Prion disease.

Genetic prion diseases (gPrDs) are caused by autosomal-dominant mutations in the prion protein gene (PRNP). Although the first PRNP mutations identified, and most since, are PRNP missense, octapeptide repeat insertions, deletion and nonsense mutations have now also been shown to cause gPrD. Based on clinicopathologic features of familial disease, gPrDs historically have been classified into three forms: familial Jakob-Creutzfeldt disease, Gerstmann-Sträussler-Scheinker disease, and fatal familial insomnia. This classification, however, occurred prior to the identification of PRNP, and although these forms are still recognized, classification now is somewhat more complex. Clinical manifestations, and even pathology, are known to be more heterogeneous and varied than the historic three phenotypic classifications. Most gPrDs either present rapidly with progression of dementia, ataxia, myoclonus, and other motor features leading to death in few months or present more slowly, declining over a few years with mild cognitive impairment, ataxia, or parkinsonism and later dementia; a few very rare mutations, however, present over years to decades with neuropsychiatric disorders and systemic symptoms (gastrointestinal disorders and neuropathy). In this chapter, we review the broad phenotypic spectrum of PRNP mutations causing gPrDs.

Genetic PrP Prion Diseases.

Genetic prion diseases (gPrDs) caused by mutations in the prion protein gene (PRNP) have been classified as genetic Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease, or fatal familial insomnia. Mutations in PRNP can be missense, nonsense, and/or octapeptide repeat insertions or, possibly, deletions. These mutations can produce diverse clinical features. They may also show varying ancillary testing results and neuropathological findings. Although the majority of gPrDs have a rapid progression with a short survival time of a few months, many also present as ataxic or parkinsonian disorders, which have a slower decline over a few to several years. A few very rare mutations manifest as neuropsychiatric disorders, with systemic symptoms that include gastrointestinal disorders and neuropathy; these forms can progress over years to decades. In this review, we classify gPrDs as rapid, slow, or mixed types based on their typical rate of progression and duration, and we review the broad spectrum of phenotypes manifested by these diseases.

Clinical Neurology and Epidemiology of the Major Neurodegenerative Diseases.

Neurodegenerative diseases are a common cause of morbidity and cognitive impairment in older adults. Most clinicians who care for the elderly are not trained to diagnose these conditions, perhaps other than typical Alzheimer's disease (AD). Each of these disorders has varied epidemiology, clinical symptomatology, laboratory and neuroimaging features, neuropathology, and management. Thus, it is important that clinicians be able to differentiate and diagnose these conditions accurately. This review summarizes and highlights clinical aspects of several of the most commonly encountered neurodegenerative diseases, including AD, frontotemporal dementia (FTD) and its variants, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and Huntington's disease (HD). For each condition, we provide a brief overview of the epidemiology, defining clinical symptoms and diagnostic criteria, relevant imaging and laboratory features, genetics, pathology, treatments, and differential diagnosis.

Genetic prion disease: Experience of a rapidly progressive dementia center in the United States and a review of the literature.

Although prion diseases are generally thought to present as rapidly progressive dementias with survival of only a few months, the phenotypic spectrum for genetic prion diseases (gPrDs) is much broader. The majority have a rapid decline with short survival, but many patients with gPrDs present as slowly progressive ataxic or parkinsonian disorders with progression over a few to several years. A few very rare mutations even present as neuropsychiatric disorders, sometimes with systemic symptoms such as gastrointestinal disorders and neuropathy, progressing over years to decades. gPrDs are caused by mutations in the prion protein gene (PRNP), and have been historically classified based on their clinicopathological features as genetic Jakob-Creutzfeldt disease (gJCD), Gerstmann-Sträussler-Scheinker (GSS), or Fatal Familial Insomnia (FFI). Mutations in PRNP can be missense, nonsense, and octapeptide repeat insertions or a deletion, and present with diverse clinical features, sensitivities of ancillary testing, and neuropathological findings. We present the UCSF gPrD cohort, including 129 symptomatic patients referred to and/or seen at UCSF between 2001 and 2016, and compare the clinical features of the gPrDs from 22 mutations identified in our cohort with data from the literature, as well as perform a literature review on most other mutations not represented in our cohort. E200K is the most common mutation worldwide, is associated with gJCD, and was the most common in the UCSF cohort. Among the GSS-associated mutations, P102L is the most commonly reported and was also the most common at UCSF. We also had several octapeptide repeat insertions (OPRI), a rare nonsense mutation (Q160X), and three novel mutations (K194E, E200G, and A224V) in our UCSF cohort. © 2016 Wiley Periodicals, Inc.

Clinical update of Jakob-Creutzfeldt disease.

PURPOSE OF REVIEW: The present review discusses recent clinical data on diagnosis, new forms, and treatment of human prion diseases, and briefly summarizes research suggesting prion-like mechanisms in other neurodegenerative diseases. RECENT FINDINGS: When proper sequences are performed, MRI has high diagnostic utility in prion disease, but there are issues with interpretation of images. The spectrum of MRI's utility for diagnosis and understanding human prion disease is still being explored. Two recent diffusion tensor imaging studies quantified changes in the gray and white matter in sporadic Jakob-Creutzfeldt disease, with unexpected results. The diagnostic utility of cerebrospinal fluid biomarkers has been controversial. A few studies showed that amplification methods can detect prions in either cerebrospinal fluid, olfactory epithelium, blood and/or urine in various human prion diseases. Additional cases of variably protease-sensitive prionopathy have led to a broader understanding of this novel sporadic prion disease. A few new mutations causing genetic prion disease, one with a very atypical presentation, have been identified. Although recent human prion disease treatment trials did not show benefit, they have improved our understanding, and led to better quantification, of the progression of these disorders. Lastly, we briefly summarize the increasing evidence that many nonprion neurodegenerative proteinopathies might spread in the brain by a prion-like mechanism. SUMMARY: New prion detection methods appear promising, but need to be replicated with larger sample sizes. Identification of novel forms of human prion disease might better elucidate the full spectrum of prion diseases and expand our understanding of their pathogenesis.

Genetic CJD with a novel E200G mutation in the prion protein gene and comparison with E200K mutation cases.

A novel point mutation resulting in a glutamate-to-glycine substitution in PRNP at codon 200, E200G with codon 129 MV polymorphism (cis valine) and type 2 PrPSc was identified in a patient with a prolonged disease course leading to pathology-proven Jakob-Creutzfeldt disease. Despite the same codon as the most common genetic form of human PRNP mutation, E200K, this novel mutation (E200G) presented with a different clinical and pathological phenotype, including prolonged duration, large vacuoles, no vacuolation in the hippocampus, severe neuronal loss in the thalamus, mild cerebellar involvement, and abundant punctate linear and curvilinear deposition of PrPSc in synaptic boutons and axonal terminals along the dendrites.

Genome-wide increase in histone H2A ubiquitylation in a mouse model of Huntington's disease.

BACKGROUND: Huntington's disease (HD) is a neurodegenerative disorder with selective vulnerability of striatal neurons and involves extensive transcriptional dysregulation early in the disease process. Previous work in cell and mouse models has shown that histone modifications are altered in HD. Specifically, monoubiquitylated histone H2A (uH2A) is present at the promoters of downregulated genes which led to the hypothesis that uH2A plays a role in transcriptional silencing in HD. OBJECTIVE: To broaden our view of uH2A function in transcription in HD, we examined genome-wide binding sites of uH2A in 12-week old striatal tissue from R6/2 transgenic HD mouse model. METHODS: We used chromatin immunoprecipitation followed by genomic promoter microarray hybridization (ChIP-chip) and then interrogated how these binding sites correlate with transcribed genes. RESULTS: Our analysis reveals that, while uH2A levels are globally increased at the genome in the transgenic (TG) striatum, uH2A localization at a gene did not strongly correlate with the absence of its transcript. Furthermore, analysis of differential ubiquitylation in wild-type (WT) and TG striata did not reveal the expected enrichment of uH2A at genes with decreased expression in the TG striatum. CONCLUSIONS: This first description of genome-wide localization of uH2A in an HD model reveals that monoubiquitylation of histone H2A may not function at the level of the individual gene but may rather influence transcription through global chromatin structure.

