PD-MitoQUANT Publications

Modeling native and seeded Synuclein aggregation and related cellular dysfunctions in dopaminergic neurons derived by a new set of isogenic iPSC lines with SNCA multiplications
Triplication of the SNCA gene, encoding the protein alpha-Synuclein (αSyn), is a rare cause of aggressive and early-onset parkinsonism. Herein, we generated iPSCs from two siblings with a recently described compact SNCA gene triplication and suffering from severe motor impairments, psychiatric symptoms, and cognitive deterioration. Using CRISPR/Cas9 gene editing, each SNCA copy was inactivated by targeted indel mutations generating a panel of isogenic iPSCs with a decremental number from 4 down to none of functional SNCA gene alleles. We differentiated these iPSC lines in midbrain dopaminergic (DA) neuronal cultures to characterize αSyn aggregation in native and seeded conditions and evaluate its associated cellular dysfunctions. Utilizing a new nanobody-based biosensor combined with super-resolved imaging, we were able to visualize and measure αSyn aggregates in early DA neurons in unstimulated conditions. Calcium dysregulation and mitochondrial alterations were the first pathological signs detectable in early differentiated DA neuronal cultures. Accelerated αSyn aggregation was induced by exposing neurons to structurally well-characterized synthetic αSyn fibrils. 4xSNCA DA neurons showed the highest vulnerability, which was associated with high levels of oxidized DA and amplified by TAX1BP1 gene disruption. Seeded DA neurons developed large αSyn deposits whose morphology and internal constituents resembled Lewy bodies commonly observed in Parkinson’s disease (PD) patient brain tissues. These findings provide strong evidence that this isogenic panel of iPSCs with SNCA multiplications offers a remarkable cellular platform to investigate mechanisms of PD and validate candidate inhibitors of native and seeded αSyn aggregation.

Neuronal hyperactivity–induced oxidant stress promotes in vivo alpha-synuclein brain spreading
Interneuronal transfer and brain spreading of pathogenic proteins are features of neurodegenerative diseases. Pathophysiological conditions and mechanisms affecting this spreading remain poorly understood. This study investigated the relationship between neuronal activity and interneuronal transfer of α-synuclein, a Parkinson-associated protein, and elucidated mechanisms underlying this relationship. In a mouse model of α-synuclein brain spreading, hyperactivity augmented and hypoactivity attenuated protein transfer. Important features of neuronal hyperactivity reported here were an exacerbation of oxidative and nitrative reactions, pronounced accumulation of nitrated α-synuclein, and increased protein aggregation. Data also pointed to mitochondria as key targets and likely sources of reactive oxygen and nitrogen species within hyperactive neurons. Rescue experiments designed to counteract the increased burden of reactive oxygen species reversed hyperactivity-induced α-synuclein nitration, aggregation, and interneuronal transfer, providing first evidence of a causal link between these pathological effects of neuronal stimulation and indicating a mechanistic role of oxidant stress in hyperactivity-induced α-synuclein spreading.

AIFM1 beyond cell death: An overview of this OXPHOS-inducing factor in mitochondrial diseases
Apoptosis-inducing factor (AIF) is a mitochondrial intermembrane space flavoprotein with diverse functions in cellular physiology. In this regard, a large number of studies have elucidated AIF’s participation to chromatin condensation during cell death in development, cancer, cardiovascular and brain disorders. However, the discovery of rare AIFM1 mutations in patients has shifted the interest of biomedical researchers towards AIF’s contribution to pathogenic mechanisms underlying inherited AIFM1-linked metabolic diseases. The functional characterization of AIF binding partners has rapidly advanced our understanding of AIF biology within the mitochondria and beyond its widely reported role in cell death. At the present time, it is reasonable to assume that AIF contributes to cell survival by promoting biogenesis and maintenance of the mitochondrial oxidative phosphorylation (OXPHOS) system. With this review, we aim to outline the current knowledge around the vital role of AIF by primarily focusing on currently reported human diseases that have been linked to AIFM1 deficiency.

