A Characterization of Neurology Consults for Inpatients with SARS-CoV-2 Infection Compared to Other Respiratory Viruses.

Introduction: Neurological consultation for patients infected with SARS-CoV-2 is common; it is currently unknown whether the neurologist's approach to inpatient consultation of patients with SARS-CoV-2 should differ from the paradigm used to evaluate hospitalized patients with similar respiratory viruses. The goal of the present study is to determine if the preponderance of new neurologic diagnoses differs between inpatients with SARS-CoV-2 and similar non-SARS-CoV-2 respiratory viruses for whom neurology is consulted. Methods: We performed a retrospective chart analysis of inpatient neurologic consultations at three major Philadelphia-based hospitals. We compared the final neurologic diagnosis of 152 patients infected with SARS-CoV-2 to 54 patients with a similar ubiquitous non-SARS-CoV-2 respiratory virus (influenza A, influenza B, respiratory syncytial virus, rhinovirus, or adenovirus, the most commonly tested respiratory viruses at our institution). Secondary metrics included age, sex, level of care, prior neurologic diagnoses, and mortality. A multinomial logistic regression model was utilized to evaluate the relative difference between diagnostic category groups on all metrics. Results: The proportion of patients with seizure who were infected with SARS-CoV-2 admitted to an intensive care unit (ICU) was significantly higher than those who were admitted to a medical-surgical floor. SARS-CoV-2 was also associated with increased risk for ICU admission compared to other common respiratory viruses. SARS-CoV-2 inpatients requiring neurologic consultation were also more likely to be older and female as compared to the non-SARS-CoV-2 cohort. In other domains, the proportion of neurologic diagnoses between SAR-CoV-2 and non-SARS-CoV-2 respiratory viruses showed no significant difference. Conclusion: Patients requiring inpatient neurologic consultation with a diagnosis of SARS-CoV-2 infection or another respiratory virus were found to be remarkably similar in terms of their ultimate neurologic diagnosis, with the exception of a larger preponderance of seizure in critical-care-level patients with SARS-CoV-2 infection. Our study suggests that the neurological approach to patients hospitalized with SARS-CoV-2 should be similar to that for patients with similar common respiratory infections, noting that seizure was seen more frequently in critically ill patients infected with SARS-CoV-2.

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.

Heme oxygenase-1 promoter (GT) n polymorphism associates with HIV neurocognitive impairment.

OBJECTIVE: To determine whether regulatory variations in the heme oxygenase-1 (HO-1) promoter (GT) n dinucleotide repeat length could identify unique population genetic risks for neurocognitive impairment (NCI) in persons living with HIV (PLWH), we genotyped 528 neurocognitively assessed PLWH of European American and African American descent and linked genotypes to cognitive status. METHODS: In this cross-sectional study of PLWH (the CNS HIV Antiretroviral Therapy Effect Research cohort), we determined HO-1 (GT) n repeat lengths in 276 African Americans and 252 European Americans. Using validated criteria for HIV-associated NCI (HIV NCI), we found associations between allele length genotypes and HIV NCI and between genotypes and plasma markers of monocyte activation and inflammation. For comparison of HO-1 (GT) n allele frequencies with another population of African ancestry, we determined HO-1 (GT) n allele lengths in African PLWH from Botswana (n = 428). RESULTS: PLWH with short HO-1 (GT) n alleles had a lower risk for HIV NCI (OR = 0.63, 95% CI: 0.42-0.94). People of African ancestry had a lower prevalence of short alleles and higher prevalence of long alleles compared with European Americans, and in subgroup analyses, the protective effect of the short allele was observed in African Americans and not in European Americans. CONCLUSIONS: Our study identified the short HO-1 (GT) n allele as partially protective against developing HIV NCI. It further suggests that this clinical protective effect is particularly relevant in persons of African ancestry, where the lower prevalence of short HO-1 (GT) n alleles may limit induction of HO-1 expression in response to inflammation and oxidative stress. Therapeutic strategies that enhance HO-1 expression may decrease HIV-associated neuroinflammation and limit HIV NCI.

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.

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.

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.

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).

Heme oxygenase-1 deficiency accompanies neuropathogenesis of HIV-associated neurocognitive disorders.

