Loss of Huntingtin Function Complemented by Small Molecules Acting as Repressor Element 1/Neuron Restrictive Silencer Element Silencer Modulators

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. 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. Huntington disease (HD) 4The abbreviations used are: HDHuntington diseaseRE1/NRSErepressor element 1/neuron restrictive silencer elementBDNFbrain-derived neurotrophic factorREST/NRSFsilencing transcription factor/neuron-restrictive silencer factorHDAChistone deacetylaseCRFcorticotropin-releasing factorRLUrelative light unitELISAenzyme-linked immunosorbent assayDAPI4′,6-diamidino-2-phenylindoleBrdUrdbromodeoxyuridineANOVAanalysis of varianceDNdominant negativeMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideTSAtrichostatin ASAHAsuberoylanilide hydroxamic acid 4The abbreviations used are: HDHuntington diseaseRE1/NRSErepressor element 1/neuron restrictive silencer elementBDNFbrain-derived neurotrophic factorREST/NRSFsilencing transcription factor/neuron-restrictive silencer factorHDAChistone deacetylaseCRFcorticotropin-releasing factorRLUrelative light unitELISAenzyme-linked immunosorbent assayDAPI4′,6-diamidino-2-phenylindoleBrdUrdbromodeoxyuridineANOVAanalysis of varianceDNdominant negativeMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideTSAtrichostatin ASAHAsuberoylanilide hydroxamic acid is an inherited dominant neurodegenerative disorder for which there is no effective treatment. Since the discovery that its genetic cause is a polyglutamine expansion in the novel protein huntingtin (1Huntington's Disease Collaborative Research GroupCell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (6893) Google Scholar), extensive efforts have been made to find its underlying mechanisms and potential therapeutic targets. Huntington disease repressor element 1/neuron restrictive silencer element brain-derived neurotrophic factor silencing transcription factor/neuron-restrictive silencer factor histone deacetylase corticotropin-releasing factor relative light unit enzyme-linked immunosorbent assay 4′,6-diamidino-2-phenylindole bromodeoxyuridine analysis of variance dominant negative 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide trichostatin A suberoylanilide hydroxamic acid Huntington disease repressor element 1/neuron restrictive silencer element brain-derived neurotrophic factor silencing transcription factor/neuron-restrictive silencer factor histone deacetylase corticotropin-releasing factor relative light unit enzyme-linked immunosorbent assay 4′,6-diamidino-2-phenylindole bromodeoxyuridine analysis of variance dominant negative 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide trichostatin A suberoylanilide hydroxamic acid A number of pathogenic mechanisms have been considered so far, which indicates that more than one target should be investigated to reach a positive therapeutic effect (2Hersch S.M. Curr. Opin. Neurol. 2003; 16: 501-506PubMed Google Scholar, 3Ryu H. Ferrante R.J. Expert Opin. Emerg. Drugs. 2005; 10: 345-363Crossref PubMed Scopus (26) Google Scholar, 4Di Prospero N.A. Fischbeck K.H. Nat. Rev. Genet. 2005; 6: 756-765Crossref PubMed Scopus (122) Google Scholar). Most of these targets have been identified by looking at disease mechanisms triggered by gain of function defects caused by mutant huntingtin (5Sipione S. Cattaneo E. Mol. Neurobiol. 2001; 23: 21-51Crossref PubMed Scopus (66) Google Scholar), and we and other groups have hypothesized that diminished wild-type huntingtin function may also contribute to the pathogenesis of HD (6Cattaneo E. Zuccato C. Tartari M. Nat. Rev. Neurosci. 2005; 6: 919-930Crossref PubMed Scopus (496) Google Scholar). This suggests that a complementary approach based on identifying compounds that mimic wild-type huntingtin activity should be considered: the administration of novel compounds capable of restoring wild-type huntingtin function, together with drugs reducing mutant huntingtin toxicity, may be an optimal therapeutic strategy. The repressor element 1/neuron restrictive silencer element (RE1/NRSE) was the first example of an affected target of wild-type huntingtin activity to be discovered and is a silencer of gene expression (7Mori N. Stein R. Sigmund O. Anderson D.J. Neuron. 1990; 4: 583-594Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 8Mori N. Schoenherr C. Vandenbergh D.J. Anderson D.J. Neuron. 1992; 9: 45-54Abstract Full Text PDF PubMed Scopus (342) Google Scholar, 9Maue R.A. Kraner S.D. Goodman R.H. Mandel G. Neuron. 1990; 4: 223-231Abstract Full Text PDF PubMed Scopus (193) Google Scholar). Bioinformatic studies indicate that more than 1000 neuronal genes in the human genome bear an RE1/NRSE sequence in their promoters (10Bruce A.W. Donaldson I.J. Wood I.C. Yerbury S.A. Sadowski M.I. Chapman M. Gottgens B. Buckley N.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10458-10463Crossref PubMed Scopus (369) Google Scholar, 11Johnson R. Gamblin R.J. Ooi L. Bruce A.W. Donaldson I.J. Westhead D.R. Wood I.C. Jackson R.M. Buckley N.J. Nucleic Acids Res. 2006; 34: 3862-3877Crossref PubMed Scopus (109) Google Scholar), of which brain-derived neurotrophic factor (BDNF) is particularly interesting, because it is a pro-survival factor for the striatal neurons that die in HD. We have previously shown that wild-type huntingtin stimulates BDNF gene transcription (12Zuccato C. Ciammola A. Rigamonti D. Leavitt B.R. Goffredo D. Conti L. MacDonald M.E. Friedlander R.M. Silani V. Hayden M.R. Timmusk T. Sipione S. Cattaneo E. Science. 2001; 293: 493-498Crossref PubMed Scopus (1025) Google Scholar) by retaining in the cytoplasm the transcription factor that binds and activates the silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) sites involved in the regulation of RE1/NRSE and located inside BDNF promoter II (13Zuccato C. Tartari M. Crotti A. Goffredo D. Valenza M. Conti L. Cataudella T. Leavitt B.R. Hayden M.R. Timmusk T. Rigamonti D. Cattaneo E. Nat. Genet. 2003; 35: 76-83Crossref PubMed Scopus (714) Google Scholar). The inactivation of RE1/NRSE sites allows BDNF gene transcription (13Zuccato C. Tartari M. Crotti A. Goffredo D. Valenza M. Conti L. Cataudella T. Leavitt B.R. Hayden M.R. Timmusk T. Rigamonti D. Cattaneo E. Nat. Genet. 2003; 35: 76-83Crossref PubMed Scopus (714) Google Scholar, 14Zuccato C. Belyaev N. Conforti P. Ooi L. Tartari M. Papadimou E. MacDonald M.E. Fossale E. Zeitlin S. Buckley N. Cattaneo E. J. Neurosci. 2007; 27: 6972-6983Crossref PubMed Scopus (210) Google Scholar). Mutant huntingtin is less capable of recruiting REST/NRSF in the cytoplasm, which leads to its pathological translocation to the nucleus and increased occupancy of its cognate RE1/NRSE genomic loci (13Zuccato C. Tartari M. Crotti A. Goffredo D. Valenza M. Conti L. Cataudella T. Leavitt B.R. Hayden M.R. Timmusk T. Rigamonti D. Cattaneo E. Nat. Genet. 2003; 35: 76-83Crossref PubMed Scopus (714) Google Scholar, 14Zuccato C. Belyaev N. Conforti P. Ooi L. Tartari M. Papadimou E. MacDonald M.E. Fossale E. Zeitlin S. Buckley N. Cattaneo E. J. Neurosci. 2007; 27: 6972-6983Crossref PubMed Scopus (210) Google Scholar). In HD, the occupied RE1/NRSE silences transcription from RE1/NRSE-regulated genes, thus contributing to neuronal dysfunction (13Zuccato C. Tartari M. Crotti A. Goffredo D. Valenza M. Conti L. Cataudella T. Leavitt B.R. Hayden M.R. Timmusk T. Rigamonti D. Cattaneo E. Nat. Genet. 2003; 35: 76-83Crossref PubMed Scopus (714) Google Scholar, 14Zuccato C. Belyaev N. Conforti P. Ooi L. Tartari M. Papadimou E. MacDonald M.E. Fossale E. Zeitlin S. Buckley N. Cattaneo E. J. Neurosci. 2007; 27: 6972-6983Crossref PubMed Scopus (210) Google Scholar). Interest in compounds acting on a transcriptional system that supports the transcription of the BDNF gene (among others) is sustained by the fact that positive outcomes have been observed in HD models after increasing BDNF levels by means of transplanting cells engineered to overexpress BDNF into a chemical model of HD (15Perez-Navarro E. Canudas A.M. Akerund P. Alberch J. Arenas E. J. Neurochem. 2000; 75: 2190-2199Crossref PubMed Scopus (171) Google Scholar) or delivering BDNF via gene therapy (16Kells A.P. Fong D.M. Dragunow M. During M.J. Young D. Connor B. Mol. Ther. 2004; 9: 682-688Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 17Bemelmans A.P. Horellou P. Pradier L. Brunet I. Colin P. Mallet J. Hum. Gene Ther. 1999; 10: 2987-2997Crossref PubMed Scopus (144) Google Scholar). Transgenic overexpression of BDNF in the cerebral cortex can delay the progression of disease phenotypes in the R6/1 HD mouse model carrying the first 63 amino acids of mutant human huntingtin protein (18Xu G. Gharami K. Russel T. 277.17/MM3 Neuroscience. 2006; Google Scholar), and the genetic reduction of BDNF in the same R6/1 mice causes a worse phenotype (19Canals J.M. Pineda J.R. Torres-Peraza J.F. Bosch M. Martin-Ibanez R. Munoz M.T. Mengod G. Ernfors P. Alberch J. J. Neurosci. 2004; 24: 7727-7739Crossref PubMed Scopus (274) Google Scholar). Furthermore, these findings are paralleled by the fact that mice lacking cortical BDNF show a hind limb clasping phenotype similar to that observed in mouse models of HD (20Baquet Z.C. Gorski J.A. Jones K.R. J. Neurosci. 2004; 24: 4250-4258Crossref PubMed Scopus (321) Google Scholar). Finally, it has been more recently demonstrated that cystamine improves the HD phenotype in mice by enhancing BDNF release from the Golgi compartment in brain cells (21Borrell-Pages M. Canals J.M. Cordelieres F.P. Parker J.A. Pineda J.R. Grange G. Bryson E.A. Guillermier M. Hirsch E. Hantraye P. Cheetham M.E. Neri C. Alberch J. Brouillet E. Saudou F. Humbert S. J. Clin. Invest. 2006; 116: 1410-1424Crossref PubMed Scopus (200) Google Scholar). We here describe the development of the first cell-based assay designed to screen for compounds that are potentially capable of restoring the loss of wild-type huntingtin function in RE1/NRSE-mediated gene transcription by identifying genetic and chemical RE1/NRSE modulators. We used this assay to identify some small molecules that inhibit RE1/NRSE activity and then assessed their efficacy on molecular and phenotype parameters in HD cellular models. Transcription Factor and Reporter Gene Constructs—The RE1/NRSEbdnf-LUC reporter construct was constructed using the enhanced luciferase reporter gene pGL3-promoter (Promega, Madison, WI) as a backbone. A 300-bp portion of the rat BDNF promoter exon II containing the RE1/NRSE sequence was obtained from the BDNF+II CAT construct (41Timmusk T. Lendahl U. Funakoshi H. Arenas E. Persson H. Metsis M. J. Cell Biol. 1995; 128: 185-199Crossref PubMed Scopus (107) Google Scholar), and into the pGL3-promoter from which was The same was used for the The 300-bp was obtained from the CAT construct T. K. Lendahl U. Metsis M. J. Biol. 1999; Full Text Full Text PDF PubMed Google Scholar). The in the RE1/NRSE are in Timmusk T. K. Lendahl U. Metsis M. J. Biol. 1999; Full Text Full Text PDF PubMed Google Scholar). The and REST/NRSF I.C. Belyaev Bruce A.W. Jones C. M. A. Buckley N.J. J. Mol. Biol. 2003; PubMed Scopus Google Scholar, A.W. Donaldson I.J. Wood I.C. Yerbury S.A. Sadowski M.I. Chapman M. Gottgens B. Buckley N.