BACKGROUND. Spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein. New SMN-enhancing therapeutics are associated with variable clinical benefits. Limited knowledge of baseline and druginduced SMN levels in disease-relevant tissues hinders efforts to optimize these treatments. METHODS. SMN mRNA and protein levels were quantified in human tissues isolated during expedited autopsies. RESULTS. SMN protein expression varied broadly among prenatal control spinal cord samples, but was restricted at relatively low levels in controls and SMA patients after 3 months of life. A 2.3-fold perinatal decrease in median SMN protein levels was not paralleled by comparable changes in SMN mRNA. In tissues isolated from nusinersen-treated SMA patients, antisense oligonucleotide (ASO) concentration and full-length (exon 7 including) SMN2 (SMN2-FL) mRNA level increases were highest in lumbar and thoracic spinal cord. An increased number of cells showed SMN immunolabeling in spinal cord of treated patients, but was not associated with an increase in whole-tissue SMN protein levels. CONCLUSIONS. A normally occurring perinatal decrease in whole-tissue SMN protein levels supports efforts to initiate SMN-inducing therapies as soon after birth as possible. Limited ASO distribution to rostral spinal and brain regions in some patients likely limits clinical response of motor units in these regions for those patients. These results have important implications for optimizing treatment of SMA patients and warrant further investigations to enhance bioavailability of intrathecally administered ASOs.
Systemically low levels of survival motor neuron-1 (SMN1) protein cause spinal muscular atrophy (SMA). α-Motor neurons of the spinal cord are considered particularly vulnerable in this genetic disorder and their dysfunction and loss cause progressive muscle weakness, paralysis and eventually premature death of afflicted individuals. Historically, SMA was therefore considered a motor neuron-autonomous disease. However, depletion of SMN in motor neurons of normal mice elicited only a very mild phenotype. Conversely, restoration of SMN to motor neurons in an SMA mouse model had only modest effects on the SMA phenotype and survival. Collectively, these results suggested that additional cell types contribute to the pathogenesis of SMA, and understanding the non-autonomous requirements is crucial for developing effective therapies. Astrocytes are critical for regulating synapse formation and function as well as metabolic support for neurons. We hypothesized that astrocyte functions are disrupted in SMA, exacerbating disease progression. Using viral-based restoration of SMN specifically to astrocytes, survival in severe and intermediate SMA mice was observed. In addition, neuromuscular circuitry was improved. Astrogliosis was prominent in end-stage SMA mice and in post-mortem patient spinal cords. Increased expression of proinflammatory cytokines was partially normalized in treated mice, suggesting that astrocytes contribute to the pathogenesis of SMA.
Gene replacement and pre-mRNA splicing modifier therapies represent breakthrough gene targeting treatments for the neuromuscular disease spinal muscular atrophy (SMA), but mechanisms underlying variable efficacy of treatment are incompletely understood. Our examination of severe infantile onset human SMA tissues obtained at expedited autopsy revealed persistence of developmentally immature motor neuron axons, many of which are actively degenerating. We identified similar features in a mouse model of severe SMA, in which impaired radial growth and Schwann cell ensheathment of motor axons began during embryogenesis and resulted in reduced acquisition of myelinated axons that impeded motor axon function neonatally. Axons that failed to ensheath degenerated rapidly postnatally, specifically releasing neurofilament light chain protein into the blood. Genetic restoration of survival motor neuron protein (SMN) expression in mouse motor neurons, but not in Schwann cells or muscle, improved SMA motor axon development and maintenance. Treatment with small-molecule SMN2 splice modifiers beginning immediately after birth in mice increased radial growth of the already myelinated axons, but in utero treatment was required to restore axonal growth and associated maturation, prevent subsequent neonatal axon degeneration, and enhance motor axon function. Together, these data reveal a cellular basis for the fulminant neonatal worsening of patients with infantile onset SMA and identify a temporal window for more effective treatment. These findings suggest that minimizing treatment delay is critical to achieve optimal therapeutic efficacy.
A pathological hallmark of spinal muscular atrophy (SMA) is severe motor neuron (MN) loss, which results in muscle weakness and often infantile or childhood mortality. Although it is well established that deficient expression of survival motor neuron (SMN) protein causes SMA, the molecular pathways that execute MN cell death are poorly defined. The c-Jun NH2-terminal kinases (JNKs) are stress-activated kinases with multiple substrates including c-Jun, which can be activated during neuronal injury and neurodegenerative disease leading to neuronal apoptosis. Recently, increased JNK-c-Jun signaling was reported in SMA raising the possibility that JNK inhibitors could be a novel treatment for this disease. We examined JNK-c-Jun activity in SMA mouse and human cultured cells and tissues. Anisomycin treatment of human SMA fibroblasts and sciatic nerve ligation in SMA mice provoked robust phosphorylated-c-Jun (p-c-Jun) expression indicating that SMN-deficiency does not prevent activation of the stress-induced JNK-c-Jun signaling pathway. Despite retained capacity to activate JNK-c-Jun, we observed no basal increase of p-c-Jun levels in SMA compared to control cultured cells, human or mouse spinal cord tissues, or mouse MNs during the period of MN loss in severe SMA model mice. In both controls and SMA, ~50% of α-MN nuclei express p-c-Jun with decreasing expression during the early postnatal period. Together these studies reveal no evidence of stress-activated JNK-c-Jun signaling in MNs of SMA mice or human tissues, but do highlight the important role of JNK-c-Jun activity during normal MN development raising caution about JNK antagonism in this pediatric neuromuscular disease.
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