Monday, November 24, 2014

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.

Thursday, August 28, 2014

miRNA, Inflammation and Coronary Heart Disease

i-FectTM Delivers miRNA for the Study of Cardiovascular Pathogenesis

We have posted over 35 publications that reference use of our i-Fect Transfection Kit to deliver siRNA, miRNA and shRNA in vitro and in vivo. Results documented in these publications prove that this kit is both non-toxic and delivers ultra-high transfection efficiency.

Here i-Fect is used to silence miR-21 microRNAs. This miRNA stimulates pro-inflammatory pathways that are at the root of Coronary Heart Disease: Guo Weizao, Liu Huichen, Li Lin, Yang Man and Du Aihua. Regulation of lovastatin on a key inflammation-related microRNA in myocardial cells. Chinese Medical Journal 2014;127(16):2977-2981:10.3760/cma.j.issn.0366-6999.20140780...... miRNA functional inhibition assay Anti-miR miRNA antagonist for miR-21 (Ambion/Life Technologies, Grand Island, NY, USA) was transfected into H9c2(2-1) cells using iFect transfection kit (Neuromics, Edina, MN, USA) according to the manufacturer's manual...

Results:Inhibition of miR-21 upregulates STAT-3 and exerts a critical role in the upregulation of cardioprotective and anti-apoptotic proteins.

Fig: Inhibition of miR-21 attenuated the up-regulation of phosphorylation of STAT3 in H9c2(2-1) cells by lovastatin (LST) in lipopolysaccharide (LPS) treated cardiomyocytes. Combination of treatments are indicated under the image, the basic comparison was 1 vs 3. 

This study demonstrates the relationship between miR-21 and the STAT3 pathway in Coronary Heart Disease. Delivering inhibitory miRNA into cardiomyocytes was key to establishing this relationship. Further study could enable discovery of STAT3 related targets for CV protective drug. In the spirit of helping researchers find the best solutions and protocols for Gene Expression Analysis Studies, we will continue to post new findings.

Wednesday, July 23, 2014

Neuromics' Transfection Kits-Genes Studied

Delivering siRNA, miRNA, Plasmids and Viral Vectors for Gene Expression Analysis.

I have shared the many genes researchers have studied using our Transfection Kits. These include: β-arrestin, ABCA, ASIC, β-arrestin, CAV1.2, CX3CR1, DOR, EHDAC2, LOVL4, IKBKAP, K+-ATPase, KV1.1, KV9.1 , neuroligin 2, The β3 subunit of the Na+,K+-ATPase, NTS1, NAV1.8, NTS1, NOV, Raf-1, RANK, SNSR1, hTert, NOV, Survivin, TLR4, Troy and TRPV1 and More!

We can now add GPNMB to this list: Lili Hou, Yanfeng Zhang, Yong Yang, Kai Xiang, Qindong Tan, Qulian Guo. Intrathecal siRNA Against GPNMB Attenuates Nociception in a Rat Model of Neuropathic Pain. Journal of Molecular Neuroscience. July 2014...Ten micrograms of siRNA1- GPNMB dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days, starting from 1 day before CCI surgery...

Abstract: Neuropathic pain is characterized by hyperalgesia, allodynia, and spontaneous pain. Recent studies have shown that glycoprotein nonmetastatic melanoma B (GPNMB) plays a pivotal role in neuronal survival and neuroprotection. However, the role of GPNMB in neuropathic pain remains unknown. The aim of the present study was to assess the role of GPNMB in neuropathic pain. In cultured spinal cord neurons, we used two small interfering RNAs (siRNAs) targeting the complementary DNA (cDNA) sequence of rat GPNMB that had potent inhibitory effects on GPNMB, and siRNA1-GPNMB was selected for further in vivo study as it had the higher inhibitory effect. After sciatic nerve injury in rats, the endogenous level of GPNMB was increased in a time-dependent manner in the spinal cord. Furthermore, the intrathecal injection of siRNA1-GPNMB inhibited the expression of GPNMB and pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia in the chronic constriction injury (CCI) model of rats. Taken together, our findings suggest that siRNA against GPNMB can alleviate the chronic neuropathic pain caused by CCI, and this effect may be mediated by attenuated expression of TNF-α, IL-1β, and IL-6 in the spinal cord of CCI rats. Therefore, inhibition of GPNMB may provide a novel strategy for the treatment of neuropathic pain.

If you would like to learn how you can optimize your gene expression analysis studies, do not hesitate to e-mail: or direct line: 612-801-1007.

