James L. Buescher

Overexpression of Monocyte Chemotactic Protein-1/CCL2 in β-Amyloid Precursor Protein Transgenic Mice Show Accelerated Diffuse β-Amyloid Deposition

Microglia accumulation at the site of amyloid plaques is a strong indication that microglia play a major role in Alzheimers disease pathogenesis. However, how microglia affect amyloid-β peptide (Aβ) deposition remains poorly understood. To address this question, we developed a novel bigenic mouse that overexpresses both amyloid precursor protein (APP) and monocyte chemotactic protein-1 (MCP-1; CCL2 in systematic nomenclature). CCL2 expression, driven by the glial fibrillary acidic protein promoter, induced mononuclear phagocyte (MP; monocyte-derived macrophage and microglial) accumulation in the brain. When APP/CCL2 transgenic mice were compared to APP mice, a fivefold increase in Aβ deposition was present despite increased MP accumulation around hippocampal and cortical amyloid plaques. Levels of full-length APP, its C-terminal fragment, and Aβ-degrading enzymes (insulin-degrading enzyme and neprilysin) in APP/CCL2 and APP mice were indistinguishable. Sodium dodecyl sulfate-insoluble Aβ (an indicator of fibrillar Aβ) was increased in APP/CCL2 mice at 5 months of age. Apolipoprotein E, which enhances Aβ deposition, was also increased (2.2-fold) in aged APP/CCL2 as compared to APP mice. We propose that although CCL2 stimulates MP accumulation, it increases Aβ deposition by reducing Aβ clearance through increased apolipoprotein E expression. Understanding the mechanisms underlying these events could be used to modulate microglial function in Alzheimers disease and positively affect disease outcomes.

Tau-tubulin kinase 1 (TTBK1), a neuron-specific tau kinase candidate, is involved in tau phosphorylation and aggregation

Neurofibrillary tangles, which are major pathological hallmarks of Alzheimers disease (AD), are composed of paired helical filaments (PHFs) containing hyperphosphorylated tau. Specific kinases regulate tau phosphorylation and are closely linked to the pathogenesis of AD. We have characterized a human tau‐tubulin kinase 1 (TTBK1) gene located on chromosome 6p21.1. TTBK1 is a serine/threonine/tyrosine kinase that is conserved among species and belongs to the casein kinase 1 superfamily. It is specifically expressed in the brain, especially in the cytoplasm of cortical and hippocampal neurons. TTBK1 phosphorylates tau proteins in both a Mg2+‐ and a Mn2+‐dependent manner. Phosphopeptide mapping and immunoblotting analysis confirmed a direct tau phosphorylation by TTBK1 at Ser198, Ser199, Ser202 and Ser422, which are also phosphorylated in PHFs. TTBK1 also induces tau aggregation in human neuronal cells in a dose‐dependent manner. We conclude that TTBK1 is a neuron‐specific dual kinase involved in tau phosphorylation at AD‐related sites and is also associated with tau aggregation.

Amyloid precursor protein-processing products affect mononuclear phagocyte activation: Pathways for sAPP- and Aβ-mediated neurotoxicity

Increasing evidence strongly supports the role of glial immunity in the pathogenesis of Alzheimers disease (AD). To investigate such events we have developed cell systems mimicking the interactions between β‐amyloid precursor protein (APP)‐expressing neurons and brain mononuclear phagocytes (MP; macrophages and microglia). MP were co‐cultured with neuronal cells expressing wild type APP or familial AD‐linked APP mutants. The latter was derived from recombinant adenoviral constructs. Neuronal APP processing products induced MP activation, reactive oxygen species, and neurotoxic activities. These occurred without the addition of pro‐inflammatory cytokines and were reversed by depletion of amyloid β‐peptide (Aβ) and secreted APP (sAPP). Neurotoxic activities were diminished by superoxide dismutase mimetics and NMDA receptor inhibitors. Microglial glutamate secretion was suppressed by the cystine‐glutamate antiporter inhibitor and its levels paralleled the depletion of sAPP and Aβ from conditioned media prepared from APP‐expressing neurons. The excitotoxins from activated MP were potent enough to evoke recombinant NMDA receptor‐mediated inward currents expressed in vitro in the Xenopus oocytes. These results demonstrate that neuronal APP‐processing products can induce oxidative neurotoxicity through microglial activation.

