Htt Fragments Research Articles

Dysregulation of non-coding RNA expression in Huntington disease model mouse

Background: Huntington disease (HD) is an inherited neurodegenerative disorder that causes chorea, psychiatric disturbances and dementia. In the brains of HD patients, neurodegeneration is most pronounced in the striatum, and medium spiny neurons (MSN) are lost. It is known that HD is caused by expanded CAG repeats encoding polyglutamine in the exon1 of Huntingtin (Htt). In the pathological situation, the N-terminal fragment of mutant Htt forms neuronal intranuclear inclusions in the brain and disrupts the activities of transcriptional machinery such as transcription factors. According to some previous studies, the expressions of some non-coding RNAs (ncRNAs) are dysregulated in HD patients.

Post-Translational Modifications (PTMs), Identified on Endogenous Huntingtin, Cluster within Proteolytic Domains between HEAT Repeats.

Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, which causes Huntington's disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, but none of them were designed to systematically characterize PTMs on the endogenous full-length Htt protein. We found that full-length endogenous Htt, which was immunoprecipitated from HD knock-in mouse and human post-mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free and mass tag labeling-based approaches, we identified near 40 PTMs, of which half are novel (data are available via ProteomeXchange with identifier PXD005753). Most PTMs were located in clusters within predicted unstructured domains rather than within the predicted α-helical structured HEAT repeats. Using quantitative mass spectrometry, we detected significant differences in the stoichiometry of several PTMs between HD and WT mouse brain. The mass-spectrometry identification and quantitation were verified using phospho-specific antibodies for selected PTMs. To further validate our findings, we introduced individual PTM alterations within full-length Htt and identified several PTMs that can modulate its subcellular localization in striatal cells. These findings will be instrumental in further assembling the Htt PTM framework and highlight several PTMs as potential therapeutic targets for HD.

The dynamics of early-state transcriptional changes and aggregate formation in a Huntington\u2019s disease cell model

BackgroundHuntington’s disease (HD) is a fatal neurodegenerative disorder caused by a CAG expansion in the Huntingtin (HTT) gene. Proteolytic cleavage of mutant huntingtin (Htt) protein with an expanded polyglutamine (polyQ) stretch results in production of Htt fragments that aggregate and induce impaired ubiquitin proteasome, mitochondrial functioning and transcriptional dysregulation. To understand the time-resolved relationship between aggregate formation and transcriptional changes at early disease stages, we performed temporal transcriptome profiling and quantification of aggregate formation in living cells in an inducible HD cell model.ResultsRat pheochromocytoma (PC12) cells containing a stably integrated, doxycycline-inducible, eGFP-tagged N-terminal human Htt fragment with an expanded polyQ domain were used to analyse gene expression changes at different stages of mutant Htt aggregation. At earliest time points after doxycycline induction no detectable aggregates and few changes in gene expression were observed. Aggregates started to appear at intermediate time points. Aggregate formation and subsequent enlargement of aggregates coincided with a rapid increase in the number of differentially expressed (DE) genes. The increase in number of large aggregates coincided with a decrease in the number of smaller aggregates whereas the transcription profile reverted towards the profile observed before mutant Htt induction. Cluster-based analysis of the 2,176 differentially expressed genes revealed fourteen distinct clusters responding differently over time. Functional enrichment analysis of the two major gene clusters revealed that genes in the up-regulated cluster were mainly involved in metabolic (antioxidant activity and cellular ketone metabolic processes) and genes in the down-regulated cluster in developmental processes, respectively. Promoter-based analysis of the identified gene clusters resulted in identification of a transcription factor network of which several previously have been linked to HD.ConclusionsWe demonstrate a time-resolved relationship between Htt aggregation and changes in the transcriptional profile. We identified two major gene clusters showing involvement of (i) mitochondrial dysfunction and (ii) developmental processes implying cellular homeostasis defects. We identified novel and known HD-linked transcription factors and show their interaction with known and predicted regulatory proteins. Our data provide a novel resource for hypothesis building on the role of transcriptional key regulators in early stages of HD and possibly other polyQ-dependent diseases.

N-Terminal Fragments of Huntingtin Longer than Residue 170 form Visible Aggregates Independently to Polyglutamine Expansion.

A hallmark of Huntington's disease is the progressive aggregation of full length and N-terminal fragments of polyglutamine (polyQ)-expanded Huntingtin (Htt) into intracellular inclusions. The production of N-terminal fragments appears important for enabling pathology and aggregation; and hence the direct expression of a variety of N-terminal fragments are commonly used to model HD in animal and cellular models. It remains unclear how the length of the N-terminal fragments relates to polyQ - mediated aggregation. We investigated the fundamental intracellular aggregation process of eight different-length N-terminal fragments of Htt in both short (25Q) and long polyQ (97Q). N-terminal fragments were fused to fluorescent proteins and transiently expressed in mammalian cell culture models. These included the classic exon 1 fragment (90 amino acids) and longer forms of 105, 117, 171, 513, 536, 552, and 586 amino acids based on wild-type Htt (of 23Q) sequence length nomenclature. N-terminal fragments of less than 171 amino acids only formed inclusions in polyQ-expanded form. By contrast the longer fragments formed inclusions irrespective of Q-length, with Q-length playing a negligible role in extent of aggregation. The inclusions could be classified into 3 distinct morphological categories. One type (Type A) was universally associated with polyQ expansions whereas the other two types (Types B and C) formed independently of polyQ length expansion. PolyQ-expansion was only required for fragments of less than 171 amino acids to aggregate. Longer fragments aggregated predominately through a non-polyQ mechanism, involving at least one, and probably more distinct clustering mechanisms.

A new Caenorhabditis elegans model of human huntingtin 513 aggregation and toxicity in body wall muscles.

Expanded polyglutamine repeats in different proteins are the known determinants of at least nine progressive neurodegenerative disorders whose symptoms include cognitive and motor impairment that worsen as patients age. One such disorder is Huntington’s Disease (HD) that is caused by a polyglutamine expansion in the human huntingtin protein (htt). The polyglutamine expansion destabilizes htt leading to protein misfolding, which in turn triggers neurodegeneration and the disruption of energy metabolism in muscle cells. However, the molecular mechanisms that underlie htt proteotoxicity have been somewhat elusive, and the muscle phenotypes have not been well studied. To generate tools to elucidate the basis for muscle dysfunction, we engineered Caenorhabditis elegans to express a disease-associated 513 amino acid fragment of human htt in body wall muscle cells. We show that this htt fragment aggregates in C. elegans in a polyglutamine length-dependent manner and is toxic. Toxicity manifests as motor impairment and a shortened lifespan. Compared to previous models, the data suggest that the protein context in which a polyglutamine tract is embedded alters aggregation propensity and toxicity, likely by affecting interactions with the muscle cell environment.

CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins.

ABSTRACTProteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms.

Polyglutamine Tract Expansion Increases S-Nitrosylation of Huntingtin and Ataxin-1.

Expansion of the polyglutamine (polyQ) tract in the huntingtin (Htt) protein causes Huntington’s disease (HD), a fatal inherited movement disorder linked to neurodegeneration in the striatum and cortex. S-nitrosylation and S-acylation of cysteine residues regulate many functions of cytosolic proteins. We therefore used a resin-assisted capture approach to identify these modifications in Htt. In contrast to many proteins that have only a single S-nitrosylation or S-acylation site, we identified sites along much of the length of Htt. Moreover, analysis of cells expressing full-length Htt or a large N-terminal fragment of Htt shows that polyQ expansion strongly increases Htt S-nitrosylation. This effect appears to be general since it is also observed in Ataxin-1, which causes spinocerebellar ataxia type 1 (SCA1) when its polyQ tract is expanded. Overexpression of nitric oxide synthase increases the S-nitrosylation of normal Htt and the frequency of conspicuous juxtanuclear inclusions of Htt N-terminal fragments in transfected cells. Taken together with the evidence that S-nitrosylation of Htt is widespread and parallels polyQ expansion, these subcellular changes show that S-nitrosylation affects the biology of this protein in vivo.

