The availability of a well characterized microarray database allows for the preliminary test of a hypothesis in-silico. Given the validity of the technology, the caveat is to avoid analyzing a broad array of targets thereby creating an over-determined statistic as we have previously reported (Gibbons et al 2005
). We surveyed expression levels in nine
HDAC genes, and accepted six
for statistical analysis. This effort is a direct extension of our ongoing examination of chromatin structure in schizophrenia and bipolar subjects (Sharma et al 2005a
, Sharma et al 2006
, Gavin et al 2007
During the preparation of this manuscript, Benes et al. (2007)
reported elevated HDAC1 levels from an independent sample of postmortem hippocampal tissue (seven normal, seven schizophrenia and seven bipolar subjects) using laser microdissected samples of GABA neurons obtained from the stratum oriens of CA2/3 layers. They reported elevated levels of HDAC1 by microarray analysis, which was validated using quantitative RT-PCR. Overall a 2 fold increase of HDAC1 in the GABA neurons of the stratum oriens of the CA2/3 region of the hippocampus from schizophrenia subjects was found.
The increase in HDAC1 in the prefrontal cortices of schizophrenia subjects (as well as the specific region of the hippocampus as described by Benes et al 2007
) is noteworthy because enhanced expression of these enzymes is predictive of chromatin that is deactylated at site specific residues and consequently repressive to transcriptional activity. Akbarian et al (2005)
have examined histone modifications in protein extracts from the prefrontal cortices of schizophrenia patients and normal subjects using Western blot. Although not finding overall differences in levels of modified histones, they report reduced levels of targeted gene expression in subsets of patients with higher levels of specific modifications including acetylation at H3 Lysine 14. We have earlier reported that histone protein obtained from the peripheral lymphocytes of clinical schizophrenia patients is less acetylated than that obtained from bipolar patients, and remains unyielding when subjects are treated with an HDAC inhibitor (Sharma et al 2006
). Other details of our analysis that should be considered preliminary gender differences particularly in the schizophrenia subsample, where female schizophrenia subjects (n=3) had almost twice the amount of HDAC1 in their prefrontal cortices than did their male counterparts (n=13) (516.86 vs. 265.33; p<0.003). Also, the confounding effects of psychotropic medication cannot be entirely resolved in this clinical sample because of multiple medications and lengths of treatment. Preliminary observations from our lab indicates that HDAC inhibitors such as Trichostatin-A and Valproic acid do not reliably change HDAC1 mRNA expression in either primary lymphocyte cultures from human subjects (Gavin and Sharma unpublished) or in NT2 dividing cells (Kundakovic and Grayson unpublished). However, MS-275, a new research HDAC inhibitor, can downregulate protein levels of HDAC1 in NT2 cells (Kundakovic and Grayson personal communication). Furthermore, the effects of the typical and atypical antipsychotics as well as antidepressant medications could not be systematically explored. The effects of valproic acid on HDAC9 are interesting and should be considered preliminary. Nonetheless, the effects of psychotropic medications on chromatin structure and its attendant enzymes will be a fruitful area for future investigation.
Prior basic work in cell lines (Kundakovic et al., 2006
), primary neuron cultures (Noh et al., 2005
; Sharma unpublished data), rodent models (Tremolizzo et al., 2002
, Simonini et al., 2006
) and peripheral blood lymphocytes from human subjects (Gavin et al., 2007
) indicate that the GAD67 promoter is regulated by epigenetic mechanisms. These studies show that GAD67 expression is profoundly impacted by the acetylation of histone proteins surrounding its promoter as well as the intensity of DNA methylation in this region. Our observation of a negative correlation between the expression of GAD67 and several HDAC enzymes is encouraging in that it provides independent validity to the relevance of these findings, and is consistent with previous published reports
HDAC1 is a nuclear protein of the Class I type (de Ruijter et al 2003
). It is ubiquitously distributed (brain as well as lymphoid tissue for example), is activated by phosphorylation and associates with larger multiprotein repressor complexes (such as Co-REST in neurons) to stabilize its enzymatic activity (Saha and Pahan 2006
). HDAC1 is also inhibited by small molecule pharmacological agents such as the widely prescribed mood stabilizer valproic acid (mM range) and trichostatin A (nM range). Deactylase activity occurs in partnership with DNA binding proteins that not only recruit the enzyme to the targeted promoter but also enhance catalytic activity. HDAC1 has two phosphorylation sites (Serine421
) which regulate catalytic activity as well as protein-protein complex formation.
Enhanced deactylase activity and restrictive chromatin in the prefrontal cortices of schizophrenia patients is a heuristic explanation for a variety of clinical findings, from reduced metabolism, cognitive disturbances, negative symptoms and low motivation. Alternatively, restrictive chromatin may be a consequence of the impoverished environmental, emotional and cognitive stimulation that is the daily experience of these subjects or even perhaps the cognitive blunting imposed by ongoing antipsychotic treatment. In either case, a catalytic enzyme serves as an ideal target for pharmacology and a realistic approach to regulating gene expression in a targeted brain region.