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Unlabelled: The proteasome and autophagy are two major intracellular protein degradation pathways and the regulation of each by ethanol metabolism affects cellular integrity. Using acute and chronic ethanol feeding to mice in vivo, and precision-cut rat liver slices (PCLS) ex vivo, we examined whether ethanol treatment altered these proteolytic pathways. In acute studies, we gave C57Bl/6 mice either ethanol or phosphate-buffered saline (PBS) by gastric intubation and sacrificed them 12h later. PCLS were exposed to 0 or 50mM ethanol for 12 and 24h with or without 4-methylpyrazole (4MP). In chronic studies we pair-fed control and ethanol liquid diets for 4-6 weeks to transgenic mice, expressing the green fluorescent protein (GFP) fused to the autophagic marker, microtubule associated protein-1 light chain 3 (LC3). Acute ethanol administration elevated autophagosomes (AVs), as judged by a 1.5-fold increase in LC3II content over PBS-gavaged control mice. Hepatic proteasome activity was unaffected by this treatment. Compared with controls, ethanol exposure for 12 and 24h to PCLS inhibited proteasome activity by 1.5- to 3-fold and simultaneously enhanced AVs by 2- to 5-fold. The decrease in proteasome activity and the rise in AVs both depended on ethanol oxidation as its inhibition by 4-methylpyrazole (4MP) blocked both proteasome inhibition and AV induction. Hepatocytes from mice chronically consuming ethanol exhibited a 1.6-fold decline in proteasome activity, and a 4-fold rise in GFP-LC3 puncta compared with pair-fed control mice. When we exposed hepatocytes from these animals to MG262, a proteasome inhibitor, LC3II puncta per cell further increased 2- to 5-fold over untreated cells., Conclusion: Our findings demonstrate that ethanol metabolism generates oxidants, the levels of which differentially influence the activities of the proteasome and autophagy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Methamphetamine abuse continues to be a worldwide problem, damaging the individual user as well as society. Only minimal information exists on molecular changes in the brain that result from methamphetamine administered in patterns typical of human abusers. In order to investigate such changes, we examined the effect of methamphetamine on the transcriptional profile in brains of monkeys. Gene expression profiling of caudate and hippocampus identified protein disulfide isomerase family member A3 (PDIA3) to be significantly up-regulated in the animals treated with methamphetamine as compared to saline treated control monkeys. Methamphetamine treatment of mice also increased striatal PDIA3 expression. Treatment of primary striatal neurons with methamphetamine revealed an up-regulation of PDIA3, showing a direct effect of methamphetamine on neurons to increase PDIA3. In vitro studies using a neuroblastoma cell line demonstrated that PDIA3 expression protects against methamphetamine-induced cell toxicity and methamphetamine-induced intracellular reactive oxygen species production, revealing a neuroprotective role for PDIA3. The current study implicates PDIA3 to be an important cellular neuroprotective mechanism against a toxic drug, and as a potential target for therapeutic investigations.

Background: The National NeuroAIDS Tissue Consortium (NNTC) performed a brain gene expression array to elucidate pathophysiologies of Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive disorders., Methods: Twenty-four human subjects in four groups were examined A) Uninfected controls; B) HIV-1 infected subjects with no substantial neurocognitive impairment (NCI); C) Infected with substantial NCI without HIV encephalitis (HIVE); D) Infected with substantial NCI and HIVE. RNA from neocortex, white matter, and neostriatum was processed with the Affymetrix® array platform., Results: With HIVE the HIV-1 RNA load in brain tissue was three log(10) units higher than other groups and over 1,900 gene probes were regulated. Interferon response genes (IFRGs), antigen presentation, complement components and CD163 antigen were strongly upregulated. In frontal neocortex downregulated neuronal pathways strongly dominated in HIVE, including GABA receptors, glutamate signaling, synaptic potentiation, axon guidance, clathrin-mediated endocytosis and 14-3-3 protein. Expression was completely different in neuropsychologically impaired subjects without HIVE. They had low brain HIV-1 loads, weak brain immune responses, lacked neuronally expressed changes in neocortex and exhibited upregulation of endothelial cell type transcripts. HIV-1-infected subjects with normal neuropsychological test results had upregulation of neuronal transcripts involved in synaptic transmission of neostriatal circuits., Interpretation: Two patterns of brain gene expression suggest that more than one pathophysiological process occurs in HIV-1-associated neurocognitive impairment. Expression in HIVE suggests that lowering brain HIV-1 replication might improve NCI, whereas NCI without HIVE may not respond in kind; array results suggest that modulation of transvascular signaling is a potentially promising approach. Striking brain regional differences highlighted the likely importance of circuit level disturbances in HIV/AIDS. In subjects without impairment regulation of genes that drive neostriatal synaptic plasticity reflects adaptation. The array provides an infusion of public resources including brain samples, clinicopathological data and correlative gene expression data for further exploration (http://www.nntc.org/gene-array-project).

Background: Substantive plasma proteomic changes follow lentiviral infection and disease pathobiology. We posit that such protein alterations are modified during drug abuse, further serving to affect the disease. To this end, we investigated the effect of opiate administration on the plasma proteome of Indian-strain rhesus monkeys infected with simian immunodeficiency virus (SIV) strain smm9., Methods: Whole blood was collected at 7 weeks prior to and 1.4 and 49 weeks after viral infection. Viral load, CD4(+) T cell subsets, and plasma protein content were measured from monkeys that did or did not receive continuous opiate administrations. The plasma proteome was identified and quantified by isobaric tags for relative and absolute quantitation labeling (iTRAQ) and mass spectrometry., Results: While substantive changes in plasma proteins were seen during SIV infection, the addition of opiates led to suppression of these changes as well as increased variance of the proteome. These changes demonstrate that opiates induce broad but variant immune suppression in SIV-infected monkeys., Conclusion: The broad suppressive changes seen in plasma of SIV-infected monkeys likely reflect reduced multisystem immune homeostatic responses induced by opiates. Such occur as a consequence of complex cell-to-cell interactions operative between the virus and the host. We conclude that such changes in plasma proteomic profiling may be underappreciated and as such supports the need for improved clinical definitions., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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.

