Expression Of Mutant Huntingtin Protein Research Articles

A novel rhesus macaque model of Huntington's disease recapitulates key neuropathological changes along with motor and cognitive decline.

We created a new nonhuman primate model of the genetic neurodegenerative disorder Huntington's disease (HD) by injecting a mixture of recombinant adeno-associated viral vectors, serotypes AAV2 and AAV2.retro, each expressing a fragment of human mutant HTT (mHTT) into the caudate and putamen of adult rhesus macaques. This modeling strategy results in expression of mutant huntingtin protein (mHTT) and aggregate formation in the injected brain regions, as well as dozens of other cortical and subcortical brain regions affected in human HD patients. We queried the disruption of cortico-basal ganglia circuitry for 30 months post-surgery using a variety of behavioral and imaging readouts. Compared to controls, mHTT-treated macaques developed working memory decline and progressive motor impairment. Multimodal imaging revealed circuit-wide white and gray matter degenerative processes in several key brain regions affected in HD. Taken together, we have developed a novel macaque model of HD that may be used to develop disease biomarkers and screen promising therapeutics.

Tolfenamic acid inhibits ROS-generating oxidase Nox1-regulated p53 activity in intrastriatal injection of malonic acid rats

It has been reported that wild-type p53-induced gene 1 (Wig1), which is downstream of p53, regulates the expression of mutant huntingtin protein (mHtt) in Huntington’s disease (HD) patients and transgenic mouse brains. Intrastriatal injection of malonic acid in rats is often used as a model to study the pathological changes of Huntington’s disease, and this model has the advantages of a fast preparation and low cost. Therefore, in this study, we used intrastriatal injections of 6 μM malonic acid in rats to evaluate the effect of tolfenamic acid on motor and cognitive deficits and the effect of 6 mg/kg and 32 mg/kg tolfenamic acid on p53 and its downstream targets, such as Wig1. The results showed that 32 mg/kg tolfenamic acid attenuated motor and spatial memory dysfunction, prevented Nox1-mediated reactive oxygen species (ROS) production, and downregulated the activity of p53 by increasing the phosphorylation level at the Ser378 site and decreasing the acetylation level at the Lys382 site. Tolfenamic acid reduced mouse double minute 2 (Mdm2), phosphatase and tensin homologue (Pten), P53-upregulated modulator of apoptosis (Puma) and Bcl2-associated X (Bax) at the mRNA level to inhibit apoptosis and downregulated sestrin 2 (Sesn2) and hypoxia inducible factor 1, alpha subunit (Hif-1α) mRNA levels to exert antioxidative stress effects. In addition, 32 mg/kg tolfenamic acid played a role in neuroprotection by decreasing the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive cell numbers. However, there was no difference in the Wig mRNA level among all groups, and tolfenamic acid could not decrease the protein level of Wig1. In conclusion, tolfenamic acid inhibited the ROS-generating oxidase Nox1-regulated p53 activity and attenuated motor and spatial memory deficits in malonic acid-injected rats.

Emerging Roles of Exosomes in Huntington's Disease.

Huntington’s disease (HD) is a rare hereditary autosomal dominant neurodegenerative disorder, which is caused by expression of mutant huntingtin protein (mHTT) with an abnormal number of glutamine repeats in its N terminus, and characterized by intracellular mHTT aggregates (inclusions) in the brain. Exosomes are small extracellular vesicles that are secreted generally by all cell types and can be isolated from almost all body fluids such as blood, urine, saliva, and cerebrospinal fluid. Exosomes may participate in the spreading of toxic misfolded proteins across the central nervous system in neurodegenerative diseases. In HD, such propagation of mHTT was observed both in vitro and in vivo. On the other hand, exosomes might carry molecules with neuroprotective effects. In addition, due to their capability to cross blood-brain barrier, exosomes hold great potential as sources of biomarkers available from periphery or carriers of therapeutics into the central nervous system. In this review, we discuss the emerging roles of exosomes in HD pathogenesis, diagnosis, and therapy.

Reducing Mutant Huntingtin Protein Expression in Living Cells by a Newly Identified RNA CAG Binder.

