polyQ-expanded Protein Research Articles

PolyQ-expanded proteins impair cellular proteostasis of ataxin-3 through sequestering the co-chaperone HSJ1 into aggregates

Polyglutamine (polyQ) expansion of proteins can trigger protein misfolding and amyloid-like aggregation, which thus lead to severe cytotoxicities and even the respective neurodegenerative diseases. However, why polyQ aggregation is toxic to cells is not fully elucidated. Here, we took the fragments of polyQ-expanded (PQE) ataxin-7 (Atx7) and huntingtin (Htt) as models to investigate the effect of polyQ aggregates on the cellular proteostasis of endogenous ataxin-3 (Atx3), a protein that frequently appears in diverse inclusion bodies. We found that PQE Atx7 and Htt impair the cellular proteostasis of Atx3 by reducing its soluble as well as total Atx3 level but enhancing formation of the aggregates. Expression of these polyQ proteins promotes proteasomal degradation of endogenous Atx3 and accumulation of its aggregated form. Then we verified that the co-chaperone HSJ1 is an essential factor that orchestrates the balance of cellular proteostasis of Atx3; and further discovered that the polyQ proteins can sequester HSJ1 into aggregates or inclusions in a UIM domain-dependent manner. Thereby, the impairment of Atx3 proteostasis may be attributed to the sequestration and functional loss of cellular HSJ1. This study deciphers a potential mechanism underlying how PQE protein triggers proteinopathies, and also provides additional evidence in supporting the hijacking hypothesis that sequestration of cellular interacting partners by protein aggregates leads to cytotoxicity or neurodegeneration.

Glial expression of disease-associated poly-glutamine proteins impairs the blood-brain barrier in Drosophila.

Expansion of poly-glutamine (polyQ) stretches in several proteins has been linked to neurodegenerative diseases. The effects of polyQ-expanded proteins on neurons have been extensively studied, but their effects on glia remain unclear. We found that expression of distinct polyQ proteins exclusively in all glia or specifically in the blood-brain barrier (BBB) and blood-retina barrier (BRB) glia caused cell-autonomous impairment of BBB/BRB integrity, suggesting that BBB/BRB glia are most vulnerable to polyQ-expanded proteins. Furthermore, we also found that BBB/BRB leakage in Drosophila is reflected in reversed waveform polarity on the basis of electroretinography (ERG), making ERG a sensitive method to detect BBB/BRB leakage. The polyQ-expanded protein Atxn3-84Q forms aggregates, induces BBB/BRB leakage, restricts Drosophila lifespan and reduces the level of Repo (a pan-glial transcriptional factor required for glial differentiation). Expression of Repo in BBB/BRB glia can rescue BBB/BRB leakage, suggesting that the reduced expression of Repo is important for the effect of polyQ on BBB/BRB impairment. Coexpression of the chaperon HSP40 and HSP70 effectively rescues the effects of Atxn3-84Q, indicating that polyQ protein aggregation in glia is deleterious. Intriguingly, coexpression of wild-type Atxn3-27Q can also rescue BBB/BRB impairment, suggesting that normal polyQ protein may have a protective function.

D6 Dna damage in lymphocytes as a predictor of illness evolution in pre-manifest and overt huntington’s disease

