Myelin forming around axons provides electrical insulation and ensures rapid and efficient transmission of electrical impulses. Disruptions to myelinated nerves often result in nerve conduction failure along with neurological symptoms and long-term disability. In the central nervous system, calpains, a family of calcium dependent cysteine proteases, have been shown to have a role in developmental myelination and in demyelinating diseases. The roles of calpains in myelination and demyelination in the peripheral nervous system remain unclear. Here, we show a transient increase of activated CAPN1, a major calpain isoform, in postnatal rat sciatic nerves when myelin is actively formed. Expression of the endogenous calpain inhibitor, calpastatin, showed a steady decrease throughout the period of peripheral nerve development. In the sciatic nerves of Trembler-J mice characterized by dysmyelination, expression levels of CAPN1 and calpastatin and calpain activity were significantly increased. In lysolecithin-induced acute demyelination in adult rat sciatic nerves, we show an increase of CAPN1 and decrease of calpastatin expression. These changes in the calpain-calpastatin system are distinct from those during central nervous system development or in acute axonal degeneration in peripheral nerves. Our results suggest that the calpain-calpastatin system has putative roles in myelination and demyelinating diseases of peripheral nerves.

Background: MicroRNAs (miRNAs) function as post-transcriptional gene expression regulators and influence the development and progression of several cancers, yet their roles in pediatric sarcomas remain poorly defined. Methods: RNA extracted from formalin-fixed paraffin-embedded tumor tissue scrolls of 108 pediatric tumors, including 32 osteosarcoma (OS), 26 Ewing’s sarcoma (EWS), and 50 rhabdomyosarcoma (RMS) cases, were analyzed for microRNA expression using the NanoString multiplex nCounter platform that yielded information on 827 human miRNAs. The expression of candidate miRNAs was validated with in situ hybridization (miRNA-ISH) and QuPath quantification on tissue microarray slides comprising an independent set of 48 OS, 17 EWS, and 104 RMS adult and pediatric cases collectively. Results: The differential expression analysis of nCounter data identified 23 miRNAs enriched in RMS, 33 in EWS, and 45 in OS (>3 fold change and p < 0.01). miR-206 was most strongly associated (>55 fold change, p < 1 × 10⁻⁹) with RMS and demonstrated the highest sensitivity and specificity in distinguishing RMS from EWS and OS; this finding was also confirmed by miRNA-ISH. A combined signature of differentially expressed miRNAs reliably separated alveolar from embryonal RMS. The expression of miR-9-5p in EWS and miR-140-5p in OS discriminated among the different tumors and correlated with adverse patient outcome. The nCounter assay exhibited greater sensitivity than miRNA-ISH in detecting miR-206 and miR-140-5p expression. Conclusions: Collectively, these findings demonstrate that distinct miRNA profiles can differentiate pediatric sarcoma types and subtypes and offer clinically relevant insights into tumor biology, prognosis, and potential diagnostic application.