Dysregulated mitophagy and mitochondrial organization in optic atrophy due to OPA1 mutations.

OBJECTIVE: To investigate mitophagy in 5 patients with severe dominantly inherited optic atrophy (DOA), caused by depletion of OPA1 (a protein that is essential for mitochondrial fusion), compared with healthy controls. METHODS: Patients with severe DOA (DOA plus) had peripheral neuropathy, cognitive regression, and epilepsy in addition to loss of vision. We quantified mitophagy in dermal fibroblasts, using 2 high throughput imaging systems, by visualizing colocalization of mitochondrial fragments with engulfing autophagosomes. RESULTS: Fibroblasts from 3 biallelic OPA1(-/-) patients with severe DOA had increased mitochondrial fragmentation and mitochondrial DNA (mtDNA)-depleted cells due to decreased levels of OPA1 protein. Similarly, in siRNA-treated control fibroblasts, profound OPA1 knockdown caused mitochondrial fragmentation, loss of mtDNA, impaired mitochondrial function, and mitochondrial mislocalization. Compared to controls, basal mitophagy (abundance of autophagosomes colocalizing with mitochondria) was increased in (1) biallelic patients, (2) monoallelic patients with DOA plus, and (3) OPA1 siRNA-treated control cultures. Mitophagic flux was also increased. Genetic knockdown of the mitophagy protein ATG7 confirmed this by eliminating differences between patient and control fibroblasts. CONCLUSIONS: We demonstrated increased mitophagy and excessive mitochondrial fragmentation in primary human cultures associated with DOA plus due to biallelic OPA1 mutations. We previously found that increased mitophagy (mitochondrial recycling) was associated with visual loss in another mitochondrial optic neuropathy, Leber hereditary optic neuropathy (LHON). Combined with our LHON findings, this implicates excessive mitochondrial fragmentation, dysregulated mitophagy, and impaired response to energetic stress in the pathogenesis of mitochondrial optic neuropathies, potentially linked with mitochondrial mislocalization and mtDNA depletion.

The thermo-TRP ion channel family: properties and therapeutic implications.

The thermo-transient receptor potentials (TRPs), a recently discovered family of ion channels activated by temperature, are expressed in primary sensory nerve terminals where they provide information about thermal changes in the environment. Six thermo-TRPs have been characterised to date: TRP vanilloid (TRPV) 1 and 2 are activated by painful levels of heat, TRPV3 and 4 respond to non-painful warmth, TRP melastatin 8 is activated by non-painful cool temperatures, while TRP ankyrin (TRPA) 1 is activated by painful cold. The thermal thresholds of many thermo-TRPs are known to be modulated by extracellular mediators, released by tissue damage or inflammation, such as bradykinin, PG and growth factors. There have been intensive efforts recently to develop antagonists of thermo-TRP channels, particularly of the noxious thermal sensors TRPV1 and TRPA1. Blockers of these channels are likely to have therapeutic uses as novel analgesics, but may also cause unacceptable side effects. Controlling the modulation of thermo-TRPs by inflammatory mediators may be a useful alternative strategy in developing novel analgesics.

Current perspectives on the modulation of thermo-TRP channels: new advances and therapeutic implications.

The thermo transient receptor potential (TRP) ion channels, a recently discovered family of ion channels activated by temperature, are expressed in primary sensory nerve terminals, where they provide information regarding thermal changes in the environment. Six thermo-TRPs have been characterized to date: TRPV1-4, which respond to different levels of warmth and heat, and TRPM8 and TRPA1, which respond to cool temperatures. We review the current state of knowledge of thermo-TRPs, and of the modulation of their thermal thresholds by a range of inflammatory mediators. Blockers of these channels are likely to have therapeutic uses as novel analgesics but may also cause unacceptable side effects. Controlling the modulation of thermo-TRPs by inflammatory mediators may be a useful alternative strategy in developing novel analgesics.

Mitochondrial free [Ca2+] levels and the permeability transition.

