Myeloid and lymphoid expression of C9orf72 regulates IL-17A signaling in mice.

A mutation in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Patients with ALS or FTD often develop autoimmunity and inflammation that precedes or coincides with the onset of neurological symptoms, but the underlying mechanisms are poorly understood. Here, we knocked out murine C9orf72 in seven hematopoietic progenitor compartments by conditional mutagenesis and found that myeloid lineage C9orf72 prevents splenomegaly, loss of tolerance, and premature mortality. Furthermore, we demonstrated that C9orf72 plays a role in lymphoid cells to prevent interleukin-17A (IL-17A) production and neutrophilia. Mass cytometry identified early and sustained elevation of the costimulatory molecule CD80 expressed on C9orf72-deficient mouse macrophages, monocytes, and microglia. Enrichment of CD80 was similarly observed in human spinal cord microglia from patients with C9ORF72-mediated ALS compared with non-ALS controls. Single-cell RNA sequencing of murine spinal cord, brain cortex, and spleen demonstrated coordinated induction of gene modules related to antigen processing and presentation and antiviral immunity in C9orf72-deficient endothelial cells, microglia, and macrophages. Mechanistically, C9ORF72 repressed the trafficking of CD80 to the cell surface in response to Toll-like receptor agonists, interferon-γ, and IL-17A. Deletion of Il17a in C9orf72-deficient mice prevented CD80 enrichment in the spinal cord, reduced neutrophilia, and reduced gut T helper type 17 cells. Last, systemic delivery of an IL-17A neutralizing antibody augmented motor performance and suppressed neuroinflammation in C9orf72-deficient mice. Altogether, we show that C9orf72 orchestrates myeloid costimulatory potency and provide support for IL-17A as a therapeutic target for neuroinflammation associated with ALS or FTD.

Downregulation of Ubiquitin-Specific Protease 15 (USP15) Does Not Provide Therapeutic Benefit in Experimental Mesial Temporal Lobe Epilepsy.

Structural epilepsies display complex immune activation signatures. However, it is unclear which neuroinflammatory pathways drive pathobiology. Transcriptome studies of brain resections from mesial temporal lobe epilepsy (mTLE) patients revealed a dysregulation of transforming growth factor ß, interferon α/ß, and nuclear factor erythroid 2-related factor 2 pathways. Since these pathways are regulated by ubiquitin-specific proteases (USP), in particular USP15, we hypothesized that USP15 blockade may provide therapeutic relief in treatment-resistant epilepsies. For validation, transgenic mice which either constitutively or inducibly lack Usp15 gene expression underwent intrahippocampal kainate injections to induce mTLE. We show that the severity of status epilepticus is unaltered in mice constitutively lacking Usp15 compared to wild types. Cell death, reactive gliosis, and changes in the inflammatory transcriptome were pronounced at 4 days after kainate injection. However, these brain inflammation signatures did not differ between genotypes. Likewise, induced deletion of Usp15 in chronic epilepsy did not affect seizure generation, cell death, gliosis, or the transcriptome. Concordantly, siRNA-mediated knockdown of Usp15 in a microglial cell line did not impact inflammatory responses in the form of cytokine release. Our data show that a lack of USP15 is insufficient to modulate the expression of relevant neuroinflammatory pathways in an mTLE mouse model and do not support targeting USP15 as a therapeutic approach for pharmacoresistant epilepsy.

Translocator protein PET imaging in temporal lobe epilepsy: A reliable test-retest study using asymmetry index.

