Mitochondria: A Promising Convergent Target for the Treatment of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons, for which current treatment options are limited. Recent studies have shed light on the role of mitochondria in ALS pathogenesis, making them an attractive therapeutic intervention target. This review contains a very comprehensive critical description of the involvement of mitochondria and mitochondria-mediated mechanisms in ALS. The review covers several key areas related to mitochondria in ALS, including impaired mitochondrial function, mitochondrial bioenergetics, reactive oxygen species, metabolic processes and energy metabolism, mitochondrial dynamics, turnover, autophagy and mitophagy, impaired mitochondrial transport, and apoptosis. This review also highlights preclinical and clinical studies that have investigated various mitochondria-targeted therapies for ALS treatment. These include strategies to improve mitochondrial function, such as the use of dichloroacetate, ketogenic and high-fat diets, acetyl-carnitine, and mitochondria-targeted antioxidants. Additionally, antiapoptotic agents, like the mPTP-targeting agents minocycline and rasagiline, are discussed. The paper aims to contribute to the identification of effective mitochondria-targeted therapies for ALS treatment by synthesizing the current understanding of the role of mitochondria in ALS pathogenesis and reviewing potential convergent therapeutic interventions. The complex interplay between mitochondria and the pathogenic mechanisms of ALS holds promise for the development of novel treatment strategies to combat this devastating disease.

Age-dependent energy metabolism and transcriptome changes in urine-derived stem cells.

The global population over 60 years old is projected to reach 1.5 billion by 2050. Understanding age-related disorders and gender-specificities is crucial for a healthy aging. Reliable age-related biomarkers are needed, preferentially obtained through non-invasive methods. Urine-derived stem cells (UDSCs) can be easily obtained, although a detailed bioenergetic characterization, according to the donor aging, remain unexplored. UDSCs were isolated from young and elderly adult women (22-35 and 70-94 years old, respectively). Surprisingly, UDSCs from elderly subjects exhibited significantly higher maximal oxygen consumption and bioenergetic health index than those from younger individuals, evaluated through oxygen consumption rate. Exploratory data analysis methods were applied to engineer a minimal subset of features for the classification and stratification of UDSCs. Additionally, RNAseq of UDSCs was performed to identify age-related transcriptional changes. Transcriptional analysis revealed downregulation of genes related to glucuronidation and estrogen metabolism, and upregulation of inflammation-related genes in UDSCs from elderly individuals. This study demonstrates unexpected differences in the UDSCs' OCR between young and elderly individuals, revealing improved bioenergetics in concurrent with an aged-like transcriptome signature. UDSCs offer a non-invasive model for studying age-related changes, holding promise for aging research and therapeutic studies.

Urine-derived stem cells in neurological diseases: current state-of-the-art and future directions.

Stem cells have potential applications in the field of neurological diseases, as they allow for the development of new biological models. These models can improve our understanding of the underlying pathologies and facilitate the screening of new therapeutics in the context of precision medicine. Stem cells have also been applied in clinical tests to repair tissues and improve functional recovery. Nevertheless, although promising, commonly used stem cells display some limitations that curb the scope of their applications, such as the difficulty of obtention. In that regard, urine-derived cells can be reprogrammed into induced pluripotent stem cells (iPSCs). However, their obtaining can be challenging due to the low yield and complexity of the multi-phased and typically expensive differentiation protocols. As an alternative, urine-derived stem cells (UDSCs), included within the population of urine-derived cells, present a mesenchymal-like phenotype and have shown promising properties for similar purposes. Importantly, UDSCs have been differentiated into neuronal-like cells, auspicious for disease modeling, while overcoming some of the shortcomings presented by other stem cells for these purposes. Thus, this review assesses the current state and future perspectives regarding the potential of UDSCs in the ambit of neurological diseases, both for disease modeling and therapeutic applications.

Recent Advances in Extracellular Vesicles in Amyotrophic Lateral Sclerosis and Emergent Perspectives.

Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease characterized by the progressive death of motor neurons, leading to paralysis and death. It is a rare disease characterized by high patient-to-patient heterogeneity, which makes its study arduous and complex. Extracellular vesicles (EVs) have emerged as important players in the development of ALS. Thus, ALS phenotype-expressing cells can spread their abnormal bioactive cargo through the secretion of EVs, even in distant tissues. Importantly, owing to their nature and composition, EVs' formation and cargo can be exploited for better comprehension of this elusive disease and identification of novel biomarkers, as well as for potential therapeutic applications, such as those based on stem cell-derived exosomes. This review highlights recent advances in the identification of the role of EVs in ALS etiopathology and how EVs can be promising new therapeutic strategies.

Integrative Profiling of Amyotrophic Lateral Sclerosis Lymphoblasts Identifies Unique Metabolic and Mitochondrial Disease Fingerprints.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with a rapid progression and no effective treatment. Metabolic and mitochondrial alterations in peripheral tissues of ALS patients may present diagnostic and therapeutic interest. We aimed to identify mitochondrial fingerprints in lymphoblast from ALS patients harboring SOD1 mutations (mutSOD1) or with unidentified mutations (undSOD1), compared with age-/sex-matched controls. Three groups of lymphoblasts, from mutSOD1 or undSOD1 ALS patients and age-/sex-matched controls, were obtained from Coriell Biobank and divided into 3 age-/sex-matched cohorts. Mitochondria-associated metabolic pathways were analyzed using Seahorse MitoStress and ATP Rate assays, complemented with metabolic phenotype microarrays, metabolite levels, gene expression, and protein expression and activity. Pooled (all cohorts) and paired (intra-cohort) analyses were performed by using bioinformatic tools, and the features with higher information gain values were selected and used for principal component analysis and Naïve Bayes classification. Considering the group as a target, the features that contributed to better segregation of control, undSOD1, and mutSOD1 were found to be the protein levels of Tfam and glycolytic ATP production rate. Metabolic phenotypic profiles in lymphoblasts from ALS patients with mutSOD1 and undSOD1 revealed unique age-dependent different substrate oxidation profiles. For most parameters, different patterns of variation in experimental endpoints in lymphoblasts were found between cohorts, which may be due to the age or sex of the donor. In the present work, we investigated several metabolic and mitochondrial hallmarks in lymphoblasts from each donor, and although a high heterogeneity of results was found, we identified specific metabolic and mitochondrial fingerprints, especially protein levels of Tfam and glycolytic ATP production rate, that may have a diagnostic and therapeutic interest.

Differences in Immune-Related Genes Underlie Temporal and Regional Pathological Progression in 3xTg-AD Mice.

The prevalence of Alzheimer's disease (AD), the most common cause of age-associated dementia, is estimated to increase over the next decades. Evidence suggests neuro-immune signaling deregulation and risk genes beyond the amyloid-ß (Aß) deposition in AD pathology. We examined the temporal profile of inflammatory mediators and microglia deactivation/activation in the brain cortex and hippocampus of 3xTg-AD mice at 3- and 9-month-old. We found upregulated APP processing, decreased expression of CD11b, CX3CR1, MFG-E8, TNF-α, IL-1ß, MHC-II and C/EBP-α and increased miR-146a in both brain regions in 3-month-old 3xTG-AD mice, suggestive of a restrictive regulation. Enhanced TNF-α, IL-1ß, IL-6, iNOS, SOCS1 and Arginase 1 were only present in the hippocampus of 9-month-old animals, though elevation of HMGB1 and reduction of miR-146a and miR-124 were common features in the hippocampus and cortex regions. miR-155 increased early in the cortex and later in both regions, supporting its potential as a biomarker. Candidate downregulated target genes by cortical miR-155 included Foxo3, Runx2 and CEBPß at 3 months and Foxo3, Runx2 and Socs1 at 9 months, which are implicated in cell survival, but also in Aß pathology and microglia/astrocyte dysfunction. Data provide new insights across AD state trajectory, with divergent microglia phenotypes and inflammatory-associated features, and identify critical targets for drug discovery and combinatorial therapies.

Regulation of hippocampal postnatal and adult neurogenesis by adenosine A2A receptor: Interaction with brain-derived neurotrophic factor.

Adenosine A2A receptor (A2A R) activation modulates several brain processes, ranging from neuronal maturation to synaptic plasticity. Most of these actions occur through the modulation of the actions of the neurotrophin brain-derived neurotrophic factor (BDNF). In this work, we studied the role of A2A Rs in regulating postnatal and adult neurogenesis in the rat hippocampal dentate gyrus (DG). Here, we show that A2A R activation with CGS 21680 promoted neural stem cell self-renewal, protected committed neuronal cells from cell death and contributed to a higher density of immature and mature neuronal cells, particularly glutamatergic neurons. Moreover, A2A R endogenous activation was found to be essential for BDNF-mediated increase in cell proliferation and neuronal differentiation. Our findings contribute to further understand the role of adenosinergic signaling in the brain and may have an impact in the development of strategies for brain repair under pathological conditions.

