Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia

Mmutations in paraplegin, a putative mitochondrial metallopeptidase of the AAA family, cause an autosomal recessive form of hereditary spastic paraplegia (HSP). Here, we analyze the function of paraplegin at the cellular level and characterize the phenotypic defects of HSP patients' cells lacking this protein. We demonstrate that paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane. These two proteins form a high molecular mass complex, which we show to be aberrant in HSP fibroblasts. The loss of this complex causes a reduced complex I activity in mitochondria and an increased sensitivity to oxidant stress, which can both be rescued by exogenous expression of wild-type paraplegin. Furthermore, complementation studies in yeast demonstrate functional conservation of the human paraplegin–AFG3L2 complex with the yeast m-AAA protease and assign proteolytic activity to this structure. These results shed new light on the molecular pathogenesis of HSP and functionally link AFG3L2 to this neurodegenerative disease.

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Variable and Tissue-Specific Subunit Composition of Mitochondrial m-AAA Protease Complexes Linked to Hereditary Spastic Paraplegia

Molecular and Cellular Biology ◽

10.1128/mcb.01470-06 ◽

2006 ◽

Vol 27 (2) ◽

pp. 758-767 ◽

Cited By ~ 121

Author(s):

Mirko Koppen ◽

Metodi D. Metodiev ◽

Giorgio Casari ◽

Elena I. Rugarli ◽

Thomas Langer

Keyword(s):

Hereditary Spastic Paraplegia ◽

Tissue Specificity ◽

Axonal Degeneration ◽

Protein Quality ◽

Brain Mitochondria ◽

Spastic Paraplegia ◽

Substrate Specificities ◽

Functional Conservation ◽

Mitochondrial Inner Membrane ◽

Different Tissues

ABSTRACT The m-AAA protease, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, controls protein quality and regulates ribosome assembly, thus exerting essential housekeeping functions within mitochondria. Mutations in the m-AAA protease subunit paraplegin cause axonal degeneration in hereditary spastic paraplegia (HSP), but the basis for the unexpected tissue specificity is not understood. Paraplegin assembles with homologous Afg3l2 subunits into hetero-oligomeric complexes which can substitute for yeast m-AAA proteases, demonstrating functional conservation. The function of a third paralogue, Afg3l1 expressed in mouse, is unknown. Here, we analyze the assembly of paraplegin into m-AAA complexes and monitor consequences of paraplegin deficiency in HSP fibroblasts and in a mouse model for HSP. Our findings reveal variability in the assembly of m-AAA proteases in mitochondria in different tissues. Homo-oligomeric Afg3l1 and Afg3l2 complexes and hetero-oligomeric assemblies of both proteins with paraplegin can be formed. Yeast complementation studies demonstrate the proteolytic activity of these assemblies. Paraplegin deficiency in HSP does not result in the loss of m-AAA protease activity in brain mitochondria. Rather, homo-oligomeric Afg3l2 complexes accumulate, and these complexes can substitute for housekeeping functions of paraplegin-containing m-AAA complexes. We therefore propose that the formation of m-AAA proteases with altered substrate specificities leads to axonal degeneration in HSP.

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ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia

Human Molecular Genetics ◽

10.1093/hmg/ddx042 ◽

2017 ◽

Vol 26 (8) ◽

pp. 1432-1443 ◽

Cited By ~ 28

Author(s):

Helen M. Cooper ◽

Yang Yang ◽

Emil Ylikallio ◽

Rafil Khairullin ◽

Rosa Woldegebriel ◽

...

Keyword(s):

Membrane Protein ◽

Hereditary Spastic Paraplegia ◽

Mitochondrial Dynamics ◽

Spastic Paraplegia ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Inner Membrane Protein

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Functional conservation of human Spastin in a Drosophila model of autosomal dominant-hereditary spastic paraplegia

Human Molecular Genetics ◽

10.1093/hmg/ddq064 ◽

2010 ◽

Vol 19 (10) ◽

pp. 1883-1896 ◽

Cited By ~ 13

Author(s):

Fang Du ◽

Emily F. Ozdowski ◽

Ingrid K. Kotowski ◽

Douglas A. Marchuk ◽

Nina Tang Sherwood

Keyword(s):

Hereditary Spastic Paraplegia ◽

Autosomal Dominant ◽

Spastic Paraplegia ◽

Functional Conservation ◽

Drosophila Model

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A single external enzyme confers alternative NADH:ubiquinone oxidoreductase activity in Yarrowia lipolytica

Journal of Cell Science ◽

10.1242/jcs.112.14.2347 ◽

1999 ◽

Vol 112 (14) ◽

pp. 2347-2354 ◽

Cited By ~ 4

Author(s):

S.J. Kerscher ◽

J.G. Okun ◽

U. Brandt

Keyword(s):

