Sirtuins Modulators Counteract Mitochondrial Dysfunction in Cellular Models of Hypoxia: Relevance to Schizophrenia.

Schizophrenia (SZ) is a neurodevelopmental-associated disorder strongly related to environmental factors, such as hypoxia. Because there is no cure for SZ or any pharmacological approach that could revert hypoxia-induced cellular damages, we evaluated whether modulators of sirtuins could abrogate hypoxia-induced mitochondrial deregulation as a neuroprotective strategy. Firstly, astrocytes from control (Wistar) and Spontaneously Hypertensive Rats (SHR), a model of both SZ and neonatal hypoxia, were submitted to chemical hypoxia. Then, cells were exposed to different concentrations of Nicotinamide (NAM), Resveratrol (Resv), and Sirtinol (Sir) for 48hrs. Our data indicate that sirtuins modulation reduces cell death increasing the acetylation of histone 3. This outcome is related to the rescue of loss of mitochondrial membrane potential, changes in mitochondrial calcium buffering capacity, decreased O2-rad levels and increased expression of metabolic regulators (Nrf-1 and Nfe2l2) and mitochondrial content. Such findings are relevant not only for hypoxia-associated conditions, named pre-eclampsia but also for SZ since prenatal hypoxia is a relevant environmental factor related to this burdensome neuropsychiatric disorder.

Haloperidol rescues the schizophrenia-like phenotype in adulthood after rotenone administration in neonatal rats.

Neuropsychiatric disorders are multifactorial disturbances that encompass several hypotheses, including changes in neurodevelopment. It is known that brain development disturbances during early life can predict psychosis in adulthood. As we have previously demonstrated, rotenone, a mitochondrial complex I inhibitor, could induce psychiatric-like behavior in 60-day-old rats after intraperitoneal injections from the 5th to the 11th postnatal day. Because mitochondrial deregulation is related to psychiatric disorders and the establishment of animal models is a high-value preclinical tool, we investigated the responsiveness of the rotenone (Rot)-treated newborn rats to pharmacological agents used in clinical practice, haloperidol (Hal), and methylphenidate (MPD). Taken together, our data show that Rot-treated animals exhibit hyperlocomotion, decreased social interaction, and diminished contextual fear conditioning response at P60, consistent with positive, negative, and cognitive deficits of schizophrenia (SZ), respectively, that were reverted by Hal, but not MPD. Rot-treated rodents also display a prodromal-related phenotype at P35. Overall, our results seem to present a new SZ animal model as a consequence of mitochondrial inhibition during a critical neurodevelopmental period. Therefore, our study is crucial not only to elucidate the relevance of mitochondrial function in the etiology of SZ but also to fulfill the need for new and trustworthy experimentation models and, likewise, provide possibilities to new therapeutic avenues for this burdensome disorder.

Increased acute blood flow induced by the aqueous extract of Euterpe oleracea Mart. fruit pulp in rats in vivo is not related to the direct activation of endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Scientific evidence supports the antioxidant, anti-inflammatory and anti-lipidemic properties of Euterpe oleracea Mart. (açaí), which all converge to reduce cardiovascular risks. Macerating the pulp of açaí fruit produces a viscous aqueous extract (AE) rich in flavonoids that is commonly used in food production. In addition to nutritional aspects, cardiovascular benefits are attributed to AE by traditional medicine. AIM OF THE STUDY: Evaluation of AE impact on blood flow in vivo in rats and investigation of the mechanism underlying this response in vitro in rat endothelial cells (RECs). MATERIALS AND METHODS: For the measurement of acute blood flow, a perivascular ultrasound probe was used in Wistar rats. The in vitro assays employed REC to evaluate: concentration (1-1000 µg/mL) and time response (2-180 min) of AE in MTT cell viability assays; nitric oxide (NO) levels measurement and intracellular calcium handling using DAF-2DA and Fluo-4-AM, respectively; cellular biopterin content by HPLC; activation of Akt pathway using western blot analysis. For the chemical analyses of AE, stock solutions of the standards (+)catechin and quercetin were used for obtaining linear calibration curves. Identification and quantification of flavonoids in AE were based on comparisons with the retention times, increase in peak area determine by co-injection of AE with standards, UV-Vis scan and standard curves of known spectra. Results were expressed as mean ± standard deviation and data were analyzed using ANOVA followed by Tukey's post-test (p < 0.05). RESULTS: Although in vivo data have revealed the participation of NO in increasing of acute blood flow on abdominal aorta, in vitro analysis demonstrated that vasodilatation AE-induced is not related to its direct action on endothelial cells inducing eNOS activation. Besides, we demonstrated in isolated endothelial cells that highest concentrations of AE caused a reduction in NO levels, effect that could be partly justified by inhibition of Akt phosphorylation which, in turn, could decrease NOS activation. The involvement of cell transduction pathways involving variations in intracellular calcium and biopterins concentration were discarded. The participation of catechin and quercetin, identified in AE, was postulated to induce the responses of AE in REC. CONCLUSIONS: Despite the responses in vitro, vasodilation prevailed in vivo, probably by activating intermediate pathways, validating a potential beneficial effect of AE in reducing cardiovascular risks.

