Marthe H.R. Ludtmann

Alpha-Synuclein Oligomers Interact with Metal Ions to Induce Oxidative Stress and Neuronal Death in Parkinson's Disease

Abstract Aims: Protein aggregation and oxidative stress are both key pathogenic processes in Parkinsons disease, although the mechanism by which misfolded proteins induce oxidative stress and neuronal death remains unknown. In this study, we describe how aggregation of alpha-synuclein (α-S) from its monomeric form to its soluble oligomeric state results in aberrant free radical production and neuronal toxicity. Results: We first demonstrate excessive free radical production in a human induced pluripotent stem-derived α-S triplication model at basal levels and on application of picomolar doses of β-sheet-rich α-S oligomers. We probed the effects of different structural species of α-S in wild-type rat neuronal cultures and show that both oligomeric and fibrillar forms of α-S are capable of generating free radical production, but that only the oligomeric form results in reduction of endogenous glutathione and subsequent neuronal toxicity. We dissected the mechanism of oligomer-induced free radical production and found that it was interestingly independent of several known cellular enzymatic sources. Innovation: The oligomer-induced reactive oxygen species (ROS) production was entirely dependent on the presence of free metal ions as addition of metal chelators was able to block oligomer-induced ROS production and prevent oligomer-induced neuronal death. Conclusion: Our findings further support the causative role of soluble amyloid oligomers in triggering neurodegeneration and shed light into the mechanisms by which these species cause neuronal damage, which, we show here, can be amenable to modulation through the use of metal chelation. Antioxid. Redox Signal. 24, 376–391.

Loss of pink1 increases the heart's vulnerability to ischemia-reperfusion injury

Objectives Mutations in PTEN inducible kinase-1 (PINK1) induce mitochondrial dysfunction in dopaminergic neurons resulting in an inherited form of Parkinson’s disease. Although PINK1 is present in the heart its exact role there is unclear. We hypothesized that PINK1 protects the heart against acute ischemia reperfusion injury (IRI) by preventing mitochondrial dysfunction. Methods and Results Over-expressing PINK1 in HL-1 cardiac cells reduced cell death following simulated IRI (29.2±5.2% PINK1 versus 49.0±2.4% control; N = 320 cells/group P<0.05), and delayed the onset of mitochondrial permeability transition pore (MPTP) opening (by 1.3 fold; P<0.05). Hearts excised from PINK1+/+, PINK1+/− and PINK1−/− mice were subjected to 35 minutes regional ischemia followed by 30 minutes reperfusion. Interestingly, myocardial infarct size was increased in PINK1−/− hearts compared to PINK1+/+ hearts with an intermediate infarct size in PINK1+/− hearts (25.1±2.0% PINK1+/+, 38.9±3.4% PINK1+/− versus 51.5±4.3% PINK1−/− hearts; N>5 animals/group; P<0.05). Cardiomyocytes isolated from PINK1−/− hearts had a lower resting mitochondrial membrane potential, had inhibited mitochondrial respiration, generated more oxidative stress during simulated IRI, and underwent rigor contracture more rapidly in response to an uncoupler when compared to PINK1+/+ cells suggesting mitochondrial dysfunction in hearts deficient in PINK1. Conclusions We show that the loss of PINK1 increases the hearts vulnerability to ischemia-reperfusion injury. This may be due, in part, to increased mitochondrial dysfunction. These findings implicate PINK1 as a novel target for cardioprotection.

A Missense Mutation in KCTD17 Causes Autosomal Dominant Myoclonus-Dystonia

Myoclonus-dystonia (M-D) is a rare movement disorder characterized by a combination of non-epileptic myoclonic jerks and dystonia. SGCE mutations represent a major cause for familial M-D being responsible for 30%-50% of cases. After excluding SGCE mutations, we identified through a combination of linkage analysis and whole-exome sequencing KCTD17 c.434 G>A p.(Arg145His) as the only segregating variant in a dominant British pedigree with seven subjects affected by M-D. A subsequent screening in a cohort of M-D cases without mutations in SGCE revealed the same KCTD17 variant in a German family. The clinical presentation of the KCTD17-mutated cases was distinct from the phenotype usually observed in M-D due to SGCE mutations. All cases initially presented with mild myoclonus affecting the upper limbs. Dystonia showed a progressive course, with increasing severity of symptoms and spreading from the cranio-cervical region to other sites. KCTD17 is abundantly expressed in all brain regions with the highest expression in the putamen. Weighted gene co-expression network analysis, based on mRNA expression profile of brain samples from neuropathologically healthy individuals, showed that KCTD17 is part of a putamen gene network, which is significantly enriched for dystonia genes. Functional annotation of the network showed an over-representation of genes involved in post-synaptic dopaminergic transmission. Functional studies in mutation bearing fibroblasts demonstrated abnormalities in endoplasmic reticulum-dependent calcium signaling. In conclusion, we demonstrate that the KCTD17 c.434 G>A p.(Arg145His) mutation causes autosomal dominant M-D. Further functional studies are warranted to further characterize the nature of KCTD17 contribution to the molecular pathogenesis of M-D.

