Alessandra Zanon

Phosphatase and Tensin Homolog (PTEN)-induced Putative Kinase 1 (PINK1)-dependent Ubiquitination of Endogenous Parkin Attenuates Mitophagy STUDY IN HUMAN PRIMARY FIBROBLASTS AND INDUCED PLURIPOTENT STEM CELL-DERIVED NEURONS

Background: The Parkinson disease-related proteins PINK1 and Parkin initiate mitophagy of damaged mitochondria. Results: Endogenous Parkin is not sufficient to induce mitophagy due to PINK1-dependent ubiquitination of Parkin. Conclusion: Mitophagy is detectable only with supraphysiological levels of Parkin and differs between fibroblasts and iPS-derived neurons. Significance: Stresses the importance of future studies in Parkinson disease-relevant tissue, i.e., dopaminergic neurons. Mutations in the E3 ubiquitin ligase Parkin and the mitochondrial PTEN-induced putative kinase 1 (PINK1) have been identified to cause autosomal recessive forms of familial Parkinson disease, with PINK1 functioning upstream of Parkin in a pathway important for the maintenance of mitochondrial function and morphology. Upon the loss of the mitochondrial membrane potential, Parkin translocates to mitochondria in a PINK1-dependent manner to ubiquitinate mitochondrial proteins. Parkin-mediated polyubiquitination of outer mitochondrial membrane (OMM) proteins recruits the ubiquitin- and LC3-binding adaptor protein p62 to mitochondria and induces mitophagy. Although previous studies examined mitophagy in established cell lines through overexpression approaches, there is an imperative to study the role of endogenous Parkin and PINK1 in human-derived and biologically relevant cell models. Here, we demonstrate in human primary fibroblasts and induced pluripotent stem-derived neurons from controls and PINK1 mutation carriers that endogenous levels of Parkin are not sufficient to initiate mitophagy upon loss of the mitochondrial membrane potential, caused by its (self-)ubiquitination, followed by degradation via the ubiquitin proteasome system. Next, we showed differential PINK1-dependent, Parkin-mediated ubiquitination of OMM proteins, which is Parkin dose-dependent and affects primarily OMM proteins of higher molecular mass. In contrast to the situation fibroblasts, we did not detect mitophagy in induced pluripotent stem-derived neurons even upon overexpression of Parkin. Taken together, our data demonstrate that mitophagy differs between human non-neuronal and neuronal cells and between “endogenous” and “Parkin-overexpressing” cellular models.

Identification of a common variant in the TFR2 gene implicated in the physiological regulation of serum iron levels

The genetic determinants of variation in iron status are actively sought, but remain incompletely understood. Meta-analysis of two genome-wide association (GWA) studies and replication in three independent cohorts was performed to identify genetic loci associated in the general population with serum levels of iron and markers of iron status, including transferrin, ferritin, soluble transferrin receptor (sTfR) and sTfR-ferritin index. We identified and replicated a novel association of a common variant in the type-2 transferrin receptor (TFR2) gene with iron levels, with effect sizes highly consistent across samples. In addition, we identified and replicated an association between the HFE locus and ferritin and confirmed previously reported associations with the TF, TMPRSS6 and HFE genes. The five replicated variants were tested for association with expression levels of the corresponding genes in a publicly available data set of human liver samples, and nominally statistically significant expression differences by genotype were observed for all genes, although only rs3811647 in the TF gene survived the Bonferroni correction for multiple testing. In addition, we measured for the first time the effects of the common variant in TMPRSS6, rs4820268, on hepcidin mRNA in peripheral blood (n = 83 individuals) and on hepcidin levels in urine (n = 529) and observed an association in the same direction, though only borderline significant. These functional findings require confirmation in further studies with larger sample sizes, but they suggest that common variants in TMPRSS6 could modify the hepcidin-iron feedback loop in clinically unaffected individuals, thus making them more susceptible to imbalances of iron homeostasis.

Overexpression of blood microRNAs 103a, 30b, and 29a in l-dopa–treated patients with PD

Objective: The aims of the present study were to profile the expression of several candidate microRNAs (miRNAs) in blood from l-dopa-treated and drug-naive patients with Parkinson disease (PD) vs unaffected controls and to interpret the miRNA expression data in a biological context. Methods: We analyzed RNAs from peripheral blood of 36 l-dopa–treated, 10 drug-naive patients with PD and unaffected controls matched 1:1 by sex and age. We evaluated expression by reverse transcription–quantitative real-time PCR, and we analyzed data using a 2-tailed paired t test. To detect miRNA targets, several miRNA resources were combined to generate an overall score for each candidate gene using weighted rank aggregation. Results: Significant overexpression of miR-103a-3p (p < 0.0001), miR-30b-5p (p = 0.002), and miR-29a-3p (p = 0.005) in treated patients with PD was observed, and promising candidate target genes for these were revealed by an integrated in silico analysis. Conclusions: We revealed 3 candidate biomarkers for PD. miRNAs 30b-5p and 29a-3p replicated a documented deregulation in PD albeit opposite to published data, while for miR-103a-3p, we demonstrated for the first time an overexpression in treated patients with PD. Expression studies in patients and/or in isolated peripheral blood mononuclear cells before and after l-dopa administration are necessary to define the involvement of l-dopa treatment in the observed overexpression. Our in silico analysis to prioritize targets of deregulated miRNAs identified candidate target genes, including genes related to neurodegeneration and PD. Despite the preliminary character of our study, the results provide a rationale for further clarifying the role of the identified miRNAs in the pathogenesis of PD and for validating their diagnostic potential.

