Sustained Smad signaling in ALS muscle promotes disease progression through a dysregulated miRNA transcriptome

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative disease of motor neurons. There is no effective treatment for ALS and the determinants of disease onset and progression remain poorly understood. ALS affects veterans disproportionately over the civilian population, and thus is a major research priority for the VA.

There are two major gaps of knowledge in ALS that this application will address: (1) The pathogenic role of the peripheral neuromusuclar system in the initiation of disease and progression, and (2) The lack of plasma biomarkers that can aid the clinician in diagnosis, progression rate, and prognosis.

Skeletal muscle and the neuromuscular junction (NMJ) are the “end organs” of motor neurons and have been a growing focus as important contributors to ALS pathogenesis and disease progression, but there remains a large knowledge gap. The earliest pathological findings in ALS are in skeletal muscle including

mitochondrial dysfunction, retraction of motor neuron termini and degeneration of the neuromuscular junction. In normal states, there is ongoing cross-talk between muscle fibers and motor neurons that maintain the health of each, including the release of growth factors by muscle such as NT-4, BMP4 and IGF1. In ALS, this

homeostasis is disrupted by the degenerative process coupled with the infiltration of immune cells into the neuromuscular system. Using skeletal muscle of ALS patients, we have characterized a molecular network of biomarkers that is induced and activated in the earliest preclinical stages of disease and progressively

increases with disease progression. This network centers around elements of the TGFβ/BMP/Smad signaling axis and prominently includes Smad8, TGF-β1, 2, 3 and BMP4. In parallel with this axis is a dysregulated microRNA (miRNA) transcriptome which we characterized by miRNA sequencing of human ALS skeletal

muscle from patient biopsies. In our preliminary studies, we have discovered that Smad8 signaling regulates many of the components of this aberrant miRNA/mRNA network and that the predicted effect, based on in silico analysis, would be impairiment of muscle homeostasis, muscle regeneration, NMJ reinnervation and

promotion of neuromuscular inflammation. In other work, we have shown that ALS patients have extensive infiltration of myeloid cells, including mast cells, neutrophils, and macrophages, in muscle and peripheral nerve that accelerate disease progression. This background and preliminary data form the basis of our overarching

hypothesis that BMP/TGFβ/Smad signaling in ALS muscle promotes disease progression through a dysregulated miRNA transcriptome which is sustained by a paracrine loop with infiltrating immune cells. Furthermore, we hypothesize that components of this molecular network reflect disease progression and thus

may serve as plasma biomarkers that can be used in the clinic to monitor disease progression. We propose 3 aims: (1) Characterize the link between activated Smad8 and dysregulated miRNAs in ALS muscle, (2) Assess the impact of muscle-based Smad8 signaling on the clinicopathological phenotype of ALS using a muscle-

specific knockout of Smad8, and (3) Assess components of the dysregulated BMP/TGFβ/Smad8 pathway for their potential as clinical biomarkers of disease activity. Signficance: This bedside-to-bench proposal represents a novel direction with compelling translational implications. It vertically integrates basic and clinical

approaches that will provide insight into the contribution of the peripheral neuromuscular system to ALS disease progression.