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

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

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.

Quantitative Assessment of Eye Phenotypes for Functional Genetic Studies Using Drosophila melanogaster.

About two-thirds of the vital genes in the Drosophila genome are involved in eye development, making the fly eye an excellent genetic system to study cellular function and development, neurodevelopment/degeneration, and complex diseases such as cancer and diabetes. We developed a novel computational method, implemented as Flynotyper software (http://flynotyper.sourceforge.net), to quantitatively assess the morphological defects in the Drosophila eye resulting from genetic alterations affecting basic cellular and developmental processes. Flynotyper utilizes a series of image processing operations to automatically detect the fly eye and the individual ommatidium, and calculates a phenotypic score as a measure of the disorderliness of ommatidial arrangement in the fly eye. As a proof of principle, we tested our method by analyzing the defects due to eye-specific knockdown of Drosophila orthologs of 12 neurodevelopmental genes to accurately document differential sensitivities of these genes to dosage alteration. We also evaluated eye images from six independent studies assessing the effect of overexpression of repeats, candidates from peptide library screens, and modifiers of neurotoxicity and developmental processes on eye morphology, and show strong concordance with the original assessment. We further demonstrate the utility of this method by analyzing 16 modifiers of sine oculis obtained from two genome-wide deficiency screens of Drosophila and accurately quantifying the effect of its enhancers and suppressors during eye development. Our method will complement existing assays for eye phenotypes, and increase the accuracy of studies that use fly eyes for functional evaluation of genes and genetic interactions.

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.

Functional genomic screen and network analysis reveal novel modifiers of tauopathy dissociated from tau phosphorylation.

A functional genetic screen using loss-of-function and gain-of-function alleles was performed to identify modifiers of tau-induced neurotoxicity using the 2N/4R (full-length) isoform of wild-type human tau expressed in the fly retina. We previously reported eye pigment mutations, which create dysfunctional lysosomes, as potent modifiers; here, we report 37 additional genes identified from ∼1900 genes screened, including the kinases shaggy/GSK-3beta, par-1/MARK, CamKI and Mekk1. Tau acts synergistically with Mekk1 and p38 to down-regulate extracellular regulated kinase activity, with a corresponding decrease in AT8 immunoreactivity (pS202/T205), suggesting that tau can participate in signaling pathways to regulate its own kinases. Modifiers showed poor correlation with tau phosphorylation (using the AT8, 12E8 and AT270 epitopes); moreover, tested suppressors of wild-type tau were equally effective in suppressing toxicity of a phosphorylation-resistant S11A tau construct, demonstrating that changes in tau phosphorylation state are not required to suppress or enhance its toxicity. Genes related to autophagy, the cell cycle, RNA-associated proteins and chromatin-binding proteins constitute a large percentage of identified modifiers. Other functional categories identified include mitochondrial proteins, lipid trafficking, Golgi proteins, kinesins and dynein and the Hsp70/Hsp90-organizing protein (Hop). Network analysis uncovered several other genes highly associated with the functional modifiers, including genes related to the PI3K, Notch, BMP/TGF-ß and Hedgehog pathways, and nuclear trafficking. Activity of GSK-3ß is strongly upregulated due to TDP-43 expression, and reduced GSK-3ß dosage is also a common suppressor of Aß42 and TDP-43 toxicity. These findings suggest therapeutic targets other than mitigation of tau phosphorylation.

Double vision: pigment genes do more than just color.

The use of P element collections led to the discovery of unanticipated effects from common genetic background mutants white, brown, and rosy in our previously reported model of tauopathy that expresses full-length human tau in the fly eye, in which mutant rosy suppresses mutant white and brown worsening of tau-induced toxicity (Ambegaokar & Jackson, 2010, Genetics, v. 186, p. 435-42). Here we discuss further possible effects of mini-white and evidence for autophagy as a mediator of white enhancement of tau toxicity.

Interaction between eye pigment genes and tau-induced neurodegeneration in Drosophila melanogaster.

Null mutations in the genes white and brown, but not scarlet, enhance a rough eye phenotype in a Drosophila melanogaster model of tauopathy; however, adding rosy mutations suppresses these effects. Interaction with nucleotide-derived pigments or increased lysosomal dysregulation are potential mechanisms. Finally, tau toxicity correlates with increased GSK-3ß activity, but not with tau phosphorylation at Ser202/Thr205.

Neurodegenerative models in Drosophila: polyglutamine disorders, Parkinson disease, and amyotrophic lateral sclerosis.

