Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling.

BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.

Mechanistic perspectives on differential mitochondrial-based neuroprotective effects of several carnitine forms in Alzheimer's disease in vitro model.

Mitochondrial deregulation has emerged as one of the earliest pathological events in Alzheimer's disease (AD), the most common age-related neurodegenerative disorder. Improvement of mitochondrial function in AD has been considered a relevant therapeutic approach. L-carnitine (LC), an amino acid derivative involved in the transport of long-chain fatty acids into mitochondria, was previously demonstrated to improve mitochondrial function, having beneficial effects in neurological disorders; moreover, acetyl-L-carnitine (ALC) is currently under phase 4 clinical trial for AD (ClinicalTrials.gov NCT01320527). Thus, in the present study, we investigated the impact of different forms of carnitines, namely LC, ALC and propionyl-L-carnitine (PLC) on mitochondrial toxicity induced by amyloid-beta peptide 1-42 oligomers (AßO; 1 µM) in mature rat hippocampal neurons. Our results indicate that 5 mM LC, ALC and PLC totally rescued the mitochondrial membrane potential and alleviated both the decrease in oxygen consumption rates and the increase in mitochondrial fragmentation induced by AßO. These could contribute to the prevention of neuronal death by apoptosis. Moreover, only ALC ameliorated AßO-evoked changes in mitochondrial movement by reducing the number of stationary mitochondria and promoting reversal mitochondrial movement. Data suggest that carnitines (LC, ALC and PLC) may act differentially to counteract changes in mitochondrial function and movement in neurons subjected to AßO, thus counteracting AD-related pathological phenotypes.

A whole brain longitudinal study in the YAC128 mouse model of Huntington's disease shows distinct trajectories of neurochemical, structural connectivity and volumetric changes.

Huntington's disease (HD) is a neurodegenerative disorder causing cognitive and motor impairments, evolving to death within 15-20 years after symptom onset. We previously established a mouse model with the entire human HD gene containing 128 CAG repeats (YAC128) which accurately recapitulates the natural history of the human disease. Defined time points in this natural history enable the understanding of longitudinal trajectories from the neurochemical and structural points of view using non-invasive high-resolution multi-modal imaging. Accordingly, we designed a longitudinal structural imaging (MRI and DTI) and spectroscopy (1H-MRS) study in YAC128, at 3, 6, 9 and 12 months of age, at 9.4 T. Structural analysis (MRI/DTI), confirmed that the striatum is the earliest affected brain region, but other regions were also identified through connectivity analysis (pre-frontal cortex, hippocampus, globus pallidus and thalamus), suggesting a striking homology with the human disease. Importantly, we found for the first time, a negative correlation between striatal and hippocampal changes only in YAC128. In fact, the striatum showed accelerated volumetric decay in HD, as opposed to the hippocampus. Neurochemical analysis of the HD striatum suggested early neurometabolic alterations in neurotransmission and metabolism, with a significant increase in striatal GABA levels, and specifically anticorrelated levels of N-acetyl aspartate and taurine, suggesting that the later is homeostatically adjusted for neuroprotection, as neural loss, indicated by the former, is progressing. These results provide novel insights into the natural history of HD and prove a valuable role for longitudinal multi-modal panels of structural and metabolite/neurotransmission in the YAC128 model.

Microbial BMAA elicits mitochondrial dysfunction, innate immunity activation, and Alzheimer's disease features in cortical neurons.