Bone marrow mononuclear cells have neurovascular tropism and improve diabetic neuropathy.

Bone marrow-derived mononuclear cells (BMNCs) have been shown to effectively treat ischemic cardiovascular diseases. Because diabetic neuropathy (DN) is causally associated with impaired angiogenesis and deficiency of angiogenic and neurotrophic factors in the nerves, we investigated whether DN can be ameliorated by local injection of BMNCs. Severe peripheral neuropathy, characterized by a significant decrease in the motor and sensory nerve conduction velocities (NCVs), developed 12 weeks after the induction of diabetes with streptozotocin in rats. The injection of BMNCs restored motor and sensory NCVs to normal levels and significantly improved vascular density and blood flow in diabetic nerves over 4 weeks. Fluorescent microscopic observation revealed that DiI-labeled BMNCs preferentially engrafted in sciatic nerves. Whole-mount fluorescent imaging and confocal microscopic evaluation demonstrated that many of the BMNCs localized following the course of the vasa nervorum in close proximity to blood vessels without incorporation into vasa nervorum as endothelial cells at a detectable level. Real-time reverse transcription-polymerase chain reaction analysis showed that the levels of angiogenic and neurotrophic factors were significantly increased in the nerves by BMNC injection. Local transplantation of BMNCs improved experimental DN by augmenting angiogenesis and increasing angiogenic and neurotrophic factors in peripheral nerves. These findings suggest that BMNC transplantation may represent a novel therapeutic option for treating DN.

Dual angiogenic and neurotrophic effects of bone marrow-derived endothelial progenitor cells on diabetic neuropathy.

BACKGROUND: Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence suggested that diabetic neuropathy is causally related to impaired angiogenesis and deficient growth factors. Accordingly, we investigated whether diabetic neuropathy could be reversed by local transplantation of EPCs. METHODS AND RESULTS: We found that motor and sensory nerve conduction velocities, blood flow, and capillary density were reduced in sciatic nerves of streptozotocin-induced diabetic mice but recovered to normal levels after hind-limb injection of bone marrow-derived EPCs. Injected EPCs were preferentially and durably engrafted in the sciatic nerves. A portion of engrafted EPCs were uniquely localized in close proximity to vasa nervorum, and a smaller portion of these EPCs were colocalized with endothelial cells. Multiple angiogenic and neurotrophic factors were significantly increased in the EPC-injected nerves. These dual angiogenic and neurotrophic effects of EPCs were confirmed by higher proliferation of Schwann cells and endothelial cells cultured in EPC-conditioned media. CONCLUSIONS: We demonstrate for the first time that bone marrow-derived EPCs could reverse various manifestations of diabetic neuropathy. These therapeutic effects were mediated by direct augmentation of neovascularization in peripheral nerves through long-term and preferential engraftment of EPCs in nerves and particularly vasa nervorum and their paracrine effects. These findings suggest that EPC transplantation could represent an innovative therapeutic option for treating diabetic neuropathy.

Altered histone monoubiquitylation mediated by mutant huntingtin induces transcriptional dysregulation.

