Neuronal innervation regulates the secretion of neurotrophic myokines and exosomes from skeletal muscle.

Myokines and exosomes, originating from skeletal muscle, are shown to play a significant role in maintaining brain homeostasis. While exercise has been reported to promote muscle secretion, little is known about the effects of neuronal innervation and activity on the yield and molecular composition of biologically active molecules from muscle. As neuromuscular diseases and disabilities associated with denervation impact muscle metabolism, we hypothesize that neuronal innervation and firing may play a pivotal role in regulating secretion activities of skeletal muscles. We examined this hypothesis using an engineered neuromuscular tissue model consisting of skeletal muscles innervated by motor neurons. The innervated muscles displayed elevated expression of mRNAs encoding neurotrophic myokines, such as interleukin-6, brain-derived neurotrophic factor, and FDNC5, as well as the mRNA of peroxisome-proliferator-activated receptor γ coactivator 1α, a key regulator of muscle metabolism. Upon glutamate stimulation, the innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs than neuron-free muscles. Consequently, biological factors secreted by innervated muscles enhanced branching, axonal transport, and, ultimately, spontaneous network activities of primary hippocampal neurons in vitro. Overall, these results reveal the importance of neuronal innervation in modulating muscle-derived factors that promote neuronal function and suggest that the engineered neuromuscular tissue model holds significant promise as a platform for producing neurotrophic molecules.

NanoLC-timsTOF-Assisted Analysis of Glycated Albumin in Diabetes-Affected Plasma and Tears.

Glycation is a spontaneous and nonenzymatic glycosylation. Glycated albumin (GA), which serves as an important biomarker in plasma in the diagnosis and characterization of diabetes, can be passively filtered from the plasma to tears. Tears are important targets for research in clinical diagnostics due to the ability to collect this biofluid noninvasively and repeatably. Therefore, the analysis of GA in tear film provides information for monitoring diabetes progression independent of blood pathologies. Due to the limited volume (1-5 µL) of natural tear film, we developed a small volume assay using a nano liquid chromatography-trapped ion mobility spectrometry-time-of-flight MS (nanoLC-timsTOF) platform for the analysis of glycated albumin in human plasma and tear films affected by diabetes. The peptides containing lysine 525, which is the main glycation site in GA, were relatively quantified and represented as the GA level. The results of the measurements showed that GA levels were significantly higher in diabetes-affected plasma and tears compared to controls with a p-value < 0.01. A strong correlation of glycated albumin levels was observed for the plasma and tear film in diabetes samples (Pearson coefficient 0.92 with a p-value 0.0012). Moreover, the number of GA glycation sites was significantly higher in diabetes-affected plasma and tear comparatively to controls. Among all the glycation sites in plasma albumin, the GA level quantified by lysine 136/137 had a strong correlation with more commonly used lysine 525, suggesting that lysine 136 /137 is an alternative diabetes biomarker in plasma. Overall, our findings demonstrate GA in tears as a biomarker for monitoring diabetes progression, highlighting new possibilities for quick and noninvasive diabetes detection and monitoring.

Biphasic Liquid Microjunction Extraction for Profiling Neuronal RNA Modifications by Liquid Chromatography-Tandem Mass Spectrometry.

RNA modifications are emerging as critical players in the spatiotemporal regulation of gene expression. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables the simultaneous quantification of numerous enzymatically modified RNAs in a biological sample, conventional RNA extraction and enzymatic digestion protocols that are employed prior to analysis have precluded the application of this technique for small-volume samples. In this study, a biphasic liquid microjunction (LMJ) extraction system using coaxial capillaries that direct and aspirate extraction solvents onto a ∼350 µm diameter sample spot was developed and applied for the extraction of RNA from individual cell clusters in the central nervous system of the marine mollusk Aplysia californica. To maximize RNA recoveries, optimized extraction solvents consisting of 10% methanol and chloroform were evaluated under dynamic and static extraction conditions. An MS-compatible RNA digestion buffer was developed to minimize the number of sample-transfer steps and facilitate the direct enzymatic digestion of extracted RNA within the sample collection tube. Compared to RNA extraction using a conventional phenol-chloroform method, the LMJ-based method provided a 3-fold greater coverage of the neuronal epitranscriptome for similar amounts of tissues and also produced mRNA of sufficient purity for reverse transcription polymerase chain reaction amplification. Using this approach, the expression of RNA-modifying enzymes in a given neuronal cell cluster can be characterized and simultaneously correlated with the LC-MS/MS analysis of RNA modifications within the same subset of neurons.