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Aged HIV-positive (HIV+) individuals represent a large proportion of the HIV population as life spans are extended significantly by successful antiretroviral therapy. Increased age with HIV infection brings a unique set of central nervous system (CNS) complications including more rapid onset and progression of age-related diseases, loss of protein quality control and accumulation of aberrant proteins, such as hyperphosphorylated Tau (hpTau). In this context, we have discovered a new signaling connection between age-related neurodegeneration and HIV involving the PINCH protein. Particularly interesting new cysteine histidine-rich protein (PINCH), an adaptor protein in neuronal cells is involved in cytoskeletal organization, cell migration and cell survival. While some of the pathophysiological aspects of the PINCH-signaling cascade in tauopathy are largely conserved among neurodegenerative diseases such as Alzheimer’s disease, HIV and others, the presence of the HIV protein Tat impacts specific key points in the PINCH pathway that exacerbate CNS cell dysfunction. In virus-infected cells, Tat regulates viral replication. Even though neurons are not permissive to viral infection, the Tat protein can enter all cell types. Our studies show that Tat interferes with key PINCH signaling partners in neurons. PINCH is robustly expressed by neurons and to a lesser degree by astrocytes in HIV infection of the CNS; whereas, in the healthy CNS, PINCH is nearly undetectable. Similarly, protein phosphatase 1α (PP1α), one of the binding partners of PINCH, is increased in the neurons of HIV encephalitis patients and co-localizes with PINCH in neurons. PINCH is a non-catalytic scaffolding protein that binds integrin-linked kinase (ILK), and PP1α and mediates Akt and GSK3β kinase activities, all of which when disrupted contribute to aberrant Tau phosphorylation. In Tat transgenic mice, increased expression levels of PINCH, PP1α and hpTau were observed. Also, in vivo manipulations of expression levels of PINCH altered the levels of hpTau, where overexpression of PINCH increased hpTau levels in Tat transgenic mouse brains and PINCH knockdown decreased hpTau formation. Furthermore, our studies show that Tat increases levels of hpTau and PINCH, interacts with PP1α and changes the subcellular distribution of PINCH and PP1α in vitro. Tat alters the levels of ILK, Akt and GSK3β, which are key kinases associated with hpTau formation. Furthermore, our preliminary data using expression plasmid for PP1α also shows that overexpression of PP1α decreases Tat-induced aberrant hpTau formation. Our studies address HIV replication-independent functions of Tat in neurons linked to PINCH signaling. These studies address a novel pathway through which Tau may be aberrantly phosphorylated. Thus, understanding new pathways of communication among Tat, PINCH, PP1α, ILK and Tau will open new directions for the study of HIV-associated tauopathy and will provide opportunities for therapeutic interventions in age- and disease-related pathologies. Several studies report associations between the PINCH protein and HIV-associated CNS disease. PINCH is detected in the cerebrospinal fluid (CSF) of HIV patients and changes in levels during disease may be indicative of changes in disease status over time. PINCH binds hpTau in the brain and CSF, but little is known about the relevance of these interactions to HIV CNS disease. In this study, PINCH and hpTau levels were assessed in three separate CSF samples collected longitudinally from 20 HIV+ participants before and after initiating antiretroviral therapy, or before and after a change in the current regimen. Correlational analyses were conducted for CSF levels of PINCH and hpTau and other variables including plasma CD4+ T-cell count, plasma and CSF viral burden, CSF Neopterin, white blood cell (WBC) count, and antiretroviral CNS penetration-effectiveness (CPE). Results suggest that in these HIV participants, changes in CSF levels of PINCH appear to correlate with changes in plasma CD4+ count and with changes in CSF hpTau levels, but not with plasma or CSF viral burden, Neopterin, or WBC, or with anti-retroviral regimen CPE. Furthermore, results from our case matched HIV brain-CSF study confirms that higher levels of PINCH and hpTau are detected in HIV encephalitis brains. Additionally, correlation between PINCH and hpTau levels in brain and other clinical parameters such as age at death, date of death suggesting the era of antiretroviral therapy, CPE score and cognition in HIV patients yielded interesting results that are currently being expanded upon by investigators in the Langford laboratory. Thus, these results suggest that PINCH may be involved in Tauopathy associated neurodegeneration in the HIV CNS disease. Therefore, understanding the contribution of PINCH to HIV-associated Tauopathy may provide a new therapeutic avenue for regulating synaptodendritic dysfunction associated with Tau. Moreover, characterizing the clinical significance of PINCH in the CSF may warrant including PINCH as a member of biomarker panel to assess severity or progression of HIV-associated neurocognitive alterations. / Biomedical Neuroscience

