BIN1 protein isoforms are differentially expressed in astrocytes, neurons, and microglia: neuronal and astrocyte BIN1 are implicated in tau pathology.

BACKGROUND: Identified as an Alzheimer's disease (AD) susceptibility gene by genome wide-association studies, BIN1 has 10 isoforms that are expressed in the Central Nervous System (CNS). The distribution of these isoforms in different cell types, as well as their role in AD pathology still remains unclear. METHODS: Utilizing antibodies targeting specific BIN1 epitopes in human post-mortem tissue and analyzing mRNA expression data from purified microglia, we identified three isoforms expressed in neurons and astrocytes (isoforms 1, 2 and 3) and four isoforms expressed in microglia (isoforms 6, 9, 10 and 12). The abundance of selected peptides, which correspond to groups of BIN1 protein isoforms, was measured in dorsolateral prefrontal cortex, and their relation to neuropathological features of AD was assessed. RESULTS: Peptides contained in exon 7 of BIN1's N-BAR domain were found to be significantly associated with AD-related traits and, particularly, tau tangles. Decreased expression of BIN1 isoforms containing exon 7 is associated with greater accumulation of tangles and subsequent cognitive decline, with astrocytic rather than neuronal BIN1 being the more likely culprit. These effects are independent of the BIN1 AD risk variant. CONCLUSIONS: Exploring the molecular mechanisms of specific BIN1 isoforms expressed by astrocytes may open new avenues for modulating the accumulation of Tau pathology in AD.

Cell-autonomous and non-cell autonomous effects of neuronal BIN1 loss in vivo.

BIN1 is the most important risk locus for Late Onset Alzheimer's Disease (LOAD), after ApoE. BIN1 AD-associated SNPs correlate with Tau deposition as well as with brain atrophy. Furthermore, the level of neuronal-specific BIN1 isoform 1 protein is decreased in sporadic AD cases in parallel with neuronal loss, despite an overall increase in BIN1 total mRNA. To address the relationship between reduction of BIN1 and neuronal cell loss in the context of Tau pathology, we knocked-down endogenous murine Bin1 via stereotaxic injection of AAV-Bin1 shRNA in the hippocampus of mice expressing Tau P301S (PS19). We observed a statistically significant reduction in the number of neurons in the hippocampus of mice injected with AAV-Bin1 shRNA in comparison with mice injected with AAV control. To investigate whether neuronal loss is due to deletion of Bin1 selectively in neurons in presence Tau P301S, we bred Bin1flox/flox with Thy1-Cre and subsequently with PS19 mice. Mice lacking neuronal Bin1 and expressing Tau P301S showed increased mortality, without increased neuropathology, when compared to neuronal Bin1 and Tau P301S-expressing mice. The loss of Bin1 isoform 1 resulted in reduced excitability in primary neurons in vitro, reduced neuronal c-fos expression as well as in altered microglia transcriptome in vivo. Taken together, our data suggest that the contribution of genetic variation in BIN1 locus to AD risk could result from a cell-autonomous reduction of neuronal excitability due to Bin1 decrease, exacerbated by the presence of aggregated Tau, coupled with a non-cell autonomous microglia activation.

BIN1 favors the spreading of Tau via extracellular vesicles.

Despite Bridging INtegrator 1 (BIN1) being the second most statistically-significant locus associated to Late Onset Alzheimer's Disease, its role in disease pathogenesis remains to be clarified. As reports suggest a link between BIN1, Tau and extracellular vesicles, we investigated whether BIN1 could affect Tau spreading via exosomes secretion. We observed that BIN1-associated Tau-containing extracellular vesicles purified from cerebrospinal fluid of AD-affected individuals are seeding-competent. We showed that BIN1 over-expression promotes the release of Tau via extracellular vesicles in vitro as well as exacerbation of Tau pathology in vivo in PS19 mice. Genetic deletion of Bin1 from microglia resulted in reduction of Tau secretion via extracellular vesicles in vitro, and in decrease of Tau spreading in vivo in male, but not female, mice, in the context of PS19 background. Interestingly, ablation of Bin1 in microglia of male mice resulted in significant reduction in the expression of heat-shock proteins, previously implicated in Tau proteostasis. These observations suggest that BIN1 could contribute to the progression of AD-related Tau pathology by altering Tau clearance and promoting release of Tau-enriched extracellular vesicles by microglia.

Single-cell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models.

