PD-MitoQUANT Publications

α-synuclein oligomers and fibrils: a spectrum of species, a spectrum of toxicities

Alam, P., Bousset, L., Melki, R., Otzen, DE. Journal of Neurochemistry (2019)

http://doi.org/10.1111/jnc.14808

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This review article provides an overview of the different species that a-synuclein aggregates can populate. It also attempts to reconcile conflicting views regarding the cytotoxic roles of oligomers versus fibrils. a-synuclein, while highly dynamic in the monomeric state, can access a large number of different assembly states. Depending on assembly conditions, these states can interconvert over different timescales. The fibrillar state is the most thermodynamically favored due to the many stabilizing interactions formed between each monomeric unit, but different fibrillar types form at different rates. The end distribution is likely to reflect kinetic partitioning as much as thermodynamic equilibra. In addition, metastable oligomeric species, some of which are on-pathway and others offpathway, can be populated for remarkably long periods of time. Chemical modifications (phosphorylation, oxidation, covalent links to ligands, etc.) perturb these physical interconversions and invariably destabilize the fibrillar state, leading to small prefibrillar assemblies which can coalesce into amorphous states. Both oligomeric and fibrillar species have been shown to be cytotoxic although firm conclusions require very careful evaluation of particle concentrations and is complicated by the great variety and heterogeneity of different experimentally observed states. The mechanistic relationship between oligomers and fibrils remains to be clarified, both in terms of assembly of oligomers into fibrils and potential dissolution of fibrils into oligomers. While oligomers are possibly implicated in the collapse of neuronal homeostasis, the fibrillar state(s) appears to be the most efficient at propagating itself both in vitro and in vivo, pointing to critical roles for multiple different aggregate species in the progression of Parkinson’s disease. 

The PINK1 kinase-driven ubiquitin ligase Parkin promotes mitochondrial protein import through the presequence pathway in living cells

M. Jacoupy, E. Hamon-Keromen, A.Ordureau, Z. Erpapazoglou1 et al. Scientific Reports 14 August 2019

https://doi.org/10.1038/s41598-019-47352-9

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Most of over a thousand mitochondrial proteins are encoded by nuclear genes and must be imported from the cytosol. Little is known about the cytosolic events regulating mitochondrial protein import, partly due to the lack of appropriate tools for its assessment in living cells. We engineered an inducible biosensor for monitoring the main presequence-mediated import pathway with a quantitative, luminescence-based readout. This tool was used to explore the regulation of mitochondrial import by the PINK1 kinase-driven Parkin ubiquitin ligase, which is dysfunctional in autosomal recessive Parkinson’s disease. We show that mitochondrial import was stimulated by Parkin, but not by disease-causing Parkin variants. This effect was dependent on Parkin activation by PINK1 and accompanied by an increase in the abundance of K11 ubiquitin chains on mitochondria and by ubiquitylation of subunits of the translocase of outer mitochondrial membrane. Mitochondrial import efficiency was abnormally low in cells from patients with PINK1- and PARK2-linked Parkinson’s disease and was restored by phosphomimetic ubiquitin in cells with residual Parkin activity. Altogether, these findings uncover a role of ubiquitylation in mitochondrial import regulation and suggest that loss of this regulatory loop may underlie the pathophysiology of Parkinson’s disease, providing novel opportunities for therapeutic intervention.

α-Synuclein conformational strains spread, seed and target neuronal cells differentially after injection into the olfactory bulb

Nolwen L. Rey, Luc Bousset, Sonia George, Zachary Madaj, Lindsay Meyerdirk, Emily Schulz, Jennifer A. Steiner, Ronald Melki and Patrik Brundin. Acta Neuropathologica Communications, 30 December 2019.

https://doi.org/10.1186/s40478-019-0859-3

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Alpha-synuclein inclusions, the hallmarks of synucleinopathies, are suggested to spread along neuronal connections in a stereotypical pattern in the brains of patients. Ample evidence now supports that pathological forms of alpha-synuclein propagate in cell culture models and in vivo in a prion-like manner. However, it is still not known why the same pathological protein targets different cell populations, propagates with different kinetics and leads to a variety of diseases (synucleinopathies) with distinct clinical features. The aggregation of the protein alpha-synuclein yields different conformational polymorphs called strains. These strains exhibit distinct biochemical, physical and structural features they are able to imprint to newly recruited alpha-synuclein. This had led to the view that the clinical heterogeneity observed in synucleinopathies might be due to distinct pathological alpha-synuclein strains.