Cocaine is a powerfully active psychostimulant which exerts its effects through blockade of dopamine, serotonin and norepinephrine transporters and resultant increases in extracellular levels of these neurotransmitters. Much of the focus on cocaine abuse in the literature has been directed towards study of the dopamine system; however, several studies have identified a role for the serotonin system in regulating the rewarding effects of cocaine as well. Specifically, the serotonin 2C (5-HT2C) receptor regulates cocaine-induced alterations in serotonin and dopamine levels in an inhibitory manner, and 5-HT2C receptor agonist treatment attenuates cocaine-induced behaviors like self-administration. In the first aspect of the current thesis study, the effects of activation of 5-HT2C receptors on cocaine-induced conditioned place preference and behavioral sensitization were assessed. It was found that pretreatment with a 5-HT2C receptor agonist, Ro 60-0175, on cocaine (10 mg/kg) conditioning days of the conditioned place preference paradigm, attenuated the development of conditioned place preference in a dose-dependent manner. These results suggest that activation of 5-HT2C receptors inhibits the euphoric effects elicited by cocaine. Behavioral sensitization studies demonstrated that pretreatment with Ro 60-0175 prior to cocaine (10 mg/kg) over a 5 day period attenuated cocaine-induced hyperactivity. When injected with a cocaine challenge injection 10 days after the last cocaine injection, mice pretreated with Ro 60-0175 demonstrated lower levels of locomotor activity as compared to saline pretreated, cocaine-injected mice. This portion of the first study demonstrated that 5-HT2C receptor activity attenuated acute cocaine-induced conditioned reward, hyperactivity and the development of long-term alterations of cocaine exposure, as measured by behavioral sensitization. The second aspect of the current study focused on the regulation of anxiety produced by withdrawal from chronic cocaine administration. Anxiety during cocaine withdrawal is a component of the negative affective state often experienced by cocaine-dependent individuals during abstinence from drug use. Anxiety during cocaine withdrawal is likely to increase an individual's susceptibility to relapse to drug use in alleviation of this negative symptom. Studies have shown a downregulation of the serotonin and dopamine systems during withdrawal that potentially contributes to anxiety symptoms. As the 5-HT2C receptor exerts inhibitory control over both the serotonin and dopamine systems, it was hypothesized that blockade of 5-HT2C receptors would attenuate anxiety-like behavior during cocaine withdrawal. Previous studies have identified co-localization of 5-HT2C receptors on inhibitory gamma-aminobutyric acid (GABA) neurons, thus it was hypothesized that a 5-HT2C receptor-GABA mediated mechanism would be involved in the regulation of anxiety during withdrawal. The dorsal raphe brain region was targeted in these studies, as this region is the primary source of serotonin for forebrain structures. The actions of cocaine on the serotonin system likely originate with influence of cocaine on the dorsal raphe neurocircuitry, with implications for dysregulation in downstream projection areas of the dorsal raphe. In this portion of the current thesis study, electrophysiology techniques were used to measure GABA activity in subregions of the dorsal raphe either 30 minutes, 25 hours, or 7 days following a 10-day chronic binge cocaine paradigm (15 mg/kg, 3 injections per day at 1 hour intervals). Controls received saline injections. Mice were tested for anxiety-like behavior on the elevated plus maze and then brain slices were collected for electrophysiology recordings. It was found that at 25 hours of withdrawal, cocaine-treated mice demonstrated heightened anxiety-like behavior on the elevated plus maze, as compared to saline controls. Mice tested during an active cocaine stage 30 minutes after the last injection, or at 7 days of withdrawal did not demonstrate increased anxiety-like behavior. Heightened GABA activity was exhibited in serotonin cells from cocaine-withdrawn mice at 25 hours of withdrawal, an effect that was normalized upon 5-HT2C receptor blockade. No differences were observed at 30 minutes after the last cocaine injection; however, there was an anatomical shift observed at 7 days of withdrawal, in that heightened GABA activity exhibited in two subregions of the dorsal raphe (dorsomedial and ventromedial aspects) at 25 hours of withdrawal shifted to the lateral wing