Nitai P. Bhattacharyya

Analysis of CAG repeats in SCA1, SCA2, SCA3, SCA6, SCA7 and DRPLA loci in spinocerebellar ataxia patients and distribution of CAG repeats at the SCA1, SCA2 and SCA6 loci in nine ethnic populations of eastern India

Abstract. To identify various subtypes of spinocerebellar ataxias (SCAs) among 57 unrelated individuals clinically diagnosed as ataxia patients we analysed the SCA1, SCA2, SCA3, SCA6, SCA7 and DRPLA loci for expansion of CAG repeats. We detected CAG repeat expansion in 6 patients (10.5%) at the SCA1 locus. Ten of the 57 patients (17.5%) had CAG repeat expansion at the SCA2 locus, while four had CAG expansion at the SCA3/MJD locus (7%). At the SCA6 locus there was a single patient (1.8%) with 21 CAG repeats. We have not detected any patient with expansion in the SCA7 and DRPLA loci. To test whether the frequencies of the large normal alleles in SCA1, SCA2 and SCA6 loci can reflect some light on prevalence of the subtypes of SCAs we studied the CAG repeat variation in these loci in nine ethnic sub-populations of eastern India from which the patients originated. We report here that the frequency of large normal alleles (>31 CAG repeats) in SCA1 locus to be 0.211 of 394 chromosomes studied. We also report that the frequency of large normal alleles (>22 CAG repeats) at the SCA2 locus is 0.038 while at the SCA6 locus frequency of large normal alleles (>13 repeats) is 0.032. We discussed our data in light of the distribution of normal alleles and prevalence of SCAs in the Japanese and white populations.

Modulation of age at onset in Huntington's disease and spinocerebellar ataxia type 2 patients originated from eastern India.

To identify the genetic modifier(s) that might alter the age at onset in Huntingtons disease (HD) we have analyzed variations in GluR6 kainate receptor (GluR6), CA150 gene, Delta2642 and polymorphic CCG repeat variation in huntingtin (htt) gene in 77 HD patients and normal individuals. In addition, variation in the RAI1 gene was analyzed in 30 spinocerebellar ataxia (SCA2) patients and normal individuals to show the possible influence on the age at onset. Multiple regression analysis indicated that variation in GluR6 and CCG repeat genotype might explain 6.2% and 3.1%, respectively, of the variability in the age at onset in HD. Similar analysis with SCA2 patients indicated that RAI1 might explain about 13% of the variability in the age at onset. Specific alleles in GluR6 and CA150 locus were only observed in HD patients.

Regulation of miR-146a by RelA/NFkB and p53 in STHdhQ111/HdhQ111 Cells, a Cell Model of Huntington's Disease

Huntingtons 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 STHdhQ111/HdhQ111 cells, a model for HD in comparison to those of wild type STHdhQ7/HdhQ7 cells. In the present manuscript, we show by luciferase reporter assays and real time PCR that decreased miR-146a expression in STHdhQ111/HdhQ111 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.

The role of intrinsically unstructured proteins in neurodegenerative diseases.

The number and importance of intrinsically disordered proteins (IUP), known to be involved in various human disorders, are growing rapidly. To test for the generalized implications of intrinsic disorders in proteins involved in Neurodegenerative diseases, disorder prediction tools have been applied to three datasets comprising of proteins involved in Huntington Disease (HD), Parkinsons disease (PD), Alzheimers disease (AD). Results show, in general, proteins in disease datasets possess significantly enhanced intrinsic unstructuredness. Most of these disordered proteins in the disease datasets are found to be involved in neuronal activities, signal transduction, apoptosis, intracellular traffic, cell differentiation etc. Also these proteins are found to have more number of interactors and hence as the proportion of disorderedness (i.e., the length of the unfolded stretch) increased, the size of the interaction network simultaneously increased. All these observations reflect that, “Moonlighting” i.e. the contextual acquisition of different structural conformations (transient), eventually may allow these disordered proteins to act as network “hubs” and thus they may have crucial influences in the pathogenecity of neurodegenerative diseases.

Caspase 8 mediated apoptotic cell death induced by β-sheet forming polyalanine peptides

Expansion of a polyalanine stretch from 10 to 12–17 residues in the N‐terminus of the protein PABP2 has been implicated in the genetically acquired disease oculopharyngeal muscular dystrophy, characterized by nuclear protein deposits. Here we report a correlation between the structural properties and cell toxicity of two peptides mimicking the N‐terminal domain of PABP2: one containing seven and the other 11 uninterrupted alanine residues. Consistent with earlier observations, the longer peptide (11‐ala) was found to adopt β‐sheet structure while the shorter one (7‐ala) formed α‐helix over a wide range of concentrations (∼20–500 μM). We observed that treatment with 11‐ala resulted in significantly enhanced death of Chinese hamster V79 cells, compared to the effect of treatment with 7‐ala, via the cytochrome c mediated apoptotic pathway. Increases in caspase 8 and caspase 3 activity were also observed in human cells (K562) treated with 11‐ala. These results indicate that the toxicity of pathogenic peptides is directly linked to their β‐sheet structure and also support recent observations that small oligomeric species of peptides and proteins are the key toxic elements in causing protein aggregation diseases.

Modulation of age at onset of Huntington disease patients by variations in TP53 and human caspase activated DNase (hCAD) genes

Variation of age at onset (AO) in Huntingtons disease (HD) cannot be explained by the number of CAG repeats alone in the mutant alleles of the gene huntingtin (Htt). Given the ability of expanded polyglutamine (poly-Q) tract present in Htt protein to interact with other proteins and increased neuronal cell death by apoptosis, variations in the genes coding for htt-interacting proteins and those involved in apoptosis are likely to alter the AO in HD. In the present investigation, we studied two single nucleotide polymorphisms (SNPs), namely, R72P in TP53 gene coding for transcription factor p53, which interacts with Htt protein and R196K in human caspase activated DNase (hCAD) gene involved in apoptosis to investigate their role as genetic modifiers of the AO of HD. Multiple linear regression analysis revealed that variations in TP53 and hCAD genes explained 12.6% and 6%, respectively, of the variance in the AO of HD after accounting for the effect of expanded CAG repeats. Statistical analysis further showed a significant effect of the interaction term between expanded CAG repeats and variations at each of TP53 and hCAD genes upon the AO. This data demonstrated that variations in TP53 and hCAD genes modulate the AO of HD.

Increased Caspase-2, Calpain Activations and Decreased Mitochondrial Complex II Activity in Cells Expressing Exogenous Huntingtin Exon 1 Containing CAG Repeat in the Pathogenic Range

Abstract(1) Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by the expansion of polymorphic CAG repeats beyond 36 at exon 1 of huntingtin gene (htt). To study cellular effects by expressing N-terminal domain of Huntingtin (Htt) in specific cell lines, we expressed exon 1 of htt that codes for 40 glutamines (40Q) and 16Q in Neuro2A and HeLa cells. (2) Aggregates and various apoptotic markers were detected at various time points after transfection. In addition, we checked the alterations of expressions of few apoptotic genes by RT-PCR. (3) Cells expressing exon 1 of htt coding 40Q at a stretch exhibited nuclear and cytoplasmic aggregates, increased caspase-1, caspase-2, caspase-8, caspase-9/6, and calpain activations, release of cytochrome c and AIF from mitochondria in a time-dependent manner. Truncation of Bid was increased, while the activity of mitochondrial complex II was decreased in such cells. These changes were significantly higher in cells expressing N-terminal Htt with 40Q than that obtained in cells expressing N-terminal Htt with 16Q. Expressions of caspase-1, caspase-2, caspase-3, caspase-7, and caspase-8 were increased while expression of Bcl-2 was decreased in cells expressing mutated Htt-exon 1. (4) Results presented in this communication showed that expression of mutated Htt-exon 1 could mimic the cellular phenotypes observed in Huntington’s disease and this cell model can be used for screening the agents that would interfere with the apoptotic pathway and aggregate formation.

Huntington’s disease: roles of huntingtin-interacting protein 1 (HIP-1) and its molecular partner HIPPI in the regulation of apoptosis and transcription

Huntingtin protein (Htt), whose mutation causes Huntington’s disease (HD), interacts with large numbers of proteins that participate in diverse cellular pathways. This observation indicates that wild‐type Htt is involved in various cellular processes and that the mutated Htt alters these processes in HD. The roles of these interacting proteins in HD pathogenesis remain largely unknown. In the present review, we present evidence that Htt‐interacting protein 1 (HIP‐1), an endocytic protein, together with its interacting partner HIPPI, regulates apoptosis and gene expression, both processes being implicated in HD. Further studies are necessary to establish whether the HIPPI–HIP‐1 complex or other interacting partners of HIPPI regulate apoptosis and gene expression that are relevant to HD.

Induction of apoptosis in cells expressing exogenous Hippi, a molecular partner of huntingtin-interacting protein Hip1

To decipher the pathway of apoptosis induction downstream to caspase-8 activation by exogenous expression of Hippi, an interactor of huntingtin-interacting protein Hip1, we studied apoptosis in HeLa and Neuro2A cells expressing GFP-tagged Hippi. Nuclear fragmentation, caspase-1, caspase-8, caspase-9/caspase-6 and caspase-3 activation were increased significantly in Hippi expressing cells. Cleavage of Bid, release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria were also increased in GFP-Hippi expressing cells. It was observed that caspase-1 and caspase-8 activation was earlier than caspase-3 activation and nuclear fragmentation. Expression of caspase-1, caspase-3 and caspase-7 was increased while anti-apoptotic gene Bcl-2 and mitochondrial genes ND1 and ND4 were reduced in Hippi expressing cells. Besides, the expression SDHA and SDHB, nuclear genes, subunits of mitochondrial complex II were decreased in GFP-Hippi expressing cells. Taken together, we concluded that Hippi expression induced apoptosis by releasing AIF and cytochrome c from mitochondria, activation of caspase-1 and caspase-3, and altering the expression of apoptotic genes and genes involved in mitochondrial complex I and II.

Huntingtin interacting protein HYPK is intrinsically unstructured

To characterize HYPK, originally identified as a novel huntingtin (Htt) interacting partner by yeast two hybrid assay, we used various biophysical and biochemical techniques. The molecular weight of the protein, determined by gel electrophoresis, was found to be about 1.3‐folds (∼22 kDa) higher than that obtained from mass spectrometric analysis (16.9 kDa). In size exclusion chromatography experiment, HYPK was eluted in three fractions, the hydrodynamic radii for which were calculated to be ∼1.5‐folds (23.06 Å) higher than that expected for globular proteins of equivalent mass (17.3 Å). The protein exhibited predominantly (63%) random coil characteristics in circular dichroism spectroscopy and was highly sensitive to limited proteolysis by trypsin and papain, indicating absence of any specific domain. Experimental evidences with theoretical analyses of amino acids composition of HYPK and comparison with available published data predicts that HYPK is an intrinsically unstructured protein (IUP) with premolten globule like conformation. In presence of increasing concentration of Ca2+, HYPK showed conformational alterations as well as concomitant reduction of hydrodynamic radius. Even though any link between the natively unfolded nature of HYPK, its conformational sensitivity towards Ca2+ and interaction with Htt is yet to be established, its possible involvement in Huntingtons disease pathogenesis is discussed. Proteins 2008.