Paola Conforti

Widespread Disruption of Repressor Element-1 Silencing Transcription Factor/Neuron-Restrictive Silencer Factor Occupancy at Its Target Genes in Huntington's Disease

Huntingtin is a protein that is mutated in Huntingtons disease (HD), a dominant inherited neurodegenerative disorder. We previously proposed that, in addition to the gained toxic activity of the mutant protein, selective molecular dysfunctions in HD may represent the consequences of the loss of wild-type protein activity. We first reported that wild-type huntingtin positively affects the transcription of the brain-derived neurotrophic factor (BDNF) gene, a cortically derived survival factor for the striatal neurons that are mainly affected in the disease. Mutation in huntingtin decreases BDNF gene transcription. One mechanism involves the activation of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) located within the BDNF promoter. We now show that increased binding of the RE1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) repressor occurs at multiple genomic RE1/NRSE loci in HD cells, in animal models, and in postmortem brains, resulting in a decrease of RE1/NRSE-mediated gene transcription. The same molecular phenotype is produced in cells and brain tissue depleted of endogenous huntingtin, thereby directly validating the loss-of-function hypothesis of HD. Through a ChIP (chromatin immunoprecipitation)-on-chip approach, we examined occupancy of multiple REST/NRSF target genes in the postmortem HD brain, providing the first example of the application of this technology to neurodegenerative diseases. Finally, we show that attenuation of REST/NRSF binding restores BDNF levels, suggesting that relief of REST/NRSF mediated repression can restore aberrant neuronal gene transcription in HD.

Systematic assessment of BDNF and its receptor levels in human cortices affected by Huntington's disease.

One cardinal feature of Huntingtons disease (HD) is the degeneration of striatal neurons, whose survival greatly depends on the binding of cortical brain‐derived neurotrophic factor (BDNF) with high‐affinity (TrkB) and low‐affinity neurotrophin receptors [p75 pan‐neurotrophin receptor (p75NTR)]. With a few exceptions, results obtained in HD mouse models demonstrate a reduction in cortical BDNF mRNA and protein, although autopsy data from a limited number of human HD cortices are conflicting. These studies indicate the presence of defects in cortical BDNF gene transcription and transport to striatum. We provide new evidence indicating a significant reduction in BDNF mRNA and protein in the cortex of 20 HD subjects in comparison with 17 controls, which supports the hypothesis of impaired BDNF production in human HD cortex. Analyses of the BDNF isoforms show that transcription from BDNF promoter II and IV is down‐regulated in human HD cortex from an early symptomatic stage. We also found that TrkB mRNA levels are reduced in caudate tissue but not in the cortex, whereas the mRNA levels of T‐Shc (a truncated TrkB isoform) and p75NTR are increased in the caudate. This indicates that, in addition to the reduction in BDNF mRNA, there is also unbalanced neurotrophic receptor signaling in HD.

RESEARCH ARTICLE: Systematic Assessment of BDNF and Its Receptor Levels in Human Cortices Affected by Huntington's Disease

One cardinal feature of Huntingtons disease (HD) is the degeneration of striatal neurons, whose survival greatly depends on the binding of cortical brain‐derived neurotrophic factor (BDNF) with high‐affinity (TrkB) and low‐affinity neurotrophin receptors [p75 pan‐neurotrophin receptor (p75NTR)]. With a few exceptions, results obtained in HD mouse models demonstrate a reduction in cortical BDNF mRNA and protein, although autopsy data from a limited number of human HD cortices are conflicting. These studies indicate the presence of defects in cortical BDNF gene transcription and transport to striatum. We provide new evidence indicating a significant reduction in BDNF mRNA and protein in the cortex of 20 HD subjects in comparison with 17 controls, which supports the hypothesis of impaired BDNF production in human HD cortex. Analyses of the BDNF isoforms show that transcription from BDNF promoter II and IV is down‐regulated in human HD cortex from an early symptomatic stage. We also found that TrkB mRNA levels are reduced in caudate tissue but not in the cortex, whereas the mRNA levels of T‐Shc (a truncated TrkB isoform) and p75NTR are increased in the caudate. This indicates that, in addition to the reduction in BDNF mRNA, there is also unbalanced neurotrophic receptor signaling in HD.

