Transmitted/founder SHIV.D replicates in the brain, causes neuropathogenesis, and persists on combination antiretroviral therapy in rhesus macaques.

A biologically relevant non-human primate (NHP) model of HIV persistence in the central nervous system (CNS) is necessary. Most current NHP/SIV models of HIV infection fail to recapitulate viral persistence in the CNS without encephalitis or fail to employ viruses that authentically represent the ongoing HIV-1 pandemic. Here, we demonstrate viral replication in the brain and neuropathogenesis after combination antiretroviral therapy (ART) in rhesus macaques (RMs) using novel macrophage-tropic transmitted/founder (TF) simian-human immunodeficiency virus SHIV.D.191,859 (SHIV.D). Quantitative immunohistochemistry (IHC) and DNA/RNAscope in situ hybridization (ISH) were performed on three brain regions from six SHIV.D-infected RMs; two necropsied while viremic, two during analytical treatment interruptions, and two on suppressive ART. We demonstrated myeloid-mediated neuroinflammation, viral replication, and proviral DNA in the brain in all animals. These results demonstrate that TF SHIV.D models native HIV-1 CNS replication, pathogenesis, and persistence on ART in rhesus macaques.

Developments in Neuroprotection for HIV-Associated Neurocognitive Disorders (HAND).

PURPOSE OF REVIEW: Reducing the risk of HIV-associated neurocognitive disorders (HAND) is an elusive treatment goal for people living with HIV. Combination antiretroviral therapy (cART) has reduced the prevalence of HIV-associated dementia, but milder, disabling HAND is an unmet challenge. As newer cART regimens that more consistently suppress central nervous system (CNS) HIV replication are developed, the testing of adjunctive neuroprotective therapies must accelerate. RECENT FINDINGS: Successes in modifying cART regimens for CNS efficacy (penetrance, chemokine receptor targeting) and delivery (nanoformulations) in pilot studies suggest that improving cART neuroprotection and reducing HAND risk is achievable. Additionally, drugs currently used in neuroinflammatory, neuropsychiatric, and metabolic disorders show promise as adjuncts to cART, likely by broadly targeting neuroinflammation, oxidative stress, aerobic metabolism, and/or neurotransmitter metabolism. Adjunctive cognitive brain therapy and aerobic exercise may provide additional efficacy. Adjunctive neuroprotective therapies, including available FDA-approved drugs, cognitive therapy, and aerobic exercise combined with improved cART offer plausible strategies for optimizing the prevention and treatment of HAND.

Regional Brain Recovery from Acute Synaptic Injury in Simian Immunodeficiency Virus-Infected Rhesus Macaques Associates with Heme Oxygenase Isoform Expression.

Brain injury occurs within days in simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV) infection, and some recovery may occur within weeks. Inflammation and oxidative stress associate with such injury, but what drives recovery is unknown. Chronic HIV infection associates with reduced brain frontal cortex expression of the antioxidant/anti-inflammatory enzyme heme oxygenase-1 (HO-1) and increased neuroinflammation in individuals with cognitive impairment. We hypothesized that acute regional brain injury and recovery associate with differences in regional brain HO-1 expression. Using SIV-infected rhesus macaques, we analyzed multiple brain regions through acute and chronic infection (90 days postinfection [dpi]) and quantified viral (SIV gag RNA), synaptic (PSD-95; synaptophysin), axonal (neurofilament/neurofilament light chain [NFL]), inflammatory, and antioxidant (enzymes, including heme oxygenase isoforms [HO-1, HO-2]) markers. PSD-95 was reduced in the brainstem, basal ganglia, neocortex, and cerebellum within 13 dpi, indicating acute synaptic injury throughout the brain. All areas except the brainstem recovered. Unchanged NFL was consistent with no acute axonal injury. SIV RNA expression was highest in the brainstem throughout infection, and it associated with neuroinflammation. Surprisingly, during the synaptic injury and recovery phases, HO-2, and not HO-1, progressively decreased in the brainstem. Thus, acute SIV synaptic injury occurs throughout the brain, with spontaneous recovery in regions other than the brainstem. Within the brainstem, the high SIV load and inflammation, along with reduction of HO-2, may impair recovery. In other brain regions, stable HO-2 expression, with or without increasing HO-1, may promote recovery. Our data support roles for heme oxygenase isoforms in modulating recovery from synaptic injury in SIV infection and suggest their therapeutic targeting for promoting neuronal recovery.IMPORTANCE Brain injury induced by acute simian (or human) immunodeficiency virus infection may persist or spontaneously resolve in different brain regions. Identifying the host factor(s) that promotes spontaneous recovery from such injury may reveal targets for therapeutic drug strategies for promoting recovery from acute neuronal injury. The gradual recovery from such injury observed in many, but not all, brain regions in the rhesus macaque model is consistent with the possible existence of a therapeutic window of opportunity for intervening to promote recovery, even in those regions not showing spontaneous recovery. In persons living with human immunodeficiency virus infection, such neuroprotective treatments could ultimately be considered as adjuncts to the initiation of antiretroviral drug therapy.