Expanded CAG trinucleotide repeats in Huntington's disease (HD) are causative for neurotoxicity. The mutant CAG repeat RNA encodes neurotoxic polyglutamine proteins and can lead to a toxic gain of function by aberrantly recruiting RNA-binding proteins. One of these is the MID1 protein, which induces aberrant Huntingtin (HTT) protein translation upon binding. Here we have identified a set of CAG repeat binder candidates by in silico methods. One of those, furamidine, reduces the level of binding of HTT mRNA to MID1 and other target proteins in vitro. Metadynamics calculations, fairly consistent with experimental data measured here, provide hints about the binding mode of the ligand. Importantly, furamidine also decreases the protein level of HTT in a HD cell line model. This shows that small molecules masking RNA-MID1 interactions may be active against mutant HTT protein in living cells.

Mutant huntingtin protein expression and blood-spinal cord barrier dysfunction in huntington disease.

The aim of the study was to assess the distribution, frequency, and specific location of mutant huntingtin protein (mHTT) aggregates-the pathological hallmark of Huntington disease (HD)-within the various compartments of the spinal cord and their potential impact on the local vasculature and blood-spinal cord barrier (BSCB). We performed a series of postmortem immunohistochemical and immunofluorescent stainings, as well as Western blot analyses, on cervical and lumbar sections of the spinal cord in patients diagnosed with HD (n = 11 of all grades of disease severity) along with sex- and age-matched healthy controls (n = 9). We observed that mHTT was preferably expressed within the anterior horn of the gray matter, in both cervical and lumbar sections. At the cellular level, mHTT aggregates were more often encountered in the extracellular matrix but could also be observed within cell bodies and neurites as well as within the endothelium of blood vessels with an increase in the density of small blood vessels in cervical sections of HD cases. These vasculature changes were accompanied with features of BSCB leakage, as assessed by the presence of increased levels of fibrinogen in the surrounding parenchyma and enhanced leukocyte infiltration. This alteration in BSCB integrity may be explained, in part, by the dysregulation we found in some of the main proteins associated with it such as junctional adhesion molecule-1 and vascular endothelial cadherin. These observations have important implications for our understanding of HD pathology and may also have significant therapeutic implications. Ann Neurol 2017;82:981-994.

Sex-dependent behavioral impairments in the HdhQ350/+ mouse line

Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disease characterized by gradual deterioration of motor and cognitive functions and development of psychiatric deficits. Animal models provide powerful means to study the pathological processes, molecular dysfunctions and symptoms associated with HD. We performed a longitudinal behavioral study of the newly developed HdhQ350/+ mouse line, a knock-in model that expresses a repeat of 350 glutamines. We found remarkable sex-dependent differences on symptom onset and severity. While both sexes lose weight and grip strength, only HdhQ350/+ males have impaired motor coordination as measured by the rotarod and alterations in gait as measured by the catwalk assay. While HdhQ350/+ females do not exhibit impairment in motor coordination, we found a reduction in dark phase locomotor activity. Male and female HdhQ350/+ mice do not show anxiety as measured by the elevated plus maze or changes in exploration as measured by the open field test. To investigate these sex-dependent differences, we performed western blot analyses of striatal tissue. We measured equal mutant huntingtin protein expression in both sexes and found evidence of aggregation. We found the expected decrease of DARPP-32 expression only in female HdhQ350/+ mice. Remarkably, we found no evidence of reduction in synaptophysin or CB1 receptors in HdhQ350/+ tissue of either sex. Our study indicates that male and female HdhQ350/+ mice differentially recapitulate select behavioral impairments commonly measured in other HD mouse models with limited sex-dependent changes in recognized histopathological markers. We conclude that expanded polyglutamine repeats influence HD pathogenesis in a sex-dependent manner.

Amyloid Precursor Protein Haploinsufficiency Preferentially Mediates Brain Iron Accumulation in Mice Transgenic for The Huntington's Disease Mutation.