BACKGROUND: After decades of research recognizing it as a complex multifactorial disorder, sporadic Alzheimer's disease (sAD) still has no known etiology. Adding to the myriad of different pathways involved, bacterial neurotoxins are assuming greater importance in the etiology and/or progression of sAD. ß-N-Methylamino-L-alanine (BMAA), a neurotoxin produced by some microorganisms namely cyanobacteria, was previously detected in the brains of AD patients. Indeed, the consumption of BMAA-enriched foods has been proposed to induce amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), which implicated this microbial metabolite in neurodegeneration mechanisms. METHODS: Freshly isolated mitochondria from C57BL/6 mice were treated with BMAA and O2 consumption rates were determined. O2 consumption and glycolysis rates were also measured in mouse primary cortical neuronal cultures. Further, mitochondrial membrane potential and ROS production were evaluated by fluorimetry and the integrity of mitochondrial network was examined by immunofluorescence. Finally, the ability of BMAA to activate neuronal innate immunity was quantified by addressing TLRs (Toll-like receptors) expression, p65 NF-κB translocation into the nucleus, increased expression of NLRP3 (Nod-like receptor 3), and pro-IL-1ß. Caspase-1 activity was evaluated using a colorimetric substrate and mature IL-1ß levels were also determined by ELISA. RESULTS: Treatment with BMAA reduced O2 consumption rates in both isolated mitochondria and in primary cortical cultures, with additional reduced glycolytic rates, decrease mitochondrial potential and increased ROS production. The mitochondrial network was found to be fragmented, which resulted in cardiolipin exposure that stimulated inflammasome NLRP3, reinforced by decreased mitochondrial turnover, as indicated by increased p62 levels. BMAA treatment also activated neuronal extracellular TLR4 and intracellular TLR3, inducing p65 NF-κB translocation into the nucleus and activating the transcription of NLRP3 and pro-IL-1ß. Increased caspase-1 activity resulted in elevated levels of mature IL-1ß. These alterations in mitochondrial metabolism and inflammation increased Tau phosphorylation and Aß peptides production, two hallmarks of AD. CONCLUSIONS: Here we propose a unifying mechanism for AD neurodegeneration in which a microbial toxin can induce mitochondrial dysfunction and activate neuronal innate immunity, which ultimately results in Tau and Aß pathology. Our data show that neurons, alone, can mount inflammatory responses, a role previously attributed exclusively to glial cells.

The NAD+-dependent deacetylase SIRT2 attenuates oxidative stress and mitochondrial dysfunction and improves insulin sensitivity in hepatocytes.

Insulin resistance is a major predictor of the development of metabolic disorders. Sirtuins (SIRTs) have emerged as potential targets that can be manipulated to counteract age-related diseases, including type 2 diabetes. SIRT2 has been recently shown to exert important metabolic effects, but whether SIRT2 regulates insulin sensitivity in hepatocytes is currently unknown. The aim of this study is to investigate this possibility and to elucidate underlying molecular mechanisms. Here, we show that SIRT2 is downregulated in insulin-resistant hepatocytes and livers, and this was accompanied by increased generation of reactive oxygen species, activation of stress-sensitive ERK1/2 kinase, and mitochondrial dysfunction. Conversely, SIRT2 overexpression in insulin-resistant hepatocytes improved insulin sensitivity, mitigated reactive oxygen species production and ameliorated mitochondrial dysfunction. Further analysis revealed a reestablishment of mitochondrial morphology, with a higher number of elongated mitochondria rather than fragmented mitochondria instigated by insulin resistance. Mechanistically, SIRT2 was able to increase fusion-related protein Mfn2 and decrease mitochondrial-associated Drp1. SIRT2 also attenuated the downregulation of TFAM, a key mtDNA-associated protein, contributing to the increase in mitochondrial mass. Importantly, we found that SIRT2 expression in PBMCs of human subjects was negatively correlated with obesity and insulin resistance. These results suggest a novel function for hepatic SIRT2 in the regulation of insulin sensitivity and raise the possibility that SIRT2 activators may offer novel opportunities for preventing or treating insulin resistance and type 2 diabetes.

Histone Deacetylase Inhibitors Protect Against Pyruvate Dehydrogenase Dysfunction in Huntington's Disease.

