Human Huntington's Disease Brain Research Articles

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

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

Metabolite mapping reveals severe widespread perturbation of multiple metabolic processes in Huntington's disease human brain

Huntington's disease (HD) is a genetically-mediated neurodegenerative disorder wherein the aetiological defect is a mutation in the Huntington's gene (HTT), which alters the structure of the huntingtin protein (Htt) through lengthening of its polyglutamine tract, thus initiating a cascade that ultimately leads to premature death. However, neurodegeneration typically manifests in HD only in middle age, and mechanisms linking the causative mutation to brain disease are poorly understood. Brain metabolism is severely perturbed in HD, and some studies have indicated a potential role for mutant Htt as a driver of these metabolic aberrations. Here, our objective was to determine the effects of HD on brain metabolism by measuring levels of polar metabolites in regions known to undergo varying degrees of damage. We performed gas-chromatography/mass spectrometry-based metabolomic analyses in a case-control study of eleven brain regions in short post-mortem-delay human tissue from nine well-characterized HD patients and nine matched controls. In each patient, we measured metabolite content in representative tissue-samples from eleven brain regions that display varying degrees of damage in HD, thus identifying the presence and abundance of 63 different metabolites from several molecular classes, including carbohydrates, amino acids, nucleosides, and neurotransmitters. Robust alterations in regional brain-metabolite abundances were observed in HD patients: these included changes in levels of small molecules that play important roles as intermediates in the tricarboxylic-acid and urea cycles, and amino-acid metabolism. Our findings point to widespread disruption of brain metabolism and indicate a complex phenotype beyond the gradient of neuropathologic damage observed in HD brain.

DNA methylation profiling in human Huntington's disease brain.

Despite extensive progress in Huntington's disease (HD) research, very little is known about the association of epigenetic variation and HD pathogenesis in human brain tissues. Moreover, its contribution to the tissue-specific transcriptional regulation of the huntingtin gene (HTT), in which HTT expression levels are highest in brain and testes, is currently unknown. To investigate the role of DNA methylation in HD pathogenesis and tissue-specific expression of HTT, we utilized the Illumina HumanMethylation450K BeadChip array to measure DNA methylation in a cohort of age-matched HD and control human cortex and liver tissues. In cortex samples, we found minimal evidence of HD-associated DNA methylation at probed sites after correction for cell heterogeneity but did observe an association with the age of disease onset. In contrast, comparison of matched cortex and liver samples revealed tissue-specific DNA methylation of the HTT gene region at 38 sites (FDR < 0.05). Importantly, we identified a novel differentially methylated binding site in the HTT proximal promoter for the transcription factor CTCF. This CTCF site displayed increased occupancy in cortex, where HTT expression is higher, compared with the liver. Additionally, CTCF silencing reduced the activity of an HTT promoter-reporter construct, suggesting that CTCF plays a role in regulating HTT promoter function. Overall, although we were unable to detect HD-associated DNA methylation alterations at queried sites, we found that DNA methylation may be correlated to the age of disease onset in cortex tissues. Moreover, our data suggest that DNA methylation may, in part, contribute to tissue-specific HTT transcription through differential CTCF occupancy.

Interactome network analysis identifies multiple caspase-6 interactors involved in the pathogenesis of HD.

Caspase-6 (CASP6) has emerged as an important player in Huntington disease (HD), Alzheimer disease (AD) and cerebral ischemia, where it is activated early in the disease process. CASP6 also plays a key role in axonal degeneration, further underscoring the importance of this protease in neurodegenerative pathways. As a protein's function is modulated by its protein-protein interactions, we performed a high-throughput yeast-2-hybrid (Y2H) screen against ∼17,000 human proteins to gain further insight into the function of CASP6. We identified a high-confidence list of 87 potential CASP6 interactors. From this list, 61% are predicted to contain a CASP6 recognition site. Of nine candidate substrates assessed, six are cleaved by CASP6. Proteins that did not contain a predicted CASP6 recognition site were assessed using a LUMIER assay approach, and 51% were further validated as interactors by this method. Of note, 54% of the high-confidence interactors identified show alterations in human HD brain at the mRNA level, and there is a significant enrichment for previously validated huntingtin (HTT) interactors. One protein of interest, STK3, a pro-apoptotic kinase, was validated biochemically to be a CASP6 substrate. Furthermore, our results demonstrate that in striatal cells expressing mutant huntingtin (mHTT), an increase in full length and fragment levels of STK3 are observed. We further show that caspase-3 is not essential for the endogenous cleavage of STK3. Characterization of the interaction network provides important new information regarding key pathways of interactors of CASP6 and highlights potential novel therapeutic targets for HD, AD and cerebral ischemia.

RNA Sequence Analysis of Human Huntington Disease Brain Reveals an Extensive Increase in Inflammatory and Developmental Gene Expression

Huntington’s Disease (HD) is a devastating neurodegenerative disorder that is caused by an expanded CAG trinucleotide repeat in the Huntingtin (HTT) gene. Transcriptional dysregulation in the human HD brain has been documented but is incompletely understood. Here we present a genome-wide analysis of mRNA expression in human prefrontal cortex from 20 HD and 49 neuropathologically normal controls using next generation high-throughput sequencing. Surprisingly, 19% (5,480) of the 28,087 confidently detected genes are differentially expressed (FDR<0.05) and are predominantly up-regulated. A novel hypothesis-free geneset enrichment method that dissects large gene lists into functionally and transcriptionally related groups discovers that the differentially expressed genes are enriched for immune response, neuroinflammation, and developmental genes. Markers for all major brain cell types are observed, suggesting that HD invokes a systemic response in the brain area studied. Unexpectedly, the most strongly differentially expressed genes are a homeotic gene set (represented by Hox and other homeobox genes), that are almost exclusively expressed in HD, a profile not widely implicated in HD pathogenesis. The significance of transcriptional changes of developmental processes in the HD brain is poorly understood and warrants further investigation. The role of inflammation and the significance of non-neuronal involvement in HD pathogenesis suggest anti-inflammatory therapeutics may offer important opportunities in treating HD.

RAN Translation in Huntington Disease

Huntington disease (HD) is caused by a CAG ⋅ CTG expansion in the huntingtin (HTT) gene. While most research has focused on the HTT polyGln-expansion protein, we demonstrate that four additional, novel, homopolymeric expansion proteins (polyAla, polySer, polyLeu, and polyCys) accumulate in HD human brains. These sense and antisense repeat-associated non-ATG (RAN) translation proteins accumulate most abundantly in brain regions with neuronal loss, microglial activation and apoptosis, including caudate/putamen, white matter, and, in juvenile-onset cases, also the cerebellum. RAN protein accumulation and aggregation are length dependent, and individual RAN proteins are toxic to neural cells independent of RNA effects. These data suggest RAN proteins contribute to HD and that therapeutic strategies targeting both sense and antisense genes may be required for efficacy in HD patients. This is the first demonstration that RAN proteins are expressed across an expansion located in an open reading frame and suggests RAN translation may also contribute to other polyglutamine diseases.

Evidence for altered cholesterol metabolism in Huntington's disease post mortem brain tissue

Cholesterol plays an essential role in membrane structure and function, being especially important in the brain. Alteration of brain cholesterol synthesis and metabolism has been demonstrated in several Huntington's disease (HD) mouse and cell models; however, less is known about these alterations in human tissue. This study aimed to identify alterations to cholesterol synthetic and metabolic pathways in human HD brain tissue. A broad range of cholesterol synthetic precursors, metabolites and oxidation products were measured by gas chromatography-tandem mass spectrometry in five regions of human post mortem HD brain and compared with age- and sex-matched control tissues. The level of enzymes that regulate cholesterol homeostasis, cholesterol 24-hydroxylase and delta(24)-sterol reductase were investigated by Western blotting and qPCR in putamen. The most significant changes were localized to the putamen, where a 60% decrease in 24(S)-hydroxycholesterol, 30% increase in cholesterol and 100-200% increase in synthetic precursors (lathosterol, zymosterol and desmosterol) was detected. The enzymes cholesterol 24-hydroxylase and delta(24)-sterol reductase were also significantly decreased in HD putamen as compared with control tissues. Free radical-generated cholesterol oxidation products 7-keto cholesterol and 7β-hydroxycholesterol were also increased by 50-70% in HD putamen. Human HD brain has significantly decreased cholesterol metabolism and disrupted cholesterol homeostasis. Our data also indicate that lipid oxidative stress accompanies HD pathology.

