DJ-1 modulates aggregation and pathogenesis in models of Huntington's disease.

The oxidation-sensitive chaperone protein DJ-1 has been implicated in several human disorders including cancer and neurodegenerative diseases. During neurodegeneration associated with protein misfolding, such as that observed in Alzheimer's disease and Huntington's disease (HD), both oxidative stress and protein chaperones have been shown to modulate disease pathways. Therefore, we set out to investigate whether DJ-1 plays a role in HD. We found that DJ-1 expression and its oxidation state are abnormally increased in the human HD brain, as well as in mouse and cell models of HD. Furthermore, overexpression of DJ-1 conferred protection in vivo against neurodegeneration in yeast and Drosophila. Importantly, the DJ-1 protein directly interacted with an expanded fragment of huntingtin Exon 1 (httEx1) in test tube experiments and in cell models and accelerated polyglutamine aggregation and toxicity in an oxidation-sensitive manner. Our findings clearly establish DJ-1 as a potential therapeutic target for HD and provide the basis for further studies into the role of DJ-1 in protein misfolding diseases.

In vitro and in vivo aggregation of a fragment of huntingtin protein directly causes free radical production.

Neurodegenerative diseases are characterized by intra- and/or extracellular protein aggregation and oxidative stress. Intense attention has been paid to whether protein aggregation itself contributes to abnormal production of free radicals and ensuing cellular oxidative damage. Although this question has been investigated in the context of extracellular protein aggregation, it remains unclear whether protein aggregation inside cells alters the redox homeostasis. To address this, we have used in vitro and in vivo (cellular) models of Huntington disease, one of nine polyglutamine (poly(Q)) disorders, and examined the causal relationship among intracellular protein aggregation, reactive oxygen species (ROS) production, and toxicity. Live imaging of cells expressing a fragment of huntingtin (httExon1) with a poly(Q) expansion shows increased ROS production preceding cell death. ROS production is poly(Q) length-dependent and not due to the httExon 1 flanking sequence. Aggregation inhibition by the MW7 intrabody and Pgl-135 treatment abolishes ROS production, showing that increased ROS is caused by poly(Q) aggregation itself. To examine this hypothesis further, we determined whether aggregation of poly(Q) peptides in vitro generated free radicals. Monitoring poly(Q) protein aggregation using atomic force microscopy and hydrogen peroxide (H(2)O(2)) production over time in parallel we show that oligomerization of httEx1Q53 results in early generation of H(2)O(2). Inhibition of poly(Q) oligomerization by the single chain antibody MW7 abrogates H(2)O(2) formation. These results demonstrate that intracellular protein aggregation directly causes free radical production, and targeting potentially toxic poly(Q) oligomers may constitute a therapeutic target to counteract oxidative stress in poly(Q) diseases.

Metallothioneins and copper metabolism are candidate therapeutic targets in Huntington's disease.

HD (Huntington's disease) is caused by a polyQ (polyglutamine) expansion in the huntingtin protein, which leads to protein misfolding and aggregation of this protein. Abnormal copper accumulation in the HD brain was first reported more than 15 years ago. Recent findings show that copper-regulatory genes are induced during HD and copper binds to an N-terminal fragment of huntingtin, supporting the involvement of abnormal copper metabolism in HD. We have demonstrated that in vitro copper accelerates the fibrillization of an N-terminal fragment of huntingtin with an expanded polyQ stretch (httExon1). As we found that copper also increases polyQ aggregation and toxicity in mammalian cells expressing httExon1, we investigated further whether overexpression of genes involved in copper metabolism, notably MTs (metallothioneins) known to bind copper, protect against httExon1 toxicity. Using a yeast model of HD, we have shown that overexpression of several genes involved in copper metabolism reduces polyQ-mediated toxicity. Overexpression of MT-3 in mammalian cells significantly reduced polyQ aggregation and toxicity. We propose that copper-binding and/or -chaperoning proteins, especially MTs, are potential therapeutic targets for HD.

Neurotoxic protein oligomerisation associated with polyglutamine diseases.

