Expanded Polyglutamine-containing N-terminal Huntingtin Fragments Are Entirely Degraded by Mammalian Proteasomes

Huntington disease is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) repeat within the protein huntingtin (Htt). N-terminal fragments of the mutant Htt (mHtt) proteins containing the polyQ repeat are aggregation-prone and form intracellular inclusion bodies. Improving the clearance of mHtt fragments by intracellular degradation pathways is relevant to obviate toxic mHtt species and subsequent neurodegeneration. Because the proteasomal degradation pathway has been the subject of controversy regarding the processing of expanded polyQ repeats, we examined whether the proteasome can efficiently degrade Htt-exon1 with an expanded polyQ stretch both in neuronal cells and in vitro. Upon targeting mHtt-exon1 to the proteasome, rapid and complete clearance of mHtt-exon1 was observed. Proteasomal degradation of mHtt-exon1 was devoid of polyQ peptides as partial cleavage products by incomplete proteolysis, indicating that mammalian proteasomes are capable of efficiently degrading expanded polyQ sequences without an inhibitory effect on the proteasomal activity.     

Pathological cell-cell interactions elicited by a neuropathogenic form of mutant Huntingtin contribute to cortical pathogenesis in HD mice

Expanded polyglutamine (polyQ) proteins in Huntington's disease (HD) as well as other polyQ disorders are known to elicit a variety of intracellular toxicities, but it remains unclear whether polyQ proteins can elicit pathological cell-cell interactions which are critical to disease pathogenesis. To test this possibility, we have created conditional HD mice expressing a neuropathogenic form of mutant huntingtin (mhtt-exon1) in discrete neuronal populations. We show that mhtt aggregation is a cell-autonomous process. However, progressive motor deficits and cortical neuropathology are only observed when mhtt expression is in multiple neuronal types, including cortical interneurons, but not when mhtt expression is restricted to cortical pyramidal neurons. We further demonstrate an early deficit in cortical inhibition, suggesting that pathological interactions between interneurons and pyramidal neurons may contribute to the cortical manifestation of HD. Our study provides genetic evidence that pathological cell-cell interactions elicited by neuropathogenic forms of mhtt can critically contribute to cortical pathogenesis in a HD mouse model.     

The Nt17 Domain and its Helical Conformation Regulate the Aggregation,Cellular Properties and Neurotoxicity of Mutant Huntingtin Exon 1

Converging evidence points to the N-terminal domain comprising the first 17 amino acids of the Huntingtin protein (Nt17) as a key regulator of its aggregation, cellular properties and toxicity. In this study, we further investigated the interplay between Nt17 and the polyQ domain repeat length in regulating the aggregation and inclusion formation of exon 1 of the Huntingtin protein (Httex1). In addition, we investigated the effect of removing Nt17 or modulating its local structure on the membrane interactions, neuronal uptake, and toxicity of monomeric or fibrillar Httex1. Our results show that the polyQ and Nt17 domains synergistically modulate the aggregation propensity of Httex1 and that the Nt17 domain plays important roles in shaping the surface properties of mutant Httex1 fibrils and regulating their poly-Q-dependent growth, lateral association and neuronal uptake. Removal of Nt17 or disruption of its transient helical conformations slowed the aggregation of monomeric Httex1 in vitro, reduced inclusion formation in cells, enhanced the neuronal uptake and nuclear accumulation of monomeric Httex1 proteins, and was sufficient to prevent cell death induced by Httex1 72Q overexpression. Finally, we demonstrate that the uptake of Httex1 fibrils into primary neurons and the resulting toxicity are strongly influenced by mutations and phosphorylation events that influence the local helical propensity of Nt17. Altogether, our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation, internalization, and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, and neuronal toxicity.     

Influence of species differences on the neuropathology of transgenic Huntington's disease animal models

Transgenic animal models have revealed much about the pathogenesis of age-dependent neurodegenerative diseases and proved to be a useful tool for uncovering therapeutic targets.Huntington's disease is ...     

Huntingtin phosphorylation sites mapped by mass spectrometry. Modulation of cleavage and toxicity

Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.     