Phenomenological equations for electron transport chain-mediated reactive oxygen species metabolism
Reactive oxygen species (ROS) are produced as metabolic by-products throughout the cell, including within the mitochondria by, among others, the electron transport chain (ETC). ROS are important signalling molecules, but their accumulation can lead to oxidative stress. As experimental protocols to measure cellular ROS levels can suffer from limitations, computational models can complement experimental measurements to elucidate mechanisms underlying ROS metabolism. Phenomenological models utilise reduced equation sets to relate aspects of ROS metabolism to measured bioenergetic parameters such as the mitochondrial membrane potential or NADH concentration, and often generate experimentally testable hypotheses. Integrating multiple bioenergetic parameters, we here describe a phenomenological equation-based model of ETC-mediated ROS metabolism implemented by expanding an existing thermodynamic model of the ETC. We demonstrate that this model can reproduce experimentally observed behaviour, and explore how the model can be applied to gain insight into ROS metabolism in the presence of pathology. Model predictions suggest that failure of scavenging may be more harmful to cells than respiratory complex impairment.
Why is this study important?
Reactive oxygen species (ROS) are a group of small, reactive, chemical molecules. ROS play many important roles in cells when at optimal levels. However, when the levels of ROS increase, they can lead to cellular ‘oxidative’ stress, which is implicated in various disease conditions. We still don’t fully understand the complete ROS lifecycle, and there are various difficulties associated with experiments to investigate ROS (such as difficulties distinguishing different ROS types or their site of origin, and varying sensitivity and specificity of different methods). Computational models (mathematical representations of the ROS system) can be used to complement experiments and analyse ROS metabolism in more detail.
Aims
Computational models can be detailed or minimal, depending on the amount of detail included. Detailed models may be closer representations of the complete biological system, but can be difficult to develop, run, and analyse. Minimal models are simplified representations of the biological system that enable analysis of more general system behaviour in specific contexts. In this paper, we aimed to design a minimal model to study the production and degradation of ROS in neurodegenerative conditions.
What did we do?
We had previously developed a computational model of the respiratory chain, the chain of enzymes responsible for producing adenosine triphosphate (ATP), the primary energy source in our cells. The respiratory chain is one of the main producers of ROS, and is also affected in neurodegenerative disorders like Parkinson’s. Here, we expanded this model to include the production of ROS by the respiratory chain. We first compared the behaviour of our model with known experiments, to ensure the model simulations were representative of the biological system. We then varied some of the parameters in the model to simulate dysfunctional activity observed in neurodegeneration, and monitored how ROS levels were predicted to be altered in these conditions.
What did we find?
We validated that the model simulations reproduce experimentally observed behaviour in several conditions. We next simulated reduced activity of the enzyme complexes along the respiratory chain, as this has been observed in Parkinson’s disease and other neurodegenerative conditions. The model predicted that reduced activity of the complexes in isolation is not sufficient to cause oxidative stress. In fact, the model predicted that malfunction in the clearance of ROS (ROS ‘scavenging’), rather than in the respiratory chain processes that produce it, are more likely to cause damaging oxidative stress.

Mechanisms and mathematical modelling of ROS production by the mitochondrial electron transport chain
Currently under embargo.
Reactive oxygen species (ROS) are recognised both as damaging molecules and intracellular signalling entities. In addition to its role in ATP generation, the mitochondrial electron transport chain (ETC) constitutes a relevant source of mitochondrial ROS, in particular during pathological conditions. Mitochondrial ROS homeostasis depends on species- and site-dependent ROS production, their bioreactivity, diffusion, and scavenging. However, our quantitative understanding of mitochondrial ROS homeostasis has thus far been hampered by technical limitations, including lack of truly site- and/or ROS-specific reporter molecules. In this context, the use of computational models is of great value to complement and interpret empirical data, as well as to predict variables that are difficult to assess experimentally. During the last decades, various mechanistic models of ETC-mediated ROS production have been developed. Although these often-complex models have generated novel insights, their parameterisation, analysis, and integration with other computational models is not straightforward. In contrast, phenomenological (sometimes termed “minimal”) models use a relatively small set of equations to describe empirical relationship(s) between ROS-related and other parameters, and generally aim to explore system behaviour and generate hypotheses for experimental validation. In this review, we first discuss ETC-linked ROS homeostasis and introduce various detailed mechanistic models. Next, we present how bioenergetic parameters (e.g. NADH/NAD+ ratio, mitochondrial membrane potential) relate to site-specific ROS production within the ETC and how these relationships can be used to design minimal models of ROS homeostasis. Finally, we illustrate how minimal models have been applied to explore pathophysiological aspects of ROS.
Why is this study important?
Reactive oxygen species (ROS) are small molecules that are important in our cells when at optimum levels. When ROS increase, however, they cause damaging ‘oxidative stress’, for example in Parkinson’s. Appropriate levels of cellular ROS (ROS homeostasis) depends on many factors, but our understanding has been hampered by a lack of specific, accurate techniques to measure ROS.
Mathematical models can be used to describe ROS production and consumption, to explore the function of ROS, and to help interpretation of experimental data.
The mitochondrial electron transport chain (ETC, a sequence of enzymes) produces adenosine triphosphate (ATP) the primary energy source of cells, but the ETC is also one of the main producers of ROS, particularly in disease.
Aims
In this review, we aimed to introduce readers to the mechanisms and mathematical modelling of ETC-linked ROS homeostasis.
What did we do?
We first described in detail the mechanisms underlying ROS production by the ETC, and the associated scavenging processes. We then introduced several detailed mathematical models of ROS homeostasis that have been published in the literature, and described how they have been applied to investigate ROS. Next, we outlined the role that key bioenergetic parameters (those parameters regulating ATP production by the ETC) play in producing ROS. We then demonstrated how these relationships can be used to design phenomenological mathematical models of ETC-mediated ROS homeostasis (a phenomenological model is based on observations, rather than on theory alone). Finally, we illustrated how these ‘minimal’ models have been applied to explore ROS in various conditions.
What does it mean?
This review can serve as a resource for the biological and computational research community interested in the mechanisms and mathematical modelling of ROS produced by the ETC. Increasing our knowledge of the processes underlying ROS homeostasis can help to understand pathways contributing to oxidative stress in disease.