Methamphetamine (Meth) is a widely abused stimulant and its users are at increased risk for multiple infectious diseases. To determine the impact of meth on the immune system, we utilized a murine model that simulates the process of meth consumption in a typical addict. Our phenotypic analysis of leukocytes from this dose escalation model revealed that meth affected key immune subsets. Meth administration led to a decrease in abundance of natural killer (NK) cells and the remaining NK cells possessed a phenotype suggesting reduced responsiveness. Dendritic cells (DCs) and Gr-1(high) monocytes/macrophages were also decreased in abundance while Gr-1(low) monocytes/macrophages appear to show signs of perturbation. CD4 and CD8 T cell subsets were affected by methamphetamine, both showing a reduction in antigen-experienced subsets. CD4 T cells also exhibited signs of activation, with increased expression of CD150 on CD226-expressing cells and an expansion of KLRG1(+), FoxP3(-) cells. These results exhibit that meth has the ability to disrupt immune homeostasis and impact key subsets of leukocytes which may leave users more vulnerable to pathogens.

We wanted to determine whether methamphetamine use affects a subset of plasma proteins in HIV-infected persons. Plasma samples from two visits were identified for subjects from four groups: HIV+, ongoing, persistent METH use; HIV+, short-term METH abstinent; HIV+, long term METH abstinence; HIV negative, no history of METH use. Among 390 proteins identified, 28 showed significant changes in expression in the HIV+/persistent METH+ group over the two visits, which were not attributable to HIV itself. These proteins were involved in complement, coagulation pathways and oxidative stress. Continuous METH use is an unstable condition, altering levels of a number of plasma proteins.

Mitochondrial dysfunction is implicated in a majority of neurodegenerative disorders and much study of neurodegenerative disease is done on cultured neurons. In traditional tissue culture, the oxygen level that cells experience is dramatically higher (21%) than in vivo conditions (1-11%). These differences can alter experimental results, especially, pertaining to mitochondria and oxidative metabolism. Our results show that primary neurons cultured at physiological oxygen levels found in the brain showed higher polarization, lower rates of ROS production, larger mitochondrial networks, greater cytoplasmic fractions of mitochondria and larger mitochondrial perimeters than those cultured at higher oxygen levels. Although neurons cultured in either physiological oxygen or atmospheric oxygen exhibit significant increases in mitochondrial reactive oxygen species (ROS) production when treated with the human immunodeficiency virus (HIV) virotoxin trans-activator of transcription, mitochondria of neurons cultured at physiological oxygen underwent depolarization with dramatically increased cell death, whereas those cultured at atmospheric oxygen became hyperpolarized with no increase in cell death. Studies with a second HIV virotoxin, negative regulation factor (Nef), revealed that Nef treatment also increased mitochondrial ROS production for both the oxygen conditions, but resulted in mitochondrial depolarization and increased death only in neurons cultured in physiological oxygen. These results indicate a role for oxidative metabolism in a mechanism of neurotoxicity during HIV infection and demonstrate the importance of choosing the correct, physiological, culture oxygen in mitochondrial studies performed in neurons.

Background: In Parkinson's disease there is progressive loss of dopamine containing neurons in the substantia nigra pars compacta. The neuronal damage is not limited to the substantia nigra but progresses to other regions of brain, leading to loss of motor control as well as cognitive abnormalities. The purpose of this study was to examine causes of progressive damage in the caudate nucleus, which plays a major role in motor coordination and cognition, in experimental Parkinson's disease., Results: Using chronic 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine treatment of rhesus monkeys to model Parkinson's disease, we found a upregulation of Cathepsin D, a lysosomal aspartic protease, in the caudate nucleus of treated monkeys. Immunofluorescence analysis of caudate nucleus brain tissue showed that the number of lysosomes increased concurrently with the increase in Cathepsin D in neurons. In vitro overexpression of Cathepsin D in a human neuroblastoma cell line led to a significant increase in the number of the lysosomes. Such expression also resulted in extralysosomal Cathepsin D and was accompanied by significant neuronal death associated with caspase activation. We examined apoptotic markers and found a strong correlation of Cathepsin D overexpression to apoptosis., Conclusions: Following damage to the substantia nigra resulting in experimental Parkinson's disease, we have identified pathological changes in the caudate nucleus, a likely site of changes leading to the progression of disease. Cathepsin D, implicated in pathogenic mechanisms in other disorders, was increased, and our in vitro studies revealed its overexpression leads to cellular damage and death. This work provides important clues to the progression of Parkinson's, and provides a new target for strategies to ameliorate the progression of this disease.

Dynamic interactions between human immunodeficiency virus-1 (HIV-1) and the macrophage govern the tempo of viral dissemination and replication in its human host. HIV-1 affects macrophage phenotype, and the macrophage, in turn, can modulate the viral life cycle. While these processes are linked to host-cell function and survival, the precise intracellular pathways involved are incompletely understood. To elucidate such dynamic virus-cell events, we employed pulsed stable isotope labeling of amino acids in cell culture. Alterations in de novo protein synthesis of HIV-1 infected human monocyte-derived macrophages (MDM) were examined after 3, 5, and 7 days of viral infection. Synthesis rates of cellular metabolic, regulatory, and DNA packaging activities were decreased, whereas, those affecting antigen presentation (major histocompatibility complex I and II) and interferon-induced antiviral activities were increased. Interestingly, enrichment of proteins linked to chromatin assembly or disassembly, DNA packaging, and nucleosome assembly were identified that paralleled virus-induced cytopathology and replication. We conclude that HIV-1 regulates a range of host MDM proteins that affect its survival and abilities to contain infection.

