Alpha-synuclein and mitochondrial dysfunction: key links between Gaucher’s disease and Parkinson’s?

Informacje na temat projektu

MitSyn

Identyfikator umowy o grant: 653434

DOI

10.3030/653434

Projekt został zamknięty

Data podpisania przez KE 23 Marca 2015

Data rozpoczęcia 13 Kwietnia 2015

Data zakończenia 12 Kwietnia 2017

Finansowanie w ramach

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Koszt całkowity

€ 183 454,80

Wkład UE

€ 183 454,80

Koordynowany przez

UNIVERSITY COLLEGE LONDON
United Kingdom

Periodic Reporting for period 1 - MitSyn (Alpha-synuclein and mitochondrial dysfunction: key links between Gaucher’s disease and Parkinson’s?)

Okres sprawozdawczy: 2015-04-13 do 2017-04-12

Podsumowanie kontekstu i ogólnych celów projektu

Mutations of a lysosomal enzyme termed glucocerebrosidase (GCase, encoded by the gene GBA) cause Gaucher’s disease (GD) but also have a strong association with Parkinson’s disease (PD). This enzyme is responsible for the recycling of a special class of lipids called glucosylcermides, which are cleaved into glucose and ceramides molecules inside the lysosomes. We have previously shown that GCase knockout in mice causes defects in the autophagy lysosomal pathway, which is responsible in cells to remove harmful macromolecules and dysfunctional organelles and recycle their primary components. These defects lead to profound mitochondrial dysfunction and accumulation of a protein termed alpha-synuclein (aS), a hallmark of PD. The mechanisms that drive impaired mitochondrial dysfunction in association with impaired activity of a lysosomal enzyme remain obscure. Thus, the basic objectives of the MitSyn project are i) to explore the mechanisms that associate mutations or deficiencies in GCase with mitochondrial dysfunction and aS accumulation, and ii) to explore the consequences of lysosomal and mitochondrial dysfunction on cell signaling pathways. To this end, we have used: i) primary neuronal and astrocytic cultures from mice in which GCase was knocked out, a model for the neuropathic form of the lysosomal storage disorder GD and ii) iPSC-derived neurons from patients with PD carrying mutations of the GBA gene. In these models, we characterized impairment of autophagy and lysosomal defects, mitochondrial dysfunction and impaired calcium homeostasis. Moreover, we modulated aS levels to study its role in mitochondrial damage.
The Mitsyn project allowed to further characterize the interplay between lysosomes and mitochondria, which seems to be crucial in the aethiopathogenesis of several neurodegenerative disorders.

Prace wykonane od początku projektu do końca okresu sprawozdawczego oraz najważniejsze dotychczasowe rezultaty

In primary neuronal culture from gba-/-, gba-/+ and gba+/+ mice, mitochondrial defects were previously assessed in the lab (Osellame et al. 2013 Cell Metabolism). However, we wished to explore the impact of this mitochondrial dysfunction on cell physiology. We therefore measured free radical production and calcium signaling in these models. Interestingly, mitochondrial calcium uptake in response to neurons stimulation through the neurotransmitter glutamate was reduced in gba-/- and gba-/+ compared gba+/+ neurons. At the same time, we found that the peak cytosolic calcium level reached after glutamate stimulation was increased in gba-/- neurons. The gba-/- neurons also showed delayed calcium deregulation, normally associated with glutamate toxicity, in response to low doses of glutamate that were innocuous for the control gba+/+ neurons. Measurements of the levels of expression of the protein components of the mitochondrial calcium uniporter (MCU) complex, the machinery responsible for the calcium uptake into mitochondria, showed that impaired calcium homeostasis is not only associated with the mitochondrial membrane potential reduction, but is accompanied by altered protein expression. mRNA expression levels of the protein components of the MCU and its regulators and of glutamate receptors also showed some dysregulation, in agreement with the calcium imaging experiments.
Moreover, we started unraveling the signaling mechanisms responsible for autophagy impairment upstream of lysosomes in gba-/- neurons, brains and mouse embryonic fibroblasts, using lipidomics approaches and studying Akt/mTOR signaling pathways.
We have also generated human iPSC lines from fibroblasts from patients with PD carrying the fascinating E326K mutation of GBA, which is linked to PD but not to GD even when homozygous, and we obtained iPSC lines derived from PD patients carrying the L444P and the N370S GBA mutations to have a more complete picture of GBA-associated PD. After the iPSC characterization, they were differentiated to dopaminergic neurons adapting a protocol published in Nature by Kriks et al. (2011). Neurons were characterized in term of neuronal and dopaminergic markers expression and in term of glutamate and high potassium response, measured as variation in cytosolic calcium.
Preliminary experiments on mitochondrial function in iPSC-derived dopaminergic neurons after their characterization were done: mitochondrial membrane potential, free radical production and cytosolic calcium levels upon high potassium stimulation were measured. Given the high variability in these cultures, more replicates will be needed to draw any conclusion.

The results obtained so far were presented at different international conferences (EBEC 2016 and GRC Lysosomal Diseases 2017) and at local/international seminars. At least two publications will be soon published summarizing the results obtained during the Mitsyn project.
A review on the mitochondrial dysfunction in lysosomal diseases was also published (Plotegher N, Duchen MR. Mitochondrial Dysfunction and Neurodegeneration in Lysosomal Storage Disorders. Trends Mol Med. 2017).

Innowacyjność oraz oczekiwany potencjalny wpływ (w tym dotychczasowe znaczenie społeczno-gospodarcze i szersze implikacje społeczne projektu)

We have demonstrated that mitochondrial dysfunction in the GBA knock out model is associated with dysregulation of calcium homeostasis and increased free radical production. We produced a new iPSC-derived neuronal models for GBA-linked PD that show mitochondrial impairment and we plan to use these cells to screen drugs and molecules able to impact on the disrupted pathways. We are setting new links between PD and lysosomal storage disorders, especially in terms of common features as mitochondrial damage and aS. These links may be helpful in understanding the aetiopathogenesis of different disorders and establish new and (maybe common) therapeutic approaches.

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