TDP-43, an ALS linked protein, regulates fat deposition and glucose homeostasis.

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.

Biochemical properties and in vivo effects of the SOD1 zinc-binding site mutant (H80G).

This study presents the initial characterization of transgenic mice with mutations in a primary zinc-binding residue (H80), either alone or with a G93A mutation. H80G;G93A superoxide dismutase 1 (SOD1) transgenic mice developed paralysis with motor neuron loss, and ubiquitin inclusion-type rather than mitochondrial vacuolar pathology. Unlike G93A SOD1-related disease, the course was not accelerated by over-expression of copper chaperone for SOD1. H80G SOD1 transgenic mice did not manifest disease at levels of SOD1 transgene expressed. The H80G mutation altered certain biochemical parameters of both human wild-type SOD1 and G93A SOD1. The H80G mutation does not substantially change the age-dependent accumulation of G93A SOD1 aggregates and hydrophobic species in spinal cord. However, both H80G;G93A SOD1 and H80G SOD1 lack dismutase activity, the ability to form homodimers, and co-operativity with copper chaperone for SOD1, indicating that their dimerization interface is abnormal. The H80G mutation also made SOD1 susceptible to protease digestion. The H80G mutation alters the redox properties of SOD1. G93A SOD1 exists in either reduced or oxidized form, whereas H80G;G93A SOD1 and H80G SOD1 exist only in a reduced state. The inability of SOD1 with an H80G mutation to take part in normal oxidation-reduction reactions has important ramifications for disease mechanisms and pathology in vivo.

Progressive motor weakness in transgenic mice expressing human TDP-43.

Familial ALS patients with TDP-43 gene mutations and sporadic ALS patients share common TDP-43 neuronal pathology. To delineate mechanisms underlying TDP-43 proteinopathies, transgenic mice expressing A315T, M337V or wild type human TDP-43 were generated. Multiple TDP-43 founders developed a severe early motor phenotype that correlated with TDP-43 levels in spinal cord. Three A315T TDP-43 lines developed later onset paralysis with cytoplasmic ubiquitin inclusions, gliosis and TDP-43 redistribution and fragmentation. The WT TDP-43 mouse line with highest spinal cord expression levels remains asymptomatic, although these mice show spinal cord pathology. One WT TDP-43 line with high skeletal muscle levels of TDP-43 developed a severe progressive myopathy. Over-expression of TDP-43 in vivo is sufficient to produce progressive motor phenotypes by a toxic gain of function paradigm. Transgenic mouse lines expressing untagged mutant and wild type TDP-43 under the same promoter represent a powerful new model system for studying TDP-43 proteinopathies in vivo.

Silencing Nogo-A promotes functional recovery in demyelinating disease.

OBJECTIVE: To determine if suppressing Nogo-A, an axonal inhibitory protein, will promote functional recovery in a murine model of multiple sclerosis (MS). METHODS: A small interfering RNA was developed to specifically suppress Nogo-A (siRNA-NogoA). The siRNA-NogoA silencing effect was evaluated in vitro and in vivo via immunohistochemistry. The siRNA was administered intravenously in 2 models of experimental autoimmune encephalomyelitis (EAE). Axonal repair was measured by upregulation of GAP43. Enzyme-linked immunosorbent assay, flow cytometry, and (3)H-thymidine incorporation were used to determine immunological changes in myelin-specific T cells in mice with EAE. RESULTS: The siRNA-NogoA suppressed Nogo-A expression in vitro and in vivo. Systemic administration of siRNA-NogoA ameliorated EAE and promoted axonal repair, as demonstrated by enhanced GAP43+ axons in the lesions. Myelin-specific T-cell proliferation and cytokine production were unchanged in the siRNA-NogoA-treated mice. INTERPRETATION: Silencing Nogo-A in EAE promotes functional recovery. The therapeutic benefit appears to be mediated by axonal growth and repair, and is not attributable to changes in the encephalitogenic capacity of the myelin-specific T cells. Silencing Nogo-A may be a therapeutic option for MS patients to prevent permanent functional deficits caused by immune-mediated axonal damage.

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 protein(1-11) 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.

Redox susceptibility of SOD1 mutants is associated with the differential response to CCS over-expression in vivo.

Over-expression of CCS in G93A SOD1 mice accelerates neurological disease and enhances mitochondrial pathology. We studied the effect of CCS over-expression in transgenic mice expressing G37R, G86R or L126Z SOD1 mutations in order to understand factors which influence mitochondrial dysfunction. Over-expression of CCS markedly decreased survival and produced mitochondrial vacuolation in G37R SOD1 mice but not in G86R or L126Z SOD1 mice. Moreover, CCS/G37R SOD1 spinal cord showed specific reductions in mitochondrial complex IV subunits consistent with an isolated COX deficiency, while no such reductions were detected in CCS/G86R or CCS/L126Z SOD1 mice. CCS over-expression increased the ratio of reduced to oxidized SOD1 monomers in the spinal cords of G37R SOD1 as well as G93A SOD1 mice, but did not influence the redox state of G86R or L126Z SOD1 monomers. The effects of CCS on disease are SOD1 mutation dependent and correlate with SOD1 redox susceptibility.