An Intein-based Strategy for the Production of Tag-free Huntingtin Exon 1 Proteins Enables New Insights into the Polyglutamine Dependence of Httex1 Aggregation and Fibril Formation

The first exon of the Huntingtin protein (Httex1) is one of the most actively studied Htt fragments because its overexpression in R6/2 transgenic mice has been shown to recapitulate several key features of Huntington disease. However, the majority of biophysical studies of Httex1 are based on assessing the structure and aggregation of fusion constructs where Httex1 is fused to large proteins, such as glutathione S-transferase, maltose-binding protein, or thioredoxin, or released in solution upon in situ cleavage of these proteins. Herein, we report an intein-based strategy that allows, for the first time, the rapid and efficient production of native tag-free Httex1 with polyQ repeats ranging from 7Q to 49Q. Aggregation studies on these proteins enabled us to identify interesting polyQ-length-dependent effects on Httex1 oligomer and fibril formation that were previously not observed using Httex1 fusion proteins or Httex1 proteins produced by in situ cleavage of fusion proteins. Our studies revealed the inability of Httex1-7Q/15Q to undergo amyloid fibril formation and an inverse correlation between fibril length and polyQ repeat length, suggesting possible polyQ length-dependent differences in the structural properties of the Httex1 aggregates. Altogether, our findings underscore the importance of working with tag-free Httex1 proteins and indicate that model systems based on non-native Httex1 sequences may not accurately reproduce the effect of polyQ repeat length and solution conditions on Httex1 aggregation kinetics and structural properties.

Studies on Domain Specific Aggregation Behavior of Huntingtin Exon1

The pathogenesis of Huntington's disease results from polyQ expansions (>36Q) in the Huntingtin (Htt) protein.1 These polyQ expansions make Htt less soluble and promote the formation of misfolded oligomers, fibrils and aggregates in Huntington's disease.2 Efforts to prevent aggregation will greatly benefit from a detailed molecular understanding of the misfolding process. However, htt misfolding has been difficult to study as the protein is tough to handle and examine spectroscopically. Here we developed a novel purification scheme to generate recombinant, clean and monomeric Htt exon1, an aggregation prone fragment of htt that is highly expressed in disease. The misfolding of spin labeled derivatives of Htt exon1 were then monitored by EPR in order to obtain site-specific folding information for residues in different regions of the protein. Our data indicate that residues in the N-terminal N17, the middle polyQ region and the C-terminal, proline rich region all undergo structural changes on different time scales. Moreover, we have also found that the aggregation kinetics and mechanisms of aggregation significantly depend on Q length. Overall, the data indicate distinct phases in which different regions of the molecule undergo conformational changes. Each of these stages may afford the design of different compounds intended at altering the aggregation process.1. Bates, GP.; Harper, PS.; Jones, L., editors. Huntington's Disease. Oxford University Press; Oxford, U.K.: 2002.2. Bates, GP.; Benn, C. The polyglutamine diseases. In: Bates, GP.; Harper, PS.; Jones, L., editors. Huntington's Disease. Oxford University Press; Oxford, U.K.: 2002. p. 429-472.

Calcium Handling by Endoplasmic Reticulum and Mitochondria in a Cell Model of Huntington's Disease.

Huntington disease (HD) is caused by the CAG (Q) expansion in exon 1 of the IT15 gene encoding a polyglutamine (poly-Q) stretch of the Huntingtin protein (Htt). In the wild type protein, the repeats specify a stretch of up 34 Q in the N-terminal portion of Htt. In the pathological protein (mHtt) the poly-Q tract is longer. Proteolytic cleavage of the protein liberates an N-terminal fragment containing the expanded poly-Q tract becomes harmful to cells, in particular to striatal neurons. The fragments cause the transcriptional dysfunction of genes that are essential for neuronal survival. Htt, however, could also have non-transcriptional effects, e.g. it could directly alter Ca2+ homeostasis and/or mitochondrial morphology and function. Ca2+ dyshomeostasis and mitochondrial dysfunction are considered important in the molecular aetiology of the disease. Here we have analyzed the effect of the overexpression of Htt fragments (18Q, wild type form, wtHtt and 150Q mutated form, mHtt) on Ca2+ homeostasis in striatal neuronal precursor cells (Q7/7). We have found that the transient overexpression of the Htt fragments increases Ca2+ transients in the mitochondria of cells stimulated with Ca2+-mobilizing agonists. The bulk Ca2+ transients in the cytosol were unaffected, but the Ca2+ content of the endoplasmic reticulum was significantly decreased in the case of mHtt expression. To rule out possible transcriptional effects due to the presence of mHtt, we have measured the mRNA level of a subunit of the respiratory chain complex II, whose expression is commonly altered in many HD models. No effects on the mRNA level was found suggesting that, in our experimental condition, transcriptional action of Htt is not occurring and that the effects on Ca2+ homeostasis were dependent to non-transcriptional mechanisms.

