Huntington’s disease (HD) is a genetic neurodegenerative disease caused by a polyglutamine expansion in the protein huntingtin. The age of symptom onset has been correlated with the length of this repeat. Adults with a repeat length of greater than 35 polyglutamines typically present symptoms between the ages of 40 and 50. Greater than 60 polyglutamine repeats causes a rapidly progressing early onset of the disease in adolescents. While both types of HD result in cognitive deficits and neuron loss, 25-35% of early-onset HD patients exhibit epileptic seizures that become less responsive to anti-epileptics over time. One of the earliest molecular changes in HD is the loss of the cannabinoid receptor 1 (CB1) before symptom onset. Activation of CB1 inhibits the release of neurotransmitters and protects neurons from cell death, whereas genetic deletion in HD models exacerbates motor symptoms. Additionally, activation of CB1 prevents the onset of seizures. The goal of this study was to determine if loss of CB1 activation increased epileptic activity, decreased survival, and whether elevating endocannabinoid (CB1 agonists) levels reversed these effects in early-onset HD. R6/2 mice (a rapidly progressing mouse model of early-onset HD) were treated with SR141716A (a CB1 antagonist), WWL123 (elevates the level of endocannabinoids), or vehicle (a control) and the frequency and severity of spontaneous and handling-induced seizures were analyzed. Preliminary results demonstrated that R6/2 mice treated with SR141716A had a decreased lifespan and more frequent seizures whereas WWL123 decreased seizure severity. This suggests that the endocannabinoid system has a regulatory effect on seizures associated with early-onset HD. Future experiments in the lab will further characterize the role of endocannabinoids and CB1 in early-onset HD seizures as a novel therapeutic pathway.

Chronic social-stress is known to lead to a number of aberrant behaviors, including anxiety and depression. Using a mouse model of social-defeat stress, this project aims to better understand the relationship between the kappa-opioid receptor (KOR), a G-protein coupled receptor (GPCR) previously shown to be activated by multiple forms of stress, and its role in generating anxiety-like and aversion behaviors in mice. Different forms of stress lead to the release of the endogenous KOR agonist, dynorphin, which activates an intracellular signal transduction cascade resulting in anxiety-like and aversion behaviors. Prior studies have demonstrated that the long-acting KOR antagonist, norbinaltorphimine (norBNI), is able to reduce social-defeat induced social avoidance behaviors when administered prior to social defeat stress exposure. Although these studies have implicated the KOR system in social-defeat induced behaviors, pretreatment of pharmacotherapies prior to a stress event is limited in therapeutic benefit.  In this research, the ability of norBNI to reverse repeated social-defeat induced changes in social behaviors was tested by administering norBNI following exposure to the social-defeat stress. The results have so far shown that post treatment with norBNI is able to effectively counter the altered social interaction behaviors caused by a repeated social-defeat stress; moreover, the reversal of the stress-induced social interaction deficits also correlates with a reduction in anxiety-like behaviors. These data suggest that social interaction deficits are due to increased anxiety in previously benign social settings, and norBNI has the therapeutic potential to reverse these social-stress induced behavior alterations.

Huntington’s disease is a hereditary neurodegenerative disease that is characterized by motor and cognitive dysfunction and currently has no known cure. One of the earliest molecular events in Huntington’s disease pathogenesis is the down-regulation of the cannabinoid 1 receptor (CB₁), occurring prior to symptom onset and loss of any other receptor. Though the exact mechanism is unclear, it has been shown that CB₁ loss occurs almost exclusively in medium spiny neurons (MSNs), cells that play a key role in movement and degenerate later in disease progression. CB₁ knockouts in mouse models also exhibit exacerbated disease phenotype. Previous studies have attempted to rescue CB₁ function in mouse models by administering chronic cannabinoid agonists, but have produced inconclusive results. Our study investigates the effects of CB₁ receptor rescue from a genetic approach, by inserting flox-stop CB₁ (fsCB₁) specifically activated in striatal MSNs of R6/2 mice, the best characterized Huntington’s disease mouse model. We first confirmed the localization and functionality of the knocked-in fsCB₁ in R6/2 and CB₁ null mice (control), and then measured motor phenotype and synaptic integrity over time. Performance of R6/2 mice with and without fsCB₁ rescue was measured using rotarod, open field, novel object recognition, and clasping behavior tests biweekly from weeks 4 to 12 of age. MSN axon integrity was measured by IHC staining for caspase-6 and NPY-Y1R, markers for Wallerian-type degeneration. By observing behavior and axon integrity from pre- through late-symptomatic stages, we hope to establish a relationship between early MSN axonal loss and impaired motor phenotype in R6/2 mice, as well as link CB₁ function to axonal integrity through R6/2 and fsCB₁-R6/2 comparisons. This research will increase the present understanding of molecular mechanisms behind Huntington’s disease and provide insight into CB₁ as a therapeutic target.