The Role of Chaperone-Mediated Autophagy in Huntingtin Degradation

Huntington Disease (HD) is caused by an abnormal expansion of polyQ tract in the protein named huntingtin (Htt). HD pathology is featured by accumulation and aggregation of mutant Htt in striatal and cortical neurons. Aberrant Htt degradation is implicated in HD pathogenesis. The aim of this study was to investigate the regulatory role of chaperone-mediated autophagy (CMA) components, heat shock protein cognate 70 (Hsc70) and lysosome-associated protein 2A (LAMP-2A) in degradation of Htt fragment 1-552aa (Htt-552). A cell model of HD was produced by overexpression of Htt-552 with adenovirus. The involvement of CMA components in degradation of Htt-552 was determined with over-expression or silencing of Hsc70 and LAMP-2A. The results confirmed previous reports that both macroautophagy and CMA were involved in degradation of Htt-552. Changing the levels of CMA-related proteins affected the accumulation of Htt-552. The lysosomal binding and luminal transport of Htt-552 was demonstrated by incubation of Htt-552 with isolated lysosomes. Expansion of the polyQ tract in Htt-552 impaired its uptake and degradation by lysosomes. Mutation of putative KFERQ motif in wild-type Htt-552 interfered with interactions between Htt-552 and Hsc70. Endogenous Hsc70 and LAMP-2A interacted with exogenously expressed Htt-552. Modulating the levels of CMA related proteins degraded endogenous full-length Htt. These studies suggest that Hsc70 and LAMP-2A through CMA play a role in the clearance of Htt and suggest a novel strategy to target the degradation of mutant Htt.     

Beta conformation of polyglutamine track revealed by a crystal structure of Huntingtin N-terminal region with insertion of three histidine residues

Huntington disease is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine (polyQ) expansion (> 35Q) in the first exon (EX1) of huntingtin protein (Htt). mHtt protein is thought to adopt one or more toxic conformation(s) that are involved in pathogenic interactions in cells . However, the structure of mHtt is not known. Here, we present a near atomic resolution structure of mHtt36Q-EX1. To facilitate crystallization, three histidine residues (3H) were introduced within the Htt36Q stretch resulting in the sequence of Q₇HQHQHQ₂₇. The Htt36Q3H region adopts α-helix, loop, β-hairpin conformations. Furthermore, we observed interactions between the backbone of the Htt36Q3H β-strand with the aromatic residues mimicking putative-toxic interactions with other proteins. Our findings support previous predictions that the expanded mHtt-polyQ region adopts a β-sheet structure. Detailed structural information about mHtt improves our understanding of the pathogenic mechanisms in HD and other polyQ expansion disorders and may form the basis for rational design of small molecules that target toxic conformations of disease-causing proteins.     

Does Huntingtin play a role in selective macroautophagy?

The accumulation of protein aggregates in neurons appears to be a basic feature of neurodegenerative disease. In huntington disease (HD), a progressive and ultimately fatal neurodegenerative disorder caused by an expansion of the polyglutamine repeat within the protein huntingtin (Htt), the immediate proximal cause of disease is well understood. However, the cellular mechanisms which modulate the rate at which fragments of Htt containing polyglutamine accumulate in neurons is a central issue in the development of approaches to modulate the rate and extent of neuronal loss in this disease. We have recently found that Htt is phosphorylated by the kinase IKK on serine (s) 13, activating its phosphorylation on S16 and its acetylation and poly-SUMOylation, modifications that modulate its clearance by the proteasome and lysosome in cells.¹ In the discussion here I suggest that Htt may have a normal function in the lysosomal mechanism of selective macroautophagy involved in its own degradation which may share some similarity with the yeast cytoplasm to vacuole targeting (Cvt) pathway. Pharmacologic activation of this pathway may be useful early in disease progression to treat HD and other neurodegenerative diseases characterized by the accumulation of disease proteins.Key words: Huntington disease, Huntingtin, polyglutamine, autophagy, IKKAn age-related reduction in protein clearance mechanisms has been implicated in the pathogenesis of neurodegenerative diseases including the polyglutamine (polyQ) repeat diseases, Alzheimer disease (AD), Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). These diseases are each associated with the accumulation of insoluble protein aggregates in diseased neurons. Huntington Disease (HD), caused by an expansion of the polyQ repeat in the protein Huntingtin (Htt), is one such disease of aging in which mutant Htt inclusions form in striatal and cortical neurons as disease progresses. Clarification of the mechanisms of Htt clearance is paramount to finding therapeutic targets to treat HD that may be broadly useful in the treatment of these currently incurable neurodegenerative diseases.     

Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila

We tested whether proteins implicated in Huntington's and other polyglutamine (polyQ) expansion diseases can cause axonal transport defects. Reduction of Drosophila huntingtin and expression of proteins containing pathogenic polyQ repeats disrupt axonal transport. Pathogenic polyQ proteins accumulate in axonal and nuclear inclusions, titrate soluble motor proteins, and cause neuronal apoptosis and organismal death. Expression of a cytoplasmic polyQ repeat protein causes adult retinal degeneration, axonal blockages in larval neurons, and larval lethality, but not neuronal apoptosis or nuclear inclusions. A nuclear polyQ repeat protein induces neuronal apoptosis and larval lethality but no axonal blockages. We suggest that pathogenic polyQ proteins cause neuronal dysfunction and organismal death by two non-mutually exclusive mechanisms. One mechanism requires nuclear accumulation and induces apoptosis; the other interferes with axonal transport. Thus, disruption of axonal transport by pathogenic polyQ proteins could contribute to early neuropathology in Huntington's and other polyQ expansion diseases.     

Dominant-negative effects of the N-terminal half of prion protein on neurotoxicity of prion protein-like protein/doppel in mice

Prion protein-like protein/doppel is neurotoxic, causing ataxia and Purkinje cell degeneration in mice, whereas prion protein antagonizes doppel-induced neurodegeneration. Doppel is homologous to the C-terminal half of prion protein but lacks the amino acid sequences corresponding to the N-terminal half of prion protein. We show here that transgenic mice expressing a fusion protein consisting of the N-terminal half, corresponding to residues 1-124, of prion protein and doppel in neurons failed to develop any neurological signs for up to 730 days in a background devoid of prion protein. In addition, the fusion protein prolonged the onset of ataxia in mice expressing exogenous doppel. These results suggested that the N-terminal part of prion protein has a neuroprotective potential acting both cis and trans on doppel. We also show that prion protein lacking the pre-octapeptide repeat (Delta25-50) or octapeptide repeat (Delta51-90) region alone could not impair the antagonistic function against doppel.     

Huntingtin associated protein 1 and its functions

Huntington's disease (HD) is caused by a polyglutamine expansion in the protein huntingtin (Htt). Several studies suggest that Htt and huntingtin associated protein 1 (HAP1) participate in intracellular trafficking and that polyglutamine expansion affects vesicular transport. Understanding the function of HAP1 and its related proteins could help elucidate the pathogenesis of HD. The present review focuses on HAP1, which has proved to be involved in intracellular trafficking. Unlike huntingtin, which is expressed ubiquitously throughout the brain and body, HAP1 is enriched in neurons, suggesting that its dysfunction could contribute to the selective neuropathology in HD. We discuss recent evidence for the involvement of HAP1 and its binding proteins in potential functions.     

Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin

Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington’s disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on “gutless” adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.     

Huntington's Disease

Huntington''s disease (HD) is the most common inherited neurodegenerative disease and is characterized by uncontrolled excessive motor movements and cognitive and emotional deficits. The mutation responsible for HD leads to an abnormally long polyglutamine (polyQ) expansion in the huntingtin (Htt) protein, which confers one or more toxic functions to mutant Htt leading to neurodegeneration. The polyQ expansion makes Htt prone to aggregate and accumulate, and manipulations that mitigate protein misfolding or facilitate the clearance of misfolded proteins tend to slow disease progression in HD models. This article will focus on HD and the evidence that it is a conformational disease.     

Modeling Huntington disease in Drosophila

Huntington disease (HD) is an inherited neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in the huntingtin (Htt) gene. Despite years of research, there is no treatment that extends life for patients with the disorder. Similarly, little is known about which cellular pathways that are altered by pathogenic Huntingtin (Htt) protein expression are correlated with neuronal loss. As part of a longstanding effort to gain insights into HD pathology, we have been studying the protein in the context of the fruitfly Drosophila melanogaster. We generated transgenic HD models in Drosophila by engineering flies that carry a 12-exon fragment of the human Htt gene with or without the toxic trinucleotide repeat expansion. We also created variants with a monomeric red fluorescent protein (mRFP) tag fused to Htt that allows in vivo imaging of Htt protein localization and aggregation. While wild-type Htt remains diffuse throughout the cytoplasm of cells, pathogenic Htt forms insoluble aggregates that accumulate in neuronal soma and axons. Aggregates can physically block transport of numerous organelles along the axon. We have also observed that aggregates are formed quickly, within just a few hours of mutant Htt expression. To explore mechanisms of neurodegeneration in our HD model, we performed in vivo and in vitro screens to search for modifiers of viability and pathogenic Htt aggregation. Our results identified several novel candidates for HD therapeutics that can now be tested in mammalian models of HD. Furthermore, these experiments have highlighted the complex relationship between aggregates and toxicity that exists in HD.     

