Organotypic Hippocampal Slice Cultures Research Articles

Development of P301S tau seeded organotypic hippocampal slice cultures to study potential therapeutics

Intracellular tau inclusions are a pathological hallmark of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and other sporadic neurodegenerative tauopathies. Recent in vitro and in vivo studies have shown that tau aggregates may spread to neighbouring cells and functionally connected brain regions, where they can seed further tau aggregation. This process is referred to as tau propagation. Here we describe an ex vivo system using organotypic hippocampal slice cultures (OHCs) which recapitulates aspects of this phenomenon. OHCs are explants of hippocampal tissue which may be maintained in culture for months. They maintain their synaptic connections and multicellular 3D architecture whilst also permitting direct control of the environment and direct access for various analysis types. We inoculated OHCs prepared from P301S mouse pups with brain homogenate from terminally ill P301S mice and then examined the slices for viability and the production and localization of insoluble phosphorylated tau. We show that following seeding, phosphorylated insoluble tau accumulate in a time and concentration dependent manner within OHCs. Furthermore, we show the ability of the conformation dependent anti-tau antibody, MC1, to compromise tau accrual in OHCs, thus showcasing the potential of this therapeutic approach and the utility of OHCs as an ex vivo model system for assessing such therapeutics.

A Highly Efficient Method for Single-Cell Electroporation in Mouse Organotypic Hippocampal Slice Culture (2605)

A Highly Efficient Method for Single-Cell Electroporation in Mouse Organotypic Hippocampal Slice Culture (2605)

Effects of a Multimerized Recombinant Autoantibody Against Amyloid-β

Alzheimer’s disease (AD) is the most common neurodegenerative disease; thus, the search for a cure or causal therapy has become necessary. Despite intense research on this topic in recent decades, there is no curative therapy up today, and also no disease-modifying treatment has been approved. As promising approach passive immunization strategies have thereby come forth. In this study, we focused on naturally occurring autoantibodies against the AD-associated peptide amyloid-β. These antibodies have already reported to show beneficial functions in vitro and in mouse models of AD. However, their availability is limited due to their low abundance in peripheral blood. In a recent study, we were able to generate four recombinant antibodies against amyloid-β. In the present study, we tested these antibodies in ELISA and SPR assays for their binding behavior and by aggregation- and phagocytosis assays as functional evidences to characterize their amyloid-β-related neutralizing and clearance abilities. Further ex vivo assay on organotypic hippocampal slice cultures gave first evidence of microglial activation and inflammatory features. The tested recombinant antibodies in IgG format showed, in comparison to naturally occurring autoantibodies against amyloid-β, insufficient binding capacities and -affinities. However, after conversion of one antibody into a single chain format multimerization of the scFv-Fc construct, the investigated binding capacity and -affinity showed improvements. Further functional assays predict a protective effect of this antibody. Although, all four recombinant antibodies showed binding to amyloid-β, promising features were only detectable after conversion into a multimeric format. The multimeric scFv-Fc antibody exhibited thereby strong impact on amyloid-β clearance and inhibition of oligomerization.

Spatial learning and memory deficits induced by prenatal glucocorticoid exposure depend on hippocampal CRHR1 and CXCL5 signaling in rats

BackgroundPrenatal synthetic glucocorticoid (sGC) exposure increases the susceptibility to cognitive and affective disorders in postnatal life. We previously demonstrated that prenatal sGC exposure results in an increase in corticotropin-releasing hormone (CRH) receptor type 1 (CRHR1) expression in the hippocampus of rats, and CRHR1 is involved in synapse formation via regulation of C-X-C chemokine ligand 5 (CXCL5) in hippocampus. We sought to investigate that the roles of CRHR1 and CXCL5 in learning and memory impairment caused by prenatal sGC exposure.MethodsPregnant rats were administered with saline or dexamethasone (DEX) from gestational day (GD) 14 to GD21. DEX offspring at 2-day old were treated with saline and CRHR1 antagonists (antalarmin and CP154526) for 7 days. Some DEX offspring received intra-hippocampal injection of AAV9 carrying CXCL5 gene. Spatial learning and memory was assessed by Morris water maze test. Immunofluorescence analysis was applied to show synapsin I and PSD95 signals in hippocampus. Synapsin I and PSD95 protein level and CXCL5 concentration were determined by western blotting and ELISA, respectively. Organotypic hippocampal slice cultures were used to investigate the effect of DEX on CXCL5 production in vitro.ResultsBoth male and female DEX offspring displayed impairment of spatial learning and memory in adulthood. Synapsin I and PSD95 signals and CXCL5 levels were decreased in DEX offspring. DEX offspring with antalarmin and CP154526 treatment showed improved spatial learning and memory. Antalarmin and CP154526 treatment increased synapsin I and PSD95 signals and CXCL5 concentration in hippocampus. Bilaterally hippocampal injection of AAV9 carrying CXCL5 gene improved the spatial learning and memory and increased CXCL5 concentration and synapsin I and PSD95 levels in hippocampus. DEX dose-dependently suppressed CXCL5 production in cultured hippocammpal slices, which was prevented by antalarmin treatment.ConclusionCRHR1 and CXCL5 signaling in the hippocampus are involved in spatial learning and memory deficits caused by prenatal DEX exposure. CRHR1 activation contributes to decreased CXCL5 production in hippocampus induced by prenatal DEX treatment. Our study provides a molecular basis of prenatal GC exposure programming spatial learning and memory.

