Thomas Henry Gillingwater

Sarcopenia is the age-related loss of muscle strength and size that leads to mobility limitations and loss of independence in older adults. The underlying cellular mechanisms remain unclear, and treatments are limited. As the critical interface between the nervous system and muscle, the neuromuscular junction (NMJ) is essential for muscle activation and force production. Here, we demonstrate that weak older individuals exhibited NMJ transmission failure that correlated with muscle weakness severity. Preclinical experiments showed similar NMJ transmission failure in aged rodents that was associated with localized loss of muscle fiber excitability at the NMJ. This excitability defect, distinct from potential synaptic cholinergic transmission abnormalities, represents a disease mechanism of sarcopenia. Across species, immunohistochemistry identified a localized reduction in the voltage-gated sodium channel specific for skeletal muscle (NaV1.4) at the postsynaptic NMJ membrane. Acute NaV1.4 inhibition with μ-conotoxin GIIIB in adult rats reproduced findings of NMJ transmission failure observed in aged rodents and humans. Finally, ClC-1 chloride ion channel inhibition enhanced muscle excitability and improved NMJ transmission and muscle function in old rodents. Together, these findings demonstrate that NMJ transmission deficits are a key, reversible driver of sarcopenia and reveal a therapeutic target for addressing muscle weakness in aging.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of survival motor neuron (SMN) protein. Several therapeutic approaches boosting SMN are approved for human patients, delivering remarkable improvements in lifespan and symptoms. However, emerging phenotypes, including neurodevelopmental comorbidities, are being reported in some treated patients with SMA, indicative of alterations in brain development. Here, using a mouse model of severe SMA, we revealed an underlying neurodevelopmental phenotype in SMA where prenatal SMN-dependent defects in translation drove disruptions in nonmotile primary cilia across the central nervous system (CNS). Low levels of SMN caused widespread perturbations in translation at E14.5 targeting genes associated with primary cilia. The density of primary cilia in vivo, as well as cilial length in vitro, was significantly decreased in prenatal SMA mice. Proteomic analysis revealed downstream perturbations in primary cilia-regulated signaling pathways, including Wnt signaling. Cell proliferation was concomitantly reduced in the hippocampus of SMA mice. Prenatal transplacental therapeutic intervention with SMN-restoring risdiplam rescued primary cilia defects in SMA mouse embryos. Thus, SMN protein is required for normal cellular and molecular development of primary cilia in the CNS. Early, systemic treatment with SMN-restoring therapies can successfully target neurodevelopmental comorbidities in SMA.

Systemic immune changes have been implicated in amyotrophic lateral sclerosis (ALS), but precise mechanisms and cellular targets remain unknown. Neuromuscular junction (NMJ) denervation is another major pathophysiological event in ALS, but it remains unclear whether immune system dysregulation contributes to this process. Here, we report leukocyte and macrophage infiltration in ALS patient-derived skeletal muscle biopsies. Immune cell infiltration was replicated across the hTDP-43, TDP-43^A315T (male only) and TDP-43^M337V mouse models, occurring from pre-symptomatic stages and targeted to NMJ-enriched muscle regions. Proteomic analysis implicated the CCL2-CCR2 axis as a driving factor. CCL2⁺ cells were enriched around NMJs in hTDP-43 mice, and in ALS patient skeletal muscle. Local treatment with CCL2-neutralising antibodies or normal IgG antibodies in hTDP-43 mice reduced leukocyte infiltration and ameliorated NMJ denervation. These results demonstrate that the CCL2-CCR2 axis drives immune cell infiltration targeting NMJs in ALS, identifying a potential avenue for therapeutic intervention to prevent NMJ denervation.

Spinal muscular atrophy (SMA) is a monogenic neurodegenerative disorder caused by decreased levels of Survival of Motor Neuron (SMN) protein. If left untreated, SMA patients have a poor prognosis, marked by the degeneration of motor neurons, progressive muscle weakness and atrophy. The approval of SMN-restoring therapies that improve symptoms and lifespan in patients with SMA has created emerging, non-neuronal phenotypes and an urgent need for deepening our understanding of disease pathogenesis. Leveraging the knowledge that SMN loss drives alterations in translation, we used multiple tissues from a mouse model of SMA to uncover early translational alterations in key mRNAs and proteins, which act as contributors to pathogenesis and hallmarks of the disease. Among hundreds of differentially translated mRNAs, Col1a1 emerged as a translation-specific manifestation of early defects in the mouse model. These findings were confirmed in fibroblasts derived from patients with varying levels of disease severity. Notably, treatment with SMN-restoring therapies rescued COL1A1 protein levels, particularly in fibroblasts from patients with the most severe forms of the disease. Overall, our study identifies COL1A1 as an indicator of disease severity in SMA, which captures early molecular alterations and respond to SMN-modifying therapies.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in the survival motor neuron 1 ( SMN1 ) gene. SMA is characterised by alpha motor neuron loss in the spinal cord and subsequent muscle atrophy. There are currently three approved SMN-directed therapies for SMA patients. While these therapies have transformed what was once a life-limiting condition into one that can be managed and even improved, they are unfortunately not cures, highlighting the need for additional supporting second-generation therapies. These should not only target the neuromuscular system but also peripheral and metabolic perturbations that are present in both SMA models and patients. Krüppel-like factor 15 ( Klf15 ) is a transcription factor that maintains metabolic homeostasis and is involved in the glucocorticoid-glucocorticoid receptor (GR) signalling pathway, in several peripheral and metabolic tissues in SMA mice. Here, we used murine and human cellular models as well as SMA mice and Caenorhabditis Elegans (C. elegans) to assess the therapeutic potential of reducing Klf15 activity with mifepristone, a glucocorticoid antagonist, combined with SMN-targeted gene therapy. We report that mifepristone reduces Klf15 expression across several in vitro models, ameliorates neuromuscular pathology in SMA smn-1(ok355) C. elegans and improves survival of SMA Smn ^2B/- mice. Furthermore, we show that combining mifepristone with an approved SMN-directed gene therapy (scAAV9- SMN1 ) results in improved tissue- and sex-specific responses to treatment. Our study demonstrates that a multi-tissue targeting SMN-independent drug, alone and in combination with an approved SMN-dependent therapy, has the potential to improve SMA disease pathology.

The motor neuron disease amyotrophic lateral sclerosis (ALS) is a devastating condition with limited treatment options. The past few years have witnessed a ramping up of translational ALS research, offering the prospect of disease-modifying therapies. Although breakthroughs using gene-targeted approaches have shown potential to treat patients with specific disease-causing mutations, the applicability of such therapies remains restricted to a minority of individuals. Therapies targeting more general mechanisms that underlie motor neuron pathology in ALS are therefore of considerable interest. ALS pathology is associated with disruption to a complex array of key cellular pathways, including RNA processing, proteostasis, metabolism and inflammation. This Review details attempts to restore cellular homeostasis by targeting these pathways in order to develop effective, broadly-applicable ALS therapeutics.