Expression Of Huntingtin Protein Research Articles

Fullerenols Prevent Neuron Death and Reduce Oxidative Stress in Drosophila Huntington's Disease Model.

Huntington's disease (HD) is one of the human neurodegenerative diseases for which there is no effective treatment. Therefore, there is a strong demand for a novel neuroprotective agent that can alleviate its course. Fullerene derivatives are considered to be such agents; however, they need to be comprehensively investigated in model organisms. In this work, neuroprotective activity of C60(OH)30 and C120O(OH)44 fullerenols was analyzed for the first time in a Drosophila transgenic model of HD. Lifespan, behavior, oxidative stress level and age-related neurodegeneration were assessed in flies with the pathogenic Huntingtin protein expression in nerve cells. Feed supplementation with hydroxylated C60 fullerene and C120O dimer oxide molecules was shown to diminish the oxidative stress level and neurodegenerative processes in the flies' brains. Thus, fullerenes displayed neuroprotective activity in this model.

An impedimetric assay for the identification of abnormal mitochondrial dynamics in living cells.

Mitochondrial dynamics (fission and fusion) plays an important role in cell functions. Disruption in mitochondrial dynamics has been associated with diseases such as neurobiological disorders and cardiovascular diseases. Analysis of mitochondrial fission/fusion has been mostly achieved through direct visualization of the fission/fusion events in live-cell imaging of fluorescently labeled mitochondria. In this study, we demonstrated a label-free, non-invasive Electrical Impedance Spectroscopy (EIS) approach to analyze mitochondrial dynamics in a genetically modified human neuroblastoma SH-SY5Y cell line with no huntingtin protein expression. Huntingtin protein has been shown to regulate mitochondria dynamics. We performed EIS studies on normal SH-SY5Y cells and two independent clones of huntingtin-null cells. The impedance data was used to determine the suspension conductivity and further cytoplasmic conductivity and relate to the abnormal mitochondrial dynamics. For instance, the cytoplasm conductivity value was increased by 11% from huntingtin-null cells to normal cells. Results of this study demonstrated that EIS is sensitive to characterize the abnormal mitochondrial dynamics that can be difficult to quantify by the conventional microscopic method.

How Do Post-Translational Modifications Influence the Pathomechanistic Landscape of Huntington's Disease? A Comprehensive Review.

Huntington’s disease (HD) is an autosomal dominant inherited neurodegenerative disorder characterized by the loss of motor control and cognitive ability, which eventually leads to death. The mutant huntingtin protein (HTT) exhibits an expansion of a polyglutamine repeat. The mechanism of pathogenesis is still not fully characterized; however, evidence suggests that post-translational modifications (PTMs) of HTT and upstream and downstream proteins of neuronal signaling pathways are involved. The determination and characterization of PTMs are essential to understand the mechanisms at work in HD, to define possible therapeutic targets better, and to challenge the scientific community to develop new approaches and methods. The discovery and characterization of a panoply of PTMs in HTT aggregation and cellular events in HD will bring us closer to understanding how the expression of mutant polyglutamine-containing HTT affects cellular homeostasis that leads to the perturbation of cell functions, neurotoxicity, and finally, cell death. Hence, here we review the current knowledge on recently identified PTMs of HD-related proteins and their pathophysiological relevance in the formation of abnormal protein aggregates, proteolytic dysfunction, and alterations of mitochondrial and metabolic pathways, neuroinflammatory regulation, excitotoxicity, and abnormal regulation of gene expression.

Huntingtin phosphorylation governs BDNF homeostasis and improves the phenotype of Mecp2 knockout mice.

