Increases of M2a macrophages and fibrosis in aging muscle are influenced by bone marrow aging and negatively regulated by muscle-derived nitric oxide.

Muscle aging is associated with changes in myeloid cell phenotype that may influence age-related changes in muscle structure. We tested whether preventing age-related reductions in muscle neuronal nitric oxide synthase (nNOS) would obviate age-related changes in myeloid cells in muscle. Our findings show that muscle aging is associated with elevations of anti-inflammatory M2a macrophages that can increase muscle fibrosis. Expression of a muscle-specific nNOS transgene in mice prevented age-related increases in M2a macrophages. Transgene expression also reduced expression of collagens and decreased muscle fibrosis. The nNOS transgene prevented age-related increases in arginase-1 but did not influence TGFß expression, indicating that the transgene may prevent age-related muscle fibrosis by inhibiting the arginase-dependent profibrotic pathway. Although aged satellite cells or fibro-adipogenic precursor (FAPs) cells also promote fibrosis, transgene expression had no effect on the expression of key signaling molecules that regulate fibrogenic activity of those cells. Finally, we tested whether increases in M2a macrophages and the associated increase in fibrosis were attributable to aging of myeloid lineage cells. Young bone marrow cells (BMCs) were transplanted into young or old mice, and muscles were collected 8 months later. Muscles of young mice receiving young BMCs showed no effect on M2a macrophage number or collagen accumulation compared to age-matched, nontransplanted controls. However, muscles of old mice receiving young BMCs showed fewer M2a macrophages and less accumulation of collagen. Thus, the age-related increase in M2a macrophages in aging muscle and the associated muscle fibrosis are determined in part by the age of bone marrow cells.

Age-related loss of nitric oxide synthase in skeletal muscle causes reductions in calpain S-nitrosylation that increase myofibril degradation and sarcopenia.

Sarcopenia, the age-related loss of muscle mass, is a highly-debilitating consequence of aging. In this investigation, we show sarcopenia is greatly reduced by muscle-specific overexpression of calpastatin, the endogenous inhibitor of calcium-dependent proteases (calpains). Further, we show that calpain cleavage of specific structural and regulatory proteins in myofibrils is prevented by covalent modification of calpain by nitric oxide (NO) through S-nitrosylation. We find that calpain in adult, non-sarcopenic muscles is S-nitrosylated but that aging leads to loss of S-nitrosylation, suggesting that reduced S-nitrosylation during aging leads to increased calpain-mediated proteolysis of myofibrils. Further, our data show that muscle aging is accompanied by loss of neuronal nitric oxide synthase (nNOS), the primary source of muscle NO, and that expression of a muscle-specific nNOS transgene restores calpain S-nitrosylation in aging muscle and prevents sarcopenia. Together, the findings show that in vivo reduction of calpain S-nitrosylation in muscle may be an important component of sarcopenia, indicating that modulation of NO can provide a therapeutic strategy to slow muscle loss during old age.

Characterization of detergent-insoluble proteins in ALS indicates a causal link between nitrative stress and aggregation in pathogenesis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages. We identified several proteins enriched in the detergent-insoluble fraction already at a preclinical stage, including intermediate filaments, chaperones and mitochondrial proteins. Aconitase, HSC70 and cyclophilin A were also significantly enriched in the insoluble fraction of spinal cords of ALS patients. Moreover, we found that the majority of proteins in mice and HSP90 in patients were tyrosine-nitrated. We therefore investigated the role of nitrative stress in aggregate formation in fALS-like murine motor neuron-neuroblastoma (NSC-34) cell lines. By inhibiting nitric oxide synthesis the amount of insoluble proteins, particularly aconitase, HSC70, cyclophilin A and SOD1 can be substantially reduced. CONCLUSION/SIGNIFICANCE: Analysis of the insoluble fractions from cellular/mouse models and human tissues revealed novel aggregation-prone proteins and suggests that nitrative stress contribute to protein aggregate formation in ALS.

Insoluble mutant SOD1 is partly oligoubiquitinated in amyotrophic lateral sclerosis mice.

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS) through an unknown gain-of-function mechanism. Mutant SOD1 aggregation may be the toxic property. In fact, proteinaceous inclusions rich in mutant SOD1 have been found in tissues from the familial form of ALS patients and in mutant SOD1 animals, before disease onset. However, very little is known of the constituents and mechanism of formation of aggregates in ALS. We and others have shown that there is a progressive accumulation of detergent-insoluble mutant SOD1 in the spinal cord of G93A SOD1 mice. To investigate the mechanism of SOD1 aggregation, we characterized by proteome technologies SOD1 isoforms in a Triton X-100-insoluble fraction of spinal cord from G93A SOD1 mice at different stages of the disease. This showed that at symptomatic stages of the disease, part of the insoluble SOD1 is unambiguously mono- and oligoubiquitinated, in spinal cord and not in hippocampus, and that ubiquitin branches at Lys(48), the major signal for proteasome degradation. At presymptomatic stages of the disease, only insoluble unmodified SOD1 is recovered. Partial ubiquitination of SOD1-rich inclusions was also confirmed by immunohistochemical and electron microscopy analysis of lumbar spinal cord sections from symptomatic G93A SOD1 mice. On the basis of these results, we propose that ubiquitination occurs only after SOD1 aggregation and that oligoubiquitination may underline alternative mechanisms in disease pathogenesis.