Mitochondrial dysfunction and mitophagy defects in LRRK2-R1441C Parkinson's disease models.

Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene have been identified as one of the most common genetic causes of Parkinson's disease (PD). The LRRK2 PD-associated mutations LRRK2G2019S and LRRK2R1441C, located in the kinase domain and in the ROC-COR domain, respectively, have been demonstrated to impair mitochondrial function. Here, we sought to further our understanding of mitochondrial health and mitophagy by integrating data from LRRK2R1441C rat primary cortical and human induced pluripotent stem cell-derived dopamine (iPSC-DA) neuronal cultures as models of PD. We found that LRRK2R1441C neurons exhibit decreased mitochondrial membrane potential, impaired mitochondrial function and decreased basal mitophagy levels. Mitochondrial morphology was altered in LRRK2R1441C iPSC-DA but not in cortical neuronal cultures or aged striatal tissue, indicating a cell-type-specific phenotype. Additionally, LRRK2R1441C but not LRRK2G2019S neurons demonstrated decreased levels of the mitophagy marker pS65Ub in response to mitochondrial damage, which could disrupt degradation of damaged mitochondria. This impaired mitophagy activation and mitochondrial function were not corrected by the LRRK2 inhibitor MLi-2 in LRRK2R1441C iPSC-DA neuronal cultures. Furthermore, we demonstrate LRRK2 interaction with MIRO1, a protein necessary to stabilize and to anchor mitochondria for transport, occurs at mitochondria, in a genotype-independent manner. Despite this, we found that degradation of MIRO1 was impaired in LRRK2R1441C cultures upon induced mitochondrial damage, suggesting a divergent mechanism from the LRRK2G2019S mutation.

Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides.

Dampening functional levels of the mitochondrial deubiquitylating enzyme Ubiquitin-specific protease 30 (USP30) has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinson's Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria, which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide-containing compound, USP30inh. Activity-based protein profiling mass spectrometry confirmed target engagement, high selectivity, and potency of USP30inh for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of USP30inh enzyme kinetics inferred slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with USP30inh, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that USP30inh binds to this thumb-palm cleft, which guides the ubiquitin C terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

Multiparameter phenotypic screening for endogenous TFEB and TFE3 translocation identifies novel chemical series modulating lysosome function.

The accumulation of toxic protein aggregates in multiple neurodegenerative diseases is associated with defects in the macroautophagy/autophagy-lysosome pathway. The amelioration of disease phenotypes across multiple models of neurodegeneration can be achieved through modulating the master regulator of lysosome function, TFEB (transcription factor EB). Using a novel multi-parameter high-throughput screen for cytoplasmic:nuclear translocation of endogenous TFEB and the related transcription factor TFE3, we screened the Published Kinase Inhibitor Set 2 (PKIS2) library as proof of principle and to identify kinase regulators of TFEB and TFE3. Given that TFEB and TFE3 are responsive to cellular stress we have established assays for cellular toxicity and lysosomal function, critical to ensuring the identification of hit compounds with only positive effects on lysosome activity. In addition to AKT inhibitors which regulate TFEB localization, we identified a series of quinazoline-derivative compounds that induced TFEB and TFE3 translocation. A novel series of structurally-related analogs was developed, and several compounds induced TFEB and TFE3 translocation at higher potency than previously screened compounds. KINOMEscan and cell-based KiNativ kinase profiling revealed high binding for the PRKD (protein kinase D) family of kinases, suggesting good selectivity for these compounds. We describe and utilize a cellular target-validation platform using CRISPRi knockdown and orthogonal PRKD inhibitors to demonstrate that the activity of these compounds is independent of PRKD inhibition. The more potent analogs induced subsequent upregulation of the CLEAR gene network and cleared pathological HTT protein in a cellular model of proteinopathy, demonstrating their potential to alleviate neurodegeneration-relevant phenotypes. Abbreviations: AD: Alzheimer disease; AK: adenylate kinase; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; HD: Huntington disease; PD: Parkinson disease; PKIS2: Published Kinase Inhibitor Set 2; PRKD: protein kinase D; TFEB: transcription factor EB.

PLCγ2 regulates TREM2 signalling and integrin-mediated adhesion and migration of human iPSC-derived macrophages.

