Protective effects of 2,4-dinitrophenol in okadaic acid-induced cellular model of Alzheimer's disease.

Alzheimer's disease (AD) research started several decades ago and despite the many efforts employed to develop new treatments or approaches to slow and/or revert disease progression, AD treatment remains an unsolved issue. Knowing that mitochondria loss of function is a central hub for many AD-associated pathophysiological processes, there has been renewed interest in exploring mitochondria as targets for intervention. In this perspective, the present study was aimed to investigate the possible beneficial effects of 2,4 dinitrophenol (DNP), a mitochondrial uncoupler agent, in an in vitro model of AD. Retinoic acid-induced differentiated SH-SY5Y cells were incubated with okadaic acid (OA), a neurotoxin often used as an AD experimental model, and/or with DNP. OA caused a decrease in neuronal cells viability, induced multiple mitochondrial anomalies including increased levels of reactive oxygen species, decreased bioenergetics and mitochondria content markers, and an altered mitochondria morphology. OA-treated cells also presented increased lipid peroxidation levels, and overactivation of tau related kinases (GSK3ß, ERK1/2 and AMPK) alongside with a significant augment in tau protein phosphorylation levels. Interestingly, DNP co-treatment ameliorated and rescued OA-induced detrimental effects not only on mitochondria but also but also reinstated signaling pathways homeostasis and ameliorated tau pathology. Overall, our results show for the first time that DNP has the potential to preserve mitochondria homeostasis under a toxic insult, like OA exposure, as well as to reestablish cellular signaling homeostasis. These observations foster the idea that DNP, as a mitochondrial modulator, might represent a new avenue for treatment of AD.

MitoTempo protects against nε-carboxymethyl lysine-induced mitochondrial dyshomeostasis and neuronal cells injury.

Enhanced formation of advanced glycation end products (AGEs) is a pivotal factor in diabetes pathophysiology, increasing the risk of diabetic complications. Nε-carboxy-methyl-lysine (CML) is one of the most relevant AGEs found in several tissues including the peripheral blood of diabetic subjects. Despite recognizing diabetes as a risk factor for neurodegenerative diseases and the documented role of mitochondrial abnormalities in this connection, the impact of CML on neuronal mitochondria and its contribution to diabetes-related neurodegeneration remain uncertain. Here, we evaluated the effects of CML in differentiated SH-SY5Y human neuroblastoma cells. Due to the association between mitochondrial dysfunction and increased production of reactive oxygen species (ROS), the possible protective effects of MitoTempo, a mitochondria-targeted antioxidant, were also evaluated. Several parameters were assessed namely cells viability, mitochondrial respiration and membrane potential, ATP and ROS production, Ca2+ levels, mitochondrial biogenesis and dynamics, mito/autophagy, endoplasmic reticulum (ER) stress and amyloidogenic and synaptic integrity markers. CML caused pronounced mitochondrial defects characterized by a significant decrease in mitochondrial respiration, membrane potential, and ATP production and an increase in ROS production. An accumulation of individual mitochondria associated with disrupted mitochondrial networks was also observed. Furthermore, CML caused mitochondrial fusion and a decrease in mitochondrial mass and induced ER stress associated with altered unfolded protein response and Ca2+ dyshomeostasis. Moreover, CML increased the protein levels of ß-secretase-1 and amyloid precursor protein, key proteins involved in Alzheimer's Disease pathophysiology. All these effects contributed to the decline in neuronal cells viability. Notable, MitoTempo was able to counteract most of CML-mediated mitochondrial defects and neuronal cells injury and death. Overall, these findings suggest that CML induces pronounced defects in neuronal mitochondria and ER stress, predisposing to neurodegenerative events. More, our observations suggest that MitoTempo holds therapeutic promise in mitigating CML-induced mitochondrial imbalance and neuronal damage and death.

Evaluation of the safety and quality of Brazil nuts (Bertholletia excelsa) using the tools of dna sequencing technology and aflatoxin profile.

