Disrupted GABAAR trafficking and synaptic inhibition in a mouse model of Huntington's disease

Growing evidence suggests that Huntington's disease (HD), a neurodegenerative movement disorder caused by the mutant huntingtin (htt) with an expanded polyglutamine (polyQ) repeat, is associated with the altered intracellular trafficking and synaptic function. GABA(A) receptors, the key determinant of the strength of synaptic inhibition, have been found to bind to the huntingtin associated protein 1 (HAP1). HAP1 serves as an adaptor linking GABA(A) receptors to the kinesin family motor protein 5 (KIF5), controlling the transport of GABA(A) receptors along microtubules in dendrites. In this study, we found that GABA(A)R-mediated synaptic transmission is significantly impaired in a transgenic mouse model of HD expressing polyQ-htt, which is accompanied by the diminished surface expression of GABA(A) receptors. Moreover, the GABA(A)R/HAP1/KIF5 complex is disrupted and dissociated from microtubules in the HD mouse model. These results suggest that GABA(A)R trafficking and function is impaired in HD, presumably due to the interference of KIF5-mediated microtubule-based transport of GABA(A) receptors. The diminished inhibitory synaptic efficacy could contribute to the loss of the excitatory/inhibitory balance, leading to increased neuronal excitotoxicity in HD.     

Huntingtin is associated with cytomatrix proteins at the presynaptic terminal

Huntington's disease (HD) is a single gene disorder produced by expansion of the gene encoding huntingtin (htt), a large protein with features of a multi-functional scaffold. Expansion of htt's polyglutamine domain induces novel, toxic interactions and likely also disrupts normal htt function. Because of its predicted role as a scaffold, pursuit of huntingtin function and HD pathogenesis has focused on identifying htt-interacting proteins. Here we present a focused screen designed to identify htt-interacting proteins in the presynaptic terminal. To identify interactions that occur in situ, synaptosomes (isolated nerve terminals) from cerebral cortices, striata and hippocampi were subjected to chemical crosslinking followed by denaturation, immunoprecipitation using an anti-htt antibody, and nano-flow liquid chromatography tandem mass spectrometry (nanoLC–MS/MS) analyses. The presynaptic cytomatrix proteins Bassoon, Piccolo/Aczonin and Ahnak were among the most consistently identified binding partners. Co-immunoprecipitation and co-fractionation studies support the conclusion that huntingtin is a component of the presynaptic cytomatrix, a complicated network of proteins that regulates the positioning and priming of synaptic vesicles. These findings implicate htt in presynaptic functioning, and suggest that aberrant organization of presynaptic components may contribute to the neurological pathology associated with HD.     

Huntingtin interacts with the receptor sorting family protein GASP2

Summary. Protein interaction networks are useful resources for the functional annotation of proteins. Recently, we have generated a highly connected protein–protein interaction network for Huntington’s disease (HD) by automated yeast two-hybrid (Y2H) screening (Goehler et al., 2004). The network included several novel direct interaction partners for the disease protein huntingtin (htt). Some of these interactions, however, have not been validated by independent methods. Here we describe the verification of the interaction between htt and GASP2 (G protein-coupled receptor associated sorting protein 2), a protein involved in membrane receptor degradation. Using membrane-based and classical coimmunoprecipitation assays we demonstrate that htt and GASP2 form a complex in cotransfected mammalian cells. Moreover, we show that the two proteins colocalize in SH-SY5Y cells, raising the possibility that htt and GASP2 interact in neurons. As the GASP protein family plays a role in G protein-coupled receptor sorting, our data suggest that htt might influence receptor trafficking via the interaction with GASP2.     

Recent insights into the molecular pathogenesis of Huntington disease

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the HD gene resulting in expression of an uninterrupted polyglutamine stretch within the N-terminus of its protein product huntingtin (htt). In this article we review the clinical, genetic, and neuropathological features of HD and discuss recent insights into the pathogenesis of HD. Examining the role of CAG repeat size on age of onset and penetrance in HD using a refined database of human HD patients has provided further support for the importance of the CAG repeat in the pathogenesis of HD and information leading to a predictive model for the likelihood of being affected by a specific age for a particular CAG expansion. In a YAC transgenic mouse model that replicates key elements of the HD phenotype, the development of selective striatal neurodegeneration is coincident with cleavage of htt and translocation of the N-terminal htt fragment into the nucleus. We also review in vitro evidence that htt is a substrate for cleavage by a group of cysteine proteases involved in apoptotic death-the caspases, and that caspase cleavage of htt results in the generation of a toxic N-terminal fragment. Inhibiting caspase cleavage of huntingtin eliminates the toxicity of the mutant htt protein. These results suggest that cleavage of huntingtin resulting in production of a truncated N-terminal fragment may be a crucial step in the pathogenesis of Huntington disease and that inhibition of this process may be a potential therapeutic strategy for this currently untreatable disorder.     