Neurodegenerative diseases encompass a large group of neurological disorders. Clinical symptoms can include memory loss, cognitive impairment, loss of movement or loss of control of movement, and loss of sensation. Symptoms are typically adult onset (although severe cases can occur in adolescents) and are reflective of neuronal and glial cell loss in the central nervous system. Neurodegenerative diseases also are considered progressive, with increased severity of symptoms over time, also reflective of increased neuronal cell death. However, various neurodegenerative diseases differentially affect certain brain regions or neuronal or glial cell types. As an example, Alzheimer disease (AD) primarily affects the temporal lobe, whereas neuronal loss in Parkinson disease (PD) is largely (although not exclusively) confined to the nigrostriatal system. Neuronal loss is almost invariably accompanied by abnormal insoluble aggregates, either intra- or extracellular. Thus, neurodegenerative diseases are categorized by (a) the composite of clinical symptoms, (b) the brain regions or types of brain cells primarily affected, and (c) the types of protein aggregates found in the brain. Here we review the methods by which Drosophila melanogaster has been used to model aspects of polyglutamine diseases, Parkinson disease, and amyotrophic lateral sclerosis and key insights into that have been gained from these models; Alzheimer disease and the tauopathies are covered elsewhere in this special issue.

Role of p21-activated kinase pathway defects in the cognitive deficits of Alzheimer disease.

Defects in dendritic spines are common to several forms of cognitive deficits, including mental retardation and Alzheimer disease. Because mutation of p21-activated kinase (PAK) can lead to mental retardation and because PAK-cofilin signaling is critical in dendritic spine morphogenesis and actin dynamics, we hypothesized that the PAK pathway is involved in synaptic and cognitive deficits in Alzheimer disease. Here, we show that PAK and its activity are markedly reduced in Alzheimer disease and that this is accompanied by reduced and redistributed phosphoPAK, prominent cofilin pathology and downstream loss of the spine actin-regulatory protein drebrin, which cofilin removes from actin. We found that beta-amyloid (Abeta) was directly involved in PAK signaling deficits and drebrin loss in Abeta oligomer-treated hippocampal neurons and in the Appswe transgenic mouse model bearing a double mutation leading to higher Abeta production. In addition, pharmacological PAK inhibition in adult mice was sufficient to cause similar cofilin pathology, drebrin loss and memory impairment, consistent with a potential causal role of PAK defects in cognitive deficits in Alzheimer disease.

Antibodies against beta-amyloid reduce Abeta oligomers, glycogen synthase kinase-3beta activation and tau phosphorylation in vivo and in vitro.

Although active and passive immunization against the beta-amyloid peptide (Abeta) of amyloid plaque-bearing transgenic mice markedly reduces amyloid plaque deposition and improves cognition, the mechanisms of neuroprotection and impact on toxic oligomer species are not understood. We demonstrate that compared to control IgG2b, passive immunization with intracerebroventricular (icv) anti-Abeta (1-15) antibody into the AD HuAPPsw (Tg2576) transgenic mouse model reduced specific oligomeric forms of Abeta, including the dodecamers that correlate with cognitive decline. Interestingly, the reduction of soluble Abeta oligomers, but not insoluble Abeta, significantly correlated with reduced tau phosphorylation by glycogen synthase kinase-3beta (GSK-3beta), a major tau kinase implicated previously in mediating Abeta toxicity. A conformationally-directed antibody against amyloid oligomers (larger than tetramer) also reduced Abeta oligomer-induced activation of GSK3beta and protected human neuronal SH-SY5Y cells from Abeta oligomer-induced neurotoxicity, supporting a role for Abeta oligomers in human tau kinase activation. These data suggest that antibodies that are highly specific for toxic oligomer subspecies may reduce toxicity via reduction of GSK-3beta, which could be an important strategy for Alzheimer's disease (AD) therapeutics.

Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo.