Mitochondrial Ca(2+) activates many processes, from mitochondrial metabolism to opening of the permeability transition pore (PTP) and apoptosis. However, there is considerable controversy regarding the free mitochondrial [Ca(2+)] ([Ca(2+)](M)) levels that can be attained during cell activation or even in mitochondrial preparations. Studies using fluorescent dyes (rhod-2 or similar), have reported that phosphate precipitation precludes [Ca(2+)](M) from increasing above 2-3 microM. Instead, using low-Ca(2+)-affinity aequorin probes, we have measured [Ca(2+)](M) values more than two orders of magnitude higher. We confirm here these values by making a direct in situ calibration of mitochondrial aequorin, and we show that a prolonged increase in [Ca(2+)](M) to levels of 0.5-1mM was actually observed at any phosphate concentration (0-10mM) during continuous perfusion of 3.5-100 microM Ca(2+)-buffers. In spite of this high and maintained (>10 min) [Ca(2+)](M), mitochondria retained functionality and the [Ca(2+)](M) drop induced by a protonophore was fully reversible. In addition, this high [Ca(2+)](M) did not induce PTP opening unless additional activators (phenyl arsine oxide, PAO) were present. PAO induced a rapid, concentration-dependent and irreversible drop in [Ca(2+)](M). In conclusion [Ca(2+)](M) levels of 0.5-1mM can be reached and maintained for prolonged periods (>10 min) in phosphate-containing medium, and massive opening of PTP requires additional pore activators.

Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules.

The secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50-100 microM Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular[Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.

Endoplasmic reticulum stress in the proapoptotic action of edelfosine in solid tumor cells.

The endoplasmic reticulum (ER) has been posited as a potential anticancer target. The synthetic antitumor alkyl-lysophospholipid analogue edelfosine accumulates in the ER of solid tumor cells. This ER accumulation of the drug leads to the inhibition of phosphatidylcholine and protein synthesis, G(2)-M arrest, depletion of ER-stored Ca(2+), Bax up-regulation and activation, transcriptional factor growth arrest and DNA damage-inducible gene 153 up-regulation, caspase-4 and caspase-8 activation, and eventually to apoptosis. Edelfosine prompted ER stress apoptotic signaling, but not the survival unfolded protein response. Edelfosine also induced persistent c-Jun NH(2)-terminal kinase (JNK) activation. Gene transfer-mediated overexpression of apoptosis signal-regulating kinase 1, which plays a crucial role in ER stress, enhanced edelfosine-induced JNK activation and apoptosis. Inhibition of JNK, caspase-4, or caspase-8 activation diminished edelfosine-induced apoptosis. Edelfosine treatment led to the generation of the p20 caspase-8 cleavage fragment of BAP31, directing proapoptotic signals between the ER and the mitochondria. bax(-/-)bak(-/-) double-knockout cells fail to undergo edelfosine-induced ER-stored Ca(2+) release and apoptosis. Wild-type and bax(-/-)bak(-/-) cells showed similar patterns of phosphatidylcholine and protein synthesis inhibition, despite their differences in drug sensitivity. Thus, edelfosine-induced apoptosis is dependent on Bax/Bak-mediated ER-stored Ca(2+) release, but phosphatidylcholine and protein synthesis inhibition is not critical. Transfection-enforced expression of Bcl-X(L), which localizes specifically in mitochondria, prevented apoptosis without inhibiting ER-stored Ca(2+) release. These data reveal that edelfosine induces an ER stress response in solid tumor cells, providing novel insights into the edelfosine-mediated antitumor activity. Our data also indicate that mitochondria are indispensable for this edelfosine-induced cell death initiated by ER stress.

Modulation of Ca(2+) release and Ca(2+) oscillations in HeLa cells and fibroblasts by mitochondrial Ca(2+) uniporter stimulation.

The recent availability of activators of the mitochondrial Ca(2+) uniporter allows direct testing of the influence of mitochondrial Ca(2+) uptake on the overall Ca(2+) homeostasis of the cell. We show here that activation of mitochondrial Ca(2+) uptake by 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca(2+) release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na(+)-Ca(2+) exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca(2+) uptake and release from mitochondria control the ability of local Ca(2+) microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). In addition, the ability of mitochondria to control Ca(2+) release from the ER allows them to modulate cytosolic Ca(2+) oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca(2+) oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca(2+) oscillations are exquisitely sensitive to the rates of mitochondrial Ca(2+) uptake and release, which precisely control the size of the local Ca(2+) microdomains around InsP(3)Rs and thus the ability to produce feedback activation or inhibition of Ca(2+) release.

The mitochondrial Na+/Ca2+ exchanger plays a key role in the control of cytosolic Ca2+ oscillations.

