Developmental Associations between Neurovascularization and Microglia Colonization.

The temporal and spatial pattern of microglia colonization and vascular infiltration of the nervous system implies critical associated roles in early stages of nervous system development. Adding to existing reviews that cover a broad spectrum of the various roles of microglia during brain development, the current review will focus on the developmental ontogeny and interdependency between the colonization of the nervous system with yolk sac derived macrophages and vascularization. Gaining a better understanding of the timing and the interdependency of these two processes will significantly contribute to the interpretation of data generated regarding alterations in either process during early development. Additionally, such knowledge should provide a framework for understanding the influence of the early gestational environmental and the impact of genetics, disease, disorders, or exposures on the early developing nervous system and the potential for long-term and life-time effects.

Quantitative fluoride imaging of teeth using CaF emission by laser induced breakdown spectroscopy.

In this work, we propose the use of molecular emission of calcium fluoride (CaF) by laser induced breakdown spectroscopy (LIBS) to obtain quantitative fluoride distribution images of teeth. LIBS has proved to be an efficient technique to detect low amounts of fluoride in solids, and human teeth have the advantage being a matrix rich in calcium. We used new calibration material from sintered hydroxyapatite pellets doped with fluoride to determine the optimized LIBS conditions of argon flow at 1 L min-1 and using the green emission bands of CaF in 530 nm, and obtained a calibration curve between 0 and 400 µg g-1, and LOD of 18 µg g-1. This methodology was applied within a rat model of fluoride exposure and showed increasing tooth-fluoride with increased exposure dose. To demonstrate applicability of this method in human teeth, we quantified fluoride distribution in teeth from three children from non-fluorinated and fluorinated water regions. Samples from children living in fluoridated water regions showed higher fluoride concentrations in dentine formed after birth, compared to a child from a non-fluoridated region. Teeth have been used as biomarkers for environmental exposure and this new method opens the opportunity in epidemiology research to study critical windows of early life exposure to fluoride as well.

Advanced glycation end-products disrupt brain microvascular endothelial cell barrier: The role of mitochondria and oxidative stress.

During diabetes mellitus, advanced glycation end-products (AGEs) are major contributors to the development of alterations in cerebral capillaries, leading to the disruption of the blood-brain barrier (BBB). Consequently, this is often associated with an amplified oxidative stress response in microvascular endothelial cells. As a model to mimic brain microvasculature, the bEnd.3 endothelial cell line was used to investigate cell barrier function. Cells were exposed to native bovine serum albumin (BSA) or modified BSA (BSA-AGEs). In the presence or absence of the antioxidant compound, N-acetyl-cysteine, cell permeability was assessed by FITC-dextran exclusion, intracellular free radical formation was monitored with H2DCF-DA probe, and mitochondrial respiratory and redox parameters were analyzed. We report that, in the absence of alterations in cell viability, BSA-AGEs contribute to an increase in endothelial cell barrier permeability and a marked and prolonged oxidative stress response. Decreased mitochondrial oxygen consumption was associated with these alterations and may contribute to reactive oxygen species production. These results suggest the need for further research to explore therapeutic interventions to restore mitochondrial functionality in microvascular endothelial cells to improve brain homeostasis in pathological complications associated with glycation.

Autotaxin downregulates LPS-induced microglia activation and pro-inflammatory cytokines production.

Inflammation is essential in defense against infection or injury. It is tightly regulated, as over-response can be detrimental, especially in immune-privileged organs such as the central nervous system (CNS). Microglia constitutes the major source of inflammatory factors, but are also involved in the regulation of the inflammation and in the reparation. Autotaxin (ATX), a phospholipase D, converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and is upregulated in several CNS injuries. LPA, a pleiotropic immunomodulatory factor, can induce multiple cellular processes including morphological changes, proliferation, death, and survival. We investigated ATX effects on microglia inflammatory response to lipopolysaccharide (LPS), mimicking gram-negative infection. Murine BV-2 microglia and stable transfected, overexpressing ATX-BV-2 (A +) microglia were treated with LPS. Tumor necrosis factor α (TNFα), interleukin (IL)-6, and IL-10 mRNA and proteins levels were examined by qRT-PCR and ELISA, respectively. Secreted LPA was quantified by a radioenzymatic assay and microglial activation markers (CD11b, CD14, B7.1, and B7.2) were determined by flow cytometry. ATX expression and LPA production were significantly enhanced in LPS treated BV-2 cells. LPS induction of mRNA and protein level for TNFα and IL-6 were inhibited in A+ cells, while IL-10 was increased. CD11b, CD14, and B7.1, and B7.2 expressions were reduced in A+ cells. Our results strongly suggest deactivation of microglia and an IL-10 inhibitory of ATX with LPS induced microglia activation.

