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High School Student with a Traditionally Underrepresented Background

HS Poster

Poster 13 - Alleviating heat stress in motor neurons via mitochondria-containing extracellular vesicles: a potential ALS therapy

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord [1-2]. Targeting this mitochondrial dysfunction presents a potential therapeutic approach as it is heavily implicated in motor neuron (MN) degeneration in ALS [2-4]. A novel approach to mitigate mitochondrial dysfunction would be to deliver functional mitochondria to the damaged motor neurons. Therefore, we propose mitochondria containing MN-derived extracellular vesicles (EVs) as a therapeutic strategy to treat ALS.

EVs are nano-sized vesicles enriched with cargo and are secreted into the extracellular environment by the cells [5]. There are different subtypes of EVs although our lab is focusing on two major subtypes: medium to large EVs (m/l EVs) and small EVs ( s EVs). We have previously reported the presence of mitochondria in a subset of EVs that are >200 nm in diameter (m/l EVs) [6-8]. EV mitochondria are transferred into recipient brain endothelial cells (BEC) resulting in increased mitochondrial function [6-8]. This study has examined the effect of heat stress to induce ALS-like oxidative stress in MNs, followed by a treatment of MN-derived EVs to determine their protective effect on the damaged MNs.

Materials and

Methods: Cell culture: We used a mouse motor neuron-like cell line—NSC-34 (Cedarlane Laboratories)—which was briefly maintained in tissue-culture flasks/multi-well plates in a humidified incubator at 37 ± 0.5℃ (Isotemp, Thermo Fisher Scientific). At ~90-95% confluency, the cells were switched from DMEM high glucose medium (HyClone Laboratories) to neurobasal medium (Gibco, Grand Island, NY)) to differentiate the NSCs into MNs.

EV Isolation: Two subsets of Differentiated NSC34-EVs (m/l EVs and s EVs) were isolated from conditioned medium by differential ultracentrifugation following the established protocol in the lab [6]. Post-isolation EVs were stored at -80℃ for long storage until used for further experiments.

EV characterization: Isolated EVs were characterized by measuring their protein content with Micro- Bicinchoninic Acid Protein (Micro BCA) Assay. Particle size, zeta potential, and polydispersity index were measured with the Dynamic Light Scattering (DLS) method by using Zetasizer Nano (Malvern Panalytical Inc., Westborough, PA) at an EV protein concentration of 0.1 mg/mL.

Heat-stress: On day 3 after differentiation, MNs were treated with 100 µl of neurobasal medium pre-heated to 50.8℃. Post-treatment, cells were re-heated in an incubator (Thermo Scientific) at 50.8℃ for 30 minutes. Post-treatment, the heated medium was replaced with 200 µL fresh differentiation medium and allowed to recover either for 4 or 24 hours in a humidified incubator at 37°C [9]. Untreated cells and cells treated with polyethyleneimine (PEI), a toxic polymer, were used as controls. Finally, a cell-titer glo luminescence assay (ATP assay) was performed to measure the relative intracellular ATP levels of the stressed motor neurons.

Results, Conclusions, and Discussions: EV characterization: Characterization studies have shown significant differences in m/l EVs and sEVs. m/l EVs have shown larger particle sizes and higher protein content compared to s EVs which matches previous findings [6]. We found that s EVs have a particle diameter of 153.2 ± 2.129 nm, a dispersity index of 0.26 ± 0.03, a zeta potential of -23.45 ± 1.47 mV, and a protein content of 2.08 mg/ml. The particle diameter, dispersity index, zeta potential, and protein content of m/l EVs were found to be 217 ± 0.47 nm, 0.28 ± 0.03, -36.58 ± 0.91 mV, and 4.12 mg/ml, respectively.

Effect of heat-stress on Differentiated NSC-34: Initial results indicate that heat stress negatively impacts cell viability suggesting that heat stress can be effectively used as a model for ALS-like oxidative stress to test the efficacy of MN-EVs (Figure 1).

Conclusions: Our experiment displays how heat is effective in inducing cellular stress and can thus be used to model ALS-like oxidative stress in motor neurons to test the effect of EVs. The results of our further experiments will help assess the therapeutic potential of mitochondria-containing EVs to treat ALS.

Acknowledgements (Optional):

References (Optional): 1. Ciervo, Y., et al., Advances, challenges and future directions for stem cell therapy in amyotrophic lateral sclerosis. Molecular neurodegeneration, 2017. 12: p. 1-22.
2. Jaiswal, M.K., Riluzole but not melatonin ameliorates acute motor neuron degeneration and moderately inhibits SOD1-mediated excitotoxicity induced disrupted mitochondrial Ca2+ signaling in amyotrophic lateral sclerosis. Frontiers in Cellular Neuroscience, 2017. 10: p. 295.
3. Calió, M., H. Elisandra, and S. Amanda, Mitochondrial Dysfunction, Neurogenesis, and Epigenetics: Putative Implications for Amyotrophic Lateral Sclerosis Neurodegeneration and Treatment. J Front Neurosci 14: 679. 2020.
4. Manfredi, G. and Z. Xu, Mitochondrial dysfunction and its role in motor neuron degeneration in ALS. Mitochondrion, 2005. 5(2): p. 77-87.
5. Ortega‐Pineda, L., et al., Designer extracellular vesicles modulate pro‐neuronal cell responses and improve intracranial retention. Advanced healthcare materials, 2022. 11(5): p. 2100805.
6. Dave, K.M., et al., Mitochondria-containing extracellular vesicles (EV) reduce mouse brain infarct sizes and EV/HSP27 protect ischemic brain endothelial cultures. Journal of Controlled Release, 2023. 354: p. 368-393.
7. D'Souza, A., et al., Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells. Journal of Controlled Release, 2021. 338: p. 505-526.
8. Dave, K.M., et al., Mitochondria-containing extracellular vesicles from mouse vs. human brain endothelial cells for ischemic stroke therapy. Journal of Controlled Release, 2024. 373: p. 803-822.
9. Robinson, M.B., et al., Exogenous Hsc70, but not thermal preconditioning, confers protection to motoneurons subjected to oxidative stress. Developmental Neurobiology, 2008. 68(1): p. 1-17.