• Ablation of Dorsomedial Striatum Patch Compartment Results in Modification to Reward-Driven Behaviors in Rats

      Ahn, Jamin Paul; School of Medicine
      The striatum is a neural structure that plays a critical role in cognitive functions, behavioral decision-making, and reward generation. The striatum exhibits a heterogeneous composition, containing neurons belonging to the patch compartment—which is thought to be involved in habitual reward-related behaviors—surrounded by neurons belonging to the matrix compartment—which is thought to be involved in adaptive motor control. Additionally, the striatum is further subdivided into the dorsolateral striatum (DLS) and the dorsomedial striatum (DMS), each with their own patch and matrix compartments. The DMS has been associated with goal-oriented behavior seen during the initial stages of addiction. Conversely, the DLS has been associated with habitual behaviors seen during late-stage addictive behaviors that are inflexible. It is thought that drug addiction is initially mediated by the DMS before DLS activity becomes predominant. Previously, it has been shown that the patch compartment of the DLS is necessary for development of habitual behavior, but the role of the patch compartment of the DMS is less clear. Our study intends to demonstrate that selective ablation of DMS patch compartment neurons results in a significant impact on the initial development of reward-driven behaviors during the early stages of drug seeking behavior. Since patch compartment neurons express a high level of mu-opioid receptors compared to the surrounding matrix, we used dermorphin-saporin to target patch compartment neurons in the DMS and DLS for ablation. Following infusion in the DMS or DLS with dermorphin-saporin or vehicle-only infusions, rats were trained to self-administer cocaine on a progressive ratio schedule of reinforcement, starting with fixed ratio of 1 and ending with a fixed ratio of 5. Ablation of the patch compartment of the DMS resulted in an increase in early-stage lever pressing, suggesting that the DMS patch compartment contributes to reward-driven behaviors in a way that offers support for the parallel model of DLS/DMS activity. Future studies may investigate the relationship between DMS and DLS patch/matrix compartments through measuring or inducing activity in the DMS patch while the animal undergoes behavioral training. Methods for such a study may include optogenetics, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), or microelectrode arrays.
    • Contributions of the N and C – Termini of Varicella – Zoster Virus Portal

      Nale Lovett, Dakota J; School of Medicine
      The VZV portal protein is a multimeric protein found at a single vertex of the viral capsid that is essential for encapsidation (packaging) of viral DNA. All viruses within the herpesvirus family contain structurally homologous portal proteins. PORT compounds have been shown to target herpesvirus portal proteins and show potential as broad-spectrum herpesvirus antivirals. We are specifically interested in the interaction of PORT compounds with the VZV portal since the activity of our compounds against VZV was shown to be in the single nanomolar range. Unfortunately, VZV portal structure has yet to be resolved by transmission electron microscopy (TEM) due to complications with size and aggregation. We aimed to observe the effects of pORF54 (VZV portal protein) terminal truncation on virus viability to determine the minimal portal protein that can be used for structural analysis. Six recombinant viruses containing N and C- terminal mutations were created using bacterial artificial chromosome (BAC) technology and targeted recombineering to create the mutant VZV strains. PCR was used to engineer stop codons at 29, 49, and 75 AAs from the C-terminal end. In addition, a second set of recombinants was constructed where the first start codon of the 769 AA pORF54 open reading frame was deleted creating a 40 AA N-terminal truncation. Sequencing of all mutant strains confirmed the expected changes for the ORF54 gene and also that no gross off-target mutations occurred. All constructs showed a similar restriction digestion pattern, upon gel electrophoresis, compared to the parent BAC, pOKA, suggesting the mutant genomes were stable. Mutagenized BACS were used to create infectious viral stocks after lipofectamine-based transfection into ARPE19 cells. Viruses with non-functional mutations in pORF54 were transfected into ARPE54 (complementing) cells, that constitutively express wild-type pORF54. The ∆29, ∆49, N40∆29, and N40∆49 viruses were replication competent in ARPE19 cells. Replication characteristics suggested that some of these viruses grew less efficiently in vitro. Overall, we confirmed that a protein of 679 AAs can form a functional viral portal. This portal multimer may represent a strong candidate for detailed structural studies.
    • Predictive Analysis of the Immunosuppressive Functionality of Human Bone Marrow Derived Mesenchymal Stem Cells as Cellular Therapeutics