Objective: Translocator protein (TSPO) targeting positron emission tomography (PET) imaging radioligands have potential utility in epilepsy to assess the efficacy of novel therapeutics for targeting neuroinflammation. However, previous studies in healthy volunteers have indicated limited test-retest reliability of TSPO ligands. Here, we examine test-retest measures using TSPO PET imaging in subjects with epilepsy and healthy controls, to explore whether this biomarker can be used as an endpoint in clinical trials for epilepsy. Methods: Five subjects with epilepsy and confirmed mesial temporal lobe sclerosis (mean age 36 years, 3 men) were scanned twice-on average 8 weeks apart-using a second generation TSPO targeting radioligand, [11C]PBR28. We evaluated the test-retest reliability of the volume of distribution and derived hemispheric asymmetry index of [11C]PBR28 binding in these subjects and compared the results with 8 (mean age 45, 6 men) previously studied healthy volunteers. Results: The mean (± SD) of the volume of distribution (VT), of all subjects, in patients living with epilepsy for both test and retest scans on all regions of interest (ROI) is 4.49 ± 1.54 vs. 5.89 ± 1.23 in healthy volunteers. The bias between test and retest in an asymmetry index as a percentage was small (-1.5%), and reliability is demonstrated here with Bland-Altman Plots (test mean 1.062, retest mean 2.56). In subjects with epilepsy, VT of [11C]PBR28 is higher in the (ipsilateral) hippocampal region where sclerosis is present than in the contralateral region. Conclusion: When using TSPO PET in patients with epilepsy with hippocampal sclerosis (HS), an inter-hemispheric asymmetry index in the hippocampus is a measure with good test-retest reliability. We provide estimates of test-retest variability that may be useful for estimating power where group change in VT represents the clinical outcome.

Divergent functional outcomes of NLRP3 blockade downstream of multi-inflammasome activation: therapeutic implications for ALS.

NOD-Like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome modulation has emerged as a potential therapeutic approach targeting inflammation amplified by pyroptotic innate immune cell death. In diseases characterized by non-cell autonomous neurodegeneration including amyotrophic lateral sclerosis (ALS), the activation of several inflammasomes has been reported. Since functional redundancy can exist among inflammasome pathways, here we investigate the effects of NLRP3 inhibition on NLRP3, NLR family CARD Domain Containing 4 (NLRC4) and non-canonical pathways to understand whether NLRP3 blockade alone can mitigate pro-inflammatory cytokine release and pyroptotic cell death in contexts where single or multiple inflammasome pathways independent of NLRP3 are activated. In this study we do not limit our insights into inflammasome biology by solely relying on the THP-1 monocytic line under the LPS/nigericin-mediated NLRP3 pathway activation paradigm. We assess therapeutic potential and limitations of NLRP3 inhibition in multi-inflammasome activation contexts utilizing various human cellular systems including cell lines expressing gain of function (GoF) mutations for several inflammasomes, primary human monocytes, macrophages, healthy and Amyotrophic Lateral Sclerosis (ALS) patient induced pluripotent stem cells (iPSC)-derived microglia (iMGL) stimulated for canonical and non-canonical inflammasome pathways. We demonstrate that NLRP3 inhibition can modulate the NLRC4 and non-canonical inflammasome pathways; however, these effects differ between immortalized, human primary innate immune cells, and iMGL. We extend our investigation in more complex systems characterized by activation of multiple inflammasomes such as the SOD1G93A mouse model. Through deep immune phenotyping by single-cell mass cytometry we demonstrate that acute NLRP3 inhibition does not ameliorate spinal cord inflammation in this model. Taken together, our data suggests that NLRP3 inhibition alone may not be sufficient to address dynamic and complex neuroinflammatory pathobiological mechanisms including dysregulation of multiple inflammasome pathways in neurodegenerative disease such as ALS.

Innate immune activation and aberrant function in the R6/2 mouse model and Huntington's disease iPSC-derived microglia.

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disease caused by CAG repeats in exon 1 of the HTT gene. A hallmark of HD along with other psychiatric and neurodegenerative diseases is alteration in the neuronal circuitry and synaptic loss. Microglia and peripheral innate immune activation have been reported in pre-symptomatic HD patients; however, what "activation" signifies for microglial and immune function in HD and how it impacts synaptic health remains unclear. In this study we sought to fill these gaps by capturing immune phenotypes and functional activation states of microglia and peripheral immunity in the R6/2 model of HD at pre-symptomatic, symptomatic and end stages of disease. These included characterizations of microglial phenotypes at single cell resolution, morphology, aberrant functions such as surveillance and phagocytosis and their impact on synaptic loss in vitro and ex vivo in R6/2 mouse brain tissue slices. To further understand how relevant the observed aberrant microglial behaviors are to human disease, transcriptomic analysis was performed using HD patient nuclear sequencing data and functional assessments were conducted using induced pluripotent stem cell (iPSC)-derived microglia. Our results show temporal changes in brain infiltration of peripheral lymphoid and myeloid cells, increases in microglial activation markers and phagocytic functions at the pre-symptomatic stages of disease. Increases in microglial surveillance and synaptic uptake parallel significant reduction of spine density in R6/2 mice. These findings were mirrored by an upregulation of gene signatures in the endocytic and migratory pathways in disease-associated microglial subsets in human HD brains, as well as increased phagocytic and migratory functions of iPSC-derived HD microglia. These results collectively suggest that targeting key and specific microglial functions related to synaptic surveillance and pruning may be therapeutically beneficial in attenuating cognitive decline and psychiatric aspects of HD.