Transcriptional deregulation and changes in mitochondrial bioenergetics, including pyruvate dehydrogenase (PDH) dysfunction, have been described in Huntington's disease (HD). We showed previously that the histone deacetylase inhibitors (HDACIs) trichostatin A and sodium butyrate (SB) ameliorate mitochondrial function in cells expressing mutant huntingtin. In this work, we investigated the effect of HDACIs on the regulation of PDH activity in striatal cells derived from HD knock-in mice and YAC128 mice. Mutant cells exhibited decreased PDH activity and increased PDH E1alpha phosphorylation/inactivation, accompanied by enhanced protein levels of PDH kinases 1 and 3 (PDK1 and PDK3). Exposure to dichloroacetate, an inhibitor of PDKs, increased mitochondrial respiration and decreased production of reactive oxygen species in mutant cells, emphasizing PDH as an interesting therapeutic target in HD. Treatment with SB and sodium phenylbutyrate, another HDACI, recovered cell viability and overall mitochondrial metabolism in mutant cells. Exposure to SB also suppressed hypoxia-inducible factor-1 (HIF-1α) stabilization and decreased the transcription of the two most abundant PDK isoforms, PDK2 and PDK3, culminating in increased PDH activation in mutant cells. Concordantly, PDK3 knockdown improved mitochondrial function, emphasizing the role of PDK3 inactivation on the positive effects achieved by SB treatment. YAC128 mouse brain presented higher mRNA levels of PDK1-3 and PDH phosphorylation and decreased energy levels that were significantly ameliorated after SB treatment. Furthermore, enhanced motor learning and coordination were observed in SB-treated YAC128 mice. These results suggest that HDACIs, particularly SB, promote the activity of PDH in the HD brain, helping to counteract HD-related deficits in mitochondrial bioenergetics and motor function.SIGNIFICANCE STATEMENT The present work provides a better understanding of mitochondrial dysfunction in Huntington's disease (HD) by showing that the pyruvate dehydrogenase (PDH) complex is a promising therapeutic target. In particular, the histone deacetylase inhibitor sodium butyrate (SB) may indirectly (through reduced hypoxia-inducible factor 1 alpha stabilization) decrease the expression of the most abundant PDH kinase isoforms (e.g., PDK3), ameliorating PDH activity and mitochondrial metabolism and further affecting motor behavior in HD mice, thus constituting a promising agent for HD neuroprotective treatment.

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.

Mitochondrial Dysfunction in Huntington's Disease.

Mitochondrial dysfunction has been described as an early pathological mechanism delineating the selective neurodegeneration that occurs in Huntington's disease (HD), a polyglutamine-expansion disorder that largely affects the striatum and the cerebral cortex. Over the years, mitochondria roles in eukaryotic cells (e.g. in neurons) have largely diverged from the classically attributed cell power source; indeed, mitochondria not only contribute for synthesis of several metabolites, but are also dynamic organelles that fragment and fuse to achieve a maximal bioenergetic performance, are transported along microtubules, regulate intracellular calcium homeostasis through the interaction with the endoplasmic reticulum, produce free radicals and participate in cell death processes. Indeed, most of these activities have been demonstrated to be affected in HD, potentially contributing for the neuronal dysfunction in pre-symptomatic stages. This chapter resumes some of the evidences that pose mitochondria as a main regulatory organelle in HD-affected neurons, uncovering some potentially therapeutic mitochondrial-based relevant targets.

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.

Expanded and Wild-type Ataxin-3 Modify the Redox Status of SH-SY5Y Cells Overexpressing α-Synuclein.

Neurodegenerative diseases are considered to be distinct clinical entities, although they share the formation of proteinaceous aggregates and several neuropathological mechanisms. Increasing evidence suggest a possible interaction between proteins that have been classically associated to distinct neurodegenerative diseases. Thus, common molecular and cellular pathways might explain similarities between disease phenotypes. Interestingly, the characteristic Parkinson's disease (PD) phenotype linked to bradykinesia is also a clinical presentation of other neurodegenerative diseases. An example is Machado-Joseph disease (MJD), with some patients presenting parkinsonism and a positive response to levodopa (L-DOPA). Protein aggregates positive for α-synuclein (α-Syn), a protein associated with PD, in the substantia nigra of MJD models made us hypothesize a putative additive biological effect induced by expression of α-Syn and ataxin-3 (Atx3), the protein affected in MJD. Hence, in this study we analysed the influence of these two proteins (α-Syn and wild-type or mutant Atx3) on modified redox signaling, a pathological process potentially linked to both diseases, and also the impact of exposure to iron and rotenone in SH-SY5Y neuroblastoma cells. Our results show that both α-Syn and mutant Atx3 overexpression per se increased oxidation of dichlorodihydrofluorescein (DCFH2), and co-expression of these proteins exhibited additive effect on intracellular oxidation, with no correlation with apoptotic features. Mutant Atx3 and α-Syn also potentiated altered redox status induced by iron and rotenone, a hint to how these proteins might influence neuronal dysfunction under pro-oxidant conditions. We further show that overexpression of wild-type Atx3 decreased intracellular DCFH2 oxidation, possibly exerting a neuroprotective role.

Alpha-synuclein-induced oxidative stress correlates with altered superoxide dismutase and glutathione synthesis in human neuroblastoma SH-SY5Y cells.

Alpha-synuclein (α-syn) is a major component of Lewy bodies found in sporadic and inherited forms of Parkinson's disease (PD). Mutations in the gene encoding α-syn and duplications and triplications of wild-type (WT) α-syn have been associated with PD. Several mechanisms have been implicated in the degeneration of dopaminergic neurons in PD, including oxidative stress and mitochondrial dysfunction. Here we defined the occurrence of oxidative stress in SH-SY5Y cells overexpressing WT α-syn in a doxycycline (Dox) regulated manner, before and after exposure to iron (500 µM), and determined the changes in proteins involved in the intracellular antioxidant defense system. Data evidenced an increase in caspase-3 activation and diminished reducing capacity of -Dox cells, associated with decreased activity of mitochondria complex I and reduced mitochondrial transcription factor A (TFAM) levels in these cells. Furthermore, total and mitochondrial reactive oxygen species levels were higher under basal conditions in cells overexpressing α-syn (-Dox) and this increase was apparently correlated with diminished levels and activities of SOD1 and SOD2 in -Dox cells. Moreover, both reduced and oxidized glutathione levels were diminished in -Dox cells under basal conditions, concomitantly with decreased activity of GCL and reduced protein levels of GCLc. The effects caused by iron (500 µM) were mostly independent of α-syn expression and triggered different antioxidant responses to possibly counterbalance higher levels of free radicals. Overall, data suggest that overexpression of α-syn modifies the antioxidant capacity of SH-SY5Y cells due to altered activity and protein levels of SOD1 and SOD2, and decreased glutathione pool.

Sirtuins: double players in Huntington's disease.

Sirtuins are a conserved family of NAD(+)-dependent class III lysine deacetylases, known to regulate longevity. In mammals, the sirtuin family has seven members (SIRT1-7), which vary in enzymatic activity, subcellular distribution and targets. Pharmacological and genetic modulation of SIRTs has been widely spread as a promising approach to slow aging and neurodegenerative processes. Huntington's disease (HD) is a neurodegenerative disorder linked to expression of polyglutamine-expanded huntingtin (HTT) protein for which there is still no disease-reversing treatment. Studies in different animal models provide convincing evidence that SIRT1 protects both cellular and animal models from mutant HTT toxicity, however controversial results were recently reported. Indeed, as a consequence of a variety of SIRT-activation pathways, either activation or inhibition of a specific SIRT appears to be neuroprotective. Therefore, this review summarizes the recent progress and knowledge in sirtuins (particularly SIRT1-3) and their implications for HD treatment.

Linking alpha-synuclein phosphorylation to reactive oxygen species formation and mitochondrial dysfunction in SH-SY5Y cells.

