Synaptic mutant huntingtin inhibits synapsin-1 phosphorylation and causes neurological symptoms

Many genetic mouse models of Huntington’s disease (HD) have established that mutant huntingtin (htt) accumulates in various subcellular regions to affect a variety of cellular functions, but whether and how synaptic mutant htt directly mediates HD neuropathology remains to be determined. We generated transgenic mice that selectively express mutant htt in the presynaptic terminals. Although it was not overexpressed, synaptic mutant htt caused age-dependent neurological symptoms and early death in mice as well as defects in synaptic neurotransmitter release. Mass spectrometry analysis of synaptic fractions and immunoprecipitation of synapsin-1 from HD CAG150 knockin mouse brains revealed that mutant htt binds to synapsin-1, a protein whose phosphorylation is critical for neurotransmitter release. We found that polyglutamine-expanded exon1 htt binds to the C-terminal region of synapsin-1 to reduce synapsin-1 phosphorylation. Our findings point to a critical role for synaptic htt in the neurological symptoms of HD, providing a new therapeutic target.     

Huntingtin forms toxic NH2-terminal fragment complexes that are promoted by the age-dependent decrease in proteasome activity

Although NH2-terminal mutant huntingtin (htt) fragments cause neurological disorders in Huntington's disease (HD), it is unclear how toxic htt fragments are generated and contribute to the disease process. Here, we report that complex NH2-terminal mutant htt fragments smaller than the first 508 amino acids were generated in htt-transfected cells and HD knockin mouse brains. These fragments constituted neuronal nuclear inclusions and appeared before neurological symptoms. The accumulation and aggregation of these htt fragments were associated with an age-dependent decrease in proteasome activity and were promoted by inhibition of proteasome activity. These results suggest that decreased proteasome activity contributes to late onset htt toxicity and that restoring the ability to remove NH2-terminal fragments will provide a more effective therapy for HD than inhibiting their production.     

Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope-specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH(2)- and COOH-terminal htt fragments of 20-100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63-64 kDa htt fragment detected by the NH(2)-domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60-61 kDa band (detected by the NH(2)-domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63-64 kDa and 60-61 kDa NH(2)-domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66-kDa, instead of the 63-64 kDa N-domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH(2)- and COOH-terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH(2)-terminal htt fragments [detected with anti(1-17) serum] compared to controls. Cortex from HD and control brains showed similar NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48-kDa COOH-terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH(2)-terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.     

A pathogenic mechanism in Huntington's disease involves small CAG-repeated RNAs with neurotoxic activity

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches.     

Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases: implications for mutant huntingtin toxicity

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (polyQ) tract in the huntingtin (htt) protein. Mutant htt toxicity is exposed after htt cleavage by caspases and other proteases release NH(2)-terminal fragments containing the polyQ expansion. Here, we show htt interacts and colocalizes with cdk5 in cellular membrane fractions. Cdk5 phosphorylates htt at Ser434, and this phosphorylation reduces caspase-mediated htt cleavage at residue 513. Reduced mutant htt cleavage resulting from cdk5 phosphorylation attenuated aggregate formation and toxicity in cells expressing the NH(2)-terminal 588 amino acids (htt588) of mutant htt. Cdk5 activity is reduced in the brains of HD transgenic mice compared with controls. This result can be accounted for by the polyQ-expanded htt fragments reducing the interaction between cdk5 and its activator p35. These data predict that the ability of cdk5 phosphorylation to protect against htt cleavage, aggregation, and toxicity is compromised in cells expressing toxic fragments of htt.     

Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity

Huntington disease (HD) is characterized by the preferential loss of striatal medium-sized spiny neurons (MSNs) in the brain. Because MSNs receive abundant glutamatergic input, their vulnerability to excitotoxicity may be largely influenced by the capacity of glial cells to remove extracellular glutamate. However, little is known about the role of glia in HD neuropathology. Here, we report that mutant huntingtin accumulates in glial nuclei in HD brains and decreases the expression of glutamate transporters. As a result, mutant huntingtin (htt) reduces glutamate uptake in cultured astrocytes and HD mouse brains. In a neuron-glia coculture system, wild-type glial cells protected neurons against mutant htt-mediated neurotoxicity, whereas glial cells expressing mutant htt increased neuronal vulnerability. Mutant htt in cultured astrocytes decreased their protection of neurons against glutamate excitotoxicity. These findings suggest that decreased glutamate uptake caused by glial mutant htt may critically contribute to neuronal excitotoxicity in HD.     