Heme oxygenase-1 (HO-1) is an inducible, detoxifying enzyme that is critical for limiting oxidative stress, inflammation, and cellular injury within the CNS and other tissues. Here, we demonstrate a deficiency of HO-1 expression in the brains of HIV-infected individuals. This HO-1 deficiency correlated with cognitive dysfunction, HIV replication in the CNS, and neuroimmune activation. In vitro analysis of HO-1 expression in HIV-infected macrophages, a primary CNS HIV reservoir along with microglia, demonstrated a decrease in HO-1 as HIV replication increased. HO-1 deficiency correlated with increased culture supernatant glutamate and neurotoxicity, suggesting a link among HIV infection, macrophage HO-1 deficiency, and neurodegeneration. HO-1 siRNA knockdown and HO enzymatic inhibition in HIV-infected macrophages increased supernatant glutamate and neurotoxicity. In contrast, increasing HO-1 expression through siRNA derepression or with nonselective pharmacologic inducers, including the CNS-penetrating drug dimethyl fumarate (DMF), decreased supernatant glutamate and neurotoxicity. Furthermore, IFN-γ, which is increased in CNS HIV infection, reduced HO-1 expression in cultured human astrocytes and macrophages. These findings indicate that HO-1 is a protective host factor against HIV-mediated neurodegeneration and suggest that HO-1 deficiency contributes to this degeneration. Furthermore, these results suggest that HO-1 induction in the CNS of HIV-infected patients on antiretroviral therapy could potentially protect against neurodegeneration and associated cognitive dysfunction.

Heme oxygenase-1 dysregulation in the brain: implications for HIV-associated neurocognitive disorders.

Heme oxygenase-1 (HO-1) is a highly inducible and ubiquitous cellular enzyme that subserves cytoprotective responses to toxic insults, including inflammation and oxidative stress. In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and multiple sclerosis, HO-1 expression is increased, presumably reflecting an endogenous neuroprotective response against ongoing cellular injury. In contrast, we have found that in human immunodeficiency virus (HIV) infection of the brain, which is also associated with inflammation, oxidative stress and neurodegeneration, HO-1 expression is decreased, likely reflecting a unique role for HO-1 deficiency in neurodegeneration pathways activated by HIV infection. We have also shown that HO-1 expression is significantly suppressed by HIV replication in cultured macrophages which represent the primary cellular reservoir for HIV in the brain. HO-1 deficiency is associated with release of neurotoxic levels of glutamate from both HIV-infected and immune-activated macrophages; this glutamate-mediated neurotoxicity is suppressed by pharmacological induction of HO-1 expression in the macrophages. Thus, HO-1 induction could be a therapeutic strategy for neuroprotection against HIV infection and other neuroinflammatory brain diseases. Here, we review various stimuli and signaling pathways regulating HO-1 expression in macrophages, which could promote neuronal survival through HO-1-modulation of endogenous antioxidant and immune modulatory pathways, thus limiting the oxidative stress that can promote HIV disease progression in the CNS. The use of pharmacological inducers of endogenous HO-1 expression as potential adjunctive neuroprotective therapeutics in HIV infection is also discussed.

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.

Adenosine triphosphate released from HIV-infected macrophages regulates glutamatergic tone and dendritic spine density on neurons.

Despite wide spread use of combination antiretroviral therapy (cART) in developed countries, approximately half of HIV-infected patients will develop impairments in cognitive function. Accumulating evidence suggests that neuronal dysfunction can be precipitated by HIV-infection of macrophages by mechanisms that involve alterations in innate and adaptive immune responses. HIV-infection of macrophages is known to increase the release of soluble neurotoxins. However, the composition of products released from infected macrophages is complex and not fully known. In this study we provide evidence that ATP and other immuno-/neuromodulatory nucleotides are exported from HIV-infected macrophages and modify neuronal structure. Supernatants collected from HIV-infected macrophages (HIV/MDM) contained large amounts of ATP, ADP, AMP and small amounts of adenosine, in addition to glutamate. Dilutions of these supernatants that were sub-threshold for glutamate receptor activation evoked rapid calcium flux in neurons that were completely inhibited by the enzymatic degradation of ATP, or by blockade of calcium permeable purinergic receptors. Applications of these highly diluted HIV/MDM onto neuronal cultures increased the amount of extracellular glutamate by mechanisms dependent on purinergic receptor activation, and downregulated spine density on neurons by mechanisms dependent on purinergic and glutamate receptor activation. We conclude from these data that ATP released from HIV-infected macrophages downregulates dendritic spine density on neurons by a mechanism that involves purinergic receptor mediated modulation of glutamatergic tone. These data suggest that neuronal function may be depressed in HIV infected individuals by mechanisms that involve macrophage release of ATP that triggers secondary effects on glutamate handling.

Dimethyl fumarate, an immune modulator and inducer of the antioxidant response, suppresses HIV replication and macrophage-mediated neurotoxicity: a novel candidate for HIV neuroprotection.