Neuroinflammation and neurodegeneration may represent two poles of brain pathology. Brain myeloid cells, particularly microglia, play key roles in these conditions. We employed single-cell mass cytometry (CyTOF) to compare myeloid cell populations in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, the R6/2 model of Huntington's disease (HD) and the mutant superoxide dismutase 1 (mSOD1) model of amyotrophic lateral sclerosis (ALS). We identified three myeloid cell populations exclusive to the CNS and present in each disease model. Blood-derived monocytes comprised five populations and migrated to the brain in EAE, but not in HD and ALS models. Single-cell analysis resolved differences in signaling and cytokine production within similar myeloid populations in EAE compared to HD and ALS models. Moreover, these analyses highlighted α5 integrin on myeloid cells as a potential therapeutic target for neuroinflammation. Together, these findings illustrate how neuropathology may differ between inflammatory and degenerative brain disease.

Microglial Physiology and Pathophysiology: Insights from Genome-wide Transcriptional Profiling.

Microglia originate from erythromyeloid progenitors (EMPs) in the yolk sac and develop in the forming CNS. Microglia are fundamental for the development and function of a healthy brain. By contrast, their role in immune host defense of the CNS remains speculative, given the immune privilege of this organ. Alterations in microglia functionality are involved in brain aging, as well as in neurodegenerative disease severity and progression. The combination of their ontogeny with the influence of the complex environment of the CNS makes microglia a unique cell population. Recent observations about microglia ontogeny combined with extensive gene expression profiling allow us to better capture the variety of nuances that microglia can manifest. Here, we provide a contemporary appraisal of microglial uniqueness based on their origin, functions, and expression profiles. Furthermore, we give an overview of the impact of aging and neurodegenerative diseases on microglia transcriptomes.

The choreography of neuroinflammation in Huntington's disease.

Currently, the concept of 'neuroinflammation' includes inflammation associated with neurodegenerative diseases, in which there is little or no infiltration of blood-derived immune cells into the brain. The roles of brain-resident and peripheral immune cells in these inflammatory settings are poorly understood, and it is unclear whether neuroinflammation results from immune reaction to neuronal dysfunction/degeneration, and/or represents cell-autonomous phenotypes of dysfunctional immune cells. Here, we review recent studies examining these questions in the context of Huntington's disease (HD), where mutant Huntingtin (HTT) is expressed in both neurons and glia. Insights into the cellular and molecular mechanisms underlying neuroinflammation in HD may provide a better understanding of inflammation in more complex neurodegenerative disorders, and of the contribution of the neuroinflammatory component to neurodegenerative disease pathogenesis.

Mutant Huntingtin promotes autonomous microglia activation via myeloid lineage-determining factors.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an extended polyglutamine repeat in the N terminus of the Huntingtin protein (HTT). Reactive microglia and elevated cytokine levels are observed in the brains of HD patients, but the extent to which neuroinflammation results from extrinsic or cell-autonomous mechanisms in microglia is unknown. Using genome-wide approaches, we found that expression of mutant Huntingtin (mHTT) in microglia promoted cell-autonomous pro-inflammatory transcriptional activation by increasing the expression and transcriptional activities of the myeloid lineage-determining factors PU.1 and C/EBPs. We observed elevated levels of PU.1 and its target genes in the brains of mouse models and individuals with HD. Moreover, mHTT-expressing microglia exhibited an increased capacity to induce neuronal death ex vivo and in vivo in the presence of sterile inflammation. These findings suggest a cell-autonomous basis for enhanced microglia reactivity that may influence non-cell-autonomous HD pathogenesis.

25-Hydroxycholesterol activates the integrated stress response to reprogram transcription and translation in macrophages.

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

Regulation of microglia activation and deactivation by nuclear receptors.

Microglia cells function as sentinels for innate immunity in the central nervous system (CNS). To perform this function, microglia express a diverse set of pattern recognition receptors (PRRs) for pathogen-associated molecular patterns (PAMPs) that include Toll-like receptors (TLRs) and inflammasomes. Several members of the TLR and inflammasome family also recognize endogenously derived molecules that are generated as a consequence of tissue injury or other pathological processes. Recognition of PAMPs or endogenous ligands by PRRs in microglia induces the robust activation of innate immune responses leading to the production of proinflammatory mediators and the activation of adaptive immunity. Activation of microglia is essential for clearance of infection and repair of tissue injury. However, uncontrolled inflammatory responses of microglia are also thought to contribute to the severity of many neurodegenerative diseases. Thus, activation of microglia must be properly and tightly regulated to maintain normal tissue homeostasis. Several mechanisms have been identified that appear to function in the active maintenance of quiescence under normal conditions and/or re-establish this state following resolution of infection or injury. These mechanisms involve communication with neurons and other glia through secreted molecules or surface expressing receptors as well as actions of members of the nuclear receptor (NR) superfamily of transcription factors. Here, we review recent advances in our understanding of the regulation of microglia activation and deactivation with a focus on counter-regulation of microglia activation by nuclear receptors.