C9orf72 suppresses systemic and neural inflammation induced by gut bacteria.

A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia1,2. The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration3-9. The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins5 before its non-canonical translation into neural-toxic dipeptide proteins3,4. The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation6-9. Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria10,11 protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system.

Inhibition of Bruton's Tyrosine Kinase Modulates Microglial Phagocytosis: Therapeutic Implications for Alzheimer's Disease.

Bruton's tyrosine kinase (BTK), a critical component of B cell receptor signaling, has recently been implicated in regulation of the peripheral innate immune response. However, the role of BTK in microglia, the resident innate immune cells of the central nervous system, and its involvement in the pathobiology of neurodegenerative disease has not been explored. Here we found that BTK is a key regulator of microglial phagocytosis. Using potent BTK inhibitors and small interfering RNA (siRNA) against BTK, we observed that blockade of BTK activity decreased activation of phospholipase gamma 2, a recently identified genetic risk factor in Alzheimer's disease (AD), and reduced phagocytosis in rodent microglia and human monocyte-derived macrophages. Inhibition of BTK signaling also decreased microglial uptake of synaptosomes but did not have major impacts on other key microglial functions such as migration and cytokine release. Similarly, blocking BTK function ex vivo in acute brain slices reduced microglial phagocytosis and maintained numbers of resting microglia. In brain tissues from the 5xFAD mouse model of AD, levels of microglial BTK were elevated while in two gene expression datasets of post-mortem AD patient brain tissues, upregulation of BTK transcript was observed. Our study provides novel insights into the role of BTK in regulating microglial phagocytosis and uptake of synaptic structures and suggests that inhibiting microglial BTK may improve cognition in AD by preventing microglial activation and synaptic loss. Graphical Abstract Microglial-mediated synapse loss has been implicated in AD pathogenesis. Inhibition of BTK decreases activation of PLCγ2, a genetic risk factor in AD, and reduces microglial phagocytosis and uptake of synaptic structures. As such BTK inhibition may represent a therapeutic route to prevent microglial activation and synapse loss in AD.

A systems-level framework for drug discovery identifies Csf1R as an anti-epileptic drug target.

The identification of drug targets is highly challenging, particularly for diseases of the brain. To address this problem, we developed and experimentally validated a general computational framework for drug target discovery that combines gene regulatory information with causal reasoning ("Causal Reasoning Analytical Framework for Target discovery"-CRAFT). Using a systems genetics approach and starting from gene expression data from the target tissue, CRAFT provides a predictive framework for identifying cell membrane receptors with a direction-specified influence over disease-related gene expression profiles. As proof of concept, we applied CRAFT to epilepsy and predicted the tyrosine kinase receptor Csf1R as a potential therapeutic target. The predicted effect of Csf1R blockade in attenuating epilepsy seizures was validated in three pre-clinical models of epilepsy. These results highlight CRAFT as a systems-level framework for target discovery and suggest Csf1R blockade as a novel therapeutic strategy in epilepsy. CRAFT is applicable to disease settings other than epilepsy.

Immunoisolation of nanoparticles containing endocytic vesicles for drug quantitation.

Cell-mediated nanoparticle delivery has recently emerged as an efficacious method of delivering therapeutic agents across physiological barriers. Use of cells as nanodelivery vehicles requires accurate assessment of their loading capacity and identification of intracellular compartments where nanoparticles are sequestered. This is of great interest since specific endocytic trafficking routes can ultimately influence the mode of nanoparticle release and their efficacy and function. Here, we describe a technique that allows for the isolation of individual populations of nanoparticle-containing endosomes for subsequent quantitative analysis and more accurate description of where nanoparticles are stored on a subcellular level.

Methods for isolation and identification of nanoparticle-containing subcellular compartments.

