significant pathological changes. Our method is ideal for studying banked samples with the intention of studying human muscle disease.

Neuromuscular Complications of SARS-CoV-2 and Other Viral Infections.

In this article we review complications to the peripheral nervous system that occur as a consequence of viral infections, with a special focus on complications of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We discuss neuromuscular complications in three broad categories; the direct consequences of viral infection, autoimmune neuromuscular disorders provoked by viral infections, and chronic neurodegenerative conditions which have been associated with viral infections. We also include discussion of neuromuscular disorders that are treated by immunomodulatory therapies, and how this affects patient susceptibility in the current context of the coronavirus disease 2019 (COVID-19) pandemic. COVID-19 is associated with direct consequences to the peripheral nervous system via presumed direct viral injury (dysgeusia/anosmia, myalgias/rhabdomyolysis, and potentially mononeuritis multiplex) and autoimmunity (Guillain Barré syndrome and variants). It has important implications for people receiving immunomodulatory therapies who may be at greater risk of severe outcomes from COVID-19. Thus far, chronic post-COVID syndromes (a.k.a: long COVID) also include possible involvement of the neuromuscular system. Whether we may observe neuromuscular degenerative conditions in the longer term will be an important question to monitor in future studies.

Transcriptome analysis from muscle biopsy tissues in late-onset myopathies identifies potential biomarkers correlating to muscle pathology.

The diagnosis of adult-onset genetic muscle diseases is challenging because of the diversity of clinical phenotypes, findings on muscle biopsy that may be nonspecific, and the large number of genetic causes. Even with thorough investigation, the diagnostic yield for genetic testing in these populations is very low, and the distinction from acquired conditions such as sporadic inclusion body myositis [sIBM] can also prove difficult. In this study, we analysed whole transcriptome data generated from RNA isolated from muscle biopsy tissues, from a cohort of 16 participants with sIBM and other histologic diagnoses. Our objective was to identify candidate RNA biomarkers that could be an adjunctive tool in differentiating these conditions. Principal component analysis was able to delineate the groups based on their histologic diagnoses. Gene ontology and pathway analyses demonstrated dysregulation of immune pathways in sIBM. In mitochondrial myopathy we observed upregulation of FGF21, GDF15, ASNS and TRIB3, which are known candidate biomarkers for mitochondrial myopathy. Novel findings included the identification of transcripts of unknown function that were dysregulated in myofibrillar myopathy [JPX], dystrophic changes [MEG3], and mitochondrial myopathy [GAS5]. We suggest future investigations with larger cohorts of participants to confirm the findings of this study, with further directed experiments to determine the role of novel transcripts in disease pathogenesis.

Multisystem Proteinopathy Due to VCP Mutations: A Review of Clinical Heterogeneity and Genetic Diagnosis.

In this work, we review clinical features and genetic diagnosis of diseases caused by mutations in the gene encoding valosin-containing protein (VCP/p97), the functionally diverse AAA-ATPase. VCP is crucial to a multitude of cellular functions including protein quality control, stress granule formation and clearance, and genomic integrity functions, among others. Pathogenic mutations in VCP cause multisystem proteinopathy (VCP-MSP), an autosomal dominant, adult-onset disorder causing dysfunction in several tissue types. It can result in complex neurodegenerative conditions including inclusion body myopathy, frontotemporal dementia, amyotrophic lateral sclerosis, or combinations of these. There is also an association with other neurodegenerative phenotypes such as Alzheimer-type dementia and Parkinsonism. Non-neurological presentations include Paget disease of bone and may also include cardiac dysfunction. We provide a detailed discussion of genotype-phenotype correlations, recommendations for genetic diagnosis, and genetic counselling implications of VCP-MSP.

Case Report: Biallelic Loss of Function ATM due to Pathogenic Synonymous and Novel Deep Intronic Variant c.1803-270T > G Identified by Genome Sequencing in a Child With Ataxia-Telangiectasia.

Ataxia-telangiectasia (AT) is a complex neurodegenerative disease with an increased risk for bone marrow failure and malignancy. AT is caused by biallelic loss of function variants in ATM, which encodes a phosphatidylinositol 3-kinase that responds to DNA damage. Herein, we report a child with progressive ataxia, chorea, and genome instability, highly suggestive of AT. The clinical ataxia gene panel identified a maternal heterozygous synonymous variant (NM_000051.3: c.2250G > A), previously described to result in exon 14 skipping. Subsequently, trio genome sequencing led to the identification of a novel deep intronic variant [NG_009830.1(NM_000051.3): c.1803-270T > G] inherited from the father. Transcript analyses revealed that c.1803-270T > G results in aberrant inclusion of 56 base pairs of intron 11. In silico tests predicted a premature stop codon as a consequence, suggesting non-functional ATM; and DNA repair analyses confirmed functional loss of ATM. Our findings highlight the power of genome sequencing, considering deep intronic variants in undiagnosed rare disease patients.