Neonatal Rotenone Administration Induces Psychiatric Disorder-Like Behavior and Changes in Mitochondrial Biogenesis and Synaptic Proteins in Adulthood.

Since psychiatric disorders are associated with changes in the development of the nervous system, an energy-dependent mechanism, we investigated whether mitochondrial inhibition during the critical neurodevelopment window in rodents would be able to induce metabolic alterations culminating in psychiatric-like behavior. We treated male Wistar rat puppies (P) with rotenone (Rot), an inhibitor of mitochondrial complex I, from postnatal days 5 to 11 (P5-P11). We demonstrated that at P60 and P120, Rot-treated animals showed hyperlocomotion and deficits in social interaction and aversive contextual memory, features observed in animal models of schizophrenia, autism spectrum disorder, and attention deficit hyperactivity disorder. During adulthood, Rot-treated rodents also presented modifications in CBP and CREB levels in addition to a decrease in mitochondrial biogenesis and Nrf1 expression. Additionally, NFE2L2-activation was not altered in Rot-treated P60 and P120 animals; an upregulation of pNFE2L2/ NFE2L2 was only observed in P12 cortices. Curiously, ATP/ADP levels did not change in all ages evaluated. Rot administration in newborn rodents also promoted modification in Rest and Mecp2 expression, and in synaptic protein levels, named PSD-95, Synaptotagmin-1, and Synaptophysin in the adult rats. Altogether, our data indicate that behavioral abnormalities and changes in synaptic proteins in adulthood induced by neonatal Rot administration might be a result of adjustments in CREB pathways and alterations in mitochondrial biogenesis and Nrf1 expression, rather than a direct deficiency of energy supply, as previously speculated. Consequently, Rot-induced psychiatric-like behavior would be an outcome of alterations in neuronal paths due to mitochondrial deregulation.

Oxygen Consumption Evaluation: An Important Indicator of Metabolic State, Cellular Function, and Cell Fate Along Neural Deregulation.

Depletion of oxygen (O2) levels and reduction in the ATP synthesis (or even its complete blockage) are important characteristics of mitochondrial dysfunction; features that are often correlated with neurodegeneration. The measurement of oxygen consumption rate (OCR) is thus essential to evaluate cellular metabolism, survival, and neuroprotective strategies. In the present chapter, we describe the oxygen consumption assay using a Clark-type oxygen electrode in different types of samples named cells suspension (from primary and established cell culture), brain slices (ex vivo), and fresh brain tissues. In addition, we demonstrate herein how the program Oxygraph can be used in order to analyze the data and different approaches to normalize it.

Mitochondrial Dysfunction, Neurogenesis, and Epigenetics: Putative Implications for Amyotrophic Lateral Sclerosis Neurodegeneration and Treatment.

Amyotrophic lateral sclerosis (ALS) is a progressive and devastating multifactorial neurodegenerative disorder. Although the pathogenesis of ALS is still not completely understood, numerous studies suggest that mitochondrial deregulation may be implicated in its onset and progression. Interestingly, mitochondrial deregulation has also been associated with changes in neural stem cells (NSC) proliferation, differentiation, and migration. In this review, we highlight the importance of mitochondrial function for neurogenesis, and how both processes are correlated and may contribute to the pathogenesis of ALS; we have focused primarily on preclinical data from animal models of ALS, since to date no studies have evaluated this link using human samples. As there is currently no cure and no effective therapy to counteract ALS, we have also discussed how improving neurogenic function by epigenetic modulation could benefit ALS. In support of this hypothesis, changes in histone deacetylation can alter mitochondrial function, which in turn might ameliorate cellular proliferation as well as neuronal differentiation and migration. We propose that modulation of epigenetics, mitochondrial function, and neurogenesis might provide new hope for ALS patients, and studies exploring these new territories are warranted in the near future.

Decreased Mitochondrial Function, Biogenesis, and Degradation in Peripheral Blood Mononuclear Cells from Amyotrophic Lateral Sclerosis Patients as a Potential Tool for Biomarker Research.