Nanoparticle-based drug delivery systems have considerable potential for improvement of drug stability, bioavailability, and reduced dosing frequency. Important technological advantages of nanoparticles include high carrier capacity across biological membranes and controlled drug release. Ultimately, success of nanodelivery systems depends on toxicologic issues associated with the understanding of the fate of nanocarriers and their polymeric constituents within the targeted cells. Here we describe a method for determining subcellular distribution of nanoparticles by isolation and identification of organelles that come in direct contact with these structures.

Biochemical and biologic characterization of exosomes and microvesicles as facilitators of HIV-1 infection in macrophages.

Exosomes and microvesicles (MV) are cell membranous sacs originating from multivesicular bodies and plasma membranes that facilitate long-distance intercellular communications. Their functional biology, however, remains incompletely understood. Macrophage exosomes and MV isolated by immunoaffinity and sucrose cushion centrifugation were characterized by morphologic, biochemical, and molecular assays. Lipidomic, proteomic, and cell biologic approaches uncovered novel processes by which exosomes and MV facilitate HIV-1 infection and dissemination. HIV-1 was "entrapped" in exosome aggregates. Robust HIV-1 replication followed infection with exosome-enhanced fractions isolated from infected cell supernatants. MV- and exosome-facilitated viral infections are affected by a range of cell surface receptors and adhesion proteins. HIV-1 containing exosomes readily completed its life cycle in human monocyte-derived macrophages but not in CD4(-) cells. The data support a significant role for exosomes as facilitators of viral infection.

Blood-borne macrophage-neural cell interactions hitchhike on endosome networks for cell-based nanozyme brain delivery.

BACKGROUND: Macrophage-carried nanoformulated catalase ('nanozyme') attenuates neuroinflammation and protects nigrostriatal neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxication. This is facilitated by effective enzyme transfer from blood-borne macrophages to adjacent endothelial cells and neurons leading to the decomposition of reactive oxygen species. MATERIALS & METHODS: We examined the intra- and inter-cellular trafficking mechanisms of nanozymes by confocal microscopy. Improved neuronal survival mediated by nanozyme-loaded macrophages was demonstrated by fluorescence-activated cell sorting. RESULTS: In macrophages, nanozymes were internalized mainly by clathrin-mediated endocytosis then trafficked to recycling endosomes. The enzyme is subsequently released in exosomes facilitated by bridging conduits. Nanozyme transfer from macrophages to adjacent cells by endocytosis-independent mechanisms diffusing broadly throughout the recipient cells. In contrast, macrophage-free nanozymes were localized in lysosomes following endocytic entry. CONCLUSION: Facilitated transfer of nanozyme from cell to cell can improve neuroprotection against oxidative stress commonly seen during neurodegenerative disease processes.

Human immunodeficiency virus type 1 endocytic trafficking through macrophage bridging conduits facilitates spread of infection.

Bridging conduits (BC) sustain communication and homeostasis between distant tethered cells. These are also exploited commonly for direct cell-to-cell transfer of microbial agents. Conduits efficiently spread infection, effectively, at speeds faster than fluid phase exchange while shielding the microbe against otherwise effective humoral immunity. Our laboratory has sought to uncover the mechanism(s) for these events for human immunodeficiency virus type one (HIV-1) infection. Indeed, in our prior works HIV-1 Env and Gag antigen and fluorescent virus tracking were shown sequestered into endoplasmic reticulum-Golgi organelles but the outcomes for spreading viral infection remained poorly defined. Herein, we show that HIV-1 specifically traffics through endocytic compartments contained within BC and directing such macrophage-to-macrophage viral transfers. Following clathrin-dependent viral entry, HIV-1 constituents bypass degradation by differential sorting from early to Rab11(+) recycling endosomes and multivesicular bodies. Virus-containing endocytic viral cargoes propelled by myosin II through BC spread to neighboring uninfected cells. Disruption of endosomal motility with cytochalasin D, nocodasole and blebbistatin diminish intercellular viral spread. These data lead us to propose that HIV-1 hijacks macrophage endocytic and cytoskeletal machineries for high-speed cell-to-cell spread.

Macrophage bridging conduit trafficking of HIV-1 through the endoplasmic reticulum and Golgi network.