The death domain-containing kinase RIP1 regulates p27(Kip1) levels through the PI3K-Akt-forkhead pathway.

Elucidating the cross-talk between inflammatory and cell proliferation pathways might provide important insights into the pathogenesis of inflammation-induced cancer. Here, we show that the receptor-interacting protein 1 (RIP1)-an essential mediator of inflammation-induced nuclear factor-kappaB (NF-kappaB) activation-regulates p27(Kip1) levels and cell-cycle progression. RIP1 regulates p27(Kip1) levels by an NF-kappaB-independent signal that involves activation of the phosphatidylinositol 3-kinase (PI3K)-Akt-forkhead pathway. Mouse embryonic fibroblasts (MEFs) from RIP1-knockout mice express high levels of p27(Kip1). Reconstitution of MEFs with RIP1 downregulates p27(Kip1) levels in a PI3K-dependent manner. RIP1 regulates p27(Kip1) at the messenger RNA level by regulating the p27(Kip1) promoter through the forkhead transcription factors. RIP1 expression blocks accumulation of cells in G(1) in response to serum starvation and favours cell-cycle progression. Finally, we show that overexpression of p27(Kip1) blocks the effects of RIP1 on the cell cycle. Thus, our study provides a new insight into how components of inflammatory and immune signalling pathways regulate cell-cycle progression.

Assessing the role of immuno-proteasomes in a mouse model of familial ALS.

The accumulation of protein aggregates is thought to be an important component in the pathogenesis of mutant SOD1-induced disease. Mutant SOD1 aggregates appear to be cleared by proteasomes, at least in vitro, suggesting a potentially important role for proteasome degradation pathways in vivo. G93A SOD1 transgenic mice show an increase in proteasome activity and induction of immuno-proteasome subunits within spinal cord as they develop neurological symptoms. To determine what role immuno-proteasomes may have in mutant SOD1-induced disease, we crossed G93A SOD1 transgenic mice with LMP2-/- mice to obtain G93A SOD1 mice lacking the LMP2 immuno-proteasome subunit. G93A SOD1/LMP2-/- mice show significant reductions in proteasome function within spinal cord compared to G93A SOD1 mice. However, G93A SOD1/LMP2-/- mice show no change in motor function decline, or survival compared to G93A SOD1 mice. These results indicate that the loss of immuno-proteasome function in vivo does not significantly alter mutant SOD1-induced disease.

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.

Non-neuronal induction of immunoproteasome subunits in an ALS model: possible mediation by cytokines.

Protein aggregation is a pathologic hallmark of familial amyotrophic lateral sclerosis caused by mutations in the Cu, Zn superoxide dismutase gene. Although SOD1-positive aggregates can be cleared by proteasomes, aggregates have been hypothesized to interfere with proteasome activity, leading to a vicious cycle that further enhances aggregate accumulation. To address this issue, we measured proteasome activity in transgenic mice expressing a G93A SOD1 mutation. We find that proteasome activity is induced in the spinal cord of such mice compared to controls but is not altered in uninvolved organs such as liver or spleen. This induction within spinal cord is not related to an overall increase in the total number of proteasome subunits, as evidenced by the steady expression levels of constitutive alpha7 and beta5 subunits. In contrast, we found a marked increase of inducible beta proteasome subunits, LMP2, MECL-1 and LMP7. This induction of immunoproteasome subunits does not occur in all spinal cord cell types but appears limited to astrocytes and microglia. The induction of immunoproteasome subunits in G93A spinal cord organotypic slices treated with TNF-alpha and interferon-gamma suggest that certain cytokines may mediate such responses in vivo. Our results indicate that there is an overall increase in proteasome function in the spinal cords of G93A SOD1 mice that correlates with an induction of immunoproteasomes subunits and a shift toward immunoproteasome composition. These results suggest that increased, rather than decreased, proteasome function is a response of certain cell types to mutant SOD1-induced disease within spinal cord.

Aggregate formation in the spinal cord of mutant SOD1 transgenic mice is reversible and mediated by proteasomes.

Cu,Zn superoxide dismutase (SOD1) mutations cause one form of familial amyotrophic lateral sclerosis by a toxic gain of function that may be related to abnormal protein folding and aggregate formation. However, the processing pathways involved in SOD1 aggregate generation within spinal cord remain unclear. We have now developed an experimental system for studying SOD1 aggregate formation and clearance in intact spinal cord tissue. Here we demonstrate that the formation of SOD1-positive aggregates in G93A SOD1 transgenic mouse spinal cord tissue involves proteasome-mediated proteolysis. Organotypic spinal cord slices from 9-day-old transgenic mice expressing G93A SOD1 develop SOD1 aggregates with proteasome inhibition. In contrast, SOD1 aggregates do not form in spinal cord slices from wild type mice or transgenic mice overexpressing wild type SOD1 following proteasome inhibition. Furthermore, SOD1 aggregate formation within G93A SOD1 spinal cord is both sensitive to small changes in overall proteasome activity and reversible with the restoration of proteasome function. Our results also establish that adult mouse spinal cord exhibits a relative deficiency in proteasome activity compared with non-CNS tissue that may help explain the propensity of spinal cord to form SOD1-positive aggregates.