CEP-1347 reduces mutant huntingtin-associated neurotoxicity and restores BDNF levels in R6/2 mice

Huntingtons disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the protein Huntingtin (Htt). We previously reported that mutant Htt expression activates the ERK1/2 and JNK pathways [Apostol, B.L., Illes, K., Pallos, J., Bodai, L., Wu, J., Strand, A., Schweitzer, E.S., Olson, J.M., Kazantsev, A., Marsh, J.L., Thompson, L.M., 2006. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum. Mol. Genet. 15, 273-285]. Chemical and genetic modulation of these pathways promotes cell survival and death, respectively. Here we test the ability of two closely related compounds, CEP-11004 and CEP-1347, which inhibit Mixed Lineage Kinases (MLKs) and are neuroprotective, to suppress mutant Htt-mediated pathogenesis in multiple model systems. CEP-11004/CEP-1347 treatment significantly decreased toxicity in mutant Htt-expressing cells that evoke a strong JNK response. However, suppression of cellular dysfunction in cell lines that exhibit only mild Htt-associated toxicity and little JNK activation was associated with activation of ERK1/2. These compounds also reduced neurotoxicity in immortalized striatal neurons from mutant knock-in mice and Drosophila expressing a mutant Htt fragment. Finally, CEP-1347 improved motor performance in R6/2 mice and restored expression of BDNF, a critical neurotrophic factor that is reduced in HD. These studies suggest a novel therapeutic approach for a currently untreatable neurodegenerative disease, HD, via CEP-1347 up-regulation of BDNF.

REST Controls Self-Renewal and Tumorigenic Competence of Human Glioblastoma Cells

The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.

Blood level of brain-derived neurotrophic factor mRNA is progressively reduced in rodent models of Huntington's disease: restoration by the neuroprotective compound CEP-1347.

Huntingtons disease (HD) is an age-related neurodegenerative disorder that is currently untreatable. A prominent feature of HD pathology is the reduction of the pro-survival neurotrophin Brain-Derived Neurotrophic Factor (BDNF). Both mRNA and protein levels of BDNF are decreased in the brains of several HD rodent models and in human HD patients. We now report for the first time that this molecular event is mirrored in blood from HD rodent models. While protein levels of BDNF are undetectable in mouse blood, mRNA levels are measurable and diminish during HD progression in transgenic mouse (R6/2) and rat models of HD. Among the eight different BDNF transcripts, only BDNF exon III is transcribed in mouse blood and its expression is progressively compromised in R6/2 mice with respect to age-matched wild-types. Assessment of BDNF mRNA in HD rat blood shows a similar result, which is reinforced by evidence that protein levels of the neurotrophin are also significantly reduced at a symptomatic stage. Finally, we demonstrate that acute and chronic treatment of R6/2 mice with CEP-1347, a mixed lineage kinase (MLK) inhibitor with neuroprotective and neurotrophic effects, leads to increased total BDNF mRNA in blood when compared to untreated R6/2 mice. Our results indicate that alterations in BDNF mRNA levels in peripheral blood are a readily accessible measurement of disease progression and drug efficacy in HD rodent models.

Rescue of gene expression by modified REST decoy oligonucleotides in a cellular model of Huntington's disease.

J. Neurochem. (2011) 116, 415–425.

NP03, a novel low-dose lithium formulation, is neuroprotective in the YAC128 mouse model of Huntington disease.

Huntington disease (HD), a neurodegenerative disorder caused by an expanded CAG repeat in the HTT gene, remains without a treatment to modify the course of the illness. Lithium, a drug widely used for the treatment of bipolar disorder, has been shown to exert neuroprotective effects in a number of models of neurological disease but may have various toxic effects at conventional therapeutic doses. We examined whether NP03, a novel low-dose lithium microemulsion, would improve the disease phenotypes in the YAC128 mouse model of HD. We demonstrate that NP03 improves motor function, ameliorates the neuropathological deficits in striatal volume, neuronal counts, and DARPP-32 expression, and partially rescues testicular atrophy in YAC128 mice. These positive effects were accompanied by improvements in multiple biochemical endpoints associated with the pathogenesis of HD, including normalization of caspase-6 activation and amelioration of deficits in BDNF levels, and with no lithium-related toxicity. Our findings demonstrate that NP03 ameliorates the motor and neuropathological phenotypes in the YAC128 mouse model of HD, and represents a potential therapeutic approach for HD.

