RNAi Screening in Drosophila Cells Identifies New Modifiers of Mutant Huntingtin Aggregation

The fruitfly Drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. Utilizing RNAi, we have carried out a high-throughput screen for modifiers of aggregate formation in Drosophila larval CNS-derived cells expressing mutant human Huntingtin exon 1 fused to EGFP with an expanded polyglutamine repeat (62Q). 7200 genes, encompassing around 50% of the Drosophila genome, were screened, resulting in the identification of 404 candidates that either suppress or enhance aggregation. These candidates were subjected to secondary screening in normal length (18Q)-expressing cells and pruned to remove dsRNAs with greater than 10 off-target effects (OTEs). De novo RNAi probes were designed and synthesized for the remaining 68 candidates. Following a tertiary round of screening, 21 high confidence candidates were analyzed in vivo for their ability to modify mutant Huntingtin-induced eye degeneration and brain aggregation. We have established useful models for the study of human HD using the fly, and through our RNAi screen, we have identified new modifiers of mutant human Huntingtin aggregation and aggregate formation in the brain. Newly identified modifiers including genes related to nuclear transport, nucleotide processes, and signaling, may be involved in polyglutamine aggregate formation and Huntington disease cascades

CAG repeat length in the androgen receptor gene is related to age at diagnosis of prostate cancer and response to endocrine therapy, but not to prostate cancer risk

The length of the polymorphic CAG repeat in the N-terminal of the androgen receptor (AR) gene is inversely correlated with the transactivation function of the AR. Some studies have indicated that short CAG repeats are related to higher risk of prostate cancer. We performed a case–control study to investigate relations between CAG repeat length and prostate cancer risk, tumour grade, tumour stage, age at diagnosis and response to endocrine therapy. The study included 190 AR alleles from prostate cancer patients and 186 AR alleles from female control subjects. All were whites from southern Sweden. The frequency distribution of CAG repeat length was strikingly similar for cases and controls, and no significant correlation between CAG repeat length and prostate cancer risk was detected. However, for men with non-hereditary prostate cancer (n = 160), shorter CAG repeats correlated with younger age at diagnosis (P = 0.03). There were also trends toward associations between short CAG repeats and high grade (P = 0.07) and high stage (P = 0.07) disease. Furthermore, we found that patients with long CAG repeats responded better to endocrine therapy, even after adjusting for pretreatment level of prostate-specific antigen and tumour grade and stage (P = 0.05). We conclude that short CAG repeats in the AR gene correlate with young age at diagnosis of prostate cancer, but not with higher risk of the disease. Selection of patients with early onset prostate cancer in case–control studies could therefore lead to an over-estimation of the risk of prostate cancer for men with short CAG repeats. An association between long CAG repeats and good response to endocrine therapy was also found, but the mechanism and clinical relevance are unclear. © 1999 Cancer Research Campaig

Polyglutamine Expansion Mutation Yields a Pathological Epitope Linked to Nucleation of Protein Aggregate: Determinant of Huntington's Disease Onset

Polyglutamine (polyQ) expansion mutation causes conformational, neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. These diseases are characterized by the aggregation of misfolded proteins, such as amyloid fibrils, which are toxic to cells. Amyloid fibrils are formed by a nucleated growth polymerization reaction. Unexpectedly, the critical nucleus of polyQ aggregation was found to be a monomer, suggesting that the rate-limiting nucleation process of polyQ aggregation involves the folding of mutated protein monomers. The monoclonal antibody 1C2 selectively recognizes expanded pathogenic and aggregate-prone glutamine repeats in polyQ diseases, including Huntington's disease (HD), as well as binding to polyleucine. We have therefore assayed the in vitro and in vivo aggregation kinetics of these monomeric proteins. We found that the repeat-length-dependent differences in aggregation lag times of variable lengths of polyQ and polyleucine tracts were consistently related to the integration of the length-dependent intensity of anti-1C2 signal on soluble monomers of these proteins. Surprisingly, the correlation between the aggregation lag times of polyQ tracts and the intensity of anti-1C2 signal on soluble monomers of huntingtin precisely reflected the repeat-length dependent age-of-onset of HD patients. These data suggest that the alterations in protein surface structure due to polyQ expansion mutation in soluble monomers of the mutated proteins act as an amyloid-precursor epitope. This, in turn, leads to nucleation, a key process in protein aggregation, thereby determining HD onset. These findings provide new insight into the gain-of-function mechanisms of polyQ diseases, in which polyQ expansion leads to nucleation rather than having toxic effects on the cells

AIB1 gene amplification and the instability of polyQ encoding sequence in breast cancer cell lines