Characteristics of Motor Dysfunction in Longstanding Human Immunodeficiency Virus.

BACKGROUND: Cognitive dysfunction in human immunodeficiency virus (HIV) has decreased, but milder forms of HIV-associated neurocognitive disorders (HAND) persist along with motor dysfunction. The HIV Motor Scale (HMS) is a validated tool that captures motor abnormalities on routine neurologic examination and which is associated with cognitive impairment in HIV. In this study, we applied a modified HMS (MHMS) to a nationwide cohort of people with longstanding HIV to characterize and understand the factors contributing to motor dysfunction. METHODS: The National NeuroAIDS Tissue Consortium is a nationwide longitudinal cohort study. Participants undergo regular assessments including neurological examination, neuropsychological testing, and immunovirologic data collection. Data from examinations were used to calculate the MHMS score, which was then correlated with history of AIDS-related central nervous system (CNS) disorders (ARCD; eg, prior CNS opportunistic infection), cerebrovascular disease (CVD), and HAND. RESULTS: Sixty-nine percent of participants showed an abnormality on the MHMS, with 27% classified as severe. Results did not vary based on demographic or immunologic variables. The most common abnormalities seen were gait (54%), followed by coordination (39%) and strength (25%), and these commonly co-occurred. CVD (P = .02), history of ARCD (P = .001), and HAND (P = .001) were all associated with higher (ie, worse) HMS in univariate analyses; CVD and ARCD persisted in multivariate analyses. CVD was also marginally associated with symptomatic HAND. CONCLUSIONS: Complex motor dysfunction remains common in HIV and is associated with CVD, ARCD, and to a lesser extent, HAND. Future studies are needed to understand the longitudinal trajectory of HIV-associated motor dysfunction, its neural substrates, and impact on quality of life.

Comparative Analysis of Gammaherpesvirus Circular RNA Repertoires: Conserved and Unique Viral Circular RNAs.

Recent studies have identified circular RNAs (circRNAs) expressed from the Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) human DNA tumor viruses. To gain initial insights into the potential relevance of EBV circRNAs in virus biology and disease, we assessed the circRNAome of the interspecies homologue rhesus macaque lymphocryptovirus (rLCV) in a naturally occurring lymphoma from a simian immunodeficiency virus (SIV)-infected rhesus macaque. This analysis revealed rLCV orthologues of the latency-associated EBV circular RNAs circRPMS1_E4_E3a and circEBNA_U. Also identified in two samples displaying unusually high lytic gene expression was a novel rLCV circRNA that contains both conserved and rLCV-specific RPMS1 exons and whose backsplice junctions flank an rLCV lytic origin of replication (OriLyt). Analysis of a lytic infection model for the murid herpesvirus 68 (MHV68) rhadinovirus identified a cluster of circRNAs near an MHV68 lytic origin of replication, with the most abundant of these, circM11_ORF69, spanning the OriLyt. Lastly, analysis of KSHV latency and reactivation models revealed the latency associated circRNA originating from the vIRF4 gene as the predominant viral circRNA. Together, the results of this study broaden our appreciation for circRNA repertoires in the Lymphocryptovirus and Rhadinovirus genera of gammaherpesviruses and provide evolutionary support for viral circRNA functions in latency and viral replication.IMPORTANCE Infection with oncogenic gammaherpesviruses leads to long-term viral persistence through a dynamic interplay between the virus and the host immune system. Critical for remodeling of the host cell environment after the immune responses are viral noncoding RNAs that modulate host signaling pathways without attracting adaptive immune recognition. Despite the importance of noncoding RNAs in persistent infection, the circRNA class of noncoding RNAs has only recently been identified in gammaherpesviruses. Accordingly, their roles in virus infection and associated oncogenesis are unknown. Here we report evolutionary conservation of EBV-encoded circRNAs determined by assessing the circRNAome in rLCV-infected lymphomas from an SIV-infected rhesus macaque, and we report latent and lytic circRNAs from KSHV and MHV68. These experiments demonstrate utilization of the circular RNA class of RNAs across 4 members of the gammaherpesvirus subfamily, and they identify orthologues and potential homoplastic circRNAs, implying conserved circRNA functions in virus biology and associated malignancies.

Neuroinflammation associates with antioxidant heme oxygenase-1 response throughout the brain in persons living with HIV.

Previous studies showed that persons living with HIV (PLWH) demonstrate higher brain prefrontal cortex neuroinflammation and immunoproteasome expression compared to HIV-negative individuals; these associate positively with HIV levels. Lower expression of the antioxidant enzyme heme oxygenase 1 (HO-1) was observed in PLWH with HIV-associated neurocognitive impairment (HIV-NCI) compared to neurocognitively normal PLWH. We hypothesized that similar expression patterns occur throughout cortical, subcortical, and brainstem regions in PLWH, and that neuroinflammation and immunoproteasome expression associate with lower expression of neuronal markers. We analyzed autopsied brains (15 regions) from 9 PLWH without HIV-NCI and 7 matched HIV-negative individuals. Using Western blot and RT-qPCR, we quantified synaptic, inflammatory, immunoproteasome, endothelial, and antioxidant biomarkers, including HO-1 and its isoform heme oxygenase 2 (HO-2). In these PLWH without HIV-NCI, we observed higher expression of neuroinflammatory, endothelial, and immunoproteasome markers in multiple cortical and subcortical regions compared to HIV-negative individuals, suggesting a global brain inflammatory response to HIV. Several regions, including posterior cingulate cortex, globus pallidus, and cerebellum, showed a distinct pattern of higher type I interferon (IFN)-stimulated gene and immunoproteasome expression. PLWH without HIV-NCI also had (i) stable or higher HO-1 expression and positive associations between (ii) HO-1 and HIV levels (CSF, plasma) and (iii) HO-1 expression and neuroinflammation, in multiple cortical, subcortical, and brainstem regions. We observed no differences in synaptic marker expression, suggesting little, if any, associated neuronal injury. We speculate that this may reflect a neuroprotective effect of a concurrent HO-1 antioxidant response despite global neuroinflammation, which will require further investigation.

BACE1 Mediates HIV-Associated and Excitotoxic Neuronal Damage Through an APP-Dependent Mechanism.

HIV-associated neurocognitive disorders (HANDs) share common symptoms with Alzheimer's disease (AD), which is characterized by amyloid-ß (Aß) plaques. Plaques are formed by aggregation of Aß oligomers, which may be the toxic species in AD pathogenesis, and oligomers are generated by cleavage of amyloid precursor protein (APP) by ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 inhibitors reverse neuronal loss and cognitive decline in animal models of AD. Although studies have also found evidence of altered APP processing in HIV+ patients, it is unknown whether increased BACE1 expression or Aß oligomer production is a common neuropathological feature of HAND. Moreover, it is unknown whether BACE1 or APP is involved in the excitotoxic, NMDAR-dependent component of HIV-associated neurotoxicity in vitro Herein, we hypothesize that HIV-associated neurotoxicity is mediated by NMDAR-dependent elevation of BACE1 and subsequent altered processing of APP. Supporting this, we observed elevated levels of BACE1 and Aß oligomers in CNS of male and female HIV+ patients. In a model of HIV-associated neurotoxicity in which rat neurons are treated with supernatants from HIV-infected human monocyte-derived macrophages, we observed NMDAR-dependent elevation of BACE1 protein. NMDA treatment also increased BACE1 and both pharmacological BACE1 inhibition and genetic loss of APP were partially neuroprotective. Moreover, in APP knock-out (APP-/-) mouse neurons, NMDA-induced toxicity was BACE1 independent, indicating that cytotoxicity of BACE1 is dependent upon APP cleavage. Our findings suggest that increased BACE1 and the resultant Aß oligomer production may contribute to HIV-associated neuropathogenesis and inhibition of BACE1 could have therapeutic potential in HANDs.SIGNIFICANCE STATEMENT HIV-associated neurocognitive disorders (HANDs) represent a range of cognitive impairments affecting ∼50% of HIV+ individuals. The specific causes of HAND are unknown, but evidence suggests that HIV-infected macrophage infiltration into the brain may cause neuronal damage. Herein, we show that neurons treated with conditioned media from HIV-infected macrophages have increased expression of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), a protein implicated in Alzheimer's disease pathogenesis. Moreover, inhibition of BACE1 prevented neuronal loss after conditioned media exposure, but had no effect on HIV-associated neurotoxicity in neurons lacking its cleavage target amyloid precursor protein. We also observed increased BACE1 expression in HIV+ patient brain tissue, confirming the potential relevance of BACE1 as a therapeutic target in HANDs.

Heme oxygenase-1 promoter region (GT)n polymorphism associates with increased neuroimmune activation and risk for encephalitis in HIV infection.

BACKGROUND: Heme oxygenase-1 (HO-1) is a critical cytoprotective enzyme that limits oxidative stress, inflammation, and cellular injury within the central nervous system (CNS) and other tissues. We previously demonstrated that HO-1 protein expression is decreased within the brains of HIV+ subjects and that this HO-1 reduction correlates with CNS immune activation and neurocognitive dysfunction. To define a potential CNS protective role for HO-1 against HIV, we analyzed a well-characterized HIV autopsy cohort for two common HO-1 promoter region polymorphisms that are implicated in regulating HO-1 promoter transcriptional activity, a (GT)n dinucleotide repeat polymorphism and a single nucleotide polymorphism (A(-413)T). Shorter HO-1 (GT)n repeats and the 'A' SNP allele associate with higher HO-1 promoter activity. METHODS: Brain dorsolateral prefrontal cortex tissue samples from an autopsy cohort of HIV-, HIV+, and HIV encephalitis (HIVE) subjects (n = 554) were analyzed as follows: HO-1 (GT)n polymorphism allele lengths were determined by PCR and capillary electrophoresis, A(-413)T SNP alleles were determined by PCR with allele specific probes, and RNA expression of selected neuroimmune markers was analyzed by quantitative PCR. RESULTS: HIV+ subjects with shorter HO-1 (GT)n alleles had a significantly lower risk of HIVE; however, shorter HO-1 (GT)n alleles did not correlate with CNS or peripheral viral loads. In HIV+ subjects without HIVE, shorter HO-1 (GT)n alleles associated significantly with lower expression of brain type I interferon response markers (MX1, ISG15, and IRF1) and T-lymphocyte activation markers (CD38 and GZMB). No significant correlations were found between the HO-1 (GT)n repeat length and brain expression of macrophage markers (CD163, CD68), endothelial markers (PECAM1, VWF), the T-lymphocyte marker CD8A, or the B-lymphocyte maker CD19. Finally, we found no significant associations between the A(-413)T SNP and HIVE diagnosis, HIV viral loads, or any neuroimmune markers. CONCLUSION: Our data suggest that an individual's HO-1 promoter region (GT)n polymorphism allele repeat length exerts unique modifying risk effects on HIV-induced CNS neuroinflammation and associated neuropathogenesis. Shorter HO-1 (GT)n alleles increase HO-1 promoter activity, which could provide neuroprotection through decreased neuroimmune activation. Therapeutic strategies that induce HO-1 expression could decrease HIV-associated CNS neuroinflammation and decrease the risk for development of HIV neurological disease.

HIV Protease Inhibitors Alter Amyloid Precursor Protein Processing via ß-Site Amyloid Precursor Protein Cleaving Enzyme-1 Translational Up-Regulation.

Mounting evidence implicates antiretroviral (ARV) drugs as potential contributors to the persistence and evolution of clinical and pathological presentation of HIV-associated neurocognitive disorders in the post-ARV era. Based on their ability to induce endoplasmic reticulum (ER) stress in various cell types, we hypothesized that ARV-mediated ER stress in the central nervous system resulted in chronic dysregulation of the unfolded protein response and altered amyloid precursor protein (APP) processing. We used in vitro and in vivo models to show that HIV protease inhibitor (PI) class ARVs induced neuronal damage and ER stress, leading to PKR-like ER kinase-dependent phosphorylation of the eukaryotic translation initiation factor 2α and enhanced translation of ß-site APP cleaving enzyme-1 (BACE1). In addition, PIs induced ß-amyloid production, indicative of increased BACE1-mediated APP processing, in rodent neuroglial cultures and human APP-expressing Chinese hamster ovary cells. Inhibition of BACE1 activity protected against neuronal damage. Finally, ARVs administered to mice and SIV-infected macaques resulted in neuronal damage and BACE1 up-regulation in the central nervous system. These findings implicate a subset of PIs as potential mediators of neurodegeneration in HIV-associated neurocognitive disorders.