Mutations in the X‐linked MECP2 gene are responsible for Rett syndrome (RTT), a severe neurological disorder for which there is no treatment. Several studies have linked the loss of MeCP2 function to alterations of brain‐derived neurotrophic factor (BDNF) levels, but non‐specific overexpression of BDNF only partially improves the phenotype of Mecp2‐deficient mice. We and others have previously shown that huntingtin (HTT) scaffolds molecular motor complexes, transports BDNF‐containing vesicles, and is under‐expressed in Mecp2 knockout brains. Here, we demonstrate that promoting HTT phosphorylation at Ser421, either by a phospho‐mimetic mutation or inhibition of the phosphatase calcineurin, restores endogenous BDNF axonal transport in vitro in the corticostriatal pathway, increases striatal BDNF availability and synaptic connectivity in vivo, and improves the phenotype and the survival of Mecp2 knockout mice—even though treatments were initiated only after the mice had already developed symptoms. Stimulation of endogenous cellular pathways may thus be a promising approach for the treatment of RTT patients.

Astrocyte transduction is required for rescue of behavioral phenotypes in the YAC128 mouse model with AAV-RNAi mediated HTT lowering therapeutics

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease caused by a CAG expansion, which translates into an elongated polyglutamine (polyQ) repeat near the amino-terminus of the huntingtin protein (HTT). This results in production of a toxic mutant huntingtin protein (mHTT) that leads to neuronal dysfunction and death. Currently, no disease-modifying treatments are available; however, numerous therapeutic strategies aimed at lowering HTT levels in the brain are under development. To date, studies have not closely examined the contribution of mHTT in neurons vs astrocytes to disease pathophysiology. To better understand the role of astrocytes in HD pathophysiology and the need for cell type specific targeting of HTT lowering therapeutic strategies, AAV capsids were employed that selectively transduce neurons, or both neurons and astrocytes. These vectors carrying miRNA sequences directed against HTT were injected into the YAC128 mouse model of HD to selectively lower HTT expression in neurons alone versus neurons and astrocytes. The results suggested that HTT lowering in neurons alone was not sufficient to rescue the motor phenotype in YAC128 mice. Furthermore, HTT lowering in both cell types was required to achieve maximal functional benefit. The study suggested that astrocyte dysfunction may play a critical role in HD pathogenesis, and thus astrocytes represent an important therapeutic target.

Transcriptional Regulation of the Huntingtin Gene.

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide expansion in the HTT gene, which encodes for an abnormal polyglutamine tract in the huntingtin protein (HTT). This review examines the known mechanisms of HTT gene regulation. We discuss HTT expression patterns, features of the HTT promoter, regulatory regions of the HTT promoter with functional significance, and HTT regulators located outside of the proximal promoter region. The factors that influence HTT expression in the brain and the mechanisms of HTT transcriptional regulation are currently poorly understood, despite continuing research. Expanding knowledge of HTT regulation will inform future studies investigating HTT function. Improving understanding of HTT expression and control may also uncover novel therapeutic approaches for HD through the development of methods to modulate mHTT levels.

Quantification of Total and Mutant Huntingtin Protein Levels in Biospecimens Using a Novel alphaLISA Assay.

The neurodegenerative Huntington’s disease (HD) is caused by a polyglutamine (polyQ) amplification in the huntingtin protein (HTT). Currently there is no effective therapy available for HD; however, several efforts are directed to develop and optimize HTT-lowering methods to improve HD phenotypes. To validate these approaches, there is an immediate need for reliable, sensitive, and easily accessible methods to quantify HTT expression. Using the AlphaLISA platform, we developed two novel sensitive and robust assays for quantification of HTT in biological samples using commercially available antibodies. The first, a polyQ-independent assay, measures the total pool of HTT, while the second, a polyQ-dependent assay, preferentially detects the mutant form of HTT. Using purified HTT protein standards and brain homogenates from an HD mouse model, we determine a lower limit of quantification of 1 and 3 pm and optimal reproducibility with CV values lower than 7% for intra- and 20% for interassay. In addition, we used the assays to quantify HTT in neural stem cells generated from patient-derived induced pluripotent stem cells in vitro and in human brain tissue lysates. Finally, we could detect changes in HTT levels in a mouse model where mutant HTT was conditionally deleted in neural tissue, verifying the potential to monitor the outcome of HTT-lowering strategies. This analytical platform is ideal for high-throughput screens and thus has an added value for the HD community as a tool to optimize novel therapeutic approaches aimed at modulating HTT protein levels.

