Mitochondrial Calcium Regulation Research Articles

Cyclophilin D regulates neuronal activity-induced filopodiagenesis by fine-tuning dendritic mitochondrial calcium dynamics.

Recent studies have highlighted the role of mitochondria in dendritic protrusion growth and plasticity. However, the detailed mechanisms that mitochondria regulate dendritic filopodia morphogenesis remain elusive. Cyclophilin D (CypD, gene name: Ppif) controls the opening of mitochondrial permeability transition pore. Although the pathological relevance of CypD has been intensively investigated, little is known about its physiological function in neurons. Here, we have found that genetic depletion of or pharmaceutical inhibition of CypD blunts the outgrowth of dendritic filopodia in response to KCl-stimulated neuronal depolarization. Further cell biological studies suggest that such inhibitory effect of CypD loss-of-function is closely associated with compromised flexibility of dendritic mitochondrial calcium regulation during neuronal depolarization, as well as the resultant changes in intradendritic calcium homeostasis, calcium signaling activation, dendritic mitochondrial motility and redistribution. Interestingly, loss of CypD attenuates oxidative stress-induced mitochondrial calcium perturbations and dendritic protrusion injury. Therefore, our study has revealed the physiological function of CypD in dendritic plasticity by acting as a fine-tuner of mitochondrial calcium homeostasis. Moreover, CypD plays distinct roles in neuronal physiology and pathology. Cover Image for this issue: doi: 10.1111/jnc.14189.

Mitochondrial calcium regulation during and following contractions in skeletal muscle

Mitochondrial calcium regulation during and following contractions in skeletal muscle

Mitochondrial Dysfunction in Airway Disease

There is increasing appreciation that mitochondria serve cellular functions beyond oxygen sensing and energy production. Accordingly, it has become important to explore noncanonical roles of mitochondria in normal and pathophysiological processes that influence airway structure and function in the context of diseases such as asthma and COPD. Mitochondria can sense upstream processes such as inflammation, infection, tobacco smoke, and environmental insults important in these diseases and in turn can respond to such stimuli through altered mitochondrial protein expression, structure, and resultant dysfunction. Conversely, mitochondrial dysfunction has downstream influences on cytosolic and mitochondrial calcium regulation, airway contractility, gene and protein housekeeping, responses to oxidative stress, proliferation, apoptosis, fibrosis, and certainly metabolism, which are all key aspects of airway disease pathophysiology. Indeed, mitochondrial dysfunction is thought to play a role even in normal processes such as aging and senescence and in conditions such as obesity, which impact airway diseases. Thus, understanding how mitochondrial structure and function play central roles in airway disease may be critical for the development of novel therapeutic avenues targeting dysfunctional mitochondria. In this case, it is likely that mitochondria of airway epithelium, smooth muscle, and fibroblasts play differential roles, consistent with their contributions to disease biology, underlining the challenge of targeting a ubiquitous cellular element of existential importance. This translational review summarizes the current state of understanding of mitochondrial processes that play a role in airway disease pathophysiology and identifying areas of unmet research need and opportunities for novel therapeutic strategies.

IP3 Receptor Determines Increased Basal Mitochondrial Calcium And Autophagy Regulation In Mdx Skeletal Muscle