When do models of NeuroAIDS faithfully imitate "the real thing"?

HIV-infected patients treated with antiretroviral medicines (ART) still face neurological challenges. HIV-associated neurocognitive disturbances (HAND) can occur, and latent viral DNA persisting in the central nervous system (CNS) prevents eradication of HIV. This communication focuses on how to develop experimental models of HAND and CNS HIV latency that best imitate the CNS pathophysiology in diseased humans, which we take to be "the real thing." Models of HIV encephalitis (HIVE) with active CNS viral replication were developed in the early years of the AIDS pandemic. The clinical relevancy of such models is in sharp decline because HIVE seldom occurs in virally suppressed patients, while HAND remains common. The search for improved models of HAND should incorporate the neurochemical, neuroimmunological and neuropathological features of virally suppressed patients. Common anomalies in these patients as established in autopsy brain specimens include brain endothelial cell activation and neurochemical imbalances of synaptic transmission; classical neurodegeneration may not be as crucial. With regard to latent HIV with viral suppression, human brain specimens show that the pool of latent proviral HIV DNA in the CNS is relatively small relative to the total body pool and does not change substantially over years. The CNS pool of latent virus probably differs from lymphoid tissues, because the mononuclear phagocyte system sustains productive infection (versus lymphocytes). These and yet-to-be discovered aspects of the human CNS of virally suppressed patients need to be better defined and addressed in experimental models. To maintain clinical relevancy, models of HAND and viral latency should faithfully emulate "the real thing."

Degradation of heme oxygenase-1 by the immunoproteasome in astrocytes: A potential interferon-γ-dependent mechanism contributing to HIV neuropathogenesis.

Induction of the detoxifying enzyme heme oxygenase-1 (HO-1) is a critical protective host response to cellular injury associated with inflammation and oxidative stress. We previously found that HO-1 protein expression is reduced in brains of HIV-infected individuals with HIV-associated neurocognitive disorders (HAND) and in HIV-infected macrophages, where this reduction associates with enhanced glutamate release and neurotoxicity. Because HIV-infected macrophages are a small component of the cellular content of the brain, the reduction of macrophage HO-1 expression likely accounts for a small portion of brain HO-1 loss in HIV infection. We therefore investigated the contribution of astrocytes, the major pool of brain HO-1. We identified immunoproteasome-mediated HO-1 degradation in astrocytes as a second possible mechanism of brain HO-1 loss in HIV infection. We demonstrate that prolonged exposure of human fetal astrocytes to interferon-gamma (IFNγ), an HIV-associated CNS immune activator, selectively reduces expression of HO-1 protein without a concomitant reduction in HO-1 RNA, increases expression of immunoproteasome subunits, and decreases expression of constitutive proteasome subunits, consistent with a shift towards increased immunoproteasome activity. In HIV-infected brain HO-1 protein reduction also associates with increased HO-1 RNA expression and increased immunoproteasome expression. Finally, we show that IFNγ treatment of astrocytic cells reduces HO-1 protein half-life in a proteasome-dependent manner. Our data thus suggest unique causal links among HIV infection, IFNγ-mediated immunoproteasome induction, and enhanced HO-1 degradation, which likely contribute to neurocognitive impairment in HAND. Such IFNγ-mediated HO-1 degradation should be further investigated for a role in neurodegeneration in inflammatory brain conditions. BRIEF SUMMARY: Kovacsics et al. identify immunoproteasome degradation of heme oxygenase-1 (HO-1) in interferon gamma-stimulated astrocytes as a plausible mechanism for the observed loss of HO-1 protein expression in the brains of HIV-infected individuals, which likely contributes to the neurocognitive impairment in HIV-associated neurocognitive disorders.

Induction of Heme Oxygenase-1 Deficiency and Associated Glutamate-Mediated Neurotoxicity Is a Highly Conserved HIV Phenotype of Chronic Macrophage Infection That Is Resistant to Antiretroviral Therapy.