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10458-10463Crossref PubMed Scopus (369) Google Scholar) expression by J. of Cell and and cells in with at in a E. Conti L. J. Neurosci. Res. PubMed Scopus Google Scholar). the the cells in at a of and of the was into an together with for the The cells using a at for and one after which they in and in To of the RE1/NRSEbdnf-LUC reporter construct and of for to one cells. the cells in and a containing was of to and The for luciferase activity in a that positive with to a and of used to cells. and of compounds of to in the developed assay was molecules to be or and other transcriptional in to that BDNF and and from and and and its from The compounds in and in at the to the cells in on the The or for the at the of was used to the and a Reporter was used to the and luciferase using the for The are as relative light after the of as a luciferase activity was using the and is as in to protein as assessed using a acid assay Gene for of of and gene expression was assessed by means of The made in for of the genes to for We used an with a system of the in a of containing of and of and The for BDNF of an at by of at at and at was the and was by means of a analysis The of target gene to a gene the same The CAT and The for and of an at by of at at and at was the and was by means of a analysis The The for of an at by of at and at was the and was by means of a analysis The The for REST/NRSF of an at by of at at and at was the and by a analysis The the of target gene to a gene to a Nucleic Acids Res. 2001; PubMed Scopus Google Scholar). in of with of The and at at and the and at The for BDNF using an as by the The by means of a acid protein assay Cell cells in into at in the with and the was by and then at the the cells to bromide and the release of was at after at for cells in with after to the the the cells in for with and then with in for at in and The cells to for at by the for The with and analysis using a with the to the cells to in the or of compound was the and the cells with for and with in for at the cells using cells with A of cells was for or an used as to the of the of the Reporter the of the luciferase construct and its to which are to endogenous REST/NRSF (13Zuccato C. Tartari M. Crotti A. Goffredo D. Valenza M. Conti L. Cataudella T. Leavitt B.R. Hayden M.R. Timmusk T. Rigamonti D. Cattaneo E. Nat. Genet. 2003; 35: 76-83Crossref PubMed Scopus (714) Google Scholar), with the reporter construct and a dominant negative REST/NRSF construct I.C. Belyaev Bruce A.W. Jones C. M. A. Buckley N.J. J. Mol. Biol. 2003; PubMed Scopus Google Scholar). The of the of the repressor and was to REST/NRSF to the luciferase reporter and increase luciferase after there was a increase in luciferase activity with increasing of in with cells or cells with an thus that overexpression endogenous REST/NRSF to in a with REST/NRSF the luciferase expression by light in the promoter the reporter a in the RE1/NRSE sequence the effect of is also that to a luciferase than because of the of the silencing activity of mutant first We therefore that the luciferase reporter is and to REST/NRSF transcription and of the Reporter Cell the was with luciferase and and the expression of the was by means of a luciferase because of the in luciferase activity The with or an and their luciferase activity was to a and luciferase of the no more than a increase in luciferase which we be to of the at the increased luciferase activity in the and we the one with the for that this to with a increase in luciferase of REST/NRSF and as a repressor A. L. Wood I.C. T. U. Buckley N.J. Mol. Biol. 2000; PubMed Scopus Google Scholar, R. C. J. P. C. Mandel G. Science. PubMed Scopus Google Scholar), the assay was in the of the transcriptional and In cells to at of to there was a increase in luciferase activity The effect of indicates that activity is for the of by and that the the luciferase reporter is to an this we as the compound for assay the assay to we the a of the of a assay that the and from positive and negative K.R. J. 1999; 4: PubMed Scopus Google Scholar). The factor from in which cells to of for was thus the of the The for the transcriptional of the developed assay was in a pilot of a of In the for a of we first used compounds that are to this We a which activates gene transcription by reducing the of silencing and and its similar of the which have previously been identified as increasing transcriptional activity in a reporter assay of mutant huntingtin cells in which the reporter was by sites S.M. Neurobiol. 