The RAGE receptor and its ligands are highly expressed in astrocytes in a grade-dependant manner in the striatum and subependymal layer in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an expansion of the CAG repeat in the huntingtin gene. One of the brain changes that occurs in HD is the expression of the receptor for advanced glycation end products (RAGE), a receptor protein capable of activating multiple signalling pathways by interacting with a range of ligands leading to either beneficial or harmful effects to the cell. Here, we demonstrate in human HD brains a high degree of co-localization of RAGE with its putative ligands S100B and N-carboxymethyllysine (CML) in the caudate nucleus (CN) and the subependymal layer (SEL). The level of co-staining for both RAGE-S100B and RAGE-CML was the highest in the astrocytes but was low in neurons and microglia. The immunostaining for RAGE, S100B and CML extended in a medio-lateral (SEL-CN) direction with increasing grade, such that any change in the expression and co-localization pattern between grades was less prominent in the lateral CN. Additionally, signalling molecules that are downstream of RAGE activation showed changes in their activation status in HD brains. A larger number of RAGE-positive astrocytic cells had NF-kB translocated to the nucleus and the level of phospho-ERK1/2 was also increased in HD brains. Interestingly, the level of mDia-1, that interacts directly with the cytoplasmic domain of RAGE, decreased in HD. Overall, the results suggest a correlation between the functions of RAGE and the HD pathology, but the influence of RAGE on astrocytes and the impact of this on HD progression requires further study. RAGE (receptor for advanced glycation end products) binds multiple types of ligand to produce either neurotrophic or neurotoxic effects. Immunohistochemical staining of HD human brains showed that both RAGE and its ligands were expressed primarily in astrocytes. The pattern of staining corresponded to the grade and region-wise pattern of neurodegeneration suggesting a possible role for RAGE in HD pathology.

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.

Partial rescue of some features of Huntington Disease in the genetic absence of caspase-6 in YAC128 mice

Huntington Disease (HD) is a progressive neurodegenerative disease caused by an elongated CAG repeat in the huntingtin (HTT) gene that encodes a polyglutamine tract in the HTT protein. Proteolysis of the mutant HTT protein (mHTT) has been detected in human and murine HD brains and is implicated in the pathogenesis of HD. Of particular importance is the site at amino acid (aa) 586 that contains a caspase-6 (Casp6) recognition motif.Activation of Casp6 occurs presymptomatically in human HD patients and the inhibition of mHTT proteolysis at aa586 in the YAC128 mouse model results in the full rescue of HD-like phenotypes. Surprisingly, Casp6 ablation in two different HD mouse models did not completely prevent the generation of this fragment, and therapeutic benefits were limited, questioning the role of Casp6 in the disease.We have evaluated the impact of the loss of Casp6 in the YAC128 mouse model of HD. Levels of the mHTT-586 fragment are reduced but not absent in the absence of Casp6 and we identify caspase 8 as an alternate enzyme that can generate this fragment. In vivo, the ablation of Casp6 results in a partial rescue of body weight gain, normalized IGF-1 levels, a reversal of the depression-like phenotype and decreased HTT levels. In the YAC128/Casp6−/− striatum there is a concomitant reduction in p62 levels, a marker of autophagic activity, suggesting increased autophagic clearance. These results implicate the HTT-586 fragment as a key contributor to certain features of HD, irrespective of the enzyme involved in its generation.

M07 Examining Conditional Caspase-6 Deficiency As A Therapeutic In Huntington Disease

