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1.
J Neurochem ; 127(1): 22-35, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23800350

ABSTRACT

Transcriptional dysregulation is a hallmark of Huntington's 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.


Subject(s)
Huntington Disease/genetics , Huntington Disease/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription, Genetic/physiology , Animals , Animals, Genetically Modified , Brain-Derived Neurotrophic Factor/metabolism , Cell Proliferation , Cells, Cultured , Chromatin Immunoprecipitation , Co-Repressor Proteins , Embryo, Nonmammalian , Enzyme-Linked Immunosorbent Assay , Humans , Huntingtin Protein , Immunohistochemistry , Luciferases/metabolism , Microinjections , Models, Molecular , Nerve Tissue Proteins/biosynthesis , Polymerase Chain Reaction , RNA, Messenger/administration & dosage , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Transcription, Genetic/genetics , Transfection , Zebrafish
2.
Neurobiol Dis ; 50: 160-70, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23089356

ABSTRACT

Huntington's 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.


Subject(s)
Cell Differentiation/genetics , Nerve Tissue Proteins/metabolism , Neural Stem Cells/cytology , Neuroglia/cytology , Neurons/cytology , Nuclear Proteins/metabolism , Animals , Fluorescent Antibody Technique , Gene Knock-In Techniques , Gene Knockout Techniques , Huntingtin Protein , Huntington Disease/genetics , Mice , Nerve Tissue Proteins/genetics , Neuroglia/metabolism , Neurons/metabolism , Nuclear Proteins/genetics , Peptides , Reverse Transcriptase Polymerase Chain Reaction , Trinucleotide Repeats
3.
Stem Cells Dev ; 20(8): 1411-23, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21275879

ABSTRACT

The potential use of neural stem cells (NSCs) in basic research, drug testing, and for the development of therapeutic strategies is dependent on their large scale in vitro amplification which, however, introduces considerable risks of genetic instability and transformation. NSCs have been derived from different sources, but the occurrence of chromosomal instability has been monitored only to a limited extent in relationship to the source of derivation, growth procedure, long-term culture, and genetic manipulation. Here we have systematically investigated the effect of these parameters on the chromosomal stability of pure populations of mouse NSCs obtained after neuralization from embryonic stem cells (ESCs) or directly from fetal or adult mouse brain. We found that the procedure of NSCs establishment is not accompanied by genetic instability and chromosomal aberration. On the contrary, we observed that a composite karyotype appears in NSCs above extensive passaging. This phenomenon is more evident in ESC- and adult sub-ventricular zone-derived NSCs and further deteriorates after genetic engineering of the cells. Fetal-derived NSCs showed the greatest euploidy state with negligible clonal structural aberrations, but persistent clonal numerical abnormalities. It was previously published that long-term passaged ESC- and adult sub-ventricular zone-derived NSCs did not show any defects in the cells' proliferative and differentiative capacity nor induced in vivo tumour formation, although we here report on the chromosomal abnormalities of these cells. Although chromosomal aberrations are known to occur less frequently in human cells, studies performed on murine stem cells provide an important complement to understand the biological events occurring in human lines.


Subject(s)
Chromosomes , Neural Stem Cells/cytology , Animals , Cell Culture Techniques/methods , Cell Differentiation , Cell Line , Cell Proliferation , Chromosomal Instability , Chromosome Aberrations , Embryo, Mammalian/cytology , Karyotype , Mice , Nervous System/embryology , Neural Stem Cells/metabolism , Neurons/cytology
4.
Curr Pharm Des ; 15(34): 3958-67, 2009.
Article in English | MEDLINE | ID: mdl-19751206

ABSTRACT

REST/NRSF is a transcription factor that represses transcription of several neuronal genes by binding to a DNA regulatory motif known as Repressor Element 1/Neuron-restrictive silencer element (RE1/NRSE). In Huntington's Disease, an inherited degenerative disease affecting the brain, REST/NRSF enters pathologically into the nucleus of affected cells, leading to the activation of the RE1/NRSE sites and causing decreased transcription of several important neuronal genes. Following this discovery, an effort has begun by some of the authors aimed at identifying compounds capable of antagonizing REST/NRSF silencing activity. Here we will review the underlying basis for focusing pharmaceutical efforts on REST/NRSF-RE1/NRSE system as well as some of the strategies for a rational drug design approach. We will highlight approaches aimed at identifying or designing small molecules able to impact REST/NRSF nuclear translocation, its DNA binding or, more generally, the formation of the REST/NRSF transcriptional complex, in the attempt to restore neuronal gene transcription in pathological conditions of the brain.


