Striatum Of Huntington's Disease Mice Research Articles

Altered activity-regulated H3K9 acetylation at TGF-beta signaling genes during egocentric memory in Huntington's disease.

Molecular mechanisms underlying cognitive deficits in Huntington's disease (HD), a striatal neurodegenerative disorder, are unknown. Here, we generated ChIPseq, 4Cseq and RNAseq data on striatal tissue of HD and control mice during striatum-dependent egocentric memory process. Multi-omics analyses showed altered activity-dependent epigenetic gene reprogramming of neuronal and glial genes regulating striatal plasticity in HD mice, which correlated with memory deficit. First, our data reveal that spatial chromatin re-organization and transcriptional induction of BDNF-related markers, regulating neuronal plasticity, were reduced since memory acquisition in the striatum of HD mice. Second, our data show that epigenetic memory implicating H3K9 acetylation, which established during late phase of memory process (e.g. during consolidation/recall) and contributed to glia-mediated, TGFβ-dependent plasticity, was compromised in HD mouse striatum. Specifically, memory-dependent regulation of H3K9 acetylation was impaired at genes controlling extracellular matrix and myelination. Our study investigating the interplay between epigenetics and memory identifies H3K9 acetylation and TGFβ signaling as new targets of striatal plasticity, which might offer innovative leads to improve HD.

Cerebrospinal fluid mutant huntingtin is a biomarker for huntingtin lowering in the striatum of Huntington disease mice

Huntington disease (HD) is a neurodegenerative disease caused by a trinucleotide repeat expansion in the HTT gene encoding an elongated polyglutamine tract in the huntingtin (HTT) protein. Expanded mutant HTT (mHTT) is toxic and leads to regional atrophy and neuronal cell loss in the brain, which occurs earliest in the striatum. Therapeutic lowering of mHTT in the central nervous system (CNS) has shown promise in preclinical studies, with multiple approaches currently in clinical development for HD. Quantitation of mHTT in the cerebrospinal fluid (CSF) is being used as a clinical pharmacodynamic biomarker of target engagement in the CNS. We have previously shown that the CNS is a major source of mHTT in the CSF. However, little is known about the specific brain regions and cell types that contribute to CSF mHTT. Therefore, a better understanding of the origins of CSF mHTT and whether therapies targeting mHTT in the striatum would be expected to be associated with significant lowering of mHTT in the CSF is needed. Here, we use complementary pharmacological and genetic-based approaches to either restrict expression of mHTT to the striatum or selectively deplete mHTT in the striatum to evaluate the contribution of this brain region to mHTT in the CSF. We show that viral expression of a mHTT fragment restricted to the striatum leads to detectable mHTT in the CSF. We demonstrate that targeted lowering of mHTT selectively in the striatum using an antisense oligonucleotide leads to a significant reduction of mHTT in the CSF of HD mice. Furthermore, using a transgenic mouse model of HD that expresses full length human mHTT and wild type HTT, we show that genetic inactivation of mHTT selectively in the striatum results in a significant reduction of mHTT in the CSF. Taken together, our data supports the conclusion that the striatum contributes sufficiently to the pool of mHTT in the CSF that therapeutic levels of mHTT lowering in the striatum can be detected by this measure in HD mice. This suggests that CSF mHTT may represent a pharmacodynamic biomarker for therapies that lower mHTT in the striatum.

Deletion of SUMO1 attenuates behavioral and anatomical deficits by regulating autophagic activities in Huntington disease

The CAG expansion of huntingtin (mHTT) associated with Huntington disease (HD) is a ubiquitously expressed gene, yet it prominently damages the striatum and cortex, followed by widespread peripheral defects as the disease progresses. However, the underlying mechanisms of neuronal vulnerability are unclear. Previous studies have shown that SUMO1 (small ubiquitin-like modifier-1) modification of mHtt promotes cellular toxicity, but the invivo role and functions of SUMO1 in HD pathogenesis are unclear. Here, we report that SUMO1 deletion in Q175DN HD-het knockin mice (HD mice) prevented age-dependent HD-like motor and neurological impairments and suppressed the striatal atrophy and inflammatory response. SUMO1 deletion caused a drastic reduction in soluble mHtt levels and nuclear and extracellular mHtt inclusions while increasing cytoplasmic mHtt inclusions in the striatum of HD mice. SUMO1 deletion promoted autophagic activity, characterized by augmented interactions between mHtt inclusions and a lysosomal marker (LAMP1), increased LC3B- and LAMP1 interaction, and decreased interaction of sequestosome-1 (p62) and LAMP1 in DARPP-32-positive medium spiny neurons in HD mice. Depletion of SUMO1 in an HD cell model also diminished the mHtt levels and enhanced autophagy flux. In addition, the SUMOylation inhibitor ginkgolic acid strongly enhanced autophagy and diminished mHTT levels in human HD fibroblasts. These results indicate that SUMO is a critical therapeutic target in HD and that blocking SUMO may ameliorate HD pathogenesis by regulating autophagy activities.

M2 cortex-dorsolateral striatum stimulation reverses motor symptoms and synaptic deficits in Huntington's disease.

Huntington's disease (HD) is a neurological disorder characterized by motor disturbances. HD pathology is most prominent in the striatum, the central hub of the basal ganglia. The cerebral cortex is the main striatal afferent, and progressive cortico-striatal disconnection characterizes HD. We mapped striatal network dysfunction in HD mice to ultimately modulate the activity of a specific cortico-striatal circuit to ameliorate motor symptoms and recover synaptic plasticity. Multimodal MRI in vivo indicates cortico-striatal and thalamo-striatal functional network deficits and reduced glutamate/glutamine ratio in the striatum of HD mice. Moreover, optogenetically-induced glutamate release from M2 cortex terminals in the dorsolateral striatum (DLS) was undetectable in HD mice and striatal neurons show blunted electrophysiological responses. Remarkably, repeated M2-DLS optogenetic stimulation normalized motor behavior in HD mice and evoked a sustained increase of synaptic plasticity. Overall, these results reveal that selective stimulation of the M2-DLS pathway can become an effective therapeutic strategy in HD.

Striatal infusion of cholesterol promotes dose-dependent behavioral benefits and exerts disease-modifying effects in Huntington's disease mice.

A variety of pathophysiological mechanisms are implicated in Huntington's disease (HD). Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from pre‐symptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. In addition, systemic injection of cholesterol‐loaded brain‐permeable nanoparticles ameliorates synaptic and cognitive function in a transgenic mouse model of HD. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of exogenous cholesterol in the HD brain, we employed osmotic mini‐pumps to infuse three escalating doses of cholesterol directly into the striatum of HD mice in a continuous and rate‐controlled manner. All tested doses prevented cognitive decline, while amelioration of disease‐related motor defects was dose‐dependent. In parallel, we found morphological and functional recovery of synaptic transmission involving both excitatory and inhibitory synapses of striatal medium spiny neurons. The treatment also enhanced endogenous cholesterol biosynthesis and clearance of mutant Huntingtin aggregates. These results indicate that cholesterol infusion to the striatum can exert a dose‐dependent, disease‐modifying effect and may be therapeutically relevant in HD.

Homeostatic regulation of STING protein at the resting state by stabilizer TOLLIP.

STING is an important innate immune protein, but its homeostatic regulation at the resting-state is unknown. Here, we identified TOLLIP as a stabilizer of STING through direct interaction to prevent its degradation. Tollip-deficiency results in reduced resting-state STING protein level in nonhematopoietic cells and tissues and renders STING protein unstable in immune cells, leading to severely dampened STING signaling capacity. The competing degradation mechanism of resting-state STING requires IRE1α and lysosomes. TOLLIP mediates clearance of Huntington’s disease (HD)-linked polyQ protein aggregates. Ectopic expression of polyQ protein in vitro or naturally occurring polyQ proteins in HD mouse striatum sequester TOLLIP away from STING, leading to reduced STING protein and dampened immune signaling. Tollip−/− also ameliorates STING-mediated autoimmune disease in Trex1−/− mice. Together, our findings reveal that resting-state STING protein level is strictly regulated by a constant tug-of-war between ‘stabilizer’ TOLLIP and ‘degrader’ IRE1α-lysosome that together maintains tissue immune homeostasis.