Characterization of huntingtin pathologic fragments in human Huntington disease, transgenic mice, and cell models

Huntington disease (HD) is caused by the expansion of a glutamine (Q) repeat near the N terminus of huntingtin (htt), resulting in altered conformation of the mutant protein to produce, most prominently in brain neurons, nuclear and cytoplasmic inclusion pathology. The inclusions and associated diffuse accumulation of mutant htt in nuclei are composed of N-terminal fragments of mutant protein. Here, we used a panel of peptide antibodies to characterize the htt protein pathologies in brain tissues from human HD, and a transgenic mouse model created by expressing the first 171 amino acids of human htt with 82Q (htt-N171-82Q). In tissues from both sources, htt pathologic features in nuclei were detected by antibodies to htt peptides 1-17 and 81-90 but not 115-129 (wild-type huntingtin numbering with 23 repeats). Human HEK 293 cells transfected with expression vectors that encode either the N-terminal 233 amino acids of human htt (htt-N233-82Q) or htt-N171-18Q accumulated smaller N-terminal fragments with antibody-binding characteristics identical to those of pathologic peptides. We conclude that the mutant htt peptides that accumulate in pathologic structures of human HD and httN171-82Q in mice are produced by similar, yet to be defined, proteolytic events in a region of the protein near or within amino acids 90-115.     

Bone marrow transplantation confers modest benefits in mouse models of Huntington's disease

Huntington's disease (HD) is caused by an expanded polyglutamine tract in the protein huntingtin (htt). Although HD has historically been viewed as a brain-specific disease, htt is expressed ubiquitously, and recent studies indicate that mutant htt might cause changes to the immune system that could contribute to pathogenesis. Monocytes from HD patients and mouse models are hyperactive in response to stimulation, and increased levels of inflammatory cytokines and chemokines are found in pre-manifest patients that correlate with pathogenesis. In this study, wild-type (WT) bone marrow cells were transplanted into two lethally irradiated transgenic mouse models of HD that ubiquitously express full-length htt (YAC128 and BACHD mice). Bone marrow transplantation partially attenuated hypokinetic and motor deficits in HD mice. Increased levels of synapses in the cortex were found in HD mice that received bone marrow transplants. Importantly, serum levels of interleukin-6, interleukin-10, CXC chemokine ligand 1, and interferon-γ were significantly higher in HD than WT mice but were normalized in mice that received a bone marrow transplant. These results suggest that immune cell dysfunction might be an important modifier of pathogenesis in HD.     

Implication of the JNK pathway in a rat model of Huntington's disease

Huntington's disease (HD) is a neurodegenerative disorder resulting from the expansion of a glutamine repeat (polyQ) in the N-terminus of the huntingtin (htt) protein. Expression of polyQ-containing proteins has been previously shown to induce various cellular stress responses. Among these, activation of the c-Jun N-terminal kinase (JNK) cascade has been observed in cellular models of HD. However, the implication of the JNK pathway has not previously been evaluated in the striatum of HD animal models. Here we report that the JNK pathway participates in HD pathology in a rat model of the disease. Increased phosphorylation of the JNK target c-Jun was observed as early as 4 weeks and persisted for 13 weeks after lentiviral-mediated expression of htt171-82Q. In order to assess the importance of this pathway in HD pathology, JNK inhibitors including dominant-negative mutants of upstream kinases (ASK1^K709R, MEKK1^D1369A), a c-Jun mutant (Δ169c-Jun) and the active domain of the scaffold protein JIP-1/IBI (IBI-JBD) were tested for their ability to mitigate the effect of htt171-82Q. The overexpression of MEKK1^D1369A and JIP-1/IBI reduced the polyQ-related loss of DARPP-32 expression, while the other inhibitors had no effect. In all cases, the formation of EM48-positive htt inclusions and P-c-Jun immunoreactivity were unaltered. These results suggest that JNK activation is involved in HD and that blockade of this pathway may be of benefit in counteracting HD-related neurotoxicity.     