UNLABELLED: Expression of the cytoprotective enzyme heme oxygenase-1 (HO-1) is significantly reduced in the brain prefrontal cortex of HIV-positive individuals with HIV-associated neurocognitive disorders (HAND). Furthermore, this HO-1 deficiency correlates with brain viral load, markers of macrophage activation, and type I interferon responses. In vitro, HIV replication in monocyte-derived macrophages (MDM) selectively reduces HO-1 protein and RNA expression and induces production of neurotoxic levels of glutamate; correction of this HO-1 deficiency reduces neurotoxic glutamate production without an effect on HIV replication. We now demonstrate that macrophage HO-1 deficiency, and the associated neurotoxin production, is a conserved feature of infection with macrophage-tropic HIV-1 strains that correlates closely with the extent of replication, and this feature extends to HIV-2 infection. We further demonstrate that this HO-1 deficiency does not depend specifically upon the HIV-1 accessory genes nef, vpr, or vpu but rather on HIV replication, even when markedly limited. Finally, antiretroviral therapy (ART) applied to MDM after HIV infection is established does not prevent HO-1 loss or the associated neurotoxin production. This work defines a predictable relationship between HIV replication, HO-1 loss, and neurotoxin production in MDM that likely reflects processes in place in the HIV-infected brains of individuals receiving ART. It further suggests that correcting this HO-1 deficiency in HIV-infected MDM could provide neuroprotection above that provided by current ART or proposed antiviral therapies directed at limiting Nef, Vpr, or Vpu functions. The ability of HIV-2 to reduce HO-1 expression suggests that this is a conserved phenotype among macrophage-tropic human immunodeficiency viruses that could contribute to neuropathogenesis. IMPORTANCE: The continued prevalence of HIV-associated neurocognitive disorders (HAND) underscores the need for adjunctive therapy that targets the neuropathological processes that persist in antiretroviral therapy (ART)-treated HIV-infected individuals. To this end, we previously identified one such possible process, a deficiency of the antioxidative and anti-inflammatory enzyme heme oxygenase-1 (HO-1) in the brains of individuals with HAND. In the present study, our findings suggest that the HO-1 deficiency associated with excess glutamate production and neurotoxicity in HIV-infected macrophages is a highly conserved phenotype of macrophage-tropic HIV strains and that this phenotype can persist in the macrophage compartment in the presence of ART. This suggests a plausible mechanism by which HIV infection of brain macrophages in ART-treated individuals could exacerbate oxidative stress and glutamate-induced neuronal injury, each of which is associated with neurocognitive dysfunction in infected individuals. Thus, therapies that rescue the HO-1 deficiency in HIV-infected individuals could provide additional neuroprotection to ART.

The complement system, neuronal injury, and cognitive function in horizontally-acquired HIV-infected youth.

The complement system (C1q/C3) is a key mediator of synaptic pruning during normal development. HIV inappropriately induces C1q and C3 production in the brain, and reduces neuronal complement inhibition. HIV may thus alter neural connectivity in the developing brain by excessively targeting synapses for elimination. The resultant pattern of neuronal injury may fundamentally alter neurodevelopmental and cognitive processes differentially across ages. This study aimed to (1) measure the association between the cerebrospinal fluid (CSF) complement factors (C1q/C3) and a marker of neuronal injury (NFL) in HIV+ subjects; (2) quantify the differences in CSF C1q/C3 between HIV+ youth and older adults; and (3) define the relationship between CSF C1q/C3 and cognitive impairment in each age group. We performed a retrospective cross-sectional study of 20 HIV+ 18-24-year-old youth and 20 HIV+ 40-46-year-old adults with varying levels of cognitive impairment enrolled in the CNS Antiretroviral Therapy Effects Research study. We quantified C3, C1q, and NFL by ELISA in paired CSF/plasma specimens. We found that CSF C1q correlates with NFL in all subjects not receiving antiretroviral therapy (n = 16, rho = 0.53, p = 0.035) when extreme NFL outliers were eliminated (n = 1). There was no difference in plasma/CSF C1q or C3 between older adults and youth. In 18-24-year-old youth, a nearly significant (p = 0.052) elevation of CSF C1q expression was observed in cognitively impaired subjects compared to cognitively normal subjects. Further investigation into the role of the CNS complement system in the neuropathogenesis of HIV is warranted and should be considered in a developmentally specific context.

E2F1 in neurons is cleaved by calpain in an NMDA receptor-dependent manner in a model of HIV-induced neurotoxicity.