2007; PubMed Google Scholar). molecules the involved in BDNF expression also in an of because an increase in levels BDNF expression P. G. Timmusk T. N. J. Neurochem. PubMed Scopus (109) Google Scholar). it is that also increase BDNF expression levels M. S. J. Neurosci. 1995; PubMed Google Scholar, Sci. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), we a and as some findings indicate that stimulates BDNF expression M.J. E. 2000; PubMed Scopus Google Scholar, J. 1999; Google Scholar), we a structural of of D. M. L. E. P. C. V. C. J. J. G. R. D. G. P. M. R. B. G. Ther. PubMed Scopus Google Scholar, G. N. A.P. G. C. R. R. Ther. 2003; PubMed Scopus Google Scholar). cells in after to the chemical in for after which luciferase activity was The compounds at and of the compounds reporter activity in for the structural of to as compound in this which to be in the was in a at nanomolar concentrations, which a increase in luciferase activity from the to the the increase in luciferase activity by of compound the of the we a and which no on luciferase expression and and and in the of This is by recently the of of on BDNF production in E. K. D.J. J. Neurosci. 2006; 23: PubMed Scopus Google Scholar). To the of compound which was identified as capable of stimulating luciferase we some similar and and to luciferase activity paralleled the effect observed in the of the positive as observed for compound then in and and similar to observed with compound the and of the three together with the to their activity luciferase and viability as assessed by To that the of the compounds was or transcriptional cells to acid and or which are to in this D. C. Conti L. Sipione S. C. E. A. Hayden M.R. S. C. Cattaneo E. J. Neurosci. 2000; PubMed Google Scholar). increased luciferase activity in the novel structural was by the in that compounds and the the reporter assay can identify compounds that inhibit the activity of the endogenous RE1/NRSE was from cells to compounds or an increase in BDNF after the administration of three compounds and an assay in the same that the of BDNF was by an increase in BDNF protein The of the BDNF protein in after to as positive and in the of the of the that the increase in endogenous BDNF transcription and is and and Gene REST/NRSF or Gene that the compounds using cells can the RE1/NRSE-regulated expression of genes other than and expression as of the neuronal genes a The from cells to the compounds using the same as was and the was using for the genes. The A and that to of compounds or to a increase in and an increase was also observed in the of the To the that the compounds as transcriptional we their on the expression of genes that are the RE1/NRSE-regulated REST/NRSF and and a increase in the cells with the the REST/NRSF and genes by compounds at that are on RE1/NRSE genes, thus that the compounds on RE1/NRSE-regulated gene transcription. and BDNF and in an HD the compounds increased BDNF gene transcription in the of mutant huntingtin using previously that a in BDNF in with their (12Zuccato C. Ciammola A. Rigamonti D. Leavitt B.R. Goffredo D. Conti L. MacDonald M.E. Friedlander R.M. Silani V. Hayden M.R. Timmusk T. Sipione S. Cattaneo E. Science. 2001; 293: 493-498Crossref PubMed Scopus (1025) Google Scholar). In a first compounds or to from mutant huntingtin mice in which an has been into the endogenous mouse gene F. Rigamonti D. P. F. Cattaneo E. MacDonald M.E. Hum. Mol. Genet. 