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  • Crosstalk between huntingtin and syntaxin 1A regulates N-type calcium channels. 16162412

    We have identified a novel interaction between huntingtin (htt) and N-type calcium channels, a channel key in coupling calcium influx with synaptic vesicle exocytosis. Htt is a widely expressed 350-kDa cytosolic protein bearing an N-terminal polyglutamine tract. Htt is proteolytically cleaved by calpains and caspases and the resultant htt N-terminal fragments have been proposed to be biologically active; however, the cellular function of htt and/or the htt fragments remains enigmatic. We show that N-terminal fragments of htt (consisting of exon1) and full-length htt associate with the synaptic protein interaction (synprint) region of the N-type calcium channel. Given that synprint has previously been shown to bind syntaxin 1A and that this association elicits inhibition of N-type calcium channels, we tested whether htt(exon1) affects the modulation of these channels. Our data indicate that htt(exon1) enhances calcium influx by blocking syntaxin 1A inhibition of N-type calcium channels and attributes a key role for htt N-terminal fragments in the fine tuning of neurotransmission.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB2166
    Nombre del producto:
    Anti-Huntingtin Protein Antibody, a.a. 181-810, clone 1HU-4C8

  • Huntingtin interacting proteins are genetic modifiers of neurodegeneration. 17500595

    Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%-4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB374
    Nombre del producto:
    Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5

  • Regulation of miR-146a by RelA/NFkB and p53 in STHdh(Q111)/Hdh(Q111) cells, a cell model of Huntington's disease. 21887328

    Huntington's disease (HD) is caused by the expansion of N-terminal polymorphic poly Q stretch of the protein huntingtin (HTT). Deregulated microRNAs and loss of function of transcription factors recruited to mutant HTT aggregates could cause characteristic transcriptional deregulation associated with HD. We observed earlier that expressions of miR-125b, miR-146a and miR-150 are decreased in STHdh(Q111)/Hdh(Q111) cells, a model for HD in comparison to those of wild type STHdh(Q7)/Hdh(Q7) cells. In the present manuscript, we show by luciferase reporter assays and real time PCR that decreased miR-146a expression in STHdh(Q111)/Hdh(Q111) cells is due to decreased expression and activity of p65 subunit of NFkB (RelA/NFkB). By reporter luciferase assay, RT-PCR and western blot analysis, we also show that both miR-150 and miR-125b target p53. This partially explains the up regulation of p53 observed in HD. Elevated p53 interacts with RelA/NFkB, reduces its expression and activity and decreases the expression of miR-146a, while knocking down p53 increases RelA/NFkB and miR-146a expressions. We also demonstrate that expression of p53 is increased and levels of RelA/NFkB, miR-146a, miR-150 and miR-125b are decreased in striatum of R6/2 mice, a mouse model of HD and in cell models of HD. In a cell model, this effect could be reversed by exogenous expression of chaperone like proteins HYPK and Hsp70. We conclude that (i) miR-125b and miR-150 target p53, which in turn regulates RelA/NFkB and miR-146a expressions; (ii) reduced miR-125b and miR-150 expressions, increased p53 level and decreased RelA/NFkB and miR-146a expressions originate from mutant HTT (iii) p53 directly or indirectly regulates the expression of miR-146a. Our observation of interplay between transcription factors and miRNAs using HD cell model provides an important platform upon which further work is to be done to establish if such regulation plays any role in HD pathogenesis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3026
    Nombre del producto:
    Anti-NFκB Antibody, p65 subunit, active subunit, clone 12H11

  • Activated caspase-6 and caspase-6-cleaved fragments of huntingtin specifically colocalize in the nucleus. 18445618

