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Decoding distinctive features of plasma extracellular vesicles in amyotrophic lateral sclerosis

Laura Pasetto, Stefano Callegaro, Deborah Ferrara, Laura Brunelli, Giovanna Sestito, Roberta Pastorelli, Elisa Bianchi, Alessandro Corbelli, Fabio Fiordaliso, Marina Cretich, Marcella Chiari, Cristina Potrich, Cristina Moglia, Massimo Corbo, Gianni Soraru, Christian Lunetta, Andrea Calvo, Adriano Chio, Gabriele Mora, Maria Pennuto, Alessandro Quattrone, Francesco Rinaldi, Vito Giuseppe D'Agostino, Manuela Basso, Valentina Bonetto

Preprint posted on August 12, 2020 https://www.medrxiv.org/content/10.1101/2020.08.06.20169300v2

Message in a bottle from the ALS brain: extracellular vesicles deliver useful information about disease and type of progression

Selected by Kristina Kuhbandner

Background

Extracellular vesicles (EVs) comprise all particles released from a cell into body fluids, with sizes ranging from about 50 to 2000 nm. Their lumen is filled with proteins, lipids and nucleic acids which are specific to the cell of origin. This cargo is protected from degradation by a lipid membrane; thus, information can be sent from cell to cell over a distance – just like a message in a bottle (1).

Importantly, the composition of their cargo changes under pathological conditions. As EVs can be isolated from biological fluids in a non-invasive manner, they are considered as valuable clinically relevant biomarkers. In the past years, this application has been extensively studied in the field of cancer (2). There is also growing evidence for a potential use of EVs as prognostic tool in neurodegenerative diseases. Given that the main pathological events in these disorders are restricted to the central nervous system (CNS), non-invasive access to biological samples is impossible and therefore pose a major diagnostic challenge. However, EVs are released from CNS cells and transported to the periphery, thereby delivering CNS information to the blood.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal neurodegenerative disease ultimately resulting in the loss of control of voluntary muscles. As symptoms are heterogenous and frequently associated with other disease conditions, early and efficient diagnosis remains extremely difficult. Up to date, there is no effective treatment available underlining the need to identify robust ALS biomarkers (3). Recently, ALS-related proteins and miRNAs have been detected in EVs from biological fluids of ALS patients (4, 5). The present study investigates the use of EVs as diagnostic and prognostic tools in a clinical setting.

Key findings

So far, no standardized protocol for EV isolation exists, and speed, purity as well as cost remain a major hurdle to overcome. Therefore, the authors first compared two methods of EV isolation: ultracentrifugation, the most common technique in research laboratories, and Nickel-based isolation, a recently described method based on electrostatic interactions with the negatively charged vesicle membrane (6, 7). To analyze the properties of isolated EVs, an array of different techniques was used. From these experiments, they conclude that Nickel-based isolation from plasma provides a high yield of intact and pure EVs of heterogenous size with preserved biochemical and biophysical features.

After establishing the Nickel-based isolation protocol, they obtained plasma-EVs from a number of ALS patients and controls, which were either healthy test persons (HC), patients with muscular dystrophies (MD) or spinal-bulbar muscular atrophy (SBMA), a motor neuron disease sharing many features with ALS. While total amount of EVs did not differ between the groups, they observed higher numbers of small particles in ALS and SBMA patients and ALS-EVs were significantly bigger than SBMA-EVs. Subsequently, these changes were verified in two genetic ALS mouse models (SOD1G93A and TDP-43Q331K). This indicates that the amount and size of particles can be used as parameters to distinguish ALS from other diseases.

Next, they investigated the presence of ALS-related proteins in plasma-EVs of patients and animal models. Importantly, Heat shock protein 90 (HSP90), chaperoning correct protein folding and reduced in ALS blood cells, was ubiquitously decreased in ALS conditions (8). Moreover, the phosphorylated form of TAR DNA-binding protein 43 (TDP-43), the pathological hallmark of ALS, was significantly enriched in EVs of ALS compared to MD patients.

Finally, having characterized the specific features of ALS-EVs, the authors used a machine learning approach to determine whether these can be used to stratify ALS from MD, SBMA and HC. This tool suggests that the size distribution of EVs is a reliable parameter to categorize patients and controls. Additional combination with protein markers further helped to correctly differentiate ALS from SBMA. Moreover, with their mathematical model, they confirmed efficient discrimination between ALS patients with slow and fast disease progression.

What I like about this preprint

EVs are promising biomarkers for neurodegenerative diseases, but in-depth characterization and evaluation is the basis for ultimate clinical application. Employing a well elaborated concept, this comprehensive study evaluated Nickel-based isolation as a fast and convenient method to obtain EVs from plasma. Using this technique, they describe ALS-specific parameters of EVs in sporadic patients and also in genetic mouse models. Of note, these features were able to discriminate between patients with ALS and SBMA, another closely related neurodegenerative disease. Ultimately, they verified the efficacy of the identified parameters in patient stratification using machine learning tools. Overall, they established an analytical protocol, which can be used in future follow-up studies with large patient cohorts, thereby paving the way to improve ALS diagnosis and prognosis.

