Author ORCID Identifier

https://orcid.org/0000-0002-5377-5657

Date of Graduation

8-2020

Document Type

Dissertation (PhD)

Program Affiliation

Neuroscience

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Cobi J. Heijnen

Committee Member

Neal Waxham

Committee Member

Pramod Dash

Committee Member

David Grosshans

Committee Member

Michael Galko

Committee Member

Annemieke Kavelaars

Abstract

Abstract

Astrocytes rescue neuronal health after cisplatin treatment through mitochondrial transfer.

Author: Krystal English

Advisory Professor: Dr. Cobi J. Heijnen, Ph.D.

Chemotherapy-induced cognitive impairments are associated with neuronal mitochondrial dysfunction. Cisplatin, a commonly used chemotherapeutic, induces neuronal mitochondrial dysfunction in vivo and in vitro. Astrocytes are key players in supporting neuronal development, synaptogenesis, axonal growth, metabolism and, potentially, mitochondrial health. We tested the hypothesis that astrocytes transfer healthy mitochondria to neurons after cisplatin treatment to restore neuronal health.

We used an in vitro system in which astrocytes with Mito-mCherry-labeled mitochondria were co-cultured with primary cortical neurons or neuronal stem cells damaged by cisplatin. Culturing primary cortical neurons with cisplatin depolarized the neuronal mitochondrial membrane potential and reduced neuronal survival. Cisplatin induced abnormalities in neuronal calcium dynamics characterized by increased resting calcium levels, reduced calcium responses to stimulation with potassium chloride (KCl), and slower calcium clearance. The same dose of cisplatin that caused neuronal damage did not affect astrocytic mitochondrial respiration or astrocyte survival. Co-culture of cisplatin-treated neurons with astrocytes was associated with the transfer of mitochondria from astrocytes to cisplatin-treated neurons leading to the restoration of neuronal mitochondrial membrane potential, normalization of neuronal calcium dynamics, and increased neuronal survival. Furthermore, the neurons that had received astrocytic mitochondria, showed a resolution of calcium dynamics.

Mitochondrial Rho-GTPase 1 (Miro-1) is an important protein for mitochondrial motility and transfer. We show that the siRNA-mediated knockdown of Miro-1 in astrocytes reduced mitochondrial transfer from astrocytes to neurons and prevented normalization of neuronal calcium dynamics.

Based on these results we questioned if astrocytes transferred mitochondria to other types of neuronal cells as well, such as neuronal stem cells (NSCs). Neuronal stem cells are important for neurogenesis, gliogenesis, and proper cognitive function. For astrocytic mitochondrial transfer to NSCs, we used an in vitro system consisting of mouse cortical astrocytes with DsRed-labeled mitochondria that were co-cultured with mouse NSCs. We observed that astrocytes transferred mitochondria to NSCs treated with cisplatin thus expanding upon the breath of astrocytic mitochondrial transfer.

In conclusion, we identified transfer of mitochondria from astrocytes to neurons and NSCs damaged by cisplatin. We propose that mitochondrial transfer by astrocytes is an important repair mechanism to protect neurons against the toxic effects of this chemotherapeutic.

Keywords

Mitochondrial transfer, mitochondria, Neurons, Astrocytes, Calcium, Miro-1, neural stem cells, cisplatin

Available for download on Friday, June 18, 2021

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