A nonlinear meccano for Alzheimer's emergence by amyloid ß-mediated glutamatergic hyperactivity.

The pathophysiological process of Alzheimer's disease (AD) is believed to begin many years before the formal diagnosis of AD dementia. This protracted preclinical phase offers a crucial window for potential therapeutic interventions, yet its comprehensive characterization remains elusive. Accumulating evidence suggests that amyloid-ß (Aß) may mediate neuronal hyperactivity in circuit dysfunction in the early stages of AD. At the same time, neural activity can also facilitate Aß accumulation through intricate feed-forward interactions, complicating elucidating the conditions governing Aß-dependent hyperactivity and its diagnostic utility. In this study, we use biophysical modeling to shed light on such conditions. Our analysis reveals that the inherently nonlinear nature of the underlying molecular interactions can give rise to the emergence of various modes of hyperactivity. This diversity in the mechanisms of hyperactivity may ultimately account for a spectrum of AD manifestations.

Spatial organization of neuron-astrocyte interactions in the somatosensory cortex.

Microcircuits in the neocortex are functionally organized along layers and columns, which are the fundamental modules of cortical information processing. While the function of cortical microcircuits has focused on neuronal elements, much less is known about the functional organization of astrocytes and their bidirectional interaction with neurons. Here, we show that Cannabinoid type 1 receptor (CB1R)-mediated astrocyte activation by neuron-released endocannabinoids elevate astrocyte Ca2+ levels, stimulate ATP/adenosine release as gliotransmitters, and transiently depress synaptic transmission in layer 5 pyramidal neurons at relatively distant synapses (˃20 µm) from the stimulated neuron. This astrocyte-mediated heteroneuronal synaptic depression occurred between pyramidal neurons within a cortical column and was absent in neurons belonging to adjacent cortical columns. Moreover, this form of heteroneuronal synaptic depression occurs between neurons located in particular layers, following a specific connectivity pattern that depends on a layer-specific neuron-to-astrocyte signaling. These results unravel the existence of astrocyte-mediated nonsynaptic communication between cortical neurons and that this communication is column- and layer-specific, which adds further complexity to the intercellular signaling processes in the neocortex.

Assessment of the Correlation and Diagnostic Accuracy between Cerebrospinal Fluid and Plasma Alzheimer's Disease Biomarkers: A Comparison of the Lumipulse and Simoa Platforms.

We compared the clinical and analytical performance of Alzheimer's disease (AD) plasma biomarkers measured using the single-molecule array (Simoa) and Lumipulse platforms. We quantified the plasma levels of amyloid beta 42 (Aß42), Aß40, phosphorylated tau (Ptau181), and total tau biomarkers in 81 patients with mild cognitive impairment (MCI), 30 with AD, and 16 with non-AD dementia. We found a strong correlation between the Simoa and Lumipulse methods. Concerning the clinical diagnosis, Simoa Ptau181/Aß42 (AUC 0.739, 95% CI 0.592-0.887) and Lumipulse Aß42 and Ptau181/Aß42 (AUC 0.735, 95% CI 0.589-0.882 and AUC 0.733, 95% CI 0.567-0.900) had the highest discriminating power. However, their power was significantly lower than that of CSF Aß42/Aß40, as measured by Lumipulse (AUC 0.879, 95% CI 0.766-0.992). Simoa Ptau181 and Lumipulse Ptau181/Aß42 were the markers most consistent with the CSF Aß42/Aß40 status (AUC 0.801, 95% CI 0.712-0.890 vs. AUC 0.870, 95% CI 0.806-0.934, respectively) at the ≥2.127 and ≥0.084 cut-offs, respectively. The performance of the Simoa and Lumipulse plasma AD assays is weaker than that of CSF AD biomarkers. At present, the analysed AD plasma biomarkers may be useful for screening to reduce the number of lumbar punctures in the clinical setting.

Endocannabinoid signaling in brain diseases: Emerging relevance of glial cells.