Mechanisms establishing TLR4-responsive activation states of inflammatory response genes.

Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early- (I/E) and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses.

Negative regulation of HIV-1 transcription by a heterodimeric NF-κB1/p50 and C-terminally truncated STAT5 complex.

Signal transducers and activator of transcription (STAT) proteins are often constitutively activated in leukocytes of HIV-1(+) individuals, which frequently show a dominant expression of a C-terminally truncated isoform of STAT5 (STAT5Δ). STAT5Δ can act as a negative regulator of human immunodeficiency virus type 1 (HIV-1) expression in both CD8-depleted primary leukocytes and chronically infected promonocytic U1 cells stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF). Activated STAT5Δ can directly bind to two consensus sequences in the HIV-1 long terminal repeat (LTR) promoter; binding impairs recruitment of RNA polymerase II (Crotti, A., Lusic, M., Lupo, R., Lievens, P. M., Liboi, E., Della Chiara, G., et al. (2007). Naturally occurring C-terminally truncated STAT5 is a negative regulator of HIV-1 expression. Blood, 109, 5380-5389). One of the STAT consensus sequences overlaps with one nuclear factor κB (NF-κB) binding site; interestingly, NF-κB1/p50 homodimers, frequently detected in monocytic cells, are negative regulators of HIV transcription. Here, we show that GM-CSF stimulation of U1 cells, while not inducing NF-κB activation, leads to STAT5Δ phosphorylation and binding to the NF-κB/STAT target sequence in the HIV LTR promoter, which already associates with p50 under unstimulated conditions. STAT5Δ was found to associate with p50, but not with RelA/p65, in both U1 cells expressing endogenous proteins and 293T cells overexpressing these factors. Furthermore, GM-CSF stimulation promoted concurrent binding of STAT5Δ and p50 at the HIV LTR promoter in U1 cells. Immunoprecipitation of chromatin from GM-CSF-stimulated U1 cells confirmed in vivo binding of p50 to the viral promoter together with STAT5Δ. Thus, cytokine-activated STAT5Δ/p50 complexes can contribute to the maintenance of HIV-1 latency in monocytic cells.

Nuclear receptors, inflammation, and neurodegenerative diseases.

Chronic inflammation is associated with many neurodegenerative diseases, including multiple sclerosis, Parkinson's disease, and Alzheimer's disease. Increasing evidence that neuroinflammation contributes to disease severity has generated considerable interest in determining whether inhibition of inflammation pathways might be of therapeutic benefit. One potential avenue of intervention is provided by members of the nuclear receptor superfamily of ligand-dependent transcription factors that exert anti-inflammatory effects in many cell types. Here, we review recent studies providing insights into the distinct mechanisms that enable nuclear receptors to modulate immune responses, describe inflammatory components of neurodegenerative diseases, and discuss recent literature relevant to roles of nuclear receptors in influencing these processes.

New players in cytokine control of HIV infection.

Cytokines are involved early in the pathogenesis of HIV infection and disease progression as a component of immunologic dysregulation and immunodeficiency and as determinants controlling virus replication. Several steps, before and after retroviral integration into host DNA in T cells and macrophages, are affected by cytokines whereas CCR5 and CXCR4 binding chemokines can interfere with viral entry. A growing number of potential players--including the gamma-common interleukin (IL)-7, IL-15, and IL-21 together with IL-17, IL-18, IL-19, IL-20, IL-23, and IL-27--are discussed in terms of their perturbation in HIV infection and of their effects on virus replication. Thus, an increasing intersection of HIV infection and the cytokine network represents a crucial determinant of virus replication and immunologic dysregulation and will likely play a key role in the development of effective strategies of HIV prevention and immunologic reconstitution.

Naturally occurring C-terminally truncated STAT5 is a negative regulator of HIV-1 expression.