Polyglutamine (polyQ) diseases are associated with a CAG/polyQ expansion mutation in unrelated proteins. Upon elongation of the glutamine tract, disease proteins aggregate within cells, mainly in the central nervous system (CNS) and this aggregation process is associated with neurotoxicity. However, it remains unclear to what extent and how this aggregation causes neuronal dysfunction in the CNS. Aiming at preventing neuronal dysfunction, it will be crucial to determine the links between aggregation and cellular dysfunction, understand the folding pathway of polyQ proteins and discover the relative neurotoxicity of polyQ protein species formed along the aggregation pathway. Here, we review what is known about conformations of polyQ peptides and proteins in their monomeric state from experimental and modelling data, how conformational changes of polyQ proteins relate to their oligomerisation and morphology of aggregates and which cellular function are impaired by oligomers, in vitro and in vivo. We also summarise the key modulatory cellular mechanisms and co-factors, which could affect the folding pathway and kinetics of polyQ aggregation. Although many studies have investigated the relationship between polyQ aggregation and toxicity, these have mainly focussed on investigating changes in the formation of the classical hallmark of polyQ diseases, i.e. microscopically visible inclusion bodies. However, recent studies in which oligomeric species have been considered start to shed light on the identity of neurotoxic oligomeric species. Initial evidence suggests that conformational changes induced by polyQ expansions and their surrounding sequence lead to the formation of particular oligomeric intermediates that may differentially affect neurotoxicity.

Polyglutamine gene function and dysfunction in the ageing brain.

The coordinated regulation of gene expression and protein interactions determines how mammalian nervous systems develop and retain function and plasticity over extended periods of time such as a human life span. By studying mutations that occur in a group of genes associated with chronic neurodegeneration, the polyglutamine (polyQ) disorders, it has emerged that CAG/glutamine stretches play important roles in transcriptional regulation and protein-protein interactions. However, it is still unclear what the many structural and functional roles of CAG and other low-complexity sequences in eukaryotic genomes are, despite being the most commonly shared peptide fragments in such proteomes. In this review we examine the function of genes responsible for at least 10 polyglutamine disorders in relation to the nervous system and how expansion mutations lead to neuronal dysfunction, by particularly focusing on Huntington's disease (HD). We argue that the molecular and cellular pathways that turn out to be dysfunctional during such diseases, as a consequence of a CAG expansion, are also involved in the ageing of the central nervous system. These are pathways that control protein degradation systems (including molecular chaperones), axonal transport, redox-homeostasis and bioenergetics. CAG expansion mutations confer novel properties on proteins that lead to a slow-progressing neuronal pathology and cell death similar to that found in other age-related conditions such as Alzheimer's and Parkinson's diseases.

Rapamycin inhibits polyglutamine aggregation independently of autophagy by reducing protein synthesis.

Accumulation of misfolded proteins and protein assemblies is associated with neuronal dysfunction and death in several neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's disease (HD). It is therefore critical to understand the molecular mechanisms of drugs that act on pathways that modulate misfolding and/or aggregation. It is noteworthy that the mammalian target of rapamycin inhibitor rapamycin or its analogs have been proposed as promising therapeutic compounds clearing toxic protein assemblies in these diseases via activation of autophagy. However, using a cellular model of HD, we found that rapamycin significantly decreased aggregation-prone polyglutamine (polyQ) and expanded huntingtin and its inclusion bodies (IB) in both autophagy-proficient and autophagy-deficient cells (by genetic knockout of the atg5 gene in mouse embryonic fibroblasts). This result suggests that rapamycin modulates the levels of misfolded polyQ proteins via pathways other than autophagy. We show that rapamycin reduces the amount of soluble polyQ protein via a modest inhibition of protein synthesis that in turn significantly reduces the formation of insoluble polyQ protein and IB formation. Hence, a modest reduction in huntingtin synthesis by rapamycin may lead to a substantial decrease in the probability of reaching the critical concentration required for a nucleation event and subsequent toxic polyQ aggregation. Thus, in addition to its beneficial effect proposed previously of reducing polyQ aggregation/toxicity via autophagic pathways, rapamycin may alleviate polyQ disease pathology via its effect on global protein synthesis. This finding may have important therapeutic implications.

Rescue from tau-induced neuronal dysfunction produces insoluble tau oligomers.