The discovery of cannabinoid receptors as the primary molecular targets of psychotropic cannabinoid Δ9 -tetrahydrocannabinol (Δ9 -THC) in late 1980s paved the way for investigations on the effects of cannabis-based therapeutics in brain pathology. Ever since, a wealth of results obtained from studies on human tissue samples and animal models have highlighted a promising therapeutic potential of cannabinoids and endocannabinoids in a variety of neurological disorders. However, clinical success has been limited and major questions concerning endocannabinoid signaling need to be satisfactorily addressed, particularly with regard to their role as modulators of glial cells in neurodegenerative diseases. Indeed, recent studies have brought into the limelight diverse, often unexpected functions of astrocytes, oligodendrocytes, and microglia in brain injury and disease, thus providing scientific basis for targeting glial cells to treat brain disorders. This Review summarizes the current knowledge on the molecular and cellular hallmarks of endocannabinoid signaling in glial cells and its clinical relevance in neurodegenerative and chronic inflammatory disorders.

27-hydroxycholesterol promotes oligodendrocyte maturation: Implications for hypercholesterolemia-associated brain white matter changes.

Oxidized cholesterol metabolite 27-hydroxycholesterol (27-OH) is a potential link between hypercholesterolemia and neurodegenerative diseases since unlike peripheral cholesterol, 27-OH is transported across the blood-brain barrier. However, the effects of high 27-OH levels on oligodendrocyte function remain unexplored. We hypothesize that during hypercholesterolemia 27-OH may impact oligodendrocytes and myelin and thus contribute to the disconnection of neural networks in neurodegenerative diseases. To test this idea, we first investigated the effects of 27-OH in cultured oligodendrocytes and found that it induces cell death of immature O4+ /GalC+ oligodendrocytes along with stimulating differentiation of PDGFR+ oligodendrocyte progenitors (OPCs). Next, transgenic mice with increased systemic 27-OH levels (Cyp27Tg) underwent behavioral testing and their brains were immunohistochemically stained and lysed for immunoblotting. Chronic exposure to 27-OH in mice resulted in increased myelin basic protein (MBP) but not 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) or myelin oligodendrocyte glycoprotein (MOG) levels in the corpus callosum and cerebral cortex. Intriguingly, we also found impairment of spatial learning suggesting that subtle changes in myelinated axons of vulnerable areas like the hippocampus caused by 27-OH may contribute to impaired cognition. Finally, we found that 27-OH levels in cerebrospinal fluid from memory clinic patients were associated with levels of the myelination regulating CNPase, independently of Alzheimer's disease markers. Thus, 27-OH promotes OPC differentiation and is toxic to immature oligodendrocytes as well as it subtly alters myelin by targeting oligodendroglia. Taken together, these data indicate that hypercholesterolemia-derived higher 27-OH levels change the oligodendrocytic capacity for appropriate myelin remodeling which is a crucial factor in neurodegeneration and aging.

Alterations of Oligodendrocyte and Myelin Energy Metabolism in Multiple Sclerosis.

Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system (CNS), characterized by demyelination and neurodegeneration. Oligodendrocytes play a vital role in maintaining the integrity of myelin, the protective sheath around nerve fibres essential for efficient signal transmission. However, in MS, oligodendrocytes become dysfunctional, leading to myelin damage and axonal degeneration. Emerging evidence suggests that metabolic changes, including mitochondrial dysfunction and alterations in glucose and lipid metabolism, contribute significantly to the pathogenesis of MS. Mitochondrial dysfunction is observed in both immune cells and oligodendrocytes within the CNS of MS patients. Impaired mitochondrial function leads to energy deficits, affecting crucial processes such as impulse transmission and axonal transport, ultimately contributing to neurodegeneration. Moreover, mitochondrial dysfunction is linked to the generation of reactive oxygen species (ROS), exacerbating myelin damage and inflammation. Altered glucose metabolism affects the energy supply required for oligodendrocyte function and myelin synthesis. Dysregulated lipid metabolism results in changes to the composition of myelin, affecting its stability and integrity. Importantly, low levels of polyunsaturated fatty acids in MS are associated with upregulated lipid metabolism and enhanced glucose catabolism. Understanding the intricate relationship between these mechanisms is crucial for developing targeted therapies to preserve myelin and promote neurological recovery in individuals with MS. Addressing these metabolic aspects may offer new insights into potential therapeutic strategies to halt disease progression and improve the quality of life for MS patients.

GABAB receptor agonist baclofen promotes central nervous system remyelination.

