Title

 

Authors

S. Corna¹, A. Mallei¹, C. Benevento¹, D. Tardito¹, G. Racagni^1,2, F.S. Lee³, M. Popoli¹.

¹ Center of Neuropharmacology, Dept. of Pharmacological Science and Centre of Excellence on Neurodegenerative Diseases, University of Milano, Italy
² I.R.C.C.S. San Giovanni di Dio-Fatebenefratelli, Brescia, Italy
³Dept. of Psychiatry, Weill Cornell Medical College of Cornell University, New York, New York 10065, USA

 

Abstract

Several lines of evidence suggest an important role for BDNF in the pathogenesis of anxiety and mood disorder. Moreover, a human polymorphism in the BDNF gene (Val66Met) that causes a Met/Val substitution in codon 66 of proBDNF was associated with major susceptibility to neuropsychiatric diseases. The BDNF Val66Met transgenic mouse is the only existing animal model that recapitulates the phenotypic effect of the human polymorphism. Indeed, both human and mice BDNF_Met allele carriers show reduced hippocampal volume and deficit in fear extinction learning (Chen et al., 2006; Soliman et al., 2010). Epigenetic mechanisms involve self-perpetuating changes in chromatin structure and function that have been shown to regulate neuronal differentiation, neurodegeneration, circadian rhythms, seizure, memory, drug addiction, and stress response. Chromatin contains regions highly condensed (heterochromatin), and regions less condensed (euchromatin). The different regions reflect distinct functional states: euchromatin is actively transcribed, while heterochromatin is usually transcriptionally inactive. Post-translational modifications of histone tail residues, such as acetylation or methylation, can remodel the structure of chromatin activating or repressing gene expression.
In order to analyze epigenetic changes induced in mice gene expression by the presence of the BDNF Val66Met polymorphism, chromatin immunoprecipitation (ChIP) of hippocampus from wild-type (BDNF^Val/Val) and transgenic (BDNF^Met/Met) mice was performed with specific antibodies directed against DNA-bound histones, methylated or acetylated. Briefly, hippocampal chromatin was cross-linked to histone proteins, and then was sheared to 300-600 bp DNA fragments by sonication, and immunoprecipitated with antibodies anti-acetyl histone H3 (Lys9/14) (marker of gene expression activation), or anti-trimethyl histone H3 (Lys27) (marker of gene expression repression). ChIP was followed by quantitative real-time PCR (qPCR) analysis of immunoprecipitated DNA to determine levels of histone modifications at promoter of selected genes implicated in neuroplasticity and synaptic functions. Specific primers for promoters of CREB, c-Fos, BDNF transcripts, NMDAR-NR1/NR2A/NR2B genes were used.The percent of input values from each qPCR data set were analyzed in two-tailed unpaired Student’s t-test to determine statistical significance. In addition, qPCR measurements with TaqMan probe were performed to confirm epigenetic histone modification of gene expression. 
We found a significant increase of trimethyl histone H3 (Lys27) for BDNF transcript VI promoter (p<0.005) and NR2A subunit of NMDAR promoter (p<0.005) in BDNF^Met/Metmice. No changes were found in Lys9/14 acetylation for these two promoters, and no changes in both epigenetic marks for the other promoters. In addition, real-time PCR analysis showed lower levels of BDNF VI mRNA in Met/Met animals (-23% vs Val/Val, p<0,05).
Moreover, we performed a global epigenetic analysis of chromatin immunoprecipitated with the same markers of activation and repression used in the ChIP study in order to identify a whole list of gene promoters with epigenetic modifications at histone H3.
Epigenetic analysis and validation by qPCR show a reduced transcription of BDNF transcript VI in Met/Met mice.The epigenetic variations observed could explain some phenotypic features of this transgenic model such as reduced dendritic arborization. Indeed, BDNF VI is one of the trascripts that showed activity-dependent targeting to distal dendrites and our results suggest that reduced dendritic targeting is involved in reduced BDNF regulated secretion previously observed in these mice (Chiaruttini et al., 2008).
 
Chen et al. (2006). Science. 314, 140–143.
Chiaruttini et al. (2008). Mol. Cell. Neurosci. 37, 11–19.
Soliman et al. (2010). Science. 327, 863–866.