Huntington's disease (HD) is an autosomal dominant disorder caused by a CAG expansion in the huntingtin gene that results in expression of mutant huntingtin protein. Iron accumulates in HD brain neurons. Amyloid precursor protein (APP) promotes neuronal iron export. However, the role of APP in brain iron accumulation in HD is unclear. To determine the effects of APP insufficiency on HD in YAC128 mice. We crossed APP hemizygous mice (APP+/-) with YAC128 mice that are transgenic (Tg) for human mutant huntingtin (hmHTT) to generate APP+/+ hmHTT-/-, APP+/- hmHTT-/-, APP+/+ hmHTT+/- and APP+/- hmHTT+/- progeny. Mice were evaluated for behavioral, biochemical and neuropathology HD outcomes at 2-12 months of age. APP heterozygosity decreased cortical APP 25% and 60% in non-Tg and Tg mice, respectively. Cerebral and striatal iron levels were increased by APP knockdown in Tg mice only. Nest-building behavior was decreased in Tg mice; APP knockdown decreased nest building in non-Tg but not Tg mice. Rota-rod endurance was decreased in Tg mice. APP+/- hHTT+/- mice demonstrated additional decreases in rota-rod endurance from 4-10 months of age. Tg mice had smaller striatal volumes and fewer striatal neurons but were not affected by APP knockdown. APP heterozygosity results in greater decreases of cortical APP in Tg versus non-Tg mice. Mutant huntingtin transgenic mice develop brain iron accumulation as a result of greater suppression of APP levels. Elevated brain iron in Tg mice was associated with a decline in motor endurance consistent with a disease promoting effect of iron in the YAC128 model of human HD.

Mutant Huntingtin Impairs BDNF Release from Astrocytes by Disrupting Conversion of Rab3a-GTP into Rab3a-GDP.

Brain-derived neurotrophic factor (BDNF) is essential for neuronal differentiation and survival. We know that BDNF levels decline in the brains of patients with Huntington's disease (HD), a neurodegenerative disease caused by the expression of mutant huntingtin protein (mHtt), and furthermore that administration of BDNF in HD mice is protective against HD neuropathology. BDNF is produced in neurons, but astrocytes are also an important source of BDNF in the brain. Nonetheless, whether mHtt affects astrocytic BDNF in the HD brain remains unknown. Here we investigated astrocytes from HD140Q knock-in mice and uncovered evidence that mHtt decreases BDNF secretion from astrocytes, which is mediated by exocytosis in astrocytes. Our results demonstrate that mHtt associates with Rab3a, a small GTPase localized on membranes of dense-core vesicles, and prevents GTP-Rab3a from binding to Rab3-GAP1, disrupting the conversion of GTP-Rab3a into GDP-Rab3a and thus impairing the docking of BDNF vesicles on plasma membranes of astrocytes. Importantly, overexpression of Rab3a rescues impaired BDNF vesicle docking and secretion from HD astrocytes. Moreover, ATP release and the number of ATP-containing dense-core vesicles docking are decreased in HD astrocytes, suggesting that the exocytosis of dense-core vesicles is impaired by mHtt in HD astrocytes. Further, Rab3a overexpression reduces reactive astrocytes in the striatum of HD140Q knock-in mice. Our results indicate that compromised exocytosis of BDNF in HD astrocytes contributes to the decreased BDNF levels in HD brains and underscores the importance of improving glial function in the treatment of HD. Huntington's disease (HD) is an inherited neurodegenerative disorder that affects one in every 10,000 Americans. To date, there is no effective treatment for HD, in part because the pathogenic mechanism driving the disease is not fully understood. The dysfunction of astrocytes is known to contribute to the pathogenesis of HD. One important role of astrocytes is to synthesize and release brain-derived neurotrophic factor (BDNF), which is vital for neuronal survival, development, and function. We found that mutant huntingtin protein (mHtt) at the endogenous level decreases BDNF secretion from astrocytes by disrupting the conversion of GTP-Rab3a into GDP-Rab3a and that overexpressing Rab3a can rescue this deficient BDNF release and early neuropathology in HD knock-in mouse brain. Our study suggests that astrocytic Rab3a is a potential therapeutic target for HD treatment.

Manifestation of Huntington's disease pathology in human induced pluripotent stem cell-derived neurons.

