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Objective: Astrocytes are proposed to be a critical reservoir of HIV in the brain. However, HIV infection of astrocytes is inefficient in vitro except for cell-to-cell transmission from HIV-infected cells. Here, we explore mechanisms by which cell-free HIV bypasses entry and postentry barriers leading to a productive infection., Methods: HIV infection of astrocytes was investigated by a variety of techniques including transfection of CD4-expressing plasmid, treatment with lysosomotropic agents or using a transwell culture system loaded with HIV-infected lymphocytes. Infection was monitored by HIV-1 p24 in culture supernatants and integrated proviral DNA was quantified by Alu-PCR., Results: Persistent HIV infection could be established in astrocytes by transfection of proviral DNA, transduction with VSV-G-pseudotyped viruses, transient expression of CD4 followed by HIV infection, or simultaneous treatment with lysosomotropic chloroquine or Tat-HA2 peptide with HIV infection. In absence of these treatments, HIV entered via endocytosis as seen by electronmicroscopy and underwent lysosomal degradation without proviral integration, indicating endocytosis is a dead end for HIV in astrocytes. Nevertheless, productive infection was observed when astrocytes were in close proximity but physically separated from HIV-infected lymphocytes in the transwell cultures. This occurred with X4 or dual tropic R5X4 viruses and was blocked by an antibody or antagonist to CXCR4., Conclusion: A CD4-independent, CXCR4-dependent mechanism of viral entry is proposed, by which immature HIV particles from infected lymphocytes might directly bind to CXCR4 on astrocytes and trigger virus--cell fusion during or after the process of viral maturation. This mechanism may contribute to the formation of brain HIV reservoirs.

Advances in antiretroviral therapy have resulted in significantly decreased HIV-related mortality. HIV-associated neurocognitive disorders, however, continue to be a major problem in infected patients. The neuropathology underlying HIV-associated neurocognitive disorders has not been well characterized, and evidence suggests different contributing mechanisms. One potential mechanism is the induction of oxidative stress. Using the HIV-1 transgenic (Tg) rat model of HIV, we found increased striatal NADPH oxidase-4 and neuronal nitric oxide synthase expression in the adult (7- to 9-month-old) Tg rat compared with control rats but not in the young (1-month-old) Tg rats. This was accompanied by increased 3-nitrotyrosine (3-NT) immunostaining in the adult Tg rats, which worsened significantly in the old Tg rats (18 to 20 months old). There was, however, no concurrent induction of the antioxidant systems because there was no change in the expression of the nuclear factor-erythroid 2-related factor 2 and its downstream targets (thioredoxin and glutathione antioxidant systems). Colocalization of 3-NT staining with neurofilament proteins and evidence of decreased tyrosine hydroxylase and dopamine transporter expression in the old rats support dopaminergic involvement. We conclude that the HIV-1 Tg rat brain shows evidence of nitrosative stress without appropriate oxidation-reduction adaptation, whereas 3-NT modification of striatal neurofilament proteins likely points to the ensuing dopaminergic neuronal loss and dysfunction in the aging HIV-1 Tg rat., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: In vivo imaging biomarkers of various HIV neuropathologies, including dopaminergic dysfunction, are still lacking. Towards developing dopaminergic biomarkers of brain involvement in HIV, we assessed the pre and postsynaptic components of the dopaminergic system in the HIV-1 transgenic rat (Tg), a well-characterized model of treated HIV+ patients, using small-animal PET imaging., Methods: Fifteen to 18 month-old Tg and wild type (WT) rats were imaged with both [18F]-FP-CMT, a dopamine transporter (DAT) ligand (n=16), and [18F]-Fallypride, a D2/D3 dopamine receptor (D2/D3DR) ligand (n=16). Five to 8 month-old Tg and WT rats (n=18) were also imaged with [18F]-FP-CMT. A subset of animals was imaged longitudinally at 7 and 17 months of age. Multiplex immunohistochemistry staining for DAT, tyrosine hydroxylase, D2DR, D3DR , GFAP, Iba1 and NeuN was performed on a subgroup of the scanned animals., Results: [18F]-FP-CMT and [18F]-Fallypride binding potential (BP ND ) values were significantly lower in 15-18 month-old Tg compared to age-matched WT rats (p<0.0001 and 0.001, respectively). [18F]-FP-CMT BP ND values in 5-8 month-old rats, however, were not significantly different. Longitudinal age-related decrease in [18F]-FP-CMT BP ND was exacerbated in the Tg rat. Immunohistochemistry showed decreased staining of dopaminergic markers in Tg rats. Rats with higher serum gp120 had lower mean BP ND values for both ligands., Conclusions: We found presynaptic and postsynaptic dopaminergic dysfunction/loss in older Tg compared to WT rats. We believe this to be related to neurotoxicity of viral proteins present in the Tg rats' serum and brain., Advances in Knowledge: Our findings confirm prior reports of neurobehavioral abnormalities suggestive of dopaminergic dysfunction in this model. They also suggest similarities between the Tg rat and HIV+ patients as far as dopaminergic dysfunction., Implications for Patient Care: The Tg rat, along with the above-described quantitative PET imaging biomarkers, can have a role in the evaluation of HIV neuroprotective therapies prior to human translation., (Published by Elsevier Inc.)

