mHTT Protein Research Articles

Mass spectrometric identification of novel posttranslational modification sites in Huntingtin

Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild-type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.

Caspase-6 Activity in a BACHD Mouse Modulates Steady-State Levels of Mutant Huntingtin Protein But Is Not Necessary for Production of a 586 Amino Acid Proteolytic Fragment

Huntington's disease (HD) is caused by a mutation in the huntingtin (htt) gene encoding an expansion of glutamine repeats at the N terminus of the Htt protein. Proteolysis of Htt has been identified as a critical pathological event in HD models. In particular, it has been postulated that proteolysis of Htt at the putative caspase-6 cleavage site (at amino acid Asp-586) plays a critical role in disease progression and pathogenesis. However, whether caspase-6 is indeed the essential enzyme that cleaves Htt at this site in vivo has not been determined. To evaluate, we crossed the BACHD mouse model with a caspase-6 knock-out mouse (Casp6(-/-)). Western blot and immunocytochemistry confirmed the lack of caspase-6 protein in Casp6(-/-) mice, regardless of HD genotype. We predicted the Casp6(-/-) mouse would have reduced levels of caspase-6 Htt fragments and increased levels of full-length Htt protein. In contrast, we found a significant reduction of full-length mutant Htt (mHtt) and fragments in the striatum of BACHD Casp6(-/-) mice. Importantly, we detected the presence of Htt fragments consistent with cleavage at amino acid Asp-586 of Htt in the BACHD Casp6(-/-) mouse, indicating that caspase-6 activity cannot fully account for the generation of the Htt 586 fragment in vivo. Our data are not consistent with the hypothesis that caspase-6 activity is critical in generating a potentially toxic 586 aa Htt fragment in vivo. However, our studies do suggest a role for caspase-6 activity in clearance pathways for mHtt protein.

Calpain and STriatal-Enriched protein tyrosine Phosphatase (STEP) activation contribute to extrasynaptic NMDA receptor localization in a Huntington's disease mouse model

In Huntington's disease (HD), the mutant huntingtin (mhtt) protein is associated with striatal dysfunction and degeneration. Excitotoxicity and early synaptic defects are attributed, in part, to altered NMDA receptor (NMDAR) trafficking and function. Deleterious extrasynaptic NMDAR localization and signalling are increased early in yeast artificial chromosome mice expressing full-length mhtt with 128 polyglutamine repeats (YAC128 mice). NMDAR trafficking at the plasma membrane is regulated by dephosphorylation of the NMDAR subunit GluN2B tyrosine 1472 (Y1472) residue by STriatal-Enriched protein tyrosine Phosphatase (STEP). NMDAR function is also regulated by calpain cleavage of the GluN2B C-terminus. Activation of both STEP and calpain is calcium-dependent, and disruption of calcium homeostasis occurs early in the HD striatum. Here, we show increased calpain cleavage of GluN2B at both synaptic and extrasynaptic sites, and elevated extrasynaptic total GluN2B expression in the YAC128 striatum. Calpain inhibition significantly reduced extrasynaptic GluN2B expression in the YAC128 but not wild-type striatum. Furthermore, calpain inhibition reduced whole-cell NMDAR current and the surface/internal GluN2B ratio in co-cultured striatal neurons, without affecting synaptic GluN2B localization. Synaptic STEP activity was also significantly higher in the YAC128 striatum, correlating with decreased GluN2B Y1472 phosphorylation. A substrate-trapping STEP protein (TAT-STEP C-S) significantly increased VGLUT1-GluN2B colocalization, as well as increasing synaptic GluN2B expression and Y1472 phosphorylation. Moreover, combined calpain inhibition and STEP inactivation reduced extrasynaptic, while increasing synaptic GluN2B expression in the YAC128 striatum. These results indicate that increased STEP and calpain activation contribute to altered NMDAR localization in an HD mouse model, suggesting new therapeutic targets for HD.

