HIV-1 mRNA knockdown with CRISPR/CAS9 enhances neurocognitive function.

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 × 109, 8.46 × 109, 1.269 × 1010, 1.692 × 1010, 2.115 × 1010, and 2.538 × 1010 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.

Microglia proliferation underlies synaptic dysfunction in the prefrontal cortex: implications for the pathogenesis of HIV-1-associated neurocognitive and affective alterations.

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.

Disrupted Decision-Making: EcoHIV Inoculation in Cocaine Dependent Rats.

Independently, chronic cocaine use and HIV-1 viral protein exposure induce neuroadaptations in the frontal-striatal circuit as evidenced by both clinical and preclinical studies; how the frontal-striatal circuit responds to HIV-1 infection following chronic drug use, however, has remained elusive. After establishing experience with both sucrose and cocaine self-administration, a pretest-posttest experimental design was utilized to evaluate preference judgment, a simple form of decision-making dependent upon the integrity of frontal-striatal circuit function. During the pretest assessment, male rats exhibited a clear preference for cocaine, whereas female animals preferred sucrose. Two posttest evaluations (3 days and 6 weeks post inoculation) revealed that, independent of biological sex, inoculation with chimeric HIV (EcoHIV), but not saline, disrupted decision-making. Prominent structural alterations in the frontal-striatal circuit were evidenced by synaptodendritic alterations in pyramidal neurons in the medial prefrontal cortex. Thus, the EcoHIV rat affords a valid animal model to critically investigate how the frontal-striatal circuit responds to HIV-1 infection following chronic drug use.

S-EQUOL: a neuroprotective therapeutic for chronic neurocognitive impairments in pediatric HIV.

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.

Diagnostic and prognostic biomarkers for HAND.

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, sAPPß, amyloid ß) 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, sAPPß, amyloid ß, 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.

Evolution of the HIV-1 transgenic rat: utility in assessing the progression of HIV-1-associated neurocognitive disorders.

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.

Selective developmental alterations in The HIV-1 transgenic rat: Opportunities for diagnosis of pediatric HIV-1.

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.

Adolescent oral oxycodone self-administration disrupts neurobehavioral and neurocognitive development.

Nonmedical use of prescription opioids peaks during late adolescence, a developmental period associated with the maturation of higher-order cognitive processes. To date, however, how chronic adolescent oxycodone (OXY) self-administration alters neurobehavioral (i.e., locomotion, startle reactivity) and/or neurocognitive (i.e., preattentive processes, intrasession habituation, stimulus-reinforcement learning, sustained attention) function has not yet been systematically evaluated. Hence, the rationale was built for establishing the dose-dependency of adolescent OXY self-administration on the trajectory of neurobehavioral and neurocognitive development. From postnatal day (PD) 35 to PD 105, an age in rats that corresponds to the adolescent and young adult period in humans, male and female F344/N rats received access to either oral OXY (0, 2, 5, or 10 mg/kg) or water under a two-bottle choice experimental paradigm. Independent of biological sex or dose, rodents voluntarily escalated their OXY intake across ten weeks. A longitudinal experimental design revealed prominent OXY-induced impairments in neurobehavioral development, characterized by dose-dependent increases in locomotion and sex-dependent increases in startle reactivity. Systematic manipulation of the interstimulus interval in prepulse inhibition supports an OXY-induced impairment in preattentive processes. Despite the long-term cessation of OXY intake, rodents with a history of chronic adolescent oral OXY self-administration exhibited deficits in sustained attention; albeit no alterations in stimulus-reinforcement learning were observed. Taken together, adolescent oral OXY self-administration induces selective long-term alterations in neurobehavioral and neurocognitive development enjoining the implementation of safer prescribing guidelines for this population.

Microglia Proliferation Underlies Synaptic Dysfunction in the Prefrontal Cortex: Implications for the Pathogenesis of HIV-1-Associated Neurocognitive and Affective Alterations.

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 predominant cell type expressing HIV-1 mRNA in the dorsolateral prefrontal cortex of postmortem HIV-1 seropositive individuals with HAND was investigated. Utilization of a combined RNAscope multiplex fluorescent and immunostaining assay 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 innoculation, 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 post synaptic density protein 95 (PSD-95), markers of pre-synaptic and post-synaptic damage, respectively. Third, regression analyses were conducted to evaluate whether microglia proliferation mechanistically underlies neuronal damage in EcoHIV and control animals. Indeed, microglia proliferation accounts 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.

SARS-CoV-2 RNA Persists in the Central Nervous System of Non-Human Primates Despite Clinical Recovery.