CD4(+) cells of most individuals infected with HIV-1 harbor a C-terminally truncated and constitutively activated form of signal transducer and activator of transcription-5 (STAT5 Delta). We report that the chronically HIV-infected U1 cell line expresses STAT5 Delta but not full-length STAT5. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of U1 cells promoted early activation of STAT5 Delta and of extracellular signal regulated kinases (ERKs), followed by later activation of activator protein 1 (AP-1) and HIV expression. Inhibition of ERK/AP-1 by PD98,059 abolished, whereas either tyrphostin AG490 or a STAT5 small interfering RNA (siRNA) enhanced, virion production in GM-CSF-stimulated U1 cells. Chromatin immunoprecipitation demonstrated the induction of STAT5 Delta binding to STAT consensus sequences in the HIV-1 promoter together with a decreased recruitment of RNA polymerase II after 1 hour of GM-CSF stimulation of U1 cells. Down-regulation of STAT5 Delta by siRNA resulted in the up-regulation of both HIV-1 gag-pol RNA and p24 Gag antigen expression in CD8-depleted leukocytes of several HIV-positive individuals cultivated ex vivo in the presence of interleukin-2 but not of interleukin-7. Thus, the constitutively activated STAT5 Delta present in the leukocytes of most HIV-positive individuals acts as a negative regulator of HIV expression.

Heterogeneity of signal transducer and activator of transcription binding sites in the long-terminal repeats of distinct HIV-1 subtypes.

HIV-1 can be subdivided into distinct subtypes; the consequences of such a genomic variability remain largely speculative. The long terminal repeats (LTR) control HIV transcription and reflect the major differences of distinct viral subtypes. Three regions in the HIV-1 subtype B LTR are close matches to the Signal Transducer and Activator of Transcription (STAT) consensus sequence. Here, we show heterogeneity in these putative STAT binding sites among HIV-1 LTR subtypes A through G. Transfection of constitutively activated STAT5 lead to transcriptional activation of HIV-1 expression in 293T cells transfected with a reporter assay driven by HIV-1 LTR subtype B. Constitutively activated STAT5 transactivated the LTR of various subtypes in U937 cells with different potency. These findings support and expand the potential relevance of STAT5 activation in HIV infection and may bear relevance for a differential regulation of latency and expression of different subtypes of HIV-1.

Nef alleles from human immunodeficiency virus type 1-infected long-term-nonprogressor hemophiliacs with or without late disease progression are defective in enhancing virus replication and CD4 down-regulation.

Infection with human immunodeficiency virus (HIV)-encoding defective nef variants may contribute to a relatively benign course of disease in a minority of long-term nonprogressors (LTNP). We have examined the functions of nef alleles from six individuals belonging to the same cohort of hemophiliacs infected with HIV-1 prior to 1985 and classified as LTNP in 1995. Three out of six individuals have progressed to HIV disease (late progressors [LP]), whereas the three remainders have maintained their LTNP status at least up to 2003. The nef alleles were obtained from both plasma virus and peripheral blood mononuclear cells of all six individuals in 1995 and 1998. The proportion of sequences containing mutations not yielding Nef expression significantly diminished in 1998 versus that in 1995. Several previously defined functional regions of intact nef alleles were highly conserved. However, the major variant obtained in 1998 from plasma RNA of five out of six individuals significantly reduced HIV infectivity/replication and impaired Nef-mediated CD4 but not major histocompatibility complex class I antigen down-modulation from the cell surface. Thus, functional alterations of the nef gene are present in both LP and LTNP, suggesting that Nef defectiveness in vitro is not necessarily associated with the long-term maintenance of LTNP status. Of interest is the fact that isolates from three out of three LP showed a dual CCR5/CXCR4 coreceptor use (R5X4), in contrast to those from LTNP, which were exclusively R5. Thus, in vivo evolution of gp120 Env to CXCR4 use appears to be associated with HIV disease progression in individuals infected with nef-defective viruses.

Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes.

Huntingtin protein is mutated in Huntington disease. We previously reported that wild-type but not mutant huntingtin stimulates transcription of the gene encoding brain-derived neurotrophic factor (BDNF; ref. 2). Here we show that the neuron restrictive silencer element (NRSE) is the target of wild-type huntingtin activity on BDNF promoter II. Wild-type huntingtin inhibits the silencing activity of NRSE, increasing transcription of BDNF. We show that this effect occurs through cytoplasmic sequestering of repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF), the transcription factor that binds to NRSE. In contrast, aberrant accumulation of REST/NRSF in the nucleus is present in Huntington disease. We show that wild-type huntingtin coimmunoprecipitates with REST/NRSF and that less immunoprecipitated material is found in brain tissue with Huntington disease. We also report that wild-type huntingtin acts as a positive transcriptional regulator for other NRSE-containing genes involved in the maintenance of the neuronal phenotype. Consistently, loss of expression of NRSE-controlled neuronal genes is shown in cells, mice and human brain with Huntington disease. We conclude that wild-type huntingtin acts in the cytoplasm of neurons to regulate the availability of REST/NRSF to its nuclear NRSE-binding site and that this control is lost in the pathology of Huntington disease. These data identify a new mechanism by which mutation of huntingtin causes loss of transcription of neuronal genes.