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.

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.

Reduced Levels of Proteasome Products in a Mouse Striatal Cell Model of Huntington's Disease.

Huntington’s disease is the result of a long polyglutamine tract in the gene encoding huntingtin protein, which in turn causes a large number of cellular changes and ultimately results in neurodegeneration of striatal neurons. Although many theories have been proposed, the precise mechanism by which the polyglutamine expansion causes cellular changes is not certain. Some evidence supports the hypothesis that the long polyglutamine tract inhibits the proteasome, a multiprotein complex involved in protein degradation. However, other studies report normal proteasome function in cells expressing long polyglutamine tracts. The controversy may be due to the methods used to examine proteasome activity in each of the previous studies. In the present study, we measured proteasome function by examining levels of endogenous peptides that are products of proteasome cleavage. Peptide levels were compared among mouse striatal cell lines expressing either 7 glutamines (STHdh Q7/Q7) or 111 glutamines in the huntingtin protein, either heterozygous (STHdh Q7/Q111) or homozygous (STHdh Q111/Q111). Both of the cell lines expressing huntingtin with 111 glutamines showed a large reduction in nearly all of the peptides detected in the cells, relative to levels of these peptides in cells homozygous for 7 glutamines. Treatment of STHdh Q7/Q7 cells with proteasome inhibitors epoxomicin or bortezomib also caused a large reduction in most of these peptides, suggesting that they are products of proteasome-mediated cleavage of cellular proteins. Taken together, these results support the hypothesis that proteasome function is impaired by the expression of huntingtin protein containing long polyglutamine tracts.

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.

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.

Proteomics of Huntington's disease-affected human embryonic stem cells reveals an evolving pathology involving mitochondrial dysfunction and metabolic disturbances.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the Huntingtin gene, where excessive (≥ 36) CAG repeats encode for glutamine expansion in the huntingtin protein. Research using mouse models and human pathological material has indicated dysfunctions in a myriad of systems, including mitochondrial and ubiquitin/proteasome complexes, cytoskeletal transport, signaling, and transcriptional regulation. Here, we examined the earliest biochemical and pathways involved in HD pathology. We conducted a proteomics study combined with immunocytochemical analysis of undifferentiated HD-affected and unaffected human embryonic stem cells (hESC). Analysis of 1883 identifications derived from membrane and cytosolic enriched fractions revealed mitochondria as the primary dysfunctional organ in HD-affected pluripotent cells in the absence of significant differences in huntingtin protein. Furthermore, on the basis of analysis of 645 proteins found in neurodifferentiated hESC, we show a shift to transcriptional dysregulation and cytoskeletal abnormalities as the primary pathologies in HD-affected cells differentiating along neural lineages in vitro. We also show this is concomitant with an up-regulation in expression of huntingtin protein in HD-affected cells. This study demonstrates the utility of a model that recapitulates HD pathology and offers insights into disease initiation, etiology, progression, and potential therapeutic intervention.

B03 Making (anti-) Sense Out Of Huntingtin Levels In Huntington Disease

<h3></h3> Huntington disease (HD) is an autosomal dominant neurodegenerative disorder, characterised 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. In this study we used a unique collection of human post-mortem HD brain tissue and fibroblast cells to investigate huntingtin mRNA and protein expression, as well as huntingtin antisense isoforms. In adult-onset HD brain samples, there was only a small but significant lower expression of mutant huntingtin mRNA compared to wild-type huntingtin mRNA, while protein expression levels were equal. Juvenile HD subjects did show a lower protein expression of mutant huntingtin compared to wild-type huntingtin protein. Additionally, in brain tissue we did not find any evidence for a reduced expression of huntingtin antisense, as we showed HTTAS_v1 expression in a homozygous HD patient. Finally, we have identified a novel huntingtin antisense isoform and named it HTTAS_v2.2. Our study shows an intricate mechanism of huntingtin RNA and protein regulation of expression with slightly less mutant huntingtin mRNA, but equal wild-type and mutant huntingtin protein levels in adult-onset HD, indicating subtle differences in huntingtin protein expression between adult-onset and juvenile HD.