Promoting remyelination is considered as a potential neurorepair strategy to prevent/limit the development of permanent neurological disability in patients with multiple sclerosis (MS). To this end, a number of clinical trials are investigating the potential of existing drugs to enhance oligodendrocyte progenitor cell (OPC) differentiation, a process that fails in chronic MS lesions. We previously reported that oligodendroglia express GABAB receptors (GABAB Rs) both in vitro and in vivo, and that GABAB R-mediated signaling enhances OPC differentiation and myelin protein expression in vitro. Our goal here was to evaluate the pro-remyelinating potential of GABAB R agonist baclofen (Bac), a clinically approved drug to treat spasticity in patients with MS. We first demonstrated that Bac increases myelin protein production in lysolecithin (LPC)-treated cerebellar slices. Importantly, Bac administration to adult mice following induction of demyelination by LPC injection in the spinal cord resulted in enhanced OPC differentiation and remyelination. Thus, our results suggest that Bac repurposing should be considered as a potential therapeutic strategy to stimulate remyelination in patients with MS.

Recombinant Integrin ß1 Signal Peptide Blocks Gliosis Induced by Aß Oligomers.

Glial cells participate actively in the early cognitive decline in Alzheimer's disease (AD) pathology. In fact, recent studies have found molecular and functional abnormalities in astrocytes and microglia in both animal models and brains of patients suffering from this pathology. In this regard, reactive gliosis intimately associated with amyloid plaques has become a pathological hallmark of AD. A recent study from our laboratory reports that astrocyte reactivity is caused by a direct interaction between amyloid beta (Aß) oligomers and integrin ß1. Here, we have generated four recombinant peptides including the extracellular domain of integrin ß1, and evaluated their capacity both to bind in vitro to Aß oligomers and to prevent in vivo Aß oligomer-induced gliosis and endoplasmic reticulum stress. We have identified the minimal region of integrin ß1 that binds to Aß oligomers. This region is called signal peptide and corresponds to the first 20 amino acids of the integrin ß1 N-terminal domain. This recombinant integrin ß1 signal peptide prevented Aß oligomer-induced ROS generation in primary astrocyte cultures. Furthermore, we carried out intrahippocampal injection in adult mice of recombinant integrin ß1 signal peptide combined with or without Aß oligomers and we evaluated by immunohistochemistry both astrogliosis and microgliosis as well as endoplasmic reticulum stress. The results show that recombinant integrin ß1 signal peptide precluded both astrogliosis and microgliosis and endoplasmic reticulum stress mediated by Aß oligomers in vivo. We have developed a molecular tool that blocks the activation of the molecular cascade that mediates gliosis via Aß oligomer/integrin ß1 signaling.

Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome.

During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12, and the tyrosine kinases of the TAM family Mer tyrosine kinase (MerTK)/Axl. In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.SIGNIFICANCE STATEMENT Microglia are the brain professional phagocytes and, in the adult hippocampal neurogenic niche, they remove newborn cells naturally undergoing apoptosis. Here we show that phagocytosis of apoptotic cells triggers a coordinated transcriptional program that alters their secretome, limiting neurogenesis both in vivo and in vitro In addition, chronic phagocytosis disruption in mice deficient for receptors P2Y12 and MerTK/Axl reduces adult hippocampal neurogenesis. In contrast, inducible MerTK downregulation transiently increases neurogenesis, suggesting that microglial phagocytosis provides a negative feedback loop that is necessary for the long-term maintenance of adult hippocampal neurogenesis. Therefore, we speculate that the effects of promoting engulfment/degradation of cell debris may go beyond merely removing corpses to actively promoting regeneration in development, aging, and neurodegenerative diseases.

GABAA Receptors Expressed in Oligodendrocytes Cultured from the Neonatal Rat Contain α3 and γ1 Subunits and Present Differential Functional and Pharmacological Properties.