The double epidemic of substance abuse and HIV infection is a multifaceted problem To investigate mechanistic clues to the effects of substance abuse on infected individuals we preformed quantitative proteomic profiling of plasma in a methamphetamine treated nonhuman primate model for AIDS. A nontargeted quantitative approach identified extracellular superoxide dismutase to be significantly upregulated by SIV and methamphetamine treatment, and targeted studies revealed an increase in expression in the antioxidant glutathione S-transferase, thus pointing to a compensatory response to increased oxidative stress in methamphetamine-treated animals.

Accessing the subarachnoid space via intrathecal needle placement can be done in nonhuman primates (NHPs) and other species to collect cerebral spinal fluid (CSF) or to deliver agents into the CSF. Researchers can use a variety of techniques for intrathecal collection and administration. Drawing on various published resources and previous experience with intracisternal CSF sampling, the authors sought to modify one collection technique to include CSF administration. Here, the authors describe how they collected CSF from the cisterna magna of rhesus macaques (Macaca mulatta) and administered substances through the cisterna magna into the CSF. They identify potential concerns that they had when developing the technique and describe how they alleviated those concerns. The authors found this technique, which relies heavily on teamwork, to be an effective method for intracisternal injection of an experimental compound.

Lentiviral replication in its target cells affects a delicate balance between cellular cofactors required for virus propagation and immunoregulation for host defense. To better elucidate cellular proteins linked to viral infection, we tested plasma from rhesus macaques infected with the simian immunodeficiency viral strain SIVsmm9, prior to, 10 days (acute), and 49 weeks (chronic) after viral infection. Changes in plasma protein content were measured by quantitative mass spectrometry by isobaric tags for absolute and relative quantitation (iTRAQ) methods. An 81 and 232% increase in SERPINA1 was seen during acute and chronic infection, respectively. Interestingly, gelsolin, vitamin D binding protein and histidine rich glycoprotein were decreased by 45% in acute conditions but returned to baseline during chronic infection. When compared to uninfected controls, a 48-103% increase in leucine rich alpha 2-glycoprotein, vitronectin, and ceruloplasmin was observed during chronic viral infection. Observed changes in plasma proteins expression likely represent a compensatory host response to persistent viral infection.

Methamphetamine (Meth) abuse increases risky behaviors that contribute to the spread of HIV infection. In addition, because HIV and Meth independently affect physiological systems including the central nervous system, HIV-induced disease may be more severe in drug users. We investigated changes in blood and brain viral load as well as differences in immune cells in chronically simian immunodeficiency virus-infected rhesus macaques that were either administered Meth or used as controls. Although Meth administration did not alter levels of virus in the plasma, viral load in the brain was significantly increased in Meth-treated animals compared with control animals. Meth treatment also resulted in an activation of natural killer cells. Given the prevalence of Meth use in HIV-infected and HIV at-risk populations, these findings reveal the likely untoward effects of Meth abuse in such individuals.

The growth and renewal of epithelial tissue is a highly orchestrated and tightly regulated process occurring in different tissue types under a variety of circumstances. We have been studying the process of pancreatic regeneration in mice. We have identified a cell surface protein, named EP1, which is expressed on the duct epithelium during pancreatic regeneration. Whereas it is not detected in the pancreas of normal mice, it is found in the intestinal epithelium of normal adult mice, as well as during pancreatic repair following cerulein-induced destruction of the acinar tissue. The distinctive situations in which EP1 is expressed, all of which share in common epithelial cell growth in the gastrointestinal tract, suggest that EP1 is involved in the growth and renewal of epithelial tissues in both the intestine and the pancreas.

Diagnosing neurodegenerative diseases, monitoring their progression and assessing responses to treatments will all be aided by the identification of molecular markers of different stages of pathology. Protein biomarkers for HIV-associated neurocognitive disorders that have been discovered using proteomics include complement C3, soluble superoxide dismutase and a prostaglandin synthase. Metabolomics has not yet been widely used for biomarker discovery, but early work shows that it has great potential.

Many facets of transcriptional and translational regulation contribute to the proper functioning of the nervous system. Dysfunctional control of mRNA and protein expression can lead to neurodegenerative conditions. Recently, a new regulatory control element--small noncoding RNAs--has been found to play a significant role in many physiologic systems. Here, we review the microRNA (miRNA) field as it pertains to discovery-based and mechanistic studies on the brain and specifically in neurodegenerative disorders. Understanding the role of miRNAs in the brain will aid to open new avenues to the field of neuroscience and, importantly, neurodegenerative disease research.

MicroRNAs (miRNAs) play important roles in regulating a plethora of physiological and pathophysiogical processes including neurodegeneration. In both HIV associated dementia in humans and its monkey model SIV encephalitis we find miR-21, a miRNA largely known for its link to oncogenesis, to be significantly upregulated in the brain. In situ hybridization of the diseased brain sections revealed induction of miR-21 in neurons. MiR-21 can be induced in neurons by prolonged N-methyl-D-aspartic acid receptor stimulation, an excitotoxic process active in HIV and other neurodegenerative diseases. Introduction of miR-21 into human neurons leads to pathological functional defects. Furthermore, we show that miR-21 specifically targets the mRNA of myocyte enhancer factor 2C (MEF2C), a transcription factor crucial for neuronal function, and reduces its expression. MEF2C is dramatically downregulated in neurons of HIV-associated dementia patients as well as monkeys with SIVE. Together, this study elucidates a novel role for miR-21 in the brain, not only as a potential signature of neurological disease but also as a crucial effector of HIV induced neuronal dysfunction and neurodegeneration.

Investigating, predicting, diagnosing, and treating HIV-1-associated neurocognitive disorder (HAND) has been hindered by the lack of disease-related molecular markers. In this study, plasma from rhesus monkeys (n = 6), before and after infection with simian immunodeficiency virus (SIV), was profiled to obtain differential fingerprints in protein expression during SIV-induced central nervous system (CNS) disease. A quantitative proteomic analysis was performed by means of isobaric tag for relative and absolute quantification (iTRAQ) labeling, using multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) run on a linear ion trap mass spectrometer in an integrated mode comprising pulsed-Q-dissociation (PQD) and CID. Among a panel of proteins showing differential expression following SIV infection, we identified afamin, a member of the albumin superfamily, to be significantly down regulated after infection. Validation by Western blot confirmed this observation and, given its potential implication in neuroprotection by transport of alpha-tocopherol (alphaTocH), provides new avenues into further understanding HIV induced CNS disease. iTRAQ-based LC-MS/MS provides a valuable platform for plasma protein profiling and has important implications in identifying molecular markers relevant for the pathogenesis of neurodegenerative diseases. Using such an approach, we show its successful application in identifying differential fingerprints in SIV/HIV induced CNS disease.

The use of antiretroviral therapy has reduced mortality and increased the quality of life of HIV-1-infected people, particularly in more developed countries where access to treatment is more widespread. However, morbidities continue, which include HIV-1-associated neurocognitive disorders (HAND). Subtle cognitive abnormalities and low-level viral replication underlie disease. The balance between robust antiviral adaptive immunity, neuronal homeostatic mechanisms, and neuroprotective factors on one hand and toxicities afforded by dysregulated immune activities on the other govern disease. New insights into the pathobiological processes for neuroimmune-linked disease and ways to modulate such activities for therapeutic gain are discussed. Better understanding of the complexities of immune regulation during HAND can improve diagnosis and disease outcomes but is also relevant for the pathogenesis of a broad range of neurodegenerative disorders.

Human immunodeficiency virus (HIV)-associated dementia (HAD) is a syndrome occurring in HIV-infected patients with advanced disease that likely develops as a result of macrophage and microglial activation as well as other immune events triggered by virus in the central nervous system. The most relevant experimental model of HAD, rhesus macaques exhibiting simian immunodeficiency virus (SIV) encephalitis (SIVE), closely reproduces the human disease and has been successfully used to advance our understanding of mechanisms underlying HAD. In this study we integrate gene expression data from uninfected and SIV-infected hippocampus with a human protein interaction network and discover modules of genes whose expression patterns distinguish these two states, to facilitate identification of neuronal genes that may contribute to SIVE/HIV cognitive deficits. Using this approach we identify several downregulated candidate genes and select one, EGR1, a key molecule in hippocampus-related learning and memory, for further study. We show that EGR1 is downregulated in SIV-infected hippocampus and that it can be downregulated in differentiated human neuroblastoma cells by treatment with CCL8, a product of activated microglia. Integration of expression data with protein interaction data to discover discriminatory modules of interacting proteins can be usefully used to prioritize differentially expressed genes for further study. Investigation of EGR1, selected in this manner, indicates that its downregulation in SIVE may occur as a consequence of the host response to infection, leading to deficits in cognition.

Themis (thymocyte-expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis had high expression in thymocytes between the pre-T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which resulted in fewer mature thymocytes. Negative selection was also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells had CD4(+)CD25(+)Foxp3(+) regulatory and CD62L(lo)CD44(hi) memory phenotypes than did wild-type T cells. In support of the idea that Themis is involved in TCR signaling, this protein was phosphorylated quickly after TCR stimulation and was needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.

Objective: Neurocognitive disorders are devastating consequences of HIV infection. Although antiretroviral regimens have been efficacious in both improving life expectancy and decreasing dementia, there has not been an effect on the overall prevalence of HIV-associated neurocognitive disorders. Whether early institution of treatment, or treatment with drugs that effectively penetrate the blood-brain barrier, would help protect from such conditions is not known. Using the simian immunodeficiency virus/macaque model, we investigated the hypothesis that early introduction of antiretroviral treatment can protect the brain., Design and Methods: Animals were inoculated with simian immunodeficiency virus, and upon resolution of the acute infection period divided into two groups and treated, or not, with combination antiretroviral therapy. Viral, immune, and physiological parameters were measured during the course of infection, followed by assessment of viral, immune, and molecular parameters in the brain., Results: We observed that even with agents that show poor penetration into the central nervous system, early antiretroviral treatment prevented characteristic neurophysiological and locomotor alterations arising after infection and resulted in a significant decrease in brain viral load. Although the number of infiltrating immune cells in the brain did not change with treatment, their phenotype did, favoring an enrichment of effector T cells. Early treatment also significantly lowered brain levels of interferon-alpha, a cytokine that can lead to neurocognitive and behavioral alterations., Conclusion: Early antiretroviral treatment prevents central nervous system dysfunction by decreasing brain viral load and interferon-alpha levels, which can have a profound impact over the course of infection.

The HIV-1-associated neurocognitive disorder occurs in approximately one-third of infected individuals. It has persisted in the current era of antiretroviral therapy, and its study is complicated by the lack of biomarkers for this condition. Since the cerebrospinal fluid is the most proximal biofluid to the site of pathology, we studied the cerebrospinal fluid in a nonhuman primate model for HIV-1-associated neurocognitive disorder. Here we present a simple and efficient liquid chromatography-coupled mass spectrometry-based proteomics approach that utilizes small amounts of cerebrospinal fluid. First, we demonstrate the validity of the methodology using human cerebrospinal fluid. Next, using the simian immunodeficiency virus-infected monkey model, we show its efficacy in identifying proteins such as alpha-1-antitrypsin, complement C3, hemopexin, IgM heavy chain, and plasminogen, whose increased expression is linked to disease. Finally, we find that the increase in cerebrospinal fluid proteins is linked to increased expression of their genes in the brain parenchyma, revealing that the cerebrospinal fluid alterations identified reflect changes in the brain itself and not merely leakage of the blood-brain or blood-cerebrospinal fluid barriers. This study reveals new central nervous system alterations in lentivirus-induced neurological disease, and this technique can be applied to other systems in which limited amounts of biofluids can be obtained.

Multiple sclerosis (MS) is an inflammatory central nervous system (CNS) disorder characterized by T cell-mediated demyelination. In MS, prolonged T cell survival and increased T cell proliferation have been linked to disease relapse and progression. Recently, the autophagy-related gene 5 (Atg5) has been shown to modulate T cell survival. In this study, we examined the expression of Atg5 using both a mouse model of autoimmune demyelination as well as blood and brain tissues from MS cases. Quantitative real-time PCR analysis of RNA isolated from blood samples of experimental autoimmune encephalomyelitis (EAE) mice revealed a strong correlation between Atg5 expression and clinical disability.Analysis of protein extracted from these cells confirmed both upregulation and post-translational modification of Atg5, the latter of which was positively correlated with EAE severity. Analysis of RNA extracted from T cells isolated by negative selection indicated that Atg5 expression was significantly elevated in individuals with active relapsing-remitting MS compared to non-diseased controls. Brain tissue sections from relapsing-remitting MS cases examined by immunofluorescent histochemistry suggested that encephalitogenic T cells are a source of Atg5 expression in MS brain samples. Together these data suggest that increased T cell expression of Atg5 may contribute to inflammatory demyelination in MS.

While methamphetamine-induced changes in brain neurotransmitters, their receptors, and transporters are well studied, the means by which methamphetamine abuse results in cognitive and behavioral abnormalities is unknown. Here, we administered methamphetamine chronically, in doses relevant to recreational usage patterns, to nonhuman primates. Neurostructural analysis revealed decreased dendritic material and loss of spines in frontal lobe neurons. Molecular examination demonstrated that type I interferons (interferon-alpha and interferon-beta) increased in the frontal lobe in response to chronic methamphetamine treatment, in correlation with the neuronal changes. Chronic methamphetamine thus results in significant changes in the primate brain, inducing cytokines and altering neuronal structure, both of which can contribute to functional abnormalities.

Many recent studies indicate that dysregulation of autophagy is a common feature of many neurodegenerative diseases. The HIV-1-associated neurological disorder is an acquired cognitive and motor disease that includes a severe neurodegenerative dementia. We find that the neurodegeneration seen in the brain in HIV-1 infection is associated with an inhibition of neuronal autophagy, leading to neuronal demise. Neurons treated with supernatants from SIV-infected microglia develop a decrease in autophagy-inducing proteins, a decrease in neuronal autophagy vesicles, and an increase in sequestosome-1/p62. Examination of brains from HIV-infected individuals and SIV-infected monkeys reveals signs of autophagy dysregulation, associated, respectively, with dementia and encephalitis. Excitotoxic and inflammatory factors could inhibit neuronal autophagy, and stimulation of autophagy with rapamycin prevents such effects. Here we amplify on these findings, and propose that in the setting of HIV-infection, the decreased neuronal autophagy sensitizes cells to pro-apoptotic and other damaging mechanisms, leading to neuronal dysfunction and death. Hence, new therapeutic approaches aimed at boosting neuronal autophagy are conceivable to treat those suffering from the neurological complications of HIV.

Since the introduction of highly active antiretroviral therapy, survival rates for human immunodeficiency virus (HIV) infection have markedly improved, but less of an effect has been found for HIV-associated neurocognitive disorders. On the basis of our previous findings, we hypothesized that increased production of osteopontin might contribute to the persistence of central nervous system (CNS) dysfunctions. We found increased levels of osteopontin in the brains of humans with HIV encephalitis and monkeys with simian immunodeficiency virus (SIV) encephalitis. In cerebrospinal fluid, osteopontin levels were found to be elevated in HIV-infected individuals, regardless of their neuropsychological status. However, plasma osteopontin levels were significantly increased in individuals with HIV-associated dementia. In addition, a longitudinal study of monkeys revealed that plasma levels of osteopontin increased before the development of SIV-induced neurological and clinical abnormalities. Thus, plasma levels of osteopontin are significantly correlated with HIV-induced CNS dysfunction in the current era of efficacious antiviral treatment, and this finding suggests that the development of interventions to modulate osteopontin production or signaling might be beneficial in the prevention or treatment of HIV-induced CNS disorders.

There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.

The success of antiretroviral therapy has reduced the incidence of severe neurological complication resulting from human immunodeficiency virus (HIV) infection. However, increased patient survival has been associated with an increased prevalence of protracted forms of HIV encephalitis leading to moderate cognitive impairment. NeuroAIDS remains a great challenge to patients, their families, and our society. Thus development of preclinical models that will be suitable for testing promising new compounds with neurotrophic and neuroprotective capabilities is of critical importance. The simian immunodeficiency virus (SIV)-infected macaque is the premiere model to study HIV neuropathogenesis. This model was central to the seminal work of Dr. Opendra "Bill" Narayan. Similar to patients with HIV encephalitis, in the SIV model there is injury to the synaptodendritic structure of excitatory pyramidal neurons and inhibitory calbindin-immunoreactive interneurons. This article, which is part of a special issue of the Journal of NeuroVirology in honor of Dr. Bill Narayan, discusses the most important neurodegenerative features in preclinical models of neuroAIDS and their potential for treatment development.

