HDAC2 and Anxiety in Alcoholism

The Impact of HDAC2 Gene Expression on Anxiety

Our i-Fect Transfection Kit continues to be a potent tool for testing the impact of altered gene expression on behavior. see: SACHIN MOONAT. The Role of Amygdaloid Chromatin and Synaptic Remodeling in Anxiety and Alcoholism. THESIS Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience in the Graduate College of the University of Illinois at Chicago, 2014.

The author hypothesized that increased HDAC2 would have a positive impact on anxiety in alchohol preferring (P) rats. Specifically, HDAC2-induced histone modifications in the amygdala may play a role in the regulation of synaptic plasticity that may underlie the behavioral phenotypes of P rats. Furthermore, it could be possible that exogenous manipulation of HDAC2 levels in the amygdala may have an effect on anxiety-like behaviors and alcohol preference in P
rats.

Figure 1. Chromatin remodeling via histone acetylation and DNA methylation regulates gene transcription associated with changes in synaptic plasticity. During gene transcriptional processes, the chromatin structure associated with DNA to be transcribed is in a relaxed chromatin conformation due to hyperacetylation of histone proteins and hypomethylation of DNA, which allows access to transcriptional machinery. This relaxed chromatin structure results in increased gene transcription, which in neurons may cause increased expression of synaptically active proteins that result in the positive modulation of synaptic plasticity, such as increased dendritic spine density (DSD). DNA methyltransferase (DNMT) methylates DNA at CpG islands, leading to hypermethylated DNA and recruiting of methyl-CpG binding domain protein (MBD) complexes which block binding of transcriptional machinery. The MBD complex can in turn recruit histone deactylases (HDAC) which remove acetyl groups from histone proteins resulting in chromatin condensation thereby decreasing gene transcription. HDACs and histone acetyltransferases (HAT) control the histone acetylation profile, such that HDACs remove acetyl groups and HATs add acetyl groups to histone proteins. In this manner, increased HDAC expression results in hypoacetylation of histones leading to a condensed chromatin structure. Chromatin condensation resulting from HDAC-induced histone deacetylation or DNMT-induced DNA methylation causes reduced gene transcription. In neuronal cells, the reduction in gene transcription may be associated with decreased expression of synaptically active proteins and negative modulation of synaptic plasticity, such as reduced DSD. Treatment with DNMT inhibitors or HDAC inhibitors may block these enzymatic processes and return chromatin to a relaxed state, resulting in increased gene transcription and synaptic plasticity (Moonat and Pandey, 2012).

Methods: P rats that had been previously cannulated for delivery of solutions directly into the CeA were infused with either HDAC2 siRNA, control siRNA or vehicle. The siRNAs were dissolved in iFect solution (Neuromics, Edina, MN), a cationic lipid-based transfection solution, such that the final concentration of the solution was 2 µg/µL. The sequence of the HDAC2 siRNA was as follows: 5’-CAAGUUUCUACGAUCAACATT-3’; 5’-UAUUGAUCGUAGAAACUUGAT-3’. Some of the HDAC2 siRNA (Qiagen, Valencia,
CA) had been modified to include a 5’ Alexa Fluor-488 fluorescent probe in order to
determine the transfection efficiency and cellular localization of transfection. The control
siRNA used was the AllStars Negative Control siRNA (Qiagen), which shows no
homology to any known mammalian gene. To prepare the vehicle, RNase-free water was
dissolved in the iFect solution in place of any siRNA. The solutions (0.5 µL) were
infused bilaterally into the CeA of P rats using an automatic infusion pump which
resulted in a dose of 1 µg of siRNA per side. The automatic pump was attached to a
microdialysis probe which seated in the guide cannula and extended 3 mm past the tip of the cannula into the CeA.

For the experiments which looked at the anxiolytic effect of HDAC2 siRNA
infusion, P rats were infused with either HDAC2 siRNA, control siRNA or vehicle at the
end of the light cycle. 16 hours after the infusion, the rats were tested for anxiety-like behaviors. Immediately following behavioral testing, rats were anaesthetized and brains
were collected for further analysis.
For the voluntary drinking experiment, P rats were infused with either HDAC2
siRNA or vehicle when the bottles were changed following the third day of 9% ethanol
exposure. The rats continued to be monitored for the intake of 9% ethanol for 7 days
following the infusion. After the final day of voluntary drinking, the rats were
anaesthetized for collection of brains and blood to confirm the cannula position and the
blood alcohol levels, respectively.

Figure. The effects of HDAC2 siRNA Infusion into the CeA of P rats on voluntary ethanol consumption as measured by the two-bottle free choice paradigm. Monitoring the voluntary ethanol consumption of alcohol-preferring (P) rats via the two bottle free choice paradigm following infusion of vehicle or histone deacetylase isoform 2 (HDAC2) siRNA into the central amygdala (CeA) demonstrates that high HDAC2 levels may mediate the high alcohol drinking behaviors of P rats. P rats were given access to water and 7% ethanol followed by water and 9% ethanol. On the sixth day of ethanol access P rats received infusion of vehicle or HDAC2 siRNA and consumption of water and 9% ethanol were monitored for sevnfusion. Total fluid intake did not significantly differ between the groups. Values are represented as the mean ± SEM of the ethanol consumption (g / kg / day) and total fluid intake (mL) plotted daily for n=6 rats per treatment group. *Significantly different between the groups.
This data suggest reduction of HDAC2 levels in the CeA leads to reduced DSD associated with a reduction in anxiety-like behaviors and alcohol preference in P rats and could prove to have therapeutic value.