A novel deep learning-based method for automatic stereology of microglia cells from low magnification images.

Microglial cells mediate diverse homeostatic, inflammatory, and immune processes during normal development and in response to cytotoxic challenges. During these functional activities, microglial cells undergo distinct numerical and morphological changes in different tissue volumes in both rodent and human brains. However, it remains unclear how these cytostructural changes in microglia correlate with region-specific neurochemical functions. To better understand these relationships, neuroscientists need accurate, reproducible, and efficient methods for quantifying microglial cell number and morphologies in histological sections. To address this deficit, we developed a novel deep learning (DL)-based classification, stereology approach that links the appearance of Iba1 immunostained microglial cells at low magnification (20×) with the total number of cells in the same brain region based on unbiased stereology counts as ground truth. Once DL models are trained, total microglial cell numbers in specific regions of interest can be estimated and treatment groups predicted in a high-throughput manner (<1 min) using only low-power images from test cases, without the need for time and labor-intensive stereology counts or morphology ratings in test cases. Results for this DL-based automatic stereology approach on two datasets (total 39 mouse brains) showed >90% accuracy, 100% percent repeatability (Test-Retest) and 60× greater efficiency than manual stereology (<1 min vs. ∼ 60 min) using the same tissue sections. Ongoing and future work includes use of this DL-based approach to establish clear neurodegeneration profiles in age-related human neurological diseases and related animal models.

SDF-1α and LPA modulate microglia potassium channels through rho gtpases to regulate cell morphology.

Microglia are the resident immune cells of the brain, which are important therapeutic targets for regulating the inflammatory responses particularly neurodegeneration in the aging human brain. The activation, chemotaxis and migration of microglia are regulated through G-protein coupled receptors by chemokines such as stromal cell-derived factor (SDF)-1α and bioactive lysophospholipids such as lysophosphatidic acid (LPA). Potassium channels play important roles in microglial function and cell fate decisions; however, the regulation of microglial potassium channels has not been fully elucidated. Here we show reciprocal action of SDF-1α and LPA, on potassium currents through Kir2.1 channels in primary murine microglia. The potassium channel modulation is mediated by the same small GTPases, Rac and Rho that regulate the actin cytoskeleton. SDF-1α rapidly increased the Kir2.1 current amplitude and cell spreading. These effects were mimicked by dialysing the cells with constitutively active Rac1 protein, and they were blocked by inhibiting the phosphatidylinositol 3-kinase (PI3K) with wortmannin. In contrast, LPA and constitutively active RhoA decreased the Kir2.1 currents and stimulated cell contraction. Thus, SDF-1α and LPA regulate both the actin cytoskeleton and the Kir2.1 potassium channels through the same Rho GTPase signaling pathways. The inhibition of Kir2.1 with chloroethylclonidine produced cell contraction independently of chemokine action. This suggests that potassium channels are essential for the morphological phenotype and functioning of microglia. In conclusion, the small GTPases, Rac and Rho, modulate Kir2.1 channels and block of Kir2.1 channels causes changes in microglia morphology.

Interleukin-6 (IL-6) receptor/IL-6 fusion protein (Hyper IL-6) effects on the neonatal mouse brain: possible role for IL-6 trans-signaling in brain development and functional neurobehavioral outcomes.