Background: There are currently no widely accepted neuro-HIV small animal models. We wanted to validate the HIV-1 Transgenic rat (Tg) as an appropriate neuro-HIV model and then establish in vivo imaging biomarkers of neuropathology, within this model, using MR structural and diffusion tensor imaging (DTI)., Methods: Young and middle-aged Tg and control rats were imaged using MRI. A subset of middle-aged animals underwent longitudinal repeat imaging six months later. Total brain volume (TBV), ventricular volume (VV) and parenchymal volume (PV = TBV-VV) were measured. Fractional anisotropy (FA) and mean diffusivity (MD) values of the corpus callosum (CC) were calculated from DTI data., Results: TBV and PV were smaller in Tg compared to control rats in young and middle-aged cohorts (p<0.0001). VV increased significantly (p = 0.005) over time in the longitudinal Tg cohort. There were lower FA (p<0.002) and higher MD (p<0.003) values in the CC of middle-aged Tg rats compared to age-matched controls. Longitudinally, MD significantly decreased over time in Tg rats (p<0.03) while it did not change significantly in the control cohort over the same period of time (p>0.05)., Conclusions: We detected brain volume loss in the Tg rat, probably due to astrocytic dysfunction/loss, loss of structural/axonal matrix and striatal neuronal loss as suggested by immunofluorescence. Increased MD and decreased FA in the CC probably reflect microstructural differences between the Tg and Control rats which could include increased extracellular space between white matter tracts, demyelination and axonal degeneration, among other pathologies. We believe that the Tg rat is an adequate model of neuropathology in HIV and that volumetric MR and DTI measures can be potentially used as biomarkers of disease progression.

The dopaminergic system is especially vulnerable to the effects of human immunodeficiency virus (HIV) infection, rendering dopaminergic deficits early surrogate markers of HIV-associated neuropathology. We quantified dopamine D2/3 receptors in young HIV-1 transgenic (Tg) (n  =  6) and age-matched control rats (n  =  7) and adult Tg (n  =  5) and age-matched control rats (n  =  5) using [18F]fallypride positron emission tomography (PET). Regional uptake was quantified as binding potential (BPND) using the two-tissue reference model with the cerebellum as the reference. Time-activity curves were generated for the ventral striatum, dorsal striatum, thalamus, and cerebellum. Whereas BPND values were significantly lower in the ventral striatum (p < .001) and dorsal striatum (p  =  .001) in the adult Tg rats compared to controls rats, they were significantly lower only in the dorsal striatum (p < .05) in the young rats. Tg rats had smaller striatal volumes on magnetic resonance imaging. We also found lower expression levels of tyrosine hydroxylase on immunohistochemistry in the Tg animals. Our findings suggest that progressive striatal D2/3 receptor deficits occur in Tg rats as they age and can be detected using small-animal PET imaging. The effectiveness of various approaches in preventing or halting this dopaminergic loss in the Tg rat can thus be measured preclinically using [18F]fallypride PET as a molecular imaging biomarker of HIV-associated neuropathology.