Human genetic studies have linked rare coding variants in microglial genes, such as TREM2, and more recently PLCG2 to Alzheimer's disease (AD) pathology. The P522R variant in PLCG2 has been shown to confer protection for AD and to result in a subtle increase in enzymatic activity. PLCγ2 is a key component of intracellular signal transduction networks and induces Ca2+ signals downstream of many myeloid cell surface receptors, including TREM2. To explore the relationship between PLCγ2 and TREM2 and the role of PLCγ2 in regulating immune cell function, we generated human induced pluripotent stem cell (iPSC)- derived macrophages from isogenic lines with homozygous PLCG2 knockout (Ko). Stimulating TREM2 signalling using a polyclonal antibody revealed a complete lack of calcium flux and IP1 accumulation in PLCγ2 Ko cells, demonstrating a non-redundant role of PLCγ2 in calcium release downstream of TREM2. Loss of PLCγ2 led to broad changes in expression of several macrophage surface markers and phenotype, including reduced phagocytic activity and survival, while LPS-induced secretion of the inflammatory cytokines TNFα and IL-6 was unaffected. We identified additional deficits in PLCγ2- deficient cells that compromised cellular adhesion and migration. Thus, PLCγ2 is key in enabling divergent cellular functions and might be a promising target to increase beneficial microglial functions.

Selection and structural characterization of anti-TREM2 scFvs that reduce levels of shed ectodomain.

Mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer's disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signaling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterized. Here, we present phage display generated antibody single-chain variable fragments (scFvs) to human TREM2 immunoglobulin-like domain. Co-crystal structures revealed the binding of two scFvs to an epitope on the TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species, which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterization of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.

A phenotypic high-content, high-throughput screen identifies inhibitors of NLRP3 inflammasome activation.

Inhibition of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome has recently emerged as a promising therapeutic target for several inflammatory diseases. After priming and activation by inflammation triggers, NLRP3 forms a complex with apoptosis-associated speck-like protein containing a CARD domain (ASC) followed by formation of the active inflammasome. Identification of inhibitors of NLRP3 activation requires a well-validated primary high-throughput assay followed by the deployment of a screening cascade of assays enabling studies of structure-activity relationship, compound selectivity and efficacy in disease models. We optimized a NLRP3-dependent fluorescent tagged ASC speck formation assay in murine immortalized bone marrow-derived macrophages and utilized it to screen a compound library of 81,000 small molecules. Our high-content screening assay yielded robust assay metrics and identified a number of inhibitors of NLRP3-dependent ASC speck formation, including compounds targeting HSP90, JAK and IKK-ß. Additional assays to investigate inflammasome priming or activation, NLRP3 downstream effectors such as caspase-1, IL-1ß and pyroptosis form the basis of a screening cascade to identify NLRP3 inflammasome inhibitors in drug discovery programs.

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages.

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an "eat-me" signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Inhibition of the NLRP3 inflammasome by HSP90 inhibitors.

Excessive and dysregulated inflammation is known to contribute to disease progression. HSP90 is an intracellular chaperone known to regulate inflammatory processes including the NLRP3 inflammasome and secretion of the pro-inflammatory cytokine interleukin(IL)-1ß. Here, primarily using an in vitro inflammasome ASC speck assay, and an in vivo model of murine peritonitis, we tested the utility of HSP90 inhibitors as anti-inflammatory molecules. We report that the HSP90 inhibitor EC144 effectively inhibited inflammatory processes including priming and activation of NLRP3 in vitro and in vivo. A specific inhibitor of the ß HSP90 isoform was ineffective suggesting the importance of the α isoform in inflammatory signalling. EC144 inhibited IL-1ß and IL-6 in vivo when administered orally, and was brain-penetrant. These data suggest that HSP90 inhibitors may be useful for targeting inflammation in diverse diseases that are worsened by the presence of inflammation.

TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages.