BackgroundHuntington’s disease (HD) is an incurable hereditary neurodegenerative disorder, which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD, existing pharmaceutical can only relieve its symptoms.ResultsHere, induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene, and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro, as evidenced by mutant huntingtin protein aggregation, increased number of lysosomes/autophagosomes, nuclear indentations, and enhanced neuronal death during cell aging. Moreover, store-operated channel (SOC) currents were detected in the differentiated neurons, and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally, the quinazoline derivative, EVP4593, reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging.ConclusionsOur data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks, providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0092-5) contains supplementary material, which is available to authorized users.

Making (anti-) sense out of huntingtin levels in Huntington disease.

BackgroundHuntington disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by motor, psychiatric and cognitive symptoms. HD is caused by a CAG repeat expansion in the first exon of the HTT gene, resulting in an expanded polyglutamine tract at the N-terminus of the huntingtin protein. Typical disease onset is around mid-life (adult-onset HD) whereas onset below 21 years is classified as juvenile HD. While much research has been done on the underlying HD disease mechanisms, little is known about regulation and expression levels of huntingtin RNA and protein.ResultsIn this study we used 15 human post-mortem HD brain samples to investigate the expression of wild-type and mutant huntingtin mRNA and protein. In adult-onset HD brain samples, there was a small but significantly lower expression of mutant huntingtin mRNA compared to wild-type huntingtin mRNA, while wild-type and mutant huntingtin protein expression levels did not differ significantly. Juvenile HD subjects did show a lower expression of mutant huntingtin protein compared to wild-type huntingtin protein. Our results in HD brain and fibroblasts suggest that protein aggregation does not affect levels of huntingtin RNA and protein. Additionally, we did not find any evidence for a reduced expression of huntingtin antisense in fibroblasts derived from a homozygous HD patient.ConclusionsWe found small differences in allelic huntingtin mRNA levels in adult-onset HD brain, with significantly lower mutant huntingtin mRNA levels. Wild-type and mutant huntingtin protein were not significantly different in adult-onset HD brain samples. Conversely, in juvenile HD brain samples mutant huntingtin protein levels were lower compared with wild-type huntingtin, showing subtle differences between juvenile HD and adult-onset HD. Since most HD model systems harbor juvenile repeat expansions, our results suggest caution with the interpretation of huntingtin mRNA and protein studies using HD cell and animal models with such long repeats. Furthermore, our huntingtin antisense results in homozygous HD cells do not support reduced huntingtin antisense expression due to an expanded CAG repeat.

Progressive Impairment of Lactate-based Gluconeogenesis in the Huntington’s Disease Mouse Model R6/2

Huntington’s disease (HD) is a neurodegenerative illness, where selective neuronal loss in the brain caused by expression of mutant huntingtin protein leads to motor dysfunction and cognitive decline in addition to peripheral metabolic changes. In this study we confirm our previous observation of impairment of lactate-based hepatic gluconeogenesis in the transgenic HD mouse model R6/2 and determine that the defect manifests very early and progresses in severity with disease development, indicating a potential to explore this defect in a biomarker context. Moreover, R6/2 animals displayed lower blood glucose levels during prolonged fasting compared to wild type animals.

Differential effects of delayed aging on phenotype and striatal pathology in a murine model of Huntington disease.

The common neurodegenerative syndromes exhibit age-related incidence, and many Mendelian neurodegenerative diseases exhibit age-related penetrance. Mutations slowing aging retard age related pathologies. To assess whether delayed aging retards the effects of a mutant allele causing a Huntington's disease (HD)-like syndrome, we generated compound mutant mice, placing a dominant HD knock-in polyglutamine allele onto the slow-aging Snell dwarf genotype. The Snell genotype did not affect mutant huntingtin protein expression. Bigenic and control mice were evaluated prospectively from 10 to 100 weeks of age. Adult HD knock-in allele mice lost weight progressively with weight loss blunted significantly in male bigenic HD knock-in/Snell dwarf mice. Impaired balance beam performance developed significantly more slowly in bigenic HD knock-in/Snell dwarf mice. Striatal dopamine receptor expression was diminished significantly and similarly in all HD-like mice, regardless of the Snell genotype. Striatal neuronal intranuclear inclusion burden was similar between HD knock-in mice with and without the Snell genotype, whereas nigral neuropil aggregates were diminished in bigenic HD knock-in/Snell dwarf mice. Compared with control mice, Snell dwarf mice exhibited differences in regional benzodiazepine and cannabinoid receptor binding site expression. These results indicate that delaying aging delayed behavioral decline with little effect on the development of striatal pathology in this model of HD but may have altered synaptic pathology. These results indicate that mutations prolonging lifespan in mice delay onset of significant phenotypic features of this model and also demonstrate dissociation between striatal pathology and a commonly used behavioral measure of disease burden in HD models.