Several reports describe that IP3 receptor (IP3R) is essential for efficient mitochondrial respiration and maintenance of cellular bioenergetics. This is due to the participation of this receptor in calcium transfer from the ER to the mitochondria. Changes in IP3R function can compromise mitochondrial function, changing ATP production and finally altering autophagy. Nevertheless until now there is no detailed study of the IP3R/calcium/autophagy axis in DMD. Our aim was to investigate the participation of IP3R in the regulation of mitochondrial calcium and autophagy in fibers from a DMD animal model, mdx. We analyzed protein levels of LC3, p62, atg5, beclin and bcl‐2 in FDB muscle of mdx (5–7 weeks), electroporated with a short‐hairpin for IP3R. We also evaluated the calcium transfer with mitochondria‐targeted probes that can be electroporated and also mitochondrial potential with TMRE in skeletal muscle fibers. The basal levels of expression of LC3II are diminished in mdx fibers compared with controls. This result correlates with an increased expression of p62 in mdx fibers. We also found differences in other autophagy proteins like atg5, beclin1 and Bcl‐2. When we performed the knockdown for IP3R we observed differences in the expression of almost all of the proteins analyzed. The expression of LC3II was increased in the electroporated mdx fibers with a decrease in p62 expression. We also confirmed that basal mitochondrial calcium level was increased in mdx fibers. Moreover, when we decreased the expression of IP3R, the basal mitochondrial calcium levels returned to normal. Similar result was obtained when we measured the mitochondrial potential in mdx fibers. Collectively, these results suggest that IP3R regulates mitochondrial calcium flux in adult skeletal muscle and is responsible for the increased mitochondrial calcium in mdx fibers. Moreover, knockdown of IP3R can regulate the expression levels of proteins associated with autophagy. Finally, we propose that some inhibitors of the IP3 pathway can be used as a new treatment for the muscle weakness observed in DMD patientsSupport or Funding InformationFONDECYT 3140491(DV), 3140532(FW), 1151293(EJ), ACT‐111 (EJ, SL), FONDAP 15130011(SL)

Regulation of mitochondrial calcium in plants versus animals.

Ca(2+) acts as an important cellular second messenger in eukaryotes. In both plants and animals, a wide variety of environmental and developmental stimuli trigger Ca(2+) transients of a specific signature that can modulate gene expression and metabolism. In animals, mitochondrial energy metabolism has long been considered a hotspot of Ca(2+) regulation, with a range of pathophysiology linked to altered Ca(2+) control. Recently, several molecular players involved in mitochondrial Ca(2+) signalling have been identified, including those of the mitochondrial Ca(2+) uniporter. Despite strong evidence for sophisticated Ca(2+) regulation in plant mitochondria, the picture has remained much less clear. This is currently changing aided by live imaging and genetic approaches which allow dissection of subcellular Ca(2+) dynamics and identification of the proteins involved. We provide an update on our current understanding in the regulation of mitochondrial Ca(2+) and signalling by comparing work in plants and animals. The significance of mitochondrial Ca(2+) control is discussed in the light of the specific metabolic and energetic needs of plant and animal cells.

Contribution of inorganic polyphosphate towards regulation of mitochondrial free calcium

BackgroundCalcium signaling plays a key role in the regulation of multiple processes in mammalian mitochondria, from cellular bioenergetics to the induction of stress-induced cell death. While the total concentration of calcium inside the mitochondria can increase by several orders of magnitude, the concentration of bioavailable free calcium in mitochondria is maintained within the micromolar range by the mitochondrial calcium buffering system. This calcium buffering system involves the participation of inorganic phosphate. However, the mechanisms of its function are not yet understood. Specifically, it is not clear how calcium-orthophosphate interactions, which normally lead to formation of insoluble precipitates, are capable to dynamically regulate free calcium concentration. Here we test the hypothesis that inorganic polyphosphate, which is a polymerized form of orthophosphate, is capable to from soluble complexes with calcium, playing a significant role in the regulation of the mitochondrial free calcium concentration. MethodsWe used confocal fluorescence microscopy to measure the relative levels of mitochondrial free calcium in cultured hepatoma cells (HepG2) with variable levels of inorganic polyphosphate (polyP). ResultsThe depletion of polyP leads to the significantly lower levels of mitochondrial free calcium concentration under conditions of pathological calcium overload. These results are coherent with previous observations showing that inorganic polyphosphate (polyP) can inhibit calcium-phosphate precipitation and, thus, increase the amount of free calcium. ConclusionsInorganic polyphosphate plays an important role in the regulation of mitochondrial free calcium, leading to its significant increase. General significanceInorganic polyphosphate is a previously unrecognized integral component of the mitochondrial calcium buffering system.

Contribution of Inorganic Polyphosphate Towards Regulation of Mitochondrial Free Calcium

Calcium signaling plays a key role in the regulation of multiple processes in mammalian mitochondria, ranging from the physiological regulation of cellular bioenergetics to the induction of stress-induced cell death. While the total concentration of calcium inside the mitochondria can increase by several orders of magnitude, the concentration of bioavailable, free calcium in mitochondria is maintained within the micromolar range by the mitochondrial calcium buffering system. It is established that this calcium buffering system involves participation of orthophosphate. However, the mechanisms of its function are not yet understood. Specifically, it is not clear how orthophosphate is capable to regulate free calcium concentration in the micromolar range. We hypothesized that inorganic polyphosphate, a biological polymer, made of up to 100 orthophosphates, plays a significant role in this regulation on mitochondria. We used confocal fluorescent microscopy to measure the relative levels of free calcium in this organelle, in cultured hepatoma (HepG2) cells with variable levels of inorganic polyphosphate. We found that the depletion of inorganic polyphosphate leads to the significantly lower levels of mitochondrial free calcium concentration, under conditions of pathological calcium overload. We conclude that inorganic polyphosphate is a previously unrecognized integral component of the mitochondrial calcium buffering system.

AMPA receptor activation causes preferential mitochondrial Ca2+ load and oxidative stress in motor neurons

It is well established that motor neurons are highly vulnerable to glutamate induced excitotoxicity. The selective vulnerability of these neurons has been attributed to AMPA receptor mediated excessive rise in cytosolic calcium and consequent mitochondrial Ca2+ loading. Earlier we have reported that in motor neurons a generic rise in [Ca2+]i does not always lead to mitochondrial Ca2+ loading and membrane depolarization but it occurs upon AMPA receptor activation. The mechanism of such specific mitochondrial involvement upon AMPA receptor activation is not known. The present study examines the mitochondrial Ca2+ regulation and oxidative stress in spinal cord neurons upon AMPA subtype of glutamate receptor activation. Stimulating the spinal neurons with AMPA exhibited a sharp rise in [Ca2+]m in both motor and other spinal neurons that was sustained up to the end of recording time of 30min. The rise in [Ca2+]m was substantially higher in motor neurons than in other spinal neurons which could be due to the differential mitochondrial homeostasis in two types of neurons. To examine this possibility, we measured AMPA induced [Ca2+]m loading in the presence of mitochondrial inhibitors. In both cell types the AMPA induced [Ca2+]m loading was blocked by mitochondrial calcium uniporter blocker ruthenium red. In motor neurons it was also inhibited substantially by CGP37157 and cyclosporine-A, the blockers of Na+/Ca2+ exchanger and mitochondrial permeability transition pore (MPTP) respectively, whereas no effect of these agents was observed in other spinal neurons. Thus in motor neurons the Ca2+ sequestration by mitochondria occurs through mitochondrial calcium uniporter as well as due to reversal of Na+/Ca2+ exchanger, in contrast the latter pathway does not contribute in other spinal neurons. The ROS formation was inhibited by nitric oxide synthase (NOS) inhibitor L-NAME in both types of neurons, however the mitochondrial complex-I inhibitor rotenone suppressed the ROS formation only in motor neurons. It appears that activation of cytoplasmic nNOS leads to ROS formation in both types of spinal neurons but mitochondria is the major source of ROS in motor neurons. Spinal neurons exhibited a significant time dependent fall in glutathione (GSH) level. The GSH level in motor neurons did not recover even at 24h after AMPA exposure, whereas the other spinal neurons exhibited a tendency to maintain the GSH after a certain level suggesting that the oxidative stress is arrested in other spinal neurons but it continues to increase in motor neurons. Thus our results demonstrate that upon AMPA receptor stimulation the motor neurons employ some additional pathways for regulation of mitochondrial calcium and oxidative stress as compared to other spinal neurons. It is suggested that such differential signaling mechanisms in motor neurons could be crucial for their selective vulnerability to excitotoxicity.