Efficient infection of organotypic hippocampal slice cultures with adenovirus carrying the transgene REST/NRSF

Organotypic hippocampal slice cultures provide a useful platform maintaining hippocampal structure and synaptic connections of the brain over weeks in culture with ease of in vitro manipulations. Gene transfer is a particularly desirable tool for using with them but current difficulties with transformation of transgenes into these cultures is a barrier to their use in research. Previous quantifications of viral infections have shown low transformation rates and have relied upon invasive microinjections. In this paper we present an efficient way of infecting organotypic cultures with adenovirus at the acute slice stage that does not require injection. We use the adenoviral delivery system to introduce the transcription factor REST and a GFP marker, providing around 41 % cellular infection spread throughout the entire slice culture and promoting transgene expression for weeks in vitro. GFP expression was observed most intensely in the slices when they were infected just a few hours after plating and was shown to infect neurons and microglia. We decided to use the transcription factor REST/NRSF as an example transgene which was delivered into cells via the adenoviral construct, conferring overexpression of REST in addition to the GFP marker. This outlines a technique whereby adenoviral infection of organotypic cultures can infect neurons with good efficiency and confer successful manipulation of genetic factors within the cell.

Tau assemblies do not behave like independently acting prion-like particles in mouse neural tissue

A fundamental property of infectious agents is their particulate nature: infectivity arises from independently-acting particles rather than as a result of collective action. Assemblies of the protein tau can exhibit seeding behaviour, potentially underlying the apparent spread of tau aggregation in many neurodegenerative diseases. Here we ask whether tau assemblies share with classical pathogens the characteristic of particulate behaviour. We used organotypic hippocampal slice cultures from P301S tau transgenic mice in order to precisely control the concentration of extracellular tau assemblies in neural tissue. Whilst untreated slices displayed no overt signs of pathology, exposure to recombinant tau assemblies could result in the formation of intraneuronal, hyperphosphorylated tau structures. However, seeding ability of tau assemblies did not titrate in a one-hit manner in neural tissue. The results suggest that seeding behaviour of tau arises at high concentrations, with implications for the interpretation of high-dose intracranial challenge experiments and the possible contribution of seeded aggregation to human disease.

Microglia-derived interleukin-10 accelerates post-intracerebral hemorrhage hematoma clearance by regulating CD36

Hematoma size after intracerebral hemorrhage (ICH) significantly affects patient outcome. However, our knowledge of endogenous mechanisms that underlie hematoma clearance and the potential role of the anti-inflammatory cytokine interleukin-10 (IL-10) is limited. Using organotypic hippocampal slice cultures and a collagenase-induced ICH mouse model, we investigated the role of microglial IL-10 in phagocytosis ex vivo and hematoma clearance in vivo. In slice culture, exposure to hemoglobin induced IL-10 expression in microglia and enhanced phagocytosis that depended on IL-10–regulated expression of CD36. Following ICH, IL-10–deficient mice had more severe neuroinflammation, brain edema, iron deposition, and neurologic deficits associated with delayed hematoma clearance. Intranasal administration of recombinant IL-10 accelerated hematoma clearance and improved neurologic function. Additionally, IL-10–deficient mice had weakened in vivo phagocytic ability owing to decreased expression of microglial CD36. Moreover, loss of IL-10 significantly increased monocyte-derived macrophage infiltration and enhanced brain inflammation in vivo. These results indicate that IL-10 regulates microglial phagocytosis and monocyte-derived macrophage infiltration after ICH and that CD36 is a key phagocytosis effector regulated by IL-10. Leveraging the innate immune response to ICH by augmenting IL-10 signaling may provide a useful strategy for accelerating hematoma clearance and improving neurologic outcome in clinical translation studies.

A hierarchy of manganese competition and entry in organotypic hippocampal slice cultures.

Contrast agents improve clinical and basic research MRI. The manganese ion (Mn2+) is an essential, endogenous metal found in cells and it enhances MRI contrast because of its paramagnetic properties. Manganese‐enhanced MRI (MEMRI) has been widely used to image healthy and diseased states of the body and the brain in a variety of animal models. There has also been some work in translating the useful properties of MEMRI to humans. Mn2+ accumulates in brain regions with high neural activity and enters cells via voltage‐dependent channels that flux calcium (Ca2+). In addition, metal transporters for zinc (Zn2+) and iron (Fe2+) can also transport Mn2+. There is also transfer through channels specific for Mn2+. Although Mn2+ accumulates in many tissues including brain, the mechanisms and preferences of its mode of entry into cells are not well characterized. The current study used MRI on living organotypic hippocampal slice cultures to detect which transport mechanisms are preferentially used by Mn2+ to enter cells. The use of slice culture overcomes the presence of the blood brain barrier, which limits inferences made with studies of the intact brain in vivo. A range of Mn2+ concentrations were used and their effects on neural activity were assessed to avoid using interfering doses of Mn2+. Zn2+ and Fe2+ were the most efficient competitors for Mn2+ uptake into the cultured slices, while the presence of Ca2+ or Ca2+ channel antagonists had a more moderate effect. Reducing slice activity via excitatory receptor antagonists was also effective at lowering Mn2+ uptake. In conclusion, a hierarchy of those agents which influence Mn2+ uptake was established to enhance understanding of how Mn2+ enters cells in a cultured slice preparation.

In-depth quantitative proteomic characterization of organotypic hippocampal slice culture reveals sex-specific differences in biochemical pathways

Sex differences in the brain of mammals range from neuroarchitecture through cognition to cellular metabolism. The hippocampus, a structure mostly associated with learning and memory, presents high vulnerability to neurodegeneration and aging. Therefore, we explored basal sex-related differences in the proteome of organotypic hippocampal slice culture, a major in vitro model for studying the cellular and molecular mechanisms related to neurodegenerative disorders. Results suggest a greater prevalence of astrocytic metabolism in females and significant neuronal metabolism in males. The preference for glucose use in glycolysis, pentose phosphate pathway and glycogen metabolism in females and high abundance of mitochondrial respiration subunits in males support this idea. An overall upregulation of lipid metabolism was observed in females. Upregulation of proteins responsible for neuronal glutamate and GABA synthesis, along with synaptic associated proteins, were observed in males. In general, the significant spectrum of pathways known to predominate in neurons or astrocytes, together with the well-known neuronal and glial markers observed, revealed sex-specific metabolic differences in the hippocampus. TEM qualitative analysis might indicate a greater presence of mitochondria at CA1 synapses in females. These findings are crucial to a better understanding of how sex chromosomes can influence the physiology of cultured hippocampal slices and allow us to gain insights into distinct responses of males and females on neurological diseases that present a sex-biased incidence.

Preparation and Culture of Organotypic Hippocampal Slices for the Analysis of Brain Metastasis and Primary Brain Tumor Growth.

Brain metastasis is a major challenge for therapy and defines the end stage of tumor progression with a very limited patients' prognosis. Experimental setups that faithfully mimic these processes are necessary to understand the mechanism of brain metastasis and to develop new improved therapeutic strategies. Here, we describe an in vitro model, which closely resembles the in vivo situation. Organotypic hippocampal brain slice cultures (OHSCs) prepared from 3- to 8-day-old mice are well suited for neuro-oncology research including brain metastasis. The original morphology is preserved in OHSCs even after culture periods of several days to weeks. Tumor cells or cells of metastatic origin can be seeded onto OHSCs to evaluate micro-tumor formation, tumor cell invasion, or treatment response. We describe preparation and culture of OHSCs including the seeding of tumor cells. Finally, we show examples of how to treat the OHSCs for life-dead or immunohistochemical staining.

Neuroprotective Effects of the FGF21 Analogue LY2405319.