Bridging conduits (BC) are tubular protrusions that facilitate cytoplasm and membrane exchanges between tethered cells. We now report that the human immunodeficiency virus type I (HIV-1) exploits these conduits to accelerate its spread and to shield it from immune surveillance. Endosome transport through BC drives HIV-1 intercellular transfers. How this occurs was studied in human monocyte-derived macrophages using proteomic, biochemical, and imaging techniques. Endosome, endoplasmic reticulum (ER), Golgi markers, and HIV-1 proteins were identified by proteomic assays in isolated conduits. Both the ER and Golgi showed elongated and tubular morphologies that extended into the conduits of polarized macrophages. Env and Gag antigen and fluorescent HIV-1 tracking demonstrated that these viral constituents were sequestered into endocytic and ER-Golgi organelles. Sequestered infectious viral components targeted the Golgi and ER by retrograde transport from early and Rab9 late endosomes. Disruption of the ER-Golgi network impaired HIV-1 trafficking in the conduit endosomes. This study provides, for the first time, mechanisms for how BC Golgi and ER direct cell-cell viral transfer.

Macrophage endocytic trafficking of antiretroviral nanoparticles.

AIM: Nanoformulated antiretroviral therapy can improve drug compliance for people infected with HIV. Additional benefits would include specific drug deliveries to viral reservoirs and reduction in systemic toxicities. METHODS: In this article, we describe mechanisms of crystalline antiretroviral nanoparticle (NP) uptake, intracellular trafficking and release in human monocyte-derived macrophages. RESULTS: Following clathrin-dependent endocytosis NPs bypassed lysosomal degradation by sorting from early endosomes to recycling endosome pathways. Disruption of this pathway by siRNAs or brefeldin-A impaired particle release. Proteomic and biological analysis demonstrated that particle recycling was primarily Rab11 regulated. Particles were released intact and retained complete antiretroviral efficacy. CONCLUSION: These results suggest possible pathways of subcellular transport of antiretroviral nanoformulations that preserve both particle integrity and antiretroviral activities demonstrating the potential utility of this approach for targeted drug delivery.

HIV-1 transforms the monocyte plasma membrane proteome.

How HIV-1 affects the monocyte proteome is incompletely understood. We posit that one functional consequence of virus-exposure to the monocyte is the facilitation of protein transformation from the cytosol to the plasma membrane (PM). To test this, cell surface labeling with CyDye fluorophores followed by 2 dimensional differential in-gel electrophoresis (2D DIGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed. Fifty three percent of HIV-1 induced proteins were PM associated. These were linked, in large measure, to cellular activation and oxidative stress. They included, but not limited to, biliverdin reductase, leukotriene hydrolase A(4), heat shock protein 70, and cystatin B. HIV-1 induced PM protein translocation was associated with cathepsin B- and caspase 9, 3-dependent apoptosis. In contrast, PMA-treated monocytes bypassed caspase 3, 9 pathways and lead to cathepsin B-dependent necrosis. These results demonstrate that HIV-1 uniquely affects monocyte activation and oxidative stress. These do not affect viral infection dynamics but are linked to stress-induced cell death.

HIV-1-infected astrocytes and the microglial proteome.

The human immunodeficiency virus (HIV) invades the central nervous system early after viral exposure but causes progressive cognitive, behavior, and motor impairments years later with the onset of immune deficiency. Although in the brain, HIV preferentially replicates productively in cells of mononuclear phagocyte (MP; blood borne macrophage and microglia), astrocytes also can be infected, at low and variable frequency, particularly in patients with encephalitis. Among their many functions, astrocytes network with microglia to provide the first line of defense against microbial infection; however, very little is known about astrocytes' consequences on MP. Here, we addressed this question using co-culture systems of HIV-infected mouse astrocytes and microglia. Pseudotyped vesicular stomatis virus/HIV was used to circumvent the absence of viral receptors and ensure cell genotypic uniformity for studies of intercellular communication. The study demonstrated that infected astrocytes show modest changes in protein elements compared to uninfected cells. In contrast, infected astrocytes induce robust changes in the proteome of HIV-1-infected microglia. Accelerated cell death and redox proteins, among others, were produced in abundance. The observations confirmed the potential of astrocytes to influence the neuropathogenesis of HIV-1 infection by specifically altering the neurotoxic potential of infected microglia and regulating viral maturation.

Proteomic modeling for HIV-1 infected microglia-astrocyte crosstalk.

