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Background: Physical exercise directly stretching the peripheral nerve promotes nerve regeneration; however, its action mechanism remains elusive. Our present study aimed to investigate the effects of mechanosensitive channel of large conductance (MscL) activated by mechanical stretching on the cultured Schwann cells (SCs) and explore the possible mechanism. Methods: Primary SCs from neonatal mice at 3–5 days of age were derived and transfected with the lentivirus vector expressing a mutant version of MscL, MscL-G22S. We first detected the cell viability and calcium ion (Ca2+) influx in the MscL-G22S-expressing SCs with low-intensity mechanical stretching and the controls. Proteomic and energy metabolomics analyses were performed to investigate the comprehensive effects of MscL-G22S activation on SCs. Measurement of glycolysis- and oxidative phosphorylation-related molecules and ATP production were respectively performed to further validate the effects of MscL-G22S activation on SCs. Finally, the roles of phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway in the mechanism of energy metabolism modulation of SCs by MscL-G22S activation was investigated. Results: Mechanical stretching-induced MscL-G22S activation significantly increased the cell viability and Ca2+ influx into the SCs. Both the proteomic and targeted energy metabolomics analysis indicated the upregulation of energy metabolism as the main action mechanism of MscL-G22S-activation on SCs. MscL-G22S-activated SCs showed significant upregulation of glycolysis and oxidative phosphorylation when SCs with stretching alone had only mild upregulation of energy metabolism than those without stimuli. MscL-G22S activation caused significant phosphorylation of the PI3K/AKT/mTOR signaling pathway and upregulation of HIF-1α/c-Myc. Inhibition of PI3K abolished the MscL-G22S activation-induced upregulation of HIF-1α/c-Myc signaling in SCs and reduced the levels of glycolysis- and oxidative phosphorylation-related substrates and mitochondrial activity. Conclusion: Mechanical stretching activates MscL-G22S to significantly promote the energy metabolism of SCs and the production of energic substrates, which may be applied to enhance nerve regeneration via the glia-axonal metabolic coupling. [ABSTRACT FROM AUTHOR]

The autoantigen La protein is an important component of telomerase and a predominantly nuclear phosphoprotein. As a telomerase subunit, La protein associates with the telomerase ribonucleoprotein and influences telomere length. In the reverse transcription, La protein stimulates enzymatic activity and increases repeated addition processivity of telomerase. As nuclear phosphoprotein, La protein binds the 3′ poly(U)‐rich elements of nascent RNA polymerase III transcripts to facilitate its correct folding and maturation. In this work, we identified a La protein homolog (TbLa) from Trypanosoma brucei (T. brucei). We revealed that TbLa interacts with ribosome‐associated protein P34/P37, 40S ribosomal protein SA, and 60S ribosomal subunit L5 in T. brucei. In the interactions between TbLa protein and (P34/P37)/L5/SA, RNA recognition motif (RRM) domain of TbLa was indicated to make the major contribution to the processes. We determined the solution structure of TbLa RRM domain. NMR chemical shift perturbations revealed that the positively charged RNA‐binding pocket of TbLa RRM domain is responsible for its interaction with ribosomal and ribosome‐associated proteins P37/L5/SA. Furthermore, depletion of TbLa affected the maturation process of 5S rRNA and ribosomal assembly, suggesting TbLa protein might play a significant role in the ribosomal biogenesis pathway in T. brucei. Taken together, our results provide a novel insight and structural basis for better understanding the roles of TbLa and RRM domain in ribosomal biogenesis in T. brucei. Database: Structural data are available in the PDB under the accession number 5ZUH. [ABSTRACT FROM AUTHOR]

DNAX activating protein of 12 kDa (DAP12)-deficiency mice showed impaired differentiation of oligodendrocytes and reduced myelin in the central nervous system. Whether DAP12 is expressed by Schwann cells and its roles in the peripheral nervous system (PNS) remains unknown. In this study, expression of DAP12 was detected in Schwann cells in vivo and in vitro. The DAP12-knockout (KO) mice showed age-related motor deficits and thinner myelin in the sciatic nerve than WT mice but significantly faster clinical recovery after sciatic nerve crush injury. In sciatic nerves of DAP12 KO and WT mice, proteomic profiles analysis identified 158 differentially expressed proteins (DEPs) at 8-week-old, 29 DEPs at 54-week-old and 33 DEPs at two weeks after crush injury. Typically, of the DEPs at 54-week-old, up-regulated Lgmn and down-regulated RecK and Yap1 were associated with myelin loss in the sciatic nerve of DAP12 KO mice. Upregulation of nicotinamide nucleotide transhydrogenase and haptoglobin were associated with the accumulation of macrophages in the crushed sciatic nerve of DAP12 KO mice. After crush injury, there were significantly more M1 macrophages at one-week and more M2 macrophages at two-week in sciatic nerve of DAP12 KO mice than WT mice, indicating that DAP12 deletion promotes the phenotype conversion of macrophages from M1 to M2. Collectively, our findings suggest that DAP12 may exert dual roles in the PNS including promoting the physiological myelin formation and maintenance of Schwann cells but delaying nerve repair after injury by modulating the recruitment of macrophages and phenotype conversion. [Display omitted] • We demonstrated the expression of DNAX activating protein of 12 kDa (DAP12) in Schwann cells. • DAP12 deletion causes age-related motor deficits in peripheral nerve. • DAP12 deletion promotes the clinical recovery of mutant mice from sciatic nerve crush injury. [ABSTRACT FROM AUTHOR]

Schwann cells (SCs) isolation is one of the basic techniques for study of peripheral nervous system and peripheral neuropathy. A combined and effective method of isolating SCs from sciatic nerves of newborn mice with high yield and purity is still lacking. Sciatic nerves from neonatal mice aged 3–5 days serve as the source of SCs. Removal of adjacent connective tissue and epineurium, treatment with arabinoside hydrochloride and differential cell detachment technique were applied to eliminate fibroblast contamination and increase the purity of SCs. Combined use of collagenase/dispase and trypsin was chosen to increase the yield of SCs. Culture dishes precoated with poly-l-lysine and laminin, culture medium supplemented with heregulin β-1 and forskolin, and reasonable cell seeding density were implemented to increase the growth and proliferation of cultured SCs. Immunostaining of S100β and p75 neurotrophin receptor was used to identify the purity of SCs. Our method is able to obtain high-yield SCs with a purity of 90% within five days and a purity more than 99% within seven days from sciatic nerves of neonatal mice. Previous SCs isolation mostly focused on rats or adult mice and have a few limitations due to fibroblasts contamination, low yield and time-consuming. Our method permits SCs isolation from neonatal mice with a high yield and purity of primary SCs within 7 days. We described a fast, efficient and step-by-step method of isolating SCs from sciatic nerves of neonatal mice with high yield and purity. [Display omitted] • A new method of isolating Schwann cells (SCs) from newborn mice was described. • A high yield and purity (> 99%) of SCs isolation were obtained. • The whole procedure can be completely accomplished within 7 days. [ABSTRACT FROM AUTHOR]

Solution structure of TbUfm1 and its binding surface to TbUba5 (marked in red). • TbUfm1 adopts a typical β-grasp fold and helixes that wrapped by the β-grasp fold. • TbUfm1 can bind to TbUba5 in vitro. • TbUfm1 interact with TbUba5 via the hydrophobic pocket formed by α1α2β1β2. Ubiquitin-like proteins are conserved in eukaryotes and involved in numerous cellular processes. Ufm1 is proved to play important roles in endoplasmic reticulum homeostasis, vesicle transportation and embryonic development. Enzyme cascade of Ufm1 is similar to that of ubiquitin. Mature Ufm1 is activated and conjugated to substrates by assistance of Ufm1 activating enzyme Uba5 (E1), Ufm1 conjugating enzyme Ufc1 (E2), and Ufm1 ligating enzyme Ufl1 (E3). Here, we determined the solution structure of TbUfm1 from Trypanosoma brucei (T. brucei) by NMR spectroscopy and explored the interactions between TbUfm1 and TbUba5/TbUfc1/TbUfl1. TbUfm1 adopts a typical β-grasp fold, which partially wraps a central α-helix and the other two helixes. NMR chemical shift perturbation indicated that TbUfm1 interacts with TbUba5 via a hydrophobic pocket formed by α1α2β1β2. Although the structure and Uba5-interaction mode of TbUfm1 are conserved in Ufm1 proteins, there are also some differences, which might reflect the potential diversity of Ufm1 in evolution and biological functions. [ABSTRACT FROM AUTHOR]