Progranulin (PGRN) is involved in wound repair

No abstract

Progranulin (PGRN) haploinsufficiency resulting from loss-of-function mutations in the PGRN gene causes frontotemporal lobar degeneration accompanied by TDP-43 accumulation, and patients with homozygous mutations in the PGRN gene present with neuronal ceroid lipofuscinosis. Although it remains unknown why PGRN deficiency causes neurodegenerative diseases, there is increasing evidence that PGRN is implicated in lysosomal functions. Here, we show PGRN is a secretory lysosomal protein that regulates lysosomal function and biogenesis by controlling the acidification of lysosomes. PGRN gene expression and protein levels increased concomitantly with the increase of lysosomal biogenesis induced by lysosome alkalizers or serum starvation. Down-regulation or insufficiency of PGRN led to the increased lysosomal gene expression and protein levels, while PGRN overexpression led to the decreased lysosomal gene expression and protein levels. In particular, the level of mature cathepsin D (CTSDmat) dramatically changed depending upon PGRN levels. The acidification of lysosomes was facilitated in cells transfected with PGRN. Then, this caused degradation of CTSDmat by cathepsin B. Secreted PGRN is incorporated into cells via sortilin or cation-independent mannose 6-phosphate receptor, and facilitated the acidification of lysosomes and degradation of CTSDmat. Moreover, the change of PGRN levels led to a cell-type-specific increase of insoluble TDP-43. In the brain tissue of FTLD-TDP patients with PGRN deficiency, CTSD and phosphorylated TDP-43 accumulated in neurons. Our study provides new insights into the physiological function of PGRN and the role of PGRN insufficiency in the pathogenesis of neurodegenerative diseases.

Neuronal and glial deposition of misfolded, proteolytically processed, polyubiquitinated and abnormally phosphorylated C‐terminal fragments (CTFs) of the TAR DNA binding protein‐43 (TDP‐43) is a pathological hallmark of frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD‐U) and certain cases of amyotrophic lateral sclerosis. We demonstrate that TDP‐43 can be proteolytically processed by caspases upon induction of apoptosis to a major 35 kDa and a minor 25 kDa CTF. These fragments are initially soluble, but over time they accumulate as insoluble and pathologically phosphorylated derivatives. However, proteolytic processing appears not to be absolutely required for the deposition of insoluble TDP‐43 species, since a caspase resistant mutant of TDP‐43 is also converted into insoluble species. Phosphorylation at S409/410 apparently occurs late during the conversion of soluble to insoluble TDP‐43, suggesting that phosphorylation is not a prerequisite for aggregation. Loss of function of the progranulin (PGRN) gene causes FTLD‐U with TDP‐43 positive inclusions and has been suggested to lead to caspase activation and subsequent TDP‐43 processing. However, siRNA‐mediated knockdown of PGRN in cell culture as well as a PGRN gene knockout in mice failed to cause the formation of the disease characterizing CTFs of TDP‐43. Our findings therefore suggest that caspase‐mediated processing generates CTFs of similar biochemical properties as those occurring in nuclear and cytoplasmic deposits of FTLD‐U patients independent of PGRN levels.

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin12. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD3. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD.

No abstract

Duchenne muscular dystrophy (DMD) is a progressive disease characterised by chronic muscle degeneration and inflammation. Our previously established DMD model rats (DMD rats) have a more severe disease phenotype than the broadly used mouse model. We aimed to investigate the role of senescence in DMD using DMD rats and patients. Senescence was induced in satellite cells and mesenchymal progenitor cells, owing to the increased expression of CDKN2A, p16- and p19-encoding gene. Genetic ablation of p16 in DMD rats dramatically restored body weight and muscle strength. Histological analysis showed a reduction of fibrotic and adipose tissues invading skeletal muscle, with increased muscle regeneration. Senolytic drug ABT263 prevented loss of body weight and muscle strength, and increased muscle regeneration in rats even at 8 months—the late stage of DMD. Moreover, senescence markers were highly expressed in the skeletal muscle of DMD patients. In situ hybridization of CDKN2A confirmed the expression of it in satellite cells and mesenchymal progenitor cells in patients with DMD. Collectively, these data provide new insights into the integral role of senescence in DMD progression.

Skeletal muscle regeneration and work-induced hypertrophy rely on molecular events responsible for activation and quiescence of resident myogenic stem cells, satellite cells. Recent studies demonstrated that hepatocyte growth factor (HGF) triggers activation and entry into the cell cycle in response to mechanical perturbation, and that subsequent expression of myostatin may signal a return to cell quiescence. However, mechanisms responsible for coordinating expression of myostatin after an appropriate time lag following activation and proliferation are not clear. Here we address the possible role of HGF in quiescence through its concentration-dependent negative-feedback mechanism following satellite cell activation and proliferation. When activated/proliferating satellite cell cultures were treated for 24 h beginning 48-h postplating with 10-500 ng/ml HGF, the percentage of bromodeoxyuridine-incorporating cells decreased down to a baseline level comparable to 24-h control cultures in a HGF dose-dependent manner. The high level HGF treatment did not impair the cell viability and differentiation levels, and cells could be reactivated by lowering HGF concentrations to 2.5 ng/ml, a concentration that has been shown to optimally stimulate activation of satellite cells in culture. Coaddition of antimyostatin neutralizing antibody could prevent deactivation and abolish upregulation of cyclin-dependent kinase (Cdk) inhibitor p21. Myostatin mRNA expression was upregulated with high concentrations of HGF, as demonstrated by RT-PCR, and enhanced myostatin protein expression and secretion were revealed by Western blots of the cell lysates and conditioned media. These results indicate that HGF could induce satellite cell quiescence by stimulating myostatin expression. The HGF concentration required (over 10-50 ng/ml), however, is much higher than that for activation, which is initiated by rapid release of HGF from its extracellular association. Considering that HGF is produced by satellite cells and spleen and liver cells in response to muscle damage, local concentrations of HGF bathing satellite cells may reach a threshold sufficient to induce myostatin expression. This time lag may delay action of the quiescence signaling program in proliferating satellite cells during initial phases of muscle regeneration followed by induction of quiescence in a subset of cells during later phases.