<h3>Background</h3> Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterised by motor, cognitive, and psychiatric symptoms. HD is caused by a CAG repeat expansion in the huntingtin (HTT) gene resulting in the production of mutant huntingtin (mHTT). Caspase-6 (C6) is a protease that plays a central role in programmed cell death and has recently been implicated in several neurodegenerative diseases. Increased C6 activation is observed in human HD brains and in mouse models of HD, and genetic or peptide inhibition of C6 activity protects neurons from degeneration. C6 is one of several proteases that cleaves mHTT, and our lab has previously demonstrated that preventing C6-mediated cleavage of mHTT at the 586 amino acid site preserves striatal volume and rescues cognitive and motor deficits in the YAC128 mouse model of HD. These data point to an essential role for C6 in the pathogenesis of HD and provide a rationale for the inhibition of C6 as a therapeutic approach. <h3>Aim</h3> The goal of this study is to investigate the effects of genetically knocking out C6 in adulthood on the phenotype of the YAC128 mouse model of HD. <h3>Method</h3> Excision of the C6 gene in YAC128 mice will be carried out using the Cre/loxP system and by administering tamoxifen at 6 weeks of age. Treated mice will be examined for behavioural, neuropathological and biochemical endpoints. <h3>Results</h3> After extensive optimisation of tamoxifen administration, a treatment protocol was established to enable adequate excision of C6 in the brain. Mice have received 19 consecutive days of IP injections at 100mg/kg to induce excision and treated mice will be examined for behavioural, neuropathological and biochemical endpoints. <h3>Conclusions</h3> Results from this study will provide insight into the effect of adult C6 inhibition on HD pathogenesis and will further validate C6 as a therapeutic target in HD.

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.

B15 Dysregulation of Gene Expression in the Striatum of Bachd Rats and Abnormalities Involved on Molecular and Protein Levels

Background Huntington disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by an expansion of CAG repeats in the gene coding for the huntingtin protein (htt). Mutant htt has been proposed to cause neuronal dysfunction and neuronal loss through multiple mechanisms. Transcriptional changes may be a core pathogenic feature of HD. Methods Utilising the Affymetrix platform we performed a genome-wide RNA expression analysis in two BACHD transgenic rat lines (TG5 and TG9) at 12 months of age, which carry full-length human mutant htt with different expression levels. Results Applying a threshold of significance of 0.01, we found 1608 genes and 871 genes differently expressed in TG5 and TG9 rats compared to wild type littermates, respectively. We only chose highly up/down regulated genes for further analysis by setting an additional threshold of 1.5 for fold change. Comparison with data from human HD brains revealed a high concordance in involved functional and IPA (Ingenuity Pathway Analysis) canonical pathways relevant in HD. In addition, we investigated the causes leading to gene expression changes on molecular and protein levels in BACHD rats including the amount of wild type htt, interaction of mhtt with polyQ containing transcription factors as well as chromatin structure. Conclusions Results of this study demonstrate that this BACHD rat model recapitulates accurately gene expression changes of the human disease and therefore supports its application in preclinical research. Analysis of causative events in BACHD rats confirmed some findings in in vitro test, and also provides a novel insight into initiators of perturbed gene transcription in HD.

Cannabinoid receptor CB2 is expressed on vascular cells, but not astroglial cells in the post-mortem human Huntington's disease brain

Huntington's disease (HD) is an inherited neurological disease with motor, cognitive and psychiatric symptoms. Characterised by neuronal degeneration, HD pathology is initially apparent in the striatum and cortex. Considerable research has recently suggested that the neurological immune response apparent in brain injury and disease may provide a valuable therapeutic target. Cannabinoid CB2 receptors are localised and up-regulated on a number of peripheral immune cell types following inflammation and injury. However, their cellular location within the human brain during inflammation has not been well characterised.The present study shows CB2 is expressed in human post-mortem striatum in HD. Quantification revealed a trend towards an increase in CB2 staining with disease, but no significant difference was measured compared to neurologically normal controls. In HD striatal tissue, there is an up-regulation of the brains' resident immune cells, with a significant increase in GFAP-positive astrocyte staining at both grade 1 (685±118%) and grade 3 (1145±163%) and Iba1-positive microglia at grade 1 (299±27%) but not grade 3 (119±48%), compared to neurologically normal controls. Both cell types exhibit irregular cell morphology, particularly at higher grades.Using double-labelled immunohistochemistry CB2 receptors are demonstrated not to be expressed on microglia or astrocytes and instead appear to be localised on CD31-positive blood vessel endothelium and vascular smooth muscle. Co-expression analysis suggests that CB2 may be more highly expressed on CD31 positive cells in HD brains than in control brains. Contrasting with evidence from rodent studies suggesting CB2 glial cell localisation, our observation that CB2 is present on blood vessel cells, with increased CD31 co-localisation in HD may represent a new context for CB2 therapeutic approaches to neurodegenerative diseases.