Yarrowia Lipolytica ◽

Complex I ◽

Nadh:Ubiquinone Oxidoreductase ◽

Inner Mitochondrial Membrane ◽

Gene Encoding ◽

Oxidoreductase Activity ◽

Mitochondrial Inner Membrane ◽

Energy Conserving ◽

And Function ◽

Complex I Activity

NADH:ubiquinone oxidoreductases catalyse the first step within the diverse pathways of mitochondrial NADH oxidation. In addition to the energy-conserving form commonly called complex I, fungi and plants contain much simpler alternative NADH:ubiquinone oxido-reductases that catalyze the same reaction but do not translocate protons across the inner mitochondrial membrane. Little is known about the distribution and function of these enzymes. We have identified YLNDH2 as the only gene encoding an alternative NADH:ubiquinone oxidoreductase (NDH2) in the obligate aerobic yeast Yarrowia lipolytica. Cells carrying a deletion of YLNDH2 were fully viable; full inhibition by piericidin A indicated that complex I activity was the sole NADH:ubiquinone oxidoreductase activity left in the deletion strains. Studies with intact mitochondria revealed that NDH2 in Y. lipolytica is oriented towards the external face of the mitochondrial inner membrane. This is in contrast to the situation seen in Saccharomyces cerevisiae, Neurospora crassa and in green plants, where internal alternative NADH:ubiquinone oxidoreductases have been reported. Phylogenetic analysis of known NADH:ubiquinone oxidoreductases suggests that during evolution conversion of an ancestral external alternative NADH:ubiquinone oxidoreductase to an internal enzyme may have paved the way for the loss of complex I in fermenting yeasts like S. cerevisiae.

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Novel mitochondrial complex I inhibitors restore glucose-handling abilities of high-fat fed mice

Journal of Molecular Endocrinology ◽

10.1530/jme-15-0225 ◽

2016 ◽

Vol 56 (3) ◽

pp. 261-271 ◽

Cited By ~ 6

Author(s):

Darren S D Martin ◽

Siobhán Leonard ◽

Robert Devine ◽

Clara Redondo ◽

Gemma K Kinsella ◽

...

Keyword(s):

Mechanism Of Action ◽

High Fat Diet ◽

Complex I ◽

Cellular Level ◽

High Fat ◽

Insulin Sensitizer ◽

Kinase Activation ◽

Drug Of Choice ◽

Protein Kinase Activation ◽

Complex I Activity

Metformin is the main drug of choice for treating type 2 diabetes, yet the therapeutic regimens and side effects of the compound are all undesirable and can lead to reduced compliance. The aim of this study was to elucidate the mechanism of action of two novel compounds which improved glucose handling and weight gain in mice on a high-fat diet. Wildtype C57Bl/6 male mice were fed on a high-fat diet and treated with novel, anti-diabetic compounds. Both compounds restored the glucose handling ability of these mice. At a cellular level, these compounds achieve this by inhibiting complex I activity in mitochondria, leading to AMP-activated protein kinase activation and subsequent increased glucose uptake by the cells, as measured in the mouse C2C12 muscle cell line. Based on the inhibition of NADH dehydrogenase (IC50 27µmolL−1), one of these compounds is close to a thousand fold more potent than metformin. There are no indications of off target effects. The compounds have the potential to have a greater anti-diabetic effect at a lower dose than metformin and may represent a new anti-diabetic compound class. The mechanism of action appears not to be as an insulin sensitizer but rather as an insulin substitute.

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Single cell morphology distinguishes genotype and drug effect in Hereditary Spastic Paraplegia

Scientific Reports ◽

10.1038/s41598-021-95995-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Gautam Wali ◽

Shlomo Berkovsky ◽

Daniel R. Whiten ◽

Alan Mackay-Sim ◽

Carolyn M. Sue

Keyword(s):

Neurodegenerative Diseases ◽

Cell Morphology ◽

Hereditary Spastic Paraplegia ◽

High Throughput Screening ◽

Cellular Level ◽

Dependent Manner ◽

Spastic Paraplegia ◽

Causative Gene ◽

Compound Libraries ◽

The Many

AbstractA central need for neurodegenerative diseases is to find curative drugs for the many clinical subtypes, the causative gene for most cases being unknown. This requires the classification of disease cases at the genetic and cellular level, an understanding of disease aetiology in the subtypes and the development of phenotypic assays for high throughput screening of large compound libraries. Herein we describe a method that facilitates these requirements based on cell morphology that is being increasingly used as a readout defining cell state. In patient-derived fibroblasts we quantified 124 morphological features in 100,000 cells from 15 people with two genotypes (SPAST and SPG7) of Hereditary Spastic Paraplegia (HSP) and matched controls. Using machine learning analysis, we distinguished between each genotype and separated them from controls. Cell morphologies changed with treatment with noscapine, a tubulin-binding drug, in a genotype-dependent manner, revealing a novel effect on one of the genotypes (SPG7). These findings demonstrate a method for morphological profiling in fibroblasts, an accessible non-neural cell, to classify and distinguish between clinical subtypes of neurodegenerative diseases, for drug discovery, and potentially for biomarkers of disease severity and progression.

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