Modelling α-Synuclein Aggregation and Neurodegeneration with Fibril Seeds in Primary Cultures of Mouse Dopaminergic Neurons
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To model α-Synuclein (αS) aggregation and neurodegeneration in Parkinson’s disease (PD), we established cultures of mouse midbrain dopamine (DA) neurons and chronically exposed them to fibrils 91 (F91) generated from recombinant human αS. We found that F91 have an exquisite propensity to seed the aggregation of endogenous αS in DA neurons when compared to other neurons in midbrain cultures. Until two weeks post-exposure, somal aggregation in DA neurons increased with F91 concentrations (0.01–0.75 μM) and the time elapsed since the initiation of seeding, with, however, no evidence of DA cell loss within this time interval. Neither toxin-induced mitochondrial deficits nor genetically induced loss of mitochondrial quality control mechanisms promoted F91-mediated αS aggregation or neurodegeneration under these conditions. Yet, a significant loss of DA neurons (~30%) was detectable three weeks after exposure to F91 (0.5 µM), i.e., at a time point where somal aggregation reached a plateau. This loss was preceded by early deficits in DA uptake. Unlike αS aggregation, the loss of DA neurons was prevented by treatment with GDNF, suggesting that αS aggregation in DA neurons may induce a form of cell death mimicking a state of trophic factor deprivation. Overall, our model system may be useful for exploring PD-related pathomechanisms and for testing molecules of therapeutic interest for this disorder.

SGPL1 stimulates VPS39 recruitment to the mitochondria in MICU1 deficient cells
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Objective
Mitochondrial “retrograde” signaling may stimulate organelle biogenesis as a compensatory adaptation to aberrant activity of the oxidative phosphorylation (OXPHOS) system. To maintain energy-consuming processes in OXPHOS deficient cells, alternative metabolic pathways are functionally coupled to the degradation, recycling and redistribution of biomolecules across distinct intracellular compartments. While transcriptional regulation of mitochondrial network expansion has been the focus of many studies, the molecular mechanisms promoting mitochondrial maintenance in energy-deprived cells remain poorly investigated.
Methods
We performed transcriptomics, quantitative proteomics and lifespan assays to identify pathways that are mechanistically linked to mitochondrial network expansion and homeostasis in Caenorhabditis elegans lacking the mitochondrial calcium uptake protein 1 (MICU-1/MICU1). To support our findings, we carried out biochemical and image analyses in mammalian cells and mouse-derived tissues.
Results
We report that micu-1(null) mutations impair the OXPHOS system and promote C. elegans longevity through a transcriptional program that is independent of the mitochondrial calcium uniporter MCU-1/MCU and the essential MCU regulator EMRE-1/EMRE. We identify sphingosine phosphate lyase SPL-1/SGPL1 and the ATFS-1-target HOPS complex subunit VPS-39/VPS39 as critical lifespan modulators of micu-1(null) mutant animals. Cross-species investigation indicates that SGPL1 upregulation stimulates VPS39 recruitment to the mitochondria, thereby enhancing mitochondria-lysosome contacts. Consistently, VPS39 downregulation compromises mitochondrial network maintenance and basal autophagic flux in MICU1 deficient cells. In mouse-derived muscles, we show that VPS39 recruitment to the mitochondria may represent a common signature associated with altered OXPHOS system.
Conclusions
Our findings reveal a previously unrecognized SGPL1/VPS39 axis that stimulates intracellular organelle interactions and sustains autophagy and mitochondrial homeostasis in OXPHOS deficient cells.