Introduction: Brazil nuts (BNs) result from sustainable extraction and are widely exploited in the Amazon region. Due to the production characteristics in the forest and the nutritional characteristics of these nuts, the occurrence of fungal contamination and the presence of aflatoxins are extensively discussed in the literature as a great aspect of interest and concern. This study aims to evaluate the microbial profile through DNA sequencing and amplification of 16S and ITS genes for bacterial and fungal analysis, respectively, and the presence of mycotoxins using high-performance liquid chromatography with fluorescence detection (HPLC-FD) from different fractions of the nuts processed. Methods: The BN samples, harvest A (HA) and harvest B (HB), from two different harvests were collected in an extractive cooperative in the Amazon region for microbiological analysis (from DNA extraction and amplification of 16S genes, bacteria analysis, and ITS for fungi) and mycotoxins (aflatoxins AFB1, AFB2, AFG1, and AFG2) using HPLC-FD/KobraCell®. Results and discussion: The samples showed a very different microbiome and aflatoxin profile. Genera such as Rothia (HA) and Cronobacter (HB) were abundant during the analysis of bacteria; as for fungi, the genera Aspergillus, Fusarium, Penicillium, and Alternaria were also considered prevalent in these samples. Soil microorganisms, including those pathogenic and related to inadequate hygienic-sanitary production practices, as well as aflatoxins, were found in the samples. However, they were within the established limits permitted by Brazilian legislation. Nuts have a diverse microbiota and are not restricted to fungi of the genus Aspergillus. The microbiological and toxicological profile can vary significantly within the same nut in the same extraction region and can be exacerbated by global climate changes. Therefore, it is necessary to advance sanitary educational actions by applying good production practices and inspection programs to ensure the sustainability and quality of the BN production chain.

Mapping the lipidome in mitochondria-associated membranes (MAMs) in an in vitro model of Alzheimer's disease.

The disruption of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) plays a relevant role in Alzheimer's disease (AD). MAMs have been implicated in neuronal dysfunction and death since it is associated with impairment of functions regulated in this subcellular domain, including lipid synthesis and trafficking, mitochondria dysfunction, ER stress-induced unfolded protein response (UPR), apoptosis, and inflammation. Since MAMs play an important role in lipid metabolism, in this study we characterized and investigated the lipidome alterations at MAMs in comparison with other subcellular fractions, namely microsomes and mitochondria, using an in vitro model of AD, namely the mouse neuroblastoma cell line (N2A) over-expressing the APP familial Swedish mutation (APPswe) and the respective control (WT) cells. Phospholipids (PLs) and fatty acids (FAs) were isolated from the different subcellular fractions and analyzed by HILIC-LC-MS/MS and GC-MS, respectively. In this in vitro AD model, we observed a down-regulation in relative abundance of some phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and lysophosphatidylethanolamine (LPE) species with PUFA and few PC with saturated and long-chain FA. We also found an up-regulation of CL, and antioxidant alkyl acyl PL. Moreover, multivariate analysis indicated that each organelle has a specific lipid profile adaptation in N2A APPswe cells. In the FAs profile, we found an up-regulation of C16:0 in all subcellular fractions, a decrease of C18:0 levels in total fraction (TF) and microsomes fraction, and a down-regulation of 9-C18:1 was also found in mitochondria fraction in the AD model. Together, these results suggest that the over-expression of the familial APP Swedish mutation affects lipid homeostasis in MAMs and other subcellular fractions and supports the important role of lipids in AD physiopathology.

Current Advances in Mitochondrial Targeted Interventions in Alzheimer's Disease.

Alzheimer's disease is the most prevalent neurodegenerative disorder and affects the lives not only of those who are diagnosed but also of their caregivers. Despite the enormous social, economic and political burden, AD remains a disease without an effective treatment and with several failed attempts to modify the disease course. The fact that AD clinical diagnosis is most often performed at a stage at which the underlying pathological events are in an advanced and conceivably irremediable state strongly hampers treatment attempts. This raises the awareness of the need to identify and characterize the early brain changes in AD, in order to identify possible novel therapeutic targets to circumvent AD's cascade of events. One of the most auspicious targets is mitochondria, powerful organelles found in nearly all cells of the body. A vast body of literature has shown that mitochondria from AD patients and model organisms of the disease differ from their non-AD counterparts. In view of this evidence, preserving and/or restoring mitochondria's health and function can represent the primary means to achieve advances to tackle AD. In this review, we will briefly assess and summarize the previous and latest evidence of mitochondria dysfunction in AD. A particular focus will be given to the recent updates and advances in the strategy options aimed to target faulty mitochondria in AD.

Therapeutic potential of glutaminyl cyclases: Current status and emerging trends.

