Hippocampal Slice Cultures Research Articles

Prolonged Hyperactivity Elicits Massive and Persistent Chloride Ion Redistribution in Subsets of Cultured Hippocampal Dentate Granule Cells.

Chloride ions play a critical role in neuronal inhibition through the activity of chloride-permeable GABAA receptor channels. Ion transporters, chloride channels, and immobile ion species tightly regulate intracellular chloride concentrations. Several studies related to epilepsy suggest that chloride extrusion function may decrease in an activity-dependent manner. Consequently, it is crucial to investigate whether intense neuronal activity, as observed during status epilepticus, could lead to sustained increases in intracellular chloride levels in neurons, which in turn could contribute to epilepsy-associated hyperexcitability. This study utilized the chloride sensitive indicator (6-Methoxyquinolinio) acetic acid ethyl ester bromide (MQAE) combined with fluorescence lifetime imaging (FLIM) to examine whether application of the convulsant, pilocarpine, a muscarinic acetylcholine receptor agonist, could induce synchronous epileptiform activity and elevate intracellular chloride concentrations in hippocampal slice cultures. Using a Gaussian mixture model, we identified a multimodal distribution of intracellular chloride levels among neurons, with a significant subset of these cells exhibiting massive and prolonged (days) chloride accumulation. The combination of multicellular imaging and statistical analysis served as a powerful tool for studying the emergence of multiple, distinct populations of neurons in pathological conditions, in contrast to homogeneous populations evident under control conditions.

Photobiomodulation improves functional recovery after mild traumatic brain injury

AbstractMild traumatic brain injury (mTBI) is a common consequence of head injury but there are no recognized interventions to promote recovery of the brain. We previously showed that photobiomodulation (PBM) significantly reduced the number of apoptotic cells in adult rat hippocampal organotypic slice cultures. In this study, we first optimized PBM delivery parameters for use in mTBI, conducting cadaveric studies to calibrate 660 and 810 nm lasers for transcutaneous delivery of PBM to the cortical surface. We then used an in vivo weight drop mTBI model in adult rats and delivered daily optimized doses of 660, 810 nm, or combined 660/810 nm PBM. Functional recovery was assessed using novel object recognition (NOR) and beam balance tests, whilst histology and immunohistochemistry were used to assess the mTBI neuropathology. We found that PBM at 810, 660 nm, or 810/660 nm all significantly improved both NOR and beam balance performance, with 810 nm PBM having the greatest effects. Histology demonstrated no overt structural damage in the brain after mTBI, however, immunohistochemistry using brain sections showed significantly reduced activation of both CD11b+ microglia and glial fibrillary acidic protein (GFAP)+ astrocytes at 3 days post‐injury. Significantly reduced cortical localization of the apoptosis marker, cleaved caspase‐3, and modest reductions in extracellular matrix deposition after PBM treatment, limited to choroid plexus and periventricular areas were also observed. Our results demonstrate that 810 nm PBM optimally improved functional outcomes after mTBI, reduced markers associated with apoptosis and astrocyte/microglial activation, and thus may be useful as a potential regenerative therapy.

Possible Role of Akt in Mossy Fiber Sprouting: Akt Activity and CA3 Mossy Fiber Sprouting in a Kainate Model of Epilepsy

The most prevalent pathological phenomenon observed in patients with epilepsy is hippocampal mossy fiber sprouting (MFS), which is thought to be associated with epileptic progression, such as worsening seizure control, cognitive function, and behavior. MFS is discovered in the dentate gyrus and the hippocampal Cornu Ammon 3 (CA3) area. The CA3 area is involved in memory, so disturbances in that area can affect memory impairment in patients with epilepsy. The mammalian target of rapamycin complex-1 (mTORC1) is also associated with MFS. Akt is an upstream activator of mTORC1 and a downstream target of mammalian target of rapamycin complex-2 (mTORC2) and plays a role in cytoskeleton organization. We analyzed Akt activity and MFS in the CA3 zone in an in vitro model of kainate-induced epilepsy. We divided organotypic hippocampal slice cultures (OHSCs) into a kainate (epilepsy) group and a control (untreated) group. On the 10th day in vitro (DIV), the kainate group was exposed to 8 µM kainic acid for 48 h, diluted in the medium. At 32 DIV, we measured Akt activity through western blotting and CA3 MFS through synaptoporin fluorescence intensity observed using confocal laser scanning microscopy. We found that Akt activity increased significantly (p = 0.000) in the kainate group, and the synaptoporin fluorescence intensity also increased in the stratum oriens of the CA3 area (p = 0.049) in the kainate group. Our findings implied that Akt may play a role in MFS development. Because Akt is a main downstream target of mTORC2, mTORC2 may also be involved in MFS development. Further research is required to clarify these findings.

