Hibiki Kawamata

Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice

Existing protocols for the neural differentiation of mouse embryonic stem (ES) cells require extended in vitro culture, yield variable differentiation results or are limited to the generation of selected neural subtypes. Here we provide a set of coculture conditions that allows rapid and efficient derivation of most central nervous system phenotypes. The fate of both fertilization- and nuclear transfer–derived ES (ntES) cells was directed selectively into neural stem cells, astrocytes, oligodendrocytes or neurons. Specific differentiation into γ-aminobutyric acid (GABA), dopamine, serotonin or motor neurons was achieved by defining conditions to induce forebrain, midbrain, hindbrain and spinal cord identity. Neuronal function of ES cell–derived dopaminergic neurons was shown in vitro by electron microscopy, measurement of neurotransmitter release and intracellular recording. Furthermore, transplantation of ES and ntES cell–derived dopaminergic neurons corrected the phenotype of a mouse model of Parkinson disease, demonstrating an in vivo application of therapeutic cloning in neural disease.

Overexpression of CCS in G93A-SOD1 mice leads to accelerated neurological deficits with severe mitochondrial pathology.

Cu, Zn superoxide dismutase (SOD1) has been detected within spinal cord mitochondria of mutant SOD1 transgenic mice, a model of familial ALS. The copper chaperone for SOD1 (CCS) provides SOD1 with copper, facilitates the conversion of immature apo-SOD1 to a mature holoform, and influences in yeast the cytosolic/mitochondrial partitioning of SOD1. To determine how CCS affects G93A-SOD1-induced disease, we generated transgenic mice overexpressing CCS and crossed them to G93A-SOD1 or wild-type SOD1 transgenic mice. Both CCS transgenic mice and CCS/wild-type-SOD1 dual transgenic mice are neurologically normal. In contrast, CCS/G93A-SOD1 dual transgenic mice develop accelerated neurological deficits, with a mean survival of 36 days, compared with 242 days for G93A-SOD1 mice. Immuno-EM and subcellular fractionation studies on the spinal cord show that G93A-SOD1 is enriched within mitochondria in the presence of CCS overexpression. Our results indicate that CCS overexpression in G93A-SOD1 mice produces severe mitochondrial pathology and accelerates disease course.

Mutant SOD1 in neuronal mitochondria causes toxicity and mitochondrial dynamics abnormalities

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by motor neuron degeneration. Mutations in Cu,Zn-superoxide dismutase (SOD1) are responsible for 20% of familial ALS cases via a toxic gain of function. In mutant SOD1 transgenic mice, mitochondria of spinal motor neurons develop abnormal morphology, bioenergetic defects and degeneration, which are presumably implicated in disease pathogenesis. SOD1 is mostly a cytosolic protein, but a substantial portion is associated with organelles, including mitochondria, where it localizes predominantly in the intermembrane space (IMS). However, whether mitochondrial mutant SOD1 contributes to disease pathogenesis remains to be elucidated. We have generated NSC34 motor neuronal cell lines expressing wild-type or mutant SOD1 containing a cleavable IMS targeting signal to directly investigate the pathogenic role of mutant SOD1 in mitochondria. We show that mitochondrially-targeted SOD1 localizes to the IMS, where it is enzymatically active. We prove that mutant IMS-targeted SOD1 causes neuronal toxicity under metabolic and oxidative stress conditions. Furthermore, we demonstrate for the first time neurite mitochondrial fragmentation and impaired mitochondrial dynamics in motor neurons expressing IMS mutant SOD1. These defects are associated with impaired maintenance of neuritic processes. Our findings demonstrate that mutant SOD1 localized in the IMS is sufficient to determine mitochondrial abnormalities and neuronal toxicity, and contributes to ALS pathogenesis.

Different regulation of wild-type and mutant Cu,Zn superoxide dismutase localization in mammalian mitochondria

The antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) is predominantly localized in the cytosol, but it is also found in mitochondria. Studies in yeast suggest that apoSOD1 is imported into mitochondria and trapped inside by folding and maturation, which is facilitated by its copper chaperone for SOD1 (CCS). Here, we show that in mammalian cells, SOD1 mitochondrial localization is dictated by its folding state, which is modulated by several interconnected factors. First, the intracellular distribution of CCS determines SOD1 partitioning in cytosol and mitochondria: CCS localization in the cytosol prevents SOD1 mitochondrial import, whereas CCS in mitochondria increases it. Second, the Mia40/Erv1 pathway for import of small intermembrane space proteins participates in CCS mitochondrial import in a respiratory chain-dependent manner. Third, CCS mitochondrial import is regulated by oxygen concentration: high (20%) oxygen prevents import, whereas physiological (6%) oxygen promotes it. Therefore, SOD1 localization responds to changes in environmental conditions following redistribution of CCS, which operates as an oxygen sensor. Fourth, all of the cysteine residues in human SOD1 are critical for its retention in mitochondria due to their involvement in intramolecular disulfide bonds and in the interaction with CCS. Mutations in SOD1 are associated with autosomal dominant familial amyotrophic lateral sclerosis. Like the wild-type protein, mutant SOD1 localizes to mitochondria, where it induces bioenergetic defects. We find that the physiological regulation of mitochondrial localization is either inefficient or absent in SOD1 pathogenic mutants. We propose misfolding and aggregation of these mutants that trap them inside mitochondria.