To date, there are no effective treatments for Alzheimer's disease (AD). Thus, a significant need for research of therapies remains. One promising pharmacological target is the hormone fibroblast growth factor 21 (FGF21), which is thought to be neuroprotective. A clinical candidate for medical use could be the FGF21 analogue LY2405319 (LY), which has a specificity and potency comparable to FGF21. The present study investigated the potential neuroprotective effect of LY via PPARγ/apoE/abca1 pathway, which is known to degrade amyloid-β (Aβ) plaques by using primary glial cells and hippocampal organotypic brain slice cultures (OBSCs) from 30- and 50-week-old transgenic APPswe/PS1dE9 (tg) mice. By LY treatment of 52-week-old tg mice with advanced Aβ deposition, we further aimed to elaborate the effect of LY on AD pathology in vivo. LY application to primary glial cells caused an upregulation of pparγ, apoE, and abca1 mRNA expression and significantly decreased number and area of Aβ plaques in OBSCs. LY treatment in tg mice increased cerebral [18F] FDG uptake and N-acetylaspartate/creatine ratio indicating enhanced neuronal activity and integrity. Although LY did not reduce the number of Aβ plaques in tg mice, the number of iba1-positive cells was significantly decreased indicating reduced microgliosis. These data identified LY in vitro as an activator of Aβ degrading genes leading to cerebral Aβ load amelioration in early and late AD pathology. Although Aβ plaque reduction by LY failed in vivo, LY may be used as therapeutic agent to treat AD-related neuroinflammation and impaired neuronal integrity.

Establishing an Adult Mouse Brain Hippocampal Organotypic Slice Culture System that Allows for Tracing and Pharmacological Manipulation of ex vivo Neurogenesis.

The function of the hippocampus depends on the process of adult hippocampal neurogenesis which underpins the exceptional neural plasticity of this structure, and is also frequently affected in CNS pathologies. Thus, manipulation of this process represents an important therapeutic goal. To identify potential strategies, organotypic adult brain slices are emerging as a valuable tool. Over the recent years, this methodology has been refined and here we present a combined protocol that brings together these refinements to enable long-term culture of adult hippocampal slices. We employ a sectioning technique that retains essential afferent inputs onto the hippocampus as well as serum-free culture conditions, so allowing an extended culture period. To sustain the neurogenic potential in the slices, we utilize the gliogenesis-inhibitor Indomethacin. Using EdU retention analysis enables us to assess the effects of pharmacological intervention on neurogenesis. With these improvements, we have established an easy and reliable method to study the effects of small molecules/drugs on proliferation and neuron formation ex vivo which will facilitate future discovery driven drug screenings.

Intrinsic DNA damage repair deficiency results in progressive microglia loss and replacement.

The DNA excision repair protein Ercc1 is important for nucleotide excision, double strand DNA break, and interstrand DNA crosslink repair. In constitutive Ercc1‐knockout mice, microglia display increased phagocytosis, proliferation and an enhanced responsiveness to lipopolysaccharide (LPS)‐induced peripheral inflammation. However, the intrinsic effects of Ercc1‐deficiency on microglia are unclear. In this study, Ercc1 was specifically deleted from Cx3cr1‐expressing cells and changes in microglia morphology and immune responses at different times after deletion were determined. Microglia numbers were reduced with approximately 50% at 2–12 months after Ercc1 deletion. Larger and more ramified microglia were observed following Ercc1 deletion both in vivo and in organotypic hippocampal slice cultures. Ercc1‐deficient microglia were progressively lost, and during this period, microglia proliferation was transiently increased. Ercc1‐deficient microglia were gradually replaced by nondeficient microglia carrying a functional Ercc1 allele. In contrast to constitutive Ercc1‐deficient mice, microglia‐specific deletion of Ercc1 did not induce microglia activation or increase their responsiveness to a systemic LPS challenge. Gene expression analysis suggested that Ercc1 deletion in microglia induced a transient aging signature, which was different from a priming or disease‐associated microglia gene expression profile.

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons.

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Calpastatin Overexpression Protects against Excitotoxic Hippocampal Injury and Traumatic Spinal Cord Injury.

Small molecule inhibitors of calcium-dependent proteases, calpains (CAPNs), protect against neurodegeneration induced by a variety of insults including excitotoxicity and spinal cord injury (SCI). Many of these compounds, however, also inhibit other proteases, which has made it difficult to evaluate the contribution of calpains to neurodegeneration. Calpastatin is a highly specific endogenous inhibitor of classical calpains, including CAPN1 and CAPN2. In the present study, we utilized transgenic mice that overexpress human calpastatin under the prion promoter (PrP-hCAST) to evaluate the hypothesis that calpastatin overexpression protects against excitotoxic hippocampal injury and contusive SCI. The PrP-hCAST organotypic hippocampal slice cultures showed reduced neuronal death and reduced calpain-dependent proteolysis (α-spectrin breakdown production, 145 kDa) at 24 h after N-methyl-D-aspartate (NMDA) injury compared with the wild-type (WT) cultures (n = 5, p < 0.05). The PrP-hCAST mice (n = 13) displayed a significant improvement in locomotor function at one and three weeks after contusive SCI compared with the WT controls (n = 9, p < 0.05). Histological assessment of lesion volume and tissue sparing, performed on the same animals used for behavioral analysis, revealed that calpastatin overexpression resulted in a 30% decrease in lesion volume (p < 0.05) and significant increases in tissue sparing, white matter sparing, and gray matter sparing at four weeks post-injury compared with WT animals. Calpastatin overexpression reduced α-spectrin breakdown by 51% at 24 h post-injury, compared with WT controls (p < 0.05, n = 3/group). These results provide support for the hypothesis that sustained calpain-dependent proteolysis contributes to pathological deficits after excitotoxic injury and traumatic SCI.

GM-CSF induces noninflammatory proliferation of microglia and disturbs electrical neuronal network rhythms in situ

BackgroundThe granulocyte-macrophage colony-stimulating factor (GM-CSF) (or CSF-2) is involved in myeloid cell growth and differentiation, and, possibly, a major mediator of inflammation in body tissues. The role of GM-CSF in the activation of microglia (CNS resident macrophages) and the consequent impacts on neuronal survival, excitability, and synaptic transmission are widely unknown, however. Here, we focused on electrical neuronal network rhythms in the gamma frequency band (30–70 Hz). Gamma oscillations are fundamental to higher brain functions, such as perception, attention, and memory, and they are exquisitely sensitive to metabolic and oxidative stress.MethodsWe explored the effects of chronic GM-CSF exposure (72 h) on microglia in male rat organotypic hippocampal slice cultures (in situ), i.e., postnatal cortex tissue lacking leukocyte invasion (adaptive immunity). We applied extracellular electrophysiological recordings of local field potential, immunohistochemistry, design-based stereology, biochemical analysis, and pharmacological ablation of microglia.ResultsGM-CSF triggered substantial proliferation of microglia (microgliosis). By contrast, the release of proinflammatory cytokines (IL-6, TNF-α) and nitric oxide, the hippocampal cytoarchitecture as well as the morphology of parvalbumin-positive inhibitory interneurons were unaffected. Notably, GM-CSF induced concentration-dependent, long-lasting disturbances of gamma oscillations, such as slowing (beta frequency band) and neural burst firing (hyperexcitability), which were not mimicked by the T lymphocyte cytokine IL-17. These disturbances were attenuated by depletion of the microglial cell population with liposome-encapsulated clodronate. In contrast to priming with the cytokine IFN-γ (type II interferon), GM-CSF did not cause inflammatory neurodegeneration when paired with the TLR4 ligand LPS.ConclusionsGM-CSF has a unique role in the activation of microglia, including the potential to induce neuronal network dysfunction. These immunomodulatory properties might contribute to cognitive impairment and/or epileptic seizure development in disease featuring elevated GM-CSF levels, blood-brain barrier leakage, and/or T cell infiltration.

Hallmarks of NLRP3 inflammasome activation are observed in organotypic hippocampal slice culture.

SummaryMicroglial inflammation driven by the NACHT, LRR and PYD domain‐containing protein 3 (NLRP3) inflammasome contributes to brain disease and is a therapeutic target. Most mechanistic studies on NLRP3 activation use two‐dimensional pure microglial cell culture systems. Here we studied the activation of the NLRP3 inflammasome in organotypic hippocampal slices, which allowed us to investigate microglial NLRP3 activation in a three‐dimensional, complex tissue architecture. Toll‐like receptor 2 and 4 activation primed microglial inflammasome responses in hippocampal slices by increasing NLRP3 and interleukin‐1β expression. Nigericin‐induced NLRP3 inflammasome activation was dynamically visualized in microglia through ASC speck formation. Downstream caspase‐1 activation, gasdermin D cleavage, pyroptotic cell death and interleukin‐1β release were also detected, and these findings were consistent when using different NLRP3 stimuli such as ATP and imiquimod. NLRP3 inflammasome pathway inhibitors were effective in organotypic hippocampal slices. Hence, we have highlighted organotypic hippocampal slice culture as a valuable ex vivo tool to allow the future study of NLRP3 inflammasomes in a representative tissue section, aiding the discovery of further mechanistic insights and drug development.