Butylphthalide mitigates traumatic brain injury by activating anti-ferroptotic AHR-CYP1B1 pathway

Ethnopharmacological relevanceIncreasing evidence suggests that ferroptosis, an iron-dependent form of cell death characterized by lipid peroxidation, may play a substantial role in the traumatic brain injury (TBI) pathophysiology. 3-n-butylphthalide (NBP), a compound extracted from the seeds of Apium graveolens Linn (Chinese celery) and used in China to treat ischemic stroke, has demonstrated encouraging anti-reactive oxygen species (ROS) effects. Ascertaining whether NBP can inhibit ferroptosis and its mechanism could potentially expand its use in models of neurological injury and neurodegenerative diseases. Methods and resultsIn this study, we used erastin-induced in vitro ferroptosis models (HT22 cells, hippocampal slices, and primary neurons) and an in vivo controlled cortical impact mouse model. Our study revealed that NBP administration mitigated erastin-induced death in HT-22 cells and decreased ROS levels, lipid peroxidation, and mitochondrial superoxide indicators, resulting in mitochondrial protection. Moreover, the ability of NBP to inhibit ferroptosis was confirmed in organotypic hippocampal slice cultures and a TBI mouse model. NBP rescued neurons, inhibited microglial activation, and reduced iron levels in the brain tissue. The protective effect of NBP can be partly attributed to the inhibition of the AHR-CYP1B1 axis, as evidenced by RNA-seq and CYP1B1 overexpression/inhibition experiments in HT22 cells and primary neurons. ConclusionsOur study underscores that NBP inhibition of the AHR-CYP1B1 axis reduces ferroptosis in neuronal damage and ameliorates brain injury.

C9orf72 dipeptides activate the NLRP3 inflammasome.

Frontotemporal dementia and amyotrophic lateral sclerosis are neurodegenerative diseases with considerable clinical, genetic and pathological overlap. The most common cause of both diseases is a hexanucleotide repeat expansion in C9orf72. The expansion is translated to produce five toxic dipeptides, which aggregate in patient brain. Neuroinflammation is a feature of frontotemporal dementia and amyotrophic lateral sclerosis; however, its causes are unknown. The nod-like receptor family, pyrin domain-containing 3 inflammasome is implicated in several other neurodegenerative diseases as a driver of damaging inflammation. The inflammasome is a multi-protein complex which forms in immune cells in response to tissue damage, pathogens or aggregating proteins. Inflammasome activation is observed in models of other neurodegenerative diseases such as Alzheimer's disease, and inflammasome inhibition rescues cognitive decline in rodent models of Alzheimer's disease. Here, we show that a dipeptide arising from the C9orf72 expansion, poly-glycine-arginine, activated the inflammasome in microglia and macrophages, leading to secretion of the pro-inflammatory cytokine, interleukin-1β. Poly-glycine-arginine also activated the inflammasome in organotypic hippocampal slice cultures, and immunofluorescence imaging demonstrated formation of inflammasome specks in response to poly-glycine-arginine. Several clinically available anti-inflammatory drugs rescued poly-glycine-arginine-induced inflammasome activation. These data suggest that C9orf72 dipeptides contribute to the neuroinflammation observed in patients, and highlight the inflammasome as a potential therapeutic target for frontotemporal dementia and amyotrophic lateral sclerosis.

Region-specific protective effects of monomethyl fumarate in cerebellar and hippocampal organotypic slice cultures following oxygen-glucose deprivation.