Alpha-synuclein (α-syn) is a soluble protein highly enriched in presynaptic terminals of neurons. Accumulation of α-syn as intracellular filamentous aggregates is a pathological feature of sporadic and familial forms of Parkinson's disease (PD). Changes in α-syn post-translational modifications, as well as mitochondrial dysfunction and oxidative stress constitute key pathogenic events of this disorder. Here we assessed the correlation between α-syn phosphorylation at serine 129 (Ser129), the formation of reactive oxygen species (ROS) and mitochondrial dysfunction in SH-SY5Y cells expressing A53T mutant or wild-type (WT) α-syn, exposed to ferrous iron (FeSO4) and rotenone (complex I inhibitor). Under basal conditions, prolonged expression of A53T mutant α-syn altered mitochondria morphology, increased superoxide formation and phosphorylation at Ser129, which was linked to decreased activity of protein phosphatase 2A (PP2A). Exposure to FeSO4 or rotenone enhanced intracellular ROS levels, including superoxide anions, in both types of cells, along with α-syn Ser129 phosphorylation and mitochondrial depolarization. Most of these changes were largely evident in A53T mutant α-syn expressing cells. Overall, the data suggest that stimuli that promote ROS formation and mitochondrial alterations highly correlate with mutant α-syn phosphorylation at Ser129, which may precede cell degeneration in PD.

Compromised mitochondrial complex II in models of Machado-Joseph disease.

Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia type 3, is an inherited dominant autosomal neurodegenerative disorder. An expansion of Cytosine-Adenine-Guanine (CAG) repeats in the ATXN3 gene is translated as an expanded polyglutamine domain in the disease protein, ataxin-3. Selective neurodegeneration in MJD is evident in several subcortical brain regions including the cerebellum. Mitochondrial dysfunction has been proposed as a mechanism of neurodegeneration in polyglutamine disorders. In this study, we used different cell models and transgenic mice to assess the importance of mitochondria on cytotoxicity observed in MJD. Transiently transfected HEK cell lines with expanded (Q84) ataxin-3 exhibited a higher susceptibility to 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II. Increased susceptibility to 3-NP was also detected in stably transfected PC6-3 cells that inducibly express expanded (Q108) ataxin-3 in a tetracycline-regulated manner. Moreover, cerebellar granule cells from MJD transgenic mice were more sensitive to 3-NP inhibition than wild-type cerebellar neurons. PC6-3 (Q108) cells differentiated into a neuronal-like phenotype with nerve growth factor (NGF) exhibited a significant decrease in mitochondrial complex II activity. Mitochondria from MJD transgenic mouse model and lymphoblast cell lines derived from MJD patients also showed a trend toward reduced complex II activity. Our results suggest that mitochondrial complex II activity is moderately compromised in MJD, which may designate a common feature in polyglutamine toxicity.

Uncovering the Early Events Associated with Oligomeric Aß-Induced Src Activation.

Soluble Aß1-42 oligomers (AßO) are formed in the early stages of Alzheimer's disease (AD) and were previously shown to trigger enhanced Ca2+ levels and mitochondrial dysfunction via the activation of N-methyl-D-aspartate receptors (NMDAR). Src kinase is a ubiquitous redox-sensitive non-receptor tyrosine kinase involved in the regulation of several cellular processes, which was demonstrated to have a reciprocal interaction towards NMDAR activation. However, little is known about the early-stage mechanisms associated with AßO-induced neurodysfunction involving Src. Thus, in this work, we analysed the influence of brief exposure to oligomeric Aß1-42 on Src activation and related mechanisms involving mitochondria and redox changes in mature primary rat hippocampal neurons. Data show that brief exposure to AßO induce H2O2-dependent Src activation involving different cellular events, including NMDAR activation and mediated intracellular Ca2+ rise, enhanced cytosolic and subsequent mitochondrial H2O2 levels, accompanied by mild mitochondrial fragmentation. Interestingly, these effects were prevented by Src inhibition, suggesting a feedforward modulation. The current study supports a relevant role for Src kinase activation in promoting the loss of postsynaptic glutamatergic synapse homeostasis involving cytosolic and mitochondrial ROS generation after brief exposure to AßO. Therefore, restoring Src activity can constitute a protective strategy for mitochondria and related hippocampal glutamatergic synapses.

