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Multiple AD risk genes are implicated in lipid metabolism, and plasma and brain lipid levels are altered in AD. Astrocytes are enriched in key lipid-related factors and are likely contributors to altered lipid homeostasis in AD. We hypothesize that APP/Aβ-related pathology and neuroimmune factors modulate astrocytic gene transcription that promote maladaptive changes in lipid pathways, including aberrant astrocytic production and release of lipids that could affect Aβ pathology and neuronal deficits. To investigate the effects of APP/Aβ and neuroinflammation on astrocytic lipid metabolism, we used homozygous mutant APP knock-in mice (APP -KI) that have progressive Aβ proteinopathy and gliosis, as well as mice injected systemically with lipopolysaccharide (LPS) to induce acute neuroinflammatory responses. Astrocyte-specific analyses were performed in vivo by either FACS-sorting astrocytes for downstream lipidomic profiling or by ribosome-tagging astrocytes in mouse models (Ribotag) for astrocyte-specific RNA sequencing. We also performed lipidomic, RNA, and protein-based analyses in primary murine astrocytes stimulated with oligomeric Aβ (oAβ) or interleukin-1 (IL-1α/β). Lipidomics in acutely sorted cortical astrocytes from APP -KI or LPS-injected mice revealed decreased levels of triglycerides and cholesterol esters. In LPS-injected mice, cortical astrocytes also had increased expression of enzymes involved in lipolysis, most notably adipose triglyceride lipase (Atgl), the principal triglyceride hydrolase. Similarly, primary astrocytes had increased levels of triglyceride lipolysis upon IL-1α/β or oAβ stimulation, leading to increased liberation of free fatty acids (FFAs) in an NF-κB and ERK1/2-dependent manner. Astrocytic FFAs generated from triglyceride lipolysis were not used for energy production via β-oxidation but instead utilized by astrocytes to produce prostaglandins (PGE ) and to facilitate secretion of extracellular vesicular bodies (EVB). Inhibiting ATGL decreased PGE production, revealing an essential role of astrocyte triglyceride lipolysis in neuroinflammatory processes. Our findings suggest that Aβ pathology and neuroinflammatory factors cause astrocytic triglyceride lipolysis, a process that liberates fatty acids to be utilized for various potentially pathogenic cascades in disease. We conclude that amyloid pathology and neuroimmune factors converge to alter astrocytic lipid-related gene transcription and lipid-based signaling.

Neuronal dysfunction due to iron accumulation in conjunction with reactive oxygen species (ROS) could represent an important, yet underappreciated, component of the epileptogenic process. However, to date, alterations in iron metabolism in the epileptogenic brain have not been addressed in detail. Iron-related neuropathology and antioxidant metabolic processes were investigated in resected brain tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE-HS), post-mortem brain tissue from patients who died after status epilepticus (SE) as well as brain tissue from the electrically induced SE rat model of TLE. Magnetic susceptibility of the presumed seizure-onset zone from three patients with focal epilepsy was compared during and after seizure activity. Finally, the cellular effects of iron overload were studied in vitro using an acute mouse hippocampal slice preparation and cultured human fetal astrocytes. While iron-accumulating neurons had a pyknotic morphology, astrocytes appeared to acquire iron-sequestrating capacity as indicated by prominent ferritin expression and iron retention in the hippocampus of patients with SE or TLE. Interictal to postictal comparison revealed increased magnetic susceptibility in the seizure-onset zone of epilepsy patients. Post-SE rats had consistently higher hippocampal iron levels during the acute and chronic phase (when spontaneous recurrent seizures are evident). In vitro, in acute slices that were exposed to iron, neurons readily took up iron, which was exacerbated by induced epileptiform activity. Human astrocyte cultures challenged with iron and ROS increased their antioxidant and iron-binding capacity, but simultaneously developed a pro-inflammatory phenotype upon chronic exposure. These data suggest that seizure-mediated, chronic neuronal iron uptake might play a role in neuronal dysfunction/loss in TLE-HS. On the other hand, astrocytes sequester iron, specifically in chronic epilepsy. This function might transform astrocytes into a highly resistant, pro-inflammatory phenotype potentially contributing to pro-epileptogenic inflammatory processes.