BACKGROUND: TREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer's disease (AD), including R47H. TREM2 signalling via SYK aids phagocytosis, chemotaxis, survival, and changes to microglial activation state. In AD mouse models, knockout (KO) of TREM2 impairs microglial clustering around amyloid and prevents microglial activation. The R47H mutation is proposed to reduce TREM2 ligand binding. We investigated cell phenotypes of the R47H mutant and TREM2 KO in a model of human microglia, and compared their transcriptional signatures, to determine the mechanism by which R47H TREM2 disrupts function. METHODS: We generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines, with homozygous R47H mutation or TREM2 knockout (KO). We firstly validated the effect of the R47H mutant on TREM2 surface and subcellular localization in pMac. To assess microglial phenotypic function, we measured phagocytosis of dead neurons, cell morphology, directed migration, survival, and LPS-induced inflammation. We performed bulk RNA-seq, comparing significant differentially expressed genes (DEGs; p < 0.05) between the R47H and KO versus WT, and bioinformatically predicted potential upstream regulators of TREM2-mediated gene expression. RESULTS: R47H modified surface expression and shedding of TREM2, but did not impair TREM2-mediated signalling, or gross phenotypes that were dysregulated in the TREM2 KO (phagocytosis, motility, survival). However, altered gene expression in the R47H TREM2 pMac overlapped by 90% with the TREM2 KO and was characterised by dysregulation of genes involved with immunity, proliferation, activation, chemotaxis, and adhesion. Downregulated mediators of ECM adhesion included the vitronectin receptor αVß3, and consequently, R47H TREM2 pMac adhered weakly to vitronectin compared with WT pMac. To counteract these transcriptional defects, we investigated TGFß1, as a candidate upstream regulator. TGFß1 failed to rescue vitronectin adhesion of pMac, although it improved αVß3 expression. CONCLUSIONS: The R47H mutation is not sufficient to cause gross phenotypic defects of human pMac under standard culture conditions. However, overlapping transcriptional defects with TREM2 KO supports the hypothesised partial loss-of-function effects of the R47H mutation. Furthermore, transcriptomics can guide us to more subtle phenotypic defects in the R47H cells, such as reduced cell adhesion, and can be used to predict targets for therapeutic intervention.

TargetDB: A target information aggregation tool and tractability predictor.

When trying to identify new potential therapeutic protein targets, access to data and knowledge is increasingly important. In a field where new resources and data sources become available every day, it is crucial to be able to take a step back and look at the wider picture in order to identify potential drug targets. While this task is routinely performed by bespoke literature searches, it is often time-consuming and lacks uniformity when comparing multiple targets at one time. To address this challenge, we developed TargetDB, a tool that aggregates public information available on given target(s) (links to disease, safety, 3D structures, ligandability, novelty, etc.) and assembles it in an easy to read output ready for the researcher to analyze. In addition, we developed a target scoring system based on the desirable attributes of good therapeutic targets and machine learning classification system to categorize novel targets as having promising or challenging tractrability. In this manuscript, we present the methodology used to develop TargetDB as well as test cases.

A polycyclic aromatic hydrocarbon-enriched environmental chemical mixture enhances AhR, antiapoptotic signaling and a proliferative phenotype in breast cancer cells.

Emerging evidence suggests the role of environmental chemicals, in particular endocrine-disrupting chemicals (EDCs), in progression of breast cancer and treatment resistance, which can impact survival outcomes. However, most research tends to focus on tumor etiology and the effect of single chemicals, offering little insight into the effects of realistic complex mixture exposures on tumor progression. Herein, we investigated the effect of a polycyclic aromatic hydrocarbon (PAH)-enriched EDC mixture in a panel of normal and breast cancer cells and in a tumor organoid model. Cells or organoids in culture were treated with EDC mixture at doses estimated from US adult intake of the top four PAH compounds within the mixture from the National Health and Nutrition Examination Survey database. We demonstrate that low-dose PAH mixture (6, 30 and 300 nM) increased aryl hydrocarbon receptor (AhR) expression and CYP activity in estrogen receptor (ER) positive but not normal mammary or ER-negative breast cancer cells, and that upregulated AhR signaling corresponded with increased cell proliferation and expression of antiapoptotic and antioxidant proteins XIAP and SOD1. We employed a mathematical model to validate PAH-mediated increases in AhR and XIAP expression in the MCF-7 ER-positive cell line. Furthermore, the PAH mixture caused significant growth increases in ER-negative breast cancer cell derived 3D tumor organoids, providing further evidence for the role of a natural-derived PAH mixture in enhancing a tumor proliferative phenotype. Together, our integrated cell signaling, computational and phenotype analysis reveals the underlying mechanisms of EDC mixtures in breast cancer progression and survival.

Selective inhibition of the K+ efflux sensitive NLRP3 pathway by Cl- channel modulation.

