Formation of Polyglutamine Inclusions in a Wide Range of Non-CNS Tissues in the HdhQ150 Knock-In Mouse Model of Huntington's Disease

Background

Huntington''s disease (HD) is an inherited progressive neurodegenerative disorder caused by a CAG repeat expansion in the ubiquitously expressed HD gene resulting in an abnormally long polyglutamine repeat in the huntingtin protein. Polyglutamine inclusions are a hallmark of the neuropathology of HD. We have previously shown that inclusion pathology is also present in the peripheral tissues of the R6/2 mouse model of HD which expresses a small N-terminal fragment of mutant huntingtin. To determine whether this peripheral pathology is a consequence of the aberrant expression of this N-terminal fragment, we extend this analysis to the genetically precise knock-in mouse model of HD, HdhQ150, which expresses mutant mouse huntingtin.

Methodology/Principal Findings

We have previously standardized the CAG repeat size and strain background of the R6/2 and HdhQ150 knock-in mouse models and found that they develop a comparable and widespread neuropathology. To determine whether HdhQ150 knock-in mice also develop peripheral inclusion pathology, homozygous Hdh ^Q150/Q150 mice were perfusion fixed at 22 months of age, and tissues were processed for histology and immunohistochemistry with the anti-huntingtin antibody S830. The peripheral inclusion pathology was almost identical to that found in R6/2 mice at 12 weeks of age with minor differences in inclusion abundance.

Conclusions/Significance

The highly comparable peripheral inclusion pathology that is present in both the R6/2 and HdhQ150 knock-in models of HD indicates that the presence of peripheral inclusions in R6/2 mice is not a consequence of the aberrant expression of an N-terminal huntingtin protein. It remains to be determined whether peripheral inclusions are a pathological feature of the human disease. Both mouse models carry CAG repeats that cause childhood disease in humans, and therefore, inclusion pathology may be a feature of the childhood rather than the adult forms of HD. It is important to establish the extent to which peripheral pathology causes the peripheral symptoms of HD from the perspective of a mechanistic understanding and future treatment options.     

Role of calnexin in the glycan-independent quality control of proteolipid protein

The endoplasmic (ER) quality control apparatus ensures that misfolded or unassembled proteins are not deployed within the cell, but are retained in the ER and degraded. A glycoprotein-specific system involving the ER lectins calnexin and calreticulin is well documented, but very little is known about mechanisms that may operate for non-glycosylated proteins. We have used a folding mutant of a non- glycosylated membrane protein, proteolipid protein (PLP), to examine the quality control of this class of polypeptide. We find that calnexin associates with newly synthesized PLP molecules, binding stably to misfolded PLP. Calnexin also binds stably to an isolated transmembrane domain of PLP, suggesting that this chaperone is able to monitor the folding and assembly of domains within the ER membrane. Notably, this glycan-independent interaction with calnexin significantly retards the degradation of misfolded PLP. We propose that calnexin contributes to the quality control of non-glycosylated polytopic membrane proteins by binding to misfolded or unassembled transmembrane domains, and discuss our findings in relation to the role of calnexin in the degradation of misfolded proteins.     

Alterations in the mouse and human proteome caused by Huntington's disease

Huntington's disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In our effort to identify proteins involved in processes upstream or downstream of the disease-causing huntingtin, we studied the proteome of a well established mouse model by large gel two-dimensional electrophoresis. We could demonstrate for the first time at the protein level that alpha1-antitrypsin and alphaB-crystalline both decrease in expression over the course of disease. Importantly, the alpha1-antitrypsin decrease in the brain precedes that in liver and testes in mice. Reduced expression of the serine protease inhibitors alpha1-antitrypsin and contraspin was found in liver, heart, and testes close to terminal disease. Decreased expression of the chaperone alphaB-crystallin was found exclusively in the brain. In three brain regions obtained post-mortem from Huntington's disease patients, alpha1-antitrypsin expression was also altered. Reduced expression of the major urinary proteins not found in the brain was seen in the liver of affected mice, demonstrating that the disease exerts its influence outside the brain of transgenic mice at the protein level. Maintaining alpha1-antitrypsin and alphaB-crystallin availability during the course of Huntington's disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.     

Cardioprotective action of CRF peptide urocortin against simulated ischemia in adult rat cardiomyocytes

The major objective of this study was to determine whether urocortin, a member of the corticotrophin-releasing factor (CRF) family, protects adult rat cardiomyocytes from ischemia that has been simulated by glucose deprivation and acidosis. When it was present during simulated ischemia, urocortin (0.1 microM) markedly attenuated the cellular injury, which was assessed by increases in creatine kinase and lactate dehydrogenase levels. This effect was comparable with that observed with adenosine (10 microM). The cardioprotective effect of urocortin was markedly attenuated by the protein kinase C inhibitor chelerythrine and by 5-hydroxydecanoate, an inhibitor of ATP-sensitive K(+) channels. Cardiomyocytes were also protected from injury by pretreatment with urocortin, either by incubation for 5 min with a subsequent 10-min recovery or incubation for 20 min with a 20-h recovery before simulated ischemia. Similar cardioprotective effects were observed with ischemic preconditioning protocols during both immediate and delayed phases. In conclusion, in adult cardiomyocytes, urocortin has immediate and delayed cardioprotective actions that mimic ischemic preconditioning. These actions are mediated via protein kinase C and ATP-sensitive K(+) channels.     