Background Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in the HTT gene that encodes huntingtin (HTT). Mutant HTT leads to abnormalities in cellular function and transcriptional dysregulation that have long been recognised as central to the pathogenesis of HD. Our previous work showed that expression for 3 h of a polyQ-expanded protein (GPF-polyQ, Ataxin-2 and Huntingtin) stimulated cellular reactive oxygen species (ROS) levels and significantly reduced the mitochondrial electrochemical gradient. This result suggest that mutant HTT-induced oxidative stress may lead to persistent activation of DNA damage response (DMR) that may represent an early, main cause of neuronal dysfunction. Methods To quantify DMR, we used cytofluorimetric analysis to detect phosphorylated H2AX (PH2AH). Blood samples were obtained by venous punctures in EDTA-tubes. Freshly isolated peripheral blood mononuclear cells (PBMC) by differential centrifugation through Ficoll were fixed 10 minutes in 4% paraformaldehyde, made permeable with ice-cold 70% ethanol for 30 min, incubated with anti-Fc to block non-specific binding of Fc receptor expressing cells and incubated with anti-PH2AX. We enrolled pre-manifest HD, stratified according to the risk of motor onset into low, medium and high groups, as previously described; symptomatic HD patients, whose disease severity was quantified according to UHDRS, and healthy subjects as controls. Exclusion criteria were: drugs HDAC inhibitors, or NADP inhibitors and other factors known to interfere with the oxidative status of the subject; concomitant serious illnesses; pregnancy and breast-feeding. Results We included 14 (female/male ratio 8/6; age F 30.0 ± 6.7; M 41.8 ± 11.68) pre-manifest HD (pre-HD; Mean Repeat: 42.8 ± 2.7); 29 (female/male ratio 11/18; age F 46.7 ± 9.4; M 55.0 ± 10.1) symptomatic HD patients (Mean Repeat: 42.66 ± 1.9) and 18 healthy subjects as controls (C) (female/male ratio 13/5; age F 49.5 ± 9.6; M 45.4 ± 14.3). We found that patients and pre-HD showed a high PH2AH index as compared to the controls (C mean: 2.9 ± 2.02; HD mean: 10.56 ± 6.9; pre-D: 10.6 ± 7.0); the odds ratio was Conclusions These data, albeit preliminary, suggest an association between a high PH2AX index and HD, being promising as biomarker to characterise HD patients and to monitor disease progression.

Early and Late Events Induced by PolyQ-expanded Proteins: IDENTIFICATION OF A COMMON PATHOGENIC PROPERTY OF POLYQ-EXPANDED PROTEINS

To find a common pathogenetic trait induced by polyQ-expanded proteins, we have used a conditional expression system in PC12 cells to tune the expression of these proteins and analyze the early and late consequences of their expression. We find that expression for 3 h of a polyQ-expanded protein stimulates cellular reactive oxygen species (ROS) levels and significantly reduces the mitochondrial electrochemical gradient. 24-36 h later, ROS induce DNA damage and activation of the checkpoint kinase, ATM. DNA damage signatures are reversible and persist as long as polyQ-expanded proteins are expressed. Transcription of neural and stress response genes is down-regulated in these cells. Selective inhibition of ATM or histone deacetylase rescues transcription and restores the expression of silenced genes. Eventually, after 1 week, the expression of polyQ-expanded protein also induces endoplasmic reticulum stress. As to the primary mechanism responsible for ROS generation, we find that polyQ-expanded proteins, including native Ataxin-2 and Huntingtin, are selectively sequestered in the lipid raft membrane compartment and interact with gp91, the membrane NADPH-oxidase subunit. Selective inhibition of NADPH oxidase or silencing of H-Ras signaling dissolves the aggregates and eliminates DNA damage. We suggest that targeting of the polyQ-expanded proteins to the lipid rafts activates the resident NADPH oxidase. This triggers a signal linking H-Ras, ROS, and ERK1/2 that maintains and propagates the ROS wave to the nucleus. This mechanism may represent the common pathogenetic signature of all polyQ-expanded proteins independently of the specific context or the function of the native wild type protein.

