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Mixed glia are infiltrated with HIV-1 virus early in the course of infection leading to the development of a persistent viral reservoir in the central nervous system. Modification of the HIV-1 genome using gene editing techniques, including CRISPR/Cas9, has shown great promise towards eliminating HIV-1 viral reservoirs; whether these techniques are capable of removing HIV-1 viral proteins from mixed glia, however, has not been systematically evaluated. Herein, the efficacy of adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing for eliminating HIV-1 messenger RNA (mRNA) from cortical mixed glia was evaluated in vitro and in vivo. In vitro, a within-subjects experimental design was utilized to treat mixed glia isolated from neonatal HIV-1 transgenic (Tg) rats with varying doses (0, 0.9, 1.8, 2.7, 3.6, 4.5, or 5.4 µL corresponding to a physical titer of 0, 4.23 × 10 9 , 8.46 × 10 9 , 1.269 × 10 10 , 1.692 × 10 10 , 2.115 × 10 10 , and 2.538 × 10 10 gc/µL) of CRISPR/Cas9 for 72 h. Dose-dependent decreases in the number of HIV-1 mRNA, quantified using an innovative in situ hybridization technique, were observed in a subset (i.e., n = 5 out of 8) of primary mixed glia. In vivo, HIV-1 Tg rats were retro-orbitally inoculated with CRISPR/Cas9 for two weeks, whereby treatment resulted in profound excision (i.e., approximately 53.2%) of HIV-1 mRNA from the medial prefrontal cortex. Given incomplete excision of the HIV-1 viral genome, the clinical relevance of HIV-1 mRNA knockdown for eliminating neurocognitive impairments was evaluated via examination of temporal processing, a putative neurobehavioral mechanism underlying HIV-1-associated neurocognitive disorders (HAND). Indeed, treatment with CRISPR/Cas9 protractedly, albeit not permanently, restored the developmental trajectory of temporal processing. Proof-of-concept studies, therefore, support the susceptibility of mixed glia to gene editing and the potential of CRISPR/Cas9 to serve as a novel therapeutic strategy for HAND, even in the absence of full viral eradication., (© 2024. The Author(s).)

Microglia, which are productively infected by HIV-1, are critical for brain development and maturation, as well as synaptic plasticity. The pathophysiology of HIV-infected microglia and their role in the pathogenesis of HIV-1-associated neurocognitive and affective alterations, however, remains understudied. Three complementary aims were undertaken to critically address this knowledge gap. First, the expression of HIV-1 mRNA in the dorsolateral prefrontal cortex of postmortem HIV-1 seropositive individuals with HAND was investigated. Utilization of immunostaining and/or RNAscope multiplex fluorescent assays revealed prominent HIV-1 mRNA in microglia of postmortem HIV-1 seropositive individuals with HAND. Second, measures of microglia proliferation and neuronal damage were evaluated in chimeric HIV (EcoHIV) rats. Eight weeks after EcoHIV inoculation, enhanced microglial proliferation was observed in the medial prefrontal cortex (mPFC) of EcoHIV rats, evidenced by an increased number of cells co-localized with both Iba1 + and Ki67 + relative to control animals. Neuronal damage in EcoHIV infected rats was evidenced by pronounced decreases in both synaptophysin and postsynaptic density protein 95 (PSD-95), markers of presynaptic and postsynaptic damage, respectively. Third, regression analyses were conducted to evaluate whether microglia proliferation mechanistically underlies neuronal damage in EcoHIV and control animals. Indeed, microglia proliferation accounted for 42-68.6% of the variance in synaptic dysfunction. Collectively, microglia proliferation induced by chronic HIV-1 viral protein exposure may underlie the profound synaptodendritic alterations in HIV-1. Understanding how microglia are involved in the pathogenesis of HAND and HIV-1-associated affective disorders affords a key target for the development of novel therapeutics., (© 2023. The Author(s) under exclusive licence to The Journal of NeuroVirology, Inc.)

HIV-1 infection affects approximately 37 million individuals, and approximately 50% of seropositive individuals will develop symptoms of clinical depression and/or apathy. Dysfunctions of both serotonergic and dopaminergic neurotransmission have been implicated in the pathogenesis of motivational alterations. The present study evaluated the efficacy of a SSRI (escitalopram) in the HIV-1 transgenic (Tg) rat. Behavioral, neurochemical, and neuroanatomical outcomes with respect to HIV-1 and sex were evaluated to determine the efficacy of chronic escitalopram treatment. Escitalopram treatment restored function in each of the behavioral tasks that were sensitive to HIV-1-induced impairments. Further, escitalopram treatment restored HIV-1-mediated synaptodendritic damage in the nucleus accumbens; treatment with escitalopram significantly increased dendritic proliferation in HIV-1 Tg rats. However, restoration did not consistently occur with the neurochemical analysis in the HIV-1 rat. Taken together, these results suggest a role for SSRI therapies in repairing long-term HIV-1 protein-mediated neuronal damage and restoring function.

