Spinal muscular atrophy (SMA) is caused by mutations of the survival of motor neuron (SMN1) gene and deficiency of full-length SMN protein (FL-SMN). All SMA patients retain one or more copies of the SMN2 gene, but the principal protein product of SMN2 lacks exon 7 (SMN⌬7) and is unable to compensate for a deficiency of FL-SMN. SMN is known to oligomerize and form a multimeric protein complex; however, the mechanisms regulating stability and degradation of FL-SMN and SMN⌬7 proteins have been largely unexplored. Using pulsechase analysis, we characterized SMN protein turnover and confirmed that SMN was ubiquitinated and degraded by the ubiquitin proteasome system (UPS). The SMN⌬7 protein had a twofold shorter half-life than FL-SMN in cells despite similar intrinsic rates of turnover by the UPS in a cell-free assay. Mutations that inhibited SMN oligomerization and complex formation reduced the FL-SMN half-life. Furthermore, recruitment of SMN into large macromolecular complexes as well as increased association with several Gemin proteins was regulated in part by protein kinase A. Together, our data indicate that SMN protein stability is modulated by complex formation. Promotion of the SMN complex formation may be an important novel therapeutic strategy for SMA.

These findings support a novel function for FSTL1 and provide the first direct evidence that a circulating cardiokine/myokine can alter myocardial and systemic energy substrate metabolism, in vivo.

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Muscle stem cells (MuSCs) hold great potential as a regenerative therapeutic but have met numerous challenges in treating systemic muscle diseases. Muscle stem cell-derived extracellular vesicles (MuSC-EVs) may overcome these limitations. We assessed the number and size distribution of extracellular vesicles (EVs) released by MuSCs ex vivo, determined the extent to which MuSC-EVs deliver molecular cargo to myotubes in vitro, and quantified MuSC-EV-mediated restoration of mitochondrial function following oxidative injury. MuSCs released an abundance of EVs in culture. MuSC-EVs delivered protein cargo into myotubes within 2 h of incubation. Fluorescent labeling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. Oxidatively injured myotubes demonstrated a significant decline in maximal oxygen consumption rate and spare respiratory capacity relative to untreated myotubes. Remarkably, subsequent treatment with MuSC-EVs significantly improved maximal oxygen consumption rate and spare respiratory capacity relative to the myotubes that were damaged but received no subsequent treatment. Surprisingly, MuSC-EVs did not affect mitochondrial function in undamaged myotubes, suggesting the cargo delivered is able to repair but does not expand the existing mitochondrial network. These data demonstrate that MuSC-EVs rapidly deliver proteins into myotubes, a portion of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged myotubes.

Modifiable cardiometabolic risk factors induce the release of pro-inflammatory cytokines and reactive oxygen species from circulating peripheral blood mononuclear cells (PBMCs) resulting in increased cardiovascular disease risk and compromised immune health. These changes may be driven by metabolic reprogramming of PBMCs resulting in impaired mitochondrial respiration; however, this has not been fully tested. We aimed to determine the independent associations between cardiometabolic risk factors, such as blood pressure, BMI, and plasma lipids with impaired mitochondrial respiration in PBMCs isolated from generally healthy individuals (n=21) across the adult lifespan (12 M/ 9 F; age: 56 ± 21 years; age-range: 22-78 year; blood pressure: 123 ± 16 / 72 ± 10 mmHg; low-density lipoprotein cholesterol, LDL-C: 111 ± 22 mg/dL; and high-density lipoprotein cholesterol, HDL-C: 62 ± 16 mg/dL). PBMCs were isolated from whole blood by density dependent centrifugation and used to assess mitochondrial function by respirometry. Primary outcomes included baseline and maximal oxygen consumption rate (OCR) which were subsequently used to determine spare respiratory capacity and OCR metabolic potential. After correcting for HDL-C, SBP, and age, LDL-C was negatively associated with maximal respiration (r=-0.61, P=0.0073), spare respiratory capacity (r=-0.65, P=0.0038) and OCR metabolic potential (r=-0.59, P=0.010), respectively. In addition, there was a strong, but non-significant positive association between HDL-C and maximal respiration (r=0.46, P=0.057) and spare respiratory capacity (r=0.43, P=0.075), after correcting for other covariates. These data suggest a link between blood cholesterol and mitochondrial health that may provide insight into how cardiometabolic risk factors contribute to impaired immune cell function.

Marek's disease (MD) is an alphaherpesvirus (Marek's disease virus, MDV)-induced pathology of chickens associated with paralysis, immunosuppression, neurological signs, and T-cell lymphomas. MD is controlled in poultry production via live attenuated vaccines. The purpose of the current study was to compare methods for precipitating exosomes from vaccinated and protected chicken sera (VEX) and tumor-bearing chicken sera (TEX) for biomarker analysis of vaccine-induced protection and MD lymphomas respectively. A standard polyethylene glycol (PEG, 8%) method was compared to a commercial reagent (total exosome isolation reagent, TEI) for exosome yield and RNA content. Although exosomes purified by PEG or TEI were comparable in size and morphology, TEI-reagent yielded 3-4-fold greater concentration. Relative expression of 8 out of 10 G. gallus - and MDV1-encoded miRNAs examined displayed significant difference depending upon the precipitation method used. Standard PEG yields comparable, albeit lower amounts of exosomes than the TEI-reagent and a distinctive miRNA composition.

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