Huntingtin-lowering strategies in Huntington's disease: antisense oligonucleotides, small RNAs, and gene editing.

The idea to lower mutant huntingtin is especially appealing in Huntington's disease (HD). It is autosomal dominant, so that expression of the mutant allele causes the disease. Advances in RNA and gene regulation provide foundations for the huntingtin gene (both normal and mutant alleles) and possibly the mutant allele only. There is much preclinical animal work to support the concept of gene and RNA silencing, but, to date, no clinical studies have been attempted in HD. Preventing expression of mutant huntingtin protein is at the cusp for a human trial. Antisense oligonucleotides delivered to patients with amyotrophic lateral sclerosis have been well tolerated; small RNAs administered to rodent and nonhuman primate brain knocked down huntingtin messenger RNA (mRNA); short-hairpin complementary DNA of microRNAs can be expressed in adeno-associated virus to provide long-term silencing of huntingtin mRNA and protein. We expect that these approaches will be ready for clinical studies in the near future, once safety has been validated. Our understanding of gene editing-changing the huntingtin gene itself-is rapidly progressing. Harnessing our knowledge of transcription and translation should push scientific creativity to new and exciting advances that overcome the lethality of the mutant gene in HD.

Targets for future clinical trials in Huntington's disease: what's in the pipeline?

The known genetic cause of Huntington's disease (HD) has fueled considerable progress in understanding its pathobiology and the development of therapeutic approaches aimed at correcting specific changes linked to the causative mutation. Among the most promising is reducing expression of mutant huntingtin protein (mHTT) with RNA interference or antisense oligonucleotides; human trials are now being planned. Zinc-finger transcriptional repression is another innovative method to reduce mHTT expression. Modulation of mHTT phosphorylation, chaperone upregulation, and autophagy enhancement represent attempts to alter cellular homeostasis to favor removal of mHTT. Inhibition of histone deacetylases (HDACs) remains of interest; recent work affirms HDAC4 as a target but questions the assumed centrality of its catalytic activity in HD. Phosphodiesterase inhibition, aimed at restoring synaptic function, has progressed rapidly to human trials. Deranged cellular signaling provides several tractable targets, but specificity and complexity are challenges. Restoring neurotrophic support in HD remains a key potential therapeutic approach. with several approaches being pursued, including brain-derived neurotrophic factor (BDNF) mimesis through tyrosine receptor kinase B (TrkB) agonism and monoclonal antibodies. An increasing understanding of the role of glial cells in HD has led to several new therapeutic avenues, including kynurenine monooxygenase inhibition, immunomodulation by laquinimod, CB2 agonism, and others. The complex metabolic derangements in HD remain under study, but no clear therapeutic strategy has yet emerged. We conclude that many exciting therapeutics are progressing through the development pipeline, and combining a better understanding of HD biology in human patients, with concerted medicinal chemistry efforts, will be crucial for bringing about an era of effective therapies.

