Complex IV subunit isoform COX6A2 protects fast-spiking interneurons from oxidative stress and supports their function.

Parvalbumin-positive (PV+ ) fast-spiking interneurons are essential to control the firing activity of principal neuron ensembles, thereby regulating cognitive processes. The high firing frequency activity of PV+ interneurons imposes high-energy demands on their metabolism that must be supplied by distinctive machinery for energy generation. Exploring single-cell transcriptomic data for the mouse cortex, we identified a metabolism-associated gene with highly restricted expression to PV+ interneurons: Cox6a2, which codes for an isoform of a cytochrome c oxidase subunit. Cox6a2 deletion in mice disrupts perineuronal nets and enhances oxidative stress in PV+ interneurons, which in turn impairs the maturation of their morphological and functional properties. Such dramatic effects were likely due to an essential role of COX6A2 in energy balance of PV+ interneurons, underscored by a decrease in the ATP-to-ADP ratio in Cox6a2-/- PV+ interneurons. Energy disbalance and aberrant maturation likely hinder the integration of PV+ interneurons into cortical neuronal circuits, leading to behavioral alterations in mice. Additionally, in a human patient bearing mutations in COX6A2, we found a potential association of the mutations with mental/neurological abnormalities.

Proteomic phenotype of cerebral organoids derived from autism spectrum disorder patients reveal disrupted energy metabolism, cellular components, and biological processes.

The way in which brain morphology and proteome are remodeled during embryonal development, and how they are linked to the cellular metabolism, could be a key for elucidating the pathological mechanisms of certain neurodevelopmental disorders. Cerebral organoids derived from autism spectrum disorder (ASD) patients were generated to capture critical time-points in the neuronal development, and metabolism and protein expression were investigated. The early stages of development, when neurogenesis commences (day in vitro 39), appeared to be a critical timepoint in pathogenesis. In the first month of development, increased size in ASD-derived organoids were detected in comparison to the controls. The size of the organoids correlates with the number of proliferating cells (Ki-67 positive cells). A significant difference in energy metabolism and proteome phenotype was also observed in ASD organoids at this time point, specifically, prevalence of glycolysis over oxidative phosphorylation, decreased ATP production and mitochondrial respiratory chain activity, differently expressed cell adhesion proteins, cell cycle (spindle formation), cytoskeleton, and several transcription factors. Finally, ASD patients and controls derived organoids were clustered based on a differential expression of ten proteins-heat shock protein 27 (hsp27) phospho Ser 15, Pyk (FAK2), Elk-1, Rac1/cdc42, S6 ribosomal protein phospho Ser 240/Ser 244, Ha-ras, mTOR (FRAP) phospho Ser 2448, PKCα, FoxO3a, Src family phospho Tyr 416-at day 39 which could be defined as potential biomarkers and further investigated for potential drug development.

Potential Retinal Biomarkers in Alzheimer's Disease.

Alzheimer's disease (AD) represents a major diagnostic challenge, as early detection is crucial for effective intervention. This review examines the diagnostic challenges facing current AD evaluations and explores the emerging field of retinal alterations as early indicators. Recognizing the potential of the retina as a noninvasive window to the brain, we emphasize the importance of identifying retinal biomarkers in the early stages of AD. However, the examination of AD is not without its challenges, as the similarities shared with other retinal diseases introduce complexity in the search for AD-specific markers. In this review, we address the relevance of using the retina for the early diagnosis of AD and the complex challenges associated with the search for AD-specific retinal biomarkers. We provide a comprehensive overview of the current landscape and highlight avenues for progress in AD diagnosis by retinal examination.

Decreased Glucose Metabolism and Glutamine Synthesis in the Retina of a Transgenic Mouse Model of Alzheimer's Disease.

