Cornelia Rüb

α-Synuclein is localized to mitochondria-associated ER membranes.

Familial Parkinson disease is associated with mutations in α-synuclein (α-syn), a presynaptic protein that has been localized not only to the cytosol, but also to mitochondria. We report here that wild-type α-syn from cell lines, and brain tissue from humans and mice, is present not in mitochondria but rather in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a structurally and functionally distinct subdomain of the ER. Remarkably, we found that pathogenic point mutations in human α-syn result in its reduced association with MAM, coincident with a lower degree of apposition of ER with mitochondria, a decrease in MAM function, and an increase in mitochondrial fragmentation compared with wild-type. Although overexpression of wild-type α-syn in mutant α-syn-expressing cells reverted the fragmentation phenotype, neither overexpression of the mitochondrial fusion/MAM-tethering protein MFN2 nor inhibition/ablation of the mitochondrial fission protein DRP1 was able to do so, implying that α-syn operates downstream of the mitochondrial fusion/fission machinery. These novel results indicate that wild-type α-syn localizes to the MAM and modulates mitochondrial morphology, and that these behaviors are impaired by pathogenic mutations in α-syn. We believe that our results have far-reaching implications for both our understanding of α-syn biology and the treatment of synucleinopathies.

Cytosolic cleaved PINK1 represses Parkin translocation to mitochondria and mitophagy

PINK1 is a mitochondrial kinase proposed to have a role in the pathogenesis of Parkinsons disease through the regulation of mitophagy. Here, we show that the PINK1 main cleavage product, PINK152, after being generated inside mitochondria, can exit these organelles and localize to the cytosol, where it is not only destined for degradation by the proteasome but binds to Parkin. The interaction of cytosolic PINK1 with Parkin represses Parkin translocation to the mitochondria and subsequent mitophagy. Our work therefore highlights the existence of two cellular pools of PINK1 that have different effects on Parkin translocation and mitophagy.

Amyloid β-peptides interfere with mitochondrial preprotein import competence by a coaggregation process

Aggregation-prone amyloid β-peptides occurring in Alzheimer’s disease (AD) inhibit the import of nuclear-encoded mitochondrial precursor proteins. The observation of insoluble coaggregates between preproteins and Aβ peptides provides a biochemical explanation for mitochondrial dysfunction typically observed in AD-affected cells.

Mitochondrial quality control by the Pink1/Parkin system

Mitochondrial dysfunction represents a prominent pathological feature in many neurodegenerative diseases, particularly in Parkinson’s disease (PD). Mutations in the genes encoding the proteins Pink1 and Parkin have been identified as genetic risk factors in familiar cases of PD. Research during the last decade has identified both proteins as crucial components of an organellar quality control system that contributes to the maintenance of mitochondrial function in healthy cells. The Pink1/Parkin system acts as a sensor for mitochondrial quality and is activated, in particular, after the loss of the electric potential across the inner mitochondrial membrane. Pink1 molecules accumulate at the surface of damaged mitochondria to recruit and activate Parkin, which, in turn, elicits a signaling pathway eventually leading to the autophagic removal of the damaged organelles. This review summarizes recent advances in our knowledge of the functional role of the Pink1/Parkin system in preventing the accumulation of damaged mitochondria by mitophagy.

Loss of the smallest subunit of cytochrome c oxidase, COX8A, causes Leigh-like syndrome and epilepsy

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patients fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease.

Biochemical properties of the kinase PINK1 as sensor protein for mitochondrial damage signalling

Defects of mitochondrial functions have been implicated in many different human diseases, in particular neurodegenerative diseases. The kinase PINK1 [phosphatase and tensin homologue (PTEN)-induced kinase 1] has been identified as a crucial player in a specific damage signalling pathway, eliminating defective mitochondria by an autophagic process. Mutations in PINK1 have been shown to cause familial cases of Parkinsons disease. In this review, we summarize the biochemical mechanisms that underlie the association of PINK1 with mitochondria under normal and pathological conditions. This unconventional mitochondrial localization pathway is discussed in the context of the role of PINK1 as a sensor of mitochondrial damage and a causative factor in Parkinsons disease.

IMiQ: a novel protein quality control compartment protecting mitochondrial functional integrity

Aggregated polypeptides accumulating inside mitochondria are sequestered in a single cellular quality compartment, called IMiQ. Its formation retains proteotoxic aggregates in a distinct cellular localization, increasing mitochondrial fitness by relieving the protein quality control system of misfolded polypeptides.

