Wininger, Katheryn M.

Abstract Description Blood-brain barrier (BBB) disruption is a putative mechanism of neurodegeneration, neuropsychiatric disorders, and long covid. Our laboratory was the first to demonstrate antigen-specific CD8 T cells induce BBB disruption. We now introduce a novel neurologic disease model in which CD8 T resident memory (TRM) cells induce BBB disruption via administration of cognate antigen, long after neurotropic virus is cleared. C57BL/6 mice intracranially infected with Theiler’s murine encephalomyelitis virus (TMEV) clear virus within 21 days. At 2 months post infection, mice received intravenous injection of viral peptide VP2(121-130) for which the largest population of CD8 TRM have T cell receptor specificity for, resulting in BBB permeability detectable by gadolinium enhanced T1 weighted MRI. A second round of BBB disruption was then initiated with VP2 peptide at 4 months post infection. One month after this second induction of BBB disruption, mice underwent behavioral assessments and whole brain flow cytometric analysis. In these mice, we found a significant inverse correlation of BBB breakdown with a shift from active to passive anxiety-like behavior to contextual fear memory compared to mock peptide controls. This shift in anxiety correlated with virus antigen specific CD8 TRM, myeloid, and NK cells. These results demonstrate memory antiviral CD8 T cells can induce BBB disruption, long after virus is cleared, a process that contributes to a shift in anxiety-like behaviors Funding Sources RO1 NS103212, R01 NS122174 Topic Categories Neuroimmunology (NEUR)

ABSTRACT Brain atrophy is a common feature of neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and more recently SARS‐CoV‐2 infection for which the neuroinflammatory mechanism is not fully understood. Nevertheless, neuroinflammation and CD8 T cell accumulation is frequently observed in postmortem brain tissue of patients with neurodegeneration. We therefore developed a murine model of brain atrophy using the Theiler's murine encephalomyelitis virus (TMEV) infection model of multiple sclerosis. We employ single major histocompatibility complex (MHC) class I molecule conditional knockout mice generated by our program to analyze the contribution of immune cell infiltration and onset of atrophy. TMEV infected C57BL/6 mice that singularly express H‐2K b or H‐2D b were evaluated. TMEV infection of these mice resulted in ventricular atrophy at 14 d.p.i. However, H‐2D b expressing mice presented with significantly greater and continuous ventricular atrophy compared to H‐2K b expressing mice and MHC class I knockout control mice. Flow cytometric analysis revealed H‐2D b expressing mice had greater brain infiltrating CD8 T cell responses at 7 and 28 dpi which correlated with the extent of ventricular atrophy. Meanwhile, H‐2K b and MHC class I knockout mice also had distinct positive correlations with ventricular atrophy and global neuroinflammation in general. These findings support the role of immune cell infiltration into the CNS as a putative mechanism of ventricular atrophy following TMEV infection, with the progressive enlargement of ventricular atrophy observed in H‐2D b mice being associated with the presence of long‐lived memory CD8 T cells residing in the brain.

Abstract Brain atrophy is a common feature of many neurological diseases as diverse as Alzheimer’s disease, cerebral palsy, Huntington’s disease, multiple sclerosis, epilepsy, encephalitis, neurosyphilis, neuroAIDS, and Covid-19 infection. We have developed a murine model of brain atrophy using the Theiler’s murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis. In this study, we investigated the contribution of the MHC class I molecule, H-2Db, in generating an immune response associated with the onset of brain atrophy using mice with a C57BL/6 background. The H-2DbClass I molecule not only contributed to significantly more ventricular enlargement in a T2-weighted MRI at 45 d.p.i., but this ventricular enlargement correlated to CD8 T cell infiltration. To further define the cellular and molecular mechanisms of MHC class I molecules in TMEV induced brain atrophy we investigated the cell specific contribution of brain resident antigen presenting cells (APCs) in ventricular atrophy. We developed novel bi-transgenic mouse lines with tamoxifen induced conditional ablation of the H-2Db class I molecule in Cx3CR1+ brain resident myeloid cells. Cx3CR1cre+/Db Lox P mice presented with significantly less ventricular atrophy at 45 d.p.i., compared to cre- littermates along with a decrease in CD8 T cell infiltration. These results strongly imply that antigen presentation by H-2Db on myeloid cells, at least in part is responsible for the development of brain atrophy and provided insight into the molecular mechanism of this poorly understood neuropathology. NS103212, NS122174