Visual changes are some of the earliest symptoms that patients with Alzheimer's disease (AD) experience. Pathophysiological processes such as amyloid-ß plaque formation, vascular changes, neuroinflammation, and loss of retinal ganglion cells (RGCs) have been detected in the retina of AD patients and animal models. However, little is known about the molecular processes that underlie retinal neurodegeneration in AD. The cellular architecture and constant sensory activity of the retina impose high metabolic demands. We thus hypothesized that energy metabolism might be compromised in the AD retina similarly to what has been observed in the AD brain. To address this question, we explored cellular alterations and retinal metabolic activity in the 5 × FAD mouse model of AD. We used 8-month-old female 5 × FAD mice, in which the AD-related pathology has been shown to be apparent. We observed that RGC density is selectively affected in the retina of 5 × FAD mice. To map retinal metabolic activity, we incubated isolated retinal tissue with [U-13C] glucose and analyzed tissue extracts by gas chromatography-mass spectrometry. We found that the retinas of 5 × FAD mice exhibit glucose hypometabolism. Moreover, we detected decreased glutamine synthesis in 5 × FAD retinas but no changes in the expression of markers of Müller glia, the main glial cell type responsible for glutamate uptake and glutamine synthesis in the retina. These findings suggest that AD presents with metabolic alterations not only in the brain but also in the retina that may be detrimental to RGC activity and survival, potentially leading to the visual impairments that AD patients suffer.

Staphylococcus aureus induces cell-surface expression of immune stimulatory NKG2D ligands on human monocytes.

Staphylococcus aureus is among the leading causes of bacterial infections worldwide. The pathogenicity and establishment of S. aureus infections are tightly linked to its ability to modulate host immunity. Persistent infections are often associated with mutant staphylococcal strains that have decreased susceptibility to antibiotics; however, little is known about how these mutations influence bacterial interaction with the host immune system. Here, we discovered that clinical S. aureus isolates activate human monocytes, leading to cell-surface expression of immune stimulatory natural killer group 2D (NKG2D) ligands on the monocytes. We found that expression of the NKG2D ligand ULBP2 (UL16-binding protein 2) is associated with bacterial degradability and phagolysosomal activity. Moreover, S. aureus-induced ULBP2 expression was linked to altered host cell metabolism, including increased cytoplasmic (iso)citrate levels, reduced glycolytic flux, and functional mitochondrial activity. Interestingly, we found that the ability of S. aureus to induce ULBP2 and proinflammatory cytokines in human monocytes depends on a functional ClpP protease in S. aureus These findings indicate that S. aureus activates ULBP2 in human monocytes through immunometabolic mechanisms and reveal that clpP inactivation may function as a potential immune evasion mechanism. Our results provide critical insight into the interplay between the host immune system and S. aureus that has evolved under the dual selective pressure of host immune responses and antibiotic treatment. Our discovery of an immune stimulatory pathway consisting of human monocyte-based defense against S. aureus suggests that targeting the NKG2D pathway holds potential for managing persistent staphylococcal infections.

The role of astrocytes in seizure generation: insights from a novel in vitro seizure model based on mitochondrial dysfunction.

Approximately one-quarter of patients with mitochondrial disease experience epilepsy. Their epilepsy is often severe and resistant towards conventional antiepileptic drugs. Despite the severity of this epilepsy, there are currently no animal models available to provide a mechanistic understanding of mitochondrial epilepsy. We conducted neuropathological studies on patients with mitochondrial epilepsy and found the involvement of the astrocytic compartment. As a proof of concept, we developed a novel brain slice model of mitochondrial epilepsy by the application of an astrocytic-specific aconitase inhibitor, fluorocitrate, concomitant with mitochondrial respiratory inhibitors, rotenone and potassium cyanide. The model was robust and exhibited both face and predictive validity. We then used the model to assess the role that astrocytes play in seizure generation and demonstrated the involvement of the GABA-glutamate-glutamine cycle. Notably, glutamine appears to be an important intermediary molecule between the neuronal and astrocytic compartment in the regulation of GABAergic inhibitory tone. Finally, we found that a deficiency in glutamine synthetase is an important pathogenic process for seizure generation in both the brain slice model and the human neuropathological study. Our study describes the first model for mitochondrial epilepsy and provides a mechanistic insight into how astrocytes drive seizure generation in mitochondrial epilepsy.