Molecular Characterization of a Putative Antiretroviral Transcriptional Factor, OTK18

Elucidation of the factors involved in host defense against human immunodeficiency viral infection remains pivotal if viral control may be achieved. Toward these ends, we investigated the function of a putative antiretroviral factor, OTK18, isolated by differential display of mRNA from HIV type 1-infected primary human monocyte-derived macrophages. Molecular and immunohistochemical analyses showed that the OTK18 nucleotide sequence contains 13 adjacent C2H2-type zinc finger motifs, a Krüppel-associated box, and is localized to both cytosol and nucleus. Mutational analyses revealed that both the Krüppel-associated box and zinc finger regions of OTK18 are responsible for the transcriptional suppressive activities of this gene. OTK18 was copiously expressed in macrophages following HIV type I infection and diminished progeny virion production. A mechanism for this antiretroviral activity was by suppression of HIV type 1 Tat-induced viral long terminal repeat promoter activity. Our findings suggest that one possible function of OTK18 is as a HIV type 1-inducible transcriptional suppresser.

Methamphetamine and Inflammatory Cytokines Increase Neuronal Na+/K+-ATPase Isoform 3: Relevance for HIV Associated Neurocognitive Disorders

Methamphetamine (METH) abuse in conjunction with human immunodeficiency virus (HIV) exacerbates neuropathogenesis and accelerates neurocognitive impairments in the central nervous system (CNS), collectively termed HIV Associated Neurocognitive Disorders (HAND). Since both HIV and METH have been implicated in altering the synaptic architecture, this study focused on investigating alterations in synaptic proteins. Employing a quantitative proteomics approach on synaptosomes isolated from the caudate nucleus from two groups of rhesus monkeys chronically infected with simian immunodeficiency virus (SIV) differing by one regimen, METH treatment, we identified the neuron specific Na+/K+-ATPase alpha 1 isoform 3 (ATP1A3) to be up regulated after METH treatment, and validated its up regulation by METH in vitro. Further studies on signaling mechanisms revealed that the activation of ATP1A3 involves the extracellular regulated kinase (ERK) pathway. Given its function in maintaining ionic gradients and emerging role as a signaling molecule, changes in ATP1A3 yields insights into the mechanisms associated with HAND and interactions with drugs of abuse.

The neuropathogenesis of HIV-1 infection.

Publisher Summary Significant neurological complications associated with HIV-1 infection occur years after an acute viral seroconversion reaction and are commonly coincident with progressive immunosuppression and high viral loads. Disease processes start soon after initial viral infection, initiated through the exchange of infected body fluids by blood transfusion, accidental needle sticks, sexual intercourse, and maternal–fetal transmission. Infection is highly restricted for varying time periods, but is usually measured in years. The evasion of the innate and acquired immune surveillance mechanisms leads to dissemination and high-level replication to regional lymphatics and HIV-1 target tissues, including the bone marrow, lung, and brain. Importantly, HIV-1 enters the central nervous system (CNS) early in the course of disease. The virus is carried into the brain principally through CD4 T lymphocytes and mononuclear phagocytes (MPs), dendritic cells, monocytes, and macrophages. Following acute infection the host engages the virus in a commensal relationship. This allows a subclinical stage in which HIV-1 persists in the infected human host but at low levels. The most severe form of HIV-1-associated tissue damage occurs in the CNS. The lack of adequate innate viral control mechanisms and adaptive immunity allows active viral replication to occur in the brain.