Neuroligin3 splice isoforms shape inhibitory synaptic function in the mouse hippocampus

Synapse formation is a dynamic process essential for the development and maturation of the neuronal circuitry in the brain. At the synaptic cleft, trans-synaptic protein-protein interactions are major biological determinants of proper synapse efficacy. The balance of excitatory and inhibitory synaptic transmission (E-I balance) stabilizes synaptic activity, and dysregulation of the E-I balance has been implicated in neurodevelopmental disorders, including autism spectrum disorders. However, the molecular mechanisms underlying the E-I balance remain to be elucidated. Here, using single-cell transcriptomics, immunohistochemistry, and electrophysiology approaches to murine CA1 pyramidal neurons obtained from organotypic hippocampal slice cultures, we investigate neuroligin (Nlgn) genes that encode a family of postsynaptic adhesion molecules known to shape excitatory and inhibitory synaptic function. We demonstrate that the NLGN3 protein differentially regulates inhibitory synaptic transmission in a splice isoform-dependent manner at hippocampal CA1 synapses. We also found that distinct subcellular localizations of the NLGN3 isoforms contribute to the functional differences observed among these isoforms. Finally, results from single-cell RNA-Seq analyses revealed that Nlgn1 and Nlgn3 are the major murine Nlgn genes and that the expression levels of the Nlgn splice isoforms are highly diverse in CA1 pyramidal neurons. Our results delineate isoform-specific effects of Nlgn genes on the E-I balance in the murine hippocampus.

Selective inhibition of mitochondrial respiratory complexes controls the transition of microglia into a neurotoxic phenotype in situ

Microglia are tissue resident macrophages (innate immunity) and universal sensors of alterations in CNS physiology. In response to pathogen or damage signals, microglia feature rapid activation and can acquire different phenotypes exerting neuroprotection or neurotoxicity. Although transcriptional aspects of microglial phenotypic transitions have been described, the underlying metabolic reprogramming is widely unknown. Employing postnatal organotypic hippocampal slice cultures, we describe that microglia transformed into a mild reactive phenotype by single TLR4 stimulation with lipopolysaccharide (LPS), which was boosted into a severe neurotoxic phenotype by IFN-γ (LPS + INF-γ). The two reactive phenotypes associated with reduction of microglial homeostatic “surveillance” markers, increase of cytokine release (IL-6, TNF-α) as well as enhancement of tissue energy demand and lactate production. These reactive phenotypes differed in the pattern of inhibition of the respiratory chain in mitochondria, however. TLR4 stimulation induced succinate dehydrogenase (complex II) inhibition by the metabolite itaconate. By contrast, TLR4 + IFN-γ receptor stimulation mainly resulted in complex IV inhibition by nitric oxide (NO) that also associated with severe oxidative stress, neuronal dysfunction and death. Notably, pharmacological depletion of microglia or treatment with itaconate resulted in effective neuroprotection reflected by well-preserved cytoarchitecture and electrical network activity, i.e., neuronal gamma oscillations (30–70 Hz) that underlie higher cognitive functions in vivo. Our findings provide in situ evidence that (i) proinflammatory microglia can substantially alter brain energy metabolism and (ii) fine-tuning of itaconate and NO metabolism determines microglial reactivity, impairment of neural network function and neurodegeneration. These data add mechanistic insights into microglial activation, with relevance to disorders featuring neuroinflammation and to drug discovery.

CNS-localized myeloid cells capture living invading T cells during neuroinflammation.

To study the role of myeloid cells in the central nervous system (CNS) in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), we used intravital microscopy, assessing local cellular interactions in vivo in EAE animals and ex vivo in organotypic hippocampal slice cultures. We discovered that myeloid cells actively engulf invading living Th17 lymphocytes, a process mediated by expression of activation-dependent lectin and its T cell-binding partner, N-acetyl-D-glucosamine (GlcNAc). Stable engulfment resulted in the death of the engulfed cells, and, remarkably, enhancement of GlcNAc exposure on T cells in the CNS ameliorated clinical EAE symptoms. These findings demonstrate the ability of myeloid cells to directly react to pathogenic T cell infiltration by engulfing living T cells. Amelioration of EAE via GlcNAc treatment suggests a novel first-defense pathway of myeloid cells as an initial response to CNS invasion and demonstrates that T cell engulfment by myeloid cells can be therapeutically exploited in vivo.