Chronic hyperglycemia impairs hippocampal neurogenesis and memory in an Alzheimer's disease mouse model.

During aging, lifestyle-related factors shape the brain's response to insults and modulate the progression of neurodegenerative pathologies such as Alzheimer's disease (AD). This is the case for chronic hyperglycemia associated with type 2 diabetes, which reduces the brain's ability to handle the neurodegenerative burden associated with AD. However, the mechanisms behind the effects of chronic hyperglycemia in the context of AD are not fully understood. Here, we show that newly generated neurons in the hippocampal dentate gyrus of triple transgenic AD (3xTg-AD) mice present increased dendritic arborization and a number of synaptic puncta, which may constitute a compensatory mechanism allowing the animals to cope with a lower neurogenesis rate. Contrariwise, chronic hyperglycemia decreases the complexity and differentiation of 3xTg-AD newborn neurons and reduces the levels of ß-catenin, a key intrinsic modulator of neuronal maturation. Moreover, synaptic facilitation is depressed in hyperglycemic 3xTg-AD mice, accompanying the defective hippocampal-dependent memory. Our data suggest that hyperglycemia evokes cellular and functional alterations that accelerate the onset of AD-related symptoms, namely memory impairment.

miRNA-31 Improves Cognition and Abolishes Amyloid-ß Pathology by Targeting APP and BACE1 in an Animal Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia worldwide, characterized by progressive memory impairment, behavioral changes, and, ultimately, loss of consciousness and death. Recently, microRNA (miRNA) dysfunction has been associated with increased production and impaired clearance of amyloid-ß (Aß) peptides, whose accumulation is one of the most well-known pathophysiological markers of this disease. In this study, we identified several miRNAs capable of targeting key proteins of the amyloidogenic pathway. The expression of one of these miRNAs, miR-31, previously found to be decreased in AD patients, was able to simultaneously reduce the levels of APP and Bace1 mRNA in the hippocampus of 17-month-old AD triple-transgenic (3xTg-AD) female mice, leading to a significant improvement of memory deficits and a reduction in anxiety and cognitive inflexibility. In addition, lentiviral-mediated miR-31 expression significantly ameliorated AD neuropathology in this model, drastically reducing Aß deposition in both the hippocampus and subiculum. Furthermore, the increase of miR-31 levels was enough to reduce the accumulation of glutamate vesicles in the hippocampus to levels found in non-transgenic age-matched animals. Overall, our results suggest that miR-31-mediated modulation of APP and BACE1 can become a therapeutic option in the treatment of AD.

Unraveling the Nanoscopic Organization and Function of Central Mammalian Presynapses With Super-Resolution Microscopy.

The complex, nanoscopic scale of neuronal function, taking place at dendritic spines, axon terminals, and other minuscule structures, cannot be adequately resolved using standard, diffraction-limited imaging techniques. The last couple of decades saw a rapid evolution of imaging methods that overcome the diffraction limit imposed by Abbe's principle. These techniques, including structured illumination microscopy (SIM), stimulated emission depletion (STED), photo-activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM), among others, have revolutionized our understanding of synapse biology. By exploiting the stochastic nature of fluorophore light/dark states or non-linearities in the interaction of fluorophores with light, by using modified illumination strategies that limit the excitation area, these methods can achieve spatial resolutions down to just a few tens of nm or less. Here, we review how these advanced imaging techniques have contributed to unprecedented insight into the nanoscopic organization and function of mammalian neuronal presynapses, revealing new organizational principles or lending support to existing views, while raising many important new questions. We further discuss recent technical refinements and newly developed tools that will continue to expand our ability to delve deeper into how synaptic function is orchestrated at the nanoscopic level.

Histamine modulates hippocampal inflammation and neurogenesis in adult mice.

Evidence points to a dual role of histamine in microglia-mediated neuroinflammation, a key pathological feature of several neurodegenerative pathologies. Moreover, histamine has been suggested as a modulator of adult neurogenesis. Herein, we evaluated the effect of histamine in hippocampal neuroinflammation and neurogenesis under physiological and inflammatory contexts. For that purpose, mice were intraperitoneally challenged with lipopolysaccharide (LPS) followed by an intrahippocampal injection of histamine. We showed that histamine per se triggered glial reactivity and induced mild long-term impairments in neurogenesis, reducing immature neurons dendritic volume and complexity. Nevertheless, in mice exposed to LPS (2 mg/Kg), histamine was able to counteract LPS-induced glial activation and release of pro-inflammatory molecules as well as neurogenesis impairment. Moreover, histamine prevented LPS-induced loss of immature neurons complexity as well as LPS-induced loss of both CREB and PSD-95 proteins (essential for proper neuronal activity). Altogether, our results highlight histamine as a potential therapeutic agent to treat neurological conditions associated with hippocampal neuroinflammation and neurodegeneration.