Although transcriptional dysregulation is a critical pathogenic mechanism in Huntington's disease (HD), it is still not known how mutant huntingtin causes it. Here we show that alteration of histone monoubiquitylation is a key mechanism. Disrupted interaction of huntingtin with Bmi-1, a component of the hPRC1L E3 ubiquitin ligase complex, increases monoubiquityl histone H2A (uH2A) levels in a cell culture model of HD. Genes with expression that is repressed in transgenic R6/2 mouse brain have increased uH2A and decreased uH2B at their promoters, whereas actively transcribed genes show the opposite pattern. Reduction in uH2A reverses transcriptional repression and inhibits methylation of histone H3 at lysine 9 in cell culture. In contrast, reduction in uH2B induces transcriptional repression and inhibits methylation of histone H3 at lysine 4. This is the first report to demonstrate hPRC1L as a huntingtin-interacting histone modifying complex and a crucial role for histone monoubiquitylation in mammalian brain gene expression, which broadens our understanding of histone code. These findings also provide a rationale for targeting histone monoubiquitylation for therapy in HD.

Dopamine release is impaired in a mouse model of DYT1 dystonia.

Early onset torsion dystonia, the most common form of hereditary primary dystonia, is caused by a mutation in the TOR1A gene, which codes for the protein torsinA. This form of dystonia is referred to as DYT1. We have used a transgenic mouse model of DYT1 dystonia [human mutant-type (hMT)1 mice] to examine the effect of the mutant human torsinA protein on striatal dopaminergic function. Analysis of striatal tissue dopamine (DA) and metabolites using HPLC revealed no difference between hMT1 mice and their non-transgenic littermates. Pre-synaptic DA transporters were studied using in vitro autoradiography with [(3)H]mazindol, a ligand for the membrane DA transporter, and [(3)H]dihydrotetrabenazine, a ligand for the vesicular monoamine transporter. No difference in the density of striatal DA transporter or vesicular monoamine transporter binding sites was observed. Post-synaptic receptors were studied using [(3)H]SCH-23390, a ligand for D(1) class receptors, [(3)H]YM-09151-2 and a ligand for D(2) class receptors. There were again no differences in the density of striatal binding sites for these ligands. Using in vivo microdialysis in awake animals, we studied basal as well as amphetamine-stimulated striatal extracellular DA levels. Basal extracellular DA levels were similar, but the response to amphetamine was markedly attenuated in the hMT1 mice compared with their non-transgenic littermates (253 +/- 71% vs. 561 +/- 132%, p < 0.05, two-way anova). These observations suggest that the mutation in the torsinA protein responsible for DYT1 dystonia may interfere with transport or release of DA, but does not alter pre-synaptic transporters or post-synaptic DA receptors. The defect in DA release as observed may contribute to the abnormalities in motor learning as previously documented in this transgenic mouse model, and may contribute to the clinical symptoms of the human disorder.

Histones associated with downregulated genes are hypo-acetylated in Huntington's disease models.

Transcriptional dysregulation plays a major role in the pathology of Huntington's disease (HD). However, the mechanisms causing selective downregulation of genes remain unknown. Histones regulate chromatin structure and thereby control gene expression; recent studies have demonstrated a therapeutic role for histone deacetylase (HDAC) inhibitors in polyglutamine diseases. This study demonstrates that despite no change in overall acetylated histone levels, histone H3 is hypo-acetylated at promoters of downregulated genes in R6/2 mice, ST14a and STHdh cells, as demonstrated by in vivo chromatin immunoprecipitation. In addition, HDAC inhibitor treatment increases association of acetylated histones with downregulated genes and corrects mRNA abnormalities. In contrast, there is a decrease in mRNA levels in wild-type cells following treatment with a histone acetyltransferase inhibitor. Although changes in histone acetylation correlate with decreased gene expression, histone hypo-acetylation may be a late event, as no hypo-acetylation is observed in 4-week-old R6/2 mice. Nevertheless, treatment with HDAC inhibitors corrects mRNA abnormalities through modification of histone proteins and may prove to be of therapeutic value in HD.

CD45 isoform expression in microglia and inflammatory cells in HIV-1 encephalitis.