Chronic neurocognitive impairments, commonly associated with pediatric human immunodeficiency virus type 1 (PHIV), are a detrimental consequence of early exposure to HIV-1 viral proteins. Strong evidence supports S-Equol (SE) as an efficacious adjunctive neuroprotective and/or neurorestorative therapeutic for neurocognitive impairments in adult ovariectomized female HIV-1 transgenic (Tg) rats. There remains, however, a critical need to assess the therapeutic efficacy of SE when treatment occurs at an earlier age (i.e., resembling a therapeutic for children with PHIV) and across the factor of biological sex. Utilization of a series of signal detection operant tasks revealed prominent, sex-dependent neurocognitive deficits in the HIV-1 Tg rat, characterized by alterations in stimulus-reinforcement learning, the response profile, and temporal processing. Early (i.e., postnatal day 28) initiation of SE treatment precluded the development of chronic neurocognitive impairments in all (i.e., 100%) HIV-1 Tg animals, albeit not for all neurocognitive domains. Most notably, the therapeutic effects of SE are generalized across the factor of biological sex, despite the presence of endogenous hormones. Results support, therefore, the efficacy of SE as a neuroprotective therapeutic for chronic neurocognitive impairments in the post-cART era; an adjunctive therapeutic that demonstrates high efficacy in both males and females. Optimizing treatment conditions by evaluating multiple factors (i.e., age, neurocognitive domains, and biological sex) associated with PHIV and HIV-1 associated neurocognitive disorders (HAND) affords a key opportunity to improve the therapeutic efficacy of SE.

In 2007, the nosology for HIV-1-associated neurocognitive disorders (HAND) was updated to a primarily neurocognitive disorder. However, currently available diagnostic tools lack the sensitivity and specificity needed for an accurate diagnosis for HAND. Scientists and clinicians, therefore, have been on a quest for an innovative biomarker to diagnose (i.e., diagnostic biomarker) and/or predict (i.e., prognostic biomarker) the progression of HAND in the post-combination antiretroviral therapy (cART) era. The present review examined the utility and challenges of four proposed biomarkers, including neurofilament light (NFL) chain concentration, amyloid (i.e., sAPPα, sAPPβ, amyloid β) and tau proteins (i.e., total tau, phosphorylated tau), resting-state functional magnetic resonance imaging (fMRI), and prepulse inhibition (PPI). Although significant genotypic differences have been observed in NFL chain concentration, sAPPα, sAPPβ, amyloid β, total tau, phosphorylated tau, and resting-state fMRI, inconsistencies and/or assessment limitations (e.g., invasive procedures, lack of disease specificity, cost) challenge their utility as a diagnostic and/or prognostic biomarker for milder forms of neurocognitive impairment (NCI) in the post-cART era. However, critical evaluation of the literature supports the utility of PPI as a powerful diagnostic biomarker with high accuracy (i.e., 86.7-97.1%), sensitivity (i.e., 89.3-100%), and specificity (i.e., 79.5-94.1%). Additionally, the inclusion of multiple CSF and/or plasma markers, rather than a single protein, may provide a more sensitive diagnostic biomarker for HAND; however, a pressing need for additional research remains. Most notably, PPI may serve as a prognostic biomarker for milder forms of NCI, evidenced by its ability to predict later NCI in higher-order cognitive domains with regression coefficients (i.e., r) greater than 0.8. Thus, PPI heralds an opportunity for the development of a brief, noninvasive diagnostic and promising prognostic biomarker for milder forms of NCI in the post-cART era.

Between 30 and 60% of HIV-seropositive individuals develop symptoms of clinical depression and/or apathy. Dopamine and serotonin are associated with motivational alterations; however, histamine is less well studied. In the present study, we used fast-scan cyclic voltammetry in HIV-1 transgenic (Tg) rats to simultaneously analyze the kinetics of nucleus accumbens dopamine (DA), prefrontal cortical serotonin (5-HT), and hypothalamic histamine (HA). For voltammetry, subjects were 15 HIV-1 Tg (7 male, 8 female) and 20 F344/N (11 male, 9 female) adult rats. Both serotonergic and dopaminergic release and reuptake kinetics were decreased in HIV-1 Tg animals relative to controls. In contrast, rates of histamine release and reuptake increased in HIV-1 Tg rats. Additionally, we used immunohistochemical (IHC) methods to identify histaminergic neurons in the tuberomammillary nucleus (TMN) of the hypothalamus. For IHC, subjects were 9 HIV-1 Tg (5 male, 4 female) and 9 F344/N (5 male, 4 female) adult rats. Although the total number of TMN histaminergic cells did not differ between HIV-1 Tg rats and F344/N controls, a significant sex effect was found, with females having an increased number of histaminergic neurons, relative to males. Collectively, these findings illustrate neurochemical alterations that potentially underlie or exacerbate the pathogenesis of clinical depression and/or apathy in HIV-1.

