Haploinsufficiency of the SLC2A1 gene and paucity of its translated product, the glucose transporter-1 (Glut1) protein, disrupt brain function and cause the neurodevelopmental disorder, Glut1 ...deficiency syndrome (Glut1 DS). There is little to suggest how reduced Glut1 causes cognitive dysfunction and no optimal treatment for Glut1 DS. We used model mice to demonstrate that low Glut1 protein arrests cerebral angiogenesis, resulting in a profound diminution of the brain microvasculature without compromising the blood-brain barrier. Studies to define the temporal requirements for Glut1 reveal that pre-symptomatic, AAV9-mediated repletion of the protein averts brain microvasculature defects and prevents disease, whereas augmenting the protein late, during adulthood, is devoid of benefit. Still, treatment following symptom onset can be effective; Glut1 repletion in early-symptomatic mutants that have experienced sustained periods of low brain glucose nevertheless restores the cerebral microvasculature and ameliorates disease. Timely Glut1 repletion may thus constitute an effective treatment for Glut1 DS.
Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through ...Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in Na+cyt (veratridine, high-K+ depolarization) and/or Ca2+cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in Ca2+mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.
Glutamate elicits Ca2+ signals and workloads that regulate neuronal fate both in physiological and pathological circumstances. Oxidative phosphorylation is required in order to respond to the ...metabolic challenge caused by glutamate. In response to physiological glutamate signals, cytosolic Ca2+ activates respiration by stimulation of the NADH malate–aspartate shuttle through Ca2+-binding to the mitochondrial aspartate/glutamate carrier (Aralar/AGC1/Slc25a12), and by stimulation of adenine nucleotide uptake through Ca2+ binding to the mitochondrial ATP-Mg/Pi carrier (SCaMC-3/Slc25a23). In addition, after Ca2+ entry into the matrix through the mitochondrial Ca2+ uniporter (MCU), it activates mitochondrial dehydrogenases. In response to pathological glutamate stimulation during excitotoxicity, Ca2+ overload, reactive oxygen species (ROS), mitochondrial dysfunction and delayed Ca2+ deregulation (DCD) lead to neuronal death. Glutamate-induced respiratory stimulation is rapidly inactivated through a mechanism involving Poly (ADP-ribose) Polymerase-1 (PARP-1) activation, consumption of cytosolic NAD+, a decrease in matrix ATP and restricted substrate supply. Glutamate-induced Ca2+-activation of SCaMC-3 imports adenine nucleotides into mitochondria, counteracting the depletion of matrix ATP and the impaired respiration, while Aralar-dependent lactate metabolism prevents substrate exhaustion. A second mechanism induced by excitotoxic glutamate is permeability transition pore (PTP) opening, which critically depends on ROS production and matrix Ca2+ entry through the MCU. By increasing matrix content of adenine nucleotides, SCaMC-3 activity protects against glutamate-induced PTP opening and lowers matrix free Ca2+, resulting in protracted appearance of DCD and protection against excitotoxicity in vitro and in vivo, while the lack of lactate protection during in vivo excitotoxicity explains increased vulnerability to kainite-induced toxicity in Aralar +/− mice. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
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•Glutamate stimulates respiration by enhancing ATP demand and by Ca2+ signaling.•Ca2+ activated Aralar and SCaMC-3 are necessary for glutamate-stimulation of respiration.•Excitotoxic PARP-1 activation causes mitochondrial ATP depletion, counteracted by SCaMC-3.•Adenine nucleotide uptake by SCaMC-3 prevents excitotoxic mitochondrial permeability transition.•Aralar and SCaMC-3 protect in vivo against kainate induced toxicity.
ARALAR/AGC1/Slc25a12, the aspartate-glutamate carrier from brain mitochondria, is the regulatory step in the malate-aspartate NADH shuttle, MAS. MAS is used to oxidize cytosolic NADH in mitochondria, ...a process required to maintain oxidative glucose utilization. The role of ARALAR was analyzed in two paradigms of glutamate-induced excitotoxicity in cortical neurons: glucose deprivation and acute glutamate stimulation. ARALAR deficiency did not aggravate glutamate-induced neuronal death in vitro, although glutamate-stimulated respiration was impaired. In contrast, the presence of L-lactate as an additional source protected against glutamate-induced neuronal death in control, but not ARALAR-deficient neurons.l-Lactate supplementation increased glutamate-stimulated respiration partially prevented the decrease in the cytosolic ATP/ADP ratio induced by glutamate and substantially diminished mitochondrial accumulation of 8-oxoguanosine, a marker of reactive oxygen species production, only in the presence, but not the absence, of ARALAR. In addition,l-lactate potentiated glutamate-induced increase in cytosolic Ca(2+), in a way independent of the presence of ARALAR. Interestingly,in vivo, the loss of half-a-dose of ARALAR in aralar(+/-)mice enhanced kainic acid-induced seizures and neuronal damage with respect to control animals, in a model of excitotoxicity in which increased L-lactate levels and L-lactate consumption have been previously proven. These results suggest that,in vivo, an inefficient operation of the shuttle in the aralar hemizygous mice prevents the protective role of L-lactate on glutamate excitotoxiciy and that the entry and oxidation of L-lactate through ARALAR-MAS pathway is required for its neuroprotective function.