The NLRP3 inflammasome regulates production of the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18, and contributes to inflammation exacerbating disease. Fenamate non-steroidal anti-inflammatory drugs (NSAIDs) were recently described as NLRP3 inflammasome inhibitors via chloride channel inhibition. Fenamate NSAIDs inhibit cyclooxygenase (COX) enzymes, limiting their potential as therapeutics for NLRP3-associated diseases due to established side effects. The aim here was to develop properties of the fenamates that inhibit NLRP3, and at the same time to reduce COX inhibition. We synthesised a library of analogues, with feedback from in silico COX docking potential, and IL-1ß release inhibitory activity. Through iterative screening and rational chemical design, we established a collection of chloride channel inhibiting active lead molecules with potent activity at the canonical NLRP3 inflammasome and no activity at COX enzymes, but only in response to stimuli that activated NLRP3 by a K+ efflux-dependent mechanism. This study identifies a model for the isolation and removal of unwanted off-target effects, with the enhancement of desired activity, and establishes a new chemical motif for the further development of NLRP3 inflammasome inhibitors.

A novel high-content imaging-based technique for measuring binding of Dickkopf-1 to low-density lipoprotein receptor-related protein 6.

INTRODUCTION: Dickkopf-related protein 1 (Dkk1) is a secreted protein ligand of low-density lipoprotein receptor-related protein 6 (LRP6), which antagonises canonical Wnt signalling. Elevated Dkk1 levels have been linked to Alzheimer's disease (AD), with protein blockade protective in pre-clinical AD models, suggesting inhibitors of Dkk1-LRP6 binding may have therapeutic utility against AD. Cell-based Dkk1-LRP6 assays reported in the literature use either modified Dkk1 protein and/or do not possess suitable throughput for drug screening. Here we report a novel immunocytochemical-based assay utilising high-content imaging (HCI) and automated data analysis suitable for the screening of protein and small-molecule inhibitors of Dkk1-LRP6 binding. METHODS: We developed an immunocytochemical (ICC) protocol to detect specific binding of exogenous human Dkk1 protein to human LRP6 transiently expressed in HEK293 cells. Images were generated using the PerkinElmer Operetta HCI System, after which quantitative data was generated using the PerkinElmer Columbus™ System. RESULTS: Our ICC technique and analysis pipeline allowed measurement of cell membrane-localised, LRP6-specific Dkk1 binding, normalised at individual cellular events. Saturation binding demonstrated concentration-dependent Dkk1 binding to LRP6, with a KD in keeping with reported values. Association kinetic experiments demonstrated the utility of the technique to investigate Dkk1 binding kinetics. Human Dkk members Dkk2 and Dkk4 fully displaced Dkk1 binding in a competition assay, while Dkk3 and Soggy-1/DkkL1 exhibited non-complete displacement of Dkk1. Finally gallocyanine, a previously reported inhibitor of Dkk1-LRP6 binding, fully displaced Dkk1 near the expected IC50. DISCUSSION: In conclusion, we provide a validated cell-based assay, suitable for the screening of inhibitors of Dkk1-LRP6 binding, and provide the basis for additional assay development, investigating Dkk1-LRP6 pharmacology.

Small vessels, dementia and chronic diseases - molecular mechanisms and pathophysiology.

Cerebral small vessel disease (SVD) is a major contributor to stroke, cognitive impairment and dementia with limited therapeutic interventions. There is a critical need to provide mechanistic insight and improve translation between pre-clinical research and the clinic. A 2-day workshop was held which brought together experts from several disciplines in cerebrovascular disease, dementia and cardiovascular biology, to highlight current advances in these fields, explore synergies and scope for development. These proceedings provide a summary of key talks at the workshop with a particular focus on animal models of cerebral vascular disease and dementia, mechanisms and approaches to improve translation. The outcomes of discussion groups on related themes to identify the gaps in knowledge and requirements to advance knowledge are summarized.

A standardized clinical evaluation of phenotypic diversity in diabetic polyneuropathy.