Prothymosin-α Interacts with Mutant Huntingtin and Suppresses Its Cytotoxicity in Cell Culture

Huntington disease (HD), a fatal neurodegenerative disorder, is caused by a lengthening of the polyglutamine tract in the huntingtin (Htt) protein. Despite considerable effort, thus far there is no cure or treatment available for the disorder. Using the approach of tandem affinity purification we recently discovered that prothymosin-α (ProTα), a small highly acidic protein, interacts with mutant Htt (mHtt). This was confirmed by co-immunoprecipitation and a glutathione S-transferase (GST) pull-down assay. Overexpression of ProTα remarkably reduced mHtt-induced cytotoxicity in both non-neuronal and neuronal cell models expressing N-terminal mHtt fragments, whereas knockdown of ProTα expression in the cells enhanced mHtt-caused cell death. Deletion of the central acidic domain of ProTα abolished not only its interaction with mHtt but also its protective effect on mHtt-caused cytotoxicity. Additionally, overexpression of ProTα inhibited caspase-3 activation but enhanced aggregation of mHtt. Furthermore, when added to cultured cells expressing mHtt, the purified recombinant ProTα protein not only entered the cells but it also significantly suppressed the mHtt-caused cytotoxicity. Taken together, these data suggest that ProTα might be a novel therapeutic target for treating HD and other polyglutamine expansion disorders.

Potent and Selective Antisense Oligonucleotides Targeting Single-Nucleotide Polymorphisms in the Huntington Disease Gene / Allele-Specific Silencing of Mutant Huntingtin

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion in the huntingtin gene (HTT) that results in a toxic gain of function in the mutant huntingtin protein (mHTT). Reducing the expression of mHTT is therefore an attractive therapy for HD. However, wild-type HTT protein is essential for development and has critical roles in maintaining neuronal health. Therapies for HD that reduce wild-type HTT may therefore generate unintended negative consequences. We have identified single-nucleotide polymorphism (SNP) targets in the human HD population for the disease-specific targeting of the HTT gene. Using primary cells from patients with HD and the transgenic YAC18 and BACHD mouse lines, we developed antisense oligonucleotide (ASO) molecules that potently and selectively silence mHTT at both exonic and intronic SNP sites. Modification of these ASOs with S-constrained-ethyl (cET) motifs significantly improves potency while maintaining allele selectively in vitro. The developed ASO is potent and selective for mHTT in vivo after delivery to the mouse brain. We demonstrate that potent and selective allele-specific knockdown of the mHTT protein can be achieved at therapeutically relevant SNP sites using ASOs in vitro and in vivo.

Forebrain Striatal-Specific Expression of Mutant Huntingtin Protein in Vivo Induces Cell-Autonomous Age-Dependent Alterations in Sensitivity to Excitotoxicity and Mitochondrial Function

HD (Huntington's disease) is characterized by dysfunction and death of striatal MSNs (medium-sized spiny neurons). Excitotoxicity, transcriptional dysregulation and mitochondrial abnormalities are among the mechanisms that are proposed to play roles in HD pathogenesis. To determine the extent of cell-autonomous effects of mhtt (mutant huntingtin) protein on vulnerability to excitotoxic insult in MSNs in vivo, we measured the number of degenerating neurons in response to intrastriatal injection of QA (quinolinic acid) in presymptomatic and symptomatic transgenic (D9-N171-98Q, also known as DE5) mice that express mhtt in MSNs but not in cortex. After QA, the number of degenerating neurons in presymptomatic DE5 mice was not significantly different from the number in WT (wild-type) controls, suggesting the early, increased vulnerability to excitotoxicity demonstrated in other HD mouse models has a largely non-cell-autonomous component. Conversely, symptomatic DE5 mice showed significantly fewer degenerating neurons relative to WT, implying the resistance to excitotoxicity observed at later ages has a primarily cell-autonomous origin. Interestingly, mitochondrial complex II respiration was enhanced in striatum of symptomatic mice, whereas it was reduced in presymptomatic mice, both relative to their age-matched controls. Consistent with the QA data, MSNs from symptomatic mice showed decreased NMDA (N-methyl-d-aspartate) currents compared with age-matched controls, suggesting that in addition to aging, cell-autonomous mechanisms mitigate susceptibility to excitotoxicity in the symptomatic stage. Also, symptomatic DE5 mice did not display some of the electrophysiological alterations present in other HD models, suggesting that blocking the expression of mhtt in cortical neurons may restore corticostriatal function in HD.

Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

BackgroundHuntington's disease is caused by aggregation of mutant huntingtin (mHtt) protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease activities may contribute to toxicity whereas others are consistent with protection. These discrepancies may be due to a number of mechanisms including distinct effects of the specific intermediate digestion products of mutant huntingtin generated by different proteases. These observations suggested a critical need to investigate the consequence of upregulation of individual lysosomal enzyme in mutant huntingtin accumulation and toxicity.ResultsIn this study, we used molecular approaches to enhance lysosomal protease activities and examined their effects on mutant huntingtin level and toxicity. We found that enhanced expression of lysosomal cathepsins D and B resulted in their increased enzymatic activities and reduced both full-length and fragmented huntingtin in transfected HEK cells. Furthermore, enhanced expression of cathepsin D or B protected against mutant huntingtin toxicity in primary neurons, and their neuroprotection is dependent on macroautophagy.ConclusionsThese observations demonstrate a neuroprotective effect of enhancing lysosomal cathepsins in reducing mutant huntingtin level and toxicity in transfected cells. They highlight the potential importance of neuroprotection mediated by cathepsin D or B through macroautophagy.

Early or Late-Stage Anti-N-Terminal Huntingtin Intrabody Gene Therapy Reduces Pathological Features in B6.HDR6/1 Mice

Huntington disease (HD) is a progressive neurodegenerative disease caused by an expansion of a polyglutamine sequence in mutant huntingtin (mhtt) that produces abnormal folding and aggregation that results in the formation of nuclear and cytoplasmic neuronal inclusion bodies. Although the precise role of mhtt aggregates in the pathogenesis is unclear, attempts to reduce accumulated mhtt protein have ameliorated the phenotype in multiple cellular and in vivo HD models. Here, we provide critical results on intracranial delivery of a single-chain Fv intrabody, C4, which targets the first 17 amino acids of the htt protein, a region of httExon1 that is increasingly being recognized as pivotal. To assess long-term efficacy and safety issues, we used adenoassociated viral vectors (AAV2/1) to deliver intrabody genes to the striatum of inbred B6.HDR6/1 mice. Treatment initiation at various stages of the disease showed that early treatment preserved the largest number of cells without nuclear aggregates and that the accumulation of aggregated material could be delayed by several months. Even when intrabody treatment was not initiated until the clinical disease stage, significant, albeit smaller, effects were seen. These data indicate that neuronal intrabodies against critical N-terminal epitopes can be safely and effectively delivered using AAV2/1 to delay the aggregation phenotype during a sustained period in this HD model, even when delivery is initiated after disease onset.

Exendin-4 Improves Glycemic Control, Ameliorates Brain and Pancreatic Pathologies, and Extends Survival in a Mouse Model of Huntington's Disease

OBJECTIVE—The aim of this study was to find an effective treatment for the genetic form of diabetes that is present in some Huntington's disease patients and in Huntington's disease mouse models. Huntington's disease is a neurodegenerative disorder caused by a polyglutamine expansion within the huntingtin protein. Huntington's disease patients exhibit neuronal dysfunction/degeneration, chorea, and progressive weight loss. Additionally, they suffer from abnormalities in energy metabolism affecting both the brain and periphery. Similarly to Huntington's disease patients, mice expressing the mutated human huntingtin protein also exhibit neurodegenerative changes, motor dysfunction, perturbed energy metabolism, and elevated blood glucose levels.RESEARCH DESIGN AND METHODS—Huntington's disease mice were treated with an FDA-approved antidiabetic glucagon-like peptide 1 receptor agonist, exendin-4 (Ex-4), to test whether euglycemia could be achieved, whether pancreatic dysfunction could be alleviated, and whether the mice showed any neurological benefit. Blood glucose and insulin levels and various appetite hormone concentrations were measured during the study. Additionally, motor performance and life span were quantified and mutant huntingtin (mhtt) aggregates were measured in both the pancreas and brain.RESULTS—Ex-4 treatment ameliorated abnormalities in peripheral glucose regulation and suppressed cellular pathology in both brain and pancreas in a mouse model of Huntington's disease. The treatment also improved motor function and extended the survival time of the Huntington's disease mice. These clinical improvements were correlated with reduced accumulation of mhtt protein aggregates in both islet and brain cells.CONCLUSIONS—Targeting both peripheral and neuronal deficits, Ex-4 is an attractive agent for therapeutic intervention in Huntington's disease patients suffering from diabetes.

Cleavage at the Caspase-6 Site Is Required for Neuronal Dysfunction and Degeneration Due to Mutant Huntingtin

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.

Mutant Huntingtin: Nuclear translocation and cytotoxicity mediated by GAPDH

The pathophysiology of Huntington's disease reflects actions of mutant Huntingtin (Htt) (mHtt) protein with polyglutamine repeats, whose N-terminal fragment translocates to the nucleus to elicit neurotoxicity. We establish that the nuclear translocation and associated cytotoxicity of mHtt reflect a ternary complex of mHtt with GAPDH and Siah1, a ubiquitin-E3-ligase. Overexpression of GAPDH or Siah1 enhances nuclear translocation of mHtt and cytotoxicity, whereas GAPDH mutants that cannot bind Siah1 prevent translocation. Depletion of GAPDH or Siah1 by RNA interference diminishes nuclear translocation of mHtt.

Intrastriatal rAAV-mediated delivery of anti-huntingtin shRNAs induces partial reversal of disease progression in R6/1 Huntington's disease transgenic mice

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the presence of an abnormally expanded polyglutamine domain in the N-terminus of huntingtin. We developed a recombinant adeno-associated viral serotype 5 (rAAV5) gene transfer strategy to posttranscriptionally suppress the levels of striatal mutant huntingtin (mHtt) in the R6/1 HD transgenic mouse via RNA interference. Transient cotransfection of HEK293 cells with plasmids expressing a portion of human mHtt derived from R6/1 transgenic HD mice and a short-hairpin RNA directed against the 5′ UTR of the mHtt mRNA (siHUNT-1) resulted in reduction in the levels of mHtt mRNA (−75%) and protein (−60%). Long-term in vivo rAAV5-mediated expression of siHUNT-1 in the striatum of R6/1 mice reduced the levels of mHtt mRNA (−78%) and protein (−28%) as determined by quantitative RT-PCR and Western blot analysis, respectively. The reduction in mHtt was concomitant with a reduction in the size and number of neuronal intranuclear inclusions and a small but significant normalization of the steady-state levels of preproenkephalin and dopamine- and cAMP-responsive phosphoprotein 32 kDa mRNA. Finally, bilateral expression of rAAV5-siHUNT-1 resulted in delayed onset of the rear paw clasping phenotype exhibited by the R6/1 mice. These results suggest that a reduction in the levels of striatal mHtt can ameliorate the HD phenotype of R6/1 mice.