Chronic immune activation is a major complication of antiretroviral therapy (ART) for HIV infection and can cause a devastating immune reconstitution inflammatory syndrome (IRIS) in the brain. The mechanism of T-cell activation in this population is not well understood. We found HIV-Tat protein and IL-17-expressing mononuclear cells in the brain of an individual with IRIS. Tat was also present in the CSF of individuals virologically controlled on ART. Hence we examined if Tat protein could directly activate T cells. Tat transcriptionally dysregulated 94 genes and induced secretion of 11 cytokines particularly activation of IL-17 signaling pathways supporting the development of a proinflammatory state. Tat increased IL-17 transcription and secretion in T cells. Tat entered the T cells rapidly by clathrin-mediated endocytosis and localized to both the cytoplasm and the nucleus. Tat activated T cells through a nonclassical pathway dependent upon vascular endothelial growth factor receptor-2 and downstream secondary signaling pathways but independent of the T-cell receptor. However, Tat stimulation of T cells did not induce T-cell proliferation but increased viral infectivity. This study demonstrates Tat's role as a virulence factor, by driving T-cell activation and contributing to IRIS pathophysiology. This supports the necessity of an anti-Tat therapy in conjunction with ART and identifies multiple targetable pathways to prevent Tat-mediated T-cell activation.

Motor and behavioral abnormalities are common presentations among individuals with HIV-1 associated neurocognitive disorders (HAND). We investigated whether longitudinal motor and behavioral performance in the HIV-1 transgenic rat (Tg), a commonly used neuro-HIV model, corresponded to in vivo neuronal death/dysfunction, by using rotarod and open field testing in parallel to [18F] 2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET). We demonstrated that age-matched non-Tg wild type (WT) rats outperformed the HIV-1 Tg rats at most time points on rotarod testing. Habituation to rotarod occurred at 8 weeks of age (fifth weekly testing session) in the WT rats but it never occurred in the Tg rats, suggesting deficits in motor learning. Similarly, in open field testing, WT rats outperformed the Tg rats at most time points, suggesting defective exploratory/motor behavior and increased emotionality in the Tg rat. Despite the neurobehavioral abnormalities, there were no concomitant deficits in 18F-FDG uptake in Tg rats on PET compared to age-matched WT rats and no significant longitudinal loss of FDG uptake in either group. The negative PET findings were confirmed using 14C- Deoxy-D-glucose autoradiography in 32 week-old Tg and WT rats. We believe that the neuropathology in the HIV-1 Tg rat is more likely a consequence of neuronal dysfunction rather than overt neurodegeneration/neuronal cell death, similar to what is seen in HIV-positive patients in the post-ART era. [ABSTRACT FROM AUTHOR]

Although cells of monocytic lineage are the primary source of human immunodeficiency virus type 1 (HIV-1) in the brain, other cell types in the central nervous system, including astrocytes, can harbor a latent or persistent HIV-1 infection. In the present study, we examined whether immature, multipotential human brain-derived progenitor cells (nestin positive) are also permissive for infection. When exposed to IIIB and NL4-3 strains of HIV-1, progenitor cells and progenitor-derived astrocytes became infected, with peak p24 levels of 100 to 500 pg/ml at 3 to 6 days postinfection. After 10 days, virus production was undetectable but could be stimulated by the addition of tumor necrosis factor alpha (TNF-α). To bypass limitations to receptor entry, we compared the fate of infection in these cell populations by transfection with the infectious HIV-1 clone, pNL4-3. Again, transfected progenitors and astrocytes produced virus for 7 days but diminished to Iow levels beyond 8 days posttransfection. During the nonproductive phase, TNF-α stimulated virus production from progenitors as late as 5 weeks posttransfection. Astrocytes produced 5- to 20-fold more infectious virus (27 ng of p24/106 cells) than progenitors at the peak of 3 days posttransfection. Differentiation of infected progenitors toward an astrocyte phenotype increased virus production to levels consistent with infected astrocytes, suggesting a phenotypic difference in viral replication. Using this cell culture system of multipotential human brain-derived progenitor cells, we provide evidence that progenitor cells may be a reservoir for HIV-1 in the brains of AIDS patients. [ABSTRACT FROM AUTHOR]