Glutaminyl cyclase (QC) activity has been identified as a key effector in distinct biological processes. Human glutaminyl-peptide cyclotransferase (QPCT) and glutaminyl-peptide cyclotransferase-like (QPCTL) are considered attractive therapeutic targets in many human disorders, such as neurodegenerative diseases, and a range of inflammatory conditions, as well as for cancer immunotherapy, because of their capacity to modulate cancer immune checkpoint proteins. In this review, we explore the biological functions and structures of QPCT/L enzymes and highlight their therapeutic relevance. We also summarize recent developments in the discovery of small-molecule inhibitors targeting these enzymes, including an overview of preclinical and clinical studies.

Normative mice retinal thickness: 16-month longitudinal characterization of wild-type mice and changes in a model of Alzheimer's disease.

Animal models of disease are paramount to understand retinal development, the pathophysiology of eye diseases, and to study neurodegeneration using optical coherence tomography (OCT) data. In this study, we present a comprehensive normative database of retinal thickness in C57BL6/129S mice using spectral-domain OCT data. The database covers a longitudinal period of 16 months, from 1 to 16 months of age, and provides valuable insights into retinal development and changes over time. Our findings reveal that total retinal thickness decreases with age, while the thickness of individual retinal layers and layer aggregates changes in different ways. For example, the outer plexiform layer (OPL), photoreceptor inner segments (ILS), and retinal pigment epithelium (RPE) thickened over time, whereas other retinal layers and layer aggregates became thinner. Additionally, we compare the retinal thickness of wild-type (WT) mice with an animal model of Alzheimer's disease (3 × Tg-AD) and show that the transgenic mice exhibit a decrease in total retinal thickness compared to age-matched WT mice, with statistically significant differences observed at all evaluated ages. This normative database of retinal thickness in mice will serve as a reference for future studies on retinal changes in neurodegenerative and eye diseases and will further our understanding of the pathophysiology of these conditions.

A Perspective on the Link between Mitochondria-Associated Membranes (MAMs) and Lipid Droplets Metabolism in Neurodegenerative Diseases.

Mitochondria interact with the endoplasmic reticulum (ER) through contacts called mitochondria-associated membranes (MAMs), which control several processes, such as the ER stress response, mitochondrial and ER dynamics, inflammation, apoptosis, and autophagy. MAMs represent an important platform for transport of non-vesicular phospholipids and cholesterol. Therefore, this region is highly enriched in proteins involved in lipid metabolism, including the enzymes that catalyze esterification of cholesterol into cholesteryl esters (CE) and synthesis of triacylglycerols (TAG) from fatty acids (FAs), which are then stored in lipid droplets (LDs). LDs, through contact with other organelles, prevent the toxic consequences of accumulation of unesterified (free) lipids, including lipotoxicity and oxidative stress, and serve as lipid reservoirs that can be used under multiple metabolic and physiological conditions. The LDs break down by autophagy releases of stored lipids for energy production and synthesis of membrane components and other macromolecules. Pathological lipid deposition and autophagy disruption have both been reported to occur in several neurodegenerative diseases, supporting that lipid metabolism alterations are major players in neurodegeneration. In this review, we discuss the current understanding of MAMs structure and function, focusing on their roles in lipid metabolism and the importance of autophagy in LDs metabolism, as well as the changes that occur in neurogenerative diseases.

Metabolic defects shared by Alzheimer's disease and diabetes: A focus on mitochondria.

Type 2 diabetes (T2D) and Alzheimer's disease (AD) are two global epidemics that share several metabolic defects, such as insulin resistance, impaired glucose metabolism, and mitochondrial defects. Importantly, strong evidence demonstrates that T2D significantly increases the risk of cognitive decline and dementia, particularly AD. Here, we provide an overview of the metabolic defects that characterize and link both pathologies putting the focus on mitochondria. The biomarker potential of mitochondrial components and the therapeutic potential of some drugs that target and modulate mitochondria are also briefly discussed.

Stage-independent biomarkers for Alzheimer's disease from the living retina: an animal study.