Lack of huntingtin promotes neural stem cells differentiation into glial cells while neurons expressing huntingtin with expanded polyglutamine tracts undergo cell death

Huntingtons disease (HD) is a neurodegenerative disorder that affects muscle coordination and diminishes cognitive abilities. The genetic basis of the disease is an expansion of CAG repeats in the Huntingtin (Htt) gene. Here we aimed to generate a series of mouse neural stem (NS) cell lines that carried varying numbers of CAG repeats in the mouse Htt gene (Hdh CAG knock-in NS cells) or that had Hdh null alleles (Hdh knock-out NS cells). Towards this end, Hdh CAG knock-in mouse ES cell lines that carried an Htt gene with 20, 50, 111, or 140 CAG repeats or that were Htt null were neuralized and converted into self-renewing NS cells. The resulting NS cell lines were immunopositive for the neural stem cell markers NESTIN, SOX2, and BLBP and had similar proliferative rates and cell cycle distributions. After 14 days in vitro, wild-type NS cells gave rise to cultures composed of 70% MAP2(+) neurons and 30% GFAP(+) astrocytes. In contrast, NS cells with expanded CAG repeats underwent neuronal cell death, with only 38%±15% of the MAP2(+) cells remaining at the end of the differentiation period. Cell death was verified by increased caspase 3/7 activity on day 14 of the neuronal differentiation protocol. Interestingly, Hdh knock-out NS cells treated using the same neuronal differentiation protocol showed a dramatic increase in the number of GFAP(+) cells on day 14 (61%±20% versus 24%±10% in controls), and a massive decrease of MAP2(+) neurons (30%±11% versus 64%±17% in controls). Both Hdh CAG knock-in NS cells and Hdh knock-out NS cells showed reduced levels of Bdnf mRNA during neuronal differentiation, in agreement with data obtained previously in HD mouse models and in post-mortem brain samples from HD patients. We concluded that Hdh CAG knock-in and Hdh knock-out NS cells have potential as tools for investigating the roles of normal and mutant HTT in differentiated neurons and glial cells of the brain.

Binding of the repressor complex REST-mSIN3b by small molecules restores neuronal gene transcription in Huntington's disease models.

Transcriptional dysregulation is a hallmark of Huntingtons disease (HD) and one cause of this dysregulation is enhanced activity of the REST‐mSIN3a‐mSIN3b‐CoREST‐HDAC repressor complex, which silences transcription through REST binding to the RE1/NRSE silencer. Normally, huntingtin (HTT) prevents this binding, allowing expressing of REST target genes. Here, we aimed to identify HTT mimetics that disrupt REST complex formation in HD. From a structure‐based virtual screening of 7 million molecules, we selected 94 compounds predicted to interfere with REST complex formation by targeting the PAH1 domain of mSIN3b. Primary screening using DiaNRSELuc8 cells revealed two classes of compounds causing a greater than two‐fold increase in luciferase. In particular, quinolone‐like compound 91 (C91) at a non‐toxic nanomolar concentration reduced mSIN3b nuclear entry and occupancy at the RE1/NRSE within the Bdnf locus, and restored brain‐derived neurotrophic factor (BDNF) protein levels in HD cells. The mRNA levels of other RE1/NRSE‐regulated genes were similarly increased while non‐REST‐regulated genes were unaffected. C91 stimulated REST‐regulated gene expression in HTT‐knockdown Zebrafish and increased BDNF mRNA in the presence of mutant HTT. Thus, a combination of virtual screening and biological approaches can lead to compounds reducing REST complex formation, which may be useful in HD and in other pathological conditions.