A human single-chain Fv intrabody preferentially targets amino-terminal Huntingtin's fragments in striatal models of Huntington's disease

Amino-terminal fragments of huntingtin (htt) appear to result from proteolytic processing of the full-length protein in Huntington's disease (HD), and fragments containing pathological expansions of polyglutamine elicit toxicity in model systems. Such fragments are sequestered into insoluble aggregates, which may initially serve a cellular protective mechanism, while soluble fragments and/or oligomers may be a more acute toxic species. Agents which enhance mutant htt clearance have shown therapeutic potential in animal models of HD. Here, we present the first evidence of an htt-specific single-chain Fv intrabody (C4) that selectively targets the soluble fraction of amino-terminal htt fragments. Our findings suggest that the C4 intrabody binds weakly, but does not alter the levels of endogenous, full-length htt. C4 appears to decrease the steady-state levels of amino-terminal htt fragments by binding to non-aggregated, but not aggregated, htt species. Intrabodies may be used as potential curative agents, and as drug discovery tools, for HD and other misfolded protein disorders.     

Nuclear localization of N-terminal mutant huntingtin is cell cycle dependent

Unlike normal huntingtin (htt) which is located predominantly in the cytoplasm, mutant htt is also found in the nucleus of affected neurons. Nuclear localization of toxic polyglutamine-containing proteins has been postulated to be necessary for the pathogenesis of triplet repeat disorders. However, little is known about the mechanism by which mutant htt enters the nucleus. We have recently reported exclusive nuclear localization of exon 1 mutant htt in striatal primary neuronal cultures from the HD94 conditional mouse model of HD. This seemed to contradict the predominant cytoplasmic localization of N-terminal htt reported from transfection experiments and prompted us to hypothesize that subcellular localization of the toxic htt fragment might be favoured in nondividing cells. To test this, we analyzed subcellular localization of mutant htt in HD94 mixed neuron-glia cultures. Subconfluent glial cells showed cytoplasmic localization. However, nuclear localization was prompted by confluence, by serum withdrawal, and by treatment with cell cycle progression inhibitors such as Ara C or lactacystin. BrdU labelling experiments further confirmed that nuclear localization does not occur in dividing cells. Our findings offer an explanation for the neuronal specific toxicity of mutant htt despite its ubiquitous expression. Unraveling the mechanism of this cell cycle arrest-dependent entrance into the nucleus may offer new opportunities for therapeutic intervention.     

Progressive and selective striatal degeneration in primary neuronal cultures using lentiviral vector coding for a mutant huntingtin fragment

A lentiviral vector expressing a mutant huntingtin protein (htt171-82Q) was used to generate a chronic model of Huntington's disease (HD) in rat primary striatal cultures. In this model, the majority of neurons expressed the transgene so that Western blot analysis and flow cytometry measurement could complement immunohistological evaluation. Mutant huntingtin produced a slowly progressing pathology characterized after 1 month by the appearance of neuritic aggregates followed by intranuclear inclusions, morphological anomalies of neurites, loss of neurofilament 160, increased expression in stress response protein Hsp70, and later loss of neuronal markers such as NeuN and MAP-2. At 2 months post-infection, a significant increase in TUNEL-positive cells confirmed actual striatal cell loss. Interestingly, cortical cultures infected with the same vector showed no sign of neuronal dysfunction despite accumulation of numerous inclusions. We finally examined whether the trophic factors CNTF and BDNF that were found neuroprotective in acute HD models could prevent striatal degeneration in a chronic model. Results demonstrated that both agents were neuroprotective without modifying inclusion formation. The present study demonstrates that viral vectors coding for mutant htt provides an advantageous system for histological and biochemical analysis of HD pathogenesis in primary striatal cultures.     

A stable G‐quartet binds to a huntingtin protein fragment containing expanded polyglutamine tracks

Huntington's disease (HD) is a progressive neurodegenerative disorder that is inherited in an autosomal dominant fashion. The disease is the result of an expanded CAG repeat in exon 1 of the HD gene, which encodes an elongated polyglutamine tract in the mutant form of the protein, huntingtin. Disease pathogenesis is linked to intracellular aggregates that form because of the tendency of the mutant protein to misfold. The role of huntingtin aggregates in disease pathology is unclear; it has been proposed that the aggregates themselves are toxic because of their ability to sequester intracellular proteins and disrupt normal cellular function. In addition, the mechanistic steps that lead to aggregate formation appear to be central to HD pathology. We have previously reported that guanosine‐rich oligonucleotides with the ability to fold into a G‐quartet are effective inhibitors of the aggregation process of a huntingtin protein fragment with an elongated polyglutamine tract, Htn 1‐171(Q58). The most active molecule is composed of 20 guanosine residues, which adopt a G‐wire conformation. Here we establish that G20 inhibits protein aggregation as judged by native gel electrophoresis, an agarose gel electrophoresis for resolving aggregates (AGERA) assay, and an immunoblotting assay. We also visualize the G20‐Htn1‐171(Q58) protein complex by using a streptavidin‐biotin pull‐down assay as well as atomic force microscopy (AFM). The G20 molecule also interacts with Htn1‐171(Q23), a fusion protein that contains 23 glutamine residues instead of 58 (Q58), but in a more degenerate and nonspecific fashion. Taken together, our data support the notion that G20 exhibits some selectivity in binding to specific protein species that assemble along the aggregation pathway. © 2009 Wiley‐Liss, Inc.     