To investigate the pathological effects of alpha-synuclein strains in vivo, we injected five different pure strains we generated de novo (fibrils, ribbons, fibrils-65, fibrils-91, fibrils-110) into the olfactory bulb of wild-type female mice. We demonstrate that they seed and propagate pathology throughout the olfactory network within the brain to different extents. We show strain-dependent inclusions formation in neurites or cell bodies. We detect thioflavin S-positive inclusions indicating the presence of mature amyloid aggregates.

In conclusion, alpha-synuclein strains seed the aggregation of their cellular counterparts to different extents and spread differentially within the central nervous system yielding distinct propagation patterns. We provide here the proof-of-concept that the conformation adopted by alpha-synuclein assemblies determines their ability to amplify and propagate in the brain in vivo. Our observations support the view that alpha-synuclein polymorphs may underlie different propagation patterns within human brains.

Differential Membrane Binding and Seeding of Distinct α-Synuclein Fibrillar Polymorphs.

AN Shrivastava, Luc Bousset, M Renner, V Redeker, J Savistchenko, A Triller, Ronald Melki. 2020 January 28. pii: S0006-3495(20)30069-2.

doi: 10.1016/j.bpj.2020.01.022

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The aggregation of the protein α-synuclein (α-Syn) leads to different synucleinopathies. We recently showed that structurally distinct fibrillar α-Syn polymorphs trigger either Parkinson’s disease or multiple system atrophy hallmarks in vivo. Here, we establish a structural-molecular basis for these observations. We show that distinct fibrillar α-Syn polymorphs bind to and cluster differentially at the plasma membrane in both primary neuronal cultures and organotypic hippocampal slice cultures from wild-type mice. We demonstrate a polymorph-dependent and concentration-dependent seeding. We show a polymorph-dependent differential synaptic redistribution of α3-Na+/K+-ATPase, GluA2 subunit containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and GluN2B-subunit containing N-methyl-D-aspartate receptors, but not GluA1 subunit containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and metabotropic glutamate receptor 5 receptors. We also demonstrate polymorph-dependent alteration in neuronal network activity upon seeded aggregation of α-Syn. Our findings bring new, to our knowledge, insight into how distinct α-Syn polymorphs differentially bind to and seed monomeric α-Syn aggregation within neurons, thus affecting neuronal homeostasis through the redistribution of synaptic proteins.

The expression level of alpha-synuclein in different neuronal populations is the primary determinant of its prion-like seeding.

Josquin Courte, Luc Bousset, Ysander Von Boxberg, Catherine Villard, Ronald Melki & Jean-Michel Peyrin. Scientific Reports volume 10, Article number: 4895 (2020) doi: https://doi.org/10.1038/s41598-020-61757-x

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Alpha-synuclein (aSyn)-rich aggregates propagate in neuronal networks and compromise cellular homeostasis leading to synucleinopathies such as Parkinson’s disease. Aggregated aSyn spread follows a conserved spatio-temporal pattern that is not solely dependent on connectivity. Hence, the differential tropism of aSyn-rich aggregates to distinct brain regions, or their ability to amplify within those regions, must contribute to this process. To better understand what underlies aSyn-rich aggregates distribution within the brain, we generated primary neuronal cultures from various brain regions of wild-type mice and mice expressing a reduced level of aSyn, and exposed them to fibrillar aSyn. We then assessed exogenous fibrillar aSyn uptake, endogenous aSyn seeding, and endogenous aSyn physiological expression levels. Despite a similar uptake of exogenous fibrils by neuronal cells from distinct brain regions, the seeded aggregation of endogenous aSyn differed greatly from one neuronal population to another. The different susceptibility of neuronal populations was linked to their aSyn expression level. Our data establish that endogenous aSyn expression level plays a key role in fibrillar aSyn prion-like seeding, supporting that endogenous aSyn expression level participates in selective regional brain vulnerability.

