No abstract

It is now well established that the conversion of the cellular prion protein, PrP C , into its anomalous conformer, PrP Sc , is central to the onset of prion disease. However, both the mechanism of prion-related neurodegeneration and the physiologic role of PrP C are still unknown. The use of animal and cell models has suggested a number of putative functions for the protein, including cell signaling, adhesion, proliferation, and differentiation. Given that skeletal muscles express significant amounts of PrP C and have been related to PrP C pathophysiology, in the present study, we used skeletal muscles to analyze whether the protein plays a role in adult morphogenesis. We employed an in vivo paradigm that allowed us to compare the regeneration of acutely damaged hind-limb tibialis anterior muscles of mice expressing, or not expressing, PrP C . Using morphometric and biochemical parameters, we provide compelling evidence that the absence of PrP C significantly slows the regeneration process compared to wild-type muscles by attenuating the stress-activated p38 pathway, and the consequent exit from the cell cycle, of myogenic precursor cells. Demonstrating the specificity of this finding, restoring PrP C expression completely rescued the muscle phenotype evidenced in the absence of PrP C .

1These authors contributed equally to this study.Abbreviations used: AEQ, aequorin; BSA, bovine serum albumin; CGN, cerebellar granule neurons; CH1, immunoglobulin heavy chain constant region 1; ER, endoplasmic reticulum; GFAP, glial fibrillary acidic protein; HA, hemagglutinin; IB, isolation buffer; KO, knock-out; KRB, Krebs-Ringer buffer; lsCSQ, leader sequence of calsequestrin; mAb, monoclonal antibody; pAb, polyclonal antibody; PBS, phosphatebuffered saline; PM, plasma membrane; PMCA, plasma membrane Ca 2+ -ATPase; PrP, prion protein; SDS, sodium dodecyl-sulphate; SER-CA, sarco-endoplasmic reticulum Ca 2+ -ATPase; SOC, store-operated channels; SOCE, store-operated Ca 2+ entry; STIM, stromal interaction molecule; Tg, transgenic; TSE, transmissible spongiform encephalopathies; VOC, voltage-operated channels; WT, wild-type. AbstractThe cellular prion protein (PrP C ) is a cell-surface glycoprotein mainly expressed in the CNS. The structural conversion of PrP C generates the prion, the infectious agent causing

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the socalled degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization.

Skeletal muscle fibers contain different isoforms of myosin heavy chain (MyHC) that define distinctive contractile properties. In light of the muscle capacity to adapt MyHC expression to pathophysiological conditions, a rapid and quantitative assessment of MyHC isoforms in small muscle tissue quantities would represent a valuable diagnostic tool for (neuro)muscular diseases. As past protocols did not meet these requirements, in the present study we applied a targeted proteomic approach based on selected reaction monitoring that allowed the absolute quantification of slow and fast MyHC isoforms in different mouse skeletal muscles with high reproducibility. This mass-spectrometry-based method was validated also in a pathological specimen, by comparison of the MyHC expression profiles in different muscles from healthy mice and a genetic mouse model of amyotrophic lateral sclerosis (ALS) expressing the SOD1(G93A) mutant. This analysis showed that terminally ill ALS mice have a fast-to-slow shift in the fiber type composition of the tibialis anterior and gastrocnemius muscles, as previously reported. These results will likely open the way to accurate and rapid diagnoses of human (neuro)muscular diseases by the proposed method. Graphical Abstract Methods for myosin heavy chain (MyHC) quantification: a comparison of classical methods and selected reaction monitoring (SRM)-based mass spectrometry approaches.