Disease progression in a transgenic model of familial amyotrophic lateral sclerosis is dependent on both neuronal and non-neuronal zinc binding proteins.

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene cause one form of familial amyotrophic lateral sclerosis, a progressive disorder of motor neurons leading to weakness and death of affected individuals. Experiments using both transgenic mice expressing mutant SOD1 and SOD1 knock-out mice have demonstrated that disease is caused by a toxic gain of function and not by a loss of normal SOD1 activity. Precise mechanisms underlying mutant SOD1 toxicity are unclear but may involve abnormal interactions between zinc and SOD1. The metallothioneins (MTs) represent a family of zinc binding proteins that can function as zinc chaperones for apo-SOD1 in vitro. We hypothesized that manipulation of metallothioneins in vivo might alter the disease phenotype of transgenic mice expressing G93A SOD1 and therefore crossed this line with MT-I and MT-II or MT-III knock-out mice. G93A SOD1 mice deficient of either MT-I and MT-II or MT-III exhibited significant reductions in survival compared with G93A SOD1 mice. In addition, motor dysfunction was markedly accelerated in G93A SOD1 mice deficient in metallothioneins with regard to onset (MT-I and MT-II) or progression (MT-III). These results indicate that the disease course in G93A SOD1 mice is dependent on levels of metallothionein expression. Because MT-I and MT-II are expressed in glia whereas MT-III is found in neurons, these results also indicate that primary changes within non-neuronal cells can affect mutant SOD1-induced disease and do so in ways distinct from primary neuronal changes.

Upregulation of autocrine-paracrine renin-angiotensin systems in chronic renovascular hypertension.

INTRODUCTION: The mechanism by which hypertension is maintained in renovascular hypertension remains poorly defined. Because plasma angiotensin II does not correlate with blood pressure in RVH, we postulated that activation of tissue-specific autocrine-paracrine renin-angiotensin systems may upregulate local production of angiotensin II and maintain hypertension in chronic RVH. METHODS: RVH was induced with a two-kidney one-clip (2K1C) rat model. Animals were killed at 1 or 12 weeks after surgery (acute or chronic RVH). Angiotensin II was quantitated with radioimmunoassay. Angiotensin II-type 1 (AT(1)) receptor density was determined with immunoblotting and immunohistochemistry. RESULTS: Blood pressure was significantly elevated in 2K1C animals compared with sham animals at 1 week (141 +/- 5 mm Hg versus 98 +/- 3 mm Hg; P <.0005) and at 12 weeks (164 +/- 14 mm Hg versus 110 +/- 7 mm Hg; P <.0005) after surgery. No significant difference was seen in plasma angiotensin II levels between 2K1C and control animals during acute (38.2 +/- 6.5 fmol/mL versus 27.6 +/- 6.8 fmol/mL; P = not significant) or chronic (40.1 +/- 17.4 fmol/mL versus 27.1 +/- 6.5 fmol/mL; P = not significant) RVH. During acute RVH, intrarenal angiotensin II was significantly increased in both the clipped (126.0 +/- 16.2 fmol/g versus 62.0 +/- 6.2 fmol/g; P <.005) and unclipped (78.9 +/- 6.3 fmol/g versus 39.9 +/- 2.5 fmol/g; P <.05) kidneys of 2K1C animals compared with control animals. Increased intrarenal angiotensin II levels persisted in chronic RVH in the clipped (147.4 +/- 37.7 fmol/g versus 59.2 +/- 8.7 fmol/g; P <.05) and unclipped (130.8 +/- 31.8 fmol/g versus 63.0 +/- 11.0 fmol/g; P <.05) kidneys of 2K1C animals compared with controls. Adrenal angiotensin II content of 2K1C animals was unchanged in acute RVH (493.7 +/- 51.4 fmol/g versus 522.6 +/- 80.5 fmol/g; P = not significant) but increased nearly three-fold over control animals during chronic RVH (1129.0 +/- 149.3 fmol/g versus 400.6 +/- 59.1 fmol/g; P <.0005). No significant difference in AT(1) receptor density was noted in renal tubules of clipped and unclipped kidneys or in the adrenal glands of 2K1C animals during acute or chronic RVH compared with control animals. CONCLUSION: Tissue angiotensin II production is upregulated in the kidneys and adrenal glands in chronic RVH, and AT(1) receptor density is maintained in these tissues, providing a potential mechanism for maintenance of hypertension in RVH.