Fus1/Tusc2 Is a Novel Regulator of Mitochondrial Calcium Handling, Ca2+-Coupled Mitochondrial Processes, and Ca2+-Dependent NFAT and NF-κB Pathways in CD4+T Cells

Fus1 has been established as mitochondrial tumor suppressor, immunomodulator, and antioxidant protein, but molecular mechanism of these activities remained to be identified. Based on putative calcium-binding and myristoyl-binding domains that we identified in Fus1, we explored our hypothesis that Fus1 regulates mitochondrial calcium handling and calcium-coupled processes. Fus1 loss resulted in reduced rate of mitochondrial calcium uptake in calcium-loaded epithelial cells, splenocytes, and activated CD4(+) T cells. The reduced rate of mitochondrial calcium uptake in Fus1-deficient cells correlated with cytosolic calcium increase and dysregulation of calcium-coupled mitochondrial parameters, such as reactive oxygen species production, ΔμH(+), mitochondrial permeability transition pore opening, and GSH content. Inhibition of calcium efflux via mitochondria, Na(+)/Ca(2+) exchanger significantly improved the mitochondrial calcium uptake in Fus1(-/-) cells. Ex vivo analysis of activated CD4(+) T cells showed Fus1-dependent changes in calcium-regulated processes, such as surface expression of CD4 and PD1/PD-L1, proliferation, and Th polarization. Fus1(-/-) T cells showed increased basal expression of calcium-dependent NF-κB and NFAT targets but were unable to fully activate these pathways after stimulation. Our results establish Fus1 as one of the few identified regulators of mitochondrial calcium handling. Our data support the idea that alterations in mitochondrial calcium dynamics could lead to the disruption of metabolic coupling in mitochondria that, in turn, may result in multiple cellular and systemic abnormalities. Our findings suggest that Fus1 achieves its protective role in inflammation, autoimmunity, and cancer via the regulation of mitochondrial calcium and calcium-coupled parameters.

LETM1 is an essential component of mitochondrial calcium uptake and regulation of cellular bioenergetics

Regulation of mitochondrial Ca2+‐dependent cellular bioenergetics has been implicated in a variety of pathophysiological settings including neurodegeneration, myocardial infarction and immunity. Recently, LETM1 was proposed as a mitochondrial Ca2+/H+ antiporter; however characterization of the functional role of LETM1‐mediated Ca2+ transfer has yet to be studied. Here we demonstrate that LETM1 knockdown significantly impairs mitochondrial Ca2+ uptake and results in bioenergetic collapse. We observed that mitochondrial Ca2+ uptake is impaired in shRNA‐mediated LETM1 knockdown and ΔEF hand LETM1 and D676AD688KLETM1 overexpression cells. Knockdown of LETM1 promotes LC3 positive multilamellar vesicle formation, indicative of autophagy induction. Furthermore, cellular bioenergetic parameters including ATP and oxygen consumption were reduced. In contrast, cellular NADH and mitochondrial membrane potential were unaltered in both control and shRNA LETM1 cells. Interestingly, knockdown of LETM1 by siRNA in primary neonatal cardiomyocytes resulted in a remarkable change in mitochondrial morphology and reduced Ca2+ uptake. We also observed that LETM1 knockdown results in increased ROS production. Our results represent the first time that LETM1 has been linked to cellular bioenergetics through regulation of mitochondrial Ca2+.Supported by NIH R01 HL086699, HL086699‐01A2S1, 1S10RR027327‐01

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.

Site-Mapping of In Vitro S-nitrosation in Cardiac Mitochondria: Implications for Cardioprotection

S-nitrosation (SNO) of mitochondrial protein cysteines can be cardioprotective. Several targets have been implicated, yet the scope and identification of specific residues has not been fully assessed. To address this, a comprehensive assessment of mitochondrial SNO-modifiable cysteines was performed to determine nitric oxide (NO) susceptible pathways and identify novel mechanisms of oxidative cardioprotection. The biotin switch assay and mass spectrometry were used on rat cardiac mitochondrial lysates treated with the nitric oxide donor, S-nitrosoglutathione, and controls (n=3) to map 83 SNO-modified cysteine residues on 60 proteins. Of these, three sites have been reported, 30 sites are new to 21 proteins previously known to be S-nitrosated but which lacked site-specific information and 50 sites were found on 39 proteins not previously implicated in SNO pathways. The SNO-modifications occurred in only a subset of available cysteines, indicating a specific targeted effect. Functional annotation and site-specificity analysis revealed a twofold greater nitric oxide-susceptibility for proteins involved in transport; including regulators of mitochondrial permeability transition suggesting SNO-regulation and a possible protective mechanism. Additionally, we identified many novel SNO-modified proteins with cardioprotective potential involved in the electron transport chain, tricarboxylic acid cycle, oxidative stress defense, fatty acid and amino acid metabolism. These findings suggest that SNO-modification may represent a novel mechanism for the regulation of oxidative phosphorylation and/or cell death. S-nitrosation of mitochondrial permeability transition-associated proteins represents an intriguing potential link to cardioprotection.