With the increased survival of human immunodeficiency virus (HIV)-infected individuals resulting from therapy, disorders in other target organs of the virus, such as the brain, are becoming more prevalent. Here the author reviews his laboratory's work on the simian immunodeficiency virus (SIV)/nonhuman model of acquired immunodeficiency syndrome (AIDS), which has revealed unique characteristics of both the virus that infects the brain, and the innate and adaptive immune response within the central nervous system (CNS) to infection. Similar to findings in humans, neurocognitive/neurobehavioral disorders during the chronic phase of infection can be detected in monkeys, and recent findings reveal potential mechanisms of CNS damage due to the virus-host interaction.

Objectives: Defects in memory CD4+ T cells correlate with development of AIDS in monkeys infected with simian immunodeficiency virus, but the early events leading to these deficits are unknown. We explored the role of cells specific to simian immunodeficiency virus and CD8 cells in the determination of CD4 failure and rapid disease course., Design and Methods: Using MamuA*01-restricted Gag and Tat epitope tetramers, we compared the kinetics of specific response in animals with regular (REG) and rapid (RAP) progression. Expressions of memory, activation and proliferation markers were examined on the global CD8 pool, as well as on CD4 T cells in those animals. In-vivo CD8 depletion in non-MamuA*01 animals was used to investigate CD8 collapse as an event leading to disease progression and CD4 deficits., Results: In animals with a rapid disease course, an initial development of cytotoxic T lymphocytes specific to simian immunodeficiency virus is followed by collapse accompanied by global changes in CD8 cells and occurs in synchrony with the characteristic CD4 deficiencies. Antibody-mediated depletion of CD8 cells early after infection with simian immunodeficiency virus induces similar changes in the CD4 cells and rapid development of AIDS., Conclusion: CD8 collapse at acute time points may result in uncontrolled viral load and development of a defective and insufficient CD4 population. Our results indicate that early breakdown in CD8 cells leads to CD4 deficits and rapid progression to AIDS and suggest that therapeutic approaches should aim at strengthening CD8 T cells early after viral infection.

HIV infiltrates the CNS soon after an individual has become infected with the virus, and can cause dementia and encephalitis in late-stage disease. Here, a global metabolomics approach was used to find and identify metabolites differentially regulated in the cerebrospinal fluid (CSF) of rhesus macaques with SIV-induced CNS disease, as we hypothesized that this might provide biomarkers of virus-induced CNS damage. The screening platform used a non-targeted, mass-based metabolomics approach beginning with capillary reverse phase chromatography and electrospray ionization with accurate mass determination, followed by novel, nonlinear data alignment and online database screening to identify metabolites. CSF was compared before and after viral infection. Significant changes in the metabolome specific to SIV-induced encephalitis were observed. Metabolites that were increased during infection-induced encephalitis included carnitine, acyl-carnitines, fatty acids, and phospholipid molecules. The elevation in free fatty acids and lysophospholipids correlated with increased expression of specific phospholipases in the brains of animals with encephalitis. One of these, a phospholipase A2 isoenzyme, is capable of releasing a number of the fatty acids identified. It was expressed in different areas of the brain in conjunction with glial activation, rather than linked to regions of SIV infection and inflammation, indicating widespread alterations in infected brains. The identification of specific metabolites as well as mechanisms of their increase illustrates the potential of mass-based metabolomics to address problems in CNS biochemistry and neurovirology, as well as neurodegenerative diseases.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infect and productively replicate in macrophages and T lymphocytes. Here, we show that SIV virions derived from macrophages have higher levels of infectivity than those derived from T cells. The lower infectivity of T-cell-derived viruses is influenced by the quantity or type of mannose residues on the virion. Our results demonstrate that the cellular origin of a virus is a major factor in viral infectivity. Cell-type-specific factors in viral infectivity, and organ-specific or disease stage-specific differences in cellular derivation of virions, can be critical in the pathogenesis of HIV and AIDS.

In order to assess the role of osteopontin (OPN) in leukocyte accumulation in inflammatory conditions, native OPN and its thrombin cleaved form (OPN+Thr) were studied in vivo using a rodent subcutaneous air pouch model (AP). Both forms of OPN-induced macrophage infiltration into the AP in wild-type mice. In animals lacking CD44, macrophage numbers were significantly reduced within the cavity, but cells still accumulated along the subcutaneous lining. In animals lacking endogenous OPN, no differences were found in exogenous OPN-induced macrophage accumulation, although macrophage exhibited increased alpha4 integrin expression. These studies reveal that both OPN and OPN+Thr attract macrophages in vivo through CD44.

Face validity in animal models of alcohol abuse and dependence is often at odds with robust demonstrations of ethanol-seeking. This study determined the relative influence of ethanol and a flavorant in maintaining ethanol intake in a nonhuman primate model of "cocktail" drinking. Four-year-old male monkeys were maintained on a 6% ethanol/6% Tang solution made available in daily (M-F) 1-h sessions. Experiments determined the effect of (1) a second daily access session, (2) concurrent presentation of the Tang vehicle, (3) sequential presentation of the vehicle in the first daily session and the ethanol solution in the second session, (4) altering the Tang concentration, (5) altering the ethanol concentration, and (6) removal of the flavorant. Mean daily intake (2.7+/-0.2 g/kg/day) was stable over 7 months. Simultaneous availability of a large, but not a low-moderate, volume of the vehicle reduced ethanol intake by about 50%. Decreasing the concentration of Tang in the first daily session reduced ethanol intake, whereas intake of the standard solution was increased in the second session. Ethanol consumption was decreased by only 27% when the flavorant was removed. In summary, alterations that reduced intake in the first daily session resulted in compensatory increases in ethanol intake in the second session, suggesting that animals sought a specific level of ethanol intake per day. It is concluded that models with excellent face validity (flavored beverages) can produce reliable ethanol intake in patterns that are highly consistent with ethanol-seeking behavior.

