Pharmacological prion protein silencing accelerates central nervous system autoimmune disease via T cell receptor signalling

The primary biological function of the endogenous cellular prion protein has remained unclear. We investigated its biological function in the generation of cellular immune responses using cellular prion protein gene-specific small interfering ribonucleic acid in vivo and in vitro. Our results were confirmed by blocking cellular prion protein with monovalent antibodies and by using cellular prion protein-deficient and -transgenic mice. In vivo prion protein gene-small interfering ribonucleic acid treatment effects were of limited duration, restricted to secondary lymphoid organs and resulted in a 70% reduction of cellular prion protein expression in leukocytes. Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity. In vivo prion protein gene-small interfering ribonucleic acid treatment promoted T cell differentiation towards pro-inflammatory phenotypes and increased survival of antigen-specific T cells. Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis. Finally, treatment of myelin basic protein1–11 T cell receptor transgenic mice with prion protein gene-small interfering ribonucleic acid resulted in spontaneous experimental autoimmune encephalomyelitis. Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses. Our findings indicate that cellular prion protein regulates T cell receptor-mediated T cell activation, differentiation and survival. Defects in autoimmunity are restricted to the immune system and not the central nervous system. Our data identify cellular prion protein as a regulator of cellular immunological homoeostasis and suggest cellular prion protein as a novel potential target for therapeutic immunomodulation

Overexpression of CCS in G93A-SOD1 mice leads to accelerated neurological deficits with severe mitochondrial pathology

Cu, Zn superoxide dismutase (SOD1) has been detected within spinal cord mitochondria of mutant SOD1 transgenic mice, a model of familial ALS. The copper chaperone for SOD1 (CCS) provides SOD1 with copper, facilitates the conversion of immature apo-SOD1 to a mature holoform, and influences in yeast the cytosolic/mitochondrial partitioning of SOD1. To determine how CCS affects G93A-SOD1-induced disease, we generated transgenic mice overexpressing CCS and crossed them to G93A-SOD1 or wild-type SOD1 transgenic mice. Both CCS transgenic mice and CCS/wild-type-SOD1 dual transgenic mice are neurologically normal. In contrast, CCS/G93A-SOD1 dual transgenic mice develop accelerated neurological deficits, with a mean survival of 36 days, compared with 242 days for G93A-SOD1 mice. Immuno-EM and subcellular fractionation studies on the spinal cord show that G93A-SOD1 is enriched within mitochondria in the presence of CCS overexpression. Our results indicate that CCS overexpression in G93A-SOD1 mice produces severe mitochondrial pathology and accelerates disease course

Metabolic acidosis regulates rat renal Na-Si cotransport activity

Recently, me cloned a cDNA (NaSi-1) localized to rat renal proximal tubules and encoding the brush-border membrane (BBM) Na gradient-dependent inorganic sulfate (S-i) transport protein (Na-S-i cotransporter). The purpose of the present study was to determine the effect of metabolic acidosis (MA) on Na-S-i cotransport activity and NaSi-1 protein and mRNA expression. In rats with MA for 24 h (but not 6 or 12 h), there was a significant increase in the fractional excretion of S-i, which was associated with a 2,4-fold decrease in BBM Na-S-i cotransport activity. The decrease in Na-S-i cotransport correlated with a 2.8-fold decrease in BBM NaSi-1 protein abundance and a 2.2-fold decrease in cortical NaSi-1 mRNA abundance. The inhibitory effect of MA on BBM Na-Si cotransport was also sustained in rats with chronic (10 days) MA. In addition, in Xenopus laevis oocytes injected with mRNA from kidney cortex, there was a significant reduction in the induced Na-S-i cotransport in rats with MA compared with control rats, suggesting that MA causes a decrease in the abundance of functional mRNA encoding the NaSi-1 cotransporter. These findings indicate that MA reduces Si reabsorption by causing decreases in BBM Na-S-i cotransport activity and that decreases in the expression of NaSi-1 protein and mRNA abundance, at least in part, play an important role in the inhibition of Na-S-i cotransport activity during MA

TDP-43, an ALS Linked Protein, Regulates Fat Deposition and Glucose Homeostasis

<div><p>The identification of proteins which determine fat and lean body mass composition is critical to better understanding and treating human obesity. TDP-43 is a well-conserved RNA-binding protein known to regulate alternative splicing and recently implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). While TDP-43 knockout mice show early embryonic lethality, post-natal conditional knockout mice show weight loss, fat depletion, and rapid death, suggesting an important role for TDP-43 in regulating energy metabolism. Here we report, that over-expression of TDP-43 in transgenic mice can result in a phenotype characterized by increased fat deposition and adipocyte hypertrophy. In addition, TDP-43 over-expression in skeletal muscle results in increased steady state levels of Tbc1d1, a RAB-GTPase activating protein involved in Glucose 4 transporter (Glut4) translocation. Skeletal muscle fibers isolated from TDP-43 transgenic mice show altered Glut4 translocation in response to insulin and impaired insulin mediated glucose uptake. These results indicate that levels of TDP-43 regulate body fat composition and glucose homeostasis in vivo.</p> </div

Tbc1d1 and Glut4 levels in skeletal muscle and white adipose tissue.