The early diagnosis of neurodegenerative disorders is still an open issue despite the many efforts to address this problem. In particular, Alzheimer's disease (AD) remains undiagnosed for over a decade before the first symptoms. Optical coherence tomography (OCT) is now common and widely available and has been used to image the retina of AD patients and healthy controls to search for biomarkers of neurodegeneration. However, early diagnosis tools would need to rely on images of patients in early AD stages, which are not available due to late diagnosis. To shed light on how to overcome this obstacle, we resort to 57 wild-type mice and 57 triple-transgenic mouse model of AD to train a network with mice aged 3, 4, and 8 months and classify mice at the ages of 1, 2, and 12 months. To this end, we computed fundus images from OCT data and trained a convolution neural network (CNN) to classify those into the wild-type or transgenic group. CNN performance accuracy ranged from 80 to 88% for mice out of the training group's age, raising the possibility of diagnosing AD before the first symptoms through the non-invasive imaging of the retina.

WWOX inhibition by Zfra1-31 restores mitochondrial homeostasis and viability of neuronal cells exposed to high glucose.

Diabetes has been associated with an increased risk of cognitive decline and dementia. However, the mechanisms underlying this association remain unclear and no effective therapeutic interventions exist. Accumulating evidence demonstrates that mitochondrial defects are a key feature of diabetes contributing to neurodegenerative events. It has also been demonstrated that the putative tumor suppressor WW domain-containing oxidoreductase 1 (WWOX) can interact with mitochondria in several pathological conditions. However, its role in diabetes-associated neurodegeneration remains unknown. So, this study aimed to evaluate the role of WWOX activation in high glucose-induced neuronal damage and death. Our experiments were mainly performed in differentiated SH-SY5Y neuroblastoma cells exposed to high glucose and treated (or not) with Zfra1-31, the specific inhibitor of WWOX. Several parameters were analyzed namely cell viability, WWOX activation (tyrosine 33 residue phosphorylation), mitochondrial function, reactive oxygen species (ROS) production, biogenesis, and dynamics, autophagy and oxidative stress/damage. The levels of the neurotoxic proteins amyloid ß (Aß) and phosphorylated Tau (pTau) and of synaptic integrity markers were also evaluated. We observed that high glucose increased the levels of activated WWOX. Interestingly, brain cortical and hippocampal homogenates from young (6-month old) diabetic GK rats showed increased levels of activated WWOX compared to older GK rats (12-month old) suggesting that WWOX plays an early role in the diabetic brain. In neuronal cells, high glucose impaired mitochondrial respiration, dynamics and biogenesis, increased mitochondrial ROS production and decreased mitochondrial membrane potential and ATP production. More, high glucose augmented oxidative stress/damage and the levels of Aß and pTau proteins and affected autophagy, contributing to the loss of synaptic integrity and cell death. Of note, the activation of WWOX preceded mitochondrial dysfunction and cell death. Importantly, the inhibition of WWOX with Zfra1-31 reversed, totally or partially, the alterations promoted by high glucose. Altogether our observations demonstrate that under high glucose conditions WWOX activation contributes to mitochondrial anomalies and neuronal damage and death, which suggests that WWOX is a potential therapeutic target for early interventions. Our findings also support the efficacy of Zfra1-31 in treating hyperglycemia/diabetes-associated neurodegeneration.

Type 2 Diabetes Induces a Pro-Oxidative Environment in Rat Epididymis by Disrupting SIRT1/PGC-1α/SIRT3 Pathway.

Diabetes mellitus type 2 (T2DM) has been associated with alterations in the male reproductive tract, especially in the epididymis. Although it is known that T2DM alters epididymal physiology, disturbing mitochondrial function and favoring oxidative stress, the mechanisms remain unknown. Sirtuin 1 (SIRT1), peroxisome proliferators-activated receptor γ coactivator 1α (PGC-1α), and sirtuin 3 (SIRT3) are key regulators of mitochondrial function and inducers of antioxidant defenses. In this study, we hypothesized that the epididymal SIRT1/PGC-1α/SIRT3 axis mediates T2DM-induced epididymis dysfunction by controlling the oxidative profile. Using 7 Goto-Kakizaki (GK) rats (a non-obese model that spontaneously develops T2DM early in life), and 7 age-matched Wistar control rats, we evaluated the protein levels of SIRT1, PGC-1α, and SIRT3, as well as the expression of mitochondrial respiratory complexes. The activities of epididymal glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) were determined, as well as the epididymal antioxidant capacity. We also evaluated protein nitration, carbonylation, and lipid peroxidation in the epididymis. The T2DM rats presented with hyperglycemia and glucose intolerance. Epididymal levels of SIRT1, PGC-1α, and SIRT3 were decreased, as well as the expression of the mitochondrial complexes II, III, and V, in the T2DM rats. We found a significant decrease in the activities of SOD, CAT, and GPx, consistent with the lower antioxidant capacity and higher protein nitration and lipid peroxidation detected in the epididymis of the T2DM rats. In sum, T2DM disrupted the epididymal SIRT1/PGC-1α/SIRT3 pathway, which is associated with a compromised mitochondrial function. This resulted in a decline of the antioxidant defenses and an increased oxidative damage in that tissue, which may be responsible for the impaired male reproductive function observed in diabetic men.

