Alonso A et al. (2023) Commun Biol "Fast detection of slender bodies in high density microscopy data."

Kirkegaard JB, Alonso A

[Commun Biol, 2023]

Computer-aided analysis of biological microscopy data has seen a massive improvement with the utilization of general-purpose deep learning techniques. Yet, in microscopy studies of multi-organism systems, the problem of collision and overlap remains challenging. This is particularly true for systems composed of slender bodies such as swimming nematodes, swimming spermatozoa, or the beating of eukaryotic or prokaryotic flagella. Here, we develop a end-to-end deep learning approach to extract precise shape trajectories of generally motile and overlapping slender bodies. Our method works in low resolution settings where feature keypoints are hard to define and detect. Detection is fast and we demonstrate the ability to track thousands of overlapping organisms simultaneously. While our approach is agnostic to area of application, we present it in the setting of and exemplify its usability on dense experiments of swimming Caenorhabditis elegans. The model training is achieved purely on synthetic data, utilizing a physics-based model for nematode motility, and we demonstrate the model's ability to generalize from simulations to experimental videos.

Perni M et al. (2018) J Neurosci Methods "Massively parallel C. elegans tracking provides multi-dimensional fingerprints for phenotypic discovery."

Casford S, Vendruscolo M, Muller T, Challa PK, Dobson CM, Perni M, Hardenberg MC, Nollen EAA, Koopman M, Fernando NW, Roode LWY, Vecchi G, Saar KL, Knowles TPJ, Sormanni P, Limbocker R, Kirkegaard JB, Habchi J

[J Neurosci Methods, 2018]

BACKGROUND: The nematode worm C. elegans is a model organism widely used for studies of genetics and of human disease. The health and fitness of the worms can be quantified in different ways, such as by measuring their bending frequency, speed or lifespan. Manual assays, however, are time consuming and limited in their scope providing a strong motivation for automation. NEW METHOD: We describe the development and application of an advanced machine vision system for characterizing the behaviour of C. elegans, the Wide Field-of-view Nematode Tracking Platform (WF-NTP), which enables massively parallel data acquisition and automated multi-parameter behavioural profiling of thousands of worms simultaneously. RESULTS: We screened more than a million worms from several established models of neurodegenerative disorders and characterised the effects of potential therapeutic molecules against Alzheimer's and Parkinson's diseases. By using very large numbers of animals we show that the sensitivity and reproducibility of behavioural assays is very greatly increased. The results reveal the ability of this platform to detect even subtle phenotypes. COMPARISON WITH EXISTING METHODS: The WF-NTP method has substantially greater capacity compared to current automated platforms that typically either focus on characterising single worms at high resolution or tracking the properties of populations of less than 50 animals. CONCLUSIONS: The WF-NTP extends significantly the power of existing automated platforms by combining enhanced optical imaging techniques with an advanced software platform. This approach will further extend the scope and utility of C. elegans as a model organism.

Perni M et al. (2018) ACS Chem Biol "Multistep Inhibition of -Synuclein Aggregation and Toxicity in Vitro and in Vivo by Trodusquemine."

Vendruscolo M, Heller GT, Barbut D, Chen SW, Cascella R, Zasloff M, Kumita JR, Cecchi C, Chiti F, Mannini B, Limbocker R, Flagmeier P, Cremades N, Knowles TPJ, Perni M, Aprile FA, Cohen SIA, Dobson CM, Kirkegaard JB, Nollen EAA, Galvagnion C, Challa PK, Meisl G

[ACS Chem Biol, 2018]

The aggregation of -synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of -synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of -synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of -synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of -synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinson's disease and related disorders.

Perni M et al. (2017) Proc Natl Acad Sci U S A "A natural product inhibits the initiation of -synuclein aggregation and suppresses its toxicity."

Heller GT, Dobson CM, Chen SW, Meisl G, Sormanni P, Cohen SI, Perni M, Zasloff M, Aprile FA, Limboker R, Muller MB, Challa PK, Bax A, Knowles TP, Chiti F, Cremades N, Cecchi C, Kirkegaard JB, Galvagnion C, Flagmeier P, Cascella R, Vendruscolo M, Maltsev A, Nollen EA

[Proc Natl Acad Sci U S A, 2017]

The self-assembly of -synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects -synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces -synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of -synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing -synuclein, observing a dramatic reduction of -synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.

Perni M et al. (2021) Front Cell Dev Biol "Comparative Studies in the A30P and A53T -Synuclein C. elegans Strains to Investigate the Molecular Origins of Parkinson's Disease."

Chen SW, Xu CK, van Ham TJ, Kirkegaard JB, Cohen SIA, Vendruscolo M, van der Goot A, Kaminski Schierle GS, Knowles TPJ, Seinstra R, Muller MBD, Nollen EAA, Laine RF, Kumita JR, Aprile FA, Perni M, Barbut D, Thijssen K, Kaminski CF, Sormanni P, Dobson CM, Flagmeier P, Ma KY, Limbocker R, Zasloff M, Fusco G, Challa PK, De Simone A, Sinnige T

[Front Cell Dev Biol, 2021]

The aggregation of -synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two <i>C. elegans</i> models of PD (PD_A30P and PD_A53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PD_WT). PD_A30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PD_A53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. -Synuclein was also observed to be less well localized into aggregates in PD_A30P worms compared to PD_A53T and PD_WT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of -synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of -synuclein and its associated toxicity in PD_A53T and PD_WT worms, but had less marked effects in PD_A30P. In addition, using an antibody that targets the N-terminal region of -synuclein, we observed a suppression of toxicity in PD_A30P, PD_A53T and PD_WT worms. These results illustrate the use of these two <i>C. elegans</i> models in fundamental and applied PD research.