Fabienne C. Fiesel

PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1

Parkinsons disease is the most common neurodegenerative movement disorder. Mutations in PINK1 and PARKIN are the most frequent causes of recessive Parkinsons disease. However, their molecular contribution to pathogenesis remains unclear. Here, we reveal important mechanistic steps of a PINK1/Parkin-directed pathway linking mitochondrial damage, ubiquitylation and autophagy in non-neuronal and neuronal cells. PINK1 kinase activity and its mitochondrial localization sequence are prerequisites to induce translocation of the E3 ligase Parkin to depolarized mitochondria. Subsequently, Parkin mediates the formation of two distinct poly-ubiquitin chains, linked through Lys 63 and Lys 27. In addition, the autophagic adaptor p62/SQSTM1 is recruited to mitochondrial clusters and is essential for the clearance of mitochondria. Strikingly, we identified VDAC1 (voltage-dependent anion channel 1) as a target for Parkin-mediated Lys 27 poly-ubiquitylation and mitophagy. Moreover, pathogenic Parkin mutations interfere with distinct steps of mitochondrial translocation, ubiquitylation and/or final clearance through mitophagy. Thus, our data provide functional links between PINK1, Parkin and the selective autophagy of mitochondria, which is implicated in the pathogenesis of Parkinsons disease.

Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6

TDP‐43 is an RNA/DNA‐binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP‐43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP‐43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP‐43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP‐43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH‐SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl‐tubulin. HDAC6 levels were restored by re‐expression of TDP‐43, dependent on RNA binding and the C‐terminal protein interaction domains. Moreover, TDP‐43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP‐43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6‐dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ‐expanded ataxin‐3 were found in TDP‐43 silenced cells. In conclusion, loss of functional TDP‐43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.

The PINK1/Parkin-mediated mitophagy is compromised by PD-associated mutations.

Mitochondrial dysfunction is an early sign of many neurodegenerative diseases. Very recently, two Parkinson disease (PD) associated genes, PINK1 and Parkin, were shown to mediate the degradation of damaged mitochondria via selective autophagy (mitophagy). PINK1 kinase activity is needed for prompt and efficient Parkin recruitment to impaired mitochondria. PD-associated Parkin mutations interfere with the process of mitophagy at distinct steps. Here we show that whole mitochondria are turned over via macroautophagy. Moreover, disease-associated PINK1 mutations also compromise the selective degradation of depolarized mitochondria. This may be due to the decreased physical binding activity of PD-linked PINK1 mutations to Parkin. Thus, PINK1 mutations abrogate autophagy of impaired mitochondria upstream of Parkin. In addition to compromised PINK1 kinase activity, reduced binding of PINK1 to Parkin leads to failure in Parkin mitochondrial translocation, resulting in the accumulation of damaged mitochondria, which may contribute to disease pathogenesis.

Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice

Mutations in the LRRK2 gene represent the most common genetic cause of late onset Parkinsons disease. The physiological and pathological roles of LRRK2 are yet to be fully determined but evidence points towards LRRK2 mutations causing a gain in kinase function, impacting on neuronal maintenance, vesicular dynamics and neurotransmitter release. To explore the role of physiological levels of mutant LRRK2, we created knock-in (KI) mice harboring the most common LRRK2 mutation G2019S in their own genome. We have performed comprehensive dopaminergic, behavioral and neuropathological analyses in this model up to 24months of age. We find elevated kinase activity in the brain of both heterozygous and homozygous mice. Although normal at 6months, by 12months of age, basal and pharmacologically induced extracellular release of dopamine is impaired in both heterozygous and homozygous mice, corroborating previous findings in transgenic models over-expressing mutant LRRK2. Via in vivo microdialysis measurement of basal and drug-evoked extracellular release of dopamine and its metabolites, our findings indicate that exocytotic release from the vesicular pool is impaired. Furthermore, profound mitochondrial abnormalities are evident in the striatum of older homozygous G2019S KI mice, which are consistent with mitochondrial fission arrest. We anticipate that this G2019S mouse line will be a useful pre-clinical model for further evaluation of early mechanistic events in LRRK2 pathogenesis and for second-hit approaches to model disease progression.

Parkin protects against tyrosinase-mediated dopamine neurotoxicity by suppressing stress-activated protein kinase pathways.