Adverse neurodevelopmental outcomes are linked to perinatal production of inflammatory mediators, including interleukin 6 (IL-6). While a pivotal role for maternal elevation in IL-6 has been established in determining neurobehavioral outcomes in the offspring and considered the primary target mediating the fetal inflammatory response, questions remain as to the specific actions of IL-6 on the developing brain. CD-1 male mice received a subdural injection of the bioactive fusion protein, hyper IL-6 (HIL-6) on postnatal-day (PND)4 and assessed from preweaning until adulthood. Immunohistochemical evaluation of astrocytes and microglia and mRNA levels for pro-inflammatory cytokines and host response genes indicated no evidence of an acute neuroinflammatory injury response. HIL-6 accelerated motor development and increased reactivity to stimulation and number of entries in a light/dark chamber, decreased ability to learn to withhold a response in passive avoidance, and effected deficits in social novelty behavior. No changes were observed in motor activity, pre-pulse startle inhibition, or learning and memory in the Morris water maze or radial arm maze, as have been reported for models of more severe developmental neuroinflammation. In young animals, mRNA levels for MBP and PLP/DM20 decreased and less complexity of MBP processes in the cortex was evident by immunohistochemistry. The non-hydroxy cerebroside fraction of cerebral lipids was increased. These results provide evidence for selective effects of IL-6 signaling, particularly trans-signaling, in the developing brain in the absence of a general neuroinflammatory response. These data contribute to our further understanding of the multiple aspects of IL-6 signaling in the developing brain.

High Content Imaging and Quantification of Microglia Phagocytosis In Vitro.

Microglia function as the tissue-specific resident macrophages of the nervous system, performing immune and non-immune functions. These functions are critical to development and to maintain homeostasis in the nervous system throughout the lifespan, and during brain injury or disease. One method by which microglia maintain homeostasis is phagocytosis of aberrant proteins, extracellular debris, synapses, or apoptotic cells. Phagocytic function can be changed by environmental or genetic risk factors that affect microglia. These protocols present a rapid and simple in vitro high-content imaging protocol for studying phagocytosis in the murine microglia BV-2 cell line. High-content imaging and analysis enable versatility of the assay, which can be used to test multiple experimental conditions, or as a screening tool. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Phagocytosis of fluorescently labeled particles Basic Protocol 2: Examining modifications to phagocytosis by test substances Basic Protocol 3: High content imaging and analysis of phagocytic cells.

Differential gene expression profiling implicates altered network development in rat postnatal day 4 cortex following 4-Methylimidazole (4-MeI) induced maternal seizures.

Compromised maternal health leading to maternal seizures can have adverse effects on the healthy development of offspring. This may be the result of inflammation, hypoxia-ischemia, and altered GABA signaling. The current study examined cortical tissue from F2b (2nd litter of the 2nd generation) postnatal day 4 (PND4) offspring of female Harlan SD rats chronically exposed to the seizuregenic compound, 4-Methylimidazole (0, 750, or 2500 ppm 4-MeI). Maternal seizures were evident only at 2500 ppm 4-MeI. GABA related gene expression as examined by qRT-PCR and whole genome microarray showed no indication of disrupted GABA or glutamatergic signaling. Canonical pathway hierarchical clustering and multi-omics combinatory genomic (CNet) plots of differentially expressed genes (DEG) showed alterations in genes associated with regulatory processes of cell development including neuronal differentiation and synaptogenesis. Functional enrichment analysis showed a similarity of cellular processes across the two exposure groups however, the genes comprising each cluster were primarily unique rather than shared and often showed different directionality. A dose-related induction of cytokine signaling was indicated however, pathways associated with individual cytokine signaling were not elevated, suggesting an alternative involvement of cytokine signaling. Pathways related to growth process and cell signaling showed a negative activation supporting an interpretation of disruption or delay in developmental processes at the 2500 ppm 4-MeI exposure level with maternal seizures. Thus, while GABA signaling was not altered as has been observed with maternal seizures, the pattern of DEG suggested a potential for alteration in neuronal network formation.

Bilateral common carotid artery ligation transiently changes brain lipid metabolism in rats.