Whole gene expression profile in blood reveals multiple pathways deregulation in R6/2 mouse model

BackgroundHuntington Disease (HD) is a progressive neurological disorder, with pathological manifestations in brain areas and in periphery caused by the ubiquitous expression of mutant Huntingtin protein. Transcriptional dysregulation is considered a key molecular mechanism responsible of HD pathogenesis but, although numerous studies investigated mRNA alterations in HD, so far none evaluated a whole gene expression profile in blood of R6/2 mouse model.FindingsTo discover novel pathogenic mechanisms and potential peripheral biomarkers useful to monitor disease progression or drug efficacy, a microarray study was performed in blood of R6/2 at manifest stage and wild type littermate mice. This approach allowed to propose new peripheral molecular processes involved in HD and to suggest different panels of candidate biomarkers. Among the discovered deregulated processes, we focused on specific ones: complement and coagulation cascades, PPAR signaling, cardiac muscle contraction, and dilated cardiomyopathy pathways. Selected genes derived from these pathways were additionally investigated in other accessible tissues to validate these matrices as source of biomarkers, and in brain, to link central and peripheral disease manifestations.ConclusionsOur findings validated the skeletal muscle as suitable source to investigate peripheral transcriptional alterations in HD and supported the hypothesis that immunological alteration may contribute to neurological degeneration. Moreover, the identification of altered signaling in mouse blood enforce R6/2 transgenic mouse as a powerful HD model while suggesting novel disease biomarkers for pre-clinical investigation.

Defective mitochondrial disulfide relay system, altered mitochondrial morphology and function in Huntington's disease

A number of studies have been conducted that link mitochondrial dysfunction (MD) to Huntington's disease (HD); however, contradicting results had resulted in a lack of a clear mechanism that links expression of mutant Huntingtin protein and MD. Mouse homozygous (HM) and heterozygous (HT) mutant striatal cells with two or one allele encoding for a mutant huntingtin protein with 111 polyGln repeats showed a significant impairment of the mitochondrial disulfide relay system (MDRS). This system (consisting of two proteins, Gfer and Mia40) is involved in the mitochondrial import of Cys-rich proteins. The Gfer-to-Mia40 ratio was significantly altered in HM cells compared with controls, along with the expression of mitochondrial proteins considered substrates of the MDRS. In progenitors and differentiated neuron-like HM cells, impairment of MDRS were accompanied by deficient oxidative phosphorylation, Complex I, IV and V activities, decreased mtDNA copy number and transcripts, accumulation of mtDNA deletions and changes in mitochondrial morphology, consistent with other MDRS-deficient biological models, thus providing a framework for the energy deficits observed in this HD model. The majority (>90%) of the mitochondrial outcomes exhibited a gene-dose dependency with the expression of mutant Htt. Finally, decreases in the mtDNA copy number, along with the accumulation of mtDNA deletions, provide a mechanism for the progressive neurodegeneration observed in HD patients.

Relationship between BDNF expression in major striatal afferents, striatum morphology and motor behavior in the R6/2 mouse model of Huntington's disease

Patients with Huntington's disease (HD) and transgenic mouse models of HD show neuronal loss in the striatum as a major feature, which contributes to cognitive and motor manifestations. Reduced expression of the neurotrophin brain-derived neurotrophic factor (BDNF) in striatal afferents may play a role in neuronal loss. How progressive loss of BDNF expression in different cortical or subcortical afferents contributes to striatal atrophy and behavioral dysfunction in HD is not known, and may best be determined in animal models. We compared age-dependent alterations of BDNF mRNA expression in major striatal afferents from the cerebral cortex, thalamus and midbrain in the R6/2 transgenic mouse model of HD. Corresponding changes in striatal morphology were quantified using unbiased stereology. Changes in motor behavior were measured using an open field, grip strength monitor, limb clasping and a rotarod apparatus. BDNF expression in cortical limbic and midbrain striatal afferents is reduced by age 4 weeks, prior to onset of motor abnormalities. BDNF expression in motor cortex and thalamic afferents is reduced by 6 weeks, coinciding with early motor dysfunction and reduced striatum volume. BDNF loss in afferents progresses until death at 13-15 weeks, correlating with progressive striatal neuronal loss and motor abnormalities. Mutant huntingtin protein expression in R6/2 mice results in progressive loss of BDNF in both cortical and subcortical striatal afferents. BDNF loss in limbic and dopaminergic striatal inputs may contribute to cognitive/psychiatric dysfunction in HD. Subsequent BDNF loss in cortical motor and thalamic afferents may accelerate striatal degeneration, resulting in progressive involuntary movements.

