Normalizing HDAC2 in brain removes repression of learning-related gene expression.

Scientists say histone deacetylase 2 (HDAC2)-selective inhibitors  may represent a therapeutic approach to slowing cognitive decline in patients with neurodegenerative disorders such as Alzheimer disease (AD). Researchers at the Massachusetts Institute of Technology’s (MIT) Picower Institute for Learning and Memory and Howard Hughes Medical Institute found increased levels of HDAC2 in two mouse models of AD and in human AD patients. These increased levels of the deacetylating enzyme were associated with the reductions in histone acetylation and expression levels of key learning-related genes.

Critically, the team subsequently showed that using RNAi to reduce HDAC2 build up in the hippocampus of a mouse neurodegenerative disease model removed this repression, reinstated neuronal structural and synaptic plasticity, and eradicated neurodegeneration-associated memory deficits. Li-Huei Tsai, Ph.D., and colleagues report their findings in Nature in a paper titled “an epigenetic blockade of cognitive functions in the neurodegenerating brain.”

Epigenetic modifications in the nervous system that are mediated by histone acetylation have been unequivocally associated with facilitating learning and memory, the researchers state. Multiple studies have in addition reported that reduced histone acetylation is associated with cognitive decline in animal models of neurodegeneration including AD.

To further investigate the role of HDAC2 in neurodegeneration-related cognition, the team looked at levels of the enzyme in CK-p25 mice. These animals can be induced to overexpress p25, in the forebrain. p25 is a truncated version of p35 and is implicated in a range of neurodegenerative diseases. Their studies showed that animals induced to overexpress p25  demosntrated significant increases in HDAC2 in neuronal nuclei, specifically  hippocampal area CA1 but not CA3 or the dentate gyrus, and also in the prefrontal cortex. In contrast, levels of the HDAC1 and HDAC3, were not changed.

The researchers then moved on to carry out chromatin immunopreciptation studies to  assess the functional consequences of HDAC2 elevation, primarily in a range of known HDAC2 targets that have been shown to be downregulated in human AD brains, including learning- and memory-related genes, and those involved in synaptic plasticity. They found elevated HDAC2 enrichment at the majority of these genes induced CK-p25 hippocampus, whereas again, HDAC1 and HDAC3 binding wasn’t affected. “Interestingly, in agreement with previous reports showing that HDAC2 can also bind to a gene’s coding region, we also found HDAC2 more abundantly bound to the coding sequence of the same genes,” they note.

Prior research has demonstrated the importance acetylation at histone residues in the promotor regions of learning-, memory-, and synaptic plasticity-related genes. When the MIT investigators assessed this in CK-p25 mice, they found different levels of hypoacetylation  for all residues at the neuroplasticity genes but no changes in acetylation of housekeeping genes. Promotor hypoacetylation in the neuroplasticity genes was in addition associated with reduced binding of activated RNA polymerase II. In fact quantitative RT-PCR assays confirmed that there was a reduced expression of all genes affected by elevated HDAC2 binding and thus reduced histone acetylation and RNA Pol II binding.  

“The effects of elevated HDAC2 levels further appear to be restricted to histones,” the authors add, “as we found no overall acetylation changes on other proteins regulated by this modification, such as tau (also known as MAPT), protein 53 (p53; also known as TP53), and tubulin nor in overall nuclear or cytoplasmic protein acetylation.”

The results thus far indicated that HDAC2 is directly involved in decreasing the expression of neuroplasticity genes, which may contribute to cognitive deficits during neurodegeneration.  In order to test this direct relationship in vivo, the researchers first designed short hairpin RNA constructs that knocked down HDAC2 in cultured cells by about 25–30%. This level of knockdown  would be enough to effectively normalize HDAC2 levels in CK-p25 mice. Viral vectors carrying the shRNA were then injected into the CA1 hippocampal region of induced CK-p25 and control mice, and HDAC2 levels in the brain assessed a month later.

Initial analyses confirmed that HDAC2 levels had in the CK-225 mice were reduced to those found in control mice, whereas protein levels of HDAC1 and HDAC3 were unchanged. Encouragingly, the lower HDAC2 levels were associated with increased H4K12 histone acetylation on most of the neuroplasticity genes evaluated, and expression that comparable or higher than that in control mice.

Encouragingly, while HDAC2 normalization didn’t have an ultimate effect on neuronal survival in the neurodegenerative CK-p25 mice, it did “reinstate morphological and synaptic plasticity in the surviving neurons.” And when the researchers evaluated cognitive capabilities using tests of hippocampus-dependent memory, they found that the associative memory of the shHDAC2-treated animals was the same as that of control animals.

The MIT team moved on to investigate potential mechanisms underlying the increase in HDAC2. Studies using cultured hippocampal neurons subjected to neurotoxic stimuli indicated that increased HDAC2 gene transcription at the mRNA level as a result of neurotoxic assault involved glucocorticioid receptor activation and its interaction with the HDAC2-glucocorticoid responsive element (GRE) in its promoter region.

Analysis of postmortem studies of human nonfamilial AD brain tissue confirmed the relevance of the mouse studies. The results showed that at all stages of AD, HDAC2 levels (but not HDAC1 or HDAC3) were significantly increased in hippocampal area CA1 and entorhinal cortex. These are two of the earliest and most severely affected brain regions in AD and are crucial for memory formation and storage, the team explains.

The overall results provide some hope that it may be possible to hold back cognitive deficits in AD patients, they state. “Our finding that HDAC2 inhibition probably reinstates transcriptional, morphological, and synaptic plasticity in the surviving neurons of the neurodegenerating brain raises hope that such plasticity is not irrevocably lost but merely constrained by the epigenetic blockade.”

The findings may also help explain why cognitive impairments in patients with AD who are involved in clinical trials persist despite successful amyloid-β  clearance. “Once the epigenetic blockade is in place, reducing amyloid-β generation and deposition alone may not be sufficient to rescue against cognitive dysfunction,” the team suggests. “A more efficacious strategy may therefore lie in the combination of amyloid-beta reduction with the inhibition of HDAC2.”

Previous articleIslet Sciences to Pick Up DiaKine Therapeutics
Next articleSequencing in the Dark