To date, apart from moderate hypothermia, there are almost no adequate interventions available for neuroprotection in cases of brain damage due to cardiac arrest. Affected persons often have severe limitations in their quality of life. The aim of this study was to investigate protective properties of the active compound of dimethyl fumarate, monomethyl fumarate (MMF), on distinct regions of the central nervous system after ischemic events. Dimethyl fumarate is an already established drug in neurology with known anti-inflammatory and antioxidant properties. In this study, we chose organotypic slice cultures of rat cerebellum and hippocampus as an ex vivo model. To simulate cardiac arrest and return of spontaneous circulation we performed oxygen-glucose-deprivation (OGD) followed by treatments with different concentrations of MMF (1-30 μM in cerebellum and 5-30 μM in hippocampus). Immunofluorescence staining with propidium iodide (PI) and 4',6-diamidine-2-phenylindole (DAPI) was performed to analyze PI/DAPI ratio after imaging with a spinning disc confocal microscope. In the statistical analysis, the relative cell death of the different groups was compared. In both, the cerebellum and hippocampus, the MMF-treated group showed a significantly lower PI/DAPI ratio compared to the non-treated group after OGD. Thus, we showed for the first time that both cerebellar and hippocampal slice cultures treated with MMF after OGD are significantly less affected by cell death.

Studying estrogen effects in an in vitro-model of traumatic brain injury (TBI)

In traumatic brain injury (TBI), mechanical forces trigger a series of detrimental processes in the affected brain, which eventually result in substantial neuronal death. TBI has thus become a leading cause of death and disability worldwide. Here we utilized organotypic hippocampal slice cultures from mice to simulate mild diffuse TBI, the most common type, in vitro. We specifically used this model to examine the potential of 17β-estradiol (E2), which is considered to be neuroprotective, to influence injury-induced events, such as astrocyte and microglia activation, and to reduce cell death, if applied acutely after TBI. We found that established consequences of mechanical brain injury are replicated in the model, as increased apoptosis was observed 8 h and PI-uptake was significantly enhanced 24 h after in vitro TBI in CA1 pyramidal layer. GFAP expression was not overall increased, but correlated with cell death, indicating a confined activation of astrocytes associated with cell injury. Similarly, no general increase of microglia was detected, but activated microglia was observed in the vicinity of dying cells. Notably, application of E2 (20 nM) increased GFAP expression after 48 h, but did not significantly reduce cell death at any of the studied time points. We conclude that the presented in vitro TBI model is generally suited to study processes triggered by diffuse mechanical forces acting on brain tissue. Our data further support a stimulating effect of E2 on GFAP expression in astrocytes, but they do not confirm a neuroprotective role of E2 in the early phase of TBI.

Ameliorative effect of vanillic acid against scopolamine-induced learning and memory impairment in rat via attenuation of oxidative stress and dysfunctional synaptic plasticity

Alzheimer’s disease (AD) is characterized by cognitive impairment, loss of learning and memory, and abnormal behaviors. Scopolamine (SCOP) is a non-selective antagonist of muscarinic acetylcholine receptors that exhibits the behavioral and molecular hallmarks of AD. Vanillic acid (VA), a phenolic compound, is obtained from the roots of a traditional plant called Angelica sinensis, and has several pharmacologic effects, including antimicrobial, anti-inflammatory, anti-angiogenic, anti-metastatic, and antioxidant properties. Nevertheless, VA’s neuroprotective potential associated with the memory has not been thoroughly investigated. Therefore, this study investigated whether VA treatment has an ameliorative effect on the learning and memory impairment induced by SCOP in rats. Behavioral experiments were utilized to assess the learning and memory performance associated with the hippocampus. Using western blotting analysis and assay kits, the neuronal damage, oxidative stress, and acetylcholinesterase activity responses of hippocampus were evaluated. Additionally, the measurement of long-term potentiation was used to determine the function of synaptic plasticity in organotypic hippocampal slice cultures. In addition, the synaptic vesicles’ density and the length and width of the postsynaptic density were evaluated using electron microscopy. Consequently, the behavioral, biochemical, electrophysiological, and ultrastructural analyses revealed that VA treatment prevents learning and memory impairments caused by SCOP in rats. The study’s findings suggest that VA has a neuroprotective effect on SCOP-induced learning and memory impairment linked to the hippocampal cholinergic system, oxidative damage, and synaptic plasticity. Therefore, VA may be a prospective therapeutic agent for treating AD.

Chromogranin A (CgA) Deficiency Attenuates Tauopathy by Altering Epinephrine-Alpha-Adrenergic Receptor Signaling.

