Alexandre Kuhn

SIRT2 inhibition achieves neuroprotection by decreasing sterol biosynthesis

Huntington’s disease (HD), an incurable neurodegenerative disorder, has a complex pathogenesis including protein aggregation and the dysregulation of neuronal transcription and metabolism. Here, we demonstrate that inhibition of sirtuin 2 (SIRT2) achieves neuroprotection in cellular and invertebrate models of HD. Genetic or pharmacologic inhibition of SIRT2 in a striatal neuron model of HD resulted in gene expression changes including significant down-regulation of RNAs responsible for sterol biosynthesis. Whereas mutant huntingtin fragments increased sterols in neuronal cells, SIRT2 inhibition reduced sterol levels via decreased nuclear trafficking of SREBP-2. Importantly, manipulation of sterol biosynthesis at the transcriptional level mimicked SIRT2 inhibition, demonstrating that the metabolic effects of SIRT2 inhibition are sufficient to diminish mutant huntingtin toxicity. These data identify SIRT2 inhibition as a promising avenue for HD therapy and elucidate a unique mechanism of SIRT2-inhibitor-mediated neuroprotection. Furthermore, the ascertainment of SIRT2’s role in regulating cellular metabolism demonstrates a central function shared with other sirtuin proteins.

Analysis of potential transcriptomic biomarkers for Huntington's disease in peripheral blood.

Highly quantitative biomarkers of neurodegenerative disease remain an important need in the urgent quest for disease-modifying therapies. For Huntingtons disease (HD), a genetic test is available (trait marker), but necessary state markers are still in development. In this report, we describe a large battery of transcriptomic tests explored as state biomarker candidates. In an attempt to exploit the known neuroinflammatory and transcriptional perturbations of disease, we measured relevant mRNAs in peripheral blood cells. The performance of these potential markers was weak overall, with only one mRNA, immediate early response 3 (IER3), showing a modest but significant increase of 32% in HD samples compared with controls. No statistically significant differences were found for any other mRNAs tested, including a panel of 12 RNA biomarkers identified in a previous report [Borovecki F, Lovrecic L, Zhou J, Jeong H, Then F, Rosas HD, Hersch SM, Hogarth P, Bouzou B, Jensen RV, et al. (2005) Proc Natl Acad Sci USA 102:11023–11028]. The present results may nonetheless inform the future design and testing of HD biomarker strategies.

Population-specific expression analysis (PSEA) reveals molecular changes in diseased brain.

Human diseases are often accompanied by histological changes that confound interpretation of molecular analyses and identification of disease-related effects. We developed population-specific expression analysis (PSEA), a computational method of analyzing gene expression in samples of varying composition that can improve analyses of quantitative molecular data in many biological contexts. PSEA of brains from individuals with Huntingtons disease revealed myelin-related abnormalities that were undetected using standard differential expression analysis.

Dysregulation of Gene Expression in Primary Neuron Models of Huntington's Disease Shows That Polyglutamine-Related Effects on the Striatal Transcriptome May Not Be Dependent on Brain Circuitry

Gene expression changes are a hallmark of the neuropathology of Huntingtons disease (HD), but the exact molecular mechanisms of this effect remain uncertain. Here, we report that in vitro models of disease comprised of primary striatal neurons expressing N-terminal fragments of mutant huntingtin (via lentiviral gene delivery) faithfully reproduce the gene expression changes seen in human HD. Neither viral infection nor unrelated (enhanced green fluorescent protein) transgene expression had a major effect on resultant RNA profiles. Expression of a wild-type fragment of huntingtin [htt171-18Q] also caused only a small number of RNA changes. The disease-related signal in htt171-82Q versus htt171-18Q comparisons was far greater, resulting in the differential detection of 20% of all mRNA probe sets. Transcriptomic effects of mutated htt171 are time- and polyglutamine-length dependent and occur in parallel with other manifestations of polyglutamine toxicity over 4–8 weeks. Specific RNA changes in htt171-82Q-expressing striatal cells accurately recapitulated those observed in human HD caudate and included decreases in PENK (proenkephalin), RGS4 (regulator of G-protein signaling 4), dopamine D1 receptor (DRD1), DRD2, CNR1 (cannabinoid CB1 receptor), and DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32; also known as PPP1R1B) mRNAs. HD-related transcriptomic changes were also observed in primary neurons expressing a longer fragment of mutant huntingtin (htt853-82Q). The gene expression changes observed in cultured striatal neurons are not secondary to abnormalities of neuronal firing or glutamatergic, dopaminergic, or brain-derived neurotrophic factor signaling, thereby demonstrating that HD-induced dysregulation of the striatal transcriptome might be attributed to intrinsic effects of mutant huntingtin.

Transcriptional changes in Huntington disease identified using genome-wide expression profiling and cross-platform analysis

Evaluation of transcriptional changes in the striatum may be an effective approach to understanding the natural history of changes in expression contributing to the pathogenesis of Huntington disease (HD). We have performed genome-wide expression profiling of the YAC128 transgenic mouse model of HD at 12 and 24 months of age using two platforms in parallel: Affymetrix and Illumina. The data from these two powerful platforms were integrated to create a combined rank list, thereby revealing the identity of additional genes that proved to be differentially expressed between YAC128 and control mice. Using this approach, we identified 13 genes to be differentially expressed between YAC128 and controls which were validated by quantitative real-time PCR in independent cohorts of animals. In addition, we analyzed additional time points relevant to disease pathology: 3, 6 and 9 months of age. Here we present data showing the evolution of changes in the expression of selected genes: Wt1, Pcdh20 and Actn2 RNA levels change as early as 3 months of age, whereas Gsg1l, Sfmbt2, Acy3, Polr2a and Ppp1r9a RNA expression levels are affected later, at 12 and 24 months of age. We also analyzed the expression of these 13 genes in human HD and control brain, thereby revealing changes in SLC45A3, PCDH20, ACTN2, DDAH1 and PPP1R9A RNA expression. Further study of these genes may unravel novel pathways contributing to HD pathogenesis. DDBJ/EMBL/GenBank accession no: GSE19677.

In vivo cell-autonomous transcriptional abnormalities revealed in mice expressing mutant huntingtin in striatal but not cortical neurons

Huntingtons disease (HD), caused by a CAG repeat expansion in the huntingtin (HTT) gene, is characterized by abnormal protein aggregates and motor and cognitive dysfunction. Htt protein is ubiquitously expressed, but the striatal medium spiny neuron (MSN) is most susceptible to dysfunction and death. Abnormal gene expression represents a core pathogenic feature of HD, but the relative roles of cell-autonomous and non-cell-autonomous effects on transcription remain unclear. To determine the extent of cell-autonomous dysregulation in the striatum in vivo, we examined genome-wide RNA expression in symptomatic D9-N171-98Q (a.k.a. DE5) transgenic mice in which the forebrain expression of the first 171 amino acids of human Htt with a 98Q repeat expansion is limited to MSNs. Microarray data generated from these mice were compared with those generated on the identical array platform from a pan-neuronal HD mouse model, R6/2, carrying two different CAG repeat lengths, and a relatively high degree of overlap of changes in gene expression was revealed. We further focused on known canonical pathways associated with excitotoxicity, oxidative stress, mitochondrial dysfunction, dopamine signaling and trophic support. While genes related to excitotoxicity, dopamine signaling and trophic support were altered in both DE5 and R6/2 mice, which may be either cell autonomous or non-cell autonomous, genes related to mitochondrial dysfunction, oxidative stress and the peroxisome proliferator-activated receptor are primarily affected in DE5 transgenic mice, indicating cell-autonomous mechanisms. Overall, HD-induced dysregulation of the striatal transcriptome can be largely attributed to intrinsic effects of mutant Htt, in the absence of expression in cortical neurons.

Genome-Wide Histone Acetylation Is Altered in a Transgenic Mouse Model of Huntington's Disease

In Huntingtons disease (HD; MIM ID #143100), a fatal neurodegenerative disorder, transcriptional dysregulation is a key pathogenic feature. Histone modifications are altered in multiple cellular and animal models of HD suggesting a potential mechanism for the observed changes in transcriptional levels. In particular, previous work has suggested an important link between decreased histone acetylation, particularly acetylated histone H3 (AcH3; H3K9K14ac), and downregulated gene expression. However, the question remains whether changes in histone modifications correlate with transcriptional abnormalities across the entire transcriptome. Using chromatin immunoprecipitation paired with microarray hybridization (ChIP-chip), we interrogated AcH3-gene interactions genome-wide in striata of 12-week old wild-type (WT) and transgenic (TG) R6/2 mice, an HD mouse model, and correlated these interactions with gene expression levels. At the level of the individual gene, we found decreases in the number of sites occupied by AcH3 in the TG striatum. In addition, the total number of genes bound by AcH3 was decreased. Surprisingly, the loss of AcH3 binding sites occurred within the coding regions of the genes rather than at the promoter region. We also found that the presence of AcH3 at any location within a gene strongly correlated with the presence of its transcript in both WT and TG striatum. In the TG striatum, treatment with histone deacetylase (HDAC) inhibitors increased global AcH3 levels with concomitant increases in transcript levels; however, AcH3 binding at select gene loci increased only slightly. This study demonstrates that histone H3 acetylation at lysine residues 9 and 14 and active gene expression are intimately tied in the rodent brain, and that this fundamental relationship remains unchanged in an HD mouse model despite genome-wide decreases in histone H3 acetylation.