Reduction of class I histone deacetylases ameliorates ER-mitochondria cross-talk in Alzheimer's disease.

Several molecular mechanisms have been described in Alzheimer's disease (AD), including repressed gene transcription and mitochondrial and endoplasmic reticulum (ER) dysfunction. In this study, we evaluate the potential efficacy of transcriptional modifications exerted by inhibition or knockdown of class I histone deacetylases (HDACs) in ameliorating ER-mitochondria cross-talk in AD models. Data show increased HDAC3 protein levels and decreased acetyl-H3 in AD human cortex, and increased HDAC2-3 in MCI peripheral human cells, HT22 mouse hippocampal cells exposed to Aß1-42 oligomers (AßO) and APP/PS1 mouse hippocampus. Tacedinaline (Tac, a selective class I HDAC inhibitor) counteracted the increase in ER-Ca2+ retention and mitochondrial Ca2+ accumulation, mitochondrial depolarization and impaired ER-mitochondria cross-talk, as observed in 3xTg-AD mouse hippocampal neurons and AßO-exposed HT22 cells. We further demonstrated diminished mRNA levels of proteins involved in mitochondrial-associated ER membranes (MAM) in cells exposed to AßO upon Tac treatment, along with reduction in ER-mitochondria contacts (MERCS) length. HDAC2 silencing reduced ER-mitochondria Ca2+ transfer and mitochondrial Ca2+ retention, while knockdown of HDAC3 decreased ER-Ca2+ accumulation in AßO-treated cells. APP/PS1 mice treated with Tac (30 mg/kg/day) also showed regulation of mRNA levels of MAM-related proteins, and reduced Aß levels. These data demonstrate that Tac normalizes Ca2+ signaling between mitochondria and ER, involving the tethering between the two organelles in AD hippocampal neural cells. Tac-mediated AD amelioration occurs through the regulation of protein expression at MAM, as observed in AD cells and animal models. Data support transcriptional regulation of ER-mitochondria communication as a promising target for innovative therapeutics in AD.

Mitochondrial and Redox Changes in Periodontitis and Type 2 Diabetes Human Blood Mononuclear Cells.

Periodontitis (PDT) and type 2 diabetes (T2D) have demonstrated a bidirectional relationship and imbalanced oxidative stress linked to mitochondrial dysfunction. Therefore, we investigated mitochondrial and redox (de)regulation in peripheral blood mononuclear cells (PBMCs) in comorbid T2D-PDT, compared to PDT, T2D patients, and control individuals. PBMCs were analyzed for mitochondrial respiration, reactive oxygen species, antioxidant proteins, and expression of Nrf2-target genes. PDT and T2D-PDT patients exhibited altered periodontal clinical markers, while T2D and T2D-PDT patients displayed increased blood HbA1c. Decreased oxygen consumption and ATP production were observed in the PDT patient's PBMCs. PDT and T2D-PDT PBMCs also evidenced increased H2O2 levels and reduced catalase levels (also detected in T2D patients), whereas a compromised glutathione cycle was observed in T2D-PDT patients. PBMCs from both T2D or T2D-PDT patients showed increased Nrf2 protein levels, enhanced GCL activity and GCL-catalytic subunit protein levels, and maintained GCLc, GST, and HO-1 mRNA levels. In contrast, the expressions of Nrf2-target genes were significantly diminished in the PDT patient's PBMCs. Decreased SOD1 and GST mRNA levels were also observed in CD3+CD8+-lymphocytes derived from PDT and T2D-PDT patients. In conclusion, PBMCs from T2D-PDT patients showed major redox changes, while mononuclear cells from PDT patients showed mitochondrial deregulation and reduced expression of Nrf2-target genes.