Profiling of Parkin-Binding Partners Using Tandem Affinity Purification

Parkinsons disease (PD) is a progressive neurodegenerative disorder affecting approximately 1–2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.

Identification of a set of endogenous reference genes for miRNA expression studies in Parkinson’s disease blood samples

BackgroundResearch on microRNAs (miRNAs) is becoming an increasingly attractive field, as these small RNA molecules are involved in several physiological functions and diseases. To date, only few studies have assessed the expression of blood miRNAs related to Parkinson’s disease (PD) using microarray and quantitative real-time PCR (qRT-PCR). Measuring miRNA expression involves normalization of qRT-PCR data using endogenous reference genes for calibration, but their choice remains a delicate problem with serious impact on the resulting expression levels. The aim of the present study was to evaluate the suitability of a set of commonly used small RNAs as normalizers and to identify which of these miRNAs might be considered reliable reference genes in qRT-PCR expression analyses on PD blood samples.ResultsCommonly used reference genes snoRNA RNU24, snRNA RNU6B, snoRNA Z30 and miR-103a-3p were selected from the literature. We then analyzed the effect of using these genes as reference, alone or in any possible combination, on the measured expression levels of the target genes miR-30b-5p and miR-29a-3p, which have been previously reported to be deregulated in PD blood samples.ConclusionsWe identified RNU24 and Z30 as a reliable and stable pair of reference genes in PD blood samples.

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinsons disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

Involvement of proprotein convertase PCSK7 in the regulation of systemic iron homeostasis

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Environmental and Genetic Variables Influencing Mitochondrial Health and Parkinson’s Disease Penetrance

There is strong evidence that impairment of mitochondrial function plays a key role in the pathogenesis of PD. The two key PD genes related to mitochondrial function are Parkin (PARK2) and PINK1 (PARK6), and also mutations in several other PD genes, including SNCA, LRRK2, DJ1, CHCHD2, and POLG, have been shown to induce mitochondrial stress. Many mutations are clearly pathogenic in some patients while carriers of other mutations either do not develop the disease or show a delayed onset, a phenomenon known as reduced penetrance. Indeed, for several mutations in autosomal dominant PD genes, penetrance is markedly reduced, whereas heterozygous carriers of recessive mutations may predispose to PD in a dominant manner, although with highly reduced penetrance, if additional disease modifiers are present. The identification and validation of such modifiers leading to reduced penetrance or increased susceptibility in the case of heterozygous carriers of recessive mutations are relevant for a better understanding of mechanisms contributing to disease onset. We discuss genetic and environmental factors as well as mitochondrial DNA alterations and protein-protein interactions, all involved in mitochondrial function, as potential causes to modify penetrance of mutations in dominant PD genes and to determine manifestation of heterozygous mutations in recessive PD genes.

SLP-2: a potential new target for improving mitochondrial function in Parkinson's disease