Global gene expression profiling of somatic motor neuron populations with different vulnerability identify molecules and pathways of degeneration and protection

Different somatic motor neuron subpopulations show a differential vulnerability to degeneration in diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and spinobulbar muscular atrophy. Studies in mutant superoxide dismutase 1 over-expressing amyotrophic lateral sclerosis model mice indicate that initiation of disease is intrinsic to motor neurons, while progression is promoted by astrocytes and microglia. Therefore, analysis of the normal transcriptional profile of motor neurons displaying differential vulnerability to degeneration in motor neuron disease could give important clues to the mechanisms of relative vulnerability. Global gene expression profiling of motor neurons isolated by laser capture microdissection from three anatomical nuclei of the normal rat, oculomotor/trochlear (cranial nerve 3/4), hypoglossal (cranial nerve 12) and lateral motor column of the cervical spinal cord, displaying differential vulnerability to degeneration in motor neuron disorders, identified enriched transcripts for each neuronal subpopulation. There were striking differences in the regulation of genes involved in endoplasmatic reticulum and mitochondrial function, ubiquitination, apoptosis regulation, nitrogen metabolism, calcium regulation, transport, growth and RNA processing; cellular pathways that have been implicated in motor neuron diseases. Confirmation of genes of immediate biological interest identified differential localization of insulin-like growth factor II, guanine deaminase, peripherin, early growth response 1, soluble guanylate cyclase 1A3 and placental growth factor protein. Furthermore, the cranial nerve 3/4-restricted genes insulin-like growth factor II and guanine deaminase protected spinal motor neurons from glutamate-induced toxicity (P < 0.001, ANOVA), indicating that our approach can identify factors that protect or make neurons more susceptible to degeneration.

Molecular signatures in post mortem brain tissue of individuals at high risk for Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative condition characterized pathologically by neuritic plaques and neurofibrillary tangles. The objective in the present transcriptional profiling study was to identify both neurosusceptibility and intrinsic neuroprotective factors at the molecular level, not confounded by the downstream consequences of pathology. We thus studied post-mortem cortical tissue in 28 cases who were non-APOE4 carriers (called the APOE3 group) and 13 cases who were APOE4 carriers. Because APOE genotype is the major genetic risk factor for late onset AD, the former group was at low risk for development of the disease and latter group was at high risk for the disease. Mean age at death was 42 years and none of the brains had histopathology diagnostic of AD at time of death. We first derived interregional difference scores in expression between cortical tissue from a region relatively invulnerable to AD (primary somatosensory cortex, BA 1/2/3) and an area known to be susceptible to AD pathology (middle temporal gyrus, BA 21). We then contrasted the magnitude of these interregional differences in between-group comparisons of the APOE3 (low-risk) and APOE4 (high-risk) genotype groups. We identified 70 transcripts that differed significantly between the groups and several Kyoto pathways that were disrupted in the APOE4 group, including those involved in mitochondrial function, calcium regulation, and cell-cycle re-entry. Using RT-qPCR, we validated multiple findings. Using pathway analyses we then found that these molecules comprised a network with multiple connections with each other and with APP and MAPT. Our results indicated that the abnormalities that we observed in single transcripts and in signaling pathways were not the consequences of diagnostic plaque and tangle pathology, but preceded it and thus may be a causative link in the long molecular prodrome that results in clinical AD.