P97 homologs from Caenorhabditis elegans, CDC‐48.1 and CDC‐48.2, suppress the aggregate formation of huntingtin exon1 containing expanded polyQ repeat

Polyglutamine (polyQ)-expanded proteins are associated with cytotoxicity in some neurodegenerative disorders such as Huntington's disease. We have reported that the aggregation of the polyQ-expanded protein is partially suppressed by co-expression of either of two homologs of an AAA chaperone p97, CDC-48.1 or CDC-48.2, in Caenorhabditis elegans, but how p97 regulates the aggregation of polyQ-expanded proteins remains unclear. Here we present direct evidence that CDC-48.1 and CDC-48.2 suppress the aggregation of a huntingtin (Htt) exon1 fragment containing an expanded polyQ repeat in vitro. CDC-48.1 and CDC-48.2 bound the Htt exon1 fragment directly, and suppressed the formation of SDS-insoluble aggregates of Htt fragments containing 53 glutamine residues (HttQ53) independently of nucleotides. CDC-48.1 and CDC-48.2 also modulated the oligomeric states of HttQ53 during the aggregate formation. In the absence of CDC-48.1 and CDC-48.2, HttQ53 formed 70-150 kDa oligomers, whereas 300-500 kDa oligomers as well as 70-150 kDa oligomers accumulated in the presence of CDC-48.1 and CDC-48.2. Taken together, these results suggest that p97 plays a protective role in neurodegenerative disorders by directly suppressing the protein aggregation as a molecular chaperone.

Polyglutamine Expansion Accelerates the Dynamics of Ataxin-1 and Does Not Result in Aggregate Formation

BackgroundPolyglutamine expansion disorders are caused by an expansion of the polyglutamine (polyQ) tract in the disease related protein, leading to severe neurodegeneration. All polyQ disorders are hallmarked by the presence of intracellular aggregates containing the expanded protein in affected neurons. The polyQ disorder SpinoCerebellar Ataxia 1 (SCA1) is caused by a polyQ-expansion in the ataxin-1 protein, which is thought to lead to nuclear aggregates.Methodology/Principal FindingsUsing advanced live cell fluorescence microscopy and a filter retardation assay we show that nuclear accumulations formed by polyQ-expanded ataxin-1 do not resemble aggregates of other polyQ-expanded proteins. Instead of being static, insoluble aggregates, nuclear accumulations formed by the polyQ-expanded ataxin-1 showed enhanced intracellular kinetics as compared to wild-type ataxin-1. During mitosis, ataxin-1 accumulations redistributed equally among daughter cells, in contrast to polyQ aggregates. Interestingly, polyQ expansion did not affect the nuclear-cytoplasmic shuttling of ataxin-1 as proposed before.Conclusions/SignificanceThese results indicate that polyQ expansion does not necessarily lead to aggregate formation, and that the enhanced kinetics may affect the nuclear function of ataxin-1. The unexpected findings for a polyQ-expanded protein and their consequences for ongoing SCA1 research are discussed.

The Length Dependence of the PolyQ-mediated Protein Aggregation

Polyglutamine (polyQ) repeat disorders are caused by the expansion of CAG tracts in certain genes, resulting in transcription of proteins with abnormally long polyQ inserts. When these inserts expand beyond 35-45 glutamines, affected proteins form toxic aggregates, leading to neuron death. Chymotrypsin inhibitor 2 (CI2) with an inserted glutamine repeat has previously been used to model polyQ-mediated aggregation in vitro. However, polyQ insertion lengths in these studies have been kept below the pathogenic threshold. We perform molecular dynamics simulations to study monomer folding dynamics and dimer formation in CI2-polyQ chimeras with insertion lengths of up to 80 glutamines. Our model recapitulates the experimental results of previous studies of chimeric CI2 proteins, showing high folding cooperativity of monomers as well as protein association via domain swapping. Surprisingly, for chimeras with insertion lengths above the pathogenic threshold, monomer folding cooperativity decreases and the dominant mode for dimer formation becomes interglutamine hydrogen bonding. These results support a mechanism for pathogenic polyQ-mediated aggregation, in which expanded polyQ tracts destabilize affected proteins and promote the formation of partially unfolded intermediates. These unfolded intermediates form aggregates through associations by interglutamine interactions.

Polyglutamine expansions: proteolysis, chaperones, and the dangers of promiscuity.

Polyglutamine expansions: proteolysis, chaperones, and the dangers of promiscuity.