Adverse neurological and psychiatric outcomes, collectively termed the post-acute sequelae of SARS-CoV-2 infection (PASC), persist in adults clinically recovered from COVID-19. Effective therapeutic interventions are fundamental to reducing the burden of PASC, necessitating an investigation of the pathophysiology underlying the debilitating neurological symptoms associated with the condition. Herein, eight non-human primates (Wild-Caught African Green Monkeys, n =4; Indian Rhesus Macaques, n =4) were inoculated with the SARS-CoV-2 isolate USA-WA1/2020 by either small particle aerosol or via multiple routes. At necropsy, tissue from the olfactory epithelium and pyriform cortex/amygdala of SARS-CoV-2 infected non-human primates were collected for ribonucleic acid in situ hybridization (i.e., RNAscope). First, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) mRNA are downregulated in the pyriform cortex/amygdala of non-human primates clinically recovered from SARS-CoV-2 inoculation relative to wildtype controls. Second, abundant SARS-CoV-2 mRNA was detected in clinically recovered non-human primates; mRNA which is predominantly harbored in pericytes. Collectively, examination of post-mortem pyriform cortex/amygdala brain tissue of non-human primates clinically recovered from SARS-CoV-2 infection revealed two early pathophysiological mechanisms potentially underlying PASC. Indeed, therapeutic interventions targeting the downregulation of ACE2, decreased expression of TMPRSS2, and/or persistent infection of pericytes in the central nervous system may effectively mitigate the debilitating symptoms of PASC.

HIV-1 mRNA Knockdown with CRISPR/Cas9 Enhances Neurocognitive Function.

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 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) of CRISPR/Cas9 for 72 hours. 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 mPFC. 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 partially 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.

Therapeutically targeting the consequences of HIV-1-associated gastrointestinal dysbiosis: Implications for neurocognitive and affective alterations.

Approximately 50 % of the individuals living with human immunodeficiency virus type 1 (HIV-1) are plagued by debilitating neurocognitive impairments (NCI) and/or affective alterations. Sizeable alterations in the composition of the gut microbiome, or gastrointestinal dysbiosis, may underlie, at least in part, the NCI, apathy, and/or depression observed in this population. Herein, two interrelated aims will be critically addressed, including: 1) the evidence for, and functional implications of, gastrointestinal microbiome dysbiosis in HIV-1 seropositive individuals; and 2) the potential for therapeutically targeting the consequences of this dysbiosis for the treatment of HIV-1-associated NCI and affective alterations. First, gastrointestinal microbiome dysbiosis in HIV-1 seropositive individuals is characterized by decreased alpha (α) diversity, a decreased relative abundance of bacterial species belonging to the Bacteroidetes phylum, and geographic-specific alterations in Bacillota (formerly Firmicutes) spp. Fundamentally, changes in the relative abundance of Bacteroidetes and Bacillota spp. may underlie, at least in part, the deficits in γ-aminobutyric acid and serotonin neurotransmission, as well as prominent synaptodendritic dysfunction, observed in this population. Second, there is compelling evidence for the therapeutic utility of targeting synaptodendritic dysfunction as a method to enhance neurocognitive function and improve motivational dysregulation in HIV-1. Further research is needed to determine whether the therapeutics enhancing synaptic efficacy exert their effects by altering the gut microbiome. Taken together, understanding gastrointestinal microbiome dysbiosis resulting from chronic HIV-1 viral protein exposure may afford insight into the mechanisms underlying HIV-1-associated neurocognitive and/or affective alterations; mechanisms which can be subsequently targeted via novel therapeutics.

Intraneuronal ß-Amyloid Accumulation: Aging HIV-1 Human and HIV-1 Transgenic Rat Brain.

The prevalence of HIV-1 associated neurocognitive disorders (HAND) is significantly greater in older, relative to younger, HIV-1 seropositive individuals; the neural pathogenesis of HAND in older HIV-1 seropositive individuals, however, remains elusive. To address this knowledge gap, abnormal protein aggregates (i.e., ß-amyloid) were investigated in the brains of aging (>12 months of age) HIV-1 transgenic (Tg) rats. In aging HIV-1 Tg rats, double immunohistochemistry staining revealed abnormal intraneuronal ß-amyloid accumulation in the prefrontal cortex (PFC) and hippocampus, relative to F344/N control rats. Notably, in HIV-1 Tg animals, increased ß-amyloid accumulation occurred in the absence of any genotypic changes in amyloid precursor protein (APP). Furthermore, no clear amyloid plaque deposition was observed in HIV-1 Tg animals. Critically, ß-amyloid was co-localized with neurons in the cortex and hippocampus, supporting a potential mechanism underlying synaptic dysfunction in the HIV-1 Tg rat. Consistent with these neuropathological findings, HIV-1 Tg rats exhibited prominent alterations in the progression of temporal processing relative to control animals; temporal processing relies, at least in part, on the integrity of the PFC and hippocampus. In addition, in post-mortem HIV-1 seropositive individuals with HAND, intraneuronal ß-amyloid accumulation was observed in the dorsolateral PFC and hippocampal dentate gyrus. Consistent with observations in the HIV-1 Tg rat, no amyloid plaques were found in these post-mortem HIV-1 seropositive individuals with HAND. Collectively, intraneuronal ß-amyloid aggregation observed in the PFC and hippocampus of HIV-1 Tg rats supports a potential factor underlying HIV-1 associated synaptodendritic damage. Further, the HIV-1 Tg rat provides a biological system to model HAND in older HIV-1 seropositive individuals.