Oligodendrocytes (OLs) express functional GABAA receptors (GABAARs) that are activated by GABA released at synaptic contacts with axons or by ambient GABA in extrasynaptic domains. In both instances, the receptors' molecular identity has not been fully defined. Furthermore, data on their structural diversity in different brain regions and information on age-dependent changes in their molecular composition are scant. This lack of knowledge has delayed access to a better understanding of the role of GABAergic signaling between neurons and OLs. Here, we used functional, and pharmacological analyses, as well as gene and protein expression of GABAAR subunits, to explore the subunit combination that could explain the receptor functional profile expressed in OLs from the neonate rat. We found that GABAAR composed of α3ß2γ1 subunits mimicked the characteristics of the endogenous receptor when expressed heterologously in Xenopus laevis oocytes. Either α3 or γ1 subunit silencing by small interfering RNA transfection changed the GABA-response characteristics in oligodendrocyte precursor cells, indicating their participation in the endogenous receptor conformation. Thus, α3 subunit silencing shifted the mean EC50 for GABA from 75.1 to 46.6 µM, whereas γ1 silencing reduced the current amplitude response by 55%. We also observed that ß-carbolines differentially enhance GABA responses in oligodendroglia as compared with those in neurons. These results contribute to defining the molecular and pharmacological properties of GABAARs in OLs. Additionally, the identification of ß-carbolines as selective enhancers of GABAARs in OLs may help to study the role of GABAergic signaling during myelination. SIGNIFICANCE STATEMENT: GABAergic signaling through GABAA receptors (GABAARs) expressed in the oligodendroglial lineage contributes to the myelination control. Determining the molecular identity and the pharmacology of these receptors is essential to define their specific roles in myelination. Using GABAAR subunit expression and silencing, we identified that the GABAAR subunit combination α3ß2γ1 conforms the bulk of GABAARs in oligodendrocytes from rat neonates. Furthermore, we found that these receptors have differential pharmacological properties that allow specific positive modulation by ß-carbolines.

Δ9 -Tetrahydrocannabinol promotes oligodendrocyte development and CNS myelination in vivo.

Δ9 -Tetrahydrocannabinol (THC), the main bioactive compound found in the plant Cannabis sativa, exerts its effects by activating cannabinoid receptors present in many neural cells. Cannabinoid receptors are also physiologically engaged by endogenous cannabinoid compounds, the so-called endocannabinoids. Specifically, the endocannabinoid 2-arachidonoylglycerol has been highlighted as an important modulator of oligodendrocyte (OL) development at embryonic stages and in animal models of demyelination. However, the potential impact of THC exposure on OL lineage progression during the critical periods of postnatal myelination has never been explored. Here, we show that acute THC administration at early postnatal ages in mice enhanced OL development and CNS myelination in the subcortical white matter by promoting oligodendrocyte precursor cell cycle exit and differentiation. Mechanistically, THC-induced-myelination was mediated by CB1 and CB2 cannabinoid receptors, as demonstrated by the blockade of THC actions by selective receptor antagonists. Moreover, the THC-mediated modulation of oligodendroglial differentiation relied on the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, as mTORC1 pharmacological inhibition prevented the THC effects. Our study identifies THC as an effective pharmacological strategy to enhance oligodendrogenesis and CNS myelination in vivo.

Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1.

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.

N-Methyl-D-Aspartate Receptor Antibodies in Autoimmune Encephalopathy Alter Oligodendrocyte Function.

OBJECTIVE: Antibodies against neuronal N-methyl-D-aspartate receptors (NMDARs) in patients with anti-NMDAR encephalitis alter neuronal synaptic function and plasticity, but the effects on other cells of the nervous system are unknown. METHODS: Cerebrospinal fluid (CSF) of patients with anti-NMDAR encephalitis (preabsorbed or not with GluN1) and a human NMDAR-specific monoclonal antibody (SSM5) derived from plasma cells of a patient, along the corresponding controls, were used in the studies. To evaluate the activity of oligodendrocyte NMDARs and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in vitro after exposure to patients' CSF antibodies or SSM5, we used a functional assay based on cytosolic Ca2+ imaging. Expression of the glucose transporter (GLUT1) in oligodendrocytes was assessed by immunocytochemistry. RESULTS: NMDAR agonist responses were robustly reduced after preincubation of oligodendrocytes with patients' CSF or SSM5 but remained largely unaltered with the corresponding controls. These effects were NMDAR specific, as patients' CSF did not alter responses to AMPA receptor agonists and was abrogated by preabsorption of CSF with HEK cells expressing GluN1 subunit. Patients' CSF also reduced oligodendrocyte expression of glucose transporter GLUT1 induced by NMDAR activity. INTERPRETATION: Antibodies from patients with anti-NMDAR encephalitis specifically alter the function of NMDARs in oligodendrocytes, causing a decrease of expression of GLUT1. Considering that normal GLUT1 expression in oligodendrocytes and myelin is needed to metabolically support axonal function, the findings suggest a link between antibody-mediated dysfunction of NMDARs in oligodendrocytes and the white matter alterations reported in patients with this disorder. ANN NEUROL 2020;87:670-676.