The neuromodulator dopamine is important for motivated behavior. Past research provides evidence that dopamine neurons encode the expected value of reward-predicting stimuli in situations with a single option. The current project investigated dopamine transmission when multiple options are present, as previous studies have arrived at conflicting conclusions. One study found that dopamine neurons encode the value of the subsequently chosen option, regardless of the expected value of the option foregone; whereas the other concluded that dopamine neurons encode the value of the greater available reward, regardless of which option was actually chosen. To address this discrepancy, we recorded changes in nucleus accumbens dopamine concentration using fast-scan cyclic voltammetry in rats performing a decision-making task in operant chambers. Sessions included single-option trials intermixed with choices between two options that differed in reward size (number of food pellets) and effort requirement (number of lever presses). When rats were presented with a low-reward/low-effort option (1 pellet for 4 presses) and a high-reward/intermediate-effort option (4 pellets for 8 presses), they preferred the high-reward/intermediate-effort option. In separate sessions, when presented with the low-reward/low-effort and a high-reward/high-effort (4 pellets for 32 presses) options, the rats preferred the low-reward/low-effort option. Preliminary voltammetry results from single option trials suggested that dopamine release was greater following the presentation of the high-reward/intermediate-effort option compared to the presentation of the low-reward/low-effort option. In the other session type, however, dopamine concentration did not differ between the high-reward/high-effort option and the low-reward/low-effort option. In two-option choice trials, dopamine release associated with each reward/effort combination was similar to that in the corresponding single-option trials. Thus far, these results are more consistent with the claim that dopamine encodes for the option subsequently chosen, but the interaction between behavioral preference and encoding of expected reward value requires further investigation.

Ubiquitin is a protein modifier that is essential for many eukaryotic cellular processes. Attachment of ubiquitin to substrate proteins (ubiquitination) occurs in a three-step cascade that involves a ubiquitin activase, ubiquitin conjugase, and ubiquitin ligase; it is the ligase that targets substrate proteins. One challenge for the ubiquitin field is identification of substrate cohorts for ubiquitin ligases. Although ubiquitination only occurs naturally in eukaryotic organisms, we have previously reconstituted the ubiqutination cascade in E. coli. Isolation of the ubiquitin pathway within a prokaryotic cell, such as E. coli, allows for the investigation and modification of individual components of the pathway and the measurement of immediate effects of these changes upon the overall process of ubiquitination. This project, which consists of the construction of a split protein assay, aims to address the abovementioned challenge by building on our previous work reconstituting ubiquitination in E. coli. The assay uses two fragments of the protein dihydrofolate reductase (DHFR) to detect the ubiquitination of proteins in E. coli, outside of the natural eukaryotic environment of ubiquitin. All of the essential components of the ubiquitination pathway, including individual fragments of DHFR fused to ubiquitin and a substrate protein of interest, are transformed into the bacterial cell. The assay tests for the covalent attachment of ubiquitin to the substrate protein through growth on selective medium, which is possible only through the combination of the separated fragments of DHFR. This reporter system allows for experimentation regarding the interaction and selectivity of the ubiquitin ligase with the substrate protein of interest. Thus, a functional reporter system will allow for the streamlined analysis and determination of the substrate cohort for a given ubiquitin ligase. Changes to the components of the system may include using different ubiquitin ligases and substrate proteins.