Interaction of the chaperones alpha B-crystallin and CHIP with fibrillar alpha-synuclein: Effects on internalization by cells and identification of interacting interfaces.

Maya Bendifallah, Virginie Redeker, Elodie Monsellier, Luc Bousset, Tracy Bellande, Ronald Melki Biochemical and Biophysical Research Communications 2020 May 16. https://doi.org/10.1016/j.bbrc.2020.04.091

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The spread of fibrillar alpha-synuclein from affected to naïve neuronal cells is thought to contribute to the progression of synucleinopathies. The binding of fibrillar alpha-synuclein to the plasma membrane is key in this process. We and others previously showed that coating fibrillar alpha-synuclein by the molecular chaperone Hsc70 affects fibrils properties. Here we assessed the effect of the two molecular chaperones alpha B-crystallin and CHIP on alpha-synuclein fibrils uptake by Neuro-2a cells. We demonstrate that both chaperones diminish fibrils take up by cells. We identify through a cross-linking and mass spectrometry strategy the interaction interfaces between alpha-synuclein fibrils and alpha B-crystallin or CHIP. Our results open the way for designing chaperone-derived polypeptide binders that interfere with the propagation of pathogenic alpha-synuclein assemblies.

AMPK preferentially depresses retrograde transport of axonal mitochondria during localised nutrient deprivation.

Orla WattersNiamh M. C. ConnollyHans-Georg KönigHeiko Düssmann and Jochen H. M. Prehn  2020. https://doi.org/10.1523/JNEUROSCI.2067-19.2020

Repository link here. Under embargo for 6 months.

Mitochondrial clusters are found at regions of high energy demand, allowing cells to meet local metabolic requirements while maintaining neuronal homeostasis. AMP-activated protein kinase (AMPK), a key energy stress sensor, responds to increases in AMP/ATP ratio by activating multiple signalling cascades to overcome the energetic deficiency. In many neurological conditions, the distal axon experiences energetic stress independent of the soma. Here, we used microfluidic devices to physically isolate these two neuronal structures and to investigate whether localised AMPK signalling influenced axonal mitochondrial transport. Nucleofection of primary cortical neurons, derived from E16 mouse embryos (both sexes), with mito-GFP allowed monitoring of the transport dynamics of mitochondria within the axon, by confocal microscopy.

Pharmacological activation of AMPK at the distal axon (0.1 mM AICAR) induced a depression of the mean frequency, velocity and distance of retrograde mitochondrial transport in the adjacent axon. Anterograde mitochondrial transport was less sensitive to local AMPK stimulus, with the imbalance of bi-directional mitochondrial transport resulting in accumulation of mitochondria at the region of energetic stress signal. Mitochondria in the axon-rich white matter of the brain rely heavily on lactate as a substrate for ATP synthesis. Interestingly, localised inhibition of lactate uptake (10 nM AR-C155858) reduced mitochondrial transport in the adjacent axon in all parameters measured, similar to that observed by AICAR treatment. Co-addition of compound C restored all parameters measured to baseline levels, confirming the involvement of AMPK. This study highlights a role of AMPK signalling in the depression of axonal mitochondrial mobility during localised energetic stress.

SIGNIFICANCE STATEMENT

As the main providers of cellular energy, the dynamic transport of mitochondria within the neuron allows for clustering at regions of high energy demand. Here we investigate whether acute changes in energetic stress signal in the spatially isolated axon would alter mitochondrial transport in this local region. Both direct and indirect activation of AMP-activated protein kinase (AMPK) isolated to the distal axon induced a rapid, marked depression in local mitochondrial transport. This work highlights the ability of acute localised AMPK signalling to affect mitochondrial mobility within the axon, with important implications for white matter injury, axonal growth and axonal degeneration.