Aggregation of highly phosphorylated tau is a hallmark of Alzheimer's disease and other tauopathies. Nevertheless, animal models demonstrate that tau-mediated dysfunction/toxicity may not require large tau aggregates but instead may be caused by soluble hyper-phosphorylated tau or by small tau oligomers. Challenging this widely held view, we use multiple techniques to show that insoluble tau oligomers form in conditions where tau-mediated dysfunction is rescued in vivo. This shows that tau oligomers are not necessarily always toxic. Furthermore, their formation correlates with increased tau levels, caused intriguingly, by either pharmacological or genetic inhibition of tau kinase glycogen-synthase-kinase-3beta (GSK-3ß). Moreover, contrary to common belief, these tau oligomers were neither highly phosphorylated, and nor did they contain beta-pleated sheet structure. This may explain their lack of toxicity. Our study makes the novel observation that tau also forms non-toxic insoluble oligomers in vivo in addition to toxic oligomers, which have been reported by others. Whether these are inert or actively protective remains to be established. Nevertheless, this has wide implications for emerging therapeutic strategies such as those that target dissolution of tau oligomers as they may be ineffective or even counterproductive unless they act on the relevant toxic oligomeric tau species.

Disruption of arterial perivascular drainage of amyloid-ß from the brains of mice expressing the human APOE ε4 allele.

Failure of elimination of amyloid-ß (Aß) from the brain and vasculature appears to be a key factor in the etiology of sporadic Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). In addition to age, possession of an apolipoprotein E (APOE) ε4 allele is a strong risk factor for the development of sporadic AD. The present study tested the hypothesis that possession of the APOE ε4 allele is associated with disruption of perivascular drainage of Aß from the brain and with changes in cerebrovascular basement membrane protein levels. Targeted replacement (TR) mice expressing the human APOE3 (TRE3) or APOE4 (TRE4) genes and wildtype mice received intracerebral injections of human Aß(40). Aß(40) aggregated in peri-arterial drainage pathways in TRE4 mice, but not in TRE3 or wildtype mice. The number of Aß deposits was significantly higher in the hippocampi of TRE4 mice than in the TRE3 mice, at both 3- and 16-months of age, suggesting that clearance of Aß was disrupted in the brains of TRE4 mice. Immunocytochemical and Western blot analysis of vascular basement membrane proteins demonstrated significantly raised levels of collagen IV in 3-month-old TRE4 mice compared with TRE3 and wild type mice. In 16-month-old mice, collagen IV and laminin levels were unchanged between wild type and TRE3 mice, but were lower in TRE4 mice. The results of this study suggest that APOE4 may increase the risk for AD through disruption and impedance of perivascular drainage of soluble Aß from the brain. This effect may be mediated, in part, by changes in age-related expression of basement membrane proteins in the cerebral vasculature.

mTOR's role in ageing: protein synthesis or autophagy?

The molecular and cellular mechanisms that regulate ageing are currently under scrutiny because ageing is linked to many human diseases. The nutrient sensing TOR pathway is emerging as a key regulator of ageing. TOR signaling is complex affecting several crucial cellular functions and two such functions, which show clear effects on ageing, are protein synthesis and autophagy. In this article we discuss the relative importance of both these processes in ageing, identify how TOR regulates translation and autophagy and speculate on links between the TOR signaling network and ageing pathways.

Amelioration of protein misfolding disease by rapamycin: translation or autophagy?

Rapamycin is an inhibitor of mTOR, a key component of the mTORC1 complex that controls the growth and survival of cells in response to growth factors, nutrients, energy balance and stresses. The downstream targets of mTORC1 include ribosome biogenesis, transcription, translation and macroautophagy. Recently it was proposed that rapamycin and its derivatives enhance the clearance (and/or reduce the accumulation) of mutant intracellular proteins causing proteinopathies such as tau, alpha-synuclein, ataxin-3, and full-length or fragments of huntingtin containing a polyglutamine (polyQ) expansion, by upregulating macroautophagy. We tested this proposal directly using macroautophagy-deficient fibroblasts. We found that rapamycin inhibits the aggregation of a fragment of huntingtin (exon 1) containing 97 polyQs similarly in macroautophagy-proficient (Atg5(+/+)) and macroautophagy-deficient (Atg5(-/-)) cells. These data demonstrate that autophagy is not the only mechanism by which rapamycin can alleviate the accumulation of misfolded proteins. Our data suggest that rapamycin inhibits mutant huntingtin fragment accumulation due to inhibition of protein synthesis. A model illustrates how a modest reduction in polyQ synthesis can lead to a long-lasting reduction in polyQ aggregation. We propose that several mechanisms exist by which rapamycin reduces the accumulation and potential toxicity of misfolded proteins in diseases caused by protein misfolding and aggregation.

Interactions of TolB with the translocation domain of colicin E9 require an extended TolB box.

The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box.