Retinal Aging in 3× Tg-AD Mice Model of Alzheimer's Disease.

The retina, as part of the central nervous system (CNS), can be the perfect target for in vivo, in situ, and noninvasive neuropathology diagnosis and assessment of therapeutic efficacy. It has long been established that several age-related brain changes are more pronounced in Alzheimer's disease (AD). Nevertheless, in the retina such link is still under-explored. This study investigates the differences in the aging of the CNS through the retina of 3× Tg-AD and wild-type mice. A dedicated optical coherence tomograph imaged mice's retinas for 16 months. Two neural networks were developed to model independently each group's ages and were then applied to an independent set containing images from both groups. Our analysis shows a mean absolute error of 0.875±1.1 × 10-2 and 1.112±1.4 × 10-2 months, depending on training group. Our deep learning approach appears to be a reliable retinal OCT aging marker. We show that retina aging is distinct in the two classes: the presence of the three mutated human genes in the mouse genome has an impact on the aging of the retina. For mice over 4 months-old, transgenic mice consistently present a negative retina age-gap when compared to wild-type mice, regardless of training set. This appears to contradict AD observations in the brain. However, the 'black-box" nature of deep-learning implies that one cannot infer reasoning. We can only speculate that some healthy age-dependent neural adaptations may be altered in transgenic animals.

ER-mitochondria communication is involved in NLRP3 inflammasome activation under stress conditions in the innate immune system.

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction, which are key events in the initiation and/or progression of several diseases, are correlated with alterations at ER-mitochondria contact sites, the so-called "Mitochondria-Associated Membranes" (MAMs). These intracellular structures are also implicated in NLRP3 inflammasome activation which is an important driver of sterile inflammation, however, the underlying molecular basis remains unclear. This work aimed to investigate the role of ER-mitochondria communication during ER stress-induced NLRP3 inflammasome activation in both peripheral and central innate immune systems, by using THP-1 human monocytes and BV2 microglia cells, respectively, as in vitro models. Markers of ER stress, mitochondrial dynamics and mass, as well as NLRP3 inflammasome activation were evaluated by Western Blot, IL-1ß secretion was measured by ELISA, and ER-mitochondria contacts were quantified by transmission electron microscopy. Mitochondrial Ca2+ uptake and polarization were analyzed with fluorescent probes, and measurement of aconitase and SOD2 activities monitored mitochondrial ROS accumulation. ER stress was demonstrated to activate the NLRP3 inflammasome in both peripheral and central immune cells. Studies in monocytes indicate that ER stress-induced NLRP3 inflammasome activation occurs by a Ca2+-dependent and ROS-independent mechanism, which is coupled with upregulation of MAMs-resident chaperones, closer ER-mitochondria contacts, as well as mitochondrial depolarization and impaired dynamics. Moreover, enhanced ER stress-induced NLRP3 inflammasome activation in the immune system was found associated with pathological conditions since it was observed in monocytes derived from bipolar disorder (BD) patients, supporting a pro-inflammatory status in BD. In conclusion, by demonstrating that ER-mitochondria communication plays a key role in the response of the innate immune cells to ER stress, this work contributes to elucidate the molecular mechanisms underlying NLRP3 inflammasome activation under stress conditions, and to disclose novel potential therapeutic targets for diseases associated with sterile inflammation.

Hypoxic Preconditioning Averts Sporadic Alzheimer's Disease-Like Phenotype in Rats: A Focus on Mitochondria.