The transcription factor E2F1 activates gene targets required for G1 -S phase progression and for apoptosis, and exhibits increased expression levels in neurons in several CNS diseases including HIV encephalitis, Alzheimer disease, and Parkinson's Disease. While E2F1 is known to regulate cell viability through activation of caspases, here we present evidence supporting the involvement of E2F1 in N-methyl-d-aspartate (NMDA) receptor-dependent, HIV-induced neuronal death mediated by calpains. Using an in vitro model of HIV-induced neurotoxicity that is dependent on NMDA receptor and calpain activation, we have shown that cortical neurons lacking functional E2F1 are less susceptible to neuronal death. In addition, we report that neuronal E2F1 is cleaved by calpain to a stable 55-kiloDalton fragment following NR2B-dependent NMDA receptor stimulation. This cleavage of E2F1 is protein conformation-dependent and involves at least two cleavage events, one at each terminus of the protein. Intriguingly, the stabilized E2F1 cleavage product is produced in post-mitotic neurons of all ages, but fails to be stabilized in cycling cells. Finally, we show that a matching E2F1 cleavage product is produced in human fetal neurons, suggesting that calpain cleavage of E2F1 may be produced in human cortical tissue. These results suggest neuronal E2F1 is processed in a novel manner in response to NMDA receptor-mediated toxicity, a mechanism implicated in HIV-associated neurocognitive disorders pathogenesis as well as several other diseases of the CNS. After crossing the blood-brain barrier, HIV-infected monocytes differentiate into macrophages and release excitotoxins and inflammatory factors including glutamate into the brain parenchyma (1). These factors stimulate neuronal N-Methyl-d-aspartate (NMDA) receptors (2), causing calcium influx (3) and subsequent activation of the cysteine protease calpain (4). Activated calpain cleaves multiple substrates including E2F1, producing a stabilized protein fragment with truncations at the N- and C-terminus (5). Calpain-cleaved E2F1 may contribute to calpain-mediated neuronal damage observed in NMDA receptor-mediated neurotoxicity (6).

Central and peripheral markers of neurodegeneration and monocyte activation in HIV-associated neurocognitive disorders.

HIV-associated neurocognitive disorders (HAND) affect up to 50 % of HIV-infected adults, independently predict HIV morbidity/mortality, and are associated with neuronal damage and monocyte activation. Cerebrospinal fluid (CSF) neurofilament subunits (NFL, pNFH) are sensitive surrogate markers of neuronal damage in several neurodegenerative diseases. In HIV, CSF NFL is elevated in individuals with and without cognitive impairment, suggesting early/persistent neuronal injury during HIV infection. Although individuals with severe cognitive impairment (HIV-associated dementia (HAD)) express higher CSF NFL levels than cognitively normal HIV-infected individuals, the relationships between severity of cognitive impairment, monocyte activation, neurofilament expression, and systemic infection are unclear. We performed a retrospective cross-sectional study of 48 HIV-infected adults with varying levels of cognitive impairment, not receiving antiretroviral therapy (ART), enrolled in the CNS Anti-Retroviral Therapy Effects Research (CHARTER) study. We quantified NFL, pNFH, and monocyte activation markers (sCD14/sCD163) in paired CSF/plasma samples. By examining subjects off ART, these correlations are not confounded by possible effects of ART on inflammation and neurodegeneration. We found that CSF NFL levels were elevated in individuals with HAD compared to cognitively normal or mildly impaired individuals with CD4+ T-lymphocyte nadirs ≤200. In addition, CSF NFL levels were significantly positively correlated to plasma HIV-1 RNA viral load and negatively correlated to plasma CD4+ T-lymphocyte count, suggesting a link between neuronal injury and systemic HIV infection. Finally, CSF NFL was significantly positively correlated with CSF pNFH, sCD163, and sCD14, demonstrating that monocyte activation within the CNS compartment is directly associated with neuronal injury at all stages of HAND.

Antiretroviral drugs induce oxidative stress and neuronal damage in the central nervous system.

HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery. Of note, potential toxicity of antiretroviral drugs in the central nervous system (CNS) remains remarkably underexplored and may contribute to the persistence of HAND in the cART era. Previous studies have shown antiretrovirals (ARVs) to be neurotoxic in the peripheral nervous system in vivo and in peripheral neurons in vitro. Alterations in lipid and protein metabolism, mitochondrial damage, and oxidative stress all play a role in peripheral ARV neurotoxicity. We hypothesized that ARVs also induce cellular stresses in the CNS, ultimately leading to neuronal damage and contributing to the changing clinical and pathological picture seen in HIV-positive patients in the cART era. In this report, we show that ARVs are neurotoxic in the CNS in both pigtail macaques and rats in vivo. Furthermore, in vitro, ARVs lead to accumulation of reactive oxygen species (ROS), and ultimately induction of neuronal damage and death. Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death. These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.