Parkinson’s disease (PD) motor symptoms are caused by degeneration of nigrostriatal dopaminergic (DAergic) neurons. The most common causes of hereditary PD are mutations in the PARKIN gene. The ubiquitin ligase parkin has been shown to mediate neuroprotection in cell culture and in vivo, but the molecular mechanisms are not well understood. We investigated the effects of parkin in a human SH‐SY5Y neuroblastoma cell culture model of PD, in which transcriptional induction of the enzyme tyrosinase causes a neurotoxic overproduction of cellular DA and its oxidative metabolites. Tyrosinase induction caused formation of reactive oxygen species in the cytosol and mitochondria, and neurotoxicity via activation of apoptotic stress‐activated protein kinases and caspase 3. Stable transfection of wild‐type parkin suppressed tyrosinase‐induced apoptosis, and PD‐associated mutations abolished the neuroprotective effect of parkin. Expression of wild‐type parkin did not affect reactive oxygen species production, but attenuated the tyrosinase‐induced activation of both c‐Jun N‐terminal kinase and p38 mitogen‐activated protein kinase as well as their cognate mitogen‐activated protein kinase kinases. PD‐associated mutations differentially affected the anti‐apoptotic signaling of parkin. Thus, parkin contributes to DAergic neuroprotection by suppression of apoptotic stress‐activated protein kinase pathways.

Suppression of dynamin GTPase decreases α-synuclein uptake by neuronal and oligodendroglial cells: a potent therapeutic target for synucleinopathy

BackgroundThe intracellular deposition of misfolded proteins is a common neuropathological hallmark of most neurodegenerative disorders. Increasing evidence suggests that these pathogenic proteins may spread to neighboring cells and induce the propagation of neurodegeneration.ResultsIn this study, we have demonstrated that α-synuclein (αSYN), a major constituent of intracellular inclusions in synucleinopathies, was taken up by neuronal and oligodendroglial cells in both a time- and concentration-dependent manner. Once incorporated, the extracellular αSYN was immediately assembled into high-molecular-weight oligomers and subsequently formed cytoplasmic inclusion bodies. Furthermore, αSYN uptake by neurons and cells of the oligodendroglial lineage was markedly decreased by the genetic suppression and pharmacological inhibition of the dynamin GTPases, suggesting the involvement of the endocytic pathway in this process.ConclusionsOur findings shed light on the mode of αSYN uptake by neuronal and oligodendroglial cells and identify therapeutic strategies aimed at reducing the propagation of protein misfolding.

Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1.

Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinsons and Huntingtons disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.

TDP-43 knockdown impairs neurite outgrowth dependent on its target histone deacetylase 6

BackgroundTrans-activation response element (TAR) DNA binding protein of 43kDa (TDP-43) is causally related to the neurodegenerative diseases frontotemporal dementia and amyotrophic lateral sclerosis being the hallmark protein in the disease-characteristic neuropathological lesions and via genetic linkage. Histone deacetylase 6 (HDAC6) is an established target of the RNA-binding protein TDP-43. HDAC6 is an unusual cytosolic deacetylase enzyme, central for a variety of pivotal cellular functions including aggregating protein turnover, microtubular dynamics and filopodia formation. All these functions are important in the context of neurodegenerative proteinopathies involving TDP-43. We have previously shown in a human embryonic kidney cell line that TDP-43 knockdown significantly impairs the removal of a toxic, aggregating polyQ ataxin-3 fusion protein in an HDAC6-dependent manner. Here we investigated the influence of TDP-43 and its target HDAC6 on neurite outgrowth.ResultsHuman neuroblastoma SH-SY5Y cells with stably silenced TDP-43 showed a significant reduction of neurite outgrowth induced by retinoic acid and brain-derived neurotrophic factor. Re-transfection with TDP-43 as well as HDAC6 rescued retinoic acid-induced neurite outgrowth. In addition, we show that silencing of HDAC6 alone is sufficient to reduce neurite outgrowth of in vitro differentiated SH-SY5Y cells.ConclusionsTDP-43 deficiency leads to impairment of neurite growth in an HDAC6-dependent manner, thereby contributing to neurodegenerative events in TDP-43 diseases.

Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinsons disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkins enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

(Patho‐)physiological relevance of PINK1‐dependent ubiquitin phosphorylation

Mutations in PINK1 and PARKIN cause recessive, early‐onset Parkinsons disease (PD). Together, these two proteins orchestrate a protective mitophagic response that ensures the safe disposal of damaged mitochondria. The kinase PINK1 phosphorylates ubiquitin (Ub) at the conserved residue S65, in addition to modifying the E3 ubiquitin ligase Parkin. The structural and functional consequences of Ub phosphorylation (pS65‐Ub) have already been suggested from in vitro experiments, but its (patho‐)physiological significance remains unknown. We have generated novel antibodies and assessed pS65‐Ub signals in vitro and in cells, including primary neurons, under endogenous conditions. pS65‐Ub is dependent on PINK1 kinase activity as confirmed in patient fibroblasts and postmortem brain samples harboring pathogenic mutations. We show that pS65‐Ub is reversible and barely detectable under basal conditions, but rapidly induced upon mitochondrial stress in cells and amplified in the presence of functional Parkin. pS65‐Ub accumulates in human brain during aging and disease in the form of cytoplasmic granules that partially overlap with mitochondrial, lysosomal, and total Ub markers. Additional studies are now warranted to further elucidate pS65‐Ub functions and fully explore its potential for biomarker or therapeutic development.