Aims: Brief episodes of sublethal hypoxia reprogram brain response to face possible subsequent lethal stimuli by triggering adaptive and prosurvival events-a phenomenon denominated hypoxic preconditioning (HP). To date, the potential therapeutic implications of HP to forestall sporadic Alzheimer's disease (sAD) pathology remain unexplored. Using a well-established protocol of HP and focusing on hippocampus as a first brain region affected in AD, this study was undertaken to investigate the potential protective effects of HP in a sAD rat model induced by the intracerebroventricular (icv) administration of streptozotocin (STZ) and to uncover the mitochondrial adaptations underlying this nonpharmacological strategy. Results: HP prevented the memory and learning deficits as well as tau pathology in the icvSTZ rat model. HP also attenuated icvSTZ-related reactive astrogliosis, as noted by increased glial fibrillary acidic protein immunoreactivity and myo-inositol levels. Notably, HP abrogated the icvSTZ-related impaired energy metabolism and oxidative damage. Particularly, HP averted increased lactate, glutamate, and succinate levels, and decreased mitochondrial respiratory chain function and mitochondrial DNA content. Concerning mitochondrial adaptations underlying HP-triggered tolerance to icvSTZ, preconditioned hippocampal mitochondria displayed an enhanced complex II-energized mitochondrial respiration, which resulted from a coordinated interaction between mitochondrial biogenesis and fusion-fission. Mitochondrial biogenesis was stimulated immediately after HP, whereas in a latter phase mitochondrial fusion-fission events are modulated favoring the generation of elongated mitochondria. Innovation and Conclusion: Overall, these results demonstrate for the first time that HP prevents the sAD-like phenotype, in part, by targeting mitochondria emerging as a preventive strategy in the context of AD. Antioxid. Redox Signal. 37, 739-757.

Oxygen Sensing and Signaling in Alzheimer's Disease: A Breathtaking Story!

Oxygen sensing and homeostasis is indispensable for the maintenance of brain structural and functional integrity. Under low-oxygen tension, the non-diseased brain has the ability to cope with hypoxia by triggering a homeostatic response governed by the highly conserved hypoxia-inducible family (HIF) of transcription factors. With the advent of advanced neuroimaging tools, it is now recognized that cerebral hypoperfusion, and consequently hypoxia, is a consistent feature along the Alzheimer's disease (AD) continuum. Of note, the reduction in cerebral blood flow and tissue oxygenation detected during the prodromal phases of AD, drastically aggravates as disease progresses. Within this scenario a fundamental question arises: How HIF-driven homeostatic brain response to hypoxia "behaves" during the AD continuum? In this sense, the present review is aimed to critically discuss and summarize the current knowledge regarding the involvement of hypoxia and HIF signaling in the onset and progression of AD pathology. Importantly, the promises and challenges of non-pharmacological and pharmacological strategies aimed to target hypoxia will be discussed as a new "hope" to prevent and/or postpone the neurodegenerative events that occur in the AD brain.

Insulin-Induced Recurrent Hypoglycemia Up-Regulates Glucose Metabolism in the Brain Cortex of Chemically Induced Diabetic Rats.

Diabetes is a chronic metabolic disease that seriously compromises human well-being. Various studies highlight the importance of maintaining a sufficient glucose supply to the brain and subsequently safeguarding cerebral glucose metabolism. The goal of the present work is to clarify and disclose the metabolic alterations induced by recurrent hypoglycemia in the context of long-term hyperglycemia to further comprehend the effects beyond brain harm. To this end, chemically induced diabetic rats underwent a protocol of repeatedly insulin-induced hypoglycemic episodes. The activity of key enzymes of glycolysis, the pentose phosphate pathway and the Krebs cycle was measured by spectrophotometry in extracts or isolated mitochondria from brain cortical tissue. Western blot analysis was used to determine the protein content of glucose and monocarboxylate transporters, players in the insulin signaling pathway and mitochondrial biogenesis and dynamics. We observed that recurrent hypoglycemia up-regulates the activity of mitochondrial hexokinase and Krebs cycle enzymes (namely, pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and succinate dehydrogenase) and the protein levels of mitochondrial transcription factor A (TFAM). Both insults increased the nuclear factor erythroid 2-related factor 2 (NRF2) protein content and induced divergent effects in mitochondrial dynamics. Insulin-signaling downstream pathways were found to be down-regulated, and glycogen synthase kinase 3 beta (GSK3ß) was found to be activated through both decreased phosphorylation at Ser9 and increased phosphorylation at Y216. Interestingly, no changes in the levels of cAMP response element-binding protein (CREB), which plays a key role in neuronal plasticity and memory, were caused by hypoglycemia and/or hyperglycemia. These findings provide experimental evidence that recurrent hypoglycemia, in the context of chronic hyperglycemia, has the capacity to evoke coordinated adaptive responses in the brain cortex that will ultimately contribute to sustaining brain cell health.