BACKGROUND: The poly Q polymorphism in AIB1 (amplified in breast cancer) gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation. The purpose of this study is to investigate the sequence variation of poly Q encoding region in breast cancer cell lines at single molecule level, and to determine if the sequence variation is related to AIB1 gene amplification. METHODS: The polymorphic poly Q encoding region of AIB1 gene was investigated at the single molecule level by PCR cloning/sequencing. The amplification of AIB1 gene in various breast cancer cell lines were studied by real-time quantitative PCR. RESULTS: Significant amplifications (5–23 folds) of AIB1 gene were found in 2 out of 9 (22%) ER positive cell lines (in BT-474 and MCF-7 but not in BT-20, ZR-75-1, T47D, BT483, MDA-MB-361, MDA-MB-468 and MDA-MB-330). The AIB1 gene was not amplified in any of the ER negative cell lines. Different passages of MCF-7 cell lines and their derivatives maintained the feature of AIB1 amplification. When the cells were selected for hormone independence (LCC1) and resistance to 4-hydroxy tamoxifen (4-OH TAM) (LCC2 and R27), ICI 182,780 (LCC9) or 4-OH TAM, KEO and LY 117018 (LY-2), AIB1 copy number decreased but still remained highly amplified. Sequencing analysis of poly Q encoding region of AIB1 gene did not reveal specific patterns that could be correlated with AIB1 gene amplification. However, about 72% of the breast cancer cell lines had at least one under represented (<20%) extra poly Q encoding sequence patterns that were derived from the original allele, presumably due to somatic instability. Although all MCF-7 cells and their variants had the same predominant poly Q encoding sequence pattern of (CAG)(3)CAA(CAG)(9)(CAACAG)(3)(CAACAGCAG)(2)CAA of the original cell line, a number of altered poly Q encoding sequences were found in the derivatives of MCF-7 cell lines. CONCLUSION: These data suggest that poly Q encoding region of AIB1 gene is somatic unstable in breast cancer cell lines. The instability and the sequence characteristics, however, do not appear to be associated with the level of the gene amplification

ZMIZ1 Preferably Enhances the Transcriptional Activity of Androgen Receptor with Short Polyglutamine Tract

The androgen receptor (AR) is a ligand-induced transcription factor and contains the polyglutamine (polyQ) tracts within its N-terminal transactivation domain. The length of polyQ tracts has been suggested to alter AR transcriptional activity in prostate cancer along with other endocrine and neurologic disorders. Here, we assessed the role of ZMIZ1, an AR co-activator, in regulating the activity of the AR with different lengths of polyQ tracts as ARQ9, ARQ24, and ARQ35 in prostate cancer cells. ZMIZ1, but not ZMIZ2 or ARA70, preferably augments ARQ9 induced androgen-dependent transcription on three different androgen-inducible promoter/reporter vectors. A strong protein-protein interaction between ZMIZ1 and ARQ9 proteins was shown by immunoprecipitation assays. In the presence of ZMIZ1, the N and C-terminal interaction of the ARQ9 was more pronounced than ARQ24 and ARQ35. Both Brg1 and BAF57, the components of SWI/SNF complexes, were shown to be involved in the enhancement of ZMIZ1 on AR activity. Using the chromatin immunoprecipitation assays (ChIP), we further demonstrated a strong recruitment of ZMIZ1 by ARQ9 on the promoter of the prostate specific antigen (PSA) gene. These results demonstrate a novel regulatory role of ZMIZ1 in modulating the polyQ tract length of AR in prostate cancer cells

Hsp40 Couples with the CSPα Chaperone Complex upon Induction of the Heat Shock Response

In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPα). CSPα is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich “string” region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPα chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Gαs. In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPα complex. Association of Hsp40 with CSPα decreases CSPα-CSPα dimerization and enhances the CSPα-induced increase in steady state GTP hydrolysis of Gαs. This newly identified CSPα-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPα in neuroprotection

Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging

Protein Quality Control (PQC) pathways are essential to maintain the equilibrium between protein folding and the clearance of misfolded proteins. In order to discover novel human PQC factors, we developed a high-content, high-throughput cell-based assay to assess PQC activity. The assay is based on a fluorescently tagged, temperature sensitive PQC substrate and measures its degradation relative to a temperature insensitive internal control. In a targeted screen of 1591 siRNA genes involved in the Ubiquitin-Proteasome System (UPS) we identified 25 of the 33 genes encoding for 26S proteasome subunits and discovered several novel PQC factors. An unbiased genome-wide siRNA screen revealed the protein translation machinery, and in particular the EIF3 translation initiation complex, as a novel key modulator of misfolded protein stability. These results represent a comprehensive unbiased survey of human PQC components and establish an experimental tool for the discovery of genes that are required for the degradation of misfolded proteins under conditions of proteotoxic stress

Histone deacetylases suppress cgg repeat-induced neurodegeneration via transcriptional silencing in models of Fragile X Tremor Ataxia Syndrome

Fragile X Tremor Ataxia Syndrome (FXTAS) is a common inherited neurodegenerative disorder caused by expansion of a CGG trinucleotide repeat in the 59UTR of the fragile X syndrome (FXS) gene, FMR1. The expanded CGG repeat is thought to induce toxicity as RNA, and in FXTAS patients mRNA levels for FMR1 are markedly increased. Despite the critical role of FMR1 mRNA in disease pathogenesis, the basis for the increase in FMR1 mRNA expression is unknown. Here we show that overexpressing any of three histone deacetylases (HDACs 3, 6, or 11) suppresses CGG repeat-induced neurodegeneration in a Drosophila model of FXTAS. This suppression results from selective transcriptional repression of the CGG repeat-containing transgene. These findings led us to evaluate the acetylation state of histones at the human FMR1 locus. In patient-derived lymphoblasts and fibroblasts, we determined by chromatin immunoprecipitation that there is increased acetylation of histones at the FMR1 locus in pre-mutation carriers compared to control or FXS derived cell lines. These epigenetic changes correlate with elevated FMR1 mRNA expression in pre-mutation cell lines. Consistent with this finding, histone acetyltransferase (HAT) inhibitors repress FMR1 mRNA expression to control levels in pre-mutation carrier cell lines and extend lifespan in CGG repeat-expressing Drosophila. These findings support a disease model whereby the CGG repeat expansion in FXTAS promotes chromatin remodeling in cis, which in turn increases expression of the toxic FMR1 mRNA. Moreover, these results provide proof of principle that HAT inhibitors or HDAC activators might be used to selectively repress transcription at the FMR1 locus.open293