Mutant huntingtin's effects on striatal gene expression in mice recapitulate changes observed in human Huntington's disease brain and do not differ with mutant huntingtin length or wild-type huntingtin dosage.

To test the hypotheses that mutant huntingtin protein length and wild-type huntingtin dosage have important effects on disease-related transcriptional dysfunction, we compared the changes in mRNA in seven genetic mouse models of Huntington's disease (HD) and postmortem human HD caudate. Transgenic models expressing short N-terminal fragments of mutant huntingtin (R6/1 and R6/2 mice) exhibited the most rapid effects on gene expression, consistent with previous studies. Although changes in the brains of knock-in and full-length transgenic models of HD took longer to appear, 15- and 22-month CHL2(Q150/Q150), 18-month Hdh(Q92/Q92) and 2-year-old YAC128 animals also exhibited significant HD-like mRNA signatures. Whereas it was expected that the expression of full-length huntingtin transprotein might result in unique gene expression changes compared with those caused by the expression of an N-terminal huntingtin fragment, no discernable differences between full-length and fragment models were detected. In addition, very high correlations between the signatures of mice expressing normal levels of wild-type huntingtin and mice in which the wild-type protein is absent suggest a limited effect of the wild-type protein to change basal gene expression or to influence the qualitative disease-related effect of mutant huntingtin. The combined analysis of mouse and human HD transcriptomes provides important temporal and mechanistic insights into the process by which mutant huntingtin kills striatal neurons. In addition, the discovery that several available lines of HD mice faithfully recapitulate the gene expression signature of the human disorder provides a novel aspect of validation with respect to their use in preclinical therapeutic trials.

Triplet repeat mutation length gains correlate with cell-type specific vulnerability in Huntington disease brain.

Huntington disease is caused by the expansion of a CAG repeat encoding an extended glutamine tract in a protein called huntingtin. Here, we provide evidence supporting the hypothesis that somatic increases of mutation length play a role in the progressive nature and cell-selective aspects of HD pathogenesis. Results from micro-dissected tissue and individual laser-dissected cells obtained from human HD cases and knock-in HD mice indicate that the CAG repeat is unstable in all cell types tested although neurons tend to have longer mutation length gains than glia. Mutation length gains occur early in the disease process and continue to accumulate as the disease progresses. In keeping with observed patterns of cell loss, neuronal mutation length gains tend to be more prominent in the striatum than in the cortex of low-grade human HD cases, less so in more advanced cases. Interestingly, neuronal sub-populations of HD mice appear to have different propensities for mutation length gains; in particular, smaller mutation length gains occur in nitric oxide synthase-positive striatal interneurons (a relatively spared cell type in HD) compared with the pan-striatal neuronal population. More generally, the data demonstrate that neuronal changes in HD repeat length can be at least as great, if not greater, than those observed in the germline. The fact that significant CAG repeat length gains occur in non-replicating cells also argues that processes such as inappropriate mismatch repair rather than DNA replication are involved in generating mutation instability in HD brain tissue.

Assessment of the relationship between pre-chip and post-chip quality measures for Affymetrix GeneChip expression data.

BACKGROUND: Gene expression microarray experiments are expensive to conduct and guidelines for acceptable quality control at intermediate steps before and after the samples are hybridised to chips are vague. We conducted an experiment hybridising RNA from human brain to 117 U133A Affymetrix GeneChips and used these data to explore the relationship between 4 pre-chip variables and 22 post-chip outcomes and quality control measures. RESULTS: We found that the pre-chip variables were significantly correlated with each other but that this correlation was strongest between measures of RNA quality and cRNA yield. Post-mortem interval was negatively correlated with these variables. Four principal components, reflecting array outliers, array adjustment, hybridisation noise and RNA integrity, explain about 75% of the total post-chip measure variability. Two significant canonical correlations existed between the pre-chip and post-chip variables, derived from MAS 5.0, dChip and the Bioconductor packages affy and affyPLM. The strongest (CANCOR 0.838, p < 0.0001) correlated RNA integrity and yield with post chip quality control (QC) measures indexing 3'/5' RNA ratios, bias or scaling of the chip and scaling of the variability of the signal across the chip. Post-mortem interval was relatively unimportant. We also found that the RNA integrity number (RIN) could be moderately well predicted by post-chip measures B_ACTIN35, GAPDH35 and SF. CONCLUSION: We have found that the post-chip variables having the strongest association with quantities measurable before hybridisation are those reflecting RNA integrity. Other aspects of quality, such as noise measures (reflecting the execution of the assay) or measures reflecting data quality (outlier status and array adjustment variables) are not well predicted by the variables we were able to determine ahead of time. There could be other variables measurable pre-hybridisation which may be better associated with expression data quality measures. Uncovering such connections could create savings on costly microarray experiments by eliminating poor samples before hybridisation.

Regional and cellular gene expression changes in human Huntington's disease brain.

Huntington's disease (HD) pathology is well understood at a histological level but a comprehensive molecular analysis of the effect of the disease in the human brain has not previously been available. To elucidate the molecular phenotype of HD on a genome-wide scale, we compared mRNA profiles from 44 human HD brains with those from 36 unaffected controls using microarray analysis. Four brain regions were analyzed: caudate nucleus, cerebellum, prefrontal association cortex [Brodmann's area 9 (BA9)] and motor cortex [Brodmann's area 4 (BA4)]. The greatest number and magnitude of differentially expressed mRNAs were detected in the caudate nucleus, followed by motor cortex, then cerebellum. Thus, the molecular phenotype of HD generally parallels established neuropathology. Surprisingly, no mRNA changes were detected in prefrontal association cortex, thereby revealing subtleties of pathology not previously disclosed by histological methods. To establish that the observed changes were not simply the result of cell loss, we examined mRNA levels in laser-capture microdissected neurons from Grade 1 HD caudate compared to control. These analyses confirmed changes in expression seen in tissue homogenates; we thus conclude that mRNA changes are not attributable to cell loss alone. These data from bona fide HD brains comprise an important reference for hypotheses related to HD and other neurodegenerative diseases.

Transcriptional dysregulation in striatal projection- and interneurons in a mouse model of Huntington's disease: neuronal selectivity and potential neuroprotective role of HAP1.

Transcriptional dysregulation has been described as a central mechanism in the pathogenesis of Huntington's disease (HD), in which medium spiny projection neurons (MSN) selectively degenerate whereas neuronal nitric-oxide-synthase-positive interneurons (nNOS-IN) survive. In order to begin to understand this differential vulnerability we compared mRNA levels of selected genes involved in N-methyl-D-aspartate (NMDA) glutamate receptor and calcium (Ca2+) signaling pathways in MSN and nNOS-IN from 12-week-old R6/2 mice, a transgenic mouse model of HD and wild-type littermates. We undertook a laser capture microdissection (LCM) study to examine the contribution of transcriptional dysregulation in candidate genes involved in these two signaling pathways in discrete populations of striatal neurons. The use of LCM in combination with quantitative real-time polymerase chain reaction (Q-PCR) allowed us to quantify the neuronal abundance of candidate mRNAs. We found different transcriptional alterations in R6/2 neurons for both MSN and nNOS-IN, indicating that global transcriptional dysregulation alone does not account for selective vulnerability. Further, we observed a striking enrichment of several mRNAs in the nNOS-IN population, including that for the NMDA receptor subunit NR2D, the postsynaptic density protein 95 (PSD-95) and the huntingtin-associated protein 1 (HAP1) as well as nitric-oxide-synthase (nNOS) mRNA itself. The higher expression levels of these molecules in nNOS-IN when compared with MSN together with an association of nNOS, NR2D and HAP1 in a protein complex with PSD-95 suggest that these proteins may be involved in protective pathways that contribute to the resistance of this interneuron population to neurodegeneration in HD.

Neostriatal and cortical quinolinate levels are increased in early grade Huntington's disease.

Huntington's disease (HD), an inherited neurodegenerative disorder, is caused by an abnormal polyglutamine expansion in the huntingtin protein. This genetic defect may result in heightened neuronal susceptibility to excitotoxic injury, a mechanism that has been postulated to play a critical role in HD. Quinolinate (QUIN) and kynurenate (KYNA), two endogenous neuroactive metabolites of the kynurenine pathway of tryptophan degradation, have been proposed to modulate excitotoxic neuronal death in HD. A third kynurenine pathway metabolite, the free radical generator 3-hydroxykynurenine (3-HK), has also been hypothesized to play a causal role in the pathogenesis of HD. We show here that the brain levels of both 3-HK and QUIN are increased three to four-fold in low-grade (grade 0/1) HD brain. These changes were seen in the neocortex and in the neostriatum, but not in the cerebellum. In contrast, brain 3-HK and QUIN levels were either unchanged or tended to decrease in grade 2 and advanced grade (grades 3-4) HD brain. Brain kynurenine and KYNA levels fluctuated only modestly as the illness progressed. These results support a possible involvement of 3-HK and QUIN in the early phases of HD pathophysiology and indicate novel therapeutic strategies against the disease.

Elevated levels of matrix metalloproteinases-9 and -1 and of tissue inhibitors of MMPs, TIMP-1 and TIMP-2 in postmortem brain tissue of progressive supranuclear palsy.

We determined the levels and tissue localization of matrix metalloproteinases (MMPs) as well as their endogenous tissue inhibitors (TIMPs) in postmortem brain tissue from 13 patients with progressive supranuclear palsy (PSP) and 8 age-matched controls. MMP-9 expression was significantly increased in both the frontal cortex (p = 0.002) and substantia nigra (p = 0.003) of PSP cases as compared to controls whereas MMP-1 levels were increased in the substantia nigra (p = 0.01) but unchanged in the frontal cortex (p = 0.41). Levels of the endogenous tissue inhibitors of MMPs, TIMP-1 and TIMP-2 were significantly elevated in the substantia nigra (TIMP-1: p = 0.004, TIMP-2: p = 0.01). Levels of TIMPs were unchanged in PSP frontal cortex as compared to control cases. Together, these data show alterations of MMPs and TIMPs in the substantia nigra as well as in the frontal cortex of PSP, consistent with the possibility that alterations in MMPs/TIMPs may contribute to disease pathogenesis.

Distribution and ultrastructural localization of torsinA immunoreactivity in the human brain.

We have examined the distribution and ultrastructural localization of torsinA, the protein product of the TOR1A gene, in the normal adult human and Macaque brain. TorsinA immunoreactivity was visualized using a monoclonal antibody raised against a fusion protein encoding exon 4 of human torsinA. Western blot analysis of brain homogenates revealed a major species of about 39 kDa, consistent with the predicted size of glycosylated torsinA protein. By light microscopy, torsinA like-immunoreactivity was enriched in gray matter in all brain regions examined. Immunoreactivity was concentrated in the neuropil and immunopositive cell bodies were not observed. Structures particularly enriched in torsinA like-immunoreactivity included the cerebral cortex, the caudate-putamen, globus pallidus, the hippocampal formation, the thalamus, the substantia nigra and molecular cell layer of the cerebellar cortex. Cell bodies of pigmented dopamine neurons in the substantia nigra pars compacta were immunonegative. Biochemical fractionation of the human striata revealed a concentration of torsinA immunoreactivity in particulate fractions. Ultrastructural studies of the human and Macaque striata further revealed an association of torsinA immunostaining with small vesicles within axons and presynaptic terminals forming symmetric synapses. These ultrastructural studies are consistent with a pre-synaptic localization of torsinA protein in the adult striatum and are consistent with a role of torsinA in modulating striatal signaling, although the widespread localization of the protein suggests it probably also participates in signaling in other regions.

TorsinA protein and neuropathology in early onset generalized dystonia with GAG deletion.

Familial, early onset, generalized torsion dystonia is the most common and severe primary dystonia. Most cases are caused by a 3-bp deletion (GAG) in the coding region of the TOR1A (DYT1) gene, which is widely expressed in human brain and encodes the protein torsinA. This study compares neuropathology and torsinA expression in the normal human brain with that in dystonia cases with and without the GAG deletion. TorsinA-like protein was expressed in neuronal cytoplasm throughout the human brain, including cerebellum, substantia nigra, hippocampus, and neostriatum, with higher levels in specific neurons. This immunostaining pattern was not discernibly different in dystonia and normal brains in midbrain and neostriatal regions. However, nigral dopaminergic neurons appeared to be larger in both GAG-deletion and non-GAG-deletion dystonia brains compared to normal, and may be more closely spaced in GAG-deletion brains. Beyond these apparent changes in neuronal size and spacing in dystonia brains, there was no indication of neuron loss, inflammation, DNA strand breaks, or altered distribution of torsin-like immunoreactivity, supporting a functional rather than degenerative etiology of early onset torsion dystonia.

Identification of nitric oxide synthase neurons for laser capture microdissection and mRNA quantification.

An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.