2000; 9: PubMed Scopus Google Scholar). the an increase after with of compounds and of compound increase the increase observed after the cells with of the same The compounds also increased BDNF protein in engineered in a as to overexpress the mutant huntingtin protein D. C. Conti L. Sipione S. C. E. A. Hayden M.R. S. C. Cattaneo E. J. Neurosci. 2000; PubMed Google Scholar) is that the effect is in a mutant huntingtin at the same as that which is in cells. Cell in HD Cell the of compound on The cells to a in the or of nanomolar of compound and viability was assessed by assay after treatment. shown in viability increased by in the of compound and an increase was observed at a of as as This increase in number in the was to increased because the of cells was increased by the compound and REST/NRSF to cells in the or of of compounds or at and using an the transcription factor the and levels of REST/NRSF using the to This in the translocation of REST/NRSF by the thus that other mechanisms at the RE1/NRSE sites may inhibit the activity of the silencer in HD cells to the The assay in this is based on a sequence RE1/NRSE silencer the BDNF silencing activity is enhanced in HD, because increased of its cognate transcription factor REST/NRSF the transcription of BDNF and other neuronal genes. The REST/NRSF phenotype is related to huntingtin because genetic of huntingtin to its overexpression or or increase REST/NRSF We have developed a a reporter construct in which luciferase activity is by a 300-bp portion of the BDNF promoter containing the The engineered of the REST/NRSF transcription factor to the the reporter as assessed by the of and its dominant negative and was therefore used for pilot of the compounds to be capable of the silencing activity of the thus to an increase in reporter this effect at low nanomolar Moreover, a assay that the identified compounds also inhibit the endogenous BDNF The increase in BDNF levels is by BDNF protein levels because this effect is also in a mutant huntingtin it is possible that the compounds may the same effect in the human Our assay was developed on the of a molecular that has been in HD models and in (12Zuccato C. Ciammola A. Rigamonti D. Leavitt B.R. Goffredo D. Conti L. MacDonald M.E. Friedlander R.M. Silani V. Hayden M.R. Timmusk T. Sipione S. Cattaneo E. Science. 2001; 293: 493-498Crossref PubMed Scopus (1025) Google Scholar, C. Tartari M. Crotti A. Goffredo D. Valenza M. Conti L. Cataudella T. Leavitt B.R. Hayden M.R. Timmusk T. Rigamonti D. Cattaneo E. Nat. Genet. 2003; 35: 76-83Crossref PubMed Scopus (714) Google Scholar) and allows the of compounds that are on RE1/NRSE-regulated genes. The for this which is by the positive effect of genetic or pharmacological BDNF administration in HD mouse is enhanced by the fact that the identified compounds may BDNF they are small which the with more their effect BDNF because they also increase the transcription of other neuronal genes RE1/NRSE in their promoter, as here in the of and The assay compounds that a reporter and on endogenous RE1/NRSE-regulated genes, because genes are for neuronal survival and of their transcription should have a positive on neuronal This the of the assay that of the identified to other than HD. The of REST/NRSF and its target genes in brain has in fact been demonstrated in a number of neurodegenerative including the to K. N. Metsis M. Timmusk T. J. Neurosci. PubMed Google Scholar, A. T. H. H. R. J. Neurosci. 2003; 23: PubMed Google Scholar), S. M. M. E. M. P. Full Text Full Text PDF PubMed Scopus Google Scholar), and some P. R. A. D. L. A. V. S. Nat. 2000; 6: PubMed Scopus Google Scholar). Our compounds increase endogenous RE1/NRSE-regulated genes and protein expression demonstrated in the are also in an HD in which they increase studies be to the activity of the the obtained in the compounds their be in to their to from HD molecular and We are to Buckley for the and for and to Timmusk for the BDNF promoter II with