Our previous studies have indicated that insulin resistance, hyperglycemia, and hypertension in aged wild-type (WT) mice can be reversed in mice lacking chromogranin-A (CgA-KO mice). These health conditions are associated with a higher risk of Alzheimer's disease (AD). CgA, a neuroendocrine secretory protein has been detected in protein aggregates in the brains of AD patients. Here, we determined the role of CgA in tauopathies, including AD (secondary tauopathy) and corticobasal degeneration (CBD, primary tauopathy). We found elevated levels of CgA in both AD and CBD brains, which were positively correlated with increased phosphorylated tau in the frontal cortex. Furthermore, CgA ablation in a human P301S tau (hTau) transgenic mice (CgA-KO/hTau) exhibited reduced tau aggregation, resistance to tau spreading, and an extended lifespan, coupled with improved cognitive function. Transcriptomic analysis of mice cortices highlighted altered levels of alpha-adrenergic receptors (Adra) in hTau mice compared to WT mice, akin to AD patients. Since CgA regulates the release of the Adra ligands epinephrine (EPI) and norepinephrine (NE), we determined their levels and found elevated EPI levels in the cortices of hTau mice, AD and CBD patients. CgA-KO/hTau mice exhibited reversal of EPI levels in the cortex and the expression of several affected genes, including Adra1 and 2, nearly returning them to WT levels. Treatment of hippocampal slice cultures with EPI or an Adra1 agonist intensified, while an Adra1 antagonist inhibited, tau hyperphosphorylation and aggregation. These findings reveal a critical role of CgA in regulation of tau pathogenesis via the EPI-Adra signaling axis.

Neuronal function spontaneously recovers in organotypic hippocampal slice cultures after repetitive exposure to occupational-level shock waves

Neuronal function spontaneously recovers in organotypic hippocampal slice cultures after repetitive exposure to occupational-level shock waves

Cellular resolution contributions to ictal population signals.

The increased amplitude of ictal activity is a common feature of epileptic seizures, but the determinants of this amplitude have not been identified. Clinically, ictal amplitudes are measured electrographically (using, e.g., electroencephalography, electrocorticography, and depth electrodes), but these methods do not enable the assessment of the activity of individual neurons. Population signal may increase from three potential sources: (1) increased synchrony (i.e., more coactive neurons); (2) altered active state, from bursts of action potentials and/or paroxysmal depolarizing shifts in membrane potential; and (3) altered subthreshold state, which includes all lower levels of activity. Here, we quantify the fraction of ictal signal from each source. To identify the cellular determinants of the ictal signal, we measured single cell and population electrical activity and neuronal calcium levels via optical imaging of the genetically encoded calcium indicator (GECI) GCaMP. Spontaneous seizure activity was assessed with microendoscopy in an APP/PS1 mouse with focal cortical injury and via widefield imaging in the organotypic hippocampal slice cultures (OHSCs) model of posttraumatic epilepsy. Single cell calcium signals were linked to a range of electrical activities by performing simultaneous GECI-based calcium imaging and whole-cell patch-clamp recordings in spontaneously seizing OHSCs. Neuronal resolution calcium imaging of spontaneous seizures was then used to quantify the cellular contributions to population-level ictal signal. The seizure onset signal was primarily driven by increased subthreshold activity, consistent with either barrages of excitatory postsynaptic potentials or sustained membrane depolarization. Unsurprisingly, more neurons entered the active state as seizure activity progressed. However, the increasing fraction of active cells was primarily driven by synchronous reactivation and not from continued recruitment of new populations of neurons into the seizure. This work provides a critical link between single neuron activity and population measures of seizure activity.

Toll-like receptor 7: A novel neuroimmune target to reduce excessive alcohol consumption

Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns in foreign pathogens and intrinsic danger/damage signals from cells. TLR7 is a nucleic acid sensing endosomal TLR that is activated by single-stranded RNAs from microbes or by small noncoding RNAs that act as endogenous ligands. TLR7 signals through the MyD88 adaptor protein and activates the transcription factor interferon regulatory factor 7 (IRF7). TLR7 is found throughout the brain and is highly expressed in microglia, the main immune cells of the brain that have also been implicated in alcohol drinking in mice. Upregulation of TLR7 mRNA and protein has been identified in postmortem hippocampus and cortex from AUD subjects that correlated positively with lifetime consumption of alcohol. Similarly, Tlr7 and downstream signaling genes were upregulated in rat hippocampal and cortical slice cultures after chronic alcohol exposure and in these regions after chronic binge-like alcohol treatment in mice. In addition, repeated administration of the synthetic TLR7 agonists imiquimod (R837) or resiquimod (R848) increased voluntary alcohol drinking in different rodent models and produced sustained upregulation of IRF7 in the brain. These findings suggest that chronic TLR7 activation may drive excessive alcohol drinking. In the brain, this could occur through increased levels of endogenous TLR7 activators, like microRNAs and Y RNAs. This review explores chronic TLR7 activation as a pathway of dysregulated neuroimmune signaling in AUD and the endogenous small RNA ligands in the brain that could perpetuate innate immune responses and escalate alcohol drinking.

