The Role of RNF114 and NF-κB in Chronic Traumatic Encephalopathy (CTE): Experimental Alteration of the Inflammatory Cascade in Cell Culture and Evaluation of a Novel Cellular Trauma Model Open Access

Giuliano, Jonathan (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/hx11xf28n?locale=en
Published

Abstract

Background: The pathological features and clinical presentation of chronic traumatic encephalopathy (CTE) have been explored, but the role of regulatory proteins in the development of CTE remains understudied. Additionally, the limitations of current models of repetitive mild traumatic brain injury (rmTBI) and CTE complicate research efforts. In the present study, we provide a review of current rmTBