Probes for KIT

HIV-1 infection within the central nervous system (CNS) includes evolution of the virus, damaging inflammatory cascades, and the involvement of multiple cell types; however, our understanding of how Env tropism and inflammation can influence CNS infectivity is incomplete. In this study, we utilize macrophage-tropic and T cell-tropic HIV-1 Env proteins to establish accurate infection profiles for multiple CNS cells under basal and interferon alpha (IFN-α) or lipopolysaccharide (LPS)-induced inflammatory states. We found that macrophage-tropic viruses confer entry advantages in primary myeloid cells, including monocyte-derived macrophage, microglia, and induced pluripotent stem cell (iPSC)-derived microglia. However, neither macrophage-tropic or T cell-tropic HIV-1 Env proteins could mediate infection of astrocytes or neurons, and infection was not potentiated by induction of an inflammatory state in these cells. Additionally, we found that IFN-α and LPS restricted replication in myeloid cells, and IFN-α treatment prior to infection with vesicular stomatitis virus G protein (VSV G) Envs resulted in a conserved antiviral response across all CNS cell types. Further, using RNA sequencing (RNA-seq), we found that only myeloid cells express HIV-1 entry receptor/coreceptor transcripts at a significant level and that these transcripts in select cell types responded only modestly to inflammatory signals. We profiled the transcriptional response of multiple CNS cells to inflammation and found 57 IFN-induced genes that were differentially expressed across all cell types. Taken together, these data focus attention on the cells in the CNS that are truly permissive to HIV-1, further highlight the role of HIV-1 Env evolution in mediating infection in the CNS, and point to limitations in using model cell types versus primary cells to explore features of virus-host interaction. IMPORTANCE The major feature of HIV-1 pathogenesis is the induction of an immunodeficient state in the face of an enhanced state of inflammation. However, for many of those infected, there can be an impact on the central nervous system (CNS) resulting in a wide range of neurocognitive defects. Here, we use a highly sensitive and quantitative assay for viral infectivity to explore primary and model cell types of the brain for their susceptibility to infection using viral entry proteins derived from the CNS. In addition, we examine the ability of an inflammatory state to alter infectivity of these cells. We find that myeloid cells are the only cell types in the CNS that can be infected and that induction of an inflammatory state negatively impacts viral infection across all cell types.

Background: The Psychiatric Ratings using Intermediate Stratified Markers (PRISM) project focuses on understanding the biological background behind social deficits, specifically social withdrawal irrespective of diagnosis. Reduced connectional integrity in fiber tracts such as Forceps minor has been indicated in low social individuals as a part of the PRISM 1 project. These fiber tracts are also involved in the Default Mode Network (DMN) and the Social network and they share a common region, the Orbitofrontal Cortex (OFC).This study aims to back-translate the clinical data to preclinical studies and associate social dysfunction in rodents with DMN and particularly OFC. Parvalbumin interneurons are targeted based on their fundamental role in maintaining Excitatory Inhibitory (E/I) balance in brain circuits. Numerous studies indicate behavioral impairment in rodents by increasing excitability of PV+ interneurons. Methods: As an initial step, we characterized the population of projection neurons within OFCs by combining Cholera Toxin subunit B (CTB) as a retrograde tracer and In situ hybridization (ISH) technique (RNAscope). We identified the expression of mRNAs marking glutamatergic (vesicular glutamate transporter [VGLUT]) and GABAergic (vesicular GABA transporter [VGAT]) by using Slc17a7 and Slc32a1 probes. CTB was injected unilaterally in the left OFC (AP=2.68, ML=-0.8, DV=2.2). after 10 days mice were perfused and RNAscope assay was performed using RNAscope™ Multiplex Fluorescent kit (ACDBio™).For inducing hypoactivation of OFC, we introduced an excitatory DREADD (designer receptors exclusively activated by designer drugs) to PV+ interneurons by using a PV-Cre mouse line. Mice were injected either AAV-hSyn-DIO-hM3D(Gq)-mCherry virus (n=12) or AAV-hSyn-DIO-mCherry (n=12) as control virus. As a novel behavioral tool, Radiofrequency identification (RFID)-assisted SocialScan combined with video tracking has been used, which provides a long-term observation of social behaviors. Monitoring the behavior in groups of four was performed for 7 days in total. After two pre-application days, Clozapine-N-oxide (CNO) was injected three times on consecutive days intraperitoneally (5mg/kg) as an activator of hM3D. application days were followed by two post-application days. Mice were perfused and RNAscope was performed to visualize c-fos mRNA expression as neuronal activity marker, and PV expression to validate our virus and mouse line efficacy. Results: ISH results indicated VGLUT1 has the highest expression within projection neurons (81%). 6% are VGAT+ and only 3% are both VGLUT1/VGAT positive neurons. Despite demonstrating the GABAergic projection neurons as a minority, their crucial role as local interneurons to moderate the excitatory neurons is indisputable.In in vivo study, CNO administration induced social dysregulation in DREAAD mice, demonstrated by a reduction in different social parameters (approach, fight, etc.) in terms of duration. During post-application days, DREAAD mice showed significantly higher social interaction in all definedparameters (Social Approach: p=0.0009, unpaired T-test) and locomotion as a non-social parameter (p= 0.0207).Results from ISH support our hypothesis that DREADD activation of PV+ interneurons is followed by high expression of neuronal activity markers in these targeted interneurons. Conclusion: This study indicates that manipulation of PV+ interneurons using artificially engineered activating protein receptors, generates in effect activation of these interneurons, and this manipulation particularly in OFC could cause social dysfunction in mice.