Inducing huntingtin inclusion formation in primary neuronal cell culture and in vivo by high-capacity adenoviral vectors expressing truncated and full-length huntingtin with polyglutamine expansion

BACKGROUND: Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (htt) gene. Vector-mediated delivery of N-terminal fragments of mutant htt has been used to study htt function in vitro and to establish HD models in rats. Due to the large size of the htt cDNA vector-mediated delivery of full-length htt has not been achieved so far. METHODS: High-capacity adenoviral (HC-Ad) vectors were generated expressing mutant and wild-type versions of N-terminal truncated and full-length htt either in vitro in primary neuronal cells or in the striatum of mice. RESULTS: In vitro these vectors were used for transduction of primary neuronal cells isolated from E17 mouse embryos. Expression of mutant htt resulted in the formation of htt inclusions, a surrogate marker of the HD pathology. Kinetics of generation and localization of htt inclusions differed between truncated and full-length htt carrying identical mutations. Following injection into the striatum vector-mediated expression of mutant truncated htt led to prominent accumulation of htt inclusions in cell nuclei, while inclusions formed upon expression of mutant full-length htt localized to the cytoplasm. CONCLUSIONS: These results indicate that HC-Ad vector-mediated in vitro and in vivo delivery of truncated and full-length mutant htt results in prominent inclusion formation in neuronal cells but in different cell compartments. These vectors will be useful tools for studying HD and may be used to generate large animal HD models.     

Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice

Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.     

Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.     

Dendritic spine loss and neurodegeneration is rescued by Rab11 in models of Huntington's disease

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein (htt) that mediates formation of intracellular protein aggregates. In the brains of HD patients and HD transgenic mice, accumulation of protein aggregates has been causally linked to lesions in axo-dendritic and synaptic compartments. Here we show that dendritic spines - sites of synaptogenesis - are lost in the proximity of htt aggregates because of functional defects in local endosomal recycling mediated by the Rab11 protein. Impaired exit from recycling endosomes (RE) and association of endocytosed protein with intracellular structures containing htt aggregates was demonstrated in cultured hippocampal neurons cells expressing a mutant htt fragment. Dendrites in hippocampal neurons became dystrophic around enlarged amphisome-like structures positive for Rab11, LC3 and mutant htt aggregates. Furthermore, Rab11 overexpression rescues neurodegeneration and dramatically extends lifespan in a Drosophila model of HD. Our findings are consistent with the model that mutant htt aggregation increases local autophagic activity, thereby sequestering Rab11 and diverting spine-forming cargo from RE into enlarged amphisomes. This mechanism may contribute to the toxicity caused by protein misfolding found in a number of neurodegenerative diseases.     

Native mutant huntingtin in human brain: evidence for prevalence of full-length monomer

Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575-850 kDa in control brain and at 650-885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1-17)) and increased when lysates were treated with denaturants (SDS, 8 M urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670-880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43-50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 M urea + DTT. Native full-length mutant htt in embryonic HD(140Q/140Q) mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer.     

Evidence for a role for transglutaminase in Huntington's disease and the potential therapeutic implications.

Transglutaminase (TGase) activity is increased in affected regions of brains from patients with Huntington's disease (HD). TGase activity is particularly elevated in the nucleus compared with the cytoplasm from these brains. Gamma-glutaminyl-lysyl cross-links have been detected in nuclear inclusions in HD brain, indicating that TGase may play a prominent role in the aggregation of huntingtin (htt). Attempts to ameliorate experimental disease, via inhibition of TGase in transgenic models of HD in mice, are under investigation.     

Histone deacetylase activity is retained in primary neurons expressing mutant huntingtin protein

Perturbation of histone acetyl-transferase (HAT) activity is implicated in the pathology of polyglutamine diseases, and suppression of the counteracting histone deacetylase (HDAC) proteins has been proposed as a therapeutic candidate for these intractable disorders. Meanwhile, it is not known whether mutant polyglutamine disease protein affects the HDAC activity in declining neurons, though the answer is essential for application of anti-HDAC drugs for polyglutamine diseases. Here, we show the effect of mutant huntingtin (htt) protein on the expression and activity of HDAC proteins in rat primary cortical neurons as well as in human Huntington's disease (HD) brains. Our findings indicate that expression and activity of HDAC proteins are not repressed by mutant htt protein. Furthermore, expression of normal and mutant htt protein slightly increased HDAC activity although the effects of normal and mutant htt were not remarkably different. In human HD cerebral cortex, HDAC5 immunoreactivity was increased in the nucleus of striatal and cortical neurons, suggesting accelerated nuclear import of this class II HDAC. Meanwhile, western blot and immunohistochemical analyses showed no remarkable change in the expression of class I HDAC proteins such as HDAC1 and HDCA8. Collectively, retained activity in affected neurons supports application of anti-HDAC drugs to the therapy of HD.     