Ca2+ is a key factor in α-synuclein-induced neurotoxicity

ABSTRACT Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinsons disease. We investigated the species-specific effects of α-synuclein on Ca2+ signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca2+ in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca2+ signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca2+, which prevented the α-synuclein-induced Ca2+ dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca2+ signalling in a structure-specific manner and the oligomeric β-sheet-rich α-synuclein species ultimately leads to Ca2+ dysregulation and Ca2+-dependent cell death. Summary: Monomeric and oligomeric α-synuclein induce Ca2+ signal in neurons and astrocytes by incorporating into the membrane.

Aggregated α -synuclein and complex I deficiency: exploration of their relationship in differentiated neurons

α-Synuclein becomes misfolded and aggregated upon damage by various factors, for example, by reactive oxygen species. These aggregated forms have been proposed to have differential toxicities and their interaction with mitochondria may cause dysfunction within this organelle that contributes to the pathogenesis of Parkinson’s disease (PD). In particular, the association of α-synuclein with mitochondria occurs through interaction with mitochondrial complex I and importantly defects of this protein have been linked to the pathogenesis of PD. Therefore, we investigated the relationship between aggregated α-synuclein and mitochondrial dysfunction, and the consequences of this interaction on cell survival. To do this, we studied the effects of α-synuclein on cybrid cell lines harbouring mutations in either mitochondrial complex I or IV. We found that aggregated α-synuclein inhibited mitochondrial complex I in control and complex IV-deficient cells. However, when aggregated α-synuclein was applied to complex I-deficient cells, there was no additional inhibition of mitochondrial function or increase in cell death. This would suggest that as complex I-deficient cells have already adapted to their mitochondrial defect, the subsequent toxic effects of α-synuclein are reduced.

Molecular pharmacology in a simple model system: implicating MAP kinase and phosphoinositide signalling in bipolar disorder.

Understanding the mechanisms of drug action has been the primary focus for pharmacological researchers, traditionally using rodent models. However, non-sentient model systems are now increasingly being used as an alternative approach to better understand drug action or targets. One of these model systems, the social amoeba Dictyostelium, enables the rapid ablation or over-expression of genes, and the subsequent use of isogenic cell culture for the analysis of cell signalling pathways in pharmacological research. The model also supports an increasingly important ethical view of research, involving the reduction, replacement and refinement of animals in biomedical research. This review outlines the use of Dictyostelium in understanding the pharmacological action of two commonly used bipolar disorder treatments (valproic acid and lithium). Both of these compounds regulate mitogen activated protein (MAP) kinase and inositol phospholipid-based signalling by unknown means. Analysis of the molecular pathways targeted by these drugs in Dictyostelium and translation of discoveries to animal systems has helped to further understand the molecular mechanisms of these bipolar disorder treatments.