P38 MAPK: critical molecule in thrombin-induced NF-kappa B-dependent leukocyte recruitment

Thrombin-stimulated endothelium synthesizes numerous adhesion molecules to recruit leukocytes; however, it is unknown which intracellular pathways are responsible for this event. A recent report from our laboratory has shown that thrombin induces E-selectin expression and that blocking nuclear factor-kappa B (NF-kappa B) activity partially blocked both E-selectin expression (60%) and leukocyte recruitment. In this study, we systematically assessed the importance of p38 MAPK in thrombin-induced NF-kappa B activation and E-selectin-dependent leukocyte recruitment. Thrombin caused phosphorylation of p38 MAPK, its substrate ATF-2, and JNK MAPK, but not ERK MAPK. The p38 MAPK inhibitors, SKF86002 and SB-203580 only reduced ATF-2 activity. We treated human umbilical vein endothelial cells with SKF86002, 1 h before thrombin stimulation, and noted inhibition of NF-kappa B mobilization and complete inhibition of leukocyte rolling and adhesion in a laminar flow chamber. Significant inhibition of leukocyte recruitment and E-selectin expression was also observed with SB-203580. SKF86002 did not affect other systems, including tumor necrosis factor-alpha-induced E-selectin-dependent leukocyte recruitment. Moreover, thrombin-induced rapid mobilization of P-selectin from Weibel Palade bodies was not p38 MAPK dependent. These data suggest that thrombin induces p38 MAPK activation, which leads to NF-kappa B mobilization to the nucleus and causes the upregulation of E-selectin and subsequent leukocyte recruitment.     

Therapeutic benefit of a dissociated glucocorticoid and the relevance of in vitro separation of transrepression from transactivation activity

Glucocorticoids (GCs) are the mainstay of asthma therapy; however, major side effects limit their therapeutic use. GCs influence the expression of genes either by transactivation or transrepression. The antiinflammatory effects of steroids are thought to be due to transrepression and the side effects, transactivation. Recently, a compound, RU 24858, has been identified that demonstrated dissociation between transactivation and transrepression in vitro. RU 24858 exerts strong AP-1 inhibition (transrepression), but little or no transactivation. We investigated whether this improved in vitro profile results in the maintenance of antiinflammatory activity (evaluated in the Sephadex model of lung edema) with reduced systemic toxicity (evaluated by loss in body weight, thymus involution, and bone turnover) compared with standard GCs. RU 24858 exhibits comparable antiinflammatory activity to the standard steroid, budesonide. However, the systemic changes observed indicate that transactivation events do occur with this GC with similar potency to the standard steroids. In addition, the GCs profiled showed no differentiation on quantitative osteopenia of the femur. These results suggest that in vitro separation of transrepression from transactivation activity does not translate to an increased therapeutic ratio for GCs in vivo or that adverse effects are a consequence of transrepression.     

Synthesis of disaccharides derived from heparin and evaluation of effects on endothelial cell growth and on binding of heparin to FGF-2

The disaccharide beta-D-GlcA-(1-->4)-alpha-D-GlcNAc-1-->OMe and other small nonsulfated oligosaccharides related to heparin/heparan sulfate have been shown to bind to FGF and activated the fibroblast growth factor (FGF) signalling pathway in (F32) cells expressing the FGF receptor. Synthetic routes to beta-D-GlcA-(1-->4)-alpha-D-GlcNAc-1-->OMe and a glucose analogue beta-D-Glc-(1-->4)-alpha-D-GlcNAc-1-->OMe are described. The effects of these disaccharides on endothelial cell growth, which is relevant to angiogenesis, were evaluated and it was found they did not mimic the inhibitory effects that were observed for heparin albumin (HA) and that have also been observed by monosaccharide conjugates. They did not alter bovine aortic endothelial cell (BAEC) proliferation, in the presence of FGF-2 in serum free medium or in absence of FGF-2 in serum free and complete medium. Disaccharides (10 microg/mL) reduced by 25-31% the inhibition caused by HA (10 microg/mL) on BAEC growth in serum-free medium but had no effect in complete medium. There was no evidence obtained for the binding of these oligosaccharides to FGF-2 in competition with HA by ELISA.     

Identification of molecular intermediates in the assembly pathway of the MUC5AC mucin

MUC5AC mucins secreted by HT-29 cells in culture are oligomeric glycoproteins with characteristics similar to the MUC5AC mucins isolated from human airway sputum (Sheehan, J. K., Brazeau, C., Kutay, S., Pigeon, H., Kirkham, S., Howard, M., and Thornton, D. J. (2000) Biochem. J. 347, 37-44). Therefore we have used this cell line as a model system to investigate the biosynthesis of this major airway mucin. Initial experiments showed that the MUC5AC mucins isolated from the cells were liable to depolymerization depending on the conditions used for their solubilization. Prevention against reduction resulted in large oligomers associated with the cells, similar to those secreted into the medium. Using a combination of density gradient centrifugation and agarose gel electrophoresis coupled with probes specific for different forms of the mucin we identified five major intracellular populations of the MUC5AC polypeptide (unglycosylated monomer and dimer, GalNAc-substituted dimer, fully glycosylated dimer, and higher order oligomers). Pulse-chase studies were performed to follow the flow of radioactivity through these various intracellular forms into the mature oligomeric mucin secreted into the medium (a process taking approximately 2-4 h). The results show that the mucin polypeptide undergoes dimerization and then becomes substituted with GalNAc residues prior to glycan elaboration to produce a mature mucin dimer, which then undergoes multimerization. These data indicate that this oligomeric mucin follows a similar assembly to the von Willebrand factor glycoprotein to yield long linear disulfide-linked chains.