How does resveratrol influence the genesis of some neurodegenerative diseases?

How does resveratrol influence the genesis of some neurodegenerative diseases?

The N17 domain mitigates nuclear toxicity in a novel zebrafish Huntington's disease model.

BackgroundAlthough the genetic cause for Huntington’s disease (HD) has been known for over 20 years, the mechanisms that cause the neurotoxicity and behavioral symptoms of this disease are not well understood. One hypothesis is that N-terminal fragments of the HTT protein are the causative agents in HD and that peptide sequences adjacent to the poly-glutamine (Q) repeats modify its toxicity. Here we test the function of the N-terminal 17 amino acids (N17) in the context of the exon 1 fragment of HTT in a novel, inducible zebrafish model of HD.ResultsDeletion of N17 coupled with 97Q expansion (mHTT-ΔN17-exon1) resulted in a robust, rapidly progressing movement deficit, while fish with intact N17 and 97Q expansion (mHTT-exon1) have more delayed-onset movement deficits with slower progression. The level of mHTT-ΔN17-exon1 protein was significantly higher than mHTT-exon1, although the mRNA level of each transgene was marginally different, suggesting that N17 may regulate HTT protein stability in vivo. In addition, cell lineage specific induction of the mHTT-ΔN17-exon1 transgene in neurons was sufficient to recapitulate the consequences of ubiquitous transgene expression. Within neurons, accelerated nuclear accumulation of the toxic HTT fragment was observed in mHTT-ΔN17-exon1 fish, demonstrating that N17 also plays an important role in sub-cellular localization in vivo.ConclusionsWe have developed a novel, inducible zebrafish model of HD. These animals exhibit a progressive movement deficit reminiscent of that seen in other animal models and human patients. Deletion of the N17 terminal amino acids of the huntingtin fragment results in an accelerated HD-like phenotype that may be due to enhanced protein stability and nuclear accumulation of HTT. These transgenic lines will provide a valuable new tool to study mechanisms of HD at the behavioral, cellular, and molecular levels. Future experiments will be focused on identifying genetic modifiers, mechanisms and therapeutics that alleviate polyQ aggregation in the nucleus of neurons.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0063-2) contains supplementary material, which is available to authorized users.

Mitochondrial division inhibitor 1 protects against mutant huntingtin-induced abnormal mitochondrial dynamics and neuronal damage in Huntington's disease.

The objective of this study was to determine the protective effects of the mitochondrial division inhibitor 1 (Mdivi1) in striatal neurons that stably express mutant Htt (STHDhQ111/Q111) and wild-type (WT) Htt (STHDhQ7/Q7). Using gene expression analysis, biochemical methods, transmission electron microscopy (TEM) and confocal microscopy methods, we studied (i) mitochondrial and synaptic activities by measuring mRNA and the protein levels of mitochondrial and synaptic genes, (ii) mitochondrial function and (iii) ultra-structural changes in mutant Htt neurons relative to WT Htt neurons. We also studied these parameters in Mdivil-treated and untreated WT and mutant Htt neurons. Increased expressions of mitochondrial fission genes, decreased expression of fusion genes and synaptic genes were found in the mutant Htt neurons relative to the WT Htt neurons. Electron microscopy of the mutant Htt neurons revealed a significantly increased number of mitochondria, indicating that mutant Htt fragments mitochondria. Biochemical analysis revealed defective mitochondrial functioning. In the Mdivil-treated mutant Htt neurons, fission genes were down-regulated, and fusion genes were up-regulated, suggesting that Mdivil decreases fission activity. Synaptic genes were up-regulated, and mitochondrial function was normal in the Mdivi1-treated mutant Htt neurons. Immunoblotting findings of mitochondrial and synaptic proteins agreed with mRNA findings. The TEM studies revealed that increased numbers of structurally intact mitochondria were present in Mdivi1-treated mutant Htt neurons. Increased synaptic and mitochondrial fusion genes and decreased fission genes were found in the Mdivi1-treated WT Htt neurons, indicating that Mdivi1 beneficially affects healthy neurons. Taken together, these findings suggest that Mdivi1 is protective against mutant Htt-induced mitochondrial and synaptic damage in HD neurons and that Mdivi1 may be a promising molecule for the treatment of HD patients.

Ectopic expression of the striatal-enriched GTPase Rhes elicits cerebellar degeneration and an ataxia phenotype in Huntington's disease

Huntington's disease (HD) is caused by an expansion of glutamine repeats in the huntingtin protein (mHtt) that invokes early and prominent damage of the striatum, a region that controls motor behaviors. Despite its ubiquitous expression, why certain brain regions, such as the cerebellum, are relatively spared from neuronal loss by mHtt remains unclear. Previously, we implicated the striatal-enriched GTPase, Rhes (Ras homolog enriched in the striatum), which binds and SUMOylates mHtt and increases its solubility and cellular cytotoxicity, as the cause for striatal toxicity in HD. Here, we report that Rhes deletion in HD mice (N171-82Q), which express the N-terminal fragment of human Htt with 82 glutamines (Rhes−/−/N171-82Q), display markedly reduced HD-related behavioral deficits, and absence of lateral ventricle dilatation (secondary to striatal atrophy), compared to control HD mice (N171-82Q). To further validate the role of GTPase Rhes in HD, we tested whether ectopic Rhes expression would elicit a pathology in a brain region normally less affected in HD. Remarkably, ectopic expression of Rhes in the cerebellum of N171-82Q mice, during the asymptomatic period led to an exacerbation of motor deficits, including loss of balance and motor incoordination with ataxia-like features, not apparent in control-injected N171-82Q mice or Rhes injected wild-type mice. Pathological and biochemical analysis of Rhes-injected N171-82Q mice revealed a cerebellar lesion with marked loss of Purkinje neuron layer parvalbumin-immunoreactivity, induction of caspase 3 activation, and enhanced soluble forms of mHtt. Similarly reintroducing Rhes into the striatum of Rhes deleted Rhes−/−Hdh150Q/150Q knock-in mice, elicited a progressive HD-associated rotarod deficit. Overall, these studies establish that Rhes plays a pivotal role in vivo for the selective toxicity of mHtt in HD.

A plant cell model of polyglutamine aggregation: Identification and characterisation of macromolecular and small-molecule anti-protein aggregation activity in vivo

In vitro studies have shown that LEA proteins from plants and invertebrates protect and stabilise other proteins under conditions of water stress, suggesting a role in stress tolerance. However, there is little information on LEA protein function in whole plants or plant cells, particularly with respect to their anti-aggregation activity. To address this, we expressed in tobacco BY-2 suspension cells an aggregation-prone protein based on that responsible for Huntington's disease (HD). In HD, abnormally long stretches of polyglutamine (polyQ) in huntingtin (Htt) protein cause aggregation of Htt fragments within cells. We constructed stably transformed BY-2 cell lines expressing enhanced green fluorescent protein (EGFP)-HttQ23 or EGFP-HttQ52 fusion proteins (encoding 23 or 52 glutamine residues, pertaining to the normal and disease states, respectively), as well as an EGFP control. EGFP-HttQ52 protein aggregated in the cytoplasm of transformed tobacco cells, which showed slow growth kinetics; in contrast, EGFP-HttQ23 or EGFP did not form aggregates and cells expressing these constructs grew normally. To test the effect of LEA proteins on protein aggregation in plant cells, we constructed cell lines expressing both EGFP-HttQ52 and LEA proteins (PM1, PM18, ZLDE-2 or AavLEA1) or a sHSP (PM31). Of these, AavLEA1 and PM31 reduced intracellular EGFP-HttQ52 aggregation and alleviated the associated growth inhibition, while PM18 and ZLDE-2 partially restored growth rates. Treatment of EGFP-HttQ52-expressing BY2 cells with the polyphenol epigallocatechin-3-gallate (EGCG) also reduced EGFP-HttQ52 aggregation and improved cell growth rate. The EGFP-HttQ52 cell line therefore has potential for characterising both macromolecular and small molecule inhibitors of protein aggregation in plant cells.