Deranged neuronal calcium signaling and Huntington disease

Huntington disease (HD) is an autosomal-dominant neurodegenerative disorder that primarily affects medium spiny striatal neurons (MSN). HD is caused by polyglutamine (polyQ) expansion (exp) in the amino-terminal region of a protein huntingtin (Htt). The connection between polyQ expansion in Httexp and MSN neurodegeneration remains elusive. Here we discuss recent data that link polyQ expansion in Httexp and deranged Ca2+ signaling in MSN neurons. Experimental evidence indicates that (1) Ca2+ homeostasis is abnormal in mitochondria isolated from lymphoblasts of HD patients and from brains of the YAC72 HD mouse model; (2) Httexp leads to potentiation of NR1/NR2B NMDA receptor activity in heterologous expression systems and in MSN from YAC72 HD mouse model; and (3) Httexp binds to the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) carboxy-terminus and causes sensitization of InsP3R1 to activation by InsP3 in planar lipid bilayers and in MSN. Based on these results we propose that Httexp-induced cytosolic and mitochondrial Ca2+ overload of MSN plays an important role in the pathogenesis of HD and that Ca2+ signaling blockers may play a beneficial role in treatment of HD.     

Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

Huntington disease (HD) is caused by expansion of a polyglutamine (polyQ) domain in the protein known as huntingtin (htt), and the disease is characterized by selective neurodegeneration. Expansion of the polyQ domain is not exclusive to HD, but occurs in eight other inherited neurodegenerative disorders that show distinct neuropathology. Yet in spite of the clear genetic defects and associated neurodegeneration seen with all the polyQ diseases, their pathogenesis remains elusive. The present review focuses on HD, outlining the effects of mutant htt in the nucleus and neuronal processes as well as the role of cell-cell interactions in HD pathology. The widespread expression and localization of mutant htt and its interactions with a variety of proteins suggest that mutant htt engages multiple pathogenic pathways. Understanding these pathways will help us to elucidate the pathogenesis of HD and to target therapies effectively.     

Reduction in Subventricular Zone-Derived Olfactory Bulb Neurogenesis in a Rat Model of Huntington’s Disease Is Accompanied by Striatal Invasion of Neuroblasts

Huntington’s disease (HD) is an inherited progressive neurodegenerative disorder caused by an expanded CAG repeat in exon 1 of the huntingtin gene (HTT). The primary neuropathology of HD has been attributed to the preferential degeneration of medium spiny neurons (MSN) in the striatum. Reports on striatal neurogenesis have been a subject of debate; nevertheless, it should be considered as an endogenous attempt to repair the brain. The subventricular zone (SVZ) might offer a close-by region to supply the degenerated striatum with new cells. Previously, we have demonstrated that R6/2 mice, a widely used preclinical model representing an early onset HD, showed reduced olfactory bulb (OB) neurogenesis but induced striatal migration of neuroblasts without affecting the proliferation of neural progenitor cell (NPCs) in the SVZ. The present study revisits these findings, using a clinically more relevant transgenic rat model of late onset HD (tgHD rats) carrying the human HTT gene with 51 CAG repeats and mimicking many of the neuropathological features of HD seen in patients. We demonstrate that cell proliferation is reduced in the SVZ and OB of tgHD rats compared to WT rats. In the OB of tgHD rats, although cell survival was reduced, the frequency of neuronal differentiation was not altered in the granule cell layer (GCL) compared to the WT rats. However, an increased frequency of dopamenergic neuronal differentiation was noticed in the glomerular layer (GLOM) of tgHD rats. Besides this, we observed a selective proliferation of neuroblasts in the adjacent striatum of tgHD rats. There was no evidence for neuronal maturation and survival of these striatal neuroblasts. Therefore, the functional role of these invading neuroblasts still needs to be determined, but they might offer an endogenous alternative for stem or neuronal cell transplantation strategies.