Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering‐induced Oma1 activation

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin‐related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin‐related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1‐knockout cells. Re‐activation of Opa1 and Mitofusin 1 in Drp1‐knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.     

MitoQ protects dopaminergic neurons in a 6-OHDA induced PD model by enhancing Mfn2-dependent mitochondrial fusion via activation of PGC-1α

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra compacta (SNc). Although mitochondrial dysfunction is the critical factor in the pathogenesis of PD, the underlying molecular mechanisms are not well understood, and as a result, effective medical interventions are lacking. Mitochondrial fission and fusion play important roles in the maintenance of mitochondrial function and cell viability. Here, we investigated the effects of MitoQ, a mitochondria-targeted antioxidant, in 6-hydroxydopamine (6-OHDA)-induced in vitro and in vivo PD models. We observed that 6-OHDA enhanced mitochondrial fission by decreasing the expression of Mfn1, Mfn2 and OPA1 as well as by increasing the expression of Drp1 in the dopaminergic (DA) cell line SN4741. Notably, MitoQ treatment particularly upregulated the Mfn2 protein and mRNA levels and promoted mitochondrial fusion in the presence of 6-OHDA in a Mfn2-dependent manner. In addition, MitoQ also stabilized mitochondrial morphology and function in the presence of 6-OHDA, which further suppressed the formation of reactive oxygen species (ROS), as well as ameliorated mitochondrial fragmentation and cellular apoptosis. Moreover, the activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) was attributed to the upregulation of Mfn2 induced by MitoQ. Consistent with these findings, administration of MitoQ in 6-OHDA-treated mice significantly rescued the decrease of Mfn2 expression and the loss of DA neurons in the SNc. Taken together, our findings suggest that MitoQ protects DA neurons in a 6-OHDA induced PD model by activating PGC-1α to enhance Mfn2-dependent mitochondrial fusion.     

Parkin‐independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain

Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease‐associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals.     

Upregulated function of mitochondria-associated ER membranes in Alzheimer disease

Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by γ-secretase, via an unknown mechanism. We recently showed that presenilin-1 and -2, the catalytic components of γ-secretase, and γ-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER–mitochondrial communication—as measured by cholesteryl ester and phospholipid synthesis, respectively—are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and γ-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER–mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD.     

Redox regulation of mitochondrial fission,protein misfolding,synaptic damage,and neuronal cell death: potential implications for Alzheimer’s and Parkinson’s diseases

Normal mitochondrial dynamics consist of fission and fusion events giving rise to new mitochondria, a process termed mitochondrial biogenesis. However, several neurodegenerative disorders manifest aberrant mitochondrial dynamics, resulting in morphological abnormalities often associated with deficits in mitochondrial mobility and cell bioenergetics. Rarely, dysfunctional mitochondrial occur in a familial pattern due to genetic mutations, but much more commonly patients manifest sporadic forms of mitochondrial disability presumably related to a complex set of interactions of multiple genes (or their products) with environmental factors (G × E). Recent studies have shown that generation of excessive nitric oxide (NO), in part due to generation of oligomers of amyloid-β (Aβ) protein or overactivity of the NMDA-subtype of glutamate receptor, can augment mitochondrial fission, leading to frank fragmentation of the mitochondria. S-Nitrosylation, a covalent redox reaction of NO with specific protein thiol groups, represents one mechanism contributing to NO-induced mitochondrial fragmentation, bioenergetic failure, synaptic damage, and eventually neuronal apoptosis. Here, we summarize our evidence in Alzheimer’s disease (AD) patients and animal models showing that NO contributes to mitochondrial fragmentation via S-nitrosylation of dynamin-related protein 1 (Drp1), a protein involved in mitochondrial fission. These findings may provide a new target for drug development in AD. Additionally, we review emerging evidence that redox reactions triggered by excessive levels of NO can contribute to protein misfolding, the hallmark of a number of neurodegenerative disorders, including AD and Parkinson’s disease. For example, S-nitrosylation of parkin disrupts its E3 ubiquitin ligase activity, and thereby affects Lewy body formation and neuronal cell death.     

Mitofusin-2 protects against cold stress-induced cell injury in HEK293 cells

Mitochondrial impairment is hypothesized to contribute to cell injury during cold stress. Mitochondria fission and fusion are closely related in the function of the mitochondria, but the precise mechanisms whereby these processes regulate cell injury during cold stress remain to be determined. HEK293 cells were cultured in a cold environment (4.0 ± 0.1 °C) for 2, 4, 8, or 12 h. Western blot analyses showed that these cells expressed decreased fission-related protein Drp1 and increased fusion-related protein Mfn2 at 4 h; meanwhile, electron microscopy analysis revealed large and long mitochondrial morphology within these cells, indicating increased mitochondrial fusion. With silencing of Mfn2 but not of Mfn1 by siRNA promoted cold-stress-induced cell death with decreased ATP production in HEK293 cells. Our results show that increased expression of Mfn2 and mitochondrial fusion are important for mitochondrial function as well as cell survival during cold stress. These findings have important implications for understanding the mechanisms of mitochondrial fusion and fission in cold-stress-induced cell injury.     

Response of mitochondrial fusion and fission protein gene expression to exercise in rat skeletal muscle

The purpose of this study was to investigate the changes in the gene expression of Mitofusion (Mfn) 1 and 2 and Fission 1 (Fis1) and mitochondrial energy metabolism in response to altered energy demand during prolonged exercise in rat skeletal muscle. Male Sprague–Dawley rats were subjected to an acute bout of treadmill running at various durations and killed immediately or during recovery. Mfn1/2 and Fis1 mRNA and protein contents, reactive oxygen species (ROS) generation, state 3 and state 4 respiration rates, trans-innermembrane potential and ATP synthase activity were measured in isolated muscle mitochondria. We found that (1) Mfn1/2 mRNA contents were progressively decreased during 150 min of exercise, along with decreased Mfn 1 protein levels. Fis1 mRNA and protein contents showed significant increases after 120–150 min of exercise. These changes persisted through the recovery period up to 24 h. (2) Mitochondrial ROS generation and state 4 respiration showed progressive increases up to 120 min, but dropped at 150 min of exercise. (3) State 3 respiration rate and respiratory control index were unchanged initially but decreased at 150 and 120 min of exercise, respectively, whereas ATP synthase activity was elevated at 45 min and returned to resting level thereafter. Our data suggested that the gene expression of mitochondrial fusion and fission proteins in skeletal muscle can respond rapidly to increased metabolic demand during prolonged exercise, which could significantly affect the efficiency of oxidative phosphorylation.     

A γ-Secretase Independent Role for Presenilin in Calcium Homeostasis Impacts Mitochondrial Function and Morphology in Caenorhabditis elegans

Mutations in the presenilin (PSEN) encoding genes (PSEN1 and PSEN2) occur in most early onset familial Alzheimer’s Disease. Despite the identification of the involvement of PSEN in Alzheimer’s Disease (AD) ∼20 years ago, the underlying role of PSEN in AD is not fully understood. To gain insight into the biological function of PSEN, we investigated the role of the PSEN homolog SEL-12 in Caenorhabditis elegans. Using genetic, cell biological, and pharmacological approaches, we demonstrate that mutations in sel-12 result in defects in calcium homeostasis, leading to mitochondrial dysfunction. Moreover, consistent with mammalian PSEN, we provide evidence that SEL-12 has a critical role in mediating endoplasmic reticulum (ER) calcium release. Furthermore, we found that in SEL-12-deficient animals, calcium transfer from the ER to the mitochondria leads to fragmentation of the mitochondria and mitochondrial dysfunction. Additionally, we show that the impact that SEL-12 has on mitochondrial function is independent of its role in Notch signaling, γ-secretase proteolytic activity, and amyloid plaques. Our results reveal a critical role for PSEN in mediating mitochondrial function by regulating calcium transfer from the ER to the mitochondria.     