C11 Transgenic Minipigs For N-terminal Part Of Human Mutated Huntingtin: Following Disease Development

Background In order to facilitate studies of pathogenesis and therapy of Huntington’s disease (HD), we have generated transgenic minipigs using microinjection of a lentiviral vector encoding N-terminal (548 aa) part of human huntingtin (Htt) containing 126 CAG/CAA repeats under the control of the human Htt promoter. The progression of HD in human patients is predominantly linked with formation of aggregates, oligomers, and fragments of Htt. Among other factors corresponding to HD is lowered BDNF (brain-derived neurotrophic factor) expression in brain, accumulation of ACBD3 (acyl-CoA binding domain containing 3), and formation of toxic Rhes/mtHtt/ACBD3 complex. Aim To follow disease development in transgenic (TgHD) minipigs using specific biomarkers of HD. Methods At present, TgHD and WT (wild-type) minipigs of F0, F1, F2 and F3 generations are being used for invasive (24 and 36 months old animals) and non-invasive approaches (up to five years old animals). We perform biochemical assays on tissues (with main focus on brain) and cells (fibroblasts, and buccal swabs) in order to follow HD development in our model. Results To date, we have not been successful in the aggregate detection, which could be due to the young age of TgHD animals. However, we have detected Htt oligomers, several Htt fragments of lower molecular weight in samples from TgHD minipigs and not from WT siblings. In addition, we monitor altered levels of proteins previously reported as markers of HD development, namely BDNF and Rhes/mtHtt/ACBD3 complex Conclusion We provide complex information about HD phenotype development in transgenic minipig model between 24–60 months of age. Support CHDI Foundation (A-5378), TACR (TA01011466), European Regional Development FundCZ.1.05/2.1.00/03.0124

C02 Molecular Analysis Of The Zq175 Knock-in Mouse Model

Background The zQ175 knock-in line was generated by replacing mouse endogenous Htt exon 1 with a mutant version of human HTT exon 1 followed by the expansion of the CAG repeat. This line is now being used extensively throughout the HD research community. Method We have performed a molecular analysis to study changes in mutant HTT and the appearance of transcriptional dysregulation. We used immunoprecipitation and western blotting to visualise HTT fragments and found that the level of the exon 1 HTT fragment decreased from two months of age. Results Seprion ELISA has indicated that there is a statistically significant level of HTT aggregation in striatum, cortex, hippocampus, cerebellum and the brain stem from 8 months of age, and the age at which these aggregates can first be detected is under investigation. We determined the expression levels of genes known to be dysregulated in HD brains in the striatum, cortex and cerebellum. The striatal genes: Pde10a and Penk1 were dysregulated by 6 months of age and Cnr1 and Darpp32 by 8 months in heterozygous zQ175 mice with good statistical power for detecting improvements. Conclusions Therefore, we have identified molecular read-outs that can be used in the preclinical validation of therapeutic targets in heterozygous zQ175 mice.

M02 Therapeutic Benefit Of A Htt-lowering Antisense Oligonucleotide Targeting The Cag-repeat In The R6/2 Huntington's Disease Mouse Model

<h3>Background</h3> Different therapeutic approaches using antisense oligonucleotides (AONs) are currently under development for Huntington’s disease (HD). Ideally, an AON-based therapeutic approach for HD would selectively lower levels of the transcript carrying the disease-causing CAG-repeat expansion, but not affect the non-expanded transcript. We have previously demonstrated preferential knockdown of the mutant transcript <i>in vitro</i>in fibroblasts derived from HD patients using a CAG-repeat targeting AON consisting of a (CUG)7 sequence. <h3>Aims</h3> Aim of this study was to gain <i>in vivo</i>proof-of-concept with our (CUG)7 AON in a relevant HD animal model. <h3>Methods/techniques</h3> R6/2 mice transgenic for an N-terminal fragment of human HTT containing ˜150 CAG repeats (n = 30) received a total of 6 weekly ICV infusions with the (CUG)7 AON or vehicle. Bodyweight was monitored and a battery of motor tests (grip strength, rotarod, open field, 3D kinematic analysis) were performed. In addition, brain volume and striatal metabolites were quantified using MRI and MRS respectively. Two weeks after the last infusion at 12 weeks of age RNA was isolated from 7 different brain regions, followed by RT-qPCR analysis of HTT transcript levels. <h3>Results/outcome</h3> A highly signficant and strong (˜90%) reduction of mutant HTT mRNA levels was observed throughout the R6/2 brain. In addition, there was a clear positive treatment effect on motor performance, indicated by an increase in rotarod latency and vertical activity in the open field. 3D kinematic analysis of fine motor skills showed a significant improvement of several parameters impaired in R6/2 mice. A trend towards an increased whole brain and cortical volume was observed using MRI volumetry as well as a significant decrease in striatal levels of the astrocytic marker inositol, known to be elevated in the striatum of R6/2 mice and HD patients. <h3>Conclusions</h3> These data demonstrate our AON targeting the CAG-repeat has therapeutic potential for HD.