areas at 7 days of withdrawal. The differential regulation of the three subregions has implications on serotonin output to projection areas and contribution to anxiety mechanisms. It was found that systemic administration and local intra-dorsal raphe administration of the 5-HT2C receptor antagonist, SB 242084, prior to elevated plus maze testing attenuated anxiety-like behavior in cocaine-withdrawn mice at 25 hours of withdrawal. Taken together, this portion of the thesis study demonstrated that 5-HT2C receptor activity, specifically within the dorsal raphe, regulates anxiety during cocaine withdrawal, through influence on the GABA inhibitory feedback system. A final aspect of the current thesis study addressed the link between dorsal raphe 5-HT2C receptor activity and activity at downstream structures in the context of cocaine withdrawal-induced anxiety, particularly the nucleus accumbens. Since the dorsal raphe is important in providing serotonin input for brain regions largely involved in regulating the effects elicited by cocaine, it is likely that dysregulation of dorsal raphe signaling during withdrawal has influence on the regulation of downstream structures in the contribution of anxiety mechanisms produced by cocaine withdrawal. It was found that dorsal raphe 5-HT2C receptor blockade attenuated cocaine withdrawal-induced reductions in cFos immunoreactivity in the nucleus accumbens. Further work is needed to investigate these interactions in the context of cocaine withdrawal-induced anxiety. Lastly, autoradiography experiments assessed the effects of cocaine withdrawal-induced anxiety on 5-HT2C receptor expression in various brain regions including the dorsal raphe, medial prefrontal cortex, nucleus accumbens, caudate putamen, and ventral tegmental area. A significant decrease in 5-HT2C receptor binding was found in the dmDR region of cocaine-withdrawn mice as compared to saline controls; however, no differences were found between groups in other regions. Future studies testing 5-HT2C receptor signaling are needed to fully understand the impact of cocaine withdrawal-induced anxiety on receptor function in these structures. In conclusion, the first portion of the current study showed that activation of 5-HT2C receptors attenuated the rewarding and locomotor sensitizing effects of cocaine, as evidenced by conditioned place preference and behavioral sensitization studies. In the second aspect of the current thesis study, we have established a role for the 5-HT2C receptor in the regulation of anxiety during cocaine withdrawal. During withdrawal, blockade of 5-HT2C receptor activity, both global as well as local dorsal raphe blockade, attenuated anxiety at 25 hours of withdrawal. This attenuation of cocaine withdrawal-induced anxiety resultant of 5-HT2C receptor blockade was likely due to a suppression of increased GABA activity evident in serotonin cells from cocaine-withdrawn mice. / Pharmacology

<p>In 1980, cholecystokinin (CCK) was demonstrated to co-exist with dopamine (DA) in certain neurons of the CNS. Since the animal studies were carried out on rodents, little was known about CCK in the brain of higher mammalian species. In the present study, CCK receptors were characterized in the brains of higher mammalian species. The pharmacological characteristics of CCK receptors appear to have been well preserved in the mammalian brain. However, marked differences in the distribution of CCK receptors were observed in several brain areas. There were basically two main types of species-specific differences. The absence or presence of CCK receptors in a given structure and differences in the distribution within a given structure. Although the reasons for such species-specific differences in receptor distribution are not clear at the present time, this evidence cautions against simple extrapolation of data obtained in animal models directly to clinical applications. Moreover, although some animals studies provided evidence of an inhibitory effect of CCK on DA function, which would be compatible with a potential antischizophrenic action, others reported a lack of modulation or an enhancement of DA function. Circling behaviour is commonly used to assess DA function. In the present study, unilateral intracranial microinjections of CCK₈ induced a dose-dependent contraversive circling bias in rats. This CCK₈-induced contraversive circling bias was attenuated by the DA receptor antagonist haloperidol. To further elucidate the mechanism(s) underlying this CCK-induced circling behaviour, the effects of CCK peptides on ligand binding at DA receptors and on DA-stimulated adenylate cyclase were investigated in the rat striatum. Under the assay conditions employed, CCK₈ has no significant acute effects on binding at the DA receptor or on DA-stimulated adenylate cyclase. It is possible that CCK induced its acute facilitatory influence on DA function in the circling behaviour paradigm by altering DA turnover or release. Another problem with the animal studies which were used as the basis for the clinical application was that the vast majority were of an acute nature. In the present study, long-term administration of CCK₈ did not significantly alter DA D₂ receptor densities nor the expression of DA D₂ receptor mRNA. In contrast, long-term haloperidol administrations significantly increased CCK binding in the nucleus accumbens, olfactory tubercle, and frontal cortex. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