Mitochondrially localized PKA reverses mitochondrial pathology and dysfunction in a cellular model of Parkinson's disease

Mutations in PTEN-induced kinase 1 (PINK1) are associated with a familial syndrome related to Parkinson's disease (PD). We previously reported that stable neuroblastoma SH-SY5Y cell lines with reduced expression of endogenous PINK1 exhibit mitochondrial fragmentation, increased mitochondria-derived superoxide, induction of compensatory macroautophagy/mitophagy and a low level of ongoing cell death. In this study, we investigated the ability of protein kinase A (PKA) to confer protection in this model, focusing on its subcellular targeting. Either: (1) treatment with pharmacological PKA activators; (2) transient expression of a constitutively active form of mitochondria-targeted PKA; or (3) transient expression of wild-type A kinase anchoring protein 1 (AKAP1), a scaffold that targets endogenous PKA to mitochondria, reversed each of the phenotypes attributed to loss of PINK1 in SH-SY5Y cells, and rescued parameters of mitochondrial respiratory dysfunction. Mitochondrial and lysosomal changes in primary cortical neurons derived from PINK1 knockout mice or subjected to PINK1 RNAi were also reversed by the activation of PKA. PKA phosphorylates the rat dynamin-related protein 1 isoform 1 (Drp1) at serine 656 (homologous to human serine 637), inhibiting its pro-fission function. Mimicking phosphorylation of Drp1 recapitulated many of the protective effects of AKAP1/PKA. These data indicate that redirecting endogenous PKA to mitochondria can compensate for deficiencies in PINK1 function, highlighting the importance of compartmentalized signaling networks in mitochondrial quality control.     

Human MIEF1 recruits Drp1 to mitochondrial outer membranes and promotes mitochondrial fusion rather than fission

Mitochondrial morphology is controlled by two opposing processes: fusion and fission. Drp1 (dynamin-related protein 1) and hFis1 are two key players of mitochondrial fission, but how Drp1 is recruited to mitochondria and how Drp1-mediated mitochondrial fission is regulated in mammals is poorly understood. Here, we identify the vertebrate-specific protein MIEF1 (mitochondrial elongation factor 1; independently identified as MiD51), which is anchored to the outer mitochondrial membrane. Elevated MIEF1 levels induce extensive mitochondrial fusion, whereas depletion of MIEF1 causes mitochondrial fragmentation. MIEF1 interacts with and recruits Drp1 to mitochondria in a manner independent of hFis1, Mff (mitochondrial fission factor) and Mfn2 (mitofusin 2), but inhibits Drp1 activity, thus executing a negative effect on mitochondrial fission. MIEF1 also interacts with hFis1 and elevated hFis1 levels partially reverse the MIEF1-induced fusion phenotype. In addition to inhibiting Drp1, MIEF1 also actively promotes fusion, but in a manner distinct from mitofusins. In conclusion, our findings uncover a novel mechanism which controls the mitochondrial fusion-fission machinery in vertebrates. As MIEF1 is vertebrate-specific, these data also reveal important differences between yeast and vertebrates in the regulation of mitochondrial dynamics.     

Genetic Insights into Sporadic Parkinson's Disease Pathogenesis

Intensive research over the last 15 years has led to the identification of several autosomal recessive and dominant genes that cause familial Parkinson’s disease (PD). Importantly, the functional characterization of these genes has shed considerable insights into the molecular mechanisms underlying the etiology and pathogenesis of PD. Collectively; these studies implicate aberrant protein and mitochondrial homeostasis as key contributors to the development of PD, with oxidative stress likely acting as an important nexus between the two pathogenic events. Interestingly, recent genome-wide association studies (GWAS) have revealed variations in at least two of the identified familial PD genes (i.e. α-synuclein and LRRK2) as significant risk factors for the development of sporadic PD. At the same time, the studies also uncovered variability in novel alleles that is associated with increased risk for the disease. Additionally, in-silico meta-analyses of GWAS data have allowed major steps into the investigation of the roles of gene-gene and gene-environment interactions in sporadic PD. The emergent picture from the progress made thus far is that the etiology of sporadic PD is multi-factorial and presumably involves a complex interplay between a multitude of gene networks and the environment. Nonetheless, the biochemical pathways underlying familial and sporadic forms of PD are likely to be shared.     

Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-Δ-prostaglandin J2

Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 μM concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTPγS or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.     

IFN‐β rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1

Mitochondrial homeostasis is essential for providing cellular energy, particularly in resource‐demanding neurons, defects in which cause neurodegeneration, but the function of interferons (IFNs) in regulating neuronal mitochondrial homeostasis is unknown. We found that neuronal IFN‐β is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN‐β induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN‐β signaling then recruits Drp1 to mitochondria, oligomerizes it, and engages INF2 to stabilize mitochondria–endoplasmic reticulum (ER) platforms. This process tethers damaged mitochondria to the ER to separate them via fission. Lack of neuronal IFN‐β in the Ifnb ^–/– model of Parkinson disease (PD) disrupts STAT5‐PGAM5‐Drp1 signaling, impairing fission and causing large multibranched, damaged mitochondria with insufficient ATP production and excessive oxidative stress to accumulate. In other PD models, IFN‐β rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN‐β activates mitochondrial fission in neurons through the pSTAT5/PGAM5/^S622Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism.     

Dynamin-related protein 1: A critical protein in the pathogenesis of neural system dysfunctions and neurodegenerative diseases

Mitochondria play a key role in the maintenance of neuronal function by continuously providing energy. Here, we will give a detailed review about the recent developments in regards to dynamin-related protein 1 (Drp1) induced unbalanced mitochondrial dynamics, excessive mitochondrial division, and neuronal injury in neural system dysfunctions and neurodegenerative diseases, including the Drp1 knockout induced mice embryonic death, the dysfunction of the Drp1-dependent mitochondrial division induced neuronal cell apoptosis and impaired neuronal axonal transportation, the abnormal interaction between Drp1 and amyloid β (Aβ) in Alzheimer's disease (AD), the mutant Huntingtin (Htt) in Huntington's disease (HD), and the Drp1-associated pathogenesis of other neurodegenerative diseases such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Drp1 is required for mitochondrial division determining the size, shape, distribution, and remodeling as well as maintaining of mitochondrial integrity in mammalian cells. In addition, increasing reports indicate that the Drp1 is involved in some cellular events of neuronal cells causing some neural system dysfunctions and neurodegenerative diseases, including impaired mitochondrial dynamics, apoptosis, and several posttranslational modification induced increased mitochondrial divisions. Recent studies also revealed that the Drp1 can interact with Aβ, phosphorylated τ, and mutant Htt affecting the mitochondrial shape, size, distribution, axonal transportation, and energy production in the AD and HD neuronal cells. These changes can affect the health of mitochondria and the function of synapses causing neuronal injury and eventually leading to the dysfunction of memory, cognitive impairment, resting tremor, posture instability, involuntary movements, and progressive muscle atrophy and paralysis in patients.     

Characterization and Molecular Profiling of PSEN1 Familial Alzheimer's Disease iPSC-Derived Neural Progenitors

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer''s disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.     

Presenilin-1 but not amyloid precursor protein mutations present in mouse models of Alzheimer's disease attenuate the response of cultured cells to γ-secretase modulators regardless of their potency and structure