YY1 and FoxD3 regulate antiretroviral zinc finger protein OTK18 promoter activation induced by HIV-1 infection

OTK18 is a C2H2 type zinc finger protein involved in the regulation of HIV-1 replication in human mononuclear phagocytes. Previously, we reported OTK18 expression in brain perivascular macrophages but not in microglia in HIV encephalitis brain. We have cloned the OTK18 promoter region proximal to the transcriptional start site and determined the region responsible (−884/+1) for the basal transcriptional activity in a microglia cell line. Sequential deletion mutation analyses reveal three important response elements: Yingyang-1 (YY1; −805/−777), an HIV-1 response element for promoter activation; FoxD3 (−743/−725), a negative regulatory element; and Ets response element (−725/−707), a basal transcriptional activity response element. HIV-1 infection-induced upregulation of YY1 and c-Ets-1 protein, binding to the promoter region as determined by immunoblotting and chromatin immunoprecipitation and polymerase chain reaction (PCR) assays, and induction of YY1 was also observed in virus-infected monocyte-derived macrophages. Silencing of FoxD3 and YY1 in the cell line by small interfering RNA duplexes specific to these molecules significantly up- and downregulated basal OTK18 promoter activity in FoxD3 and YY1 response element-dependent manners, respectively. On the other hand, infection of primary cultured human microglia significantly reduced YY1 expression and induced FoxD3 as determined by immunoblotting and reverse transcription real-time PCR. These data suggest that HIV-1 induces OTK18 expression through a YY1-mediated manner in human macrophages, although its gene expression is suppressed by FoxD3 upregulation and YY1 downregulation in human microglia. This mechanism may explain the perivascular macrophage-specific expression of OTK18 in HIV encephalitis brains.

OTK18 levels in plasma and cerebrospinal fluid correlate with viral load and CD8 T-cells in normal and AIDS patients

OTK18 is a C2H2 type zinc finger protein expressed by human macrophages following HIV infection. OTK18 possesses antiretroviral activity, and its processing products accumulate in the cytoplasm of perivascular brain macrophages in advanced HIV encephalitis cases. Since the regulation of OTK18 expression in living patients following human immunodeficiency virus-1 (HIV-1) infection is unknown, our objective is to investigate the first cohort study on OTK18 protein levels in living patients. We assessed OTK18 levels in plasma and cerebrospinal fluid (CSF) in 44 living patients with or without HIV-1 infection, with diverse demographic and clinical background. A novel high-sensitivity OTK18 ELISA system was developed to measure OTK18 levels in CSF and plasma using custom made biotinylated monoclonal antibodies against OTK18. The correlation of OTK18 levels with epidemiological parameters was statistically analyzed. Multiple linear regression modeling suggested that plasma OTK18 levels for HIV-1-positive subjects were only about one sixth of that for HIV-1-negative subjects. Higher CD8 T-cell counts were associated with higher levels of OTK18. Using proportional odds logistic regression, we showed that HIV-1-positive patients have significantly lower OTK18 in CSF samples, but we did not observe significant correlation between CD8 T-cell counts and CSF OTK18 levels. OTK18 levels in both plasma and CSF are significantly lower in HIV-1-positive subjects as compared to HIV-1-negative subjects. Plasma OTK18 levels are positively correlated to CD8 T-cell counts, independent of HIV-1 status.

Isolation of Synaptosomes from Archived Brain Tissues

Synapses in the central nervous system serve as communication points between neurons and are critical regulators of neurotransmission and synaptic plasticity, the latter refers to a process of experience dependent changes in synaptic connectivity, where neurons undergo extensive sculpting and rewiring. Research on understanding the changes at the level of the synapse holds great promise into understanding the biological basis of many neurodegenerative and neuropsychiatric disorders in which brain wiring goes awry. One such approach to understand the changes occurring at the synapse is by isolating synaptosomes. Here, we describe the isolation of synaptosomes from archived human brain tissue using subcellular fractionation, which when combined to high-throughput “omics”-based approaches could yield vital clues into understanding the underlying bases of neurodegeneration.