Questions to the authors

  • Based on your findings in genetic mouse models, do you expect differences between EVs from familial compared to sporadic ALS patients?
  • Does medication influence EV properties?
  • Why is phosphorylated TDP-43 in ALS-EVs significantly lower in comparison to MD patients, but does not majorly differ compared to HC?

 

References

  1. Yáñez-Mó, M., Siljander, P. R. M., Andreu, Z., Bedina Zavec, A., Borràs, F. E., Buzas, E. I., … & Colás, E. (2015). Biological properties of extracellular vesicles and their physiological functions. Journal of extracellular vesicles, 4(1), 27066.
  2. Makler, A., & Asghar, W. (2020). Exosomal biomarkers for cancer diagnosis and patient monitoring. Expert Review of Molecular Diagnostics, 20(4), 387-400.
  3. Paganoni, S., Macklin, E. A., Lee, A., Murphy, A., Chang, J., Zipf, A., … & Atassi, N. (2014). Diagnostic timelines and delays in diagnosing amyotrophic lateral sclerosis (ALS). Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 15(5-6), 453-456.
  4. Sproviero, D., La Salvia, S., Giannini, M., Crippa, V., Gagliardi, S., Bernuzzi, S., … & Cereda, C. (2018). Pathological proteins are transported by extracellular vesicles of sporadic amyotrophic lateral sclerosis patients. Frontiers in neuroscience, 12, 487.
  5. Saucier, D., Wajnberg, G., Roy, J., Beauregard, A. P., Chacko, S., Crapoulet, N., … & O’Connell, C. (2019). Identification of a circulating miRNA signature in extracellular vesicles collected from amyotrophic lateral sclerosis patients. Brain research, 1708, 100-108.
  6. Basso, M., Pozzi, S., Tortarolo, M., Fiordaliso, F., Bisighini, C., Pasetto, L., … & Bendotti, C. (2013). Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes IMPLICATIONS FOR DISEASE SPREADING AND MOTOR NEURON PATHOLOGY IN AMYOTROPHIC LATERAL SCLEROSIS. Journal of Biological Chemistry, 288(22), 15699-15711.
  7. Notarangelo, M., Zucal, C., Modelska, A., Pesce, I., Scarduelli, G., Potrich, C., … & Pasini, L. (2019). Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood. EBioMedicine, 43, 114-126.
  8. Filareti, M., Luotti, S., Pasetto, L., Pignataro, M., Paolella, K., Messina, P., … & Fuda, G. (2017). Decreased levels of Foldase and chaperone proteins are associated with an early-onset amyotrophic lateral sclerosis. Frontiers in molecular neuroscience, 10, 99.

 

 

Tags: amyotrophic lateral sclerosis, biomarker, extracellular vesicles

Posted on: 10th September 2020 , updated on: 14th September 2020

doi: https://doi.org/10.1242/prelights.24654

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  • Author's response

    Valentina Bonetto and Manuela Basso shared

    Thank you Kristina for your questions and for your very nice report of our findings.

    Valentina Bonetto and I thought to write down our answers. We are keen to discuss more about this work.

    Q: Based on your findings in genetic mouse models, do you expect differences between EVs from familial compared to sporadic ALS patients?
    A: We do not. We analyzed the plasma EV of sporadic patients and two ALS mouse models bearing genetic mutations in SOD1 (G93A) and TDP-43 (Q331K). Unexpectedly, the trend for size reduction and amount increase was very similar in patients and mice, suggesting that ‘not yet uncovered’ mechanisms involved in EV release are altered in ALS in general. We looked with extreme interest into the decrease of plasma EV size and increase in the amount at the presymptomatic stage of SOD1 G93A mice. It is tempting to hypothesize that EV alterations are already present at the presymptomatic stage, during the prodromal phase of the disease. We will investigate that further.

    Q: Does medication influence EV properties?
    A: We don’t have any indication that it does. We are starting a prospective study in which we are recruiting patients at time zero, which means during the first neurological assessment, before diagnosis to answer this question.

    Q: Why is phosphorylated TDP-43 in ALS-EVs significantly lower in comparison to MD patients, but does not majorly differ compared to HC?
    A: It may be due to the age of MD patients. They are usually younger than ALS patients and HC. Phosphorylated TDP-43 could be increased in people older than 65 years, as reported by others (Arnold et al., 2013; Uchino et al., 2017) or in the prodromal phase of Alzheimer’s disease (Wilson et al., 2011; Kadokura et al., 2009). It is striking that if we compare ALS to SBMA or MD, phosphorylated TDP-43 is indubiously higher.

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