There is increasing evidence that mitochondria play an important role in the control of cytosolic Ca2+ signaling. We show here that the main mitochondrial Ca2+-exit pathway, the mitochondrial Na+/Ca2+ exchanger, controls the pattern of cytosolic Ca2+ oscillations in non-excitable cells. In HeLa cells, the inhibitor of the mitochondrial Na+/Ca2+ exchanger CGP37157 changed the pattern of the oscillations induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the average Ca2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent, disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial [Ca2+] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated histamine-induced Ca2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca2+ peak induced by histamine in HeLa cells. Our results suggest that the mitochondrial Na+/Ca2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca2+ release and in that way controls cytosolic Ca2+ oscillations.

Calcium dynamics in catecholamine-containing secretory vesicles.

We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic [Ca(2+)] and decreased secretory granule [Ca(2+)] ([Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic [Na(+)] (0-140 mM) or [Ca(2+)] (0-10 microM) did not modify either ([Ca(2+)](SG)). Instead, [Ca(2+)](SG) was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca(2+)](SG), while cytosolic alcalinization reversibly increased [Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on [Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers [Ca(2+)](SG) changes in the long-term but allows highly dynamic free [Ca(2+)](SG) changes to occur in seconds or minutes.

Modulation of mitochondrial Ca(2+) uptake by estrogen receptor agonists and antagonists.

Ca(2+) uptake by mitochondria is a key element in the control of cellular Ca(2+) homeostasis and Ca(2+)-dependent phenomena. It has been known for many years that this Ca(2+) uptake is mediated by the mitochondrial Ca(2+) uniporter, a specific Ca(2+) channel of the inner mitochondrial membrane. We have shown previously that this channel is strongly activated by a series of natural phytoestrogenic flavonoids. We show here that several agonists and antagonists of estrogen receptors (ERs) also modulate the activity of the uniporter. The specific alpha-ER agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was the strongest activator, increasing the rate of mitochondrial Ca(2+) uptake in permeabilized HeLa cells by 10-fold at 2 microM. Consistently, PPT largely increased the histamine-induced mitochondrial [Ca(2+)] peak and reduced the cytosolic one. Diethylstilbestrol and 17-beta-estradiol (but not 17-alpha-estradiol) were active at pharmacological concentrations while the beta-estrogen-receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) was little effective. The ER modulators tamoxifen and 4-hydroxy-tamoxifen inhibited mitochondrial Ca(2+) uptake (IC(50) 2.5+/-1.5 and 2.5+/-1.4 microM, mean+/-s.d., respectively) both in the presence and in the absence of PPT, but raloxifene and the pure estrogen antagonist ICI 182,780 produced no effect. Activation by PPT was immediate and inhibition by tamoxifen or 4-hydroxy-tamoxifen required only 5 min to reach maximum. Tamoxifen did not modify mitochondrial membrane potential and PPT induced a slow mitochondrial depolarization at higher concentrations than those required to activate mitochondrial Ca(2+) uptake. These results suggest that some kind of ER or related protein located in mitochondria controls the activity of the Ca(2+) uniporter by a nongenomic mechanism. This novel mechanism of action of estrogen agonists and antagonists can provide a new interpretation for several previously reported effects of these compounds.

Direct activation of the mitochondrial calcium uniporter by natural plant flavonoids.

During cell activation, mitochondria play an important role in Ca2+ homoeostasis due to the presence of a fast and specific Ca2+ channel in its inner membrane, the mitochondrial Ca2+ uniporter. This channel allows mitochondria to buffer local cytosolic [Ca2+] changes and controls the intramitochondrial Ca2+ levels, thus modulating a variety of phenomena from respiratory rate to apoptosis. We have described recently that SB202190, an inhibitor of p38 MAPK (mitogen-activated protein kinase), strongly activated the uniporter. We show in the present study that a series of natural plant flavonoids, widely distributed in foods, produced also a strong stimulation of the mitochondrial Ca2+ uniporter. This effect was of the same magnitude as that induced by SB202190 (an approx. 20-fold increase in the mitochondrial Ca2+ uptake rate), developed without measurable delay and was rapidly reversible. In intact cells, the mitochondrial Ca2+ peak induced by histamine was also largely increased by the flavonoids. Stimulation of the uniporter by either flavonoids or SB202190 did not require ATP, suggesting a direct effect on the uniporter or an associated protein which is not mediated by protein phosphorylation. The most active compound, kaempferol, increased the rate of mitochondrial Ca2+ uptake by 85+/-15% (mean+/-S.E.M., n=4) and the histamine-induced mitochondrial Ca2+ peak by 139+/-19% (mean+/-S.E.M., n=5) at a concentration of 1 microM. Given that flavonoids can reach this concentration range in plasma after ingestion of flavonoid-rich food, these compounds could be modulating the uniporter under physiological conditions.