Lactate now stands as a metabolite necessary for multiple functions in the brain and is an alternative energy source during excitotoxic brain injury. Here we find that the absence of a functional malate-aspartate NADH shuttle caused by aralar/AGC1 disruption causes a block in lactate utilization by neurons, which prevents the protective role of lactate on excitotoxicity, but not glutamate excitotoxicity itself. Thus, failure to use lactate is detrimental and is possibly responsible for the exacerbated in vivo excitotoxicity in aralar(+/-)mice.
GDAP1 is an outer mitochondrial membrane protein that acts as a regulator of mitochondrial dynamics. Mutations of the GDAP1 gene cause Charcot–Marie–Tooth (CMT) neuropathy. We show that GDAP1 ...interacts with the vesicle-organelle trafficking proteins RAB6B and caytaxin, which suggests that GDAP1 may participate in the mitochondrial movement within the cell. GDAP1 silencing in the SH-SY5Y cell line induces abnormal distribution of the mitochondrial network, reduces the contact between mitochondria and endoplasmic reticulum (ER) and alters the mobilization of mitochondria towards plasma membrane upon depletion of ER-Ca2+ stores. GDAP1 silencing does not affect mitochondrial Ca2+ uptake, ER-Ca2+, or Ca2+ flow from ER to mitochondria, but reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) following mobilization of ER-Ca2+ and SOCE-driven Ca2+ entry in mitochondria. Our studies suggest that the pathophysiology of GDAP1-related CMT neuropathies may be associated with abnormal distribution and movement of mitochondria throughout cytoskeleton towards the ER and subplasmalemmal microdomains, resulting in a decrease in SOCE activity and impaired SOCE-driven Ca2+ uptake in mitochondria.
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•GDAP1 participates in the cell trafficking and mobilization of mitochondria.•Silencing of GDAP1 gene affects the mitochondrial network distribution.•GDAP1 depletion reduces of Ca2+ entry through store-operated Ca2+ channels (SOCE).•Abnormal mitochondrial distribution and altered SOCE could be the basis of the GDAP1-associated CMT disease pathophysiology.
Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca(2+) signal or to Ca(2+) itself. Na(+)i, Ca(2+)i and ATPi dynamics ...in intact neurons exposed to different workloads in the absence and presence of Ca(2+) clearly showed that Ca(2+)-stimulation of coupled respiration is required to maintain ATPi levels. Ca(2+) may regulate respiration by activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or after Ca(2+) entry in mitochondria through the calcium uniporter (MCU). Two Ca(2+)-regulated mitochondrial metabolite transporters are expressed in neurons, the aspartate-glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate-aspartate shuttle, and the ATP-Mg/Pi exchanger SCaMC-3/APC2/Slc25a23, with S0.5 for Ca(2+) of 300nM and 3.4μM, respectively. The lack of SCaMC-3 results in a smaller Ca(2+)-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca(2+)-dependent responses to all workloads: a reduction of about 65-70% in the response to the high workload imposed by veratridine, and completely suppression of the OCR responses to moderate (K(+)-depolarization) and small (carbachol) workloads, effects reverted by pyruvate supply. For K(+)-depolarization, this occurs in spite of the presence of large Ca(2+)mit signals and increased formation of mitochondrial NAD(P)H. These results show that ARALAR-MAS is a major contributor of Ca(2+)-stimulated respiration in neurons by providing increased pyruvate supply to mitochondria. In its absence and under moderate workloads, matrix Ca(2+) is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions. This article was invited for a Special Issue entitled: 18th European Bioenergetic Conference.