Synaptic dysfunction is associated with alterations in the initiation of goal-directed behaviors: Implications for HIV-1-associated apathy.

Individuals living with human immunodeficiency virus type 1 (HIV-1) exhibit an increased prevalence of neuropsychiatric comorbities (e.g., apathy) relative to their seronegative counterparts. Given the profound functional consequences associated with apathy, characterizing the multidimensional neuropsychiatric syndrome, and associated neural mechanisms, following chronic HIV-1 viral protein exposure remains a critical need. HIV-1-associated apathy was examined by quantifying goal-directed behaviors, indexed using voluntary wheel running, during the diurnal and nocturnal cycle. Apathetic behaviors in the HIV-1 transgenic (Tg) rat were characterized by a profound decrease in the number of running bouts during both the diurnal and nocturnal cycle, supporting a prominent deficit in the self-initiation of spontaneous behaviors. Additionally, HIV-1 Tg animals exhibited a decreased reinforcing efficacy of voluntary wheel running during the nocturnal cycle. Following the completion of voluntary wheel running, synaptic dysfunction in medium spiny neurons (MSNs) of the nucleus accumbens core (NAcc) was examined as a potential neural mechanism underlying HIV-1-associated apathy. HIV-1 Tg animals displayed prominent synaptic dysfunction in MSNs of the NAcc, characterized by enhanced dendritic branching complexity and a population shift towards an immature dendritic spine phenotype relative to control animals. Synaptic dysfunction, which accounted for 42.0% to 68.5% of the variance in the number of running bouts, was strongly associated with the self-initiation of spontaneous behaviors. Establishment of the relationship between synaptic dysfunction and apathy affords a key target for the development of novel therapeutics and cure strategies for affective alterations associated with HIV-1.

Neurodevelopmental Processes in the Prefrontal Cortex Derailed by Chronic HIV-1 Viral Protein Exposure.

Due to the widespread access to, and implementation of, combination antiretroviral therapy, individuals perinatally infected with human immunodeficiency virus type 1 (HIV-1) are living into adolescence and adulthood. Perinatally infected adolescents living with HIV-1 (pALHIV) are plagued by progressive, chronic neurocognitive impairments; the pathophysiological mechanisms underlying these deficits, however, remain understudied. A longitudinal experimental design from postnatal day (PD) 30 to PD 180 was utilized to establish the development of pyramidal neurons, and associated dendritic spines, from layers II-III of the medial prefrontal cortex (mPFC) in HIV-1 transgenic (Tg) and control animals. Three putative neuroinflammatory markers (i.e., IL-1ß, IL-6, and TNF-α) were evaluated early in development (i.e., PD 30) as a potential mechanism underlying synaptic dysfunction in the mPFC. Constitutive expression of HIV-1 viral proteins induced prominent neurodevelopmental alterations and progressive synaptodendritic dysfunction, independent of biological sex, in pyramidal neurons from layers II-III of the mPFC. From a neurodevelopmental perspective, HIV-1 Tg rats exhibited prominent deficits in dendritic and synaptic pruning. With regards to progressive synaptodendritic dysfunction, HIV-1 Tg animals exhibited an age-related population shift towards dendritic spines with decreased volume, increased backbone length, and decreased head diameter; parameters associated with a more immature dendritic spine phenotype. There was no compelling evidence for neuroinflammation in the mPFC during early development. Collectively, progressive neuronal and dendritic spine dysmorphology herald synaptodendritic dysfunction as a key neural mechanism underlying chronic neurocognitive impairments in pALHIV.

A Rat Model of EcoHIV Brain Infection.

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Microglial HIV-1 Expression: Role in HIV-1 Associated Neurocognitive Disorders.