Mitochondrial dysfunction and intracellular calcium dysregulation in ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that affects the aging population. A progressive loss of motor neurons in the spinal cord and brain leads to muscle paralysis and death. As in other common neurodegenerative diseases, aging-related mitochondrial dysfunction is increasingly being considered among the pathogenic factors. Mitochondria are critical for cell survival: they provide energy to the cell, buffer intracellular calcium, and regulate apoptotic cell death. Whether mitochondrial abnormalities are a trigger or a consequence of the neurodegenerative process and the mechanisms whereby mitochondrial dysfunction contributes to disease are not clear yet. Calcium homeostasis is a major function of mitochondria in neurons, and there is ample evidence that intracellular calcium is dysregulated in ALS. The impact of mitochondrial dysfunction on intracellular calcium homeostasis and its role in motor neuron demise are intriguing issues that warrants in depth discussion. Clearly, unraveling the causal relationship between mitochondrial dysfunction, calcium dysregulation, and neuronal death is critical for the understanding of ALS pathogenesis. In this review, we will outline the current knowledge of various aspects of mitochondrial dysfunction in ALS, with a special emphasis on the role of these abnormalities on intracellular calcium handling.

Neurotoxicity and behavioral deficits associated with Septin 5 accumulation in dopaminergic neurons

Septin 5, a parkin substrate, is a vesicle‐ and membrane‐associated protein that plays a significant role in inhibiting exocytosis. The regulatory function of Septin 5 in dopaminergic (DAergic) neurons of substantia nigra (SN), maintained at relatively low levels, has not yet been delineated. As loss of function mutations of parkin are the principal cause of a familial Parkinsons disease, a prevailing hypothesis is that the loss of parkin activity results in accumulation of Septin 5 which confers neuron‐specific toxicity in SN‐DAergic neurons. In vitro and in vivo models were used to support this hypothesis. In our well‐characterized DAergic SN4741 cell model, acute accumulation of elevated levels of Septin 5, but not synphilin‐1 (another parkin substrate), resulted in cytotoxic cell death that was markedly reduced by parkin co‐transfection. A transgenic mouse model expressing a dominant negative parkin mutant accumulated moderate levels of Septin 5 in SN‐DAergic neurons. These mice acquired a progressive l‐DOPA responsive motor dysfunction that developed despite a 25% higher than normal level of striatal dopamine (DA) and no apparent loss of DAergic neurons. The phenotype of this animal, increased striatal dopamine and reduced motor function, was similar to that observed in parkin knockout animals, suggesting a common DAergic alteration. These data suggest that a threshold level of Septin 5 accumulation is required for DAergic cell loss and that l‐DOPA‐responsive motor deficits can occur even in the presence of elevated DA.

Abnormal Intracellular Calcium Signaling and SNARE-Dependent Exocytosis Contributes to SOD1G93A Astrocyte-Mediated Toxicity in Amyotrophic Lateral Sclerosis

Motor neurons are progressively and predominantly degenerated in ALS, which is not only induced by multiple intrinsic pathways but also significantly influenced by the neighboring glial cells. In particular, astrocytes derived from the SOD1 mutant mouse model of ALS or from human familial or sporadic ALS patient brain tissue directly induce motor neuron death in culture; however, the mechanisms of pathological astroglial secretion remain unclear. Here we investigated abnormal calcium homeostasis and altered exocytosis in SOD1G93A astrocytes. We found that purinergic stimulation induces excess calcium release from the ER stores in SOD1G93A astrocytes, which results from the abnormal ER calcium accumulation and is independent of clearance mechanisms. Furthermore, pharmacological studies suggested that store-operated calcium entry (SOCE), a calcium refilling mechanism responsive to ER calcium depletion, is enhanced in SOD1G93A astrocytes. We found that oxidant-induced increased S-glutathionylation and calcium-independent puncta formation of the ER calcium sensor STIM1 underlies the abnormal SOCE response in SOD1G93A astrocytes. Enhanced SOCE contributes to ER calcium overload in SOD1G93A astrocytes and excess calcium release from the ER during ATP stimulation. In addition, ER calcium release induces elevated ATP release from SOD1G93A astrocytes, which can be inhibited by the overexpression of dominant-negative SNARE. Selective inhibition of exocytosis in SOD1G93A astrocytes significantly prevents astrocyte-mediated toxicity to motor neurons and delays disease onset in SOD1G93A mice. Our results characterize a novel mechanism responsible for calcium dysregulation in SOD1G93A astrocytes and provide the first in vivo evidence that astrocyte exocytosis contributes to the pathogenesis of ALS.