Glutamate excitotoxicity is caused by sustained activation of neuronal NMDA receptors causing a large Ca(2+) and Na(+) influx, activation of poly(ADP ribose) polymerase-1 (PARP-1), and delayed Ca(2+) ...deregulation. Mitochondria undergo early changes in membrane potential during excitotoxicity, but their precise role in these events is still controversial. Using primary cortical neurons derived from mice, we show that NMDA exposure results in a rapid fall in mitochondrial ATP in neurons deficient in SCaMC-3/Slc25a23, a Ca(2+)-regulated mitochondrial ATP-Mg/Pi carrier. This fall is associated with blunted increases in respiration and a delayed decrease in cytosolic ATP levels, which are prevented by PARP-1 inhibitors or by SCaMC-3 activity promoting adenine nucleotide uptake into mitochondria. SCaMC-3 KO neurons show an earlier delayed Ca(2+) deregulation, and SCaMC-3-deficient mitochondria incubated with ADP or ATP-Mg had reduced Ca(2+) retention capacity, suggesting a failure to maintain matrix adenine nucleotides as a cause for premature delayed Ca(2+) deregulation. SCaMC-3 KO neurons have higher vulnerability to in vitro excitotoxicity, and SCaMC-3 KO mice are more susceptible to kainate-induced seizures, showing that early PARP-1-dependent fall in mitochondrial ATP levels, counteracted by SCaMC-3, is an early step in the excitotoxic cascade.
GDAP1 is an outer mitochondrial membrane protein involved in Charcot-Marie-Tooth (CMT) disease. Lack of GDAP1 gives rise to altered mitochondrial networks and endoplasmic reticulum (ER)-mitochondrial ...interactions resulting in a decreased ER-Ca
levels along with a defect on store-operated calcium entry (SOCE) related to a misallocation of mitochondria to subplasmalemmal sites. The defect on SOCE is mimicked by MCU silencing or mitochondrial depolarization, which prevent mitochondrial calcium uptake. Ca
release from de ER and Ca
inflow through SOCE in neuroblastoma cells result in a Ca
-dependent upregulation of respiration which is blunted in GDAP1 silenced cells. Reduced SOCE in cells with CMT recessive missense mutations in the α-loop of GDAP1, but not dominant mutations, was associated with smaller SOCE-stimulated respiration. These cases of GDAP1 deficiency also resulted in a decreased ER-Ca
levels which may have pathological implications. The results suggest that CMT neurons may be under energetic constraints upon stimulation by Ca
mobilization agonists and point to a potential role of perturbed mitochondria-ER interaction related to energy metabolism in forms of CMT caused by some of the recessive or null mutations of GDAP1.
The ATPase Inhibitory Factor 1 (IF1) is an inhibitor of the mitochondrial H+-ATP synthase that regulates the activity of both oxidative phosphorylation (OXPHOS) and cell death. Here, we have ...developed transgenic Tet-On and Tet-Off mice that express a mutant active form of hIF1 in the hepatocytes to restrain OXPHOS in the liver to investigate the relevance of mitochondrial activity in hepatocarcinogenesis. The expression of hIF1 promotes the inhibition of OXPHOS in both Tet-On and Tet-Off mouse models and induces a state of metabolic preconditioning guided by the activation of the stress kinases AMPK and p38 MAPK. Expression of the transgene significantly augmented proliferation and apoptotic resistance of carcinoma cells, which contributed to an enhanced diethylnitrosamine-induced liver carcinogenesis. Moreover, the expression of hIF1 also diminished acetaminophen-induced apoptosis, which is unrelated to differences in permeability transition pore opening. Mechanistically, cell survival in hIF1-preconditioned hepatocytes results from a nuclear factor-erythroid 2-related factor (Nrf2)-guided antioxidant response. The results emphasize in vivo that a metabolic phenotype with a restrained OXPHOS in the liver is prone to the development of cancer.
Abstract
GDAP1 is an outer mitochondrial membrane protein involved in Charcot-Marie-Tooth (CMT) disease. Lack of GDAP1 gives rise to altered mitochondrial networks and endoplasmic reticulum ...(ER)-mitochondrial interactions resulting in a decreased ER-Ca
2+
levels along with a defect on store-operated calcium entry (SOCE) related to a misallocation of mitochondria to subplasmalemmal sites. The defect on SOCE is mimicked by MCU silencing or mitochondrial depolarization, which prevent mitochondrial calcium uptake. Ca
2+
release from de ER and Ca
2+
inflow through SOCE in neuroblastoma cells result in a Ca
2+
-dependent upregulation of respiration which is blunted in GDAP1 silenced cells. Reduced SOCE in cells with CMT recessive missense mutations in the α-loop of GDAP1, but not dominant mutations, was associated with smaller SOCE-stimulated respiration. These cases of GDAP1 deficiency also resulted in a decreased ER-Ca
2+
levels which may have pathological implications. The results suggest that CMT neurons may be under energetic constraints upon stimulation by Ca
2+
mobilization agonists and point to a potential role of perturbed mitochondria-ER interaction related to energy metabolism in forms of CMT caused by some of the recessive or null mutations of GDAP1.
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