Dopamine transmission in DYT1 dystonia: a biochemical and autoradiographical study.

Indices of dopamine transmission were measured in the postmortem striatum of DYT1 dystonia brains. A significant increase in the striatal 3,4-dihydroxyphenylacetic acid/dopamine ratio was found. Quantitative autoradiography revealed no differences in the density of dopamine transporter or vesicular monoamine transporter-2 binding; however, there was a trend toward a reduction in D(1) receptor and D(2) receptor binding. One brain with DYT1 parkinsonism was similarly evaluated and marked reductions in striatal dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid content as well as the density of binding of all four dopaminergic ligands were measured.

Molecular cloning and expression of rat torsinA in the normal and genetically dystonic (dt) rat.

Deletions within the TOR1A gene cause early-onset (DYT1) torsion dystonia. We have cloned and sequenced the rat cDNA homologue of TOR1A and found a 91% identity with the human sequence. Northern blot analysis detects a single transcript of approximately 1.5 kb. In situ hybridization reveals a widespread distribution of torsinA mRNA within brain. No mutations were identified in the coding region of the gene in the genetically dystonic (dt) rat.

Expression and activity of antioxidants in the brain in progressive supranuclear palsy.

Recent evidence implicates oxidative stress in the pathophysiology of progressive supranuclear palsy (PSP). Thus, we undertook a study of the activity and localization of two essential antioxidant systems (superoxide dismutase [SOD] enzymes and total glutathione) in the human post-mortem PSP and control brain. Marked increases in SOD1 (Cu/ZnSOD) activity and glutathione levels were measured within most PSP brain regions examined, whereas, only the subthalamic nucleus exhibited a significant increase (+68%) in SOD2 (MnSOD) activity. Two additional cases with mild pathological abnormalities were studied. The first (case A) may represent an example of an asymptomatic PSP case, while the second (case B) had mild pathological abnormalities consistent with typical PSP. In case A, only the STN had elevated levels of SOD activity, in the absence of an increase in tissue glutathione content. In case B, SOD activities and tissue glutathione content were elevated in several regions. Immunolocalization of the SOD1 and SOD2 proteins in paraffin-embedded tissue sections revealed a marked increase in the density of SOD immunopositive profiles (particularly glia) in the typical PSP brain, particularly within the white matter. Together, our data argues strongly in favor of the involvement of oxidative stress in the etiology and progression of PSP, and suggests that deficit in SOD or glutathione metabolism are not causative.

Contrasting Effects of Raclopride and SCH 23390 on the Cellular Content of Preproenkephalin A mRNA in Rat Striatum: A Quantitative Non-radioactive In Situ Hybridization Study.

The effects of acute i.p. administration of selective dopamine (DA) receptor antagonists on the expression of preproenkephalin A (PPE A) mRNA was investigated in the adult rat striatum. Animals were injected with either (a) a selective D1 receptor antagonist SCH 23390 (0.25 mg/kg), (b) a selective D2 receptor antagonist raclopride (5 mg/kg), or (c) SCH 23388 (0.25 mg/kg), the (S)-enantiomer of SCH 23390. Control naive animals did not receive an injection. At specific time points following drug administration (1, 3 or 9 h), rats were killed and striatal tissue processed for in situ hybridization with an alkaline phosphatase-labelled oligonucleotide probe complementary to a portion of the rat PPE A cDNA. Treatment of rats with SCH 23388 did not affect the content of PPE A mRNA expressed by striatal cells at any time point. However, 1 h after SCH 23390 administration, a significant decrease in striatal PPE A mRNA was detected, reflected by a decrease in the cellular content of mRNA. No significant changes in PPE A mRNA were detected in raclopride-treated sections at this time point. In contrast, both 3 and 9 h after an injection of raclopride a significant increase in the cellular content of PPE A mRNA was detected in the striatum. No change in the cellular content of mRNA was detected in SCH 23390-treated rats at these two latter time points. Throughout the striatum approximately 46% of neurons were found to express PPE A mRNA, with the highest percentage of cells (55%) being detected in the mid-caudal striatum. No significant differences in striatal DA content were detected with any drug treatment using HPLC electrochemical detection methods. These results demonstrate that acute administration of the DA D1 and D2 receptor antagonists has contrasting effects on the cellular content of PPE A mRNA in the adult rat striatum. These effects may reflect changes in the rate of mRNA transcription which may be mediated by cAMP.