Abstract Tuberous sclerosis complex (TSC) is a monogenetic disease that arises due to mutations in either the TSC1 or TSC2 gene and affects multiple organ systems. One of the hallmark manifestations of TSC are cortical malformations referred to as cortical tubers. These tubers are frequently associated with treatment-resistant epilepsy. Some of these patients are candidates for epilepsy surgery. White matter abnormalities, such as loss of myelin and oligodendroglia, have been described in a small subset of resected tubers but mechanisms underlying this phenomenon are unclear. Herein, we analyzed a variety of neuropathologic and immunohistochemical features in gray and white matter areas of resected cortical tubers from 46 TSC patients using semi-automated quantitative image analysis. We observed divergent amounts of myelin basic protein as well as numbers of oligodendroglia in both gray and white matter when compared with matched controls. Analyses of clinical data indicated that reduced numbers of oligodendroglia were associated with lower numbers on the intelligence quotient scale and that lower amounts of myelin-associated oligodendrocyte basic protein were associated with the presence of autism-spectrum disorder. In conclusion, myelin pathology in cortical tubers extends beyond the white matter and may be linked to cognitive dysfunction in TSC patients.

Gangliogliomas (GGs) are low-grade brain tumours that cause intractable focal epilepsy in children and adults. In GG, as in epileptogenic focal malformations ( i.e., tuberous sclerosis complex, TSC), there is evidence of sustained neuroinflammation with involvement of the pro-inflammatory cytokine IL-1β. On the other hand, anti-inflammatory mediators are less studied but bear relevance for understanding seizure mechanisms. Therefore, we investigated the effect of the key anti-inflammatory cytokine IL-10 on GABAergic neurotransmission in GG. We assessed the IL-10 dependent signaling by transcriptomic analysis, immunohistochemistry and performed voltage-clamp recordings on Xenopus oocytes microtransplanted with cell membranes from brain specimens, to overcome the limited availability of acute GG slices. We report that IL-10-related mRNAs were up-regulated in GG and slightly in TSC. Moreover, we found IL-10 receptors are expressed by neurons and astroglia. Furthermore, GABA currents were potentiated significantly by IL-10 in GG. This effect was time and dose-dependent and inhibited by blockade of IL-10 signaling. Notably, in the same tissue, IL-1β reduced GABA current amplitude and prevented the IL-10 effect. These results suggest that in epileptogenic tissue, pro-inflammatory mechanisms of hyperexcitability prevail over key anti-inflammatory pathways enhancing GABAergic inhibition. Hence, boosting the effects of specific anti-inflammatory molecules could resolve inflammation and reduce intractable seizures.

The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.

Mitochondrial reactive oxygen species (mROS), such as superoxide and hydrogen peroxide (H O ), are implicated in aging-associated neurological disorders, including Alzheimer's Disease and frontotemporal dementia. Mitochondrial complex III of the respiratory chain has the highest capacity for mROS production and generates mROS toward the cytosol, poising it to regulate intracellular signaling and disease mechanisms. However, the exact triggers of complex III-derived ROS (CIII-ROS), its downstream molecular targets, and its functional roles in dementia-related pathogenesis remain unclear. Here, we investigated the drivers and consequences of CIII-ROS production in primary mouse astrocytes and mouse models of dementia-linked proteinopathy using site-selective mROS suppressors and genetic approaches together with live-cell imaging of sub-compartmental H O dynamics, stoichiometric redox proteomics, and transcriptomics. We found that specific disease-related factors transiently increase astrocytic CIII-ROS levels, and this effect is dependent on a mitochondrial sodium-calcium exchanger. CIII-ROS oxidized specific cysteines on astrocytic proteins associated with disease, amplified STAT3 phosphorylation and nuclear translocation, and promoted gene expression changes linked to STAT3 and related neuroimmune pathways. Inhibition of other sites of ROS production, including mitochondrial complex I and NADPH oxidase, had no effects on mROS responses or STAT3 signaling, demonstrating the specificity of CIII-ROS induction and its context-dependent modulation of STAT3 activities. Blockade of CIII-ROS in transgenic mouse models of dementia with a site-selective and brain-penetrant suppressor reduced astrocytic alterations, neuropathology, and premature mortality. Our data suggest that dementia-associated factors induce site-specific mROS production and that CIII-ROS promote precise post-translational redox modifications and amplify STAT3-linked signaling and changes in gene expression. Together, our findings reveal CIII-ROS as an important node of mitochondrial-nuclear communication in pathogenic conditions, whereby mROS transients are converted into long-lasting changes in gene transcription and cell function. CIII-ROS and redox signaling offer new therapeutic opportunities for aging-associated neurological disorders.