Despite antiretroviral therapy (ART), HIV infection promotes cognitive dysfunction and neurodegeneration through persistent inflammation and neurotoxin release from infected and/or activated macrophages/microglia. Furthermore, inflammation and immune activation within both the CNS and periphery correlate with disease progression and morbidity in ART-treated individuals. Accordingly, drugs targeting these pathological processes in the CNS and systemic compartments are needed for effective, adjunctive therapy. Using our in vitro model of HIV-mediated neurotoxicity, in which HIV-infected monocyte-derived macrophages release excitatory neurotoxins, we show that HIV infection dysregulates the macrophage antioxidant response and reduces levels of heme oxygenase-1 (HO-1). Furthermore, restoration of HO-1 expression in HIV-infected monocyte-derived macrophages reduces neurotoxin release without altering HIV replication. Given these novel observations, we have identified dimethyl fumarate (DMF), used to treat psoriasis and showing promising results in clinical trials for multiple sclerosis, as a potential neuroprotectant and HIV disease-modifying agent. DMF, an immune modulator and inducer of the antioxidant response, suppresses HIV replication and neurotoxin release. Two distinct mechanisms are proposed: inhibition of NF-κB nuclear translocation and signaling, which could contribute to the suppression of HIV replication, and induction of HO-1, which is associated with decreased neurotoxin release. Finally, we found that DMF attenuates CCL2-induced monocyte chemotaxis, suggesting that DMF could decrease recruitment of activated monocytes to the CNS in response to inflammatory mediators. We propose that dysregulation of the antioxidant response during HIV infection drives macrophage-mediated neurotoxicity and that DMF could serve as an adjunctive neuroprotectant and HIV disease modifier in ART-treated individuals.

NMDA receptor modulation by the neuropeptide apelin: implications for excitotoxic injury.

Excitotoxic neuronal damage via over-activation of the NMDA receptor has been implicated in many neurodegenerative diseases. In vitro modeling of excitotoxic injury has shown that activation of G-protein coupled receptors (GPCRs) counteracts such injury through modulation of neuronal pro-survival pathways and/or NMDA receptor signaling. We have previously demonstrated that the GPCR APJ and its endogenous neuropeptide ligand apelin can protect neurons against excitotoxicity, but the mechanism(s) of this neuroprotection remain incompletely understood. We hypothesized that apelin can promote neuronal survival by activating pro-survival signaling as well as inhibiting NMDA receptor-mediated excitotoxic signaling cascades. Our results demonstrate that (i) apelin activates pro-survival signaling via inositol trisphosphate (IP(3) ), protein kinase C (PKC), mitogen-activated protein kinase kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase-1/2 (ERK1/2) to protect against excitotoxicity, and (ii) apelin inhibits excitotoxic signaling by attenuating NMDA receptor and calpain activity, and by modulating NMDA receptor subunit NR2B phosphorylation at serine 1480. These studies delineate a novel apelinergic signaling pathway that concurrently promotes survival and limits NMDA receptor-mediated injury to protect neurons against excitotoxicity. Defining apelin-mediated neuroprotection advances our understanding of neuroprotective pathways and will potentially improve our ability to develop therapeutics for excitotoxicity-associated neurodegenerative disorders.

Soybean-derived Bowman-Birk inhibitor inhibits neurotoxicity of LPS-activated macrophages.

BACKGROUND: Lipopolysaccharide (LPS), the major component of the outer membrane of gram-negative bacteria, can activate immune cells including macrophages. Activation of macrophages in the central nervous system (CNS) contributes to neuronal injury. Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, has anti-inflammatory properties. In this study, we examined whether BBI has the ability to inhibit LPS-mediated macrophage activation, reducing the release of pro-inflammatory cytokines and subsequent neurotoxicity in primary cortical neural cultures. METHODS: Mixed cortical neural cultures from rat were used as target cells for testing neurotoxicity induced by LPS-treated macrophage supernatant. Neuronal survival was measured using a cell-based ELISA method for expression of the neuronal marker MAP-2. Intracellular reactive oxygen species (ROS) production in macrophages was measured via 2', 7'-dichlorofluorescin diacetate (DCFH2DA) oxidation. Cytokine expression was determined by quantitative real-time PCR. RESULTS: LPS treatment of macrophages induced expression of proinflammatory cytokines (IL-1ß, IL-6 and TNF-α) and of ROS. In contrast, BBI pretreatment (1-100 µg/ml) of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS. Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures. BBI, when directly added to the neuronal cultures (1-100 µg/ml), had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment. In addition, BBI (100 µg/ml) had no effect on N-methyl-D-aspartic acid (NMDA)-mediated neurotoxicity. CONCLUSIONS: These findings demonstrate that BBI, through its anti-inflammatory properties, protects neurons from neurotoxicity mediated by activated macrophages.

Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration.

