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.
Light control of living systems is an emerging field in bioelectronics, in regenerative medicine and cell‐based therapy. Herein, the design of a semitransparent bio–photoelectrolytic platform for ...control of a neuroblastoma cell line via light pulses is laid out. The platform is based on conjugated polymer films interfaced with a biological electrolyte solution confined in a compact chamber. Human SH‐SY5Y neuroblastoma cells are cultured for 3 days on the organic semiconductor and subjected to a pulsed light protocol. At the end of the culture time, proliferative activity of cells on the polymer film subjected to light pulses is reduced by 50% compared to the cultures kept in dark. An increase in intracellular Ca2+ level is observed, indicating a significant perturbation of the equilibrium potential of the cells. It is shown that the platform, in a sandwich‐type closed architecture with two transparent electrodes, can provide a tool for the initial recording of bioelectrical photovoltage signals (mV) that can complement analysis with more sophisticated electrophysiological tools. Obtained results can pave the way to new noninvasive photomanipulation techniques to stimulate/control living cells and their proliferation through both optical and electrical stimulation and probes, for application in the fields of biosensing and biomedicine.
A bio–photoelectrolytic platform allows cell culture on organic semiconducting polymers and measurement of photoelectrical cellular response by employing a novel device that can be used for both stimulation and recording. Pulsed‐light illumination results in inhibition of proliferation of a cancer cell line with increase in intracellular calcium, mainly due to plasma membrane channel opening as indicated by photovoltage signals.
Human induced pluripotent stem cells (hiPSCs) represent an unlimited cell source for the generation of patient-specific dopaminergic (DA) neurons, overcoming the hurdle of restricted accessibility to ...disease-affected tissue for mechanistic studies on Parkinson's disease (PD). However, the complexity of the human brain is not fully recapitulated by existing monolayer culture methods. Neurons differentiated in a three dimensional (3D)
culture system might better mimic the
cellular environment for basic mechanistic studies and represent better predictors of drug responses
. In this work we established a new
cell culture system based on the microencapsulation of hiPSCs in small alginate/fibronectin beads and their differentiation to DA neurons. Optimization of hydrogel matrix concentrations and composition allowed a high viability of embedded hiPSCs. Neural differentiation competence and efficiency of DA neuronal generation were increased in the 3D cultures compared to a conventional 2D culture methodology. Additionally, electrophysiological parameters and metabolic switching profile confirmed increased functionality and an anticipated metabolic resetting of neurons grown in alginate scaffolds with respect to their 2D counterpart neurons. We also report long-term maintenance of neuronal cultures and preservation of the mature functional properties. Furthermore, our findings indicate that our 3D model system can recapitulate mitochondrial superoxide production as an important mitochondrial phenotype observed in neurons derived from PD patients, and that this phenotype might be detectable earlier during neuronal differentiation. Taken together, these results indicate that our alginate-based 3D culture system offers an advantageous strategy for the reliable and rapid derivation of mature and functional DA neurons from hiPSCs.
...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. ...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). ...flies with reduced SLP-2 expression exhibited reduced flight ability and wing posture phenotypes indicative of muscle dysfunction, as it has been observed with the loss of PD genes like parkin. ...SLP-2 provides an interesting new target for the development of rescue strategies by specifically targeting the mitochondrial dysfunction in neurons implicated in the pathogenesis of PD. Our data on the rescue of functional defects due to Parkin-deficiency by SLP-2 overexpression places SLP-2 in a pathway downstream or in parallel to Parkin.
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.
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.
Objective: Objective of this pilot study was to evaluate the reliability and internal consistency of the Columbia Impairment Scale questionnaire, as a tool to provide a global measure of impairment ...functioning in an Italian adolescents sample.
Methods: The questionnaire is composed by 4 sections of functioning (interpersonal relations, broad psychopathological domains, functioning in schoolwork, use of leisure time) for a total of 13 items. It was administered twice in the same sample belonging to a Professional School in Frosinone (Central Italy), to 120 adolescents on the first administration and to 108 on the second time. Reliability analysis was performed and Cronbach’s alpha was used as a measure of internal consistency. Statistical analysis was performed using SPSS 19.0.
Results: Considering all 13 items, Cronbach’s alpha amounted to 0,762 on the first time and to 0.826 on the second time, showing a very satisfactory internal validity. In the sample selected, the answers to the questionnaire showed adolescents have a low impairment functioning.
Conclusion: The results of the pilot study indicate that the questionnaire presents a good reliability property and in terms of internal consistency and validity shows a good performance. The results are promising and suggest that this tool cood be used in the Italian setting for future research targeted to adequately capture the impaired functioning in adolescents.
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.
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.
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.
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.
Imbalances of iron and dopamine metabolism along with mitochondrial dysfunction have been linked to the pathogenesis of Parkinson's disease (PD). We have previously suggested a direct link between ...iron homeostasis and dopamine metabolism, as dopamine can increase cellular uptake of iron into macrophages thereby promoting oxidative stress responses. In this study, we investigated the interplay between iron, dopamine, and mitochondrial activity in neuroblastoma SH-SY5Y cells and human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons differentiated from a healthy control and a PD patient with a mutation in the α-synuclein (SNCA) gene. In SH-SY5Y cells, dopamine treatment resulted in increased expression of the transmembrane iron transporters transferrin receptor 1 (TFR1), ferroportin (FPN), and mitoferrin2 (MFRN2) and intracellular iron accumulation, suggesting that dopamine may promote iron uptake. Furthermore, dopamine supplementation led to reduced mitochondrial fitness including decreased mitochondrial respiration, increased cytochrome c control efficiency, reduced mtDNA copy number and citrate synthase activity, increased oxidative stress and impaired aconitase activity. In dopaminergic neurons derived from a healthy control individual, dopamine showed comparable effects as observed in SH-SY5Y cells. The hiPSC-derived PD neurons harboring an endogenous SNCA mutation demonstrated altered mitochondrial iron homeostasis, reduced mitochondrial capacity along with increased oxidative stress and alterations of tricarboxylic acid cycle linked metabolic pathways compared with control neurons. Importantly, dopamine treatment of PD neurons promoted a rescue effect by increasing mitochondrial respiration, activating antioxidant stress response, and normalizing altered metabolite levels linked to mitochondrial function. These observations provide evidence that dopamine affects iron homeostasis, intracellular stress responses and mitochondrial function in healthy cells, while dopamine supplementation can restore the disturbed regulatory network in PD cells.
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•Dopamine alters iron homeostasis in neuronal cell models.•Mitochondria are susceptible to dopamine-iron interaction.•Dopamine-mediated iron perturbations induce oxidative stress under physiological conditions.•In PD hiPSC-derived neurons, dopamine restores altered intracellular iron distribution and mitochondrial function.
Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's 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.
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