Understanding the progression of HIV-1-associated neurocognitive disorders (HAND) is a critical need as the prevalence of HIV-1 in older individuals (>50 years) is markedly increasing due to the great success of combination antiretroviral therapy (cART). Longitudinal experimental designs, in comparison to cross-sectional studies, provide an opportunity to establish age-related disease progression in HAND. The HIV-1 transgenic (Tg) rat, which has been promoted for investigating the effect of long-term HIV-1 viral protein exposure, was used to examine two interrelated goals. First, to establish the integrity of sensory and motor systems through the majority of the animal's functional lifespan. Strong evidence for intact sensory and motor system function through advancing age in HIV-1 Tg and control animals was observed in cross-modal prepulse inhibition (PPI) and locomotor activity. The integrity of sensory and motor system function suggested the utility of the HIV-1 Tg rat in investigating the progression of HAND. Second, to assess the progression of neurocognitive impairment, including temporal processing and long-term episodic memory, in the HIV-1 Tg rat; the factor of biological sex was integral to the experimental design. Cross-modal PPI revealed significant alterations in the development of temporal processing in HIV-1 Tg animals relative to controls; alterations which were more pronounced in female HIV-1 Tg rats relative to male HIV-1 Tg rats. Locomotor activity revealed deficits in intrasession habituation, suggestive of a disruption in long-term episodic memory, in HIV-1 Tg animals. Understanding the progression of HAND heralds an opportunity for the development of an advantageous model of progressive neurocognitive deficits in HIV-1 and establishes fundamental groundwork for the development of neurorestorative treatments.

Since the advent of combination antiretroviral therapy (cART), pediatric HIV-1 (PHIV) has evolved from a fatal disease to a chronic disease as children perinatally infected with HIV-1 survive into adulthood. The HIV-1 transgenic (Tg) rat, which expresses 7 of the 9 HIV-1 genes constitutively throughout development, was used to model the early development of chronic neurological impairment in PHIV. Male and female Fischer HIV-1 Tg and F344 N control rats, sampled from 35 litters, were repeatedly assessed during early development using multiple experimental paradigms, including somatic growth, locomotor activity, cross-modal prepulse inhibition (PPI) and gap-prepulse inhibition (gap-PPI). Later eye opening was observed in HIV-1 Tg animals relative to controls. HIV-1 Tg animals exhibited a shift in the development of locomotor activity implicating alterations in the maturation of the forebrain cholinergic inhibitory system. Alterations in the development of PPI and perceptual sharpening were observed in both auditory and visual PPI as indexed by a relative insensitivity to the dimension of time (msec for ISI; days of age for perceptual sharpening) as a function of the HIV-1 transgene. Presence of the HIV-1 transgene was diagnosed with 97.1 % accuracy using auditory and visual PPI measurements from PD 17 and 21. Early selective developmental alterations observed in the HIV-1 Tg rats provide an opportunity for the development of a point-of-care screening tool, which would permit the early diagnosis of PHIV and improve the long-term outcome for children perinatally infected with HIV-1.

Neurologic impairments associated with human immunodeficiency virus (HIV) infection in pediatric patients may affect quality of life, and can develop despite antiretroviral therapy (ART). Behavioral changes observed in clinical studies of HIV-infected children suggest alterations in dopaminergic neurotransmission. Findings from our model of choice, the HIV-1 transgenic rat, reveal a significant increase in phosphorylated tyrosine hydroxylase protein expression and a decrease in dopamine transporter mRNA, without changes in tyrosine hydroxylase (TH) or dopamine transporter (DAT) protein or in more general markers of protein and gene expression levels in the HIV-1 transgenic rat midbrain. Thus these findings suggest selective vulnerability of the dopamine system in developing brains to HIV-1 infection.

With the advent of highly active antiretroviral therapy, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) is becoming a more chronic, manageable disease; nevertheless, the prevalence of neurological complications of AIDS is increasing. In this study, protein levels of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the substantia nigra of HIV-infected brains and -seronegative controls were determined by immunoblotting. The immunoreactivity of neuronal specific enolase (NSE) was used to assess cell loss. Although there were no changes in levels of immunoreactive DAT or NSE proteins in HIV brains, levels of immunoreactive TH were significantly reduced, relative to controls. These results suggest that decreases in TH, the rate-limiting enzyme of dopamine synthesis, may be a factor in the neurological manifestations of HIV infection.