Effects of Platelet-Rich Plasma on Cellular Populations of the Central Nervous System: The Influence of Donor Age.

Platelet-rich plasma (PRP) is a biologic therapy that promotes healing responses across multiple medical fields, including the central nervous system (CNS). The efficacy of this therapy depends on several factors such as the donor's health status and age. This work aims to prove the effect of PRP on cellular models of the CNS, considering the differences between PRP from young and elderly donors. Two different PRP pools were prepared from donors 65‒85 and 20‒25 years old. The cellular and molecular composition of both PRPs were analyzed. Subsequently, the cellular response was evaluated in CNS in vitro models, studying proliferation, neurogenesis, synaptogenesis, and inflammation. While no differences in the cellular composition of PRPs were found, the molecular composition of the Young PRP showed lower levels of inflammatory molecules such as CCL-11, as well as the presence of other factors not found in Aged PRP (GDF-11). Although both PRPs had effects in terms of reducing neural progenitor cell apoptosis, stabilizing neuronal synapses, and decreasing inflammation in the microglia, the effect of the Young PRP was more pronounced. In conclusion, the molecular composition of the PRP, conditioned by the age of the donors, affects the magnitude of the biological response.

Mitochondrial division inhibitor 1 disrupts oligodendrocyte Ca2+ homeostasis and mitochondrial function.

Mitochondrial fission mediated by cytosolic dynamin related protein 1 (Drp1) is essential for mitochondrial quality control but may contribute to apoptosis as well. Blockade of Drp1 with mitochondrial division inhibitor 1 (mdivi-1) provides neuroprotection in several models of neurodegeneration and cerebral ischemia and has emerged as a promising therapeutic drug. In oligodendrocytes, overactivation of AMPA-type ionotropic glutamate receptors (AMPARs) induces intracellular Ca2+ overload and excitotoxic death that contributes to demyelinating diseases. Mitochondria are key to Ca2+ homeostasis, however it is unclear how it is disrupted during oligodendroglial excitotoxicity. In the current study, we have analyzed mitochondrial dynamics during AMPAR activation and the effects of mdivi-1 on excitotoxicity in optic nerve-derived oligodendrocytes. Sublethal AMPAR activation triggered Drp1-dependent mitochondrial fission, whereas toxic AMPAR activation produced Drp1-independent mitochondrial swelling. Accordingly, mdivi-1 efficiently inhibited Drp1-mediated mitochondrial fission and did not prevent oligodendrocyte excitotoxicity. Unexpectedly, mdivi-1 also induced mitochondrial depolarization, ER Ca2+ depletion and modulation of AMPA-induced Ca2+ signaling. These off-target effects of mdivi-1 sensitized oligodendrocytes to excitotoxicity and ER stress and eventually produced oxidative stress and apoptosis. Interestingly, in cultured astrocytes mdivi-1 induced nondetrimental mitochondrial depolarization and oxidative stress that did not cause toxicity or sensitization to apoptotic stimuli. In summary, our results provide evidence of Drp1-mediated mitochondrial fission during activation of ionotropic glutamate receptors in oligodendrocytes, and uncover a deleterious and Drp1-independent effect of mdivi-1 on mitochondrial and ER function in these cells. These off-target effects of mdivi-1 limit its therapeutic potential and should be taken into account in clinical studies.

Aß1-42 triggers the generation of a retrograde signaling complex from sentinel mRNAs in axons.