Abstract Brain atrophy is a common feature of many neurological diseases as diverse as Alzheimer’s disease, cerebral palsy, Huntington’s disease, Krabbe disease, multiple sclerosis, Pick’s disease, epilepsy, encephalitis, neurosyphilis, neuroAIDS, and Covid-19 infection. We have developed a murine model of brain atrophy using the Theiler’s murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis. In this study, we investigated the contribution of the MHC class I molecule, H-2Db, in generating an immune response associated with the onset of brain atrophy. To define the roll of the MHC class I molecule in the cellular and molecular mechanisms of brain atrophy, we developed a novel bi-transgenic mouse lines with tamoxifen induced conditional ablation of the H-2Db class I molecule in Cx3CR1+ brain resident myeloid cells on the C57BL/6 background. Interestingly, both CX3CR1cre/Db LoxP mice and their cre- littermate controls experienced short-term memory loss using novel object recognition tests. However, Cx3CR1cre+/Db LoxP mice presented with significantly less brain atrophy as assessed by analysis of lateral ventricular volume at 45 d.p.i. using T2-weighted MRI, compared to cre- littermates. This change in ventricular volume was sustained at 6 months post infection. These results strongly imply a requirement for antigen presentation by H-2Db on myeloid cells for the development of brain atrophy and provided insight into the molecular mechanism of this poorly understood neuropathology. Supported by NIH (RFI NS122174)

AbstractDisruptions in circadian rhythms are risk factors for excessive alcohol drinking. The ethanol‐sensitive adenosine equilibrative nucleoside transporter type 1 (ENT1, slc29a1) regulates ethanol‐related behaviors, sleep, and entrainment of circadian rhythms. However, the mechanism underlying the increased ethanol consumption in ENT1 knockout (KO) mice in constant light (LL) and whether there are sex differences in ethanol consumption in ENT1 mice are less studied. Here, we investigated the effects of loss of ENT1, LL, and sex on ethanol drinking using two‐bottle choice. In addition, we monitored the locomotor activity rhythms. We found that LL increased ethanol drinking and reduced accumbal ENT1 expression and adenosine levels in male but not female mice, compared with control mice. Interestingly, only LL‐exposed male, not female, ENT1 KO mice exhibited higher ethanol drinking and a longer circadian period with a higher amplitude compared with wild‐type (WT) mice. Furthermore, viral‐mediated rescue of ENT1 expression in the NAc of ENT1 KO mice reduced ethanol drinking, demonstrating a possible causal link between ENT1 expression and ethanol drinking in males. Together, our findings indicate that deficiency of ENT1 expression contributes to excessive ethanol drinking in a sex‐dependent manner.

Abstract Waiting impulsivity is a risk factor for many psychiatric disorders including alcohol use disorder (AUD). Highly impulsive individuals are vulnerable to alcohol abuse. However, it is not well understood whether chronic alcohol use increases the propensity for impulsive behavior. Here, we establish a novel experimental paradigm demonstrating that continuous binge‐like ethanol exposure progressively leads to maladaptive impulsive behavior. To test waiting impulsivity, we employed the 5‐choice serial reaction time task (5‐CSRTT) in C57BL/6J male mice. We assessed premature responses in the fixed and variable intertrial interval (ITI) 5‐CSRTT sessions. We further characterized our ethanol‐induced impulsive mice using Open Field, y‐maze, two‐bottle choice, and an action‐outcome task. Our results indicate that continuous binge‐like ethanol exposure significantly increased premature responses when mice were tested in variable ITI sessions even during a prolonged abstinent period. Ethanol‐induced impulsive mice exhibited anxiety‐like behavior during chronic exposures. This behavior was also observed in a separate cohort that was subjected to 20 days of abstinence. Ethanol‐treated mice were less motivated for a sucrose reward compared with air‐exposed control mice, while also demonstrating reduced responding during action‐outcome testing. Overall, ethanol‐treated mice demonstrated increased impulsive behavior, but a reduced motivation for a sucrose reward. Although waiting impulsivity has been hypothesized to be a trait or risk factor for AUD, our findings indicate that maladaptive impulse control can also be potentiated or induced by continuous chronic ethanol exposure in mice.

AbstractAcamprosate is an FDA‐approved medication for the treatment of alcoholism that is unfortunately only effective in certain patients. Although acamprosate is known to stabilize the hyper‐glutamatergic state in alcoholism, pharmacological mechanisms of action in brain tissue remains unknown. To investigate the mechanism of acamprosate efficacy, the authors employ a pharmacoproteomics approach using an animal model of alcoholism, type 1 equilibrative nucleoside transporter (ENT1) null mice. The results demonstrate that acamprosate treatment significantly decreased both ethanol drinking and preference in ENT1 null mice compared to that of wild‐type mice. Then, to elucidate acamprosate efficacy mechanism in ENT1 null mice, the authors utilize label‐free quantification proteomics comparing both genotype and acamprosate treatment effects in the nucleus accumbens (NAc). A total of 1040 protein expression changes are identified in the NAc among 3634 total proteins detected. The proteomics and Western blot result demonstrate that acamprosate treatment decreased EAAT expression implicating stabilization of the hyper‐glutamatergic condition in ENT1 null mice. Pathway analysis suggests that acamprosate treatment in ENT1 null mice seems to rescue glutamate toxicity through restoring of RTN4 and NF‐κB medicated neuroimmune signaling compared to wild‐type mice. Overall, pharmacoproteomics approaches suggest that neuroimmune restoration is a potential efficacy mechanism in the acamprosate treatment of certain sub‐populations of alcohol dependent subjects.