Doctor of Philosophy, Karolinska Institutet (2012)
Doctor of Medicine, Uppsala Universitet (2005)
Thomas Sudhof, Postdoctoral Research Mentor
Mitochondrial dysfunction is implicated in aging and degenerative disorders such as Parkinson's disease (PD). Continuous fission and fusion of mitochondria shapes their morphology and is essential to maintain oxidative phosphorylation. Loss-of-function mutations in PTEN-induced kinase1 (PINK1) or Parkin cause a recessive form of PD and have been linked to altered regulation of mitochondrial dynamics. More specifically, the E3 ubiquitin ligase Parkin has been shown to directly regulate the levels of mitofusin 1 (Mfn1) and Mfn2, two homologous outer membrane large GTPases that govern mitochondrial fusion, but it is not known whether this is of relevance for disease pathophysiology. Here, we address the importance of Mfn1 and Mfn2 in midbrain dopamine (DA) neurons in vivo by characterizing mice with DA neuron-specific knockout of Mfn1 or Mfn2. We find that Mfn1 is dispensable for DA neuron survival and motor function. In contrast, Mfn2 DA neuron-specific knockouts develop a fatal phenotype with reduced weight, locomotor disturbances and death by 7 weeks of age. Mfn2 knockout DA neurons have spherical and enlarged mitochondria with abnormal cristae and impaired respiratory chain function. Parkin does not translocate to these defective mitochondria. Surprisingly, Mfn2 DA neuron-specific knockout mice have normal numbers of midbrain DA neurons, whereas there is a severe loss of DA nerve terminals in the striatum, accompanied by depletion of striatal DA levels. These results show that Mfn2, but not Mfn1, is required for axonal projections of DA neurons in vivo.
View details for DOI 10.1093/hmg/dds352
View details for Web of Science ID 000310369000002
View details for PubMedID 22914740
A variety of observations support the hypothesis that deficiency of complex I [reduced nicotinamide-adenine dinucleotide (NADH):ubiquinone oxidoreductase] of the mitochondrial respiratory chain plays a role in the pathophysiology of Parkinson's disease (PD). However, recent data from a study using mice with knockout of the complex I subunit NADH:ubiquinone oxidoreductase iron-sulfur protein 4 (Ndufs4) has challenged this concept as these mice show degeneration of non-dopamine neurons. In addition, primary dopamine (DA) neurons derived from such mice, reported to lack complex I activity, remain sensitive to toxins believed to act through inhibition of complex I. We tissue-specifically disrupted the Ndufs4 gene in mouse heart and found an apparent severe deficiency of complex I activity in disrupted mitochondria, whereas oxidation of substrates that result in entry of electrons at the level of complex I was only mildly reduced in intact isolated heart mitochondria. Further analyses of detergent-solubilized mitochondria showed the mutant complex I to be unstable but capable of forming supercomplexes with complex I enzyme activity. The loss of Ndufs4 thus causes only a mild complex I deficiency in vivo. We proceeded to disrupt Ndufs4 in midbrain DA neurons and found no overt neurodegeneration, no loss of striatal innervation and no symptoms of Parkinsonism in tissue-specific knockout animals. However, DA homeostasis was abnormal with impaired DA release and increased levels of DA metabolites. Furthermore, Ndufs4 DA neuron knockouts were more vulnerable to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Taken together, these findings lend in vivo support to the hypothesis that complex I deficiency can contribute to the pathophysiology of PD.
View details for DOI 10.1093/hmg/ddr537
View details for Web of Science ID 000300242000010
View details for PubMedID 22090423
Degeneration of midbrain dopamine neurons causes the striatal dopamine deficiency responsible for the hallmark motor symptoms of Parkinson's disease (PD). Intraparenchymal delivery of neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), is a possible future therapeutic approach. In animal PD models, GDNF can both ameliorate neurodegeneration and promote recovery of the dopamine system following a toxic insults. However, clinical studies have generated mixed results and GDNF has not been efficacious in genetic animal models based on alpha-synuclein overexpression. We have tested the response to GDNF in a genetic mouse PD model with progressive degeneration of dopamine neurons caused by mitochondrial impairment. We find that GDNF, delivered to the striatum by either an adeno-associated virus or via mini-osmotic pumps, partially alleviates the progressive motor symptoms without modifying the rate of neurodegeneration. These behavioral changes are accompanied by increased levels of dopamine in the midbrain, but not in striatum. At high levels, GDNF may instead reduce striatal dopamine levels. These results demonstrate the therapeutic potential of GDNF in a progressively impaired dopamine system.