BACKGROUND: HIV-1-infected and immune competent brain mononuclear phagocytes (MP; macrophages and microglia) secrete cellular and viral toxins that affect neuronal damage during advanced disease. In contrast, astrocytes can affect disease by modulating the nervous system's microenvironment. Interestingly, little is known how astrocytes communicate with MP to influence disease. METHODS AND FINDINGS: MP-astrocyte crosstalk was investigated by a proteomic platform analysis using vesicular stomatitis virus pseudotyped HIV infected murine microglia. The microglial-astrocyte dialogue was significant and affected microglial cytoskeleton by modulation of cell death and migratory pathways. These were mediated, in part, through F-actin polymerization and filament formation. Astrocyte secretions attenuated HIV-1 infected microglia neurotoxicity and viral growth linked to the regulation of reactive oxygen species. CONCLUSIONS: These observations provide unique insights into glial crosstalk during disease by supporting astrocyte-mediated regulation of microglial function and its influence on the onset and progression of neuroAIDS. The results open new insights into previously undisclosed pathogenic mechanisms and open the potential for biomarker discovery and therapeutics that may influence the course of HIV-1-mediated neurodegeneration.

Nitrated alpha-synuclein and microglial neuroregulatory activities.

Microglial neuroinflammatory responses affect the onset and progression of Parkinson's disease (PD). We posit that such neuroinflammatory responses are, in part, mediated by microglial interactions with nitrated and aggregated alpha-synuclein (alpha-syn) released from Lewy bodies as a consequence of dopaminergic neuronal degeneration. As disease progresses, secretions from alpha-syn-activated microglia can engage neighboring glial cells in a cycle of autocrine and paracrine amplification of neurotoxic immune products. Such pathogenic processes affect the balance between a microglial neurotrophic and neurotoxic signature. We now report that microglia secrete both neurotoxic and neuroprotective factors after exposure to nitrated alpha-syn (N-alpha-syn). Proteomic (surface enhanced laser desorption-time of flight, 1D sodium dodecyl sulfate electrophoresis, and liquid chromatography-tandem mass spectrometry) and limited metabolomic profiling demonstrated that N-alpha-syn-activated microglia secrete inflammatory, regulatory, redox-active, enzymatic, and cytoskeletal proteins. Increased extracellular glutamate and cysteine and diminished intracellular glutathione and secreted exosomal proteins were also demonstrated. Increased redox-active proteins suggest regulatory microglial responses to N-alpha-syn. These were linked to discontinuous cystatin expression, cathepsin activity, and nuclear factor-kappa B activation. Inhibition of cathepsin B attenuated, in part, N-alpha-syn microglial neurotoxicity. These data support multifaceted microglia functions in PD-associated neurodegeneration.

Nitrated alpha-synuclein-activated microglial profiling for Parkinson's disease.

Microglial neuroinflammatory processes play a primary role in dopaminergic neurodegeneration for Parkinson's disease (PD). This can occur, in part, by modulation of glial function following activation by soluble or insoluble modified alpha-synuclein (alpha-syn), a chief component of Lewy bodies that is released from affected dopaminergic neurons. alpha-Syn is nitrated during oxidative stress responses and in its aggregated form, induces inflammatory microglial functions. Elucidation of these microglial function changes in PD could lead to new insights into disease mechanisms. To this end, PD-associated inflammation was modeled by stimulation of microglia with aggregated and nitrated alpha-syn. These activated microglia were ameboid in morphology and elicited dopaminergic neurotoxicity. A profile of nitrated, aggregated alpha-syn-stimulated microglia was generated using combinations of genomic (microarrays) and proteomic (liquid chromatography-tandem mass spectrometry, differential gel electrophoresis, and protein array) assays. Genomic studies revealed a substantive role for nuclear factor-kappa B transcriptional activation. Qualitative changes in the microglial proteome showed robust increases in inflammatory, redox, enzyme, and cytoskeletal proteins supporting the genomic tests. Autopsy brain tissue acquired from substantia nigra and basal ganglia of PD patients demonstrated that parallel nuclear factor-kappa B-related inflammatory processes were, in part, active during human disease. Taken together, the transcriptome and proteome of nitrated alpha-syn activated microglia, shown herein, provide new potential insights into disease mechanisms.