HIV Infection Induces Extracellular Cathepsin B Uptake and Damage to Neurons.

HIV-associated neurocognitive disorders prevail in 20-50 percent of infected individuals. Macrophages transmigrate through the blood brain barrier during HIV-1 infection, triggering neuronal dysfunction. HIV-infected macrophages secrete cathepsin B (CATB), and serum amyloid p component (SAPC), inducing neuronal apoptosis by an unknown mechanism. We hypothesized that HIV infection facilitates CATB/SAPC secretion from macrophages followed by neuronal internalization, promoting dysfunction. SK-N-SH neuronal cells were exposed to active recombinant histidine-tagged cathepsin B (His-CATB). His-CATB entry was tracked by intracellular flow cytometry, and neuronal dysfunction was verified by western blot. Macrophage-derived extracellular vesicles (EVs) were tested for the presence of CATB and SAPC. Neurons internalized His-CATB, an effect that was partially decreased by pre-treatment with anti-CATB antibody. Pre-treatment with CATB and SAPC antibodies decreased cleavage of caspase-3 and restored synaptophysin in neurons. Neurons exposed to macrophage-conditioned media differentially internalized His-CATB, dependent on the HIV replication levels. Finally, CATB and SAPC were secreted in EVs. We report for the first time that CATB is secreted from macrophages both free and in EVs, and is internalized by neurons. Moreover, HIV-replication levels modulate the amount of CATB neuronal uptake, and neuronal dysfunction can be decreased with CATB antibodies. In conclusion, the CATB/SAPC complex represents a novel target against HIV-associated neurocognitive disorders.

Microwave & magnetic proteomics of macrophages from patients with HIV-associated cognitive impairment.

OBJECTIVE: HIV-infected monocytes can infiltrate the blood brain barrier as differentiated macrophages to the central nervous system, becoming the primary source of viral and cellular neurotoxins. The final outcome is HIV-associated cognitive impairment (HACI), which remain prevalent today, possibly due to the longer life-span of the patients treated with combined anti-retroviral therapy. Our main goal was to characterize the proteome of monocyte-derived macrophages (MDM) from HACI patients, and its association with their cognitive status, to find novel targets for therapy. METHODS: MDM were isolated from the peripheral blood of 14 HIV-seropositive women characterized for neurocognitive function, including: four normal cognition (NC), five asymptomatic (A), and five with cognitive impaired (CI). Proteins from macrophage lysates were isobaric-labeled with the microwave and magnetic (M2) sample preparation method followed by liquid chromatography-tandem mass spectrometry-based protein identification and quantification. Differences in protein abundance across groups classified by HACI status were determined using analysis of variance. RESULTS: A total of 2,519 proteins were identified with 2 or more peptides and 28 proteins were quantified as differentially expressed. Statistical analysis revealed increased abundance of 17 proteins in patients with HACI (p<0.05), including several enzymes associated to the glucose metabolism. Western blot confirmed increased expression of 6-Phosphogluconate dehydrogenase and L-Plastin in A and CI patients over NC and HIV seronegatives. CONCLUSIONS: This is the first quantitative proteomics study exploring the changes in protein abundance of macrophages isolated from patients with HACI. Further studies are warranted to determine if these proteins may be target candidates for therapy development against HACI.

Different levels of HIV DNA copy numbers in cerebrospinal fluid cellular subsets.

Inequities in the incidence of HIV infection and AIDS with continued persistence of HIV-associated neurocognitive disorders (HAND) exist in populations in Hawaii (HI) and Puerto Rico (PR). We previously reported that peripheral monocyte HIV DNA levels are high in patients in Hawaii with HAND and we now hypothesize that similar findings would be observed in the cerebrospinal fluid (CSF) cellular subsets. Cerebrospinal fluid cells were obtained from patients from PR and HI undergoing neurocognitive testing and sorted into monocytes (CD14+) and lymphocytes (CD14-) and HIV DNA was measured. From six PR subjects (three HAND, three normal cognition, NC) and six HI subjects (three HAND, three NC), HIV DNA burden in CD14+ cells was higher in HAND than NC patients; NC patients had higher HIV DNA burden in CD14-cells versus HAND. Differences in HIV DNA burden in particular CSF cellular subsets suggest that HIV DNA burden may play a role in HAND neuropathogenesis.

Antioxidant enzyme dysfunction in monocytes and CSF of Hispanic women with HIV-associated cognitive impairment.

HIV-associated cognitive neurological disorders (HAND) prevail in the antiretroviral therapy era. Proteomics analysis of CSF revealed expression of Cu/Zn superoxide dismutase (Cu/Zn SOD) in Hispanic women with cognitive impairment (CI). We tested the hypothesis that there is reduced capacity of antioxidant enzymes in CI by measures of expression and activity of Cu/Zn SOD, catalase, and Se-glutathione peroxidase in HAND. Our results showed that the function of these antioxidants was decreased in the CSF and monocytes of women with CI. These findings have important implications regarding their possible contribution to oxidative stress and in the diagnosis and therapy for HAND.

Characterization of peripheral blood human immunodeficiency virus isolates from Hispanic women with cognitive impairment.

Human immunodeficiency virus type 1 (HIV-1) tropism plays an important role in HIV-associated dementia. In this study, aimed at determining if the tropism and coreceptor usage of circulating viruses correlates with cognitive function, the authors isolated and characterized HIV from the peripheral blood of 21 Hispanic women using antiretroviral therapy. Macrophage tropism was determined by inoculation of HIV isolates onto monocyte-derived macrophages and lymphocyte cultures. To define coreceptor usage, the HIV isolates were inoculated onto the U87.CD4 glioma cell lines with specific CCR5 and CXCR4 coreceptors. HIV isolates from cognitively impaired patients showed higher levels of replication in mitogen-stimulated peripheral blood mononuclear cells than did isolates from patients with normal cognition (P < .05). The viral growth of HIV primary isolates in macrophages and lymphocytes did not differ between patients with and those without cognitive impairment. However, isolates from the cognitively impaired women preferentially used the X4 coreceptor (P < .05). These phenotypic studies suggest that cognitively impaired HIV-infected women receiving treatment may have a more highly replicating and more pathogenic X4 virus in the circulation that could contribute to their neuropathogenesis.