KATP channel blockade protects midbrain dopamine neurons by repressing a glia‐to‐neuron signaling cascade that ultimately disrupts mitochondrial calcium homeostasis

While K(ATP) channels serve primarily as metabolic gatekeepers in excitable cells, they might also participate in other important cellular functions. Here, we demonstrate that K(ATP) channel blockade with the sulfonylurea derivative glibenclamide provided robust protection to dopamine neurons undergoing spontaneous and selective degeneration in midbrain cultures. Unexpectedly, glibenclamide operated not by a direct effect on dopamine neurons but instead by halting the proliferation of a population of immature glial cells lacking astrocytic and microglial markers. The antimitotic effect of glibenclamide appeared essential to unmask a prosurvival phosphoinositide 3-kinase (PI3K)/Akt-dependent signaling pathway that controlled shuttling of calcium from endoplasmic reticulum to mitochondria in dopamine neurons. Preventing integrin-ligand interactions with a decoy ligand, the Arg-Gly-Asp-Ser sequence peptide, reproduced survival promotion by glibenclamide via a mechanism that also required PI3K/Akt-dependent regulation of mitochondrial calcium. Noticeably, Arg-Gly-Asp-Ser did not cause a reduction in glial cell numbers indicating that it prevented the death process downstream of the level at which glibenclamide intervenes. Based on these results, we propose that K(ATP) channel blockade protected dopamine neurons by inhibiting a glia-to-neuron signaling pathway that propagates through integrin/ligand interactions and ultimately disrupts PI3K/Akt-dependent signaling and mitochondrial calcium homeostasis.

Molecular signatures in post-mortem brain tissue of younger individuals at high risk for Alzheimer's disease as based on APOE genotype

Alzheimer's disease (AD) is a neurodegenerative condition characterized histopathologically by neuritic plaques and neurofibrillary tangles. The objective of this transcriptional profiling study was to identify both neurosusceptibility and intrinsic neuroprotective factors at the molecular level, not confounded by the downstream consequences of pathology. We thus studied post-mortem cortical tissue in 28 cases that were non-APOE4 carriers (called the APOE3 group) and 13 cases that were APOE4 carriers. As APOE genotype is the major genetic risk factor for late-onset AD, the former group was at low risk for development of the disease and the latter group was at high risk for the disease. Mean age at death was 42 years and none of the brains had histopathology diagnostic of AD at the time of death. We first derived interregional difference scores in expression between cortical tissue from a region relatively invulnerable to AD (primary somatosensory cortex, BA 1/2/3) and an area known to be susceptible to AD pathology (middle temporal gyrus, BA 21). We then contrasted the magnitude of these interregional differences in between-group comparisons of the APOE3 (low risk) and APOE4 (high risk) genotype groups. We identified 70 transcripts that differed significantly between the groups. These included EGFR, CNTFR, CASP6, GRIA2, CTNNB1, FKBPL, LGALS1 and PSMC5. Using real-time quantitative PCR, we validated these findings. In addition, we found regional differences in the expression of APOE itself. We also identified multiple Kyoto pathways that were disrupted in the APOE4 group, including those involved in mitochondrial function, calcium regulation and cell-cycle reentry. To determine the functional significance of our transcriptional findings, we used bioinformatics pathway analyses to demonstrate that the molecules listed above comprised a network of connections with each other, APOE, and APP and MAPT. Overall, our results indicated that the abnormalities that we observed in single transcripts and in signaling pathways were not the consequences of diagnostic plaque and tangle pathology, but preceded it and thus may be a causative link in the long molecular prodrome that results in clinical AD.

Superoxide Anion, Uncoupling Proteins and Alzheimer’s Disease

Superoxide anion is the first generated reactive oxygen species (ROS) after oxygen enters living cells. It was once considered to be highly deleterious to cell functions and aging. Therefore, antioxidants were suggested to prevent aging and degenerative diseases. However, superoxide signaling has been shown in many physiological responses such as transcriptional regulation, protein activation, bioenergy output, cell proliferation and apoptosis. The uncoupling proteins (UCPs) are a family of mitochondrial anion-carrier proteins located in the inner mitochondrial membrane and are considered to reduce the generation of superoxide anion through the mitochondrial mild uncoupling. UCPs are important in prevention of mitochondrial excessive generation of ROS, transfer of mitochondrial substrates, mitochondrial calcium uniport and regulation of thermogenesis. Superoxide anion and uncoupling proteins are linked to Alzheimer’s disease in mitochondria. Simultaneous disorders of superoxide and uncoupling proteins create the conditions for neuronal oxidative damages. On the one hand, sustained oxidative damage causes neuronal apoptosis and eventually, accumulated neuronal apoptosis, leading to exacerbations of Alzheimer’s disease. On the other hand, our study has shown that UCP2 and UCP4 have important impact on mitochondrial calcium concentration of nerve cells, suggesting that their abnormal expression may involve in the pathogenesis of Alzheimer’s disease.