Chronic stress triggers divergent dendritic alterations in immature neurons of the adult hippocampus, depending on their ultimate terminal fields.

Chronic stress, a suggested precipitant of brain pathologies, such as depression and Alzheimer's disease, is known to impact on brain plasticity by causing neuronal remodeling as well as neurogenesis suppression in the adult hippocampus. Although many studies show that stressful conditions reduce the number of newborn neurons in the adult dentate gyrus (DG), little is known about whether and how stress impacts on dendritic development and structural maturation of these newborn neurons. We, herein, demonstrate that chronic stress impacts differentially on doublecortin (DCX)-positive immature neurons in distinct phases of maturation. Specifically, the density of the DCX-positive immature neurons whose dendritic tree reaches the inner molecular layer (IML) of DG is reduced in stressed animals, whereas their dendritic complexity is increased. On the contrary, no change on the density of DCX-positive neurons whose dendritic tree extends to the medial/outer molecular layer (M/OML) of the DG is found under stress conditions, whereas the dendritic complexity of these cells is diminished. In addition, DCX+ cells displayed a more complex and longer arbor in the dendritic compartments located in the granular cell layer of the DG under stress conditions; on the contrary, their dendritic segments localized into the M/OML were shorter and less complex. These findings suggest that the neuroplastic effects of chronic stress on dendritic maturation and complexity of DCX+ immature neurons vary based on the different maturation stage of DCX-positive cells and the different DG sublayer, highlighting the complex and dynamic stress-driven neuroplasticity of immature neurons in the adult hippocampus.

Coriolus versicolor biomass increases dendritic arborization of newly-generated neurons in mouse hippocampal dentate gyrus.

Brain cognitive reserve refers to the ability of the brain to manage different challenges that arise throughout life, making it resilient to neuropathology. Hippocampal adult neurogenesis has been considered to be a relevant contributor for brain cognitive reserve and brain plasticity. Coriolus versicolor (CV), a common healthful mushroom, has been receiving increasing attention by its antitumoral, anti-inflammatory, antioxidant, antibacterial, and immunomodulatory properties, including in the hippocampus. Herein, we evaluated whether CV biomass oral administration for 2.5 months enhances hippocampal neurogenic reserve under normal/physiological conditions, by quantifying hippocampal dentate gyrus (DG) granular cell layer (GCL) and subgranular zone (SGZ) volumes, proliferation, number and dendritic complexity features of hippocampal newly-generated neurons. We also analyzed ß-catenin levels in DG newly-generated immature neurons, because it plays a major role in neurogenesis. Although no differences were observed in the volume of GCL and SGZ layers, in proliferation and in the number of newly-generated neurons of controls and CV-administered mice, we found that CV administration promotes a significant increase in dendritic length and branching and total dendritic volume of immature neurons, suggesting a positive effect of oral CV administration in the hippocampal neurogenic reserve. We also observed that ß-catenin levels are increased both in the nucleus and cytoplasm of DG immature neurons, suggesting that Wnt/ß-catenin signalling may play an important role in the CV positive effect on the differentiation of these cells. These data unveil a so far unexplored neurogenic potential of CV supplementation, which emerges as a possible preventive strategy for different neurological conditions.

Characterization of subventricular zone-derived progenitor cells from mild and late symptomatic YAC128 mouse model of Huntington's disease.