    Proteolysis of mutant huntingtin is crucial to the development of Huntington disease (HD). Specifically preventing proteolysis at the capase-6 (C6) consensus sequence at amino acid 586 of mutant huntingtin prevents the development of behavioural, motor and neuropathological features in a mouse model of HD. However, the mechanism underlying the selective toxicity of the 586 amino acid cleavage event is currently unknown. We have examined the subcellular localization of different caspase proteolytic fragments of huntingtin using neo-epitope antibodies. Our data suggest that the nucleus is the primary site of htt cleavage at amino acid 586. Endogenously cleaved 586 amino acid fragments are enriched in the nucleus of immortalized striatal cells and primary striatal neurons where they co-localize with active C6. Cell stress induced by staurosporine results in the nuclear translocation and activation of C6 and an increase in 586 amino acid fragments of huntingtin in the nucleus. In comparison, endogenous caspase-2/3-generated huntingtin 552 amino acid fragments localize to the perinuclear region. The different cellular itineraries of endogenously generated caspase products of huntingtin may provide an explanation for the selective toxicity of huntingtin fragments cleaved at amino acid 586.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB2166
    Nombre del producto:
    Anti-Huntingtin Protein Antibody, a.a. 181-810, clone 1HU-4C8

  • Elucidating a normal function of huntingtin by functional and microarray analysis of huntingtin-null mouse embryonic fibroblasts. 18412970

    The polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington's disease (HD). Htt is an essential gene as deletion of the mouse Htt gene homolog (Hdh) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD.To systematically search for a mouse Htt function, we took advantage of the Hdh +/- and Hdh-floxed mice and generated four mouse embryonic fibroblast (MEF) cells lines which contain a single copy of the Hdh gene (Hdh-HET) and four MEF lines in which the Hdh gene was deleted (Hdh-KO). The function of Htt in calcium (Ca2+) signaling was analyzed in Ca2+ imaging experiments with generated cell lines. We found that the cytoplasmic Ca2+ spikes resulting from the activation of inositol 1,4,5-trisphosphate receptor (InsP3R) and the ensuing mitochondrial Ca2+ signals were suppressed in the Hdh-KO cells when compared to Hdh-HET cells. Furthermore, in experiments with permeabilized cells we found that the InsP3-sensitivity of Ca2+ mobilization from endoplasmic reticulum was reduced in Hdh-KO cells. These results indicated that Htt plays an important role in modulating InsP3R-mediated Ca2+ signaling. To further evaluate function of Htt, we performed genome-wide transcription profiling of generated Hdh-HET and Hdh-KO cells by microarray. Our results revealed that 106 unique transcripts were downregulated by more than two-fold with p less than 0.05 and 173 unique transcripts were upregulated at least two-fold with p less than 0.05 in Hdh-KO cells when compared to Hdh-HET cells. The microarray results were confirmed by quantitative real-time PCR for a number of affected transcripts. Several signaling pathways affected by Hdh gene deletion were identified from annotation of the microarray results.Functional analysis of generated Htt-null MEF cells revealed that Htt plays a direct role in Ca2+ signaling by modulating InsP3R sensitivity to InsP3. The genome-wide transcriptional profiling of Htt-null cells yielded novel and unique information about the normal function of Htt in cells, which may contribute to our understanding and treatment of HD.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Partial depletion of CREB-binding protein reduces life expectancy in a mouse model of Huntington disease. 20448484

    Previous studies have reported that mutant huntingtin (htt) interferes with cyclic AMP response element binding protein binding protein (CBP)-mediated transcription, possibly by inhibiting the acetylation of histones. In Drosophila models that express fragments of mutant htt, histone deacetylase inhibitors reverse deficits in histone acetylation, rescue photoreceptor degeneration, and prolong their survival. These compounds also improve motor deficits in a transgenic mouse model of Huntington disease (HD). To determine whether endogenous CBP depletion contributes to HD pathogenesis, we crossed HD-N171-82Q transgenic mice with mice harboring a disrupted CBP gene and produced mice with partial (50%) depletion of CBP. This reduction of CBP levels decreased the life expectancy of the HD-N171-82Q Line 6 mouse model. The loss of CBP had no obvious impact on the severity of motor impairment, degeneration of the striatum, mutant htt inclusion formation, or global levels of acetylated histones H3 or H4 in brain. In cell models, we confirmed that mutant htt inclusions recruit human CBP but found no evidence for interactions between soluble forms of mutant htt and CBP. Although we identified no neurological explanation for the decreased life expectancy of HD-N171-82Q mice with partial depletion of CBP, the data are consistent with the notion that CBP function mitigates mutant htt toxicity by a currently unidentified mechanism.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis. 22556411

    Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB2166
    Nombre del producto:
    Anti-Huntingtin Protein Antibody, a.a. 181-810, clone 1HU-4C8

  • Inhibition of mitochondrial fragmentation diminishes Huntington's disease-associated neurodegeneration. 24231356

    Huntington's disease (HD) is the result of expression of a mutated Huntingtin protein (mtHtt), and is associated with a variety of cellular dysfunctions including excessive mitochondrial fission. Here, we tested whether inhibition of excessive mitochondrial fission prevents mtHtt-induced pathology. We developed a selective inhibitor (P110-TAT) of the mitochondrial fission protein dynamin-related protein 1 (DRP1). We found that P110-TAT inhibited mtHtt-induced excessive mitochondrial fragmentation, improved mitochondrial function, and increased cell viability in HD cell culture models. P110-TAT treatment of fibroblasts from patients with HD and patients with HD with iPS cell-derived neurons reduced mitochondrial fragmentation and corrected mitochondrial dysfunction. P110-TAT treatment also reduced the extent of neurite shortening and cell death in iPS cell-derived neurons in patients with HD. Moreover, treatment of HD transgenic mice with P110-TAT reduced mitochondrial dysfunction, motor deficits, neuropathology, and mortality. We found that p53, a stress gene involved in HD pathogenesis, binds to DRP1 and mediates DRP1-induced mitochondrial and neuronal damage. Furthermore, P110-TAT treatment suppressed mtHtt-induced association of p53 with mitochondria in multiple HD models. These data indicate that inhibition of DRP1-dependent excessive mitochondrial fission with a P110-TAT-like inhibitor may prevent or slow the progression of HD.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Identification of distinct conformations associated with monomers and fibril assemblies of mutant huntingtin. 29912367

    The N-terminal fragments of mutant huntingtin (mHTT) misfold and assemble into oligomers, which ultimately bundle into insoluble fibrils. Conformations unique to various assemblies of mHTT remain unknown. Knowledge on the half-life of various multimeric structures of mHTT is also scarce. Using a panel of four new antibodies named PHP1-4, we have identified new conformations in monomers and assembled structures of mHTT. PHP1 and PHP2 bind to epitopes within the proline-rich domain (PRD), whereas PHP3 and PHP4 interact with motifs formed at the junction of polyglutamine (polyQ) and polyproline (polyP) repeats of HTT. The PHP1- and PHP2-reactive epitopes are exposed in fibrils of mHTT exon1 (mHTTx1) generated from recombinant proteins and mHTT assemblies, which progressively accumulate in the nuclei, cell bodies and neuropils in the brains of HD mouse models. Notably, electron microscopic examination of brain sections of HD mice revealed that PHP1- and PHP2-reactive mHTT assemblies are present in myelin sheath and in vesicle-like structures. Moreover, PHP1 and PHP2 antibodies block seeding and subsequent fibril assembly of mHTTx1 in vitro and in a cell culture model of HD. PHP3 and PHP4 bind to epitopes in full-length and N-terminal fragments of monomeric mHTT and binding diminishes as the mHTTx1 assembles into fibrils. Interestingly, PHP3 and PHP4 also prevent the aggregation of mHTTx1 in vitro highlighting a regulatory function for the polyQ-polyP motifs. These newly detected conformations may affect fibril assembly, stability and intercellular transport of mHTT.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Polyglutamine- and temperature-dependent conformational rigidity in mutant huntingtin revealed by immunoassays and circular dichroism spectroscopy. 25464275

    In Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearinggreater than 35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays.By performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein.The temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington's disease.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB2166
    Nombre del producto:
    Anti-Huntingtin Protein Antibody, a.a. 181-810, clone 1HU-4C8