Amelioration of Duchenne muscular dystrophy in mdx mice by elimination of matrix-associated fibrin-driven inflammation coupled to the αMß2 leukocyte integrin receptor.

In Duchenne muscular dystrophy (DMD), a persistently altered and reorganizing extracellular matrix (ECM) within inflamed muscle promotes damage and dysfunction. However, the molecular determinants of the ECM that mediate inflammatory changes and faulty tissue reorganization remain poorly defined. Here, we show that fibrin deposition is a conspicuous consequence of muscle-vascular damage in dystrophic muscles of DMD patients and mdx mice and that elimination of fibrin(ogen) attenuated dystrophy progression in mdx mice. These benefits appear to be tied to: (i) a decrease in leukocyte integrin α(M)ß(2)-mediated proinflammatory programs, thereby attenuating counterproductive inflammation and muscle degeneration; and (ii) a release of satellite cells from persistent inhibitory signals, thereby promoting regeneration. Remarkably, Fib-gamma(390-396A) (Fibγ(390-396A)) mice expressing a mutant form of fibrinogen with normal clotting function, but lacking the α(M)ß(2) binding motif, ameliorated dystrophic pathology. Delivery of a fibrinogen/α(M)ß(2) blocking peptide was similarly beneficial. Conversely, intramuscular fibrinogen delivery sufficed to induce inflammation and degeneration in fibrinogen-null mice. Thus, local fibrin(ogen) deposition drives dystrophic muscle inflammation and dysfunction, and disruption of fibrin(ogen)-α(M)ß(2) interactions may provide a novel strategy for DMD treatment.

Modification of brain lipids but not phenotype in alpha-synucleinopathy transgenic mice by long-term dietary n-3 fatty acids.

Transgenic mice expressing both wild mouse alpha-synuclein and the Parkinson's disease associated A53T mutated human alpha-synuclein were subjected to long-term diets impoverished in n-3 or diets impoverished in n-3 and supplemented with docosahexaenoic acid (DHA) for 6 months. Transgenic mice evidenced mild phenotype characterized by increased total alpha-synuclein expression, truncated alpha-synuclein forms, and abnormal solubility and aggregation, in the absence of Lewy bodies and neurites, and lack of apparent neuronal loss, astrocytosis and microgliosis. These diets produced a reduction in the content of linolenic, n-3 docosapentaenoic and total polyunsaturated fatty acids, leading to significantly lower double bond and peroxidizability indexes as well as to lower protein oxidative damage, with no effects in alpha-synuclein expression and with no modifications in the number of cortical astrocytes and microglial cells. The present results show that diets may modify brain lipid composition and susceptibility to oxidative damage that do not interfere with phenotype in models with a genetic susceptibility to develop alpha-synucleinopathy.

TAR DNA-Binding protein 43 accumulation in protein aggregate myopathies.

Protein aggregate myopathies, including myofibrillar myopathies and sporadic inclusion body myositis (sIBM), are characterized by abnormal protein aggregates composed of various muscular and ectopic proteins. Previous studies have shown the crucial role ofdysregulated transcription factors such as neuron-restrictive silencerfactor in the expression of aberrant proteins in myotilinopathies. Here, we assessed possible aberrant expression of TAR DNA-bindingprotein 43 (TDP-43), another factor involved in transcription regulation. TDP-43-immunoreactive intracytoplasmic inclusions were seen in all cases examined of myotilinopathy, desminopathy, and sIBM, and in 1 case of inclusion body myositis with Paget disease of bone and frontotemporal degeneration (IBMPFD). TAR DNA-binding protein 43 colocalized with myotilin and valosin in myotilinopathies and IBMPFD, respectively, but only occasionally colocalized with ubiquitin in myotilinopathies, desminopathies, sIBM, and IBMPFD; this indicates that accumulated TDP-43 is largely not ubiquitinated. Moreover, phosphorylated TDP-43 at Ser403/404 and Ser409/410 accumulated in the cytoplasm of vulnerable fibers but did not always colocalize with nonphosphorylated TDP-43. Cytoplasmic deposition was accompanied by decreased TDP-43 localization in the nuclei of affected fibers. These findings indicate that TDP-43 not only is another protein accumulated in myofibrillar myopathies, sIBM, and IBMPFD but also likely has a role through altered microRNA processing in the abnormal protein production, modification, and accumulation in protein aggregate myopathies.

Desmin is oxidized and nitrated in affected muscles in myotilinopathies and desminopathies.

Degenerative diseases with abnormal protein aggregates are characterized by the accumulation of proteins with variable posttranslational modifications including phosphorylation, glycoxidation, oxidation, and nitration. Myofibrillar myopathies, including myotilinopathies and desminopathies, are characterized by the intracytoplasmic focal accumulation of proteins in insoluble aggregates in muscle cells. By using single immunohistochemistry, monodimensional gel electrophoresis and Western blotting, and bidimensional gel electrophoresis, in-gel digestion, and mass spectometry, desmin was demonstrated to be a major target of oxidation and nitration in both desminopathies and myotilinopathies. Because oxidized and nitrated proteins may have toxic effects and may impair ubiquitin-proteasomal function, modified desmin can be considered to be an additional element in the pathogenesis of myofibrillar myopathies. In addition to desmin, pyruvate kinase muscle splice form M1 is oxidized, thus supporting complemental mitochondrial damage, at least in some cases of myotilinopathy.

Oxidative stress in desminopathies and myotilinopathies: a link between oxidative damage and abnormal protein aggregation.

Myotilinopathies and desminopathies are subgroups of myofibrillar myopathies (MFM) caused by mutations in myotilin and desmin genes, respectively. They are characterized by the presence of protein aggregates in muscle cells. As oxidation of proteins facilitates their aggregation and makes them more resistant to proteolysis, the present study was geared to analyze oxidative stress in MFM. For this purpose, markers of glycoxidation, lipoxidation and nitration were examined with gel electrophoresis and Western blotting, single immunohistochemistry, and double- and triple-labeling immunofluorescence and confocal microscopy in muscle biopsies from patients suffering from myotilinopathy and desminopathy. Increased levels of glycation-end products (AGEs), N-carboxymethyl-lysine (CML) and N-carboxyethyl-lysine (CEL), malondialdehyde-lysine (MDAL), 4-hydroxynonenal (HNE) and nitrotyrosine (N-tyr) were found in MFM. Furthermore, aberrant expression of AGE, CML, CEL, MDAL and HNE, as well as of neuronal, inducible and endothelial nitric oxide synthases (nNOS, iNOS, eNOS), and superoxide dismutase 2 (SOD2), was found in muscle fibers containing protein aggregates in myotilinopathies and desminopathies. AGE, ubiquitin and p62 co-localized in several muscle fibers in MFM. As oxidized proteins are vulnerable to misfolding and are resistant to degradation by the UPS, the present observations support a link between oxidative stress, protein aggregation and abnormal protein deposition in MFMs.