Intra-i-Fect Tissue Specific siRNA Kits

I have published content here on our collaborator’s success using i-Fect to delivery siRNA in vivo and in-vitro. These kits have been used for gene expression analysis studies of DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, Survivin,  Flaviviruses and more.

I am excited about another iteration of capablilities with our new:
Intra-i-Fect Tissue Specific siRNA Kits.

These kits are designed to deliver siRNA in vivo via intravenous injections with high efficiency to specific tissue in rats and mice. The protocol involves these simple steps: prep, mix, dry, hydrate and inject.

They are developed using a proprietary platform that uses nano-particles as the delivery vehicle. This platform enables:

  • Effective delivery (60%+ knockdown) with no toxicity.
  • Scalable to high throughput siRNA based gene screening.
  • Consistent and reproducible results

Delivering 27mer DsiRNAs to Mice DRGs

I have been a proponent of using 27mer DsiRNAs (Dicer Substrate Small Interfering RNAs) with our i-Fect kits to deliver siRNA to the CNS for gene expression analysis. The potency of this platform was highlighted in my profile of Dr. Mark Behlke.

It was further confirmed  in Studies conducted by Dr. Philippe Serrat and his team at University of Sherbrooke.

Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); Jul;16(7):1331-9. Epub 2008 Jun 3 doi:10.1038/mt.2008.98.

Using ultra low dose of DsiRNAs complexed with Neuromics’  i-Fect , they were able to successfully reduce NTS2 gene expression by up to 86% in rat lumbar Dorsal Root Ganglia after only two intrathecal injections. This was confirmed by Western Blot and qPCR analysis.

We now have further confirmation of the capabilities of this delivery platform in a just released publication by Dr. Jeffrey Mogil and team:

Michael L. LaCroix-Fralish, Gary Mo, Shad B. Smith, Susana G. Sotocinal, Jennifer Ritchie, Jean-Sebastien Austin, Kara Melmed, Ara Schorscher-Petcu, Audrey C. Laferriere, Tae Hoon Lee, Dmitry Romanovsky, Guochun Liao, Mark A. Behlke, David J. Clark, Gary Peltz, Philippe Séguéla, Maxim Dobretsov and Jeffrey S. Mogil. The β3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test. doi:10.1016/j.pain.2009.04.028.

IT Delivery of siRNA in vivo supplement

Knockdown of rSNSR1 in vivo

I have featured successes with delivering siRNA in vivo in this blog. These included stories on Dr. Philipe Serrat and his team at the University of Sherbrooke and Dr. Mark Behlke’s work at Integrated DNA and Dicerna.

I am pleased to report the parade of success with use our i-FectTM in vivo grows. 

Here’s the most recent study:

Christian Ndong, Amynah Pradhan, Carole Puma, Jean-Pierre Morello, Cyrla Hoffert, Thierry Groblewski , Dajan O’Donnell, Jennifer M.A. Laird. Role of rat sensory neuron-specific receptor (rSNSR1) in inflammatory pain: Contribution of TRPV1 to SNSR signaling in the pain pathway. PAIN 143 (2009) 130–137.
…For experiments in which siRNA was delivered by bolus injections, 10 ul of siRNA or vehicle was injected directly into the intrathecal catheter once daily for 4 days. In this case, siRNAs were prepared immediately prior to administration by mixing the RNA solution (200 uM in annealing buffer) with the transfection reagent i-FectTM (Neuromics) at a ratio of 1:4 (w:v) for a final siRNA/ lipid complex concentration of 2 ug/10 ul…

Related Data:


Images: in vivo characterization of knockdown produced by rSNSR1 siRNA. (A) A dose-dependent decrease in rSNSR1 mRNA levels measured in lumbar L3/L4/L5 DRGs was
observed when rSNSR1 siRNA (n = 7–14/group) or MM siRNA (n = 6/group) was delivered by four daily bolus injections. *p < 0.05; **p < 0.01; ***p < 0.001 as determined by oneway analysis of variance followed by sequential testing. (B) rSNSR1 immunoreactivity in dorsal horn of the spinal cord was visibly reduced in rSNSR1 siRNA-treated animals (5 lg/day, left panel). Immunoreactivity with neuron-specific isolectin B4 (IB4; right panel) did not change between treatment groups, showing the integrity of each dorsal horn analyzed (n = 6/group). (C) A semi-quantitative score of rSNSR1 immunoreactivity showed that siRNA treatment greatly decreased rSNSR1 protein levels compared to MM and control groups. A blinded observer scored 9–12 individual sections taken from a 1 cm segment of the spinal cord.