Huntington's disease (HD) is caused by an expansion of CAG repeats in the HTT gene, leading to expression of mutant huntingtin (mHTT) and selective striatal neuronal loss, frequently associated with mitochondrial dysfunction and decreased support of brain-derived neurotrophic factor (BDNF). New neurons derived from the subventricular zone (SVZ) are apparently not able to rescue HD pathological features. Thus, we analyzed proliferation, migration and differentiation of adult SVZ-derived neural stem/progenitor cells (NSPC) from mild (6month-old (mo)) and late (10mo) symptomatic HD YAC128 mice expressing full-length (FL)-mHTT versus age-matched wild-type (WT) mice. SVZ cells derived from 6mo YAC128 mice exhibited higher migratory capacity and a higher number of MAP2+ and synaptophysin+cells, compared to WT cells; MAP2 labeling was enhanced after exposure to BDNF. However, BDNF-evoked neuronal differentiation was not observed in 10mo YAC128 SVZ-derived cells. Interestingly, 6mo YAC128 SVZ-derived cells showed increased intracellular Ca2+ levels in response to KCl, which was potentiated by BDNF, evidencing the presence of differentiated neurons. In contrast, KCl depolarization-induced intracellular Ca2+ increase in 10mo YAC128 SVZ-derived cells was shown to be increased only in BDNF-treated YAC128 SVZ-derived cells, suggestive of decreased differentiation capacity. In addition, BDNF-untreated NSPC from 10mo YAC128 mice exhibited lower mitochondrial membrane potential and increased mitochondrial Ca2+ accumulation, in relation with NSPC from 6mo YAC128 mice. Data evidence age-dependent reduced migration and decreased acquisition of a neuronal phenotype, accompanied by decreased mitochondrial membrane potential in SVZ-derived cells from YAC128 mice through HD symptomatic phases.

Transcription factor NRF2 controls the fate of neural stem cells in the subgranular zone of the hippocampus.

Neural stem/progenitor cells (NSPCs) located at the subgranular zone (SGZ) of the hippocampus participate in the maintenance of synaptic networks that ensure cognitive functions during life. Although it is known that this neurogenic niche losses activity with oxidative stress and ageing, the molecular events involved in its regulation are largely unknown. Here, we studied the role of transcription factor Nuclear Factor-Erythroid 2-Related Factor 2 (NRF2) in the control of NSPCs destinies in the SGZ. We first describe that NRF2-knockout (Nrf2-/-) mice exhibit impaired long term potentiation, a function that requires integrity of the SGZ, therefore suggesting a cognitive deficit that might be linked to hippocampal neurogenesis. Then, we found a reduction in NSCs from birth to adulthood that was exacerbated in Nrf2-/- vs. Nrf2+/+ mice. The clonogenic and proliferative capacity of SGZ-derived NSPCs from newborn and 3-month-old Nrf2-/- mice was severely reduced as determined in neurosphere cultures. Nrf2-deficiency also impaired neuronal differentiation both the SGZ, and in neurosphere differentiation assays, leading to an abnormal production of astrocytes and oligodendrocytes vs. neurons. Rescue of Nrf2-/- NSPCs by ectopic expression of NRF2 attenuated the alterations in clonogenic, proliferative and differentiating capacity of hippocampal NSPCs. In turn, knockdown of the NRF2 gene in wild type NSPCs reproduced the data obtained with Nrf2-/- NSPCs. Our findings demonstrate the importance of NRF2 in the maintenance of proper proliferation and differentiation rates of hippocampal NSPCs and suggest that interventions to up-regulate NRF2 might provide a mechanism to preserve the neurogenic functionality of the hippocampus.

Mitochondrial Ca2+ handling in Huntington's and Alzheimer's diseases - Role of ER-mitochondria crosstalk.

Mitochondria play a relevant role in Ca2+ buffering, governing energy metabolism and neuronal function. Huntington's disease (HD) and Alzheimer's disease (AD) are two neurodegenerative disorders that, although clinically distinct, share pathological features linked to selective brain damage. These include mitochondrial dysfunction, intracellular Ca2+ deregulation and mitochondrial Ca2+ handling deficits. Both diseases are associated with misfolding and aggregation of specific proteins that physically interact with mitochondria and interfere with endoplasmic reticulum (ER)/mitochondria-contact sites. Cumulating evidences indicate that impairment of mitochondrial Ca2+ homeostasis underlies the susceptibility to selective neuronal death observed in HD and AD; however data obtained with different models and experimental approaches are not always consistent. In this review, we explore the recent literature on deregulation of mitochondrial Ca2+ handling underlying the interplay between mitochondria and ER in HD and AD-associated neurodegeneration.

Interventions for age-related diseases: Shifting the paradigm.

Over 60% of people aged over 65 are affected by multiple morbidities, which are more difficult to treat, generate increased healthcare costs and lead to poor quality of life compared to individual diseases. With the number of older people steadily increasing this presents a societal challenge. Age is the major risk factor for age-related diseases and recent research developments have led to the proposal that pharmacological interventions targeting common mechanisms of ageing may be able to delay the onset of multimorbidity. Here we review the state of the knowledge of multimorbidity, appraise the available evidence supporting the role of mechanisms of ageing in the development of the most common age-related diseases and assess potential molecules that may successfully target those key mechanisms.