Amyotrophic lateral sclerosis (ALS) is a multifactorial and progressive neurodegenerative disease of unknown etiology. Due to ALS's unpredictable onset and progression rate, the search for biomarkers that allow the detection and tracking of its development and therapeutic efficacy would be of significant medical value. Considering that alterations of energy supply are one of ALS's main hallmarks and that a correlation has been established between gene expression in human brain tissue and peripheral blood mononuclear cells (PBMCs), the present work investigates whether changes in mitochondrial function could be used to monitor ALS. To achieve this goal, PBMCs from ALS patients and control subjects were used; blood sampling is a quite non-invasive method and is cost-effective. Different parameters were evaluated, namely cytosolic calcium levels, mitochondrial membrane potential, oxidative stress, and metabolic compounds levels, as well as mitochondrial dynamics and degradation. Altogether, we observed lower mitochondrial calcium uptake/retention, mitochondria depolarization, and redox homeostasis deregulation, in addition to a decrease in critical metabolic genes, a diminishment in mitochondrial biogenesis, and an augmentation in mitochondrial fission and autophagy-related gene expression. All of these changes can contribute to the decreased ATP and pyruvate levels observed in ALS PBMCs. Our data indicate that PBMCs from ALS patients show a significant mitochondrial dysfunction, resembling several findings from ALS' neural cells/models, which could be exploited as a powerful tool in ALS research. Our findings can also guide future studies on new pharmacological interventions for ALS since assessments of brain samples are challenging and represent a relevant limited strategy. Graphical abstract.

Human Induced Pluripotent Stem Cell Models of Neurodegenerative Disorders for Studying the Biomedical Implications of Autophagy.

Autophagy is an intracellular degradation process that is essential for cellular survival, tissue homeostasis, and human health. The housekeeping functions of autophagy in mediating the clearance of aggregation-prone proteins and damaged organelles are vital for post-mitotic neurons. Improper functioning of this process contributes to the pathology of myriad human diseases, including neurodegeneration. Impairment in autophagy has been reported in several neurodegenerative diseases where pharmacological induction of autophagy has therapeutic benefits in cellular and transgenic animal models. However, emerging studies suggest that the efficacy of autophagy inducers, as well as the nature of the autophagy defects, may be context-dependent, and therefore, studies in disease-relevant experimental systems may provide more insights for clinical translation to patients. With the advancements in human stem cell technology, it is now possible to establish disease-affected cellular platforms from patients for investigating disease mechanisms and identifying candidate drugs in the appropriate cell types, such as neurons that are otherwise not accessible. Towards this, patient-derived human induced pluripotent stem cells (hiPSCs) have demonstrated considerable promise in constituting a platform for effective disease modeling and drug discovery. Multiple studies have utilized hiPSC models of neurodegenerative diseases to study autophagy and evaluate the therapeutic efficacy of autophagy inducers in neuronal cells. This review provides an overview of the regulation of autophagy, generation of hiPSCs via cellular reprogramming, and neuronal differentiation. It outlines the findings in various neurodegenerative disorders where autophagy has been studied using hiPSC models.

Mitochondrial Dysfunction and Changes in High-Energy Compounds in Different Cellular Models Associated to Hypoxia: Implication to Schizophrenia.

Schizophrenia (SZ) is a multifactorial mental disorder, which has been associated with a number of environmental factors, such as hypoxia. Considering that numerous neural mechanisms depends on energetic supply (ATP synthesis), the maintenance of mitochondrial metabolism is essential to keep cellular balance and survival. Therefore, in the present work, we evaluated functional parameters related to mitochondrial function, namely calcium levels, mitochondrial membrane potential, redox homeostasis, high-energy compounds levels and oxygen consumption, in astrocytes from control (Wistar) and Spontaneously Hypertensive Rats (SHR) animals exposed both to chemical and gaseous hypoxia. We show that astrocytes after hypoxia presented depolarized mitochondria, disturbances in Ca2+ handling, destabilization in redox system and alterations in ATP, ADP, Pyruvate and Lactate levels, in addition to modification in NAD+/NADH ratio, and Nfe2l2 and Nrf1 expression. Interestingly, intrauterine hypoxia also induced augmentation in mitochondrial biogenesis and content. Altogether, our data suggest that hypoxia can induce mitochondrial deregulation and a decrease in energy metabolism in the most prevalent cell type in the brain, astrocytes. Since SHR are also considered an animal model of SZ, our results can likewise be related to their phenotypic alterations and, therefore, our work also allow an increase in the knowledge of this burdensome disorder.

