BY SAM POWELL
Recent large-scale, international, collaborative projects like ENCODE (Encyclopedia of DNA Elements) and FANTOM (Functional Annotation of the Mammalian Genome) have revealed that much of our genomes are in fact transcribed. While approximately 1.5 percent of the human genome consists of protein-coding genes, anywhere from 70 to 90 percent of the human genome is, at some point in time, in at least one type of cell, transcribed into an RNA transcript. The fact that a region of the genome is transcribed does not mean that the particular RNA transcript is necessarily functional, but several recent studies have indicated crucially important roles for over 120 different long non-protein-coding RNAs (lnc-RNA) in human health and disease. Because of this, a strong interest in lnc-RNAs has emerged, and new studies are continuously emerging that reveal novel roles for these new non-protein coding transcripts. Natural Antisense Transcripts (NATs) are a major type of lnc-RNA, and are transcripts that have reverse complementary sequence to a protein-coding, or “sense” gene.
A single sense gene can have one or several NATs and current estimates suggest that anywhere from 25 to 40 percent of all protein-coding genes have at least one NAT. NATs act through a variety of different mechanisms to regulate the expression of their corresponding sense-gene partners, and a given NAT can either increase or decrease the expression of the mRNA to which it binds.
Addiction is a debilitating psychiatric disorder characterized by the compulsive intake of a drug of abuse despite adverse consequences to the individual’s physical, mental, psycho-social and occupational health. Chronic intake of cocaine is known to cause lasting changes in the structure and function of the circuits in the brain that process rewarding stimuli, regulate mood and drive, and form memories. These changes are facilitated by widespread alterations in patterns of gene expression in these brain areas brought about by cocaine. These gene expression changes come about by what are called “epigenetic” mechanisms, which, broadly defined, are processes that alter gene expression without changing the sequence of the gene itself.
Knowing that Natural Antisense Transcripts regulate gene expression in an epigenetic fashion, I wanted to see if NATs have any sort of role in facilitating many of the gene expression changes caused by cocaine, and if NATs could be targeted to block and/or treat drug-seeking behavior in animal models of addiction.
The first step in this ongoing project was to test how simply exposing mice to cocaine affected the expression of NATs to genes that are known to be involved in the molecular mechanisms of cocaine addiction. After screening a transcriptomic database called Aceview to identify which cocaine-related genes have NATs that are endogenously expressed in the mouse brain, I injected mice with cocaine for a period of two weeks, which is a “chronic” period for a mouse. After doing so, I sacrificed the animals and isolated a brain region called the Nucleus Accumbens (NAc), which is considered the reward center of the brain. I extracted the RNA from these tissue samples and used real-time, quantitative PCR (rt-PCR) to compare the expression levels of over 60 NATs in saline-treated versus cocaine-treated mice. I found that the expression of several NATs was changed in some way by cocaine, and decided to focus on a particular transcript — the NAT for brain-derived neurotrophic factor (Bdnf).
Bdnf is an important addiction-related gene. Previous studies have shown that drugs of abuse increase the expression of Bdnf, and that blocking this effect either attenuates or completely prevents the acquisition of addiction-like behavior in animal models. I found that chronic exposure to cocaine decreased the expression of the natural antisense transcripts to Bdnf, and the next step, which I am working on now, is to tease out the mechanism of how this transcript may affect Bdnf expression. For this part of the project, I am currently working in an in vitro cell culture model. Using mouse neuronal precursor cells called N2a cells, I am knocking down the expression of the Bdnf antisense transcript that I found was affected by cocaine. Following this, I will see how knockdown of the Bdnf antisense transcript affects the expression of Bdnf mRNA and protein. From there, I will do further studies to figure out how the Bdnf antisense transcript alters the expression of Bdnf, and may even transition to behavioral models to test how knockdown or forced overexpression of the Bdnf antisense transcript affects drug-seeking behavior in animal models of addiction. This series of studies is the first to implicate natural antisense transcripts in the molecular mechanisms of cocaine addiction, and may even pave the way for innovative treatments that target these transcripts.