Previous work has demonstrated that the surface glycoprotein (gp120) of human immunodeficiency virus-1 (HIV-1) can induce damage and apoptosis of neurons both in vitro and in vivo. In this report, we provide evidence that double-stranded RNA-activated protein kinase (PKR), a stress kinase, is involved in HIV/gp120-associated neurodegeneration. In cultures of mixed cortical cells, HIV/gp120 increased the protein level of PKR. Additionally, PKR was phosphorylated in neurons but not glia after exposure to gp120. The use of two independent pharmacological inhibitors of PKR activity abrogated neuronal cell death induced by gp120. Cortical neurons from PKR knock-out mice were significantly protected from neurotoxicity induced by gp120, further validating the pivotal proapoptotic function of PKR. gp120-induced phosphorylated PKR localized prominently to neuronal nuclei; PKR inhibition or the NMDA receptor antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate] abrogated this effect. PKR inactivation also inhibited gp120-induced caspase-3 activation, consistent with its neuroprotective effect. Finally, brain tissue from individuals diagnosed with HIV-associated dementia (HAD), but not HIV infection alone, contained the activated form of PKR, which localized predominantly to neuronal nuclei. Together, these results identify PKR as a critical mediator of gp120 neurotoxicity, suggesting that activation of PKR contributes to the neuronal injury and cell death observed in HAD.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme, has been implicated in the pathogenesis of various neurological disorders. IDO expression is induced by IFN-gamma and leads to neurotoxicity by generating quinolinic acid. Additionally, it inhibits the immune response through both tryptophan depletion and generating other tryptophan catabolites. IL-4 and IL-13 have been shown to control IDO expression by antagonizing the effects of IFN-gamma in different cell types. Here, we investigated the effects of these cytokines on IDO expression in microglia. Interestingly, we observed that both IL-4 and IL-13 greatly enhanced IFN-gamma-induced IDO expression. However, tryptophanyl-tRNA synthetase (WRS), which is coinduced with IDO by IFN-gamma, is downregulated by IL-4 and IL-13. The effect of IL-4 and IL-13 was independent of STAT-6. Modulation of IDO but not WRS was eliminated by inhibition of protein phosphatase 2A (PP2A) activity. The phosphatidylinositol 3-kinase (PI3K) pathway further differentiated the regulation of these two enzymes, as inhibiting the PI3K pathway eliminated IFN-gamma induction of IDO, whereas such inhibition greatly enhanced WRS expression. These findings show discordance between modulations of expression of two distinct enzymes utilizing tryptophan as a common substrate, and raise the possibility of their involvement in regulating immune responses in various neurological disorders., (Copyright (c) 2007 Wiley-Liss, Inc.)

In monkeys infected with simian immunodeficiency virus (SIV), changes in body temperature and locomotor activity occur after the acute retroviral syndrome stage of the disease. However, alterations to the circadian rhythm of these factors in SIV-infected monkeys have not been reported. To determine whether the circadian rhythm of body temperature and locomotor activity are disrupted during SIV infection, we analyzed the temperature and activity patterns of SIV-infected monkeys through different stages of the disease, progressing to SIV encephalitis by using the cosinor model for circadian oscillation. We found that SIV infection resulted in significant impairments of the amplitude and mean of the circadian rhythm of body temperature and activity and in the acrophase of the circadian rhythm for temperature. These alterations were not related to changes observed in the acute febrile response induced after viral inoculation. In animals killed once marked circadian anomalies were evident, microglia infiltration and macrophage accumulation in the hypothalamus were observed. Together, these results clearly demonstrate that SIV infection compromises aspects of circadian regulation in monkeys, with important implications for physiological functions, including cognition, in HIV-infected individuals.

Cells of the monocyte/macrophage lineage have been shown to be the principal targets for productive HIV-1 replication within the CNS. In addition, HIV-1-associated dementia (HAD) has been shown to correlate with macrophage abundance in the brain. Although increased entry of monocytes into the brain is thought to initiate this process, mechanisms that prevent macrophage egress from the brain and means that prevent macrophage death may also contribute to cell accumulation. We hypothesized that osteopontin (OPN) was involved in the accumulation of macrophages in the brain in neuroAIDS. Using in vitro model systems, we have demonstrated the role of OPN in two distinct aspects of macrophage accumulation: prevention from recirculation and protection from apoptosis. In these unique mechanisms, OPN would aid in macrophage survival and accumulation in the brain, the pathological substrate of HAD.

The host reaction to infection of the brain contributes to a number of CNS pathologies including neuro-AIDS. In this study, we have identified the accumulation of SIV-specific CTL in the brains of SIV-infected animals who have neurophysiological abnormalities but are otherwise asymptomatic. SIV-specific CTL enter the brain early after viral infection and are maintained in the brain even when those reactive with an immunodominant epitope in Tat are lost from the rest of the body. The specialized CNS environment contributes to this unique outcome. Following SIV infection, brain levels of IL-15 were significantly elevated whereas IL-2 was absent, creating an environment that favors CTL persistence. Furthermore, in response to IL-15, brain-derived CD8(+) T cells could expand in greater numbers than those from spleen. The accumulation, persistence, and maintenance of CTL in the brain are closely linked to the increased levels of IL-15 in the absence of IL-2 in the CNS following SIV infection.

"Metabolomics", the measurement of metabolite concentrations and fluxes in cell systems, is an emerging science that has enormous potential and several unique characteristics. The current applications for this field are, primarily, toxicological profiling and biomarker studies. This review of metabolomics research highlights the identification of reliable biomarkers with emphasis on neuroAIDS. Such identification of candidate markers will be advantageous for tracking the progression of human immunodeficiency virus/central nervous system (HIV/CNS) disease to gain maximal benefit from antiretroviral treatment and to provide insight into the mechanism of related neuropathogenesis.