Effect of the Anti-depressant Sertraline, the Novel Anti-seizure Drug Vinpocetine and Several Conventional Antiepileptic Drugs on the Epileptiform EEG Activity Induced by 4-Aminopyridine.

Seizures are accompanied by an exacerbated activation of cerebral ion channels. 4-aminopyridine (4-AP) is a pro-convulsive agent which mechanism of action involves activation of Na(+) and Ca(2+) channels, and several antiepileptic drugs control seizures by reducing these channels permeability. The antidepressant, sertraline, and the anti-seizure drug vinpocetine are effective inhibitors of cerebral presynaptic Na(+) channels. Here the effectiveness of these compounds to prevent the epileptiform EEG activity induced by 4-AP was compared with the effectiveness of seven conventional antiepileptic drugs. For this purpose, EEG recordings before and at three intervals within the next 30 min following 4-AP (2.5 mg/kg, i.p.) were taken in anesthetized animals; and the EEG-highest peak amplitude values (HPAV) calculated. In control animals, the marked increase in the EEG-HPAV observed near 20 min following 4-AP reached its maximum at 30 min. Results show that this epileptiform EEG activity induced by 4-AP is prevented by sertraline and vinpocetine at a dose of 2.5 mg/kg, and by carbamazepine, phenytoin, lamotrigine and oxcarbazepine at a higher dose (25 mg/kg). In contrast, topiramate (25 mg/kg), valproate (100 mg/kg) and levetiracetam (100 mg/kg) failed to prevent the epileptiform EEG activity induced by 4-AP. It is concluded that 4-AP is a useful tool to elicit the mechanism of action of anti-seizure drugs at clinical meaningful doses. The particular efficacy of sertraline and vinpocetine to prevent seizures induced by 4-AP is explained by their high effectiveness to reduce brain presynaptic Na(+) and Ca(2+) channels permeability.

Preserved blood-brain barrier and neurovascular coupling in female 5xFAD model of Alzheimer's disease.

Introduction: Dysfunction of the cerebral vasculature is considered one of the key components of Alzheimer's disease (AD), but the mechanisms affecting individual brain vessels are poorly understood. Methods: Here, using in vivo two-photon microscopy in superficial cortical layers and ex vivo imaging across brain regions, we characterized blood-brain barrier (BBB) function and neurovascular coupling (NVC) at the level of individual brain vessels in adult female 5xFAD mice, an aggressive amyloid-ß (Aß) model of AD. Results: We report a lack of abnormal increase in adsorptive-mediated transcytosis of albumin and preserved paracellular barrier for fibrinogen and small molecules despite an extensive load of Aß. Likewise, the NVC responses to somatosensory stimulation were preserved at all regulatory segments of the microvasculature: penetrating arterioles, precapillary sphincters, and capillaries. Lastly, the Aß plaques did not affect the density of capillary pericytes. Conclusion: Our findings provide direct evidence of preserved microvascular function in the 5xFAD mice and highlight the critical dependence of the experimental outcomes on the choice of preclinical models of AD. We propose that the presence of parenchymal Aß does not warrant BBB and NVC dysfunction and that the generalized view that microvascular impairment is inherent to Aß aggregation may need to be revised.

Prevention of Cell Death by Activation of Hydroxycarboxylic Acid Receptor 1 (GPR81) in Retinal Explants.