53. Post-Transcriptional Suppression of Striatal Mutant Huntingtin Leads to Mild Phenotypic Improvements in the R6/1 Mouse

Top of pageAbstract We developed a recombinant adeno-associated viral (rAAV) gene transfer strategy to post-transcriptionally suppress the levels of striatal mutant huntingtin (m-Htt) in the R6/1 HD transgenic mouse. Transient co-transfection of 293 cells with a mammalian expression vector containing the R6/1 HD transgene and a short-hairpin RNA (shRNA) directed against nucleotides 262|[ndash]|280 of the human huntingtin mRNA (siHUNT-1) resulted in |[sim]|58% and |[sim]|75% reduction in the levels of m-Htt mRNA and protein, respectively, when compared to cells receiving either a GFP-vector or a shRNA vector against the dog rhodopsin mRNA (siRho-1). Long-term in vivo rAAV5-mediated expression of siHUNT-1 in the striatum of R6/1 mice resulted in a loss of both m-Htt mRNA and protein as determined by quantitative RT-PCR and immunoblotting analysis of transduced striatal tissue. This was concomitant with a significant reduction in the size and number of neuronal intranuclear inclusions as assessed by immunohistochemistry analysis of transduced versus non-transduced regions. We also observed a small (|[sim]|20%) but significant increase in the steady-state levels of pre-pro-enkephalin (ppEnk) and dopamine- and cAMP-responsive phosphoprotein of 32 kDa (DARPP-32) mRNAs throughout the striatum of rAAV5-siHUNT-1 injected transgenic mice compared to rAAV5-GFP injected controls. Finally, bilateral expression of rAAV5-siHUNT-1 resulted in a delayed on the onset of the rear paw clasping phenotype exhibited by the R6/1 mice. These results suggest that reduced levels of striatal m-Htt can lead to the amelioration of the HD phenotype in the R6/1 mouse.

954. Demonstration of In Vivo Off-Targeting Using rAAV-Delivered Ribozymes and shRNA Molecules in a Mouse Model of Huntington's Disease

Top of pageAbstract The recent advances in RNA interference (RNAi) technology have led to the rapid development of nucleic acid-based gene therapies for the treatment of disease. We designed two hammerhead ribozymes (HD6 and HD7) and a short-hairpin RNA (siHUNT-2) directed against a region between nucleotides 342-363 of the human huntingtin mRNA in order to suppress the expression of striatal m-Htt in the R6/1 transgenic model of HD. The binding sites for the HD6 and HD7 ribozymes overlapped the siHUNT-2 targeted site. Long-term rAAV5-mediated expression of siHUNT-2 in R6/1 mice led to a significant decrease in the levels of m-Htt mRNA and protein as well as a remarkable reduction in the size and numbers of neuronal intranuclear inclusions. However, we were unable to detect in vivo knock-down of m-Htt after the expression of either HD6 or HD7 hammerhead ribozymes. Contrary to the expected result, rAAV5-mediated expression of siHUNT-2 resulted in a dramatic reduction in the levels of pre-pro-enkephalin (ppEnk), dopamine- and cAMP-regulated phosphoprotein of 32-kDa (DARPP-32) and the dompamine type-2 receptor (D2R) mRNAs in both transgenic and wild-type mice. This reduction was not due to an overall suppression in neuronal transcriptional activity since expression of the nerve growth factor-inducible A (NGFi-A) transcript was induced to 20% above wild-type levels in both R6/1 and wild-type mice. These results were also observed in HD6 or HD7 treated animals. Long-term expression ( 12 months) of siHUNT-2 in the striatum of wild-type mice led to a loss in DARPP-32 immunoreactivity as well as an enlargement of the lateral ventricles. These data demonstrate that in vivo use of RNAi could lead to off-targeting effects that would only be detected using gene expression profiling techniques such as in situ hybridization, qRT-PCR and gene chip analysis. Moreover, the region in the human Htt gene targeted by these two anti-huntingtin ribozymes and the shRNA, must be similar to a sequence in another unknown gene potentially involved in the regulation of a specific neuronal transcriptional pathway.