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

Dorotea Rigamonti,Daniele Bolognini,Cesare Mutti,Chiara Zuccato,Marzia Tartari,Francesco Sola,Marta Valenza,Aleksey G Kazantsev,Elena Cattaneo

doi:10.1074/jbc.m609885200

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.

Highlights

Huntington disease (HD)4 is an inherited dominant neurodegenerative disorder for which there is no effective treatment
We have previously shown that wild-type huntingtin stimulates brainderived neurotrophic factor (BDNF) gene transcription [12] 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 [13]
Forty-eight hours after transfection, there was a proportional increase in luciferase activity with increasing doses of dominant negative REST/NRSF construct (DN):REST in comparison with untransfected cells or cells treated with an empty vector (Fig. 1A), indicating that DN:REST overexpression attenuates endogenous REST/NRSF binding to RE1/NRSEbdnf in a dose-dependent manner

Summary

EXPERIMENTAL PROCEDURES

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. The amplification cycles for total BDNF consisted of an initial 10-min denaturing cycle at 95 °C, followed by 40 cycles of 10 s at 95 °C, 10 s at 56 °C, and 20 s at 72 °C. At the indicated time points, the cells were fixed in paraformaldehyde 4% for 15 min, washed with 1ϫ phosphate-buffered saline and permeabilized with 0.5% Triton X-100 in 1ϫ phosphate-buffered saline. They were blocked for 1 h at room temperature in 1ϫ phosphate-buffered saline, 5% nonfat milk, and 5% goat serum. Statistical Analysis—Bonferroni’s test or an ANOVA t test were used as indicated to evaluate the statistical significance of the results

RESULTS

PCR showed an increase in total

Receptor Gene Expression but Do

DISCUSSION