The persistence of HIV-1 viral reservoirs in the brain, despite treatment with combination antiretroviral therapy (cART), remains a critical roadblock for the development of a novel cure strategy for HIV-1. To enhance our understanding of viral reservoirs, two complementary studies were conducted to (1) evaluate the HIV-1 mRNA distribution pattern and major cell type expressing HIV-1 mRNA in the HIV-1 transgenic (Tg) rat, and (2) validate our findings by developing and critically testing a novel biological system to model active HIV-1 infection in the rat. First, a restricted, region-specific HIV-1 mRNA distribution pattern was observed in the HIV-1 Tg rat. Microglia were the predominant cell type expressing HIV-1 mRNA in the HIV-1 Tg rat. Second, we developed and critically tested a novel biological system to model key aspects of HIV-1 by infusing F344/N control rats with chimeric HIV (EcoHIV). In vitro, primary cultured microglia were treated with EcoHIV revealing prominent expression within 24 h of infection. In vivo, EcoHIV expression was observed seven days after stereotaxic injections. Following EcoHIV infection, microglia were the major cell type expressing HIV-1 mRNA, results that are consistent with observations in the HIV-1 Tg rat. Within eight weeks of infection, EcoHIV rats exhibited neurocognitive impairments and synaptic dysfunction, which may result from activation of the NogoA-NgR3/PirB-RhoA signaling pathway and/or neuroinflammation. Collectively, these studies enhance our understanding of HIV-1 viral reservoirs in the brain and offer a novel biological system to model HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse) in rats.

HIV-Associated Apathy/Depression and Neurocognitive Impairments Reflect Persistent Dopamine Deficits.

Individuals living with human immunodeficiency virus type 1 (HIV-1) are often plagued by debilitating neurocognitive impairments and affective alterations;the pathophysiology underlying these deficits likely includes dopaminergic system dysfunction. The present review utilized four interrelated aims to critically examine the evidence for dopaminergic alterations following HIV-1 viral protein exposure. First, basal dopamine (DA) values are dependent upon both brain region andexperimental approach (i.e., high-performance liquid chromatography, microdialysis or fast-scan cyclic voltammetry). Second, neurochemical measurements overwhelmingly support decreased DA concentrations following chronic HIV-1 viral protein exposure. Neurocognitive impairments, including alterations in pre-attentive processes and attention, as well as apathetic behaviors, provide an additional line of evidence for dopaminergic deficits in HIV-1. Third, to date, there is no compelling evidence that combination antiretroviral therapy (cART), the primary treatment regimen for HIV-1 seropositive individuals, has any direct pharmacological action on the dopaminergic system. Fourth, the infection of microglia by HIV-1 viral proteins may mechanistically underlie the dopamine deficit observed following chronic HIV-1 viral protein exposure. An inclusive and critical evaluation of the literature, therefore, supports the fundamental conclusion that long-term HIV-1 viral protein exposure leads to a decreased dopaminergic state, which continues to persist despite the advent of cART. Thus, effective treatment of HIV-1-associated apathy/depression and neurocognitive impairments must focus on strategies for rectifying decreases in dopamine function.

S-Equol mitigates motivational deficits and dysregulation associated with HIV-1.

Motivational deficits (e.g., apathy) and dysregulation (e.g., addiction) in HIV-1 seropositive individuals, despite treatment with combination antiretroviral therapy, necessitates the development of innovative adjunctive therapeutics. S-Equol (SE), a selective estrogen receptor ß agonist, has been implicated as a neuroprotective and/or neurorestorative therapeutic for HIV-1 associated neurocognitive disorders (HAND); its therapeutic utility for motivational alterations, however, has yet to be systematically evaluated. Thus, HIV-1 transgenic (Tg) and control animals were treated with either a daily oral dose of SE (0.2 mg) or vehicle and assessed in a series of tasks to evaluate goal-directed and drug-seeking behavior. First, at the genotypic level, motivational deficits in HIV-1 Tg rats treated with vehicle were characterized by a diminished reinforcing efficacy of, and sensitivity to, sucrose. Motivational dysregulation was evidenced by enhanced drug-seeking for cocaine relative to control animals treated with vehicle. Second, treatment with SE ameliorated both motivational deficits and dysregulation in HIV-1 Tg rats. Following a history of cocaine self-administration, HIV-1 Tg animals treated with vehicle exhibited lower levels of dendritic branching and a shift towards longer dendritic spines with decreased head diameter. Treatment with SE, however, led to long-term enhancements in dendritic spine morphology in HIV-1 Tg animals supporting a potential underlying basis by which SE exerts its therapeutic effects. Taken together, SE restored motivated behavior in the HIV-1 Tg rat, expanding the potential clinical utility of SE to include both neurocognitive and affective alterations.

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.