γ-Secretase modulators (GSMs) inhibit the generation of amyloidogenic Aβ42 peptides and are promising agents for treatment or prevention of Alzheimer's disease (AD). Recently, a second generation of GSMs with favorable pharmacological properties has emerged, but preclinical studies to assess their efficacy in vivo are lacking. Such studies rely on transgenic mouse models that express amyloid precursor protein (APP) and presenilin (PSEN) mutations associated with early-onset familial AD. Previously, we have shown that certain PSEN1 mutations attenuated the response of cultured cells to GSMs and potentially confound in vivo studies in AD mouse models. However, different combinations of familial AD mutations might have synergistic or opposing effects, and we have now systematically determined the response of APP and PSEN1 mutations present in current AD models. Using a potent acidic GSM, we found that APP mutations, either single mutations or in combination, did not affect the potency of GSMs. In contrast, all PSEN1 mutations that have been used to accelerate pathological changes in AD models strongly attenuated the Aβ42-lowering activity of GSMs with two exceptions (M146L, A246E). Similar results were obtained with potent non-acidic GSMs indicating that the attenuating effect of PSEN1 mutations cannot simply be overcome by increased potency or structural changes. Notably, two non-acidic compounds fully compensated the attenuating effect of the PSEN1-G384A mutation. Taken together, our findings indicate that most AD models with rapid pathology and advanced phenotypes are unsuitable for preclinical GSM studies. However, we also provide evidence that additional compound screens could discover GSMs that are able to break the attenuating effects of PSEN mutations.     

Mutations in Fis1 disrupt orderly disposal of defective mitochondria

Mitochondrial fission is mediated by the dynamin-related protein Drp1 in metazoans. Drp1 is recruited from the cytosol to mitochondria by the mitochondrial outer membrane protein Mff. A second mitochondrial outer membrane protein, named Fis1, was previously proposed as recruitment factor, but Fis1^−/− cells have mild or no mitochondrial fission defects. Here we show that Fis1 is nevertheless part of the mitochondrial fission complex in metazoan cells. During the fission cycle, Drp1 first binds to Mff on the surface of mitochondria, followed by entry into a complex that includes Fis1 and endoplasmic reticulum (ER) proteins at the ER–mitochondrial interface. Mutations in Fis1 do not normally affect fission, but they can disrupt downstream degradation events when specific mitochondrial toxins are used to induce fission. The disruptions caused by mutations in Fis1 lead to an accumulation of large LC3 aggregates. We conclude that Fis1 can act in sequence with Mff at the ER–mitochondrial interface to couple stress-induced mitochondrial fission with downstream degradation processes.     

Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30–35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer''s disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.     

Protective effect of ursodeoxycholic acid on liver mitochondrial function in rats with alloxan-induced diabetes: link with oxidative stress

We investigated the effects of ursodeoxycholic acid (UDCA) on mitochondrial functions and oxidative stress and evaluated their relationships in the livers of rats with alloxan-induced diabetes. Diabetes was induced in male Wistar rats by a single alloxan injection (150 mg kg^− 1 b.w., i.p.). UDCA (40 mg kg^− 1 b.w., i.g., 30 days) was administered from the 5th day after the alloxan treatment. Mitochondrial functions were evaluated by oxygen consumption with Clark oxygen electrode using succinate, pyruvate + malate or palmitoyl carnitine as substrates and by determination of succinate dehydrogenase and NADH dehydrogenase activities. Liver mitochondria were used to measure chemiluminiscence enhanced by luminol and lucigenin, reduced liver glutathione and the end-products of lipid peroxidation. The activities of both NADH dehydrogenase and succinate dehydrogenase as well as the respiratory control (RC) value with all the substrates and the ADP/O ratio with pyruvate + malate and succinate as substrates were significantly decreased in diabetic rats. UDCA developed the beneficial effect on the mitochondrial respiration and oxidative phosphorylation parameters in alloxan-treated rats, whereas the activities of mitochondrial enzymes were increased insignificantly after the administration of UDCA. The contents of polar carbonyls and MDA as well as the chemiluminescence with luminol were elevated in liver mitochondria of diabetic rats. The treatment with UDCA normalized all the above parameters measured except the MDA content. UDCA administration prevents mitochondrial dysfunction in rats treated with alloxan and this process is closely connected with inhibition of oxidative stress by this compound.     

Defects in Mitochondrial Fission Protein Dynamin-Related Protein 1 Are Linked to Apoptotic Resistance and Autophagy in a Lung Cancer Model

Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549) cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype–mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy). A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.