Mitochondrial fission in Huntington's disease mouse striatum disrupts ER-mitochondria contacts leading to disturbances in Ca2+ efflux and Reactive Oxygen Species (ROS) homeostasis

Mitochondria-associated membranes (MAMs) are dynamic structures that communicate endoplasmic reticulum (ER) and mitochondria allowing calcium transfer between these two organelles. Since calcium dysregulation is an important hallmark of several neurodegenerative diseases, disruption of MAMs has been speculated to contribute to pathological features associated with these neurodegenerative processes. In Huntington's disease (HD), mutant huntingtin induces the selective loss of medium spiny neurons within the striatum. The cause of this specific susceptibility remain unclear. However, defects on mitochondrial dynamics and bioenergetics have been proposed as critical contributors, causing accumulation of fragmented mitochondria and subsequent Ca2+ homeostasis alterations. In the present work, we show that aberrant Drp1-mediated mitochondrial fragmentation within the striatum of HD mutant mice, forces mitochondria to place far away from the ER disrupting the ER-mitochondria association and therefore causing drawbacks in Ca2+ efflux and an excessive production of mitochondria superoxide species. Accordingly, inhibition of Drp1 activity by Mdivi-1 treatment restored ER-mitochondria contacts, mitochondria dysfunction and Ca2+ homeostasis.In sum, our results give new insight on how defects on mitochondria dynamics may contribute to striatal vulnerability in HD and highlights MAMs dysfunction as an important factor involved in HD striatal pathology.

Small molecule modulator of protein disulfide isomerase attenuates mutant huntingtin toxicity and inhibits endoplasmic reticulum stress in a mouse model of Huntington's disease.

Huntington's disease (HD) is caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat expansion in the huntingtin (HTT) gene encoding an elongated polyglutamine tract within the N-terminal of the huntingtin protein (Htt) and leads to Htt misfolding, aberrant protein aggregation, and progressive appearance of disease symptoms. Chronic activation of endoplasmic reticulum (ER) stress by mutant Htt (mHtt) results in cellular dysfunction and ultimately cell death. Protein disulfide isomerase (PDI) is a chaperone protein located in the ER. Our previous studies demonstrated that mHtt caused PDI to accumulate at mitochondria-associated ER membranes and triggered cell death, and that modulating PDI activity using small molecules protected cells again mHtt toxicity in cell and brain slice models of HD. In this study, we demonstrated that PDI is upregulated in the HD human brain, in cell and mouse models. Chronic administration of a reversible, brain penetrable small molecule PDI modulator, LOC14 (20 mg/kg/day), significantly improved motor function, attenuated brain atrophy and extended survival in the N171-82Q HD mice. Moreover, LOC14 preserved medium spiny neuronal marker dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32000 (DARPP32) levels in the striatum of HD mice. Mechanistic study revealed that LOC14 suppressed mHtt-induced ER stress, indicated by repressing the abnormally upregulated ER stress proteins in HD models. These findings suggest that LOC14 is promising to be further optimized for clinical trials of HD, and modulation of signaling pathways coping with ER stress may constitute an attractive approach to reduce mHtt toxicity and identify new therapeutic targets for treatment of HD.

2,4 DNP improves motor function, preserves medium spiny neuronal identity, and reduces oxidative stress in a mouse model of Huntington's disease

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the gene huntingtin. There is no treatment to prevent or delay the disease course of HD currently. Oxidative stress and mitochondrial dysfunction have emerged as key determinants of the disease progression in HD. Therefore, counteracting mutant huntingtin (mHtt)-induced oxidative stress and mitochondrial dysfunction appears as a new approach to treat this devastating disease. Interestingly, mild mitochondrial uncoupling improves neuronal resistance to stress and facilitates neuronal survival. Mild mitochondrial uncoupling can be induced by the proper dose of 2,4-dinitrophenol (DNP), a proton ionophore that was previously used for weight loss. In this study, we evaluated the effects of chronic administration of DNP at three doses (0.5, 1, 5mg/kg/day) on mHtt-induced behavioral deficits and cellular abnormalities in the N171-82Q HD mouse model. DNP at a low dose (1mg/kg/day) significantly improved motor function and preserved medium spiny neuronal marker DARPP32 and postsynaptic protein PSD95 in the striatum of HD mice. Further mechanistic study suggests that DNP at this dose reduced oxidative stress in HD mice, which was indicated by reduced levels of F2-isoprostanes in the brain of HD mice treated with DNP. Our data indicated that DNP provided behavioral benefit and neuroprotective effect at a weight neutral dose in HD mice, suggesting that the potential value of repositioning DNP to HD treatment is warranted in well-controlled clinical trials in HD.

Effects of Exogenous NUB1 Expression in the Striatum of HDQ175/Q7 Mice.

Reducing mutant huntingtin (mHTT) in neurons may be a therapy for Huntington's disease (HD). Elevating NUB1 protein reduced mHTT levels in cell and fly models of HD through a proteasome dependent mechanism. To examine the effects of augmenting NUB1 in HD mouse striatum on mHTT levels. Striata of HDQ175/Q7 mice were injected at 3 months of age with recombinant AAV2/9 coding for NUB1 or GFP under the control of the neuron specific human synapsin 1 promoter and examined 6 months post-injection for levels of huntingtin, the striatal markers DARPP32 and PDE10A, the astrocyte marker GFAP, and the autophagy and mHTT aggregate marker P62 using immunolabeling of brain sections and Western blot assay of striatal subcellular fractions. By Western blot human HD brain had only one of the two variants of NUB1 present in human control brain. In striatum of WT and HD mice NUB1 was localized in medium size neurons and enriched in the nucleus of large neurons. In the striatum of NUB1 injected HD mice, there was widespread neuronal distribution of exogenous NUB1 labeling and protein levels were ∼2.5-fold endogenous levels. DARPP32 and GFAP distribution and levels were unchanged but PDE10A levels were lower in crude homogenates and P62 was increased in nuclear enriched P1 fractions. Elevating NUB1 did not change levels of full-length mHTT or the number and size of mHTT (S830) positive nuclear inclusions. Findings suggest that increasing NUB1 protein in striatal neurons of HDQ175/Q7 mice in vivo may be relatively safe but is ineffective in reducing mHTT. Increased NUB1 expression in HD striatum alters PDE10A and P62 which are known to be influenced by mHTT.

AMPK activation protects from neuronal dysfunction and vulnerability across nematode, cellular and mouse models of Huntington's disease.

The adenosine monophosphate activated kinase protein (AMPK) is an evolutionary-conserved protein important for cell survival and organismal longevity through the modulation of energy homeostasis. Several studies suggested that AMPK activation may improve energy metabolism and protein clearance in the brains of patients with vascular injury or neurodegenerative disease. However, in Huntington's disease (HD), AMPK may be activated in the striatum of HD mice at a late, post-symptomatic phase of the disease, and high-dose regiments of the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide may worsen neuropathological and behavioural phenotypes. Here, we revisited the role of AMPK in HD using models that recapitulate the early features of the disease, including Caenorhabditis elegans neuron dysfunction before cell death and mouse striatal cell vulnerability. Genetic and pharmacological manipulation of aak-2/AMPKα shows that AMPK activation protects C. elegans neurons from the dysfunction induced by human exon-1 huntingtin (Htt) expression, in a daf-16/forkhead box O-dependent manner. Similarly, AMPK activation using genetic manipulation and low-dose metformin treatment protects mouse striatal cells expressing full-length mutant Htt (mHtt), counteracting their vulnerability to stress, with reduction of soluble mHtt levels by metformin and compensation of cytotoxicity by AMPKα1. Furthermore, AMPK protection is active in the mouse brain as delivery of gain-of-function AMPK-γ1 to mouse striata slows down the neurodegenerative effects of mHtt. Collectively, these data highlight the importance of considering the dynamic of HD for assessing the therapeutic potential of stress-response targets in the disease. We postulate that AMPK activation is a compensatory response and valid approach for protecting dysfunctional and vulnerable neurons in HD.

Neuronal identity genes regulated by super-enhancers are preferentially down-regulated in the striatum of Huntington's disease mice.

Huntington's disease (HD) is a neurodegenerative disease associated with extensive down-regulation of genes controlling neuronal function, particularly in the striatum. Whether altered epigenetic regulation underlies transcriptional defects in HD is unclear. Integrating RNA-sequencing (RNA-seq) and chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq), we show that down-regulated genes in HD mouse striatum associate with selective decrease in H3K27ac, a mark of active enhancers, and RNA Polymerase II (RNAPII). In addition, we reveal that decreased genes in HD mouse striatum display a specific epigenetic signature, characterized by high levels and broad patterns of H3K27ac and RNAPII. Our results indicate that this signature is that of super-enhancers, a category of broad enhancers regulating genes defining tissue identity and function. Specifically, we reveal that striatal super-enhancers display extensive H3K27 acetylation within gene bodies, drive transcription characterized by low levels of paused RNAPII, regulate neuronal function genes and are enriched in binding motifs for Gata transcription factors, such as Gata2 regulating striatal identity genes. Together, our results provide evidence for preferential down-regulation of genes controlled by super-enhancers in HD striatum and indicate that enhancer topography is a major parameter determining the propensity of a gene to be deregulated in a neurodegenerative disease.

Interrogation of brain miRNA and mRNA expression profiles reveals a molecular regulatory network that is perturbed by mutant huntingtin

Emerging evidence indicates that microRNAs (miRNAs) may play an important role in the pathogenesis of Huntington's disease (HD). To identify the individual miRNAs that are altered in HD and may therefore regulate a gene network underlying mutant huntingtin-induced neuronal dysfunction in HD, we performed miRNA array analysis combined with mRNA profiling in the cerebral cortex from N171-82Q HD mice. Expression profiles of miRNAs as well as mRNAs in HD mouse cerebral cortex were analyzed and confirmed at different stages of disease progression; the most significant changes of miRNAs in the cerebral cortex were also detected in the striatum of HD mice. Our results revealed a significant alteration of miR-200 family members, miR-200a, and miR-200c in the cerebral cortex and the striatum, at the early stage of disease progression in N171-82Q HD mice. We used a coordinated approach to integrate miRNA and mRNA profiling, and applied bioinformatics to predict a target gene network potentially regulated by these significantly altered miRNAs that might be involved in HD disease progression. Interestingly, miR-200a and miR-200c are predicted to target genes regulating synaptic function, neurodevelopment, and neuronal survival. Our results suggest that altered expression of miR-200a and miR-200c may interrupt the production of proteins involved in neuronal plasticity and survival, and further investigation of the involvement of perturbed miRNA expression in HD pathogenesis is warranted, and may lead to reveal novel approaches for HD therapy.

Modulation of lipid peroxidation and mitochondrial function improves neuropathology in Huntington’s disease mice

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. Oxidative damage has been associated with pathological neuronal loss in HD. The therapeutic modulation of oxidative stress and mitochondrial function using low molecular weight compounds may be an important strategy for delaying the onset and slowing the progression of HD. In the present study, we found a marked increase of 4-hydroxy-2-nonenal (4-HNE) adducts, a lipid peroxidation marker, in the caudate and putamen of HD brains and in the striatum of HD mice. Notably, 4-HNE immunoreactivity was colocalized with mutant huntingtin inclusions in the striatal neurons of R6/2 HD mice. Administration of nordihydroguaiaretic acid (NDGA), an antioxidant that functions by inhibiting lipid peroxidation, markedly reduced 4-HNE adduct formation in the nuclear inclusions of R6/2 striatal neurons. NDGA also protected cultured neurons against oxidative stress-induced cell death by improving ATP generation and mitochondrial morphology and function. In addition, NDGA restored mitochondrial membrane potential, mitochondrial structure, and synapse structure in the striatum of R6/2 mice and increased their lifespan. The present findings suggest that further therapeutic studies using NDGA are warranted in HD and other neurodegenerative diseases characterized by increased oxidative stress and altered mitochondrial function.