Experimental mutagenesis of huntingtin to map cleavage sites: different outcomes in cell and mouse models.

N-terminal cleavage products of mutant huntingtin (htt) generate pathologic neuronal inclusion bodies. The precise length of the htt fragment, termed Cp-A/1, that produces HD pathologic inclusions is unknown. We sought to elucidate the protein sequence elements within the N-terminus of htt that mediate its proteolysis based on a model in which engineered htt fragments terminating at residue 171 are cleaved to produce Cp-A/1 fragments. We expressed htt N171 cDNAs harboring a series of experimental mutations in the presumptive cleavage site that generates Cp-A/1 in cells to identify cleavage resistant mutants of htt N171. One of these constructs was expressed in mice, followed by analysis using immunoblots of brain extracts and immunohistochemistry of transgenic mouse brain tissues. Using the HEK293 cell model, mutagenesis studies mapped the cleavage site in htt N171 to sequences between residues 105-114. Mutation of 8 positively charged residues (H, K, R) located between residues 88 and 114 to alanine to neutralize the charge also blocked the generation of Cp-A/1 like fragments. Transgenic mice expressing this latter construct, termed N171-82Q-N8, developed phenotypes similar to previously characterized N171-82Q transgenic mice, including rotarod deficiency, intranuclear inclusions, and premature death. Surprisingly, the N171-82Q-N8 protein was efficiently cleaved in vivo to produce Cp-A/1 fragments that accumulated as insoluble inclusions. Mutagenesis of htt to identify critical amino acids that direct its cleavage predicted a role for charged residues in the sequence flanking the presumptive cleavage site. However, the role for these residues could not be confirmed in vivo. The basis for the discrepancy between predicted outcomes in HEK293 cells and the mouse models remain unresolved, but the data provide another validation of the hypothesis that Cp-A/1 fragments of mutant htt can induce HD-like phenotypes.

Soluble N-terminal fragment of mutant Huntingtin protein impairs mitochondrial axonal transport in cultured hippocampal neurons

Huntington's disease (HD) is an autosomal dominant, progressive, neurodegenerative disorder caused by an unstable expansion of CAG repeats (>35 repeats) within exon 1 of the interesting transcript 15 (IT15) gene. This gene encodes a protein called Huntingtin (Htt), and mutation of the gene results in a polyglutamine (polyQ) near the N-terminus of Htt. The N-terminal fragments of mutant Htt (mHtt), which tend to aggregate, are sufficient to cause HD. Whether these aggregates are causal or protective for HD remains hotly debated. Dysfunctional mitochondrial axonal transport is associated with HD. It remains unknown whether the soluble or aggregated form of mHtt is the primary cause of the impaired mitochondrial axonal transport in HD pathology. Here, we investigated the impact of soluble and aggregated N-terminal fragments of mHtt on mitochondrial axonal transport in cultured hippocampal neurons. We found that the N-terminal fragment of mHtt formed aggregates in almost half of the transfected neurons. Overexpression of the N-terminal fragment of mHtt decreased the velocity of mitochondrial axonal transport and mitochondrial mobility in neurons regardless of whether aggregates were formed. However, the impairment of mitochondrial axonal transport in neurons expressing the soluble and aggregated N-terminal fragments of mHtt did not differ. Our findings indicate that both the soluble and aggregated N-terminal fragments of mHtt impair mitochondrial axonal transport in cultured hippocampal neurons. We predict that dysfunction of mitochondrial axonal transport is an early-stage event in the progression of HD, even before mHtt aggregates are formed.