Alzheimer's disease (AD) involves amyloid beta (Abeta) accumulation, oxidative damage, and inflammation, and risk is reduced with increased antioxidant and anti-inflammatory consumption. The phenolic yellow curry pigment curcumin has potent anti-inflammatory and antioxidant activities and can suppress oxidative damage, inflammation, cognitive deficits, and amyloid accumulation. Since the molecular structure of curcumin suggested potential Abeta binding, we investigated whether its efficacy in AD models could be explained by effects on Abeta aggregation. Under aggregating conditions in vitro, curcumin inhibited aggregation (IC(50) = 0.8 microM) as well as disaggregated fibrillar Abeta40 (IC(50) = 1 microM), indicating favorable stoichiometry for inhibition. Curcumin was a better Abeta40 aggregation inhibitor than ibuprofen and naproxen, and prevented Abeta42 oligomer formation and toxicity between 0.1 and 1.0 microM. Under EM, curcumin decreased dose dependently Abeta fibril formation beginning with 0.125 microM. The effects of curcumin did not depend on Abeta sequence but on fibril-related conformation. AD and Tg2576 mice brain sections incubated with curcumin revealed preferential labeling of amyloid plaques. In vivo studies showed that curcumin injected peripherally into aged Tg mice crossed the blood-brain barrier and bound plaques. When fed to aged Tg2576 mice with advanced amyloid accumulation, curcumin labeled plaques and reduced amyloid levels and plaque burden. Hence, curcumin directly binds small beta-amyloid species to block aggregation and fibril formation in vitro and in vivo. These data suggest that low dose curcumin effectively disaggregates Abeta as well as prevents fibril and oligomer formation, supporting the rationale for curcumin use in clinical trials preventing or treating AD.

Insulin-degrading enzyme as a downstream target of insulin receptor signaling cascade: implications for Alzheimer's disease intervention.

Insulin-degrading enzyme (IDE) is one of the proteins that has been demonstrated to play a key role in degrading beta-amyloid (Abeta) monomer in vitro and in vivo, raising the possibility of upregulating IDE as an approach to reduce Abeta. Little is known, however, about the cellular and molecular regulation of IDE protein. Because one of the main functions of IDE is to degrade insulin, we hypothesized that there is a negative feedback mechanism whereby stimulation of insulin receptor-mediated signaling upregulates IDE to prevent chronic activation of the pathway. We show that treatment of primary hippocampal neurons with insulin increased IDE protein levels by approximately 25%. Insulin treatment also led to phosphatidylinositol-3 (PI3) kinase activation evidenced by Akt phosphorylation, which was blocked by PI3 kinase inhibitors, wortmannin and LY 294002. Inhibition of PI3 kinase abolished the IDE upregulation by insulin, indicating a cause-effect relationship between insulin signaling and IDE upregulation. Further support for this link was provided by the findings that deficient insulin signaling (decreased PI3 kinase subunit P85) was correlated with reduced IDE in Alzheimer's disease (AD) brains and in Tg2576 Swedish amyloid precursor protein transgenic mice fed a safflower oil-enriched ("Bad") diet used to accelerate pathogenesis. Consistent with IDE function in the degradation of Abeta monomer, the IDE decrease in the Bad diet-fed Tg2576 mice was associated with increased Abeta monomer levels. These in vitro and in vivo analyses validate the use of enhanced CNS insulin signaling as a potential strategy for AD intervention to correct the IDE defects occurring in AD.

Curcumin inhibits dose-dependently and time-dependently neuroglial cell proliferation and growth.

OBJECTIVES: Curcumin (CUR), the active chemical of the Asian spice turmeric, has strong anti-oxidant and anti-inflammatory properties. CUR inhibits proliferation and growth of several cell types, e.g. cancer cells. While CUR inhibitory effects on microglial cells are demonstrated, little is known of its effects on neuroglia, astrocytes (AST) and oligodendrocytes (OLG). Our work focuses on CUR's effects on neuroglial proliferation and growth in vitro, utilizing C-6 rat glioma 2B-clone cells, a mixed colony of both neuroglial cells, in 6 day trials. METHODS: The doses studied included 4, 5, 10, 15, and 20 microM - concentrations slightly smaller than those shown to stimulate protein expression in ASTs. Automated particle counter was used to determine proliferation, and marker enzyme assays were used to determine AST and OLG activity. RESULTS: CUR inhibited neuroglial proliferation, with the degree of inhibition correlated directly with the CUR concentration. Proliferative inhibition was observed after a concentration as low as 5 microM by day 6, while inhibition of 20 microM doses occurred by day 2 of culture. Proliferative inhibition is associated with morphological changes, e.g. cell elongation and neurite prolongation, and increased activity of a marker enzyme corresponding to differentiation of OLG and with a reduced activity of the marker enzyme for AST. CONCLUSIONS: Our data suggests CUR acts continuously over a period of time, with low doses being as effective as higher doses given a longer period of treatment. It has been suggested that CUR's anti-inflammatory and anti-oxidant actions may be useful in the prevention-treatment of neurodegenerative diseases, e.g. Alzheimer's and Parkinson's Diseases. Given neuroglial involvement in these diseases, and CUR's observed actions on neuroglia, the data presented here may provide further explanations of CUR's preventative-therapeutic role in these diseases.