Neurons frequently encounter neurodegenerative signals first in their periphery. For example, exposure of axons to oligomeric Aß1-42 is sufficient to induce changes in the neuronal cell body that ultimately lead to degeneration. Currently, it is unclear how the information about the neurodegenerative insult is transmitted to the soma. Here, we find that the translation of pre-localized but normally silenced sentinel mRNAs in axons is induced within minutes of Aß1-42 addition in a Ca2+-dependent manner. This immediate protein synthesis following Aß1-42 exposure generates a retrograde signaling complex including vimentin. Inhibition of the immediate protein synthesis, knock-down of axonal vimentin synthesis, or inhibition of dynein-dependent transport to the soma prevented the normal cell body response to Aß1-42 These results establish that CNS axons react to neurodegenerative insults via the local translation of sentinel mRNAs encoding components of a retrograde signaling complex that transmit the information about the event to the neuronal soma.

Contribution of P2X4 Receptors to CNS Function and Pathophysiology.

The release and extracellular action of ATP are a widespread mechanism for cell-to-cell communication in living organisms through activation of P2X and P2Y receptors expressed at the cell surface of most tissues, including the nervous system. Among ionototropic receptors, P2X4 receptors have emerged in the last decade as a potential target for CNS disorders such as epilepsy, ischemia, chronic pain, anxiety, multiple sclerosis and neurodegenerative diseases. However, the role of P2X4 receptor in each pathology ranges from beneficial to detrimental, although the mechanisms are still mostly unknown. P2X4 is expressed at low levels in CNS cells including neurons and glial cells. In normal conditions, P2X4 activation contributes to synaptic transmission and synaptic plasticity. Importantly, one of the genes present in the transcriptional program of myeloid cell activation is P2X4. Microglial P2X4 upregulation, the P2X4+ state of microglia, seems to be common in most acute and chronic neurodegenerative diseases associated with inflammation. In this review, we summarize knowledge about the role of P2X4 receptors in the CNS physiology and discuss potential pitfalls and open questions about the therapeutic potential of blocking or potentiation of P2X4 for different pathologies.

Sephin1 Protects Neurons against Excitotoxicity Independently of the Integrated Stress Response.

Sephin1 is a derivative of guanabenz that inhibits the dephosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α) and therefore may enhance the integrated stress response (ISR), an adaptive mechanism against different cellular stresses, such as accumulation of misfolded proteins. Unlike guanabenz, Sephin1 provides neuroprotection without adverse effects on the α2-adrenergic system and therefore it is considered a promising pharmacological therapeutic tool. Here, we have studied the effects of Sephin1 on N-methyl-D-aspartic acid (NMDA) receptor signaling which may modulate the ISR and contribute to excitotoxic neuronal loss in several neurodegenerative conditions. Time-course analysis of peIF2α levels after NMDA receptor overactivation showed a delayed dephosphorylation that occurred in the absence of activating transcription factor 4 (ATF4) and therefore independently of the ISR, in contrast to that observed during endoplasmic reticulum (ER) stress induced by tunicamycin and thapsigargin. Similar to guanabenz, Sephin1 completely blocked NMDA-induced neuronal death and was ineffective against AMPA-induced excitotoxicity, whereas it did not protect from experimental ER stress. Interestingly, both guanabenz and Sephin1 partially but significantly reduced NMDA-induced cytosolic Ca2+ increase, leading to a complete inhibition of subsequent calpain activation. We conclude that Sephin1 and guanabenz share common strong anti-excitotoxic properties with therapeutic potential unrelated to the ISR.

Microglial immune response is impaired against the neurotropic fungus Lomentospora prolificans.

Lomentospora (Scedosporium) prolificans is an opportunistic pathogen capable of causing invasive infections in immunocompromised patients. The fungus is able to disseminate via the bloodstream finally arriving at the central nervous system producing neurological symptoms and, in many cases, patient death. In this context, microglial cells, which are the resident immune cells in the central nervous system, may play an important role in these infections. However, this aspect of anti-L. prolificans immunity has been poorly researched to date. Thus, the interactions and activity of microglial cells against L. prolificans were analysed, and the results show that there was a remarkable impairment in their performance regarding phagocytosis, the development of oxidative burst, and in the production of pro-inflammatory cytokines, compared with macrophages. Interestingly, L. prolificans displays great growth also when challenged with immune cells, even when inside them. We also proved that microglial phagocytosis of the fungus is highly dependent on mannose receptor and especially on dectin-1. Taken together, these data provide evidence for an impaired microglial response against L. prolificans and contribute to understanding the pathobiology of its neurotropism.

Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.