BACKGROUND: Progressive retinal ganglion cell (RGC) dysfunction and death are common characteristics of retinal neurodegenerative diseases. Recently, hydroxycarboxylic acid receptor 1 (HCA1R, GPR81) was identified as a key modulator of mitochondrial function and cell survival. Thus, we aimed to test whether activation of HCA1R with 3,5-Dihydroxybenzoic acid (DHBA) also promotes RGC survival and improves energy metabolism in mouse retinas. METHODS: Retinal explants were treated with 5 mM of the HCA1R agonist, 3,5-DHBA, for 2, 4, 24, and 72 h. Additionally, explants were also treated with 15 mM of L-glutamate to induce toxicity. Tissue survival was assessed through lactate dehydrogenase (LDH) viability assays. RGC survival was measured through immunohistochemical (IHC) staining. Total ATP levels were quantified through bioluminescence assays. Energy metabolism was investigated through stable isotope labeling and gas chromatography-mass spectrometry (GC-MS). Lactate and nitric oxide levels were measured through colorimetric assays. RESULTS: HCA1R activation with 3,5-DHBAincreased retinal explant survival. During glutamate-induced death, 3,5-DHBA treatment also increased survival. IHC analysis revealed that 3,5-DHBA treatment promoted RGC survival in retinal wholemounts. 3,5-DHBA treatment also enhanced ATP levels in retinal explants, whereas lactate levels decreased. No effects on glucose metabolism were observed, but small changes in lactate metabolism were found. Nitric oxide levels remained unaltered in response to 3,5-DHBA treatment. CONCLUSION: The present study reveals that activation of HCA1R with 3,5-DHBA treatment has a neuroprotective effect specifically on RGCs and on glutamate-induced retinal degeneration. Hence, HCA1R agonist administration may be a potential new strategy for rescuing RGCs, ultimately preventing visual disability.

Neuronal Loss of the Glutamate Transporter GLT-1 Promotes Excitotoxic Injury in the Hippocampus.

GLT-1, the major glutamate transporter in the mammalian central nervous system, is expressed in presynaptic terminals that use glutamate as a neurotransmitter, in addition to astrocytes. It is widely assumed that glutamate homeostasis is regulated primarily by glutamate transporters expressed in astrocytes, leaving the function of GLT-1 in neurons relatively unexplored. We generated conditional GLT-1 knockout (KO) mouse lines to understand the cell-specific functions of GLT-1. We found that stimulus-evoked field extracellular postsynaptic potentials (fEPSPs) recorded in the CA1 region of the hippocampus were normal in the astrocytic GLT-1 KO but were reduced and often absent in the neuronal GLT-1 KO at 40 weeks. The failure of fEPSP generation in the neuronal GLT-1 KO was also observed in slices from 20 weeks old mice but not consistently from 10 weeks old mice. Using an extracellular FRET-based glutamate sensor, we found no difference in stimulus-evoked glutamate accumulation in the neuronal GLT-1 KO, suggesting a postsynaptic cause of the transmission failure. We hypothesized that excitotoxicity underlies the failure of functional recovery of slices from the neuronal GLT-1 KO. Consistent with this hypothesis, the non-competitive NMDA receptor antagonist MK801, when present in the ACSF during the recovery period following cutting of slices, promoted full restoration of fEPSP generation. The inclusion of an enzymatic glutamate scavenging system in the ACSF conferred partial protection. Excitotoxicity might be due to excess release or accumulation of excitatory amino acids, or to metabolic perturbation resulting in increased vulnerability to NMDA receptor activation. Previous studies have demonstrated a defect in the utilization of glutamate by synaptic mitochondria and aspartate production in the synGLT-1 KO in vivo, and we found evidence for similar metabolic perturbations in the slice preparation. In addition, mitochondrial cristae density was higher in synaptic mitochondria in the CA1 region in 20-25 weeks old synGLT-1 KO mice in the CA1 region, suggesting compensation for loss of axon terminal GLT-1 by increased mitochondrial efficiency. These data suggest that GLT-1 expressed in presynaptic terminals serves an important role in the regulation of vulnerability to excitotoxicity, and this regulation may be related to the metabolic role of GLT-1 expressed in glutamatergic axon terminals.

Role of histaminergic system in blood-brain barrier dysfunction associated with neurological disorders.

Blood-brain barrier (BBB) disruption has been associated with several acute and chronic brain disorders such as Alzheimer's disease, Parkinson's disease and epilepsy. This represents a critical situation because damaged integrity of the BBB is related to the influx of immune mediators, plasma proteins and other outside elements from blood to the central nervous system (CNS) that may trigger a cascade of events that leads to neuroinflammation. In this review, evidence that mast cells and the release of factors such as histamine play an important role in the neuroinflammatory process associated with brain disorders such as Alzheimer's disease, Parkinson's disease and epilepsy is presented.