The influence of the branched-chain fatty acids pristanic acid and Refsum disease-associated phytanic acid on mitochondrial functions and calcium regulation of hippocampal neurons, astrocytes, and oligodendrocytes

Pristanic acid and phytanic acid are branched-chain fatty acids, which play an important role in diseases with peroxisomal impairment, like Refsum disease (MIM 266500), Zellwegers syndrome and α-methylacyl-CoA racemase deficiency (MIM 604489). Several studies revealed that the toxic activity of phytanic acid is mediated by multiple mitochondrial dysfunctions. However, the action of pristanic acid on brain cells is still completely unknown. Here, we exposed astrocytes, oligodendrocytes and neurons in mixed culture to pristanic acid and phytanic acid to analyse cellular consequences. Pristanic acid exerts a strong cytotoxic activity on brain cells, displayed by dramatic Ca 2+ deregulation, in situ mitochondrial depolarization and cell death. Interestingly, pristanic acid strongly induced generation of reactive oxygen species (ROS), whereas phytanic acid exerts weaker effects on ROS production. In conclusion, pristanic acid as well as phytanic acid induced a complex array of toxic activities with mitochondrial dysfunction and Ca 2+ deregulation.

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca 2+, competing with Ca 2+ transporters like the sarcoplasmic reticulum (SR) Ca 2+-ATPase. Rapid mitochondrial [Ca 2+] transients have been reported to be synchronized with normal cytosolic [Ca 2+] i transients. However, most intra-mitochondrial free [Ca 2+] ([Ca 2+] mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca 2+] mito in single rat myocytes using the ratiometric Ca 2+ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca 2+] mito dependence on [Ca 2+] i (with 5 mM EGTA) was sigmoid with [Ca 2+] mito < [Ca 2+] i for [Ca 2+] i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca 2+] i (± 15 mM Na +) cyclical spontaneous SR Ca 2+ release occurred (5–15/min). Changes in [Ca 2+] mito during individual [Ca 2+] i transients were small (∼ 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca 2+ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca 2+] mito and spontaneous Ca 2+ transients (confirming that SR Ca 2+ releases caused the [Ca 2+] mito rise). Confocal imaging of local [Ca 2+] mito (using rhod-2) showed that [Ca 2+] mito rose rapidly with a delay after SR Ca 2+ release (with amplitude up to 10 nM), but declined much more slowly than [Ca 2+] i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca 2+ uptake for larger [Ca 2+] mito transients was ∼ 0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca 2+ buffering), consistent with prior indirect estimates from [Ca 2+] i measurements, and corresponds to ∼ 1% of the SR Ca 2+ uptake during a normal Ca 2+ transient. Thus small phasic [Ca 2+] mito transients and gradually integrating [Ca 2+] mito signals occur during repeating [Ca 2+] i transients.

Association between mitochondrial DNA 10398A&gt;G polymorphism and the volume of amygdala

Mitochondrial calcium regulation plays a number of important roles in neurons. Mitochondrial DNA (mtDNA) is highly polymorphic, and its interindividual variation is associated with various neuropsychiatric diseases and mental functions. An mtDNA polymorphism, 10398A>G, was reported to affect mitochondrial calcium regulation. Volume of hippocampus and amygdala is reportedly associated with various mental disorders and mental functions and is regarded as an endophenotype of mental disorders. The present study investigated the relationship between the mtDNA 10398A>G polymorphism and the volume of hippocampus and amygdala in 118 right-handed healthy subjects. The brain morphometry using magnetic resonance images employed both manual tracing volumetry in the native space and voxel-based morphometry (VBM) in the spatially normalized space. Amygdala volume was found to be significantly larger in healthy subjects with 10398A than in those with 10398G by manual tracing, which was confirmed by the VBM. Brain volumes in the other gray matter regions and all white matter regions showed no significant differences associated with the polymorphism. These provocative findings might provide a clue to the complex relationship between mtDNA, brain structure and mental disorders.