Diabetic polyneuropathy (DPN) is a major cause of neuropathic pain and a frequent target condition in analgesic treatment trials. Differences in the clinical symptoms and signs associated with DPN suggest distinct pathophysiological mechanisms underlying nerve damage and dysfunction that are likely to have therapeutic relevance. The aim of this study was to develop a tool for the bedside assessment of painful neuropathies such as DPN that captures the diversity of phenotypes. Sixty-one patients with type 2 diabetes and painful neuropathy, 19 patients with painless DPN, 25 patients with type 2 diabetes but no clinical evidence of neuropathy, and 20 healthy control subjects completed a structured interview (47 items) and a standardized physical examination (39 items). After analyzing critical features of pain and painless symptoms and examining the outcome of physical tests of sensory function, we determined principal components of the phenotypic variance among patients. Increased sensitivity to mechanical or thermal stimuli and, to a lesser extent, the sensory quality of pain or paresthesia were the most discriminating elements of DPN phenotypes. Correlation patterns of symptoms and signs indicated the involvement of functionally distinct nerve fiber populations. We combined interview questions and physical tests identifying these differences in a shortened assessment protocol that we named Standardized Evaluation of Pain and Somatosensory Function (StEPS). The protocol StEPS generates a phenotypic profile of patients with neuropathy. Separate intensity ratings for spontaneous painful symptoms and pain evoked by standard stimuli support a detailed documentation of neuropathic pain and its response to analgesic treatment.

Down-regulation of the ATP-binding cassette transporter 2 (Abca2) reduces amyloid-ß production by altering Nicastrin maturation and intracellular localization.

Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-ß (Aß) generation. In vitro studies revealed altered γ-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered γ-secretase processing of APP, and thus Aß generation, without affecting Notch cleavage.

Xenon reduces activation of transient receptor potential vanilloid type 1 (TRPV1) in rat dorsal root ganglion cells and in human TRPV1-expressing HEK293 cells.

AIMS: Xenon provides effective analgesia in several pain states at sub-anaesthetic doses. Our aim was to examine whether xenon may mediate its analgesic effect, in part, through reducing the activity of transient receptor potential vanilloid type 1 (TRPV1), a receptor known to be involved in certain inflammatory pain conditions. MAIN METHODS: We studied the effect of xenon on capsaicin-evoked cobalt uptake in rat cultured primary sensory neurons and in human TRPV1 (hTRPV1)-expressing human embryonic kidney 293 (HEK293) cells. We also examined xenon's effect on the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the rat spinal dorsal horn evoked by hind-paw injection of capsaicin. KEY FINDINGS: Xenon (75%) reduced the number of primary sensory neurons responding to the TRPV1 agonist, capsaicin (100 nM-1 µM) by ~25% to ~50%. Xenon reduced the number of heterologously-expressed hTRPV1 activated by 300 nM capsaicin by ~50%. Xenon (80%) reduced by ~40% the number of phosphorylated ERK1/2-expressing neurons in rat spinal dorsal horn resulting from hind-paw capsaicin injection. SIGNIFICANCE: Xenon substantially reduces the activity of TRPV1 in response to noxious stimulation by the specific TRPV1 agonist, capsaicin, suggesting a possible role for xenon as an adjunct analgesic where hTRPV1 is an active contributor to the excitation of primary afferents which initiates the pain sensation.

Neuroprotection by the selective iNOS inhibitor GW274150 in a model of Parkinson disease.

Neuroinflammation and the activation of inducible nitric oxide synthase (iNOS) have been proposed to play a role in the pathogenesis of Parkinson disease (PD). In this study we investigated the effects of the selective iNOS inhibitor GW274150 in the 6-OHDA model of PD. 6-OHDA administration was associated with increased numbers of cells expressing iNOS. Administration of the iNOS inhibitor twice daily for 7 days, beginning 2 days after the 6-OHDA lesioning, led to a significant neuroprotection as shown by assessment of the integrity of the nigrostriatal system by tyrosine hydroxylase immunocytochemistry and HPLC assessment of striatal dopamine content. However, GW274150 displayed a bell-shaped neuroprotective profile, being ineffective at high doses. 6-OHDA lesioning was associated with an increase in microglial activation as assessed by the MHC II antigen OX-6 and the number of matrix metalloproteinase 9 (MMP-9)-immunopositive cells. NO is a known modulator of MMP-9, and iNOS inhibition was associated with decreased numbers of MMP-9-immunopositive cells, culminating in a reduction in the numbers of reactive microglia. Withdrawal of GW274150 for a further 7 days negated any neuroprotective effects of iNOS inhibition, suggesting that the damaging effects of inflammation last beyond 7 days in this model and the continued administration of the drug may be required.