Age-dependent changes in the glutamate-nitric oxide pathway in the hippocampus of the triple transgenic model of Alzheimer's disease: implications for neurometabolic regulation.

Age-dependent changes in nitric oxide ((•)NO) concentration dynamics may play a significant role in both decaying synaptic and metabolic functions in Alzheimer's disease (AD). This neuromodulator acts presynaptically to increase vesicle release and glutamatergic transmission and also regulates mitochondrial function. Under conditions of altered intracellular redox environment, (•)NO may react and produce reactive species such as peroxynitrite. Using the triple transgenic mouse model of AD (3xTgAD), we investigated age-dependent changes in the glutamate-(•)NO axis in the hippocampus. Direct measurement of (•)NO concentration dynamics revealed a significant increase in N-methyl-D-aspartate type receptor-evoked peak (•)NO in the 3xTgAD model at an early age. Aging produced a decrease in peak (•)NO accompanied by significant decrease in production and decay rates in the transgenic model. Evaluation of energy metabolism revealed age-dependent decrease in basal oxygen consumption rate, a general decrease in mitochondrial oxidative phosphorylation parameters, and loss in mitochondrial sparing capacity in both genotypes. Finally, we observed age-dependent increase in 3-nitrotyrosine residues in the hippocampus, consistent with a putative shift in (•)NO bioactivity toward oxidative chemistry associated with neurotoxicity.

Ataxin-3 phosphorylation decreases neuronal defects in spinocerebellar ataxia type 3 models.

Different neurodegenerative diseases are caused by aberrant elongation of repeated glutamine sequences normally found in particular human proteins. Although the proteins involved are ubiquitously distributed in human tissues, toxicity targets only defined neuronal populations. Changes caused by an expanded polyglutamine protein are possibly influenced by endogenous cellular mechanisms, which may be harnessed to produce neuroprotection. Here, we show that ataxin-3, the protein involved in spinocerebellar ataxia type 3, also known as Machado-Joseph disease, causes dendritic and synapse loss in cultured neurons when expanded. We report that S12 of ataxin-3 is phosphorylated in neurons and that mutating this residue so as to mimic a constitutive phosphorylated state counters the neuromorphologic defects observed. In rats stereotaxically injected with expanded ataxin-3-encoding lentiviral vectors, mutation of serine 12 reduces aggregation, neuronal loss, and synapse loss. Our results suggest that S12 plays a role in the pathogenic pathways mediated by polyglutamine-expanded ataxin-3 and that phosphorylation of this residue protects against toxicity.

Aß and NMDAR activation cause mitochondrial dysfunction involving ER calcium release.

Early cognitive deficits in Alzheimer's disease (AD) seem to be correlated to dysregulation of glutamate receptors evoked by amyloid-beta (Aß) peptide. Aß interference with the activity of N-methyl-d-aspartate receptors (NMDARs) may be a relevant factor for Aß-induced mitochondrial toxicity and neuronal dysfunction. To evaluate the role of mitochondria in NMDARs activation mediated by Aß, we followed in situ single-cell simultaneous measurement of cytosolic free Ca(2+)(Cai(2+)) and mitochondrial membrane potential in primary cortical neurons. Our results show that direct exposure to Aß + NMDA largely increased Cai(2+) and induced immediate mitochondrial depolarization, compared with Aß or NMDA alone. Mitochondrial depolarization induced by rotenone strongly inhibited the rise in Cai(2+) evoked by Aß or NMDA, suggesting that mitochondria control Ca(2+) entry through NMDARs. However, incubation with rotenone did not preclude mitochondrial Ca(2+) (mitCa(2+)) retention in cells treated with Aß. Aß-induced Cai(2+) and mitCa(2+) rise were inhibited by ifenprodil, an antagonist of GluN2B-containing NMDARs. Exposure to Aß + NMDA further evoked a higher mitCa(2+) retention, which was ameliorated in GluN2B(-/-) cortical neurons, largely implicating the involvement of this NMDAR subunit. Moreover, pharmacologic inhibition of endoplasmic reticulum (ER) inositol-1,4,5-triphosphate receptor (IP3R) and mitCa(2+) uniporter (MCU) evidenced that Aß + NMDA-induced mitCa(2+) rise involves ER Ca(2+) release through IP3R and mitochondrial entry by the MCU. Altogether, data highlight mitCa(2+) dyshomeostasis and subsequent dysfunction as mechanisms relevant for early neuronal dysfunction in AD linked to Aß-mediated GluN2B-composed NMDARs activation.