Short-term striatal gene expression responses to brain-derived neurotrophic factor are dependent on MEK and ERK activation

Background Brain-derived neurotrophic factor (BDNF) is believed to be an important regulator of striatal neuron survival, differentiation, and plasticity. Moreover, reduction of BDNF delivery to the striatum has been implicated in the pathophysiology of Huntingtons disease. Nevertheless, many essential aspects of BDNF responses in striatal neurons remain to be elucidated. Methodology/Principal Findings In this study, we assessed the relative contributions of multipartite intracellular signaling pathways to the short-term induction of striatal gene expression by BDNF. To identify genes regulated by BDNF in these GABAergic cells, we first used DNA microarrays to quantify their transcriptomic responses following 3 h of BDNF exposure. The signal transduction pathways underlying gene induction were subsequently dissected using pharmacological agents and quantitative real-time PCR. Gene expression responses to BDNF were abolished by inhibitors of TrkB (K252a) and calcium (chelator BAPTA-AM and transient receptor potential cation channel [TRPC] antagonist SKF-96365). Interestingly, inhibitors of mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) and extracellular signal-regulated kinase ERK also blocked the BDNF-mediated induction of all tested BDNF-responsive genes. In contrast, inhibitors of nitric oxide synthase (NOS), phosphotidylinositol-3-kinase (PI3K), and CAMK exhibited less prevalent, gene-specific effects on BDNF-induced RNA expression. At the nuclear level, the activation of both Elk-1 and CREB showed MEK dependence. Importantly, MEK-dependent activation of transcription was shown to be required for BDNF-induced striatal neurite outgrowth, providing evidence for its contribution to striatal neuron plasticity. Conclusions These results show that the MEK/ERK pathway is a major mediator of neuronal plasticity and other important BDNF-dependent striatal functions that are fulfilled through the positive regulation of gene expression.

Cross-species and cross-platform gene expression studies with the Bioconductor-compliant R package 'annotationTools'.

BackgroundThe variety of DNA microarray formats and datasets presently available offers an unprecedented opportunity to perform insightful comparisons of heterogeneous data. Cross-species studies, in particular, have the power of identifying conserved, functionally important molecular processes. Validation of discoveries can now often be performed in readily available public data which frequently requires cross-platform studies.Cross-platform and cross-species analyses require matching probes on different microarray formats. This can be achieved using the information in microarray annotations and additional molecular biology databases, such as orthology databases. Although annotations and other biological information are stored using modern database models (e.g. relational), they are very often distributed and shared as tables in text files, i.e. flat file databases. This common flat database format thus provides a simple and robust solution to flexibly integrate various sources of information and a basis for the combined analysis of heterogeneous gene expression profiles.ResultsWe provide annotationTools, a Bioconductor-compliant R package to annotate microarray experiments and integrate heterogeneous gene expression profiles using annotation and other molecular biology information available as flat file databases. First, annotationTools contains a specialized set of functions for mining this widely used database format in a systematic manner. It thus offers a straightforward solution for annotating microarray experiments. Second, building on these basic functions and relying on the combination of information from several databases, it provides tools to easily perform cross-species analyses of gene expression data.Here, we present two example applications of annotationTools that are of direct relevance for the analysis of heterogeneous gene expression profiles, namely a cross-platform mapping of probes and a cross-species mapping of orthologous probes using different orthology databases. We also show how to perform an explorative comparison of disease-related transcriptional changes in human patients and in a genetic mouse model.ConclusionThe R package annotationTools provides a simple solution to handle microarray annotation and orthology tables, as well as other flat molecular biology databases. Thereby, it allows easy integration and analysis of heterogeneous microarray experiments across different technological platforms or species.

Combined transcriptome analysis of fetal human and mouse cerebral cortex exposed to alcohol

Fetal exposure to environmental insults increases the susceptibility to late-onset neuropsychiatric disorders. Alcohol is listed as one of such prenatal environmental risk factors and known to exert devastating teratogenetic effects on the developing brain, leading to complex neurological and psychiatric symptoms observed in fetal alcohol spectrum disorder (FASD). Here, we performed a coordinated transcriptome analysis of human and mouse fetal cerebral cortices exposed to ethanol in vitro and in vivo, respectively. Up- and down-regulated genes conserved in the human and mouse models and the biological annotation of their expression profiles included many genes/terms related to neural development, such as cell proliferation, neuronal migration and differentiation, providing a reliable connection between the two species. Our data indicate that use of the combined rodent and human model systems provides an effective strategy to reveal and analyze gene expression changes inflicted by various physical and chemical environmental exposures during prenatal development. It also can potentially provide insight into the pathogenesis of environmentally caused brain disorders in humans.