Sphingolipid changes in Parkinson L444P GBA mutation fibroblasts promote α-synuclein aggregation
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Intraneuronal accumulation of aggregated α-synuclein is a pathological hallmark of Parkinson’s disease. Therefore, mechanisms capable of promoting α-synuclein deposition bear important pathogenetic implications. Mutations of the glucocerebrosidase 1 (GBA) gene represent a prevalent Parkinson’s disease risk factor. They are associated with loss of activity of a key enzyme involved in lipid metabolism, glucocerebrosidase, supporting a mechanistic relationship between abnormal α-synuclein–lipid interactions and the development of Parkinson pathology.
In this study, the lipid membrane composition of fibroblasts isolated from control subjects, patients with idiopathic Parkinson’s disease and Parkinson’s disease patients carrying the L444P GBA mutation (PD-GBA) was assayed using shotgun lipidomics.
The lipid profile of PD-GBA fibroblasts differed significantly from that of control and idiopathic Parkinson’s disease cells. It was characterized by an overall increase in sphingolipid levels. It also featured a significant increase in the proportion of ceramide, sphingomyelin and hexosylceramide molecules with shorter chain length and a decrease in the percentage of longer-chain sphingolipids. The extent of this shift was correlated to the degree of reduction of fibroblast glucocerebrosidase activity. Lipid extracts from control and PD-GBA fibroblasts were added to recombinant α-synuclein solutions. The kinetics of α-synuclein aggregation were significantly accelerated after addition of PD-GBA extracts as compared to control samples. Amyloid fibrils collected at the end of these incubations contained lipids, indicating α-synuclein–lipid co-assembly. Lipids extracted from α-synuclein fibrils were also analysed by shotgun lipidomics. Data revealed that the lipid content of these fibrils was significantly enriched by shorter-chain sphingolipids. In a final set of experiments, control and PD-GBA fibroblasts were incubated in the presence of the small molecule chaperone ambroxol. This treatment restored glucocerebrosidase activity and sphingolipid levels and composition of PD-GBA cells. It also reversed the pro-aggregation effect that lipid extracts from PD-GBA fibroblasts had on α-synuclein.
Taken together, the findings of this study indicate that the L444P GBA mutation and consequent enzymatic loss are associated with a distinctly altered membrane lipid profile that provides a biological fingerprint of this mutation in Parkinson fibroblasts. This altered lipid profile could also be an indicator of increased risk for α-synuclein aggregate pathology.

CEST-2.2 overexpression alters lipid metabolism and extends longevity of mitochondrial mutants
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Mitochondrial dysfunction can either extend or decrease Caenor-habditis elegans lifespan, depending on whether transcriptionally regulated responses can elicit durable stress adaptation to other-wise detrimental lesions. Here, we test the hypothesis that enhanced metabolic flexibility is sufficient to circumvent bioenergetic abnormalities associated with the phenotypic threshold effect, thereby transforming short-lived mitochondrial mutants into long-lived ones. We find that CEST-2.2, a carboxylesterase mainly localizes in the intestine, may stimulate the survival of mitochondrial deficient animals. We report that genetic manipulation of cest-2.2expression has a minor lifespan impact on wild-type nematodes, whereas its overexpression markedly extends the life span of complex I-deficient gas–1(fc21)mutants. We profile the transcriptome and lipidome of cest-2.2overexpressing animals and show that CEST-2.2 stimulates lipid metabolism and fatty acid beta-oxidation, thereby enhancing mitochondrial respiratory capacity through complex II and LET-721/ETFDH, despite the inherited genetic lesion of complex I. Together, our findings unveil a metabolic pathway that, through the tissue-specific mobilization of lipid deposits, may influence the longevity of mitochondrial mutants elegans.

Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution through tunneling nanotubes
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TP53INP1 exerts neuroprotection under ageing and Parkinson’s disease-related stress condition
Emilie Dinh, Thomas Rival, Alice Carrier, Noemi Asfogo, Olga Corti, Christophe Melon, Pascal Salin, Sylviane Lortet and Lydia Kerkerian-Le Goff, Cell Death Dis 12, 460 (2021). https://doi.org/10.1038/s41419-021-03742-4
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TP53INP1 is a stress-induced protein, which acts as a dual positive regulator of transcription and of autophagy and whose deficiency has been linked with cancer and metabolic syndrome. Here, we addressed the unexplored role of TP53INP1 and of its Drosophila homolog dDOR in the maintenance of neuronal homeostasis under chronic stress, focusing on dopamine (DA) neurons under normal ageing- and Parkinson’s disease (PD)-related context. Trp53inp1−/− mice displayed additional loss of DA neurons in the substantia nigra compared to wild-type (WT) mice, both with ageing and in a PD model based on targeted overexpression of α-synuclein. Nigral Trp53inp1 expression of WT mice was not significantly modified with ageing but was markedly increased in the PD model. Trp53inp2 expression showed similar evolution and did not differ between WT and Trp53inp1−/− mice. In Drosophila, pan-neuronal dDOR overexpression improved survival under paraquat exposure and mitigated the progressive locomotor decline and the loss of DA neurons caused by the human α-synuclein A30P variant. dDOR overexpression in DA neurons also rescued the locomotor deficit in flies with RNAi-induced downregulation of dPINK1 or dParkin. Live imaging, confocal and electron microscopy in fat bodies, neurons, and indirect flight muscles showed that dDOR acts as a positive regulator of basal autophagy and mitophagy independently of the PINK1-mediated pathway. Analyses in a mammalian cell model confirmed that modulating TP53INP1 levels does not impact mitochondrial stress-induced PINK1/Parkin-dependent mitophagy. These data provide the first evidence for a neuroprotective role of TP53INP1/dDOR and highlight its involvement in the regulation of autophagy and mitophagy in neurons.

The differential solvent exposure of N-terminal residues provides ‘fingerprints’ of alpha-synuclein fibrillar polymorphs
Landureau M, Redeker V, Bellande T, Eyquem S, Melki R, Journal of Biological Chemistry (2021), https://doi.org/10.1016/j.jbc.2021.100737.
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Synucleinopathies are neurodegenerative diseases characterized by the presence of intracellular deposits containing the protein alpha-synuclein (aSYN) within patients’ brains. It has been shown that aSYN can form structurally distinct fibrillar assemblies, also termed polymorphs. We previously showed that distinct aSYN polymorphs assembled in vitro, named fibrils, ribbons and fibrils 91, differentially bind to and seed the aggregation of endogenous aSYN in neuronal cells, which suggests that distinct synucleinopathies may arise from aSYN polymorphs. In order to better understand the differential interactions of aSYN polymorphs with their partner proteins, we mapped aSYN polymorphs surfaces. We used limited proteolysis, hydrogen-deuterium exchange, and differential antibody accessibility to identify amino-acids on their surfaces. We showed that the aSYN Cterminal region spanning residues 94-140 exhibited similarly high solvent accessibility in these three polymorphs. However, the N-terminal amino acid residues 1-38 of fibrils were exposed to the solvent, while only residues 1-18 within fibrils 91 were exposed, and no N-terminal residues within ribbons were solvent-exposed. It is likely that these differences in surface accessibility contribute to the differential binding of distinct aSYN polymorphs to partner proteins. We thus posit that the polypeptides exposed on the surface of distinct aSYN fibrillar polymorphs are comparable to fingerprints. Our findings have diagnostic and therapeutic potential, particularly in the prion-like propagation of fibrillar aSYN, as they can facilitate the design of ligands that specifically bind and distinguish between fibrillar polymorphs.
What is alpha-synuclein and why is it important in Parkinson’s disease?
Alpha-synuclein is a highly flexible protein that adopts multiple shapes. Prof. Ronald Melki has illustrated the dynamic properties of this protein in a cartoon made by France Parkinson and available on youtube. Among the many shapes this protein adopts, there are forms that can interact with molecules of similar shape to form stacks that are harmful to neurons (nerve cells in the brain), in particular the neurons that synthesise dopamine. Dopamine is a chemical that transmits nerve impulses across the connections (synapses) between neurons.
These stacks of alpha-synuclein are normally removed but the clearance system is not 100% efficient and its efficiency decreases with time. Problems with the clearance system happen as we age or in people with neurodegenerative diseases, like Parkinson’s. The result is that the alpha-synuclein stacks remain and accumulate over time, leading to neuronal stress through synaptic and mitochondrial dysfunction, and ultimately to neuronal death.
There is evidence that alpha-synuclein aggregates spread from affected neurons to healthy neurons. This contamination process contributes to disease progression. Stopping this process should slow disease progression, so understanding the effects of alpha-synuclein on the brain is an area of interest for drug development for neurodegenerative diseases.
What did this study find?
We have demonstrated recently that different forms of alpha-synuclein aggregate into stacks that bind differentially to neurons, spread differentially in the brain of animal models and yield Parkinson’s characteristic Lewy bodies or inclusions in oligodendrocytes (a type of cell that supports neurons) that are the hallmark of multiple system atrophy, another pathology associated with alpha-synuclein (synucleinopathy). In this study, we identified the surfaces of distinct alpha-synuclein stacks as they define binding to neurons and interaction with cellular proteins or organelles, in particular the mitochondria.
So what do the study results mean?
One part of alpha-synuclein distinguishes one pathological stack from another, while another part characterises all pathogenic alpha-synuclein stacks. Finally, we singled out parts that are not exposed at all at the surfaces of alpha-synuclein pathogenic aggregates.
What is next?
The differential recognition of different alpha-synuclein stacks has early diagnostic, as well as therapeutic, potential. We will be validating our finding in brain tissues donated by patients to brain bio-banks. We aim to develop specific binders for distinct alpha-synuclein pathogenic aggregates. We also aim to identify the mitochondrial proteins that interact with the alpha-synuclein regions that we have identified in this paper. The latter will be done within the PD-MitoQUANT project.