<p>(<b>A</b>) Quantification of TBC1D1 mRNA levels in muscle (6 weeks, N=4). Data are fold gene expression normalized with cyclophilin and expressed as mean ± S.E.M. (<b>B</b>) Western blot showing protein level of Tbc1d1 in non-transgenic and A315T line 23 and line 61 mice. 50 µg of total protein loaded per lane. (<b>C</b>) Quantification of TBC1D11 mRNA levels in white adipose tissue. Data are fold gene expression normalized with cyclophilin and expressed as mean ± S.E.M. (<b>D</b>) Quantification of Glut1 and Glut4 mRNA levels in muscle (6 weeks, N=4). Data are fold gene expression normalized with cyclophilin and expressed as mean ± S.E.M. (<b>E</b>) Western blot showing protein level of Glut4 in skeletal muscle of non-transgenic, A315T line 23, and A315T line 61 mice. 50 µg of total protein loaded. Actin was used as a loading control. (<b>F</b>) Quantification of Glut1 and Glut4 mRNA levels in white adipose tissue (6 weeks, N=4). Data are fold gene expression normalized with cyclophilin and expressed as mean±S.E.M. (<b>G</b>) Western blot showing protein level of Glut4 in WAT of non-transgenic, A315T line 23, and A315T line 61. 50 µg of total protein loaded. GAPDH was used as a loading control. Representative western blots are shown, each sample was run ≥ 2 times with N ≥ 2 mice in each group analyzed. Statistical significance between non-transgenic and A315T mice: *P<0.05.</p

Metabolic analysis of A315T line 61 mice.

<p>(<b>A</b>–<b>D</b>) Cumulative values from individual mice over 96 hours for horizontal X/Y activity (<b>A</b> and <b>B</b>), vertical Z activity (<b>C</b>) and food intake (<b>D</b>) from non-transgenic and A315T line 61 mice in metabolic cages. (<b>E</b>) Food intake normalized to weight for A315T line 61 and non-transgenic controls for a 96 hour period. (<b>F</b>) Energy expenditure normalized to lean mass (10 weeks of age; N=6 each group). Bar =mean±S.E.M. (<b>G</b>-<b>K</b>) Fasting blood values for glucose (<b>G</b>), cholesterol (<b>H</b>), triglycerides (<b>I</b>), gamma glutamyltransferase (<b>J</b>), and creatine kinase (<b>K</b>) in non-transgenic and A315T line 61 mice (8-16 weeks, N= 4-9). (<b>L</b>) Glucose tolerance test in non-transgenic and A315T line 61 mice (N=5-12).</p

White adipose tissue and muscle in A315T line 23 and line 61 have increased TDP-43 levels.

<p>(<b>A</b>) Quantification of TDP-43 endogenous and transgene mRNA levels in WAT (6 weeks, N=4). Data are fold gene expression normalized with cyclophilin and expressed as mean ± S.E.M. *P<0.05, **P<0.001. (<b>B</b>) TDP-43 protein levels in WAT from 6 week old mice probed with polyclonal antibody that recognizes both human and mouse TDP-43. (<b>C</b>) TDP-43 immuno-reactivity in WAT and BAT of non-transgenic (16 weeks). A315T line 23 (6 weeks) and A315T line 61 mice (16 weeks). Magnification=400X. (<b>D</b>) TDP-43 protein levels in spinal cord from non-transgenic, A315T line 23 and A315T line 61(12 week old) mice using monoclonal antibody which recognizes human TDP-43 only. (<b>E</b>) Quantification of TDP-43 endogenous and transgene mRNA levels in skeletal muscle (6 weeks, N=4). Data are fold gene expression normalized with cyclophilin and expressed as mean ± S.E.M. *P<0.05, **P<0.005. (<b>F</b>) TDP-43 protein levels in skeletal muscle of non-transgenic, A315T line 23, A315T line 61 mice (12 weeks of age) using polyclonal antibody. (<b>G</b>) TDP-43 immunoflorescence and H&E stained sections of skeletal muscle of non-transgenic and A315T line 61 (16 weeks of age). Magnification: 400X for immunoflorescence,200X for H&E. 5 µg of total protein loaded per lane for spinal cord and muscle western blot. 10 µg of total protein loaded per lane for WAT western blot. 1 µg of total protein loaded per lane for muscle western blot. Tubulin, actin or GAPDH were used as loading controls. Representative western blots are shown, each sample was run ≥ 2 times with N≥ 2 mice in each group analyzed.</p