Neurocognitive deficits seen in HIV-associated neurocognitive disorders (HANDs) are attributed to the release of soluble factors from CNS-resident, HIV-infected and/or activated macrophages and microglia. To study HIV-associated neurotoxicity, we used our in vitro model in which primary rat neuronal/glial cultures are treated with supernatants from cultured human monocyte-derived macrophages, infected with a CNS-isolated HIV-1 strain (HIV-MDM). We found that neuronal damage, detected as a loss of microtubule-associated protein-2 (MAP2), begins as early as 2h and is preceded by a loss of mitochondrial membrane potential (Δψ(m)). Interestingly, inhibitors of calpains, but not inhibitors of caspases, blocked MAP2 loss, however neither type of inhibitor prevented the loss of Δψ(m). To facilitate throughput for these studies, we refined a MAP2 cell-based-ELISA whose data closely compare with our standardized method of hand counting neurons. In addition, we developed a tetramethyl rhodamine methyl ester (TMRM)-based multi-well fluorescent plate assay for the evaluation of whole culture Δψ(m). Together, these findings indicate that calpain activation and loss of Δψ(m) may be parallel pathways to death in HIV-MDM-treated neurons and also demonstrate the validity of plate assays for assessing multiple experimental parameters as is useful for screening neurotherapeutics for neuronal damage and death.

HIV and SIV induce alterations in CNS CaMKII expression and activation: a potential mechanism for cognitive impairment.

The molecular mechanisms underlying learning and memory impairment in patients with HIV-associated neurological disease have remained unclear. Calcium/calmodulin-dependent kinase II (CaMKII) has key roles in synaptic potentiation and memory storage in neurons and also may have immunomodulatory functions. To determine whether HIV and simian immunodeficiency virus (SIV) induce alterations in CaMKII expression and/or activation (autophosphorylation) in the brain, we measured CaMKII alterations by quantitative immunoblotting in both an in vitro HIV/neuronal culture model and in vivo in an SIV-infected macaque model of HIV-associated neurological damage. Using primary rat hippocampal neuronal cultures treated with culture supernatants harvested from HIV-1-infected human monocyte-derived macrophages (HIV/MDM), we found that CaMKII activation declined after exposure of neurons to HIV/MDM. Consistent with our in vitro measurements, a significant decrease in CaMKII activation was present in both the hippocampus and frontal cortex of SIV-infected macaques compared with uninfected animals. In SIV-infected animals, total CaMKII expression in the hippocampus correlated well with levels of synaptophysin. Furthermore, CaMKII expression in both the hippocampus and frontal cortex was inversely correlated with viral load in the brain. These findings suggest that alterations in CaMKII may compromise synaptic function in the early phases of chronic neurodegenerative processes induced by HIV.

Propionibacterium acnes brain abscess appearing 10 years after neurosurgery.

OBJECTIVE: To describe a case of Propionibacterium acnes infection arising 10 years after neurosurgery and to review the literature regarding similar cases and their treatment. DESIGN: Case report. SETTING: Hospital of the University of Pennsylvania. PATIENT: A 70-year-old man with an intracerebral abscess and 2 biopsies culture positive for P acnes 10 years after subdural hematoma evacuation. Intervention Surgical biopsy followed by 6 weeks of intravenous vancomycin. MAIN OUTCOME MEASURES: Magnetic resonance imaging, neurologic examination, and microbiology culture results. RESULTS: Biopsies obtained from abscesses grew only P acnes. Magnetic resonance imaging and serial neurological examinations showed marked improvement after 6 weeks of intravenous vancomycin. CONCLUSIONS: Infection by P acnes can complicate neurosurgical procedures as late as 10 years after surgery and therefore should be considered in the evaluation of patients presenting with neurologic signs and symptoms with a history of neurosurgery.

Expression and regulation of antiviral protein APOBEC3G in human neuronal cells.

Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) has recently been identified as a potent antiviral protein. Here, we examined the expression and regulation of APOBEC3G in human brain tissues and the cells of central nervous system (CNS). Similar to the immune cells, human brain tissue and the CNS cells expressed APOBEC3G at both mRNA and protein levels. The expression of APOBEC3G could be up-regulated in human neuronal cells (NT2-N) and astrocytes (U87-MG) by interferons (IFN-alpha, beta and gamma), interleukin-1 (IL-1), and tumor necrosis factor. Other cytokines (IL-4, IL-6 and transforming growth factor beta1) and CC-chemokines (CCL3, 4 and 5), however, had little impact on the expression of APOBEC3G. In addition, pseudotyped HIV-1 infection and cytokine/chemokine-enriched supernatants from lipopolysaccharide-stimulated macrophage cultures induced APOBEC3G expression in NT2-N cells. APOBEC3G expressed in the neuronal cells and astrocytes was biologically functional, as the suppression of APOBEC3G expression by the specific siRNA led to increase of pseudotyped HIV-1 replication in these cells. These findings provide direct and compelling evidence that there is intracellular expression and regulation of functional APOBEC3G in the neuronal cells, which may be one of innate defense mechanisms involved in the neuronal protection in the CNS.