AMPK preferentially depresses retrograde transport of axonal mitochondria during localised nutrient deprivation.
Orla Watters, Niamh M. C. Connolly, Hans-Georg König, Heiko Düssmann and Jochen H. M. Prehn Journal of Neuroscience 11 May 2020. https://doi.org/10.1523/JNEUROSCI.2067-19.2020
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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.
What are mitochondria and why are they important for people with neurological diseases?
The axon is a long, cable-like part of a nerve cell that helps carry information from our brain to other nerve cells and to our muscles throughout our bodies. Mitochondria act like a digestive system for the cell, taking in nutrients and breaking them down into energy. This energy is used as fuel to move them along the axon. It’s not just a one-way system though – these cells and mitochondria are constantly communicating and responding to the differing needs of each individual cell. This means that mitochondria can travel throughout the cell redirecting their energy to where it’s most needed, which is vital for healthy brain function.
There is increasing evidence that impaired mitochondrial function could be associated with many neurological diseases including stroke, amyotrophic lateral sclerosis (ALS), Alzheimer’s and Parkinson’s, but detailed understanding of the cause and effect of this impairment is lacking.
What did this study find?
This study shows that when energy levels are critically low in a region of the axon, mitochondria can build up at the site of this energy stress. As energy is required for the movement of mitochondria, it is unsurprising that we found that when cells are under stress and running low on energy, mitochondrial movement slows down. We show that this is a result of a special protein, called AMP-activated protein kinase or AMPK becoming active. By blocking this protein, we were able to reverse the effect, increasing mitochondrial movement back to its normal levels. Our most interesting finding is that AMPK activity has a bigger effect on slowing down the movement of mitochondria away from the site of energy stress than those moving towards this region. This means that there are more mitochondria at the site of energy stress, where they work to produce more energy to try to restore the proper functioning of this region of the cell.
So what do the study results mean?
This study shows that mitochondrial transport is altered in response to local energy stress in nerve cells. We don’t know how important this will be yet, but this research is exciting as energy stress, mitochondrial impairment and ultimately cell death play a role in the development of neurological diseases.
What’s next?
We are now continuing this research to study whether changes in mitochondrial transport are a piece of the puzzle in how impaired mitochondrial function contributes to neurological diseases as part of the PD-MitoQUANT project.

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.

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.

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. Biophysical Journal. 2020 January 28. pii: S0006-3495(20)30069-2.
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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.

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.
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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.

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
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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)
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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.