CD45 is a membrane tyrosine phosphatase that modulates the function of the hematopoietic cells. In vitro, agonist antibodies to CD45RO or CD45RB isoforms have been shown to suppress microglial activation, but whether microglia in vivo express these isoforms in HIV encephalitis (HIVE) is unknown. Brain sections from control and HIVE were immunostained for CD45 isoforms using exon-specific antibodies (RA, RB, RC and RO). RA and RC were limited to rare lymphocytes, while RB expression was robust in microglia and inflammatory cells. RO was low in control microglia, but increased in HIVE. RO was also localized to macrophages and CD8+ T cells. Targeting CD45 in vivo with isoform-specific antibodies remains a therapeutic option for neuroinflammatory diseases.

Anti-CD45RO suppresses human immunodeficiency virus type 1 replication in microglia: role of Hck tyrosine kinase and implications for AIDS dementia.

Macrophages and microglia are productively infected by HIV-1 and play a pivotal role in the pathogenesis of AIDS dementia. Although macrophages and microglia express CD45, a transmembrane protein tyrosine phosphatase, whether modulation of its activity affects human immunodeficiency virus type 1 (HIV-1) replication is unknown. Here, we report that of the five human CD45 isoforms, microglia express CD45RB and CD45RO (RB > RO) and treatment of microglia with a CD45 agonist antibody alphaCD45RO (UCHL-1) inhibits HIV-1 replication. alphaCD45RO prevented HIV-1 negative factor (Nef)-induced autophosphorylation of hematopoietic cell kinase (Hck), a myeloid lineage-specific Src kinase. Recombinant CD45 protein also inhibited HIV-1-induced Hck phosphorylation in microglia. Antennapedia-mediated delivery of Hck Src homology domain 3 (SH3), a domain that binds to the Nef PxxP motif with high affinity, reduced HIV-1-induced Hck phosphorylation and HIV-1 production in microglia. HIV-1-induced LTR transactivation was observed in U38 cells stably overexpressing wild-type Hck but not kinase-inactive Hck. In microglia, alphaCD45RO reduced activation of transcription factors (NF-kappaB and CCAAT enhancer binding protein) necessary for LTR transactivation in macrophages. These results establish that in myeloid lineage cells, Nef interacts with the Hck SH3 domain, resulting in autophosphorylation of Hck and an increase in HIV-1 transcription. alphaCD45RO-mediated inhibition of HIV-1 replication in microglia identifies the CD45 protein tyrosine phosphatase as a potential therapeutic target for HIV-1 infection/AIDS dementia.

Inhibition of granulocyte-macrophage colony-stimulating factor signaling and microglial proliferation by anti-CD45RO: role of Hck tyrosine kinase and phosphatidylinositol 3-kinase/Akt.

Increasing evidence suggests that CD45, a transmembrane protein tyrosine phosphatase, is an important modulator of macrophage activation. Microglia, resident brain macrophages, express CD45 and proliferate under pathologic conditions. In this study, we examined the role of CD45 in modulating GM-CSF-induced proliferation and signal transduction in primary human microglial cultures. Soluble, but not immobilized anti-CD45RO induced tyrosine phosphatase activity and inhibited GM-CSF-induced microglial proliferation. Microglial proliferation was also inhibited by PP2 (Src inhibitor), LY294002 (PI3K inhibitor), and U0126 (MEK inhibitor). GM-CSF induced phosphorylation of Jak2, Stat5, Hck (the myeloid-restricted Src kinase), Akt, Stat3, and Erk MAPKs in microglia. Of these, anti-CD45RO inhibited phosphorylation of Hck and Akt, and PP2 inhibited phosphorylation of Hck and Akt. In a macrophage cell line stably overexpressing wild-type or kinase-inactive Hck, GM-CSF increased proliferation of the control (empty vector) and wild-type but not kinase-inactive cells, and this was inhibited by anti-CD45RO. Together, these results demonstrate that, in macrophages, Hck tyrosine kinase is activated by GM-CSF, and that Hck plays a pivotal role in cell proliferation and survival by activating the PI3K/Akt pathway. Ab-mediated activation of macrophage and microglial CD45 tyrosine phosphatase may have therapeutic implications for CNS inflammatory diseases.

