Author ORCID Identifier

0000-0002-6982-8485

Date of Graduation

9-2017

Document Type

Dissertation (PhD)

Program Affiliation

Cell and Regulatory Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Michael Xi Zhu

Committee Member

Jeffrey A. Frost

Committee Member

Kartik Venkatachalam

Committee Member

Guangwei Du

Committee Member

Zhizhong Pan

Abstract

Cell death is not only an essential phenomenon in normal development and homeostasis, but also crucial in various pathologies. It is now clear that many types of cell death can be regulated by pharmacological or genetic interventions. These were largely achieved by identifying the molecular mechanisms underlying the regulated cell death (RCD). While in the immune system, RCD needs to be facilitated to help the clearance of pathogens and tumors, in healthy cells, especially the terminally differentiated neurons in the nervous system, it is more desirable to protect cells from dying due to stress under pathological conditions. Thus, understating the inhibitory and the activating signals for RCD in different systems is important. In this thesis, I will discuss the study of two key molecules involved in RCD: programmed death 1 (PD-1, as inhibitor for RCD) and serine/threonine kinase receptor interaction protein 3 (RIP3, as promoter for RCD) in two different systems.

First, I studied the role of PD-1 signaling in the cytotoxicity of natural killer (NK) cells. In recent years, PD-1 has become a hot target of immunotherapy. However, because most studies on PD-1 have focused on T cells, the precise mechanism by which PD-1 mediates its effects on NK cells remains poorly characterized. Using NK cell lines that express PD-1, I found that PD-1 activation blocked NK cell cytotoxicity. However, PD-1 signaling did not inhibit cell-cell conjugation between NK and target cells as NK cells that expressed PD-1 formed stable immunological synapse (IS) with target cells that expressed the PD-1 ligand, PD-L1. Instead, the PD-1 engagement by PD-L1 blocked lytic granule (LG) polarization via disruption of the integrin ‘outside-in’ signaling pathway and abolished the calcium signaling for degranulation. Similar to T cells, the immunoreceptor tyrosine-based switch motif (ITSM) but not immunoreceptor tyrosine-based inhibitory motif (ITIM) of PD-1 was crucial for its inhibitory effect on cytotoxicity.

In the second part, I examined the involvement of RIP3 in acidosis-induced cell death, which critically contributes to ischemic brain injury and was recently shown to occur through necroptosis, a new form of programmed necrosis. Although RIP3 has been proven to be an important mediator of necroptosis, whether acidosis-induced neuronal death requires RIP3 is not clear. Here, I show that RIP3 is required for acidosis-induced cell death, in which serum starvation is another critical contributing factor. There are at least two key downstream molecules of RIP3 for acidosis/starvation-induced cell death: mixed lineage kinase domain-like pseudokinase (MLKL) and mitochondrial apoptosis-inducing factor (AIF). Inhibiting RIP3 by an FDA approved anti-cancer drug, dabrafenib, reduced both acid-induced death of mouse cortical neurons in vitro and brain infarction in mice subjected to middle cerebral artery occlusion (MCAO) in vivo.

Depending on the specific scenarios and the physiological systems involved, the goals for pharmacological and/or genetic modification of RCD can be quite different. Thus, a better understanding of both the positive (e.g., RIP3) and negative (e.g., PD-1) regulators of RCD is of significant values for both basic sciences and therapeutic development in treating different diseases in the future.

Keywords

Natural killer cell, PD-1, Necroptosis, Ischemic stroke, RIP3, ASIC1a

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