Brain lipid metabolism was studied in rats following permanent bilateral common carotid artery ligation (BCCL), a model for chronic cerebral hypoperfusion. Unesterified (free) fatty acids (uFA) and acyl-CoA concentrations were measured 6 h, 24 h, and 7 days after BCCL or sham surgery, in high energy-microwaved brain. In BCCL compared to sham rats, cytosolic phospholipase A(2) (cPLA(2)) immunoreactivity in piriform cortex, and concentrations of total uFA and arachidonoyl-CoA, an intermediate for arachidonic acid reincorporation into phospholipids, were increased only at 6 h. At 24 h, immunoreactivity for secretory phospholipase A(2) (sPLA(2)), which may regulate blood flow, was increased near cortical and hippocampal blood vessels. BCCL did not affect levels of brain IB(4)+ microglia, glial fibrillary acidic protein (GFAP)+ astrocytes, cyclooxygenase-2 (COX-2) immunoreactivity at any time, but increased cPLA(2) immunoreactivity in one region at 6 h. Thus, BCCL affected brain lipid metabolism transiently, likely because of compensatory sPLA(2)-mediated vasodilation, without producing evidence of neuroinflammation.

Imaging Inflammasome Activation in Microglia.

Inflammasomes are multiprotein complexes that play key roles in the host's innate immune response to insult. The assembly of an inflammatory complex is initiated with the oligomerization of the upstream inflammasome-forming sensor and then follows a well-orchestrated multi-step process leading to downstream effector functions that are critical in the innate immune response. The final assembly of these steps provides a detectable readout of inflammasome complex activation in the form of an apoptosis-associated speck-like protein containing a CARD (ASC) speck. Inflammasome activation-and the release of IL-1ß and ASC specks from the microglia, the brain resident immune cell-have been implicated in various neurological and neurodegenerative disorders. Protocols exist for the generation of fluorescent inflammasome indicator peripheral macrophages. Building upon these protocols, we describe here a protocol that details the generation of fluorescent inflammasome indicator microglia cells using recombinant retroviruses to transduce murine BV-2 cells. In this protocol, the cells are established in a manner to allow for experimental control of the initial priming step of the inflammasome activation process. We then provide a series of steps for using these reporter cells within an inflammasome activation assay and use real-time imaging of ASC-speck formation as an indicator of inflammasome activation. In addition, we describe strategies for using these cells for examining the effects of a test substance on inflammasome activation. This protocol offers an effective approach conducive to screening for and examining modifications of microglia inflammasome activation due to exposure to chemicals or pharmacological agents. © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Production of retroviruses to express inflammasome indicator Basic Protocol 2: Generation of inflammasome indicator BV-2 cells Basic Protocol 3: Priming and activation of BV-2-ASC-Cerulean cells for inflammasome activation assay Basic Protocol 4: Examining modifications to inflammasome activation by test substances Basic Protocol 5: Imaging and analysis of ASC speck formation.

Roadbumps at the Crossroads of Integrating Behavioral and In Vitro Approaches for Neurotoxicity Assessment.

With the appreciation that behavior represents the integration and complexity of the nervous system, neurobehavioral phenotyping and assessment has seen a renaissance over the last couple of decades, resulting in a robust database on rodent performance within various testing paradigms, possible associations with human disorders, and therapeutic interventions. The interchange of data across behavior and other test modalities and multiple model systems has advanced our understanding of fundamental biology and mechanisms associated with normal functions and alterations in the nervous system. While there is a demonstrated value and power of neurobehavioral assessments for examining alterations due to genetic manipulations, maternal factors, early development environment, the applied use of behavior to assess environmental neurotoxicity continues to come under question as to whether behavior represents a sensitive endpoint for assessment. Why is rodent behavior a sensitive tool to the neuroscientist and yet, not when used in pre-clinical or chemical neurotoxicity studies? Applying new paradigms and evidence on the biological basis of behavior to neurobehavioral testing requires expertise and refinement of how such experiments are conducted to minimize variability and maximize information. This review presents relevant issues of methods used to conduct such test, sources of variability, experimental design, data analysis, interpretation, and reporting. It presents beneficial and critical limitations as they translate to the in vivo environment and considers the need to integrate across disciplines for the best value. It proposes that a refinement of behavioral assessments and understanding of subtle pronounced differences will facilitate the integration of data obtained across multiple approaches and to address issues of translation.

Integrating Environment and Aging Research: Opportunities for Synergy and Acceleration.