A novel action of minocycline: inhibition of human immunodeficiency virus type 1 infection in microglia.

Human immunodeficiency virus type 1 (HIV-1) infection of the brain produces a characteristic disease called acquired immunodeficiency syndrome (AIDS) dementia in which productive infection and inflammatory activation of microglia and macrophages play a central role. In this report, the authors demonstrate that minocycline (MC), a second-generation tetracycline with proven safety and penetration to the central nervous system, potently inhibited viral production from microglia. Inhibition of viral release was sustained through the entire course of infection and even when the drug exposure was limited to the first day of infection. Minocycline was effective even at low viral doses, and against R5- and X4R5-HIV, as well as in single-cycle reporter virus assays. Electrophoretic mobility shift analysis showed that minocycline inhibited nuclear factor (NF)-kappaB activation in microglia. HIV-1 long terminal repeat (LTR)-promoter activity in U38 cells was also inhibited. These results, combined with recently demonstrated in vivo anti-inflammatory effects of MC on microglia, suggest a potential utility for MC as an effective adjunct therapy for AIDS dementia.

Regulation of RANTES/CCL5 expression in human astrocytes by interleukin-1 and interferon-beta.

In the CNS, astrocytes are significant sources of RANTES/CCL5 (regulated upon activation, normal T cell expressed and secreted), a CC-chemokine with important biological function. Astrocyte RANTES/CCL5 has been shown to be induced by interleukin-1 (IL-1), with interferon-gamma (IFNgamma) as a primer, but whether type I interferons play any role in the expression of RANTES/CCL5 is not known. In this report, we studied the detailed mechanism of RANTES/CCL5 induction in primary human astrocytes activated with IL-1 and IFNbeta. Ribonuclease protection assay and ELISA showed that IFNbeta, although not effective alone, increased IL-1-induced RANTES/CCL5 expression, but did not antagonize IFNgamma. IL-1 or IL-1/IFNbeta-induced RANTES/CCL5 expression was inhibited by the super-repressor IkappaBalpha or inhibitors of p38 or c-Jun N-terminal kinase (JNK) MAPKs (mitogen-activated protein kinases), but not by extracellular signal regulated kinases (ERK) inhibitors. IFNbeta enhanced IL-1-induced phosphorylation of p38 MAPK, but was not effective alone. Transfection with mutated RANTES/CCL5 promoter-reporter constructs revealed that kappaB, interferon-stimulated response element (ISRE) and CAATT-enhancer binding protein-beta (C/EBPbeta) sites all contributed to IL-1/IFNbeta-induced RANTES/CCL5 transcription. IFNbeta synergized with IL-1 to induce nuclear accumulation of C/EBPbeta protein. They also synergized to form nuclear ISRE complexes with Stat1, Stat2 and interferon regulatory factor-1 (IRF-1) proteins. Together, our results demonstrate that IFNbeta plays a positive regulatory role in the expression of RANTES/CCL5 in human astrocytes through several distinct mechanisms.

Modulation of astrocyte inducible nitric oxide synthase and cytokine expression by interferon beta is associated with induction and inhibition of interferon gamma-activated sequence binding activity.