Mechanism of allele-selective inhibition of huntingtin expression by duplex RNAs that target CAG repeats: function through the RNAi pathway.

Huntington’s disease is an incurable neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat within one allele of the huntingtin (HTT) gene. Agents that block expression of mutant HTT and preserve expression of wild-type HTT target the cause of the disease and are an alternative for therapy. We have previously demonstrated that mismatch-containing duplex RNAs complementary to the expanded trinucleotide repeat are potent and allele-selective inhibitors of mutant HTT expression, but the mechanism of allele selectivity was not explored. We now report that anti-CAG duplex RNA preferentially recruits argonaute 2 (AGO2) to mutant rather than wild-type HTT mRNA. Efficient inhibition of mutant HTT protein expression requires less AGO2 than needed for inhibiting wild-type expression. In contrast, inhibiting the expression of mutant HTT protein is highly sensitive to reduced expression of GW182 (TNRC6A) and its two paralogs, a protein family associated with miRNA action. Allele-selective inhibition may involve cooperative binding of multiple protein–RNA complexes to the expanded repeat. These data suggest that allele-selective inhibition proceeds through an RNA interference pathway similar to that used by miRNAs and that discrimination between mutant and wild-type alleles of HTT mRNA is highly sensitive to the pool of AGO2 and GW182 family proteins inside cells.

Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin

Huntington's disease (HD) is a fatal, autosomal dominant neurodegenerative disorder caused by an expanded trinucleotide (CAG) repeat in exon 1 of the huntingtin gene (Htt). This expansion creates a toxic polyglutamine tract in the huntingtin protein (HTT). Currently, there is no treatment for either the progression or prevention of the disease. RNA interference (RNAi) technology has shown promise in transgenic mouse models of HD by reducing expression of mutant HTT and slowing disease progression. The advancement of RNAi therapies to human clinical trials is hampered by problems delivering RNAi to affected neurons in a robust and sustainable manner. Mesenchymal stem cells (MSC) have demonstrated a strong safety profile in both completed and numerous ongoing clinical trials. MSC exhibit a number of innate therapeutic effects, such as immune system modulation, homing to injury, and cytokine release into damaged microenvironments. The ability of MSC to transfer larger molecules and even organelles suggested their potential usefulness as delivery vehicles for therapeutic RNA inhibition. In a series of model systems we have found evidence that MSC can transfer RNAi targeting both reporter genes and mutant huntingtin in neural cell lines. MSC expressing shRNA antisense to GFP were found to decrease expression of GFP in SH-SY5Y cells after co-culture when assayed by flow cytometry. Additionally MSC expressing shRNA antisense to HTT were able to decrease levels of mutant HTT expressed in both U87 and SH-SY5Y target cells when assayed by Western blot and densitometry. These results are encouraging for expanding the therapeutic abilities of both RNAi and MSC for future treatments of Huntington's disease.

D-β-Hydroxybutyrate Is Protective in Mouse Models of Huntington's Disease

Abnormalities in mitochondrial function and epigenetic regulation are thought to be instrumental in Huntington's disease (HD), a fatal genetic disorder caused by an expanded polyglutamine track in the protein huntingtin. Given the lack of effective therapies for HD, we sought to assess the neuroprotective properties of the mitochondrial energizing ketone body, D-β-hydroxybutyrate (DβHB), in the 3-nitropropionic acid (3-NP) toxic and the R6/2 genetic model of HD. In mice treated with 3-NP, a complex II inhibitor, infusion of DβHB attenuates motor deficits, striatal lesions, and microgliosis in this model of toxin induced-striatal neurodegeneration. In transgenic R6/2 mice, infusion of DβHB extends life span, attenuates motor deficits, and prevents striatal histone deacetylation. In PC12 cells with inducible expression of mutant huntingtin protein, we further demonstrate that DβHB prevents histone deacetylation via a mechanism independent of its mitochondrial effects and independent of histone deacetylase inhibition. These pre-clinical findings suggest that by simultaneously targeting the mitochondrial and the epigenetic abnormalities associated with mutant huntingtin, DβHB may be a valuable therapeutic agent for HD.