Parkinson’s disease (PD) is a progressive neurodegenerative disease, which is generally considered a multifactorial disorder that arises owing to a combination of genes and environmental factors. While most cases are idiopathic, in about 10% of the patients a genetic cause can be detected, ascribable to mutations in more than a dozen genes. PD is characterized clinically by tremor, rigidity, reduced motor activity (bradykinesia), and postural instability and pathologically by loss of dopaminergic (DA) neurons in the substantia nigra pars compacta, loss of DA innervation in the striatum, and the presence of α-synuclein positive aggregates in the form of Lewy bodies. The symptomatic treatment of PD with levodopa, which aims at replacing dopamine, remains the gold standard, and no neuroprotective or disease-modifying therapy is available. During treatment, the disease continues to progress, and long-term use of levodopa has important limitations including motor complications termed dyskinesias. Therefore, a pharmacological therapy able to prevent or halt the neurodegenerative process is urgently required. PARK2 (the gene encoding Parkin) mutations are the most common known cause of early-onset PD, accounting for up to 77% of the familial cases with an age of onset < 30 years (Lücking et al., 2000), and Parkin dysfunction represents a risk factor for sporadic PD. The Parkin protein functions as an E3 ubiquitin ligase, transferring activated ubiquitin to lysine residues of protein substrates. It shows an amino-terminal ubiquitin-like domain, which is involved in substrate recognition, proteasome association, and the regulation of Parkin expression levels and activity, followed by an atypical RING domain, named RING0 or the unique Parkin domain. The carboxy-terminal domain consists of the RING-between-RING domain, comprising RING1 and RING2, and one in-between-ring domain and is responsible for the interaction with the ubiquitination machinery. At steady state, Parkin exists in an inactive state, repressed by several mechanisms of autoinhibition. By studying the function of PARK2 mutations, a direct role of mitochondrial dysfunction in the onset of PD has emerged. Parkin, in concert with PTEN-induced putative kinase 1 (PINK1), another recessively linked PD gene, has been implicated in the degradation of dysfunctional, depolarized mitochondria, a process known as mitophagy. Parkin translocates in a PINK1-dependent manner from the cytosol to dysfunctional mitochondria and ubiquitinates mitochondrial outer membrane proteins to initiate selective autophagy. In addition, Parkin plays a crucial role in the degradative pathways mediated by the ubiquitin-proteasome system, which is required for the clearance of Parkin substrates, like PARIS, which acts as a transcriptional repressor of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), a transcriptional coactivator and master regulator of mitochondrial biogenesis. Moreover, Parkin mediates nondegradative ubiquitination involved in cell death processes and is implicated in the regulation of inflammatory signaling (Winklhofer, 2014). Since the full repertoire of Parkin-binding proteins is purely defined, and to shed further light on the diverse spectrum of Parkin functions, we have characterized the profile of binding partners of Parkin, particularly at the mitochondrial level, by tandem affinity purification/mass spectrometry interaction screens (Zanon et al., 2013). We identified a total of 203 candidate Parkin-binding proteins involved in cell death processes, protein folding and response to unfolded protein, the fission/fusion machinery, and the mitophagy pathway. In a subsequent study, we have investigated the functional relevance of the Parkin interaction with one of the newly identified binding partners Stomatin-like protein 2 (SLP-2) in human neurons and Drosophila melanogaster (Zanon et al., 2017). SLP-2 is a member of the stomatin family (comprising stomatin, SLP-1, SLP-2, SLP-3, and podocin), characterized by the presence of a conserved stomatin domain, which is further related to the SPFH (stomatin, prohibitin, flotillin, HflC/K) protein superfamily (Lapatsina et al., 2012). SPFH proteins are scaffolds, which assemble into ring-like structures and are present in lipid raft microdomains of diverse cellular membranes. The SLP-2 protein is distinguished within the stomatin protein family by its localization at the inner mitochondrial membrane, and an N-terminal mitochondrial leading sequence replaces the hydrophobic membrane anchor of the other family members (Lapatsina et al., 2012). There is increasing evidence for a fine-scale organization also of the inner mitochondrial membrane into functional microdomains, and proteins of the SPFH family assist their formation by specific protein-protein and protein-lipid interactions. SLP-2 was shown to bind to cardiolipin and to prohibitins (PHB-1 and PHB-2) (Christie et al., 2011) and forms cardiolipin and prohibitin-enriched microdomains in the inner mitochondrial membrane, which facilitate the assembly of respiratory chain complexes and their function in T-cells (Christie et al., 2012). Cardiolipin synthesis is increased in SLP-2 overexpressing cells, which translates into increased mitochondrial membrane formation and biogenesis (Christie et al., 2011), and PHB2 has recently been identified as an inner mitochondrial membrane receptor required for Parkin-induced mitophagy by binding the autophagosomal membrane-associated protein LC3 upon mitochondrial depolarization and rupture of the outer membrane (Wei et al., 2017). In addition, SLP-2 was described as part of a new mitochondrial protein complex in the inner mitochondrial membrane composed of the rhomboid protease PARL and the i-AAA protease YME1L named SPY, which facilitates the cleavage of PINK1 by PARL and limits the activity of the mitochondrial protease OMA1, resulting in the protection of OPA1 and stress-induced hyperfusion under stress conditions (Wai et al., 2016). It was suggested that SLP-2, acting as a membrane scaffold, defines the lipid environment for proteolysis and modulates substrate accessibility (Wai et al., 2016). Furthermore, SLP-2 is known to form a complex with mitofusin-2 (MFN2), which is a mitochondrial outer membrane fusion protein and a Parkin ubiquitination substrate, and it was identified in a proteomic screen performed in brain synaptosomes as a binding partner of monomeric α-synuclein (Betzer et al., 2015), a presynaptic neuronal protein that is genetically and neuropathologically linked to PD. Ultimately, the expression level of SLP-2 affects the composition of detergent-resistant membranes in macrophages and the signaling underlying the innate immune response (Chowdhury et al., 2015) (Figure 1). In our recent work, we have shown that SLP-2and Parkin-depleted cells exhibit similar defects in mitochondrial function regarding reduced complex I activity, adenosine triphosphate (ATP) production as well as a fragmented mitochondrial network morphology in neuroblastoma SH-SY5Y cells and induced pluripotent stem cell (iPSC)-derived neurons harboring PD-causing PARK2 mutations (Zanon et al., 2017). In addition, knockdown of SLP2 in Drosophila mirrors several phenotypes associated with the loss of Parkin function. Reduced levels of SLP-2 resulted in a clear disruption of the mitochondrial network structure accompanied by a significant reduction in ATP levels in energy-demanding flight muscles. Accordingly, flies with reduced SLP-2 expression exhibited reduced flight ability and wing posture phenotypes in-