Subject(s)
Huntington Disease/genetics , Huntington Disease/metabolism , Neuroprotective Agents/pharmacology , Repressor Proteins/metabolism , Genetic Therapy , Humans , Repressor Proteins/genetics
5.
Bioorg Med Chem ; 16(10): 5695-703, 2008 May 15.
Article in English | MEDLINE | ID: mdl-18406155

ABSTRACT

REST/NRSF is a multifunctional transcription factor that represses or silences many neuron-specific genes in both neural and non-neural cells by recruitment to its cognate RE1/NRSE regulatory sites. An increase in RE1/NRSE genomic binding is found in Huntington's disease (HD), resulting in the repression of REST/NRSF regulated gene transcription, among which BDNF, thus representing one of the possible detrimental effectors in HD. Three 2-aminothiazole derivatives were recently identified as potent modulators of the RE1/NRSE silencing activity through a cell-based gene reporter assay. In this study, the structure-activity relationships (SAR) of a library of commercially available 2-aminoisothiazoles diversely substituted at the amino group or at position 4 has been evaluated. A quantitative structure-activity relationship analysis performed using the Phase strategy yielded highly predictive 3D-QSAR pharmacophore model for in silico drug screening.


Subject(s)
Huntington Disease/genetics , Thiazoles/chemistry , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Gene Library , Gene Silencing/drug effects , Huntington Disease/drug therapy , Models, Molecular , Molecular Structure , Quantitative Structure-Activity Relationship , Repressor Proteins/drug effects , Repressor Proteins/genetics , Reproducibility of Results , Stereoisomerism , Structure-Activity Relationship , Thiazoles/pharmacology , Thiazoles/therapeutic use , Transcription, Genetic/drug effects
6.
J Biol Chem ; 282(34): 24554-62, 2007 Aug 24.
Article in English | MEDLINE | ID: mdl-17565993

ABSTRACT

Increased levels of the repressor element 1/neuron restrictive silencer element (RE1/NRSE) silencing activity promoter, and a consequent reduction in the transcription of many RE1/NRSE-bearing neuronal genes, including brain-derived neurotrophic factor (BDNF), have been demonstrated in Huntington disease (HD) and represent one possible effector of its selective neuronal vulnerability. Restoring the expression levels of neuronal genes in diseased neurons therefore seems to be an attractive therapeutic approach. To this end, we have developed a cell-based reporter assay for monitoring RE1/NRSE silencing activity and validated it by genetically inactivating the RE1/NRSE or pharmacologically stimulating global transcription. In a pilot compound screen, we identified three closely related structural analogues that up-regulate reporter expression at low nanomolar concentrations, and follow-up studies have shown that they efficaciously increase endogenous BDNF levels in HD cells. Moreover, one of the compounds increases the viability of HD cells. Our findings suggest a new avenue for the development of drugs for HD and other neurodegenerative disorders based on the pharmacological up-regulation of the production of the neuronal survival factor BDNF and of other RE1/NRSE-regulated neuronal genes.


Subject(s)
Nerve Tissue Proteins/physiology , Nuclear Proteins/physiology , Animals , Brain-Derived Neurotrophic Factor/metabolism , Cell Survival , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation , Gene Silencing , Huntingtin Protein , Immunohistochemistry , Luciferases/metabolism , Models, Chemical , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Nuclear Proteins/metabolism , Peptides/chemistry , Rats , Transcription Factors/metabolism
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