View details for PubMedID 23051605
Mitochondrial dysfunction is heavily implicated in Parkinson disease (PD) as exemplified by the finding of an increased frequency of respiratory chain-deficient dopamine (DA) neurons in affected patients. An inherited form of PD is caused by impaired function of Parkin, an E3 ubiquitin ligase reported to translocate to defective mitochondria in vitro to facilitate their clearance. We have developed a reporter mouse to assess mitochondrial morphology in DA neurons in vivo and show here that respiratory chain deficiency leads to fragmentation of the mitochondrial network and to the formation of large cytoplasmic bodies derived from mitochondria. Surprisingly, the dysfunctional mitochondria do not recruit Parkin in vivo, and neither the clearance of defective mitochondria nor the neurodegeneration phenotype is affected by the absence of Parkin. We also show that anterograde axonal transport of mitochondria is impaired in respiratory chain-deficient DA neurons, leading to a decreased supply of mitochondria to the axonal terminals.
View details for DOI 10.1073/pnas.1103295108
View details for Web of Science ID 000293385700078
View details for PubMedID 21768369
LRPPRC (also called LRP130) is an RNA-binding pentatricopeptide repeat protein. LRPPRC has been recognized as a mitochondrial protein, but has also been shown to regulate nuclear gene transcription and to bind specific RNA molecules in both the nucleus and the cytoplasm. We here present a bioinformatic analysis of the LRPPRC primary sequence, which reveals that orthologs to the LRPPRC gene are restricted to metazoan cells and that all of the corresponding proteins contain mitochondrial targeting signals. To address the subcellular localization further, we have carefully analyzed LRPPRC in mammalian cells and identified a single isoform that is exclusively localized to mitochondria. The LRPPRC protein is imported to the mitochondrial matrix and its mitochondrial targeting sequence is cleaved upon entry.
View details for DOI 10.1016/j.bbrc.2010.07.019
View details for Web of Science ID 000280901300025
View details for PubMedID 20633537
The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), specific to neurons and muscle, supplies aspartate to the cytosol and, as a component of the malate-aspartate shuttle, enables mitochondrial oxidation of cytosolic NADH, thought to be important in providing energy for neurons in the central nervous system. We describe AGC1 deficiency, a novel syndrome characterized by arrested psychomotor development, hypotonia, and seizures in a child with a homozygous missense mutation in the solute carrier family 25, member 12, gene SLC25A12, which encodes the AGC1 protein. Functional analysis of the mutant AGC1 protein showed abolished activity. The child had global hypomyelination in the cerebral hemispheres, suggesting that impaired efflux of aspartate from neuronal mitochondria prevents normal myelin formation.
View details for Web of Science ID 000268443900009
View details for PubMedID 19641205
LRRK2, alpha-synuclein, UCH-L1 and DJ-1 are implicated in the etiology of Parkinson's disease. We show for the first time that increase in striatal alpha-synuclein levels induce increased Lrrk2 mRNA levels while Dj-1 and Uch-L1 are unchanged. We also demonstrate that a mouse strain lacking the dopamine signaling molecule DARPP-32 has significantly reduced levels of both Lrrk2 and alpha-synuclein, while mice carrying a disabling mutation of the DARPP-32 phosphorylation site T34A or lack alpha-synuclein do not show any changes. To test if striatal dopamine depletion influences Lrrk2 or alpha-synuclein expression, we used the neurotoxin 6-hydroxydopamine in rats and MitoPark mice in which there is progressive degeneration of dopamine neurons. Because striatal Lrrk2 and alpha-synuclein levels were not changed by dopamine depletion, we conclude that Lrrk2 and alpha-synuclein mRNA levels are possibly co-regulated, but they are not influenced by striatal dopamine levels.