Despite significant overlaps in mission, the fields of environmental health sciences and aging biology are just beginning to intersect. It is increasingly clear that genetics alone does not predict an individual's neurological aging and sensitivity to disease. Accordingly, aging neuroscience is a growing area of mutual interest within environmental health sciences. The impetus for this review came from a workshop hosted by the National Academies of Sciences, Engineering, and Medicine in June of 2020, which focused on integrating the science of aging and environmental health research. It is critical to bridge disciplines with multidisciplinary collaborations across toxicology, comparative biology, epidemiology to understand the impacts of environmental toxicant exposures and age-related outcomes. This scoping review aims to highlight overlaps and gaps in existing knowledge and identify essential research initiatives. It begins with an overview of aging biology and biomarkers, followed by examples of synergy with environmental health sciences. New areas for synergistic research and policy development are also discussed. Technological advances including next-generation sequencing and other-omics tools now offer new opportunities, including exposomic research, to integrate aging biomarkers into environmental health assessments and bridge disciplinary gaps. This is necessary to advance a more complete mechanistic understanding of how life-time exposures to toxicants and other physical and social stressors alter biological aging. New cumulative risk frameworks in environmental health sciences acknowledge that exposures and other external stressors can accumulate across the life course and the advancement of new biomarkers of exposure and response grounded in aging biology can support increased understanding of population vulnerability. Identifying the role of environmental stressors, broadly defined, on aging biology and neuroscience can similarly advance opportunities for intervention and translational research. Several areas of growing research interest include expanding exposomics and use of multi-omics, the microbiome as a mediator of environmental stressors, toxicant mixtures and neurobiology, and the role of structural and historical marginalization and racism in shaping persistent disparities in population aging and outcomes. Integrated foundational and translational aging biology research in environmental health sciences is needed to improve policy, reduce disparities, and enhance the quality of life for older individuals.

Prolonged, Low-Level Exposure to the Marine Toxin, Domoic Acid, and Measures of Neurotoxicity in Nonhuman Primates.

BACKGROUND: The excitotoxic molecule, domoic acid (DA), is a marine algal toxin known to induce overt hippocampal neurotoxicity. Recent experimental and epidemiological studies suggest adverse neurological effects at exposure levels near the current regulatory limit (20 ppm, ∼0.075-0.1mg/kg). At these levels, cognitive effects occur in the absence of acute symptoms or evidence of neuronal death. OBJECTIVES: This study aimed to identify adverse effects on the nervous system from prolonged, dietary DA exposure in adult, female Macaca fascicularis monkeys. METHODS: Monkeys were orally exposed to 0, 0.075, and 0.15mg/kg per day for an average of 14 months. Clinical blood counts, chemistry, and cytokine levels were analyzed in the blood. In-life magnetic resonance (MR) imaging assessed volumetric and tractography differences in and between the hippocampus and thalamus. Histology of neurons and glia in the fornix, fimbria, internal capsule, thalamus, and hippocampus was evaluated. Hippocampal RNA sequencing was used to identify differentially expressed genes. Enrichment of gene networks for neuronal health, excitotoxicity, inflammation/glia, and myelin were assessed with Gene Set Enrichment Analysis. RESULTS: Clinical blood counts, chemistry, and cytokine levels were not altered with DA exposure in nonhuman primates. Transcriptome analysis of the hippocampus yielded 748 differentially expressed genes (fold change≥1.5; p≤0.05), reflecting differences in a broad molecular profile of intermediate early genes (e.g., FOS, EGR) and genes related to myelin networks in DA animals. Between exposed and control animals, MR imaging showed comparable connectivity of the hippocampus and thalamus and histology showed no evidence of hypomyelination. Histological examination of the thalamus showed a larger microglia soma size and an extension of cell processes, but suggestions of a GFAP+astrocyte response showed no indication of astrocyte hypertrophy. DISCUSSION: In the absence of overt hippocampal excitotoxicity, chronic exposure of Macaca fascicularis monkeys to environmentally relevant levels of DA suggested a subtle shift in the molecular profile of the hippocampus and the microglia phenotype in the thalamus that was possibly reflective of an adaptive response due to prolonged DA exposure. https://doi.org/10.1289/EHP10923.

Exposure to an organometal compound stimulates adipokine and cytokine expression in white adipose tissue.