Mitochondrial Dysfunction and Decreased Cytochrome c in Cell and Animal Models of Machado-Joseph Disease.

Mitochondrial dysfunction has been described in many neurodegenerative disorders; however, there is less information regarding mitochondrial deficits in Machado-Joseph disease (MJD), a polyglutamine (polyQ) disorder caused by CAG repeat expansion in the ATXN3 gene. In the present study, we characterized the changes in mitochondrial function and biogenesis markers in two MJD models, CMVMJD135 (MJD135) transgenic mice at a fully established phenotype stage and tetracycline-regulated PC6-3 Q108 cell line expressing mutant ataxin-3 (mATXN3). We detected mATXN3 in the mitochondrial fractions of PC6-3 Q108 cells, suggesting the interaction of expanded ATXN3 with the organelle. Interestingly, in both the cerebella of the MJD135 mouse model and in PC6-3 Q108 cells, we found decreased mitochondrial respiration, ATP production and mitochondrial membrane potential, strongly suggesting mitochondrial dysfunction in MJD. Also, in PC6-3 Q108 cells, an additional enhanced glycolytic flux was observed. Supporting the functional deficits observed in MJD mitochondria, MJD135 mouse cerebellum and PC6-3 Q108 cells showed reduced cytochrome c mRNA and protein levels. Overall, our findings show compromised mitochondrial function associated with decreased cytochrome c levels in both cell and animal models of MJD.

Extracellular vesicles improve GABAergic transmission in Huntington's disease iPSC-derived neurons.

Background: Extracellular vesicles (EVs) carry bioactive molecules associated with various biological processes, including miRNAs. In both Huntington's disease (HD) models and human samples, altered expression of miRNAs involved in synapse regulation was reported. Recently, the use of EV cargo to reverse phenotypic alterations in disease models with synaptopathy as the end result of the pathophysiological cascade has become an interesting possibility. Methods: Here, we assessed the contribution of EVs to GABAergic synaptic alterations using a human HD model and studied the miRNA content of isolated EVs. Results: After differentiating human induced pluripotent stem cells into electrophysiologically active striatal-like GABAergic neurons, we found that HD-derived neurons displayed reduced density of inhibitory synapse markers and GABA receptor-mediated ionotropic signaling. Treatment with EVs secreted by control (CTR) fibroblasts reversed the deficits in GABAergic synaptic transmission and increased the density of inhibitory synapses in HD-derived neuron cultures, while EVs from HD-derived fibroblasts had the opposite effects on CTR-derived neurons. Moreover, analysis of miRNAs from purified EVs identified a set of differentially expressed miRNAs between manifest HD, premanifest, and CTR lines with predicted synaptic targets. Conclusion: The EV-mediated reversal of the abnormal GABAergic phenotype in HD-derived neurons reinforces the potential role of EV-miRNAs on synapse regulation.

Mitochondrial function and dynamics in neural stem cells and neurogenesis: Implications for neurodegenerative diseases.

Mitochondria have been largely described as the powerhouse of the cell and recent findings demonstrate that this organelle is fundamental for neurogenesis. The mechanisms underlying neural stem cells (NSCs) maintenance and differentiation are highly regulated by both intrinsic and extrinsic factors. Mitochondrial-mediated switch from glycolysis to oxidative phosphorylation, accompanied by mitochondrial remodeling and dynamics are vital to NSCs fate. Deregulation of mitochondrial proteins, mitochondrial DNA, function, fission/fusion and metabolism underly several neurodegenerative diseases; data show that these impairments are already present in early developmental stages and NSC fate decisions. However, little is known about mitochondrial role in neurogenesis. In this Review, we describe the recent evidence covering mitochondrial role in neurogenesis, its impact in selected neurodegenerative diseases, for which aging is the major risk factor, and the recent advances in stem cell-based therapies that may alleviate neurodegenerative disorders-related neuronal deregulation through improvement of mitochondrial function and dynamics.