Metabolic Alteration and Amyotrophic Lateral Sclerosis Outcome: A Systematic Review.

Background: The development of strategies that could not only efficiently detect the onset of Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disorder with no cure but also predict its development and evaluate therapeutic intervention would be of great value. In this respect, the metabolic status of ALS patients has called attention. Hence, this study aimed to investigate the potential correlation between changes in ALS's metabolic parameters with the disease outcome in a systematic review. Methods: The manuscripts were manually searched within different databases (PubMed, Web of Science and Cochrane). The inclusion criteria were original articles and reviews about individuals with ALS and its survival, disease prognosis and metabolism (weight, cholesterol, hypertension, BMI, and glycaemia). The authors also established three different exclusion criteria: studies including ALS and other degenerative disorders, works including animal models and published before the year 2000. Results: In total, 29 papers were selected. From all manuscripts, only 82.8% ensured the participation of sALS patients. Also, 27.6% of selected studies described the presence of a genetic mutation. Regarding ALS prognosis, patient's age, the age of ALS onset, ALS duration and survival, <50% of the papers addressed these issues. Specifically, regarding metabolism, 65.5% of articles mentioned BMI, 20.7% mentioned any data concerning hypertension, 6.89% cardiovascular risk, 10.3% obesity, 13.78% diabetes and 10.3% glycaemia. Concerning lipid metabolism, more results were gathered, but still, they did not suffice to establish a correlation with ALS development. Conclusions: Altogether, the authors concluded that available information is not enough to establish a link between ALS and metabolism. In reality, less than half of the manuscripts evaluated show an association between both factors. Nonetheless, it is worth mentioning that metabolism does influence ALS, but not in a unique manner. There is a debate about patients' hypo- and hypermetabolism. Thus, to provide a reliable record, a public policy in which all research and clinical centers might assess the parameters discussed herein is suggested. Accordingly, this systematic review attempts to provide a comprehensible database to facilitate multicentered collaboration, validation, and clinical translation.

Gene Expression Control by Glucocorticoid Receptors during Innate Immune Responses.

Glucocorticoids (GCs) are potent anti-inflammatory compounds that have been extensively used in clinical practice for several decades. GC's effects on inflammation are generally mediated through GC receptors (GRs). Signal transduction through these nuclear receptors leads to dramatic changes in gene expression programs in different cell types, typically due to GR binding to DNA or to transcription modulators. During the last decade, the view of GCs as exclusive anti-inflammatory molecules has been challenged. GR negative interference in pro-inflammatory gene expression was a landmark in terms of molecular mechanisms that suppress immune activity. In fact, GR can induce varied inhibitory molecules, including a negative regulator of Toll-like receptors pathway, or subject key transcription factors, such as NF-κB and AP-1, to a repressor mechanism. In contrast, the expression of some acute-phase proteins and other players of innate immunity generally requires GR signaling. Consequently, GRs must operate context-dependent inhibitory, permissive, or stimulatory effects on host defense signaling triggered by pathogens or tissue damage. This review aims to disclose how contradictory or comparable effects on inflammatory gene expression can depend on pharmacological approach (including selective GC receptor modulators; SEGRMs), cell culture, animal treatment, or transgenic strategies used as models. Although the current view of GR-signaling integrated many advances in the field, some answers to important questions remain elusive.

CD36 is expressed in a defined subpopulation of neurons in the olfactory epithelium.

The sensory neurons in the olfactory epithelium (OSNs) are equipped with a large repertoire of olfactory receptors and the associated signal transduction machinery. In addition to the canonical OSNs, which express odorant receptors (ORs), the epithelium contains specialized subpopulations of sensory neurons that can detect specific information from environmental cues and relay it to relevant neuronal circuitries. Here we describe a subpopulation of mature OSNs in the main olfactory epithelium (MOE) which expresses CD36, a multifunctional receptor involved in a series of biological processes, including sensory perception of lipid ligands. The Cd36 expressing neurons coexpress markers of mature OSNs and are dispersed throughout the MOE. Unlike several ORs analyzed in our study, we found frequent coexpression of the OR Olfr287 in these neurons, suggesting that only a specific set of ORs may be coexpressed with CD36 in OSNs. We also show that CD36 is expressed in the cilia of OSNs, indicating a possible role in odorant detection. CD36-deficient mice display no signs of gross changes in the organization of the olfactory epithelium, but show impaired preference for a lipid mixture odor. Our results show that CD36-expressing neurons represent a distinct population of OSNs, which may have specific functions in olfaction.