Dopamine Signaling in Neuropsychiatric Disorders and Addiction

Dopamine (DA) signaling in the brain is requisite for a number of key behaviors, including motivation, reward, motor function, and learning. Multiple neurological and neuropsychiatric disorders exhibit aberrant DA signaling, including addiction, schizophrenia, autism spectrum disorder (ASD), Parkinson's disease, and attention-deficit/hyperactivity disorder (ADHD). Despite the association of these disorders with dopaminergic dysfunction, the molecular mechanisms and neuronal circuits involved in these processes are not well defined. In order to investigate these pressing questions, we leverage a variety of approaches that span from molecules to behavior in mouse models, including in vivo monitoring of neuronal activity and DA signaling using genetically encoded tools. We currently are pursuing multiple lines of investigation:

Regulation of the Cocaine-Sensitive DA Transporter (DAT):  Our laboratory is interested in the circuit- and molecular-specific mechanisms that regulate DA signaling and DA-dependent behaviors. Once released, extracellular DA is temporally and spatially restricted by presynaptic DA reuptake facilitated by the DA transporter (DAT). In addition to its central role in basal synaptic transmission, DAT is the primary target for addictive psychostimulants, cocaine and amphetamine, as well as therapeutic psychoactive drugs, such as methylphenidate (Ritalin) and bupropion (Wellbutrin/Zyban).  These agents block DAT activity and thereby enhance extracellular DA levels and drive dysfunction in DA-depndent behaviors. 

Given DAT’s importance in DAergic neurotransmission and as a psychoactive drug target, cellular mechanisms that impact DAT function are likely to have significant impact on DA signaling and neuropsychiatric disorders.  Multiple DAT coding variants have been identified in ADHD and autism patients, further supporting that altered DAT function is linked to significant behavioral consequences.  Work from our lab investigates the cellular and molecular mechanisms that regulate DAT.  Endocytic trafficking dynamically controls DAT plasma membrane availability, and a variety of cellular signaling pathways and psychostimulant drugs rapid alter DAT trafficking, surface expression and function. We have identified multiple key players that govern DAT trafficking.  Using a variety of cutting edge approaches, such as viral-mediated gene expression, gene silencing (RNAi), optogenetics and chemogenetics, we are investigating how DAT regulation impacts DA neurotransmission and DA-associated behaviors.

Role of modulatory glutamate signaling in motor function, novelty, and reward: Glutamate is the major excitatory neurotransmitter in the brain, but also has a modulatory role by signaling through metabotropic glutamate receptors (mGluRs). Recent work from our laboratory revealed that selective expression of mGluR5 in DA neurons is required for several DA-dependent behaviors and DA signaling. However, the circuit- and mechanistic-specific underpinnings of these processes have not been elucidated. Using a novel conditional knockout model, we are leveraging several intersectional approaches to determine how DAergic mGluR5 impacts DA neuron function and DA-dependent behaviors.

1. Parkinson's Disease: Signaling Cascades that lead to dopamine neuron degeneration

Parkinson's disease (PD) is the 2nd most prevalent neurodegenerative disease and affects >1 million individuals in the U.S., with a 50% higher occurance in males vs. females. Dopamine (DA) is required for movement initiation and PD is the result of DA neuron (DAN) cell death, leading to freezing, gait instability, and resting tremor. Therapy options for PD patients are limited to approaches that replace DA. These are effective in early disease stages, but wane in efficacy as PD progresses. Importantly, none of these treatment strategies target the root cause of PD: DAN cell death. The lack of progress in developing impactful treatments is due, in part, to a lack of understanding the cellular signaling dysfunction that leads to DAN cell death in PD. Moreover, it is completely unknown why males are more susceptible to PD than females.

Rit2 (AKA: Ras-like in neurons; Rin) is a small neuronal GTPase that is highly enriched in DANs, and is a PD risk allele. Importantly, a recent single-cell RNA sequencing study from idiopathic PD patients revealed a patient subpopulation in which Rit2 was markedly decreased in DANs. While intriguing, it was yet unknown whether Rit2 losses were simply due to DAN cell death, or actually causative. In collaboration with the UMASS Chan Gene Therapy Center, we recently developed a novel, AAV- and shRNA-mediated approach to silence Rit2 in mouse DANs and found that Rit2 knockdown (Rit2-KD) leads to a significant, striking, and progressive PD phenotype that develops more rapidly in males than females. This new PD mouse model presents a unique opportunity to probe multiple questions regarding Rit2 and PD. What gene expression changes occur downstream of Rit2 loss? Are any of these druggable targets? What are the differences between males and females that make males more susceptible to DAN cell death? Is there an intervention window in which DAN viability can be rescued after Rit2 loss insult? There are 2 rotation projects to tackle these questions.

Project 1a. Identify transcriptional changes that occur in DANs downstream of Rit2-KD. Together with the Thomson laboratory (Dept. of Neurobiology), we are performing bulk and single-cell RNA sequencing to identify specific gene expression changes that occur downstream of Rit2-KD in DANs. Students will learn to handle mice and harvest DANs, and to analyze RNA sequencing data.

Project 1b: Identify a therapeutic window to rescue motor function in Rit2-KD mice. Students will perform a motor learning assay on control vs. Rit2-KD mice after suppressing Rit2 shRNA expression. In addition, students will design and generate a transgene construct to rescue Rit2 expression and test its efficacy using an in vitro assay.