Wednesday, June 25, 2014

Gene Expression Analysis For Neuroscientists

In Vitro and In Vivo Studies

Neuromics has a strong line up of Transfection Tools designed specifically for Neuroscientists. Neurons, Glia and Astrocytes are notoriously hard to transfect. We are proud of our track record.

This is an excellent study for Neuroscience Researchers interested in using best methods:  Here researchers delivered 14-3-3 siRNA sub units + i-Fect intrathecally. Here's specific knockdown results:

Figure: A. Dose-dependent inhibition of 14-3-3-zeta (z) expression with anti-14-3-3-z siRNA as measured with qRT-PCR in cultured spinal neurons (n = 3 independent experiments). B. Immunolabeling for 14-3-3-z is visualized in the dorsal horn of naive rats after intrathecal injection of mismatch RNA (a, mmRNA; 2 µg in 10µL i-Fect reagent) or anti-14-3-3-z siRNA (b, anti-14-3-3-z siRNA; 2 µg in 10µL i-Fect reagent). Bar: 50 µm C. Detection in the spinal cord (a, SC) and lumbar dorsal root ganglia (b, DRG) of intrathecally injected fluorescent siRNA. Staining is seen in the dorsal horn (open star) but not in the dorsal root ganglia (filled star). Bar: 50 µm D1. Quantification of 14-3-3-z mRNA levels with qRT-PCR in the ipsilateral lumbar (L4 and L5) dorsal spinal cord of three groups of rats (n=4 in each group): sham, neuropathic (same data as Figure 1B), and neuropathic with 3 intrathecal injections of anti-14-3-3-z siRNA. The upregulation of 14-3-3 mRNA in neuropathic conditions is abolished after anti-14-3-3-z siRNA injections. D2. Same quantitative procedure carried out in the ipsilateral lumbar (L4 and L5) dorsal root ganglia. No modification was induced by intrathecal injections of anti-14-3-3-z siRNA (n=4 in each group, same data as Figure S1B for Sham and SNL).

Our customers have successfully studied many genes with our tools. Here's a sampling: ABCA, ASIC, β-arrestin, CAV, CX3CR1, DOR, ELOVL4, IKBKAP, K+-ATPase, KV1.1, KV9.1 ,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV1.8, NTS1, NOV, Raf-1, RANK, SNSR1, hTert,  NOV, Survivin, TLR4, Troy and  TRPV1. Here're the related publications.

Tuesday, March 11, 2014

i-Fect, BDNF and Irritable Bowel Syndrome (IBS)

Gene Expression Analysis Determines BDNF's Role in IBS

In this study, Researchers used Neuromics'  i-FectTM Transfection Kit to deliver BDNF to determine effect on visceral hypersensitivity (VHS): J. H. Winston1 Q. Li1, S. K. Sarna1. Chronic prenatal stress epigenetically modifies spinal cord BDNF expression to induce sex-specific visceral hypersensitivity in offspring. Article first published online: 4 MAR 2014. Neurogastroenterology & Motility. DOI: 10.1111/nmo.12326. The Journal of Pain, Volume 14, Issue 11, November 2013, Pages 1485–1491

Intrathecal treatment with brain-derived neurotrophic factor (BDNF) antagonists reduced VMR to colorectal distension (CRD) in female chronic prenatal stress (CPS)+HeICS rats. (A) Graph shows that intrathecal administration of BDNF antagonist trkBFc in female CPS rats significantly decreased VMR to CRD, 24 h following adult 29 HeICS (two-way repeated measures ANOVA found a significant main effect of treatment, F1,53 = 10.4, p = 0.015; post hoc tests found significant differences at 30, 40, 50, and 60 mmHg, n = 4). (B) Graph shows that intrathecal administration of BDNF siRNA in female CPS rats significantly decreased VMR to CRD, 24 h following adult 29 HeICS (two-way repeated measures ANOVA: treatment 9 pressure interaction, F1,77 = 3.49, p = 0.008, tukey post hoc tests found significance at 30 mmHg, p = 0.013 and at 40, 50 50, 80 mmHg, p < 0.001, n = 7 Ctr., n = 6 BDNF siRNA). (C) Western blot shows a significant decrease in spinal cord BDNF protein expression in rats treated with BDNF siRNA (*p < 0.05).

Conclusion: Chronic prenatal stress followed by a second exposure to HeICS in adult offspring exacerbated visceral hypersensitivity (VHS) greater in female offspring that persisted longer than in male offspring. Chronic prenatal stress up-regulated BDNF expression in the lumbar-sacral dorsal horn that correlated with the exacerbation of VHS in female, but not in male offspring by increasing RNA Pol II binding and histone H3 acetylation, and decreasing histone deacetylase 1 association with the core promoter of BDNF in female offspring.