Kinase signalling in Huntington's disease.

Alterations in numerous signal transduction pathways and aberrant activity of specific kinases have been identified in multiple cell and mouse models of Huntington's disease (HD), as well as in human HD brain. The balance and integration of a network of kinase signalling pathways is paramount for the regulation of a wide range of cellular and physiological processes, such as proliferation, differentiation, inflammation, neuronal plasticity and apoptosis. Unbalanced activity within these pathways provides a potential mechanism for many of the pathological phenotypes associated with HD, such as transcriptional dysregulation, inflammation and ultimately neurodegeneration. The characterisation of aberrant kinase signalling regulation in HD has been inconsistent and may be a result of failure to consider integration between multiple signalling pathways, as well as alterations that may occur over time with both age and disease progression. Collating the information about the effect of mHTT on signalling pathways demonstrates that it has wide ranging effects on multiple pro- and anti-apoptotic kinases, resulting in the dysregulation of numerous complex interactions within a dynamic network.

DJ-1 modulates aggregation and pathogenesis in models of Huntington's disease

The oxidation-sensitive chaperone protein DJ-1 has been implicated in several human disorders including cancer and neurodegenerative diseases. During neurodegeneration associated with protein misfolding, such as that observed in Alzheimer's disease and Huntington's disease (HD), both oxidative stress and protein chaperones have been shown to modulate disease pathways. Therefore, we set out to investigate whether DJ-1 plays a role in HD. We found that DJ-1 expression and its oxidation state are abnormally increased in the human HD brain, as well as in mouse and cell models of HD. Furthermore, overexpression of DJ-1 conferred protection in vivo against neurodegeneration in yeast and Drosophila. Importantly, the DJ-1 protein directly interacted with an expanded fragment of huntingtin Exon 1 (httEx1) in test tube experiments and in cell models and accelerated polyglutamine aggregation and toxicity in an oxidation-sensitive manner. Our findings clearly establish DJ-1 as a potential therapeutic target for HD and provide the basis for further studies into the role of DJ-1 in protein misfolding diseases.

Imbalance of p75NTR/TrkB protein expression in Huntington’s disease: implication for neuroprotective therapies

Neuroprotective therapies based on brain-derived neurotrophic factor (BDNF) administration have been proposed for Huntington's disease (HD) treatment. However, our group has recently reported reduced levels of TrkB in HD mouse models and HD human brain suggesting that besides a decrease on BDNF levels a reduction of TrkB expression could also contribute to diminished neurotrophic support in HD. BDNF can also bind to p75 neurotrophin receptor (p75NTR) modulating TrkB signaling. Therefore, in this study we have analyzed the levels of p75NTR in several HD models, as well as in HD human brain. Our data demonstrates a p75NTR/TrkB imbalance in the striatum of two different HD mouse models, HdhQ111/111 homozygous knockin mice and R6/1 mice that was also manifested in the putamen of HD patients. The imbalance between TrkB and p75NTR levels in a HD cellular model did not affect BDNF-mediated TrkB activation of prosurvival pathways but induced activation of apoptotic cascades as demonstrated by increased JNK phosphorylation. Moreover, BDNF failed to protect mutant huntingtin striatal cells transfected with p75NTR against NMDA-mediated excitotoxicity, which was associated with decreased Akt phosphorylation. Interestingly, lack of Akt activation following BDNF and NMDA treatment correlated with increased PP1 levels. Accordingly, pharmacological inhibition of PP1 by okadaic acid (OA) prevented mutant huntingtin striatal cell death induced by NMDA and BDNF. Altogether, our findings demonstrate that the p75NTR/TrkB imbalance induced by mutant huntingtin in striatal cells associated with the aberrant activity of PP1 disturbs BDNF neuroprotection likely contributing to increasing striatal vulnerability in HD. On the basis of this data we hypothesize that normalization of p75NTR and/or TrkB expression or their signaling will improve BDNF neuroprotective therapies in HD.