Monomeric Alpha-Synuclein Exerts a Physiological Role on Brain ATP Synthase

Misfolded α-synuclein is a key factor in the pathogenesis of Parkinsons disease (PD). However, knowledge about a physiological role for the native, unfolded α-synuclein is limited. Using brains of mice lacking α-, β-, and γ-synuclein, we report that extracellular monomeric α-synuclein enters neurons and localizes to mitochondria, interacts with ATP synthase subunit α, and modulates ATP synthase function. Using a combination of biochemical, live-cell imaging and mitochondrial respiration analysis, we found that brain mitochondria of α-, β-, and γ-synuclein knock-out mice are uncoupled, as characterized by increased mitochondrial respiration and reduced mitochondrial membrane potential. Furthermore, synuclein deficiency results in reduced ATP synthase efficiency and lower ATP levels. Exogenous application of low unfolded α-synuclein concentrations is able to increase the ATP synthase activity that rescues the mitochondrial phenotypes observed in synuclein deficiency. Overall, the data suggest that α-synuclein is a previously unrecognized physiological regulator of mitochondrial bioenergetics through its ability to interact with ATP synthase and increase its efficiency. This may be of particular importance in times of stress or PD mutations leading to energy depletion and neuronal cell toxicity. SIGNIFICANCE STATEMENT Misfolded α-synuclein aggregations in the form of Lewy bodies have been shown to be a pathological hallmark in histological staining of Parkinsons disease (PD) patient brains. It is known that misfolded α-synuclein is a key driver in PD pathogenesis, but the physiological role of unfolded monomeric α-synuclein remains unclear. Using neuronal cocultures and isolated brain mitochondria of α-, β-, and γ-synuclein knock-out mice and monomeric α-synuclein, this current study shows that α-synuclein in its unfolded monomeric form improves ATP synthase efficiency and mitochondrial function. The ability of monomeric α-synuclein to enhance ATP synthase efficiency under physiological conditions may be of importance when α-synuclein undergoes the misfolding and aggregation reported in PD.

Hypoxia signaling controls postnatal changes in cardiac mitochondrial morphology and function

Fetal cardiomyocyte adaptation to low levels of oxygen in utero is incompletely understood, and is of interest as hypoxia tolerance is lost after birth, leading to vulnerability of adult cardiomyocytes. It is known that cardiac mitochondrial morphology, number and function change significantly following birth, although the underlying molecular mechanisms and physiological stimuli are undefined. Here we show that the decrease in cardiomyocyte HIF-signaling in cardiomyocytes immediately after birth acts as a physiological switch driving mitochondrial fusion and increased postnatal mitochondrial biogenesis. We also investigated mechanisms of ATP generation in embryonic cardiac mitochondria. We found that embryonic cardiac cardiomyocytes rely on both glycolysis and the tricarboxylic acid cycle to generate ATP, and that the balance between these two metabolic pathways in the heart is controlled around birth by the reduction in HIF signaling. We therefore propose that the increase in ambient oxygen encountered by the neonate at birth acts as a key physiological stimulus to cardiac mitochondrial adaptation.

Protein kinase C signalling during miracidium to mother sporocyst development in the helminth parasite, Schistosoma mansoni

For schistosomes, development of the miracidium to mother sporocyst within a compatible molluscan host requires considerable physiological and morphological changes by the parasite. The molecular mechanisms controlling such development have not been explored extensively. To begin to elucidate the importance of kinase-mediated signal transduction to this process, the phosphorylation (activation) of protein kinase C (PKC) in larval stages of Schistosoma mansoni undergoing in vitro transformation was explored. Mining of the S. mansoni genomic database revealed two S. mansoni PKC proteins with high homology to human PKCbeta and containing the conserved autophosphorylation (activation) site represented by serine 660 of human PKCbeta(II). Western blotting with anti-phosphospecific antibodies directed to this site demonstrated that miracidia freshly-hatched from eggs possessed PKC (78kDa) which was phosphorylated (activated) when miracidia were exposed to phorbol ester, and dephosphorylated (inhibited) following exposure to the PKC inhibitor GF109203X. Miracidia treated with the phospholipase C (PLC) inhibitor U73122 also displayed decreased PKC phosphorylation. S. mansoni PKC was phosphorylated during the initial 24h development of miracidia into mother sporocysts; after 31h and 48h development, phosphorylation was reduced by 72% and 86%, respectively. Confocal microscopy of miracidia revealed phosphorylated PKC associated with the neural mass, excretory vesicle, tegument, ciliated plates, terebratorium and germinal cells; in larvae undergoing transformation for 31h, phosphorylated PKC was only occasionally detected, being present in regions likely corresponding to the ridge cyton. Inhibition of PKC in miracidia by GF109230X resulted in accelerated transformation, particularly to the postmiracidium stage; ciliated plates were also shed from developing larvae more rapidly. These results highlight the dynamic nature of PKC signalling during S. mansoni postembryonic development and support a role for active PKC in restricting transformation of S. mansoni miracidia into mother sporocysts.

Naringenin inhibits the growth of Dictyostelium and MDCK-derived cysts in a TRPP2 (polycystin-2)- dependent manner

Identifying and characterizing potential new therapeutic agents to target cell proliferation may provide improved treatments for neoplastic disorders such as cancer and polycystic diseases.