Structural and Functional Alterations in Mitochondria-Associated Membranes (MAMs) and in Mitochondria Activate Stress Response Mechanisms in an In Vitro Model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the accumulation of extracellular plaques composed by amyloid-ß (Aß) and intracellular neurofibrillary tangles of hyperphosphorylated tau. AD-related neurodegenerative mechanisms involve early changes of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) and impairment of cellular events modulated by these subcellular domains. In this study, we characterized the structural and functional alterations at MAM, mitochondria, and ER/microsomes in a mouse neuroblastoma cell line (N2A) overexpressing the human amyloid precursor protein (APP) with the familial Swedish mutation (APPswe). Proteins levels were determined by Western blot, ER-mitochondria contacts were quantified by transmission electron microscopy, and Ca2+ homeostasis and mitochondria function were analyzed using fluorescent probes and Seahorse assays. In this in vitro AD model, we found APP accumulated in MAM and mitochondria, and altered levels of proteins implicated in ER-mitochondria tethering, Ca2+ signaling, mitochondrial dynamics, biogenesis and protein import, as well as in the stress response. Moreover, we observed a decreased number of close ER-mitochondria contacts, activation of the ER unfolded protein response, reduced Ca2+ transfer from ER to mitochondria, and impaired mitochondrial function. Together, these results demonstrate that several subcellular alterations occur in AD-like neuronal cells, which supports that the defective ER-mitochondria crosstalk is an important player in AD physiopathology.

Longitudinal normative OCT retinal thickness data for wild-type mice, and characterization of changes in the 3×Tg-AD mice model of Alzheimer's disease.

Mice are widely used as models for many diseases, including eye and neurodegenerative diseases. However, there is a lack of normative data for retinal thickness over time, especially at young ages. In this work, we present a normative thickness database from one to four-months-old, for nine layers/layer-aggregates, including the total retinal thickness, obtained from the segmentation of spectral-domain optical coherence tomography (SD-OCT) data from the C57BL6/129S mouse strain. Based on fifty-seven mice, this normative database provides an opportunity to study the ageing of control mice and characterise disease models' ageing, such as the triple transgenic mouse model of Alzheimer's disease (3×Tg-AD) used in this work. We report thickness measurements, the differences in thickness per layer, demonstrate a nasal-temporal asymmetry, and the variation of thickness as a function to the distance to the optic disc centre. Significant differences were found between the transgenic group's thickness and the normative database for the entire period covered in this study. Even though it is well accepted that retinal nerve fibre layer (RNFL) thinning is a hallmark of neurodegeneration, our results show a thicker RNFL-GCL (RNFL-Ganglion cell layer) aggregate for the 3×Tg-AD mice until four-months-old.

IGF1R Deficiency Modulates Brain Signaling Pathways and Disturbs Mitochondria and Redox Homeostasis.

Insulin-like growth factor 1 receptor (IGF1R)-mediated signaling pathways modulate important neurophysiological aspects in the central nervous system, including neurogenesis, synaptic plasticity and complex cognitive functions. In the present study, we intended to characterize the impact of IGF1R deficiency in the brain, focusing on PI3K/Akt and MAPK/ERK1/2 signaling pathways and mitochondria-related parameters. For this purpose, we used 13-week-old UBC-CreERT2; Igf1rfl/fl male mice in which Igf1r was conditionally deleted. IGF1R deficiency caused a decrease in brain weight as well as the activation of the IR/PI3K/Akt and inhibition of the MAPK/ERK1/2/CREB signaling pathways. Despite no alterations in the activity of caspases 3 and 9, a significant alteration in phosphorylated GSK3ß and an increase in phosphorylated Tau protein levels were observed. In addition, significant disturbances in mitochondrial dynamics and content and altered activity of the mitochondrial respiratory chain complexes were noticed. An increase in oxidative stress, characterized by decreased nuclear factor E2-related factor 2 (NRF2) protein levels and aconitase activity and increased H2O2 levels were also found in the brain of IGF1R-deficient mice. Overall, our observations confirm the complexity of IGF1R in mediating brain signaling responses and suggest that its deficiency negatively impacts brain cells homeostasis and survival by affecting mitochondria and redox homeostasis.