Facets of the immune response early after human immunodeficiency virus (HIV) infection influence the course of disease. In the simian immunodeficiency virus (SIV)-rhesus monkey system, a global dysfunction of CD4(+) T cell cytokine secretion was reported to develop early after infection [McKay PF, Barouch DH, Schmitz JE, Veazey RS, Gorgone DA, Lifton MA, Williams KC, and Letvin NL: J Virol 2003;77:4695-4702]. Because differences have been found in SIV pathogenesis depending on the origin of the monkeys, we investigated the correlation between animal background, defined by country of origin (India or China), and circulating T cell cytokine secretion as well as cycling ability within the first 3 mo of SIV infection. An early loss of CD4(+) T cells that produce interferon (IFN)-gamma and interleukin (IL)-2, those that produce IFN-gamma but not tumor necrosis factor (TNF)-alpha, as well as those that do not express IFN-gamma but can express IL-2 or TNF-alpha, was observed in animals of Indian, but not of Chinese, origin after SIV infection. After infection CD4(+) T cells in Chinese macaques developed an increased proliferating pool of T cells compared with Indian animals. These data reveal host diversity in the global effects of SIV infection on functional subsets of immune cells, which can add to a better understanding of differences observed in populations from diverse ethnic origins.

CNS abnormalities can be detected during chronic human immunodeficiency virus (HIV) infection, before the development of opportunistic infections or other sequelae of immunodeficiency. However, although end-stage dementia caused by HIV has been linked to the presence of infected and activated macrophages and microglia in the brain, the nature of the changes resulting in the motor and cognitive disorders in the chronic stage is unknown. Using simian immunodeficiency virus-infected rhesus monkeys, we sought the molecular basis for CNS dysfunction. In the chronic stable stage, nearly 2 years after infection, all animals had verified CNS functional abnormalities. Both virus and infiltrating lymphocytes (CD8+ T-cells) were found in the brain. Molecular analysis revealed that the expression of several immune response genes was increased, including CCL5, which has pleiotropic effects on neurons as well as immune cells. CCL5 was significantly upregulated throughout the course of infection, and in the chronic phase was present in the infiltrating lymphocytes. We have identified an altered state of the CNS at an important stage of the viral-host interaction, likely arising to protect against the virus but in the long term leading to damaging processes.

It has long been postulated that drugs of abuse may represent significant cofactors in the progression of human immunodeficiency virus (HIV)-induced disease. Both HIV infection and drugs of abuse have significant effect on the immune system as well as on the nervous system. In HIV infection, abnormalities in these systems intersect to lead to a constellation of symptoms known as neuroAIDS. Drugs of abuse may synergize with such damage, acting on immune and/or neural cells. However, definitive epidemiological evidence for such an interaction is lacking. Here we review such studies as well as the use of the nonhuman primate/simian immunodeficiency virus system to investigate the interaction of neuroAIDS with drugs of abuse. Furthermore, recent findings on mechanisms of actions of selected drugs reveal the possibility of protective as well as detrimental effects on the central nervous system damage induced by HIV.

The use of China-derived monkeys in AIDS research has been limited by reports of reduced susceptibility to SIV. We performed a serial passage of SIV in Chinese macaques, which resulted in a viral stock capable of inducing simian AIDS and high levels of replication in these animals. Similar to HIV in humans, SIV pathogenesis in non-human primates is not limited by geographical origin. Chinese macaques are useful in pathogenesis, vaccine, and therapeutic studies in AIDS.

Injuries to the central nervous system (CNS) trigger an inflammatory reaction with potentially devastating consequences. In this report we compared the characteristics of the inflammatory response on spinal cord injury (SCI) caused by a stab wound between the T7 and T9 vertebrae and spontaneous experimental autoimmune encephalomyelitis (EAE). SCI and EAE were compared in two types of myelin basic protein Ac1-11-specific T-cell receptor transgenic mice: T/R+ mice harbor regulatory T cells, and T/R- mice lack regulatory T cells. Our results show that 8 days after SCI, T/R- mice developed a strong T-cell infiltrate in the spinal cord, with remarkable down-modulation of CD4 expression that was accompanied by a local increase in Mac-3+ and F4/80+ reactivity and diffuse local and distal astrogliosis. In contrast, T/R+ mice exhibited a modest increase in CD4+ cells localized to the site of injury, without CD4 down-modulation; focal astrogliosis was restricted to the site of the lesion, although Mac-3+ and F4/80+ cells were also present. Similarly to T/R- mice that underwent SCI, T cells displaying down-modulated CD4 expression were found in the CNS of older T/R- mice afflicted by spontaneous EAE. Overall, our results suggest that common mechanisms regulate T-cell accumulation in CNS lesions of different causes, such as mechanic lesion or autoimmune-mediated damage.

HIV is found in the brains of most infected individuals but only 30% develop neurological disease. Both viral and host factors are thought to contribute to the motor and cognitive disorders resulting from HIV infection. Here, using the SIV/rhesus monkey system, we characterize the salient characteristics of the virus from the brain of animals with neuropathological disorders. Nine unique molecular clones of SIV were derived from virus released by microglia cultured from the brains of two macaques with SIV encephalitis. Sequence analysis revealed a remarkably high level of similarity between their env and nef genes as well as their 3' LTR. As this genotype was found in the brains of two separate animals, and it encoded a set of distinct amino acid changes from the infecting virus, it demonstrates the convergent evolution of the virus to a unique brain-adapted genotype. This genotype was distinct from other macrophage-tropic and neurovirulent strains of SIV. Functional characterization of virus derived from representative clones showed a robust in vitro infection of 174xCEM cells, primary macrophages and primary microglia. The infectious phenotype of this virus is distinct from that shown by other strains of SIV, potentially reflecting the method by which the virus successfully infiltrates and infects the CNS. Positive in vivo selection of a brain-adapted strain of SIV resulted in a near-homogeneous strain of virus with distinct properties that may give clues to the viral basis of neuroAIDS.