      lipat, ariel joy mann; School of Medicine
      Human Mesenchymal Stem/Stromal Cells (MSCs) of bone marrow carry immunomodulatory and regenerative properties and are being tested as a cellular therapy for inflammatory and degenerative disorders. They are involved with the paracrine secretion of anti-inflammatory cytokines and chemokines and the promotion of anti-inflammation in tissue microenvironments by dampening inflammatory T-cells. However, the mechanism of action of MSCs on T-cells has yet to be understood. Here we aim to identify the pattern of chemokine secretion in human bone marrow MSCs and their regulation and functions on T-cell responses and immune suppression. MSCs were derived from healthy human bone marrow aspirates. MSC secretome was collected systematically under defined cell densities and subjected to multiplex secretome analysis with or without exogenous stimulation to identify inherently secreted MSC chemokines. MSC derived chemokines’ immunosuppressive role on T-cells was further determined with a PBMC and MSC coculture and siRNA chemokine transfection strategies. MSC secretome was further tested on human peripheral blood mononuclear cells (PBMCs) derived from blood and early phosphorylation of signaling molecules in T-cells were specifically analyzed utilizing PhosflowTM technology in flow cytometry. Of thirty tested chemokines nine (CXCL16, CCL2, CXCL6, CCL7, CXCL1, CCL13, CCL5 CXCL2 and CCL1) are secreted inherently by MSCs suggesting that MSC potency and immunosuppressive potential can be determined by the presence of these chemokines. In addition, MSC mediated blocking of T cell proliferation predominantly inversely correlates with chemokines. Knockdown of chemokines have demonstrated that MSC sourced inherent chemokines do not actively play a role in T cell suppression and thus are the bystander predictors of T cell suppression. The present analysis of MSC’s matrix chemokine responses can be deployed in the advanced potency determination of MSCs. As well, little difference was seen between chemokine levels from intestinal organoid secretome samples from IBD and non-IBD cultures. Seven signaling molecules [PLCγ1, PLCγ2, PKCα, JNK, P38 MAPK, Erk ½, pAkt (pS473)] were analyzed for phosphorylation events in T-cells when stimulated with MSC secretome. Our results provided evidence that MSC derived chemokines and secretome predicts T-cell suppression. These mechanistic understandings will help us to improve MSC based cellular therapy.
    • Treatment of Neuropathic Pain Using 1-O-Hexyl-2,3,5- Trimethylhydroquinone (HTHQ)

      Shoaga, Elizabeth Omolara; School of Medicine
      Neuropathic pain is caused by a primary lesion or injury to the nervous system and can be spontaneous, with no obvious peripheral stimulus. Pain results from the complex interplay between signaling systems and individual perception. Neuropathic and chronic pain effect more than 100 million Americans while costing the country billions of dollars annually to treat and manage. The purpose of this research study was to discover if preemptive or post-injury treatment with 1-O-Hexyl-2,3,5- trimethylhydroquinone (HTHQ), a hydroquinone monoalkyl ether known for its antioxidative abilities, effectively alleviates neuropathic pain induced by partial sciatic nerve ligation in rats. In order to test the effectiveness of HTHQ in reducing neuropathic pain, we began by taking preliminary baseline behavior assessments to test animal pain response prior to injury. Some animals were treated with HTHQ for three consecutive days prior to the performance of partial sciatic nerve ligation (pSNL) surgery, while some were treated each day beginning four days after the induction of injury. Animal behavior was observed again for 10 days after nerve injury and drug treatment. The levels of antioxidant and pro-inflammatory proteins were analyzed using the western blot technique in order to determine the effectiveness of HTHQ at treating neuropathic pain. Preliminary findings suggests that treatment with HTHQ three days prior to nerve ligation surgery showed an increase in antioxidant catalase, an enzyme responsible for converting hydrogen peroxide to water and oxygen, in the spinal cords of injured rats compared to those that were not treated 10-days after nerve injury. Our findings also demonstrate that rats treated with HTHQ three days prior to nerve injury showed an increase in antioxidative protein SOD2 but also decreases the expression of pro-inflammatory protein IL-1ß compared to vehicle treated neuropathic rats. There were no therapeutic changes seen in rats treated with HTHQ four days after pSNL surgery.