Although interferon (IFN)-beta is firmly established as a therapeutic agent for multiple sclerosis, information regarding its role in astrocyte cytokine production is limited. In primary cultures of human astrocytes, we determined the effects of IFN-beta on astrocyte cytokine [tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6] and inducible nitric oxide synthase (iNOS) expression by ribonuclease protection assay and ELISA. We found that IFN-beta inhibited astrocyte cytokine/iNOS induced by IL-1 plus IFN-gamma, but in the absence of IFN-gamma, IFN-beta enhanced IL-1-induced cytokine/iNOS expression. Electrophoretic mobility shift analysis (EMSA) demonstrated that IFN-gamma induced sustained IFN-gamma-activated sequence (GAS) binding, while IFN-beta induced transient GAS binding. When used together, IFN-beta inhibited IFN-gamma-induced GAS binding activity. Nuclear factor-kappa B (NF-kappaB) activation was not altered by either IFNs, whereas IFN stimulated response element (ISRE) was only activated by IFN-beta and not IFN-gamma. These results suggest that IFN-beta can both mimic and antagonize the effect of IFN-gamma by modulating induction of nuclear GAS binding activity. Our results demonstrating differential regulation of astrocyte cytokine/iNOS induction by IFN-beta are novel and have implications for inflammatory diseases of the human CNS.

Vpr- and Nef-dependent induction of RANTES/CCL5 in microglial cells.

Microglia are pivotal in the pathogenesis of AIDS dementia, as they serve as the major target of HIV infection in the CNS. In addition, activation of microglia correlates best with clinical dementia. Although the beta-chemokine RANTES/CCL5 is important in modulating HIV infection as well as cellular activation, no information is available regarding how its expression is regulated in microglia by HIV-1. Here we report that RANTES/CCL5 expression is induced in microglia by HIV-1, but that this requires infection by HIV-1. This conclusion was supported by (1) the delayed kinetics coinciding with viral replication; (2) the lack of effect of X4 viruses; (3) inhibition by the reverse transcriptase inhibitor AZT, and (4) the lack of effect of cytokine antagonists or antibodies. Interestingly, RANTES/CCL5 production was dependent on the viral accessory protein Vpr, in addition to Nef, demonstrating a novel role for Vpr in chemokine induction in primary macrophage-type cells. Furthermore, the specific p38 MAP kinase inhibitor SB203580 augmented chemokine expression in microglia, indicating a negative role played by p38. These data suggest unique features of RANTES/CCL5 regulation by HIV-1 in human microglial cells.

Interferon-beta activates multiple signaling cascades in primary human microglia.

Microglia, the resident brain macrophages, are the principal cells involved in the regulation of inflammatory and antimicrobial responses in the CNS. Interferon-beta (IFNbeta) is an antiviral cytokine induced by viral infection or following non-specific inflammatory challenges of the CNS. Because of the well-known anti-inflammatory properties of IFNbeta, it is also used to treat multiple sclerosis, an inflammatory CNS disease. Despite the importance of IFNbeta signaling in CNS cells, little has been studied, particularly in microglia. In this report, we investigated the molecular mechanisms underlying IFNbeta-induced beta-chemokine expression in primary human fetal microglia. Multiple signaling cascades are activated in microglia by IFNbeta, including nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and Jak/Stat. IFNbeta induced IkappaBalpha degradation and NF-kappaB (p65:p50) DNA binding. Inhibition of NF-kappaB by either adenoviral transduction of a super repressor IkappaBalpha, or an antioxidant inhibitor of NF-kappaB reduced expression of the beta-chemokines, regulated upon activation, normal T-cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP)-1beta. IFNbeta also induced phosphorylation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase, and the MAP kinase kinase 1 (MEK1) inhibitor PD98059 dose-dependently inhibited beta-chemokine mRNA and protein expression. PD98059 did not inhibit NF-kappaB binding, demonstrating that ERK was not responsible for NF-kappaB activation. Two downstream targets of ERK were identified in microglia: AP-1 and Stat1. IFNbeta induced AP-1 nuclear binding activity in microglia and this was suppressed by PD98059. Additionally, IFNbeta induced Stat1 phosphorylation at both tyrosine 701 (Y701) and serine 727 (S727) residues. S727 phosphorylation of Stat1, which is known to be required for maximal transcriptional activation, was inhibited by PD98059. Our results demonstrating multiple signaling cascades initiated by IFNbeta in primary human microglia are novel and have implications for inflammatory and infectious diseases of the CNS.