Stewart D. Clark, Ph.D. - US grants

The mesolimbic pathway through the release of dopamine from neurons in the ventral tegmental area (VTA), which project to the nucleus accumbens (NAc), is thought to have a role in motivation and reward processes. Some addictive drugs produce their potent effects on behavior by enhancing mesolimbic dopamine activity. Release of dopamine from VTA neurons is controlled by various factors and neuronal structures. One pathway providing regulatory input to the VTA originates in the mesopontine nuclei of the brainstem. We have previously shown that the mesopontine-VTA-NAc pathway is modulated by the novel neuropeptide urotensin II (Uil). To facilitate our studies we have developed and validated a fusion of diphtheria toxin with UN (Dtx-Uli). This toxin is able to selectively ablate Ull-R expressing neurons of the mesopontine without damage to surrounding tissue. Through the combination ofthis toxin with microdialysis and behavioral paradigms (self-administration and conditioned place preference), we will establish whether Uil can modulate reinforced behaviors. In addition, to better understand the function ofthe mesopontine nuclei, the Dtx-Uli will be used to ablate neurons of this region. Subsequently, different aspects of proposed mesopontine function will be tested ( ex. prepulse inhibition, conditioned place preference). Drug addiction is a multifactorial condition. This heterogeneity is partly due to genetic predisposition. Any gene that is expressed in the neuronal circuitry known to modulate the acquisition and maintenance of addiction could impact the development of an addiction, although that gene may not be the direct target of the drug. Uil is an emerging neuromodulator which may have implications in addiction and the development of addiction. Furthermore, in other work UN has been shown to produce cellular remolding, which may point to a role in neuroplasticity. Therefore, future studies will include the investigation ofthe role of Ull-R in molecular neurobiological changes and the development of addiction.

Project Summary Substance abuse disorders have a devastating impact on individuals, families, and society. Present pharmacological treatments do not fully ameliorate patients' symptoms and in many cases are unable to treat the full spectrum of symptoms. Therefore, it is paramount to identify new targets for pharmacological intervention. To do this we must better understand the neural mechanisms underpinning drug-taking behavior and drug-mediated reward. The Neuropeptide S (NPS) system when activated in mice enhances memory formation and produces both anxiolytic-like and hyperlocomotor effects. This is a unique behavioral profile, and if pharmacologically targeted could have multiple therapeutic benefits. Based on research conducted in our lab, we have identified the first and only biased NPSR agonist (RTI-263). It retains full agonist properties in calcium mobilization assays, but has attenuated ability to increase cAMP levels. Importantly, in mice, RTI-263 evokes similar anxiolytic-like and memory enhancing effects as NPS. However, RTI-263 is unable to produce the pronounced increase in locomotor activity and actually blocks hyperlocomotion induced by exogenously administered NPS. Other compounds that block this hyperlocomotion, NPSR antagonists, are known to reduce cocaine seeking in rodents. Therefore, we hypothesize that RTI-263 will display dual properties whereby it will likely simultaneously produce anxiolysis and curb cocaine intake. This has obvious benefit as relapse into the cycle of drug taking in humans is often associated with stress and anxiety producing life events. The goals of this project are to better elucidate the circuitry of the NPS-system by determining the role of particular subpopulations of NPSR-expressing neurons in core behaviors it is known to modulate. In addition, we will show proof-of-concept that NPSR ligands with similar pharmacological profiles as RTI-263 have potential as therapeutics for cocaine abuse. NPSR is a potential target for the development of therapeutics for addiction and other neuropsychiatric disorders related to the dysregulation of stress and anxiety.

Summary Progressive Supranuclear Palsy (PSP) is a debilitating disease with aggregates of tau protein in multiple brain areas and severe mental and motor deficits. PSP is often misdiagnosed as Parkinson's Disease (rate of 50%) and there are no drugs that help PSP sufferers. Those with PSP have a life expectancy of only 6-8 years, suggestive that the neurodegeneration is already far advanced when symptomology becomes evident. Therefore, there is a need to i) increase the accuracy of diagnosis, ii) find biomarkers or behavioral deficits that predate the symptoms, and iii) find therapeutics. To facilitate these goals, we need to understand 1) from where within the brain does the disease originate, 2) which neural pathways when degenerated produce which symptoms, and 3) the topographical progression of pathogenesis. Based on strong preliminary data, we propose that the accumulation of tau protein in cholinergic pedunculopontine tegmentum (PPT) neurons in the hindbrain will produce tau aggregates in brain regions impacted in PSP, progress from a disease-free state to a PSP-like end stage, and produce PSP-like behavioral deficits (e.g. dysexecutive frontal syndrome and motor deficits). To produce PSP-like pathology in rats we use a genetically engineered virus to selectively over-express the isoform of the tau protein that predominates in PSP (1N4R) in cholinergic PPT neurons. At 5 months post- infection, the model is consistent with PSP: i) a loss of cholinergic neurons, ii) loss of substantia nigra dopaminergic neurons, iii) increased number of hyperphosphorylated tau-positive neurons, iv) acoustic startle reflex deficit, and v) motor deficits. Animals with tau protein over-expression will be compared to those that have the over-expression of a benign protein at 5 month intervals until old age. We will complete extensive postmortem histochemical analysis, Magnetic Resonance Imaging (MRI), REM sleep recordings, and behavioral testing (e.g. cognitive & motor) to establish whether the pathology progresses to a condition consistent with late-stage PSP. Once it is established that accumulation of tau in the cholinergic PPT neurons is sufficient to produce late stage PSP-like pathology and behavior deficits, future work would include: 1) identifying strategies that ameliorate symptomology and disease progression, 2) the discovery of early markers of disease onset, and 3) molecular mechanistic studies (e.g. knockdown of specific targets).