Suppression of neurodegeneration and increased neurotransmission caused by expanded full-length huntingtin accumulating in the cytoplasm

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expansion of a translated CAG repeat in the N terminus of the huntingtin (htt) protein. Here we describe the generation and characterization of a full-length HD Drosophila model to reveal a previously unknown disease mechanism that occurs early in the course of pathogenesis, before expanded htt is imported into the nucleus in detectable amounts. We find that expanded full-length htt (128Qhtt(FL)) leads to behavioral, neurodegenerative, and electrophysiological phenotypes. These phenotypes are caused by a Ca2+-dependent increase in neurotransmitter release efficiency in 128Qhtt(FL) animals. Partial loss of function in synaptic transmission (syntaxin, Snap, Rop) and voltage-gated Ca2+ channel genes suppresses both the electrophysiological and the neurodegenerative phenotypes. Thus, our data indicate that increased neurotransmission is at the root of neuronal degeneration caused by expanded full-length htt during early stages of pathogenesis.     

Mitochondria in Huntington's disease

Huntington's disease (HD) is an inherited progressive neurodegenerative disorder associated with involuntary abnormal movements (chorea), cognitive deficits and psychiatric disturbances. The disease is caused by an abnormal expansion of a CAG repeat located in exon 1 of the gene encoding the huntingtin protein (Htt) that confers a toxic function to the protein. The most striking neuropathological change in HD is the preferential loss of medium spiny GABAergic neurons in the striatum. The mechanisms underlying striatal vulnerability in HD are unknown, but compelling evidence suggests that mitochondrial defects may play a central role. Here we review recent findings supporting this hypothesis. Studies investigating the toxic effects of mutant Htt in cell culture or animal models reveal mitochondrial changes including reduction of Ca²⁺ buffering capacity, loss of membrane potential, and decreased expression of oxidative phosphorylation (OXPHOS) enzymes. Striatal neurons may be particularly vulnerable to these defects. One hypothesis is that neurotransmission systems such as dopamine and glutamate exacerbate mitochondrial defects in the striatum. In particular, mitochondrial dysfunction facilitates impaired Ca²⁺ homeostasis linked to the glutamate receptor-mediated excitotoxicity. Also dopamine receptors modulate mutant Htt toxicity, at least in part through regulation of the expression of mitochondrial complex II. All these observations support the hypothesis that mitochondria, acting as “sensors” of the neurochemical environment, play a central role in striatal degeneration in HD.     

Choosing and using Drosophila models to characterize modifiers of Huntington's disease

HD (Huntington's disease) is a fatal inherited gain-of-function disorder caused by a polyQ (polyglutamine) expansion in the htt (huntingtin protein). Expression of mutant htt in model organisms is sufficient to recapitulate many of the cellular defects found in HD patients. Many groups have independently developed Drosophila models of HD, taking advantage of its rapid life cycle, carefully annotated genome and well-established molecular toolkits. Furthermore, unlike simpler models, Drosophila have a complex nervous system, displaying a range of carefully co-ordinated behaviours which offer an exquisitely sensitive readout of neuronal disruption. Measuring HD-associated changes in behaviour in Drosophila therefore offers a window into the earliest stages of HD, when therapeutic interventions might be particularly effective. The present review describes a number of recently developed Drosophila models of HD and offers practical guidance on the advantages and disadvantages of various experimental approaches that can be used to screen these models for modifiers of mutant htt-mediated toxicity.     

rAAV-mediated shRNA ameliorated neuropathology in Huntington disease model mouse

Huntington disease (HD) is a fatal progressive neurodegenerative disorder associated with expansion of a CAG repeat in the first exon of the gene coding the protein huntingtin (htt). Although the feasibility of RNA interference (RNAi)-mediated reduction of htt expression to attenuate HD-associated symptoms is suggested, the effects of post-symptomatic RNAi treatment in the HD model mice have not yet been certified. Here we show the effects of recombinant adeno-associated virus (rAAV)-mediated delivery of RNAi into the HD model mouse striatum after the onset of disease. Neuropathological abnormalities associated with HD, such as insoluble protein accumulation and down-regulation of DARPP-32 expression, were successfully ameliorated by the RNAi transduction. Importantly, neuronal aggregates in the striatum were reduced after RNAi transduction in the animals comparing to those at the time point of RNAi transduction. These results suggest that the direct inhibition of mutant gene expression by rAVV would be promising for post-symptomatic HD therapy.