High aggregation sensitivity of mammalian mitochondrial elongation factor Tu (Tufm) as a sensor for organellar stress

As proteins in mammalian cells exhibit optimal stability at natural temperatures, small temperature variations may cause unfolding and subsequent non-specific aggregation. As this process leads to a loss of function of the affected polypeptides as well as to further cytotoxic stress, aggregate formation has been recognized as a major pathogenic factor in human diseases. In this study we determined the impact of physiological heat stress on mammalian mitochondria on a proteomic level. The overall solubility of endogenous mitochondrial proteins was only marginally affected by a treatment at elevated temperatures. However, we identified a small subset of polypeptides that exhibited an exceptionally high sensitivity to heat stress. The mitochondrial translation elongation factor Tu (Tufm), a protein essential for organellar protein biosynthesis, was highly aggregation-prone and lost its solubility already under mild heat stress conditions. In parallel, mitochondrial translation as well as the import of cytosolic proteins was defective in heat stressed mitochondria. We propose that a shutdown of endogenous protein synthesis concomitant with a reduced preprotein import has a protective function by attenuating the proteotoxic stress caused by an accumulation of nascent polypeptides with a high tendency to misfold.As proteins in mammalian cells exhibit optimal stability at natural temperatures, small temperature variations may cause unfolding and subsequent non-specific aggregation. As this process leads to a loss of function of the affected polypeptides as well as to further cytotoxic stress, aggregate formation has been recognized as a major pathogenic factor in human diseases. In this study we determined the impact of physiological heat stress on mammalian mitochondria on a proteomic level. The overall solubility of endogenous mitochondrial proteins was only marginally affected by a treatment at elevated temperatures. However, we identified a small subset of polypeptides that exhibited an exceptionally high sensitivity to heat stress. The mitochondrial translation elongation factor Tu (Tufm), a protein essential for organellar protein biosynthesis, was highly aggregation-prone and lost its solubility already under mild heat stress conditions. In parallel, mitochondrial translation as well as the import of cytosolic proteins was defective in heat stressed mitochondria. Both types of nascent polypeptides, derived from translation as well as from import exhibited a strong heat-induced aggregation tendency. We propose a model that a quick and specific inactivation of elongation factors may prevent an accumulation of misfolded nascent polypeptides and thereby attenuate proteotoxicity under stress.

Analysis of heat-induced protein aggregation in human mitochondria

Proteins in mammalian cells exhibit optimal stability at physiological temperatures, and even small temperature variations may cause unfolding and nonspecific aggregation. Because this process leads to a loss of function of the affected polypeptides and to cytotoxic stress, formation of protein aggregates has been recognized as a major pathogenic factor in human diseases. In this study, we determined the impact of physiological heat stress on mitochondria isolated from HeLa cells. We found that the heat-stressed mitochondria had lower membrane potential and ATP level and exhibited a decreased production of reactive oxygen species. An analysis of the mitochondrial proteome by 2D PAGE showed that the overall solubility of endogenous proteins was only marginally affected by elevated temperatures. However, a small subset of polypeptides exhibited an high sensitivity to heat stress. The mitochondrial translation elongation factor Tu (Tufm), a protein essential for organellar protein biosynthesis, was highly aggregation-prone and lost its solubility already under mild heat-stress conditions. Moreover, mitochondrial translation and the import of cytosolic proteins were defective in the heat-stressed mitochondria. Both types of nascent polypeptides, produced by translation or imported into the mitochondria, exhibited a strong tendency to aggregate in the heat-exposed mitochondria. We propose that a fast and specific inactivation of elongation factors may prevent the accumulation of misfolded nascent polypeptides and may thereby attenuate proteotoxicity under heat stress.

Chaperones and Proteases of Mitochondria: From Protein Folding and Degradation to Mitophagy

In eukaryotic cells, mitochondria fulfill a multitude of essential functions. Under both normal and stress conditions, a complex system of molecular chaperones and protease enzymes is at work to maintain mitochondrial biogenesis and protein quality control (PQC), summarized as “mitochondrial protein homeostasis.” Mitochondrial chaperones of the heat shock protein Hsp60 and Hsp70 families play main roles in the import and folding of nuclear-encoded polypeptides, representing the majority of the mitochondrial proteome. These enzymes together with chaperones of the Clp family prevent the accumulation of aggregated or superfluous polypeptides in a close cooperation with specific PQC proteases of the AAA+ (adenosine triphosphatases associated with diverse cellular activities) family. Recent evidence demonstrated the removal of terminally damaged mitochondria as a whole by a variation of autophagy, termed mitophagy, as an additional level of mitochondrial quality control. The details of mitochondrial protein homeostasis, comprising the functional contribution of this broad set of chaperones and proteases will be discussed here.