Date of Award

Spring 4-4-2024

Embargo Period

4-4-2029

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

Additional College

College of Medicine

First Advisor

Besim Ogretmen

Second Advisor

Shikhar Mehrotra

Third Advisor

Lori McMahon

Fourth Advisor

Michael Ostrowski

Fifth Advisor

Wenjian Gan

Abstract

Sphingolipids are bioactive molecules involved in several cellular processes, from maintaining cellular and subcellular structural integrity and barrier function to regulating and transducing signaling cascades in various cellular contexts. In this dissertation, I describe the involvement of two key sphingolipids – ceramide and sphingosine-1-phosphate (S1P) – in T-cell metabolism, function, and signaling.

The first part of the dissertation builds upon the recently discovered role of ceramide synthase 6 (CerS6)-generated C14/16-ceramide in inducing mitophagy in aging T-cells, which in turn attenuates T-cell anti-tumor function. This ultimately contributes to the reduced cancer resistance in the aging population. Interestingly, epidemiological studies have shown that cancer incidence is significantly less in aging Alzheimer’s Disease (AD) patients without much mechanistic insight. I aimed to understand this cancer resistance mechanism linked to AD and exploit it for broader cancer therapies. A hallmark of AD is the accumulation of amyloid precursor protein (APP) in neuronal mitochondria, inhibiting mitophagy, the clearance of dysfunctional mitochondria, and contributing to AD pathology. Unlike neurons, attenuation of ceramide-dependent mitophagy would play a favorable role in aging T-cells, as it would enhance their anti-tumor function. Given that APP is also processed in T-cells, I hypothesized that APP accumulates aberrantly in aging Alzheimer’s T-cells, inhibiting mitophagy and enhancing T-cell anti-tumor activity. Employing techniques of lipidomics, metabolomics, imaging, molecular biology, and genetic models, I show that AD T-cells from a mouse model and human patients are protected against aging-associated ceramide-dependent mitophagy, which enhanced their anti-tumor functions. The excessive localization of APP to the T-cell mitochondria, where it inhibits the accumulation of CerS6 and C14/C16-ceramides, attenuated the levels of mitophagy and protected the Alzheimer’s T-cells from metabolic defects observed in aging, namely protecting against the exhaustion of fumarate pools. Both in vitro and in vivo re-supplementation of fumarate to aging WT mouse T-cells effectively rescued their tumor killing capacity both in vitro and in adoptive T-cell transfers to mice with melanoma, functionally mimicking the AD phenotype. Moreover, I show that Alzheimer’s mice are protected against the growth of both carcinogen-induced and allografted tumors. Finally, transfer of mitochondria from AD T-cells to aging WT T-cells improved their metabolic fitness and anti-cancer functions. In summary, I show that AD-associated APP improves anti-tumor immunity of aging T-cells by inhibiting ceramide-mediated lethal mitophagy and restoring mitochondrial fumarate metabolism. These data provide a novel mechanism to explain reduced cancer incidences in AD patients.

In the second part of this dissertation, I turn my attention to another molecule at the center of sphingolipid metabolism: S1P, a bioactive lysophospholipid generated by two isoenzymes of the sphingosine kinase protein (Sphk1 and Sphk2), which plays an important role in T-cell function from activation and differentiation to egress and migration. While the role of Sphk1-generated S1P has been studied before, the role of Sphk2 in modulating T-cell functionality remains unclear and poorly defined. I show here that Sphk2 interacts with the Zeta Chain of T Cell Receptor Associated Protein Kinase 70 (ZAP70), a kinase essential for the initiation of canonical biochemical signaling pathways upon activation via T-cell antigen receptor (TCR) stimulation. ZAP70 activity is strictly regulated by phosphorylation and dephosphorylation events which act as an on-off switch that keeps the downstream signaling cascades in check. This is especially interesting as Sphk2-derived S1P has been shown to mimic protein phosphorylation in other proteins, and I hypothesized that it might be playing a similar role upon interacting with ZAP70 in T-cells. Using both pharmacological inhibition of Sphk2 as well as Sphk2 knock-out mice, I show a reduction in the time and intensity of ZAP70 phosphorylation and downstream signal propagation upon TCR activation in the absence of Sphk2. Molecular docking predictions and mutational studies identified the amino acid R160 of ZAP70 to be interacting with Sphk2-derived S1P, which in turn supports ZAP70 activity and functionality by mimicking its phosphorylation at T130 or Y126 residues upon T-cell receptor activation. This work gives new insights into the previously unexplored role of endogenous S1P, generated by SphK2, in the regulation of T-cell activation via regulating ZAP70 signaling, which plays key roles in inflammation and autoimmune diseases.

Rights

Copyright is held by the author. All rights reserved.

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Available for download on Wednesday, April 04, 2029

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