B01 Development Of Aptamers For The Specific Detection Of The Amino-terminal Region Of The Huntingtin Protein

Introduction The huntingtin protein (HTT) has 3144 amino acids and undergoes numerous post-translational modifications. Its amino-terminal end is highly conserved and seems to be involved in huntingtin-huntingtin interactions. Several antibodies have been developed for detecting the amino-terminal domain of HTT (including N17, the CAG tract and the polyProline region), but few have been shown to detect endogeneous HTT expression using histological means. Objectives To develop “custom-made” aptamers that can be used to detect HTT in neuritic aggregates in human and rodent histological material. Methods Aptamers are synthetic DNA-based polymers selected in vitro from random-nucleotide combination libraries. For this project, we used single-chain oligonucleotides due to their flexible nature. These oligonucleotides have been selected against a synthetic albumin-bound peptide and with a polypeptide corresponding to the first exon of HTT (67-amino acid-long polypeptide plus a poly-Q stretch of 23 glutamine residues) bound to glutathione-S-transferase (GST) protein. Results The aptamers obtained varied in the recognition of the recombinant and the native protein in Western-Blots and immunocytochemical analysis of histological sections. An aptamer against the first 12 AA of HTT (termed HTT5) was obtained, which is able to recognise not only normal HTT in control brain sections but also mutant HTT in neuritic aggregates and intranuclear inclusions in histological brain sections from Huntington’s disease patients. Conclusions Aptamer HTT5 is a new tool for the detection of normal and mutant HTT that can be used for the specific recognition of this protein in cells and tissues from animal models and HD-affected 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.

Six-month partial suppression of Huntingtin is well tolerated in the adult rhesus striatum.

Huntington's disease is caused by expression of a mutant form of Huntingtin protein containing an expanded polyglutamine repeat. One possible treatment for Huntington's disease may be to reduce expression of mutant Huntingtin in the brain via RNA interference. Unless the therapeutic molecule is designed to be allele-specific, both wild-type and mutant protein will be suppressed by an RNA interference treatment. A key question is whether suppression of wild-type as well as mutant Huntingtin in targeted brain regions can be tolerated and result in a net benefit to patients with Huntington's disease. Whether Huntingtin performs essential functions in the adult brain is unclear. Here, we tested the hypothesis that the adult primate brain can tolerate moderately reduced levels of wild-type Huntingtin protein for an extended period of time. A serotype 2 adeno-associated viral vector encoding for a short hairpin RNA targeting rhesus huntingtin messenger RNA (active vector) was bilaterally injected into the striatum of four adult rhesus monkeys. Four additional animals received a comparable vector encoding a scrambled control short hairpin RNA (control vector). General health and motor behaviour were monitored for 6 months. Upon termination, brain tissues were sampled and assessed blindly for (i) huntingtin messenger RNA knockdown; (ii) Huntingtin protein expression; and (iii) neuropathological changes. Reduction in wild-type huntingtin messenger RNA levels averaging ∼30% was measured in the striatum of active vector recipients 6 months post-injection. A widespread reduction in Huntingtin protein levels was also observed by immunohistochemistry in these animals, with an average protein reduction of ∼45% relative to controls measured by western blot analysis in the putamen of active vector recipients. As with control vector recipients, no adverse effects were observed behaviourally, and no neurodegeneration was found on histological examination of active vector recipients. Our results suggest that long-term partial suppression of wild-type Huntingtin may be safe, and thus if a comparable level of suppression of mutant Huntingtin is beneficial, then partial suppression of both wild-type and mutant Huntingtin may result in a net benefit in patients with heterozygous Huntington's disease.

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.