Search

BackgroundSarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in the efferent nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve in aging mice.MethodsSciatic nerve and gastrocnemius muscle were obtained from young adult, middleaged, old, and sarcopenic (5,18, 21 and 24 months old, respectively) C57BL/6J female mice (n=6 per age group). Sciatic nerve RNA was extracted and subjected to RNA sequencing (RNA-seq), with real-time quantitative reverse transcription PCR (qRT-PCR) validation of differentially expressed genes (DEGs). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups was performed. Sarcopenia was confirmed with qRT-PCR of previously established markers of sarcopenia onset in gastrocnemius muscle.ResultsWe detected 33 significant DEGs in sciatic nerve of 18-month-old mice compared to 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05) which we validated with qRT-PCR of the three top up- and down-regulated genes. Up-regulated genes were associated with circadian rhythm and the AMPK signaling pathway, while down-regulated genes were associated with biosynthesis and metabolic pathways and circadian rhythm. Strikingly, we detected a significant increase in Myog expression (log2 fold change = 18.93, FDR q-value = 1.54×10−12) in sciatic nerve of 18-month-old mice, before up-regulation in muscle was observed. We identified seven clusters of genes with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in sarcopenia initiation including extracellular matrix organization and circadian regulation of gene expression.ConclusionsGene expression changes in mouse peripheral nerve can be detected prior to overt clinical onset of sarcopenia. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies will validate which of the key changes we reported have disease modifying and/or biomarker potential.

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

Despite a recent increase in e-cigarette use, the adverse human health effects of exposure to e-cigarette aerosol, especially on the central nervous system (CNS), remain unclear. Multiple neurotoxic metals have been identified in e-cigarette aerosol. However, it is unknown whether those metals accumulate in the CNS at biologically meaningful levels. To answer this question, two groups of mice were whole-body exposed twice a day, 5 days a week, for two months, to either a dose of e-cigarette aerosol equivalent to human secondhand exposure, or a 5-fold higher dose. After the last exposure, the olfactory bulb, anterior and posterior frontal cortex, striatum, ventral midbrain, cerebellum, brainstem, remaining brain tissue and spinal cord were collected for metal quantification by inductively coupled plasma mass spectrometry and compared to tissues from unexposed control mice. The two-month exposure caused significant accumulation of several neurotoxic metals in various brain areas - for some metals even at the low exposure dose. The most striking increases were measured in the striatum. For several metals, including Cr, Cu, Fe, Mn, and Pb, similar accumulations are known to be neurotoxic in mice. Decreases in some essential metals were observed across the CNS. Our findings suggest that chronic exposure to e-cigarette aerosol could lead to CNS neurotoxic metal deposition and endogenous metal dyshomeostasis, including potential neurotoxicity. We conclude that e-cigarette-mediated metal neurotoxicity may pose long-term neurotoxic and neurodegenerative risks for e-cigarette users and bystanders.