A novel method for detecting 7-methyl guanine reveals aberrant methylation levels in Huntington disease

Guanine methylation is a ubiquitous process affecting DNA and various RNA species. N-7 guanine methylation (7-MG), although relatively less studied, could have a significant role in normal transcriptional regulation as well as in the onset and development of pathological conditions. The lack of a sensitive method to accurately quantify trace amounts of altered bases such as 7-MG has been a major deterrent in delineating its biological function(s). Here we report the development of methods to detect trace amounts of 7-MG in biological samples using electrochemical detection combined with high-performance liquid chromatography (HPLC) separation of compounds. We further sought to assess global alterations in DNA methylation in Huntington disease (HD), where transcriptional dysregulation is a major factor in pathogenesis. The developed method was used to study guanine methylation in cytoplasmic and nuclear nucleic acids from human and transgenic mouse HD brain and controls. Significant differences were observed in the guanine methylation levels in mouse and human samples, consistent with the known transcriptional pathology of HD. The sensitivity of the method makes it capable of detecting subtle aberrations. Identification of changes in methylation pattern will provide insights into the molecular mechanism changes that translate into onset and/or development of symptoms in diseases such as HD.

Regional vulnerability in Huntington's disease: fMRI-guided molecular analysis in patients and a mouse model of disease

Although the huntingtin gene is expressed in brain throughout life, phenotypically Huntington's disease (HD) begins only in midlife and affects specific brain regions. Here, to investigate regional vulnerability in the disease, we used functional magnetic resonance imaging (fMRI) to translationally link studies in patients with a mouse model of disease. Using fMRI, we mapped cerebral blood volume (CBV) in three groups: HD patients, symptom-free carriers of the huntingtin genetic mutation, and age-matched controls. In contrast to a region in the anterior caudate, in which dysfunction was linked to genotype independent of phenotype, a region in the posterior body of the caudate was differentially associated with disease phenotype. Guided by these observations, we harvested regions from the anterior and posterior body of the caudate in postmortem control and HD human brain tissue. Gene-expression profiling identified two molecules whose expression levels were most strongly correlated with regional vulnerability — protein phosphatase 1 regulatory subunit 7 (PPP1R7) and Wnt inhibitory factor-1 (WIF-1). To verify and potentially extend these findings, we turned to the YAC128 (C57BL/6J) HD transgenic mice. By fMRI we longitudinally mapped CBV in transgenic and wildtype (WT) mice, and over time, abnormally low fMRI signal emerged selectively in the dorsal striatum. A relatively unaffected brain region, primary somatosensory cortex (S1), was used as a control. Both dorsal striatum and S1 were harvested from transgenic and WT mice and molecular analysis confirmed that PPP1R7 deficiency was strongly correlated with the phenotype. Together, converging findings in human HD patients and this HD mouse model suggest a functional pattern of caudate vulnerability and that variation in expression levels of herein identified molecules correlate with this pattern of vulnerability.

A Novel BACHD Transgenic Rat Exhibits Characteristic Neuropathological Features of Huntington Disease

Huntington disease (HD) is an inherited progressive neurodegenerative disorder, characterized by motor, cognitive, and psychiatric deficits as well as neurodegeneration and brain atrophy beginning in the striatum and the cortex and extending to other subcortical brain regions. The genetic cause is an expansion of the CAG repeat stretch in the HTT gene encoding huntingtin protein (htt). Here, we generated an HD transgenic rat model using a human bacterial artificial chromosome (BAC), which contains the full-length HTT genomic sequence with 97 CAG/CAA repeats and all regulatory elements. BACHD transgenic rats display a robust, early onset and progressive HD-like phenotype including motor deficits and anxiety-related symptoms. In contrast to BAC and yeast artificial chromosome HD mouse models that express full-length mutant huntingtin, BACHD rats do not exhibit an increased body weight. Neuropathologically, the distribution of neuropil aggregates and nuclear accumulation of N-terminal mutant huntingtin in BACHD rats is similar to the observations in human HD brains. Aggregates occur more frequently in the cortex than in the striatum and neuropil aggregates appear earlier than mutant htt accumulation in the nucleus. Furthermore, we found an imbalance in the striatal striosome and matrix compartments in early stages of the disease. In addition, reduced dopamine receptor binding was detectable by in vivo imaging. Our data demonstrate that this transgenic BACHD rat line may be a valuable model for further understanding the disease mechanisms and for preclinical pharmacological studies.