<p>The pharmacology of 2-(¹²⁵I) iodomelatonin ((¹²⁵I) MEL) binding was investigated in Syrian hamster brain regions. Both kinetic and saturation binding analysis revealed a site with an affinity of ≅2 nM in the hypothalamus. Correlation of the pharmacological profiles in different brain regions indicated this site also to be present in the cerebral cortex and hippocampus. This binding site appeared to be present throughout the CNS and peripheral tissues of the hamster and is distinct from the picomolar-affinity melatonin receptor. Using the technique of radiation inactivation, the molecular sizes of the chick retinal picomolar-affinity melatonin receptor (Mᵣ = 44 kDa) and hamster hypothalamic low (nanomolar)-affinity binding site (Mᵣ = 30 kDa) were found to be significantly (p<0.04) different. This, along with the pharmacological differences, supports the notion of two separate melatonin binding sites. Binding studies indicated that the continuous cell line RPMI 1846 (Syrian hamster melanoma) possesses a melatonin binding site similar to that found in the hamster CNS. A high correlation was observed for a series of compounds between the Kᵢ in hamster hypothalamic membranes vs RPMI 1846 membranes. Scatchard-Rosenthal analysis of saturation binding of (¹²⁵I) MEL to membranes indicated a site of similar affinity to that in brain. This cell line was used to study potential signal transduction mechanisms for the site. No effect of melatonin was seen on basal or forskolin-stimulated membrane adenylate cyclase activity. Using (³H) adenine and (³H) myo-inositol prelabelling of cells, melatonin was found to be without effect on in situ basal or stimulated cAMP or basal IP₃ levels. In ⁴⁵Ca-flux experiments, melatonin was without effect on basal or K⁺-stimulated uptake in hamster brain synaptosomes or basal uptake in RPMI 1846 cells. This nanomolar-affinity melatonin binding site therefore does not seem to be coupled to the adenylate cyclase or inositol phosphate second messenger systems and its biochemical and physiological function(s) remain(s) unknown.</p> / Doctor of Philosophy (PhD)

The Uncinate Fasciculus (UF) is a white matter association tract responsible for connecting the frontal and temporal lobes of the brain. Its location, general shape, and estimated projections have been visualized and analyzed at low-resolution via diffusion tensor imaging (DTI) in many studies, allowing for a basic understanding of the pathway and its potential functions. Despite this, the microstructural properties of this white matter highway are still relatively unknown as we can estimate characteristics such as myelination and pathway density from low-resolution imaging, but we cannot gain a deeper understanding of the fine characteristics that allow the pathway to function in health and how those features are disrupted in disease. To address this gap, we examined six post-mortem tissue samples, featuring approximately 12,000 axons of the temporal segment of the UF (adjacent to the amygdala and hippocampus) from neurotypical controls. We used high-resolution microscopy to systematically sample and study the microstructure of myelinated axons to characterize the UF for future comparisons to disease states, like Autism Spectrum Disorder (ASD). We provide novel characterization of the microstructure of the UF in both hemispheres, examining density, size, myelination, and trajectories of axons in healthy adult tissue. Our data revealed hemispheric trends of axon characteristics that align with previous imaging studies, including slight left hemispheric asymmetry in regard to density and myelination and a small increase in trajectory variability in the right hemisphere. In addition, like values found previously in the Corpus Callosum, the distribution of thin and medium axons was much higher than thick axons. Our findings provide a more in-depth understanding of the microstructural characteristics of the UF at the level of single axons, providing context for white matter anisotropy and diffusivity estimates from previous DTI studies through values such as myelin thickness, area fraction, and axon trajectory. This structural framework of this pathway can be used to compare with results from in vivo imaging studies, and to provide a basis for comparison with pathological states in the future. / 2026-06-06T00:00:00Z