2. Modulatory glutamate signaling: impact on motor function, novelty, and cocaine addiction

Glutamate is the major excitatory neurotransmitter in the brain, but also has a modulatory role by signaling through metabotropic glutamate receptors (mGluRs). Recent work from our laboratory revealed that mGluR5 expression in DA neurons is required for DA signaling and several DA-dependent behaviors including motor learning and cocaine reward. However, the circuit- and mechanistic-specific underpinnings of these processes are unkonwn. Using a novel conditional knockout model, we are leveraging genetically encoded DA sensors to determine how DAergic mGluR5 impacts DA neuron function and DA-dependent behaviors in mice. Rotation students will learn to behaviorally assess cocaine addiction in this mouse model, and will learn immunohistochemical approaches to validate viral expression

Cocaine Fails to increase DA in mGluR5-DA-silenced mice. Fiber photometry using genetically-encoded DA sensors during cocaine response. Control or mGluR5-DA-silenced mice were injected with 7.5 mg/kg cocaine and DA signals were recorded in the nucleus accumbens using dLight 1.2 sensor. Control mice: Cocaine caused a significant increase in the total DA signal from 3-5 min post-injection as compared to saline-injections. mGluR5-DA-silenced mice: Cocaine failed to significantly increase DA signals. *p<0.05, paired Student's t test, n=4-5.

3. Nociceptin: A neuropeptide that impacts cocaine sensitivity

Nociceptin is an endogenous opiate-like neuropeptide that is implicated in pain and reward motivation. Recent studies suggest that nociceptin has the capacity to block cocaine's initial addictive properties, but the mechanisms underlying nociceptin's actions are largely unknown. Work in our lab has discovered that nociceptin blocks cocaine reward, and does so by acting directly on DA axon terminals in the nucleus accumbens, the brain's "reward center". In addition, our results suggest that nociceptin likely shifts cocaine sensitivity, and does so by controlling reward tone differentially in males vs. females. These exciting results may pave the way towards harnesing nociceptin signaling as a potential addiction therapeutic. However, the mechanisms underlying nociceptin's actions are unknown, nor is it known whether nociceptin specificallly impacts cocaine reward, or other drugs of abuse. There are two possible rotation projects:

Project 3a: Impact of nociceptin signaling on other drugs of abuse. Students will conduct addiction behavioral assays while infusing nociceptin directly into the nucleus accumbens in mice. Students will learn mouse handling and stereotaxic surgery, and mouse behavioral assays.

Project 3b: Sex-specific differences in nociceptin signalig. Students will use RT-qPCR to test whether males and female mouse midbrain neurons express differing amounts of the nociceptin receptor, NOPR. Students will learn mouse handling skills, brain harvesting, and RT-qPCR.

Nociceptin infusion into the nucleus accumbens blocks cocaine preference in females. Conditioned Place Preference Assay (CPP). Wildtype female mice received either saline or 10mg/kg cocaine (I.P.) and were trained for 3 days in a 3-chambered CPP aparatus, while being infused with either vehicle or 500nM nociceptin. Preference scores were determined on test days. Control-infused mice exhibited signifiant preference to the cocaine-paired chamber as compared to the saline-paired chamber (****p<0.001), whereas mice infused with nociceptin failed to establish cocaine preference (p=0.74). Two-way ANOVA with Tukey's multiple comparison test, n=4-10.

4. Dopamine Transporter (DAT) Trafficking in Cocaine Addiction

DAT is a membrane protein in dopamine (DA) axon teminals whose primary function is to remove released dopamine and thereby terminate synpatic transmission. Addictive psychostimulants, such as cocaine, block DAT function, which increases extracellular DA and is responsible for cocaine's addictive properties. DAT is not static at the plasma membrane, but is dynamically regulated by membrane traffficking. This project is to determine whether DAT membrane trafficking is required for cocaine addiction. Our lab has developed a novel, AAV-mediated, in vivo molecular replacement strategy, that replaces wildytpe DAT with DAT trafficking dysregulated mutants in adult mice. There are 2 possible rotation projects:

Project 1a: Rotation students will assist in behaviorally assessing replacement mice as compared to controls, and will additionally use immumohistochemical approaches to validate mutant protein expression in dopamine neurons in situ. Students will gain experience in mouse behavior, brain dissection, preparation of brain slices,  and immunhistochemical techniques in mouse brain.

Project 1b: Rotation students will develop a CRISPR/Cas9 guide RNA in order to perform gene editing and mutate DAT in vivo, and will test its efficacy in vitro. Students will learn guide RNA design, and mammalian tissue culture, and genomic sequencing.

The Melikian Lab seeks a creative and motivated PhD neurobiologist to join our investigative team to study the mechanisms that govern addictive and motivated behaviors, with a focus on circuits that converge on and modulate dopaminergic terminals. We leverage intersectional approaches spanning from molecules to behavior, and utilize mouse genetic, viral, optogenetic and photometric methods.

We are seeking postdoctoral applicants with a strong track record of productivity, who will take the lead on a novel project. Applicants will optimally have experience in mouse handling and modern neurobiology approaches (e.g. optogenetics, photometry), but other strong candidates will also be considered.

The Neurobiology Department at UMASS Chan Medical School is the hub of a vibrant multidisciplinary neuroscience community, with research strengths in neuropsychiatric disorders, neurodegeneration, neuroinflammation, neuroplasticity, and neurodevelopment. Under the leadership of our recently appointed Chair, Dr. Danny Winder, the department provides an exceptional and interactive training environment that emphasizes team science in a diverse and inclusive space.

Interested applicants should submit a CV and names of three references to Dr. Melikian: haley(dot)melikian(at)umassmed(dot)edu.