Kv1.3 potassium channel regulation of microglial inflammatory response 公开

Bowen, Christine (Fall 2024)

Permanent URL: https://etd.library.emory.edu/concern/etds/vm40xt09m?locale=zh

Published

Abstract

Microglia are the resident macrophages in the brain. During the lifespan, microglia have a distinct heterogeneity, where microglia can transition to different states over time. In disease conditions, like Alzheimer’s Disease, microglia shift toward a disease associated state, referred to as disease associated microglia (DAM). DAM have distinct expression profiles, where there is an activation of the TLR2 response, resulting an increase in inflammatory cytokines (e.g. TNF-α, IL-1β, STAT1). The transition to this state reduces the ability to clear Aβ plaques and promote brain homeostasis, while increasing the detrimental effects of AD. Our lab shows that the Kv1.3 potassium channel is highly present on the cell surface of these DAM. Treatment via blockade of the channel results in a reduction of the detrimental components of DAM. However, it remains unclear how Kv1.3 influences the inflammatory response of microglia. We hypothesize that Kv1.3 directly interacts with key signaling proteins during a microglial immune response. To test this hypothesis, we utilized proximity-labeling and mass spectrometry to evaluate proteins interacting with Kv1.3. We determined that around 200 proteins interact with the N-terminus of Kv1.3 and is largely responsible for protein processing and trafficking Kv1.3 to the mitochondria. During an immune response, the C-terminus of Kv1.3 interacts with key immune signaling proteins (e.g. TLR2, STAT1, C3). Some of the C-terminal interactors (around 70 proteins) are dependent on the PDZ-binding domain, such that these proteins no longer interact with Kv1.3 when the PDZ-binding domain is removed. Overall, we highlight that Kv1.3 directly interacts with key immune signaling proteins during a microglial inflammatory response. This thesis will enable better understanding for how Kv1.3 influences microglial response and can be used to evaluate how targeting Kv1.3 could be beneficial for the treatment of neuroinflammatory diseases.

TABLE OF CONTENTS

I. INTRODUCTION………………………………………………………….…………1

i. Microglia are the immune cells of the brain……………………………………....1

a. Microglia are derived from macrophage progenitors in early development…………………………………………………………2

b. Molecular and functional heterogeneity of microglia……………….2

c. Microglia nomenclature and terminology………...…………………5

d. Microglia during inflammation have a distinct phenotype………….7

e. In vitro models for microglial activation and induction of inflammatory response………………………………………………8

f. Lipopolysaccharide (LPS) as a model for inflammation…………..11

g. Microglia in Neuroinflammatory disease and inflammation………12

h. Immune Cells contribute significantly to the disease progression of Alzheimer’s Disease……………………………………………….13

i. Microglia have a distinct disease-associated state in AD………….14

j. Mouse models of AD have similar features to human AD………...16

ii. Kv1.3 potassium channel influences microglial response……………………….18

a. The Kv1.3 potassium channel is highly expressed by microglia in neuroinflammatory states…………………………………………….18

b. The Kv1.3 potassium channel allows for efflux of potassium in immune cells…………………………………………………………19

c. Kv1.3 is present in the mitochondria and alters metabolism in cells...20

d. Kv1.3 activity increases in T-lymphocyte and macrophage inflammatory responses……………………………………………...22

e. Kv1.3 channels in the brain …………………………………………23

f. Knock out (KO) of Kv1.3 in mouse models highlights a reduction of inflammatory response……………………………………………….24

g. Blockade of Kv1.3 alters inflammatory responses…………………..25

h. KV1.3 interactors in immune cells…………………………………..26

i. Kv1.3 as a potential for the treatment of AD………………………...27

j. Current knowledge gaps related to Kv1.3 channels in microglia……27

k. Central Hypothesis: Kv1.3 directly interacts with immune signaling proteins in microglia during neuroinflammatory diseases…………...31

II. CHAPTER II: Proximity labeling proteomics reveals Kv1.3 potassium channel immune interactors in microglia……………………………………………………..31

i. Abstract…………………………………………………………………………..32

ii. Graphical Abstract……………………………………………………………….33

iii. Introduction………………………………………………………………………33

iv. Experimental Procedures………………………………………………………...36

v. Results………………………………………………….………………………...59

a. Validation of N and C terminus TurboID fusion constructs for mapping the Kv1.3 interactome in HEK-293 cells………………………………...59

b. Kv1.3 Amino and Carboxyl terminal fusions with TurboID identifies distinct domain-interacting proteins in HEK-293 cells……………….…63

c. Generation and validation of stably transduced BV-2 microglial lines expressing N and C term Kv1.3-TurboID fusions……………………….67

d. Identification of Kv1.3 channel domain-specific protein interactors and molecular pathways in BV-2 microglia…………………………………70

e. Pro-inflammatory activation preferentially modifies the C-terminal interactome of Kv1.3 channels in microglia……………………………..74

f. Kv1.3 C-terminal inflammatory interactors dependent on the PDZ binding domain……………………………………………………………………75

g.Kv1.3 channel is present in mitochondria-enriched fractions in microglia ……………………………………………………………………………78

h. Verification of interactions of Kv1.3 with C3 and pSTAT1 via the C terminal domain………………………………………………………….79

vi. Discussion…………………………………………………………………….….85

vii. Conclusion……………………………………………………………………….90

viii. Supplemental Figures…………………………………………………………….91

III. CHAPTER III: DISCUSSION AND FUTURE DIRECTIONS……………….……………………………………………………….99

i. Kv1.3 interacting proteins………………………………………………………100

a. Kv1.3 is associated with cell signaling in HEK-293 cells……………..101

b. Proximity labeling shows that Kv1.3 interacts with many endoplasmic reticulum associated proteins…………………………………………...102

c. Proximity labeling of proteins interacting with Kv1.3 highlight the presence of Kv1.3 in the mitochondria……………………………..…..103

d. Kv1.3 interactome contains proteins that are dependent on the PDZ-binding domain to interact with Kv1.3………………..………..………105

e. The C-termini of Kv1.3 interacts with key immune signaling during LPS stimulation in microglia………………………………………………...106

f. Implications of Kv1.3 interactors in microglia…………………………107

g. Limitations of TurboID-based proximity labeling for Kv1.3 channel interactome studies…………………………………………..………….107

ii. Future Applications…………………………………………..…………………108

a. Establishing Kv1.3 Potassium Channel interactors in T-cells………....108

b. Utilizing the Split-TurboID method to further evaluate interactors with the Kv1.3 complex in microglia……………………………………...…109

c. Pharmacological blockade of Kv1.3 influences Microglial inflammatory response…………………………………………………………..…….109

d. Developing a microglial-specific Kv1.3 conditional Knock Out…..….111

IV. CHAPTER IV: REFERENCES…………………………………………..………...112TABLE OF CONTENTS