Impairment of PGC-1alpha expression, neuropathology and hepatic steatosis in a transgenic mouse model of Huntington's disease following chronic energy deprivation

We investigated the ability of AMP-activated protein kinase (AMPK) to activate PPARgamma coactivator-1alpha (PGC-1alpha) in the brain, liver and brown adipose tissue (BAT) of the NLS-N171-82Q transgenic mouse model of Huntington's disease (HD). In the striatum of the HD mice, the baseline levels of PGC-1alpha, NRF1, NRF2, Tfam, COX-II, PPARdelta, CREB and ERRalpha mRNA and mitochondrial DNA (mtDNA), were significantly reduced. Administration of the creatine analog beta guanidinopropionic acid (GPA) reduced ATP and PCr levels and increased AMPK mRNA in both the cerebral cortex and striatum. Treatment with GPA significantly increased expression of PGC-1alpha, NRF1, Tfam and downstream genes in the striatum and cerebral cortex of wild-type (WT) mice, but there was no effect on these genes in the HD mice. The striatum of the untreated HD mice showed microvacuolation in the neuropil, as well as gliosis and huntingtin aggregates, which were exacerbated by treatment with GPA. GPA treatment produced a significant increase in mtDNA in the cerebral cortex and striatum of WT mice, but not in HD mice. The HD mice treated with GPA had impaired activation of liver PGC-1alpha and developed hepatic steatosis with accumulation of lipids, degeneration of hepatocytes and impaired activation of gluconeogenesis. The BAT in the HD mice showed vacuolation due to accumulation of neutral lipids, and age-dependent impairment of UCP-1 activation and temperature regulation. Impaired activation of PGC-1alpha, therefore, plays an important role in the behavioral phenotype, metabolic disturbances and pathology of HD, which suggests the possibility that agents that enhance PGC-1alpha function will exert therapeutic benefits in HD patients.

Early Increase in Extrasynaptic NMDA Receptor Signaling and Expression Contributes to Phenotype Onset in Huntington's Disease Mice

N-methyl-D-aspartate receptor (NMDAR) excitotoxicity is implicated in the pathogenesis of Huntington's disease (HD), a late-onset neurodegenerative disorder. However, NMDARs are poor therapeutic targets, due to their essential physiological role. Recent studies demonstrate that synaptic NMDAR transmission drives neuroprotective gene transcription, whereas extrasynaptic NMDAR activation promotes cell death. We report specifically increased extrasynaptic NMDAR expression, current, and associated reductions in nuclear CREB activation in HD mouse striatum. The changes are observed in the absence of dendritic morphological alterations, before and after phenotype onset, correlate with mutation severity, and require caspase-6 cleavage of mutant huntingtin. Moreover, pharmacological block of extrasynaptic NMDARs with memantine reversed signaling and motor learning deficits. Our data demonstrate elevated extrasynaptic NMDAR activity in an animal model of neurodegenerative disease. We provide a candidate mechanism linking several pathways previously implicated in HD pathogenesis and demonstrate successful early therapeutic intervention in mice.

Remodeling of striatal NMDA receptors by chronic A2A receptor blockade in Huntington's disease mice

Excitotoxicity plays a major role in the pathogenesis of Huntington disease (HD), a fatal neurodegenerative disorder. Adenosine A2A receptors (A2ARs) modulate excitotoxicity and have been suggested to play a pathogenetic role in HD. The main aim of this study was to evaluate the effect of A2AR blockade on the expression and functions of NMDA receptors in the striatum of HD mice (R6/2). We found that 3 weeks' treatment with SCH 58261 (0.01 mg/kg/day i.p. from the 8th week of age) modified NR1 and NR2A/NR2B expression in the striatum of R6/2 (Western blotting) while had no effect on NMDA-induced toxicity in corticostriatal slices (electrophysiological experiments). In conclusion, in vivo A2AR blockade induced a remodeling of NMDA receptors in the striatum of HD mice. Even though the functional relevance of the above effect remains to be fully elucidated, these results add further evidence to the modulatory role of A2ARs in HD.

Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin.

Mutant huntingtin accumulates in the neuronal nuclei and processes, which suggests that its subcellular localization is critical for the pathology of Huntington's disease (HD). However, the contribution of cytoplasmic mutant huntingtin and its aggregates in neuronal processes (neuropil aggregates) has not been rigorously explored. We generated an intracellular antibody (intrabody) whose binding to a unique epitope of human huntingtin is enhanced by polyglutamine expansion. This intrabody decreases the cytotoxicity of mutant huntingtin and its distribution in neuronal processes. When expressed in the striatum of HD mice via adenoviral infection, the intrabody reduces neuropil aggregate formation and ameliorates neurological symptoms. Interaction of the intrabody with mutant huntingtin increases the ubiquitination of cytoplasmic huntingtin and its degradation. These findings suggest that the intrabody reduces the specific neurotoxicity of cytoplasmic mutant huntingtin and its associated neurological symptoms by preventing the accumulation of mutant huntingtin in neuronal processes and promoting its clearance in the cytoplasm.

Abnormal Sensitivity to Cannabinoid Receptor Stimulation Might Contribute to Altered Gamma-Aminobutyric Acid Transmission in the Striatum of R6/2 Huntington’s Disease Mice

One of the earliest neurochemical alterations observed in both Huntington's disease (HD) patients and HD animal models is the dysregulation of the endocannabinoid system, an alteration that precedes the development of identifiable striatal neuropathology. How this alteration impacts striatal synaptic transmission is unknown. We measured the effects of cannabinoid receptor stimulation on gamma-aminobutyric acid (GABA)-ergic synaptic currents recorded from striatal neurons of R6/2 HD mice in the early phase of their disease. The sensitivity of striatal GABA synapses to cannabinoid receptor stimulation is severely impaired in R6/2 HD mice. In particular, whereas in control animals activation of cannabinoid CB1 receptors results in a significant inhibition of both evoked and spontaneous GABA-mediated synaptic events by a presynaptic mechanism, in R6/2 mice this treatment fails to reduce GABA currents but causes, in contrast, a slight increase of spontaneous inhibitory postsynaptic currents (sIPSCs). Experimental HD was also associated with enhanced frequency of sIPSCs, a result consistent with the conclusion that loss of cannabinoid-mediated control of GABA transmission might contribute to hyperactivity of GABA synapses in the striatum of HD mice. Accordingly, spontaneous excitatory postsynaptic currents, which were not upregulated in R6/2 mice, were still sensitive to cannabinoid receptor stimulation.

Huntingtin inclusions do not deplete polyglutamine-containing transcription factors in HD mice.

A pathological hallmark of polyglutamine diseases is the presence of inclusions or aggregates of the expanded polyglutamine protein. Polyglutamine inclusions are present in the neuronal nucleus in a number of inherited neurodegenerative disorders, including Huntington disease (HD). Recent studies suggest that polyglutamine inclusions may sequester polyglutamine-containing transcription factors and deplete their concentration in the nucleus, leading to altered gene expression. To test this hypothesis, we examined the expression and localization of the polyglutamine-containing or glutamine-rich transcription factors TBP, CBP and Sp1 in HD mouse models. All three transcription factors were diffusely distributed in the nucleus, despite the presence of abundant intranuclear inclusions. There were no differences in the nuclear staining of these transcription factors between HD and wild-type mouse brains. Although some CBP staining appeared as dots in the selective brain regions (e.g. hypothalamus and amygdala), double labeling showed that most CBP was not co-localized with huntingtin nuclear inclusions. Electron microscopy confirmed that CBP was diffusely distributed in the nucleus. Western blots showed that these transcription factors were not trapped in huntingtin inclusions. In the striatum of HD mice, which suffers a significant reduction in the expression of a number of genes, mutant huntingtin was present in both an aggregated and a diffuse form. These findings suggest that altered gene expression may result from the interactions of soluble mutant huntingtin with nuclear transcription factors, rather than from the depletion of transcription factors by nuclear inclusions.