Chronic inflammation and the role for cofactors (hepatitis C, drug abuse, antiretroviral drug toxicity, aging) in HAND persistence.

HIV-associated neurocognitive disorders (HAND) is a group of syndromes of varying degrees of cognitive impairment affecting up to 50 % of HIV-infected individuals. The neuropathogenesis of HAND is thought to be driven by HIV invasion and productive replication within brain perivascular macrophages and endogenous microglia, and to some degree by restricted infection of astrocytes. The persistence of HAND in individuals experiencing suppression of systemic HIV viral load with antiretroviral therapy (ART) is incompletely explained, and suggested factors include chronic inflammation, persistent HIV replication in brain macrophages, effects of aging on brain vulnerability, and co-morbid conditions including hepatitis C (HCV) co-infection, substance abuse, and CNS toxicity of ART, among other factors. This review discusses several of these conditions: chronic inflammation, co-infection with HCV, drugs of abuse, aging, and antiretroviral drug effects. Effectively managing these co-morbid conditions in individuals with and without HAND is critical for improving neurocognitive outcomes and decreasing HIV-associated morbidity.

Dimethyl fumarate modulation of immune and antioxidant responses: application to HIV therapy.

The persistence of chronic immune activation and oxidative stress in human immunodeficiency virus (HIV)-infected, antiretroviral drug-treated individuals are major obstacles to fully preventing HIV disease progression. The immune modulator and antioxidant dimethyl fumarate (DMF) is effective in treating immune-mediated diseases and it also has potential applications to limiting HIV disease progression. Among the relevant effects of DMF and its active metabolite monomethyl fumarate (MMF) are induction of a Th1 to Th2 lymphocyte shift, inhibition of pro-inflammatory cytokine signaling, inhibition of NF-κB nuclear translocation, inhibition of dendritic cell maturation, suppression of lymphocyte and endothelial cell adhesion molecule expression, and induction of the Nrf2-dependent antioxidant response element (ARE) and effector genes. Associated with these effects are reduced lymphocyte and monocyte infiltration into psoriatic skin lesions in humans and immune-mediated demyelinating brain lesions in rodents, which confirms potent systemic and central nervous system (CNS) effects. In addition, DMF and MMF limit HIV infection in macrophages in vitro, albeit by unknown mechanisms. Finally, DMF and MMF also suppress neurotoxin production from HIV-infected macrophages, which drives CNS neurodegeneration. Thus, DMF might protect against systemic and CNS complications in HIV infection through its effective suppression of immune activation, oxidative stress, HIV replication, and macrophage-associated neuronal injury.

Calpain-mediated degradation of MDMx/MDM4 contributes to HIV-induced neuronal damage.

Neuronal damage in HIV-associated Neurocognitive Disorders (HAND) has been linked to inflammation induced by soluble factors released by HIV-infected, and non-infected, activated macrophages/microglia (HIV M/M) in the brain. It has been suggested that aberrant neuronal cell cycle activation determines cell fate in response to these toxic factors. We have previously shown increased expression of cell cycle proteins such as E2F1 and phosphorylated pRb in HAND midfrontal cortex in vivo and in primary neurons exposed to HIV M/M supernatants in vitro. In addition, we have previously shown that MDMx (also referred to as MDM4), a negative regulator of E2F1, was decreased in the brain in a primate model of HIV-induced CNS neurodegeneration. Thus, we hypothesized that MDMx provides indirect neuroprotection from HIV-induced neurodegeneration in our in vitro model. In this report, we found significant reductions in MDMx protein levels in the mid-frontal cortex of patients with HAND. In addition, treatment of primary rat neuroglial cultures with HIV M/M led to NMDA receptor- and calpain-dependent degradation of MDMx and decreased neuronal survival, while overexpression of MDMx conferred partial protection from HIV M/M toxicity in vitro. Further, our results demonstrate that MDMx is a novel and direct calpain substrate. Finally, blocking MDMx activity led to neuronal death in vitro in the absence of toxic stimulus, which was reversed by calpain inhibition. Overall, our results indicate that MDMx plays a pro-survival role in neurons, and that strategies to stabilize and/or induce MDMx can provide neuroprotection in HAND and in other neurodegenerative diseases where calpain activation contributes to neuropathogenesis.