View details for DOI 10.1016/j.mcn.2008.08.001
View details for Web of Science ID 000261541600009
View details for PubMedID 18790059
Heteroplasmic mitochondrial DNA (mtDNA) mutations (mutations present only in a subset of cellular mtDNA copies) arise de novo during the normal ageing process or may be maternally inherited in pedigrees with mitochondrial disease syndromes. A pathogenic mtDNA mutation causes respiratory chain deficiency only if the fraction of mutated mtDNA exceeds a certain threshold level. These mutations often undergo apparently random mitotic segregation and the levels of normal and mutated mtDNA can vary considerably between cells of the same tissue. In human ageing, segregation of somatic mtDNA mutations leads to mosaic respiratory chain deficiency in a variety of tissues, such as brain, heart and skeletal muscle. A similar pattern of mutation segregation with mosaic respiratory chain deficiency is seen in patients with mitochondrial disease syndromes caused by inherited pathogenic mtDNA mutations. We have experimentally addressed the role of mosaic respiratory chain deficiency in ageing and mitochondrial disease by creating mouse chimeras with a mixture of normal and respiratory chain-deficient neurons in cerebral cortex. We report here that a low proportion (>20%) of respiratory chain-deficient neurons in the forebrain are sufficient to cause symptoms, whereas premature death of the animal occurs only if the proportion is high (>60-80%). The presence of neurons with normal respiratory chain function does not only prevent mortality but also delays the age at which onset of disease symptoms occur. Unexpectedly, respiratory chain-deficient neurons have adverse effect on normal adjacent neurons and induce trans-neuronal degeneration. In summary, our study defines the minimal threshold level of respiratory chain-deficient neurons needed to cause symptoms and also demonstrate that neurons with normal respiratory chain function ameliorate disease progression. Finally, we show that respiratory chain-deficient neurons induce death of normal neurons by a trans-neuronal degeneration mechanism. These findings provide novel insights into the pathogenesis of mosaic respiratory chain deficiency in ageing and mitochondrial disease.
View details for DOI 10.1093/hmg/ddn030
View details for Web of Science ID 000255523400007
View details for PubMedID 18245781
Complex I, the main entry point for electrons to the respiratory chain, is of critical importance for cellular energy homeostasis. In this issue of Cell Metabolism, Kruse and coworkers (2008) describe the first mouse knockout for a complex I structural subunit, thus advancing our understanding of complex I in disease.
View details for DOI 10.1016/j.cmet.2008.03.011
View details for Web of Science ID 000254867000002
View details for PubMedID 18396129
Mitochondrial dysfunction is implicated in the pathophysiology of Parkinson's disease (PD), a common age-associated neurodegenerative disease characterized by intraneuronal inclusions (Lewy bodies) and progressive degeneration of the nigrostriatal dopamine (DA) system. It has recently been demonstrated that midbrain DA neurons of PD patients and elderly humans contain high levels of somatic mtDNA mutations, which may impair respiratory chain function. However, clinical studies have not established whether the respiratory chain deficiency is a primary abnormality leading to inclusion formation and DA neuron death, or whether generalized metabolic abnormalities within the degenerating DA neurons cause secondary damage to mitochondria. We have used a reverse genetic approach to investigate this question and created conditional knockout mice (termed MitoPark mice), with disruption of the gene for mitochondrial transcription factor A (Tfam) in DA neurons. The knockout mice have reduced mtDNA expression and respiratory chain deficiency in midbrain DA neurons, which, in turn, leads to a parkinsonism phenotype with adult onset of slowly progressive impairment of motor function accompanied by formation of intraneuronal inclusions and dopamine nerve cell death. Confocal and electron microscopy show that the inclusions contain both mitochondrial protein and membrane components. These experiments demonstrate that respiratory chain dysfunction in DA neurons may be of pathophysiological importance in PD.
View details for DOI 10.1073/pnas.0605208103
View details for Web of Science ID 000243849900039
View details for PubMedID 17227870