Astrocytes, key regulators of brain homeostasis, interact with neighboring glial cells, neurons and the vasculature through complex processes involving different signaling pathways. It is not entirely clear how these interactions change in the ageing brain and which factors influence astrocyte ageing. Here, we investigate the role of endocannabinoid signaling, because it is an important modulator of neuron and astrocyte functions, as well as brain ageing. We demonstrate that mice with a specific deletion of CB1 receptors on GABAergic neurons (GABA-Cnr1-/- mice), which show a phenotype of accelerated brain ageing, affects age-related changes in the morphology of astrocytes in the hippocampus. Thus, GABA-Cnr1-/- mice showed a much more pronounced age-related and layer-specific increase in GFAP-positive areas in the hippocampus compared to wild-type animals. The number of astrocytes, in contrast, was similar between the two genotypes. Astrocytes in the hippocampus of old GABA-Cnr1-/- mice also showed a different morphology with enhanced GFAP-positive process branching and a less polarized intrahippocampal distribution. Furthermore, astrocytic TNFα levels were higher in GABA-Cnr1-/- mice, indicating that these morphological changes were accompanied by a more pro-inflammatory function. These findings demonstrate that the disruption of endocannabinoid signaling on GABAergic neurons is accompanied by functional changes in astrocyte activity, which are relevant to brain ageing.

Tuberous sclerosis complex (TSC) is a congenital disorder characterized by cortical malformations and concomitant epilepsy caused by loss‐of‐function mutations in the mTOR suppressors TSC1 or TSC2. While the underlying molecular changes caused by mTOR activation in TSC have previously been investigated, the drivers of these transcriptional change have not been fully elucidated. A better understanding of the perturbed transcriptional regulation could lead to the identification of novel pathways for therapeutic intervention not only in TSC, but other genetic epilepsies in which mTOR activation plays a key role, such as focal cortical dysplasia 2b (FCD). Here, we analyzed RNA sequencing data from cortical tubers and a tsc2−/− zebrafish. We identified differential expression of the transcription factors (TFs) SPI1/PU.1, IRF8, GBX2, and IKZF1 of which SPI1/PU.1 and IRF8 targets were enriched among the differentially expressed genes. Furthermore, for SPI1/PU.1 these findings were conserved in TSC zebrafish model. Next, we confirmed overexpression of SPI1/PU.1 on the RNA and protein level in a separate cohort of surgically resected TSC tubers and FCD tissue, in fetal TSC tissue, and a Tsc1GFAP−/− mouse model of TSC. Subsequently, we validated the expression of SPI1/PU.1 in dysmorphic cells with mTOR activation in TSC tubers. In fetal TSC, we detected SPI1/PU.1 expression prenatally and elevated RNA Spi1 expression in Tsc1GFAP−/− mice before the development of seizures. Finally, in vitro, we identified that in astrocytes and neurons SPI1 transcription was driven by H2O2‐induced oxidative stress, independent of mTOR. We identified SPI1/PU.1 as a novel TF involved in the pro‐inflammatory gene expression of malformed cells in TSC and FCD 2b. This transcriptional program is activated in response to oxidative stress and already present prenatally. Importantly, SPI1/PU.1 protein appears to be strictly limited to malformed cells, as we did not find SPI1/PU.1 protein expression in mice nor in our in vitro models. Using RNA sequencing data from surgically resected tubers from tuberous sclerosis complex (TSC) patients and tissue from a tsc2−/− zebrafish knockout model we identified the transcription factor SPI1/PU.1 to be upregulated and its targets to be enriched among the differentially expressed genes. Furthermore, we validated these findings in a separate cohort of TSC as well as in the histopathologically similar focal cortical dysplasia (FCD) type 2b, in fetal TSC tissue and an experimental TSC model on the RNA and protein levels. We identified SPI1/PU.1 as a novel transcription factor involved in the pro‐inflammatory gene expression of malformed cells in TSC and FCD 2b and found in vitro evidence for its regulation by oxidative stress.