Long-term Rapamycin Treatment Inhibit AKT Activity and Lower Intracellular Calcium Expression in Organotypic Hippocampal Slice Cultures Model of Epilepsy

Despite the development of anti-epilepsy drugs, drug-refractory epilepsy still becomes a challenging problem along with an increased incidence of epilepsy. To face that challenge and increase patients’ quality of life, treatment of epilepsy must effectively prevent epileptogenesis, not only symptomatic treatment. AKT signaling pathway was proven to have important roles in epilepsy through its function in the synaptic plasticity, neurogenesis, axon guidance, modulation of the glutamate transporter, and activation of the Ca2+ channel. AKT also activated mTOR signaling pathway as activator of mTORC1 and also effector of mTORC2. Several studies showed the ability of long-term rapamycin treatment to inhibit mTORC2. This study used organotypic hippocampal slice cultures (OHSC) and long-term rapamycin treatment was administered for 3, 5, 8, and 10 days at a dose of 20 nM after induction of epilepsy by low-Mg2+ medium administration for 40 minutes. Low-Mg2+ medium administration induced seizure activity in OHSC showed by significant increase in intracellular Ca2+expressionand also significantly increase AKT activity. After administration of long-term rapamycin treatment AKT activity and intracellular Ca2+expression were significantly reduced. The longer the treatment of rapamycin, the lower the AKT activity and intracellular Ca2+expression. Long-term rapamycin treatment has the potential to become a novel epilepsy drug through its ability to attenuate AKT activity and suppress the seizures proven by lower intracellular Ca2+expression.

In Rat Organotypic Hippocampal Slice Cultures, Conventional Antiepileptic Medications are Unable to Inhibit Epileptiform Activity

Context and Goal: Prior research has shown that organotypic hippocampal slice cultures (OHSCs) can elicit tonic-clinic seizure-like events (SLEs), which resemble the electroencephalographic correlates of limbic seizures in humans and animals. We have investigated whether OHSCs can be used as in vitro models of limbic seizures to research seizures processes and vetting novel antiepileptic substances. Experimental Strategy: The interface method was used to cultivate OHSCs. Under submerged conditions, the levels of extracellular potassium and neuronal activity were observed. Lowering magnesium concentrations or administering the potassium channel blocker 4-aminopyridine both caused SLEs. Analysis was done on the impact of common antiepileptic medications (AEDs) on SLEs, including carbamazepine, phenytoin, valproic acid, clonazepam, diazepam, and phenobarbital sodium. Important Outcomes: AEDs did not stop the over 93% of OHSCs from happening induction of SLEs or cease current seizure activity even in the presence of hazardous dosages. The postnatal age at explanation (P2–P10), the manner of seizure provocation, and the length of in vitro cultivation (2 months) had no bearing on this chemoresistance. GABAA-agonist muscimol or glutamate antagonists were able to reversibly block SLEs. Inferences and Conclusions: Unlike animal models of thermoresistant seizures, where responders and nonresponses may only be distinguished after an experiment, we describe an easy to set up in vitro model of tonic-clinic SLEs that is a priori thermoresistant. OHSCs may be useful for investigating the mechanisms underlying medication-resistant seizures and for the discovery of novel anticonvulsive substances that may one day be used to treat drugresistant epilepsy.

Regional Microglial Response in Entorhino-Hippocampal Slice Cultures to Schaffer Collateral Lesion and Metalloproteinases Modulation.

Microglia and astrocytes are essential in sustaining physiological networks in the central nervous system, with their ability to remodel the extracellular matrix, being pivotal for synapse plasticity. Recent findings have challenged the traditional view of homogenous glial populations in the brain, uncovering morphological, functional, and molecular heterogeneity among glial cells. This diversity has significant implications for both physiological and pathological brain states. In the present study, we mechanically induced a Schaffer collateral lesion (SCL) in mouse entorhino-hippocampal slice cultures to investigate glial behavior, i.e., microglia and astrocytes, under metalloproteinases (MMPs) modulation in the lesioned area, CA3, and the denervated region, CA1. We observed distinct response patterns in the microglia and astrocytes 3 days after the lesion. Notably, GFAP-expressing astrocytes showed no immediate changes post-SCL. Microglia responses varied depending on their anatomical location, underscoring the complexity of the hippocampal neuroglial network post-injury. The MMPs inhibitor GM6001 did not affect microglial reactions in CA3, while increasing the number of Iba1-expressing cells in CA1, leading to a withdrawal of their primary branches. These findings highlight the importance of understanding glial regionalization following neural injury and MMPs modulation and pave the way for further research into glia-targeted therapeutic strategies for neurodegenerative disorders.

Kaempferol-3-O-(2″-O-galloyl-β-D-glucopyranoside): a novel neuroprotective agent from Diospryros kaki against cerebral ischemia-induced brain injury.

Our previous study demonstrated neuroprotective and therapeutic effects of a standardized flavonoid extract from leaves of Diospyros kaki L.f. (DK) on middle cerebral artery occlusion-and-reperfusion (MCAO/R)-induced brain injury and its underlying mechanisms. This study aimed to clarify flavonoid components responsible for the effects of DK using in vitro and in vivo transient brain ischemic models. Organotypic hippocampal slice cultures (OHSCs) subjected to oxygen- and glucose-deprivation (OGD) were performed to evaluate in vitro neuroprotective activity of DK extract and nine isolated flavonoid components. MCAO/R mice were employed to elucidate in vivo neuroprotective effects of the flavonoid component that exhibited the most potent neuroprotective effect in OHSCs. DK extract and seven flavonoids [quercetin, isoquercetin, hyperoside, quercetin-3-O-(2″-O-galloyl-β-D-galactopyranoside), kaempferol, astragalin, and kaempferol-3-O-(2″-O-galloyl-β-D-glucopyranoside) compound (9)] attenuated OGD-induced neuronal cell damage and compound (9) possessed the most potent neuroprotective activity in OHSCs. The MCAO/R mice showed cerebral infarction, massive weight loss, characteristic neurological symptoms, and deterioration of neuronal cells in the brain. Compound (9) and a reference drugs, edaravone, significantly attenuated these physical and neurological impairments. Compound (9) mitigated the blood-brain barrier dysfunction and the change of glutathione and malondialdehyde content in the MCAO mouse brain. Edaravone suppressed the oxidative stress but did not significantly affect the blood-brain barrier permeability. The present results indicated that compound (9) is a flavonoid constituent of DK with a potent neuroprotective activity against transient ischemia-induced brain damage and this action, at least in part, via preservation of blood-brain barrier integrity and suppression of oxidative stress caused by ischemic insult.

Maternal suboptimal selenium intake and low-level lead exposure affect offspring’s microglial immune profile and its reactivity to a subsequent inflammatory hit

Micronutrients such as selenium (Se) are essentials since prenatal life to support brain and cognitive development. Se deficiency, which affects up to 1 billion people worldwide, can interact with common adverse environmental challenges including (Pb), exacerbating their toxic effects. Exploiting our recently validated rat model of maternal Se restriction and developmental low Pb exposure, our aims were to investigate: (i) the early consequences of suboptimal Se intake and low-Pb exposure on neuroinflammation in neonates’ whole brains; (ii) the potential priming effect of suboptimal Se and low-Pb exposure on offspring’s glial reactivity to a further inflammatory hit. To these aims female rats were fed with suboptimal (0.04 mg/kg; Subopt) and optimal (0.15 mg/kg; Opt) Se dietary levels throughout pregnancy and lactation and exposed or not to environmentally relevant Pb dose in drinking water (12.5 µg/mL) since 4 weeks pre-mating. We found an overall higher basal expression of inflammatory markers in neonatal brains, as well as in purified microglia and organotypic hippocampal slice cultures, from the Subopt Se offspring. Subopt/Pb cultures were highly activated than Subopt cultures and showed a higher susceptibility to the inflammatory challenge lipopolysaccharide than cultures from the Opt groups. We demonstrate that even a mild Se deficiency and low-Pb exposure during brain development can influence the neuroinflammatory tone of microglia, exacerbate the toxic effects of Pb and prime microglial reactivity to subsequent inflammatory stimuli. These neuroinflammatory changes may be responsible, at least in part, for adverse neurodevelopmental outcomes.

C9orf72 dipeptides trigger neuroinflammation via the NLRP3 inflammasome

AbstractBackgroundThe most common cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a repeat expansion in C9orf72. The expansion is translated to produce five toxic dipeptides (DPRs), which aggregate in patient brain. Neuroinflammation is observed in patients, with increased microglial activation and elevated levels of pro‐inflammatory cytokines in CSF. However, the causes of this inflammation are unknown. Here, we investigated whether DPRs activate immune cells and contribute to neuroinflammation.MethodPrimary macrophages or microglia were treated with synthetic C9orf72 dipeptides and levels of pro‐inflammatory cytokines released into the culture media were analysed by ELISA and Western blot. Cytotoxicity was measured by LDH assay. Mouse organotypic hippocampal slice cultures were also treated with DPRs and cytokine release measured.ResultOne of the DPRs, GR, caused secretion of the pro‐inflammatory cytokine, interleukin (IL)‐1β, from macrophages, microglia and in hippocampal slice cultures. GR also caused significant toxicity. IL‐1β secretion was prevented by pre‐treatment with MCC950, a drug which inhibits the NLRP3 inflammasome. The inflammasome is a protein complex which forms in the cytosol of immune cells is response to “danger” stimuli, resulting in cleavage of caspase‐1 and subsequent release of IL‐1β. We confirmed that caspase‐1 was cleaved in macrophages following GR treatment. In addition, knockout of NLRP3 prevented IL‐1β secretion in response to GR. Finally, we showed that several drugs which are clinically approved for use in other conditions rescued GR‐induced NLRP3 activation.ConclusionHere, we demonstrate that C9orf72 dipeptides could directly contribute to neuroinflammation in FTD/ALS via the NLRP3 inflammasome. Furthermore, we demonstrate that this can be inhibited pharmacologically, highlighting NLRP3 as a potential therapeutic target in FTD/ALS.

The cytosolic antibody receptor TRIM21 is required for effective tau immunotherapy

AbstractBackgroundUsing a variety of cell and animal model systems we demonstrate that the cytosolic antibody receptor and E3 ligase TRIM21 plays a critical role in antibody‐mediated protection against tau pathologyMethodExperiments with organotypic hippocampal slice cultures and in vivo studies in mouse models were conducted to study the role of TRIM21 in tau immunotherapy.ResultWe demonstrate that tau:antibody complexes were internalised to the cytosol of neurons, enabling TRIM21 engagement and thereby protecting against seeded aggregation. Crucially, antibody‐mediated protection against tau pathology was lost in mice lacking TRIM21ConclusionThe cytosolic compartment provides a site of immunotherapeutic protection, which can help in the design of antibody‐based therapies in neurodegenerative disease.

Immunotherapy against tau pathology in mouse models requires cytosolic Fc receptor TRIM21

AbstractBackgroundThe passive transfer of monoclonal antibodies reduces tau pathology in mouse models and is under evaluation as a potential immunotherapeutic strategy against Alzheimer’s disease and other tauopathies. The mechanisms that drive therapeutic protection of anti‐tau antibodies are not well understood.MethodP301S tau transgenic animals were treated with antibodies against pS422 and analysed for tau pathology by biochemical fractionation. Organotypic hippocampal slice cultures were used as a model of tau seeding and neutralisation. AAV serotypes were used to deliver tau‐selective degraders.ResultGenetic deletion of the cytosolic antibody receptor and E3 ubiquitin ligase TRIM21 rendered tau immunotherapy ineffective in animal models. In organotypic slices, expression of TRIM21 was essential for protection against seeded aggregation of tau. Cytosolic antibody‐tau complexes were visible inside neurons in complex with TRIM21 shortly after application of tau assemblies and antibodies to the cell exterior. Nanobody‐based degraders were developed using TRIM21 and found to protect against tau pathology.ConclusionAnti‐tau antibodies can engage extracellular tau seeds and enter neurons as a complex. Following cytosolic entry, TRIM21 engages the antibodies and mediates neutralisation of tau seeding activity. The results reveal a mechanism that can be optimised for future immunotherapeutics. In this direction, AAV‐delivered degraders reveal a route by which tau aggregates may be selectively removed from the brain.