Selective degeneration in YAC mouse models of Huntington disease

Huntington disease (HD) is one of at least nine polyglutamine disorders caused by a CAG expansion in the coding region of a disease-causing gene. These disorders are characterized by selective degeneration of different regions of the brain, which is not explained by the expression pattern of the mutant protein. In HD, degeneration primarily occurs in the striatum and cortex. To examine the mechanisms responsible for the selective neuronal loss in HD, we have generated yeast artificial chromosome (YAC) transgenic models of HD that express full length mutant huntingtin (htt) from a YAC. These mice have appropriate tissue-specific and temporal expression of mutant htt and accordingly recapitulate the motor deficits, cognitive impairment and selective degeneration of HD. As in human patients, mutant htt expression is not increased in the affected regions of the brain. In contrast, detection of mutant htt in the nucleus is earliest and greatest in the striatum, the region most affected in HD, suggesting that selective nuclear localization of mutant htt may contribute to the region specific atrophy in these mice. Selective phosphorylation of mutant htt on serine 421 may also contribute, as phosphorylation of mutant htt reduces its toxicity and is decreased in the striatum compared to other regions of the brain. Finally, the fact that mutant htt expression increases the susceptibility of striatal neurons to excitotoxicity but not neurons from the cerebellum, suggests that altered sensitization to excitotoxic death may also contribute to selective degeneration in YAC mice. Overall, YAC mice recapitulate the region specific damage that occurs in HD and provide a suitable model for examining the mechanisms underlying of selective degeneration.     

Omi / HtrA2 is relevant to the selective vulnerability of striatal neurons in Huntington's disease

Selective vulnerability of neurons is a critical feature of neurodegenerative diseases, but the underlying molecular mechanisms remain largely unknown. We here report that Omi/HtrA2, a mitochondrial protein regulating survival and apoptosis of cells, decreases selectively in striatal neurons that are most vulnerable to the Huntington's disease (HD) pathology. In microarray analysis, Omi/HtrA2 was decreased under the expression of mutant huntingtin (htt) in striatal neurons but not in cortical or cerebellar neurons. Mutant ataxin-1 (Atx-1) did not affect Omi/HtrA2 in any type of neuron. Western blot analysis of primary neurons expressing mutant htt also confirmed the selective reduction of the Omi/HtrA2 protein. Immunohistochemistry with a mutant htt-transgenic mouse line and human HD brains confirmed reduction of Omi/HtrA2 in striatal neurons. Overexpression of Omi/HtrA2 by adenovirus vector reverted mutant htt-induced cell death in primary neurons. These results collectively suggest that the homeostatic but not proapoptotic function of Omi/HtrA2 is linked to selective vulnerability of striatal neurons in HD pathology.     

Huntingtin Protein Colocalizes with Lesions of Neurodegenerative Diseases: An Investigation in Huntington''s, Alzheimer''s, and Pick''s Diseases

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease associated with a CAG trinucleotide repeat expansion in a large gene on chromosome 4. The gene encodes the protein huntingtin with a polyglutamine tract encoded by the CAG repeat at the N-terminus. The number of CAG repeats in HD are significantly increased (36 to 120+) compared with the normal population (8–39). The pathological mechanism associated with the expanded CAG repeat in HD is not clear but there is evidence that polyglutamine is directly neurotoxic. We have immunolocalized huntingtin with an in-house, well-characterised, polyclonal antibody in HD, Alzheimer's disease (AD), and Picks disease (PiD) brains. Control brain tissue sections were from head injured and cerebral ischaemia cases. In HD, huntingtin was immunopositive in the surviving but damaged neurons and reactive astrocytes of the caudate and putamen. However, in AD and PiD the immunostaining was largely restricted to the characteristic intracellular inclusion bodies associated with the disease process in each case. In AD, huntingtin was localized only in the intracellular neurofibrillary tangles and dystrophic neurites within the neuritic amyloid plaques but not with the amyloid. In PiD, strongly positive huntingtin immunostaining was present within cytoplasmic Pick bodies. Our findings suggest huntingtin selectively accumulates in association with abnormal intracytoplasmic and cytoskeletal filaments of neurons and glia in neurodegenerative diseases such as HD, AD, and PiD. Cells in the CNS appear sensitive to damage by the aggregated, toxic levels of huntingtin and evidence of its interaction with neurofilaments could provide information about its potential role in the aetiology of HD.