Mitochondria and endoplasmic reticulum crosstalk in amyotrophic lateral sclerosis

Physical and functional interactions between mitochondria and the endoplasmic reticulum (ER) are crucial for cell life. These two organelles are intimately connected and collaborate to essential processes, such as calcium homeostasis and phospholipid biosynthesis. The connections between mitochondria and endoplasmic reticulum occur through structures named mitochondria associated membranes (MAMs), which contain lipid rafts and a large number of proteins, many of which serve multiple functions at different cellular sites. Growing evidence strongly suggests that alterations of ER-mitochondria interactions are involved in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a devastating and rapidly fatal motor neuron disease. Mutations in proteins that participate in ER-mitochondria interactions and MAM functions are increasingly being associated with genetic forms of ALS and other neurodegenerative diseases. This evidence strongly suggests that, rather than considering the two organelles separately, a better understanding of the disease process can derive from studying the alterations in their crosstalk. In this review we discuss normal and pathological ER-mitochondria interactions and the evidence that link them to ALS.

The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation

A decline in α‐ketoglutarate dehydrogenase complex (KGDHC) activity has been associated with neurodegeneration. Provision of succinyl‐CoA by KGDHC is essential for generation of matrix ATP (or GTP) by substrate‐level phosphorylation catalyzed by succinyl‐CoA ligase. Here, we demonstrate ATP consumption in respiration‐impaired isolated and in situ neuronal somal mitochondria from transgenic mice with a deficiency of either dihydrolipoyl succinyltransferase (DLST) or dihydrolipoyl dehydrogenase (DLD) that exhibit a 20–48% decrease in KGDHC activity. Import of ATP into the mitochondrial matrix of transgenic mice was attributed to a shift in the reversal potential of the adenine nucleotide translocase toward more negative values due to diminished matrix substrate‐level phosphorylation, which causes the translocase to reverse prematurely. Immunoreactivity of all three subunits of succinyl‐CoA ligase and maximal enzymatic activity were unaffected in transgenic mice as compared to wild‐type littermates. Therefore, decreased matrix substrate‐level phosphorylation was due to diminished provision of succinyl‐CoA. These results were corroborated further by the finding that mitochondria from wild‐type mice respiring on substrates supporting substrate‐level phosphorylation exhibited ~30% higher ADP‐ATP exchange rates compared to those obtained from DLST+/– or DLD+/– littermates. We propose that KGDHC‐associated pathologies are a consequence of the inability of respiration‐impaired mitochondria to rely on “in‐house” mitochondrial ATP reserves.—Kiss, G., Konrad, C., Doczi, J., Starkov, A. A., Kawamata, H., Manfredi, G., Zhang, S. F., Gibson, G. E., Beal, M. F., Adam‐Vizi, V., Chinopoulos, C. The negative impact of α‐ketoglutarate dehydrogenase complex deficiency on matrix substrate‐level phosphorylation. FASEB J. 27, 2392–2406 (2013). www.fasebj.org

adPEO mutations in ANT1 impair ADP–ATP translocation in muscle mitochondria

Mutations in the heart and muscle isoform of adenine nucleotide translocator 1 (ANT1) are associated with autosomal-dominant progressive external opthalmoplegia (adPEO) clinically characterized by exercise intolerance, ptosis and muscle weakness. The pathogenic mechanisms underlying the mitochondrial myopathy caused by ANT1 mutations remain largely unknown. In yeast, expression of ANT1 carrying mutations corresponding to the human adPEO ones causes a wide range of mitochondrial abnormalities. However, functional studies of ANT1 mutations in mammalian cells are lacking, because they have been hindered by the fact that ANT1 expression leads to apoptotic cell death in commonly utilized replicating cell lines. Here, we successfully express functional ANT1 in differentiated mouse myotubes, which naturally contain high levels of ANT1, without causing cell death. We demonstrate, for the first time in these disease-relevant mammalian cells, that mutant human ANT1 causes dominant mitochondrial defects characterized by decreased ADP-ATP exchange function and abnormal translocator reversal potential. These abnormalities are not due to ANT1 loss of function, because knocking down Ant1 in myotubes causes functional changes different from ANT1 mutants. Under certain physiological conditions, mitochondria consume ATP to maintain membrane potential by reversing the ADP-ATP transport. The modified properties of mutant ANT1 can be responsible for disease pathogenesis in adPEO, because exchange reversal occurring at higher than normal membrane potential can cause excessive energy depletion and nucleotide imbalance in ANT1 mutant muscle cells.