OBJECTIVE: White adipose tissue (WAT) is now considered a defined tissue capable of interactions with other organ systems. WAT role in elevating the level of systemic chronic inflammation suggests that alterations in this tissue as the result of disease or environmental factors may influence the development and progression of various obesity-related pathologies. This study investigated WAT cell-specific responses to an organometal compound, trimethyltin (TMT), to determine possible contribution to induced inflammation. METHODS: Human primary mature adipocytes and macrophage differentiated THP-1 cells were cultured in TMT presence and relative toxicities and different adipokine levels were determined. The inflammatory response was examined in TMT presence for primary cells from obese ob/ob mice WAT, and after TMT injection in ob/ob mice. RESULTS: Both adipocytes and macrophages were resistant to cell death induced by TMT. However, adipocytes cultured in TMT presence showed increased expression of TNFα and IL-6, and modified leptin levels. In macrophage cultures, TMT also increased TNFα and IL-6, while MCP-1 and MIP-1α were decreased. In vivo, a single injection of TMT in ob/ob mice, elevated TNFα, MIP-1α and adiponectin in WAT. CONCLUSIONS: Elevation of the inflammatory related products can be induced by chemical exposure in adipocytes and macrophages, as well as murine WAT. These data suggest that numerous factors, including a systemic chemical exposure, can induce an inflammatory response from the WAT. Furthermore, when characterizing both chemical-induced toxicity and the progression of the chronic inflammation associated with elevated WAT content, such responses in this target tissue should be taken into consideration.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

BACKGROUND: Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin (IL)-1ß. IL-1ß plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders. OBJECTIVE: The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and IL-1ß processing. METHOD: Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, IL-1ß enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays. RESULTS: TETBr and TMTOH induced inflammasome aggregate formation and IL-1ß release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or LPS+ATP challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways. CONCLUSION: Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314.

Altered cerebellar development in nuclear receptor TAK1/ TR4 null mice is associated with deficits in GLAST(+) glia, alterations in social behavior, motor learning, startle reactivity, and microglia.

Previously, deficiency in the expression of the nuclear orphan receptor TAK1 was found to be associated with delayed cerebellar granule cell migration and Purkinje cell maturation with a permanent deficit in foliation of lobules VI­VII, suggesting a role for TAK1 in cerebellum development. In this study, we confirm that TAK1-deficient (TAK1(−/−)) mice have a smaller cerebellum and exhibit a disruption of lobules VI­VII. We extended these studies and show that at postnatal day 7, TAK1(−/−) mice exhibit a delay in monolayer maturation of dysmorphic calbindin 28K-positive Purkinje cells. The astrocyte-specific glutamate transporter (GLAST) was expressed within Bergmann fibers and internal granule cell layer at significantly lower levels in the cerebellum of TAK1(−/−) mice. At PND21, Golgi-positive Purkinje cells in TAK1(−/−) mice displayed a smaller soma (18%) and shorter distance to first branch point (35%). Neuronal death was not observed in TAK1(−/−) mice at PND21; however, activated microglia were present in the cerebellum, suggestive of earlier cell death. These structural deficits in the cerebellum were not sufficient to alter motor strength, coordination, or activity levels; however, deficits in acoustic startle response, prepulse startle inhibition, and social interactions were observed. Reactions to a novel environment were inhibited in a light/dark chamber, open-field, and home-cage running wheel. TAK1(−/−) mice displayed a plateau in performance on the running wheel, suggesting a deficit in learning to coordinate performance on a motor task. These data indicate that TAK1 is an important transcriptional modulator of cerebellar development and neurodevelopmentally regulated behavior.

An association between mitochondria and microglia effector function. What do we think we know?

While resident innate immune cells of the central nervous system, the microglia, represent a cell population unique in origin, microenvironment, and longevity, they assume many properties displayed by peripheral macrophages. One prominent shared property is the ability to undergo a metabolic switch towards glycolysis and away from oxidative phosphorylation (OXPHOS) upon activation by the pro-inflammatory stimuli lipopolysaccharide. This shift serves to meet specific cellular demands and allows for cell survival, similar to the Warburg effect demonstrated in cancer cells. In contrast, normal survelliance phenotype or stimulation to a non-proinflammatory phenotype relies primarily on OXPHOS and fatty acid oxidation. Thus, mitochondria appear to function as a pivotal signaling platform linking energy metabolism and macrophage polarization upon activation. These unique shifts in cell bioenergetics in response to different stimuli are essential for proper effector responses at sites of infection, inflammation, or injury. Here we present a summary of recent developments as to how these dynamics characterized in peripheral macrophages are displayed in microglia. The new insights provided by an increased understanding of metabolic reprogramming in macrophages may allow for translation to the CNS and a better understanding of microglia heterogeneity, regulation, and function.

Mitochondrial-related effects of pentabromophenol, tetrabromobisphenol A, and triphenyl phosphate on murine BV-2 microglia cells.

The predominant reliance on bromated flame retardants (BFRs) is diminishing with expanded use of alternative organophosphate flame retardants. However, exposure related issues for susceptible populations, the developing, infirmed, or aged, remain given environmental persistence and home-environment detection. In this regard, reports of flame retardant (FR)-related effects on the innate immune system suggest process by which a spectrum of adverse health effects could manifest across the life-span. As representative of the nervous system innate immune system, the current study examined changes in microglia following exposure to representative FRs, pentabromophenol (PBP), tetrabromobisphenol A (2,2',6,6',-tetrabromo-4,4'-isopropylidine diphenol; TBBPA) and triphenyl phosphate (TPP). Following 18hr exposure of murine BV-2 cells, at dose levels resulting in ≥80% viability (10 and 40 µM), limited alterations in pro-inflammatory responses were observed however, changes were observed in mitochondrial respiration. Basal respiration was altered by PBP; ATP-linked respiration by PBP and TBBPA, and maximum respiration by all three FRs. Basal glycolytic rate was altered by PBP and TBBPA and compensatory glycolysis by all three. Phagocytosis was decreased for PBP and TBBPA. NLRP3 inflammasome activation was assessed using BV-2-ASC (apoptosis-associated speck-like protein containing a CARD) reporter cells to visualize aggregate formation. PBP, showed a direct stimulation of aggregate formation and properties as a NLRP3 inflammasome secondary trigger. TBBPA showed indications of possible secondary triggering activity while no changes were seen with TPP. Thus, the data suggests an effect of all three FRs on mitochondria metabolism yet, different functional outcomes including, phagocytic capability and NLRP3 inflammasome activation.

Age-Related Decrease in Tyrosine Hydroxylase Immunoreactivity in the Substantia Nigra and Region-Specific Changes in Microglia Morphology in HIV-1 Tg Rats.

Animal models have been used to study cellular processes related to human immunodeficiency virus-1 (HIV-1)-associated neurocognitive disorders (HAND). The HIV-1 transgenic (Tg) rat expresses HIV viral genes except the gag-pol replication genes and exhibits neuropathological features similar to HIV patients receiving combined antiretroviral therapy (cART). Using this rat, alterations in dopaminergic function have been demonstrated; however, the data for neuroinflammation and glial reactivity is conflicting. Differences in behavior, tyrosine hydroxylase (TH) immunoreactivity, neuroinflammation, and glia reactivity were assessed in HIV-1 Tg male rats. At 6 and 12 weeks of age, rotarod performance was diminished, motor activity was not altered, and active avoidance latency performance and memory were diminished in HIV-1 Tg rats. TH+ immunoreactivity in the substantia nigra (SN) was decreased at 8 months but not at 2-5 months. At 5 months, astrocyte and microglia morphology was not altered in the cortex, hippocampus, or SN. In the striatum, astrocytes were unaltered, microglia displayed slightly thickened proximal processes, mRNA levels for Iba1 and Cd11b were elevated, and interleukin (Il)1α,Cxcr3, and cell adhesion molecule, Icam, decreased. In the hippocampus, mRNA levels for Tnfa and Cd11b were slightly elevated. No changes were observed in the cortex or SN. The data support an age-related effect of HIV proteins upon the nigrostriatal dopaminergic system and suggest an early response of microglia in the terminal synaptic region with little evidence of an associated neuroinflammatory response across brain regions.