The goal of our research is to identify regulatory mechanisms of key genes implicated in behavior in human and mouse models of mental illness that can be targeted to normalize long-term gene expression for effective and long-lasting treatment of these illnesses. Our fundamental research has identified the molecular mechanisms that regulate the 5-HT1A receptor gene, a key regulator of the serotonin system implicated in anxiety and depression. We have cloned novel 5-HT1A regulators (Lemonde et al; Ou et al., J Neurosci 2003), and identified and characterized the rs6295 gene polymorphism that affects this regulation in vitro, in mouse models and in humans (Lemonde et al. J Neurosci 2003; J Neurochem 2004; IJNP 2004; Szewczyk 2010, 2014 IJNP). In addition, we have recently examined the DNA methylation patterns of the 5-HT1A promoter sites, and upstream sites recognized by these transcription factors (Le Francois, in revision). Understanding the proteins that regulate the 5-HT1A promoter will lead to insights on receptor regulation and provide new therapeutic targets relevant to depression, anxiety, and other mental illnesses (Albert, Mol Brain 2011; CPD 2013; Front Beh Neuro 2014;PTRSB 2012). Finally, we have generated stress- and stroke models of depression, and are using optogenetic approaches to directly modify the behavioral phenotype.
Knockout/Optogenetic Mouse Models
We are now testing in vivo the effect of knockout of these repressors on 5-HT1A expression and 5-HT activity. These include Deaf1 (Yip, Nature Imm 2009; Czesak JBC 2012; Ordereau JBC 2013) , Hes proteins (Jacobsen MCN 2008), Freud-1, and Pet-1 (Jacobsen JNeurochem 2008). We are focusing on conditional knockout models for Freud-1, Deaf1, and 5-HT1A that will allow us to assess the importance of these regulators in depression, anxiety, and responses to antidepressant treatment. We are also testing conditional transgenic mice expressing light-activated channel-rhodopsins for in vivo optogenetic modification of serotonin circuits involved anxiety and depression.
Functional Genetic Polymorphisms
We are continuing to evaluate the functional significance of the polymorphism in depression using knockout models, and by in vitro assessments of the role of the C(-1019)G polymorphism in 5-HT1A expression (Albert 2014). We have further identified and studied novel functional polymorphisms in human disease in TPH2 (Zhang Mol Psych 2011), dopamine D2 receptor (Rogaeva JBC 2007), and transcription factors (Nurr1 (Jacobsen Neurobiol Dis 2008), Deaf1, etc.).
Environmental Stress/Stroke Models
In addition to risk alleles that alter genetic regulation, environment appears also to alter gene regulation by inducing changes in DNA methylation at specific sites. We are examining the role of stressful environment on the developmental and epigenetic regulation of the 5-HT1A receptor and on behavioral depression in the adult (Szewczyk, IJNP 2014; Le Francois, Neurobiol. Disease 2015). We have also generated a new mouse model of post-stroke depression to reveal whether similar changes are found in stress- or stroke-induced depression.
Multi-target transcriptional regulators
Freud-1, a strong repressor of the 5-HT1A receptor, also represses the dopamine D2 receptor gene (Rogaeva JBC 2007), and suggests that a single transcription factor may coordinately regulate several neurotransmitter systems. We are examining their roles in vivo using conditional knockout mice.
Multi-disease risk alleles
The likelihood of depression following stroke is about 2-fold increased, while stroke is more likely in depressed subjects. Thus, a given genetic polymorphism may influence predisposition to many illnesses. We are conducting association of the functional genetic polymorphisms implicated in depression with post-stroke depression and stroke recovery (Vahid-Ansari, 2014).
Autoregulation of 5-HT1A receptor expression
We are examining the role of specific transcription factors in the down-regulation of 5-HT1A receptors upon chronic stimulation by 5-HT. This down-regulation appears crucial to allow antidepressants to enhance 5-HT activity and improve depressed behavior.
Structure-function analysis of 5-HT1A and dopamine D2 receptor signaling
We have identified the C-terminal portion of the i2 domain as critical for the signaling of the 5-HT1A receptor to multiple Gβγ pathways, and phosphorylation of this domain desensitizes the receptor (Kushwaha Mol Pharm 2006; Thibault JBC 2011; Wu EJP 2013). Using conditional expression approaches we are currently testing selected mutants for signaling in vivo and role in behavior and antidepressant actions in vivo. Based on specific of mutations to Ci2, we are generating several new reagents that can specifically alter the interaction of Ci2 with specific G-protein subunits to disrupt specific signaling pathways.
IMPACT
Our seminal studies have been highly cited and represent the first detailed mechanism for functional effect of a polymorphism associated with depression or suicide: even the well-studied SERT-LPR lacks a specific transcription mechanism. Other research has spurred numerous studies that associated the C(-1019)G polymorphism with other mental illnesses, 5-HT1A PET signal, phenotypes and as a marker to identify new polymorphisms. The concept that differential regulation of pre- and post-synaptic 5-HT1A expression is necessary for effective antidepressants that we proposed (Albert NPP 1996) is now well supported and matches the actions of Deaf1 regulation.