Constitutive activation of the mTOR pathway, as observed in Tuberous Sclerosis Complex (TSC), leads to glial dysfunction and subsequent epileptogenesis. Although astrocytes are considered important mediators for synaptic clearance and phagocytosis, little is known on how astrocytes contribute to the epileptogenic network. We employed singlenuclei RNA sequencing and a hybrid fetal calf serum (FCS)/FCS-free cell culture model to explore the capacity of TSC-derived astrocytes to maintain glutamate homeostasis and clear debris in their environment. We found that TSC astrocytes show reduced maturity on RNA and protein level as well as the inability to clear excess glutamate through the loss of both enzymes and transporters complementary to a reduction of phagocytic capabilities. Our study provides evidence of mechanistic alterations in TSC astrocytes, underscoring the significant impairment of their supportive functions. These insights enhance our understanding of TSC pathophysiology and hold potential implications for future therapeutic interventions.

Microglia, the resident immune cells of the brain, play important roles in defending the brain against pathogens and supporting neuronal circuit plasticity. Chronic or excessive pro-inflammatory responses of microglia damage neurons, therefore their activity is tightly regulated. Pharmacological and genetic studies revealed that cannabinoid type 1 (CB1) receptor activity influences microglial activity, although microglial CB1 receptor expression is very low and activity-dependent. The CB1 receptor is mainly expressed on neurons in the central nervous system (CNS)-with an especially high level on GABAergic interneurons. Here, we determined whether CB1 signaling on this neuronal cell type plays a role in regulating microglial activity. We compared microglia density, morphology and cytokine expression in wild-type (WT) and GABAergic neuron-specific CB1 knockout mice (GABA/CB1 ) under control conditions (saline-treatment) and after 3 h, 24 h or repeated lipopolysaccharide (LPS)-treatment. Our results revealed that hippocampal microglia from saline-treated GABA/CB1 mice resembled those of LPS-treated WT mice: enhanced density and larger cell bodies, while the size and complexity of their processes was reduced. No further reduction in the size or complexity of microglia branching was detected after LPS-treatment in GABA/CB1 mice, suggesting that microglia in naïve GABA/CB1 mice were already in an activated state. This result was further supported by correlating the level of microglial tumor necrosis factor α (TNFα) with their size. Acute LPS-treatment elicited in both genotypes similar changes in the expression of pro-inflammatory cytokines (TNFα, interleukin-6 (IL-6) and interleukin 1β (IL-1β)). However, TNFα expression was still significantly elevated after repeated LPS-treatment in WT, but not in GABA/CB1 mice, indicating a faster development of tolerance to LPS. We also tested the possibility that the altered microglia activity in GABA/CB1 mice was due to an altered expression of neuron-glia interaction proteins. Indeed, the level of fractalkine (CX3CL1), a neuronal protein involved in the regulation of microglia, was reduced in hippocampal GABAergic neurons in GABA/CB1 mice, suggesting a disturbed neuronal control of microglial activity. Our result suggests that CB1 receptor agonists can modulate microglial activity indirectly, through CB1 receptors on GABAergic neurons. Altogether, we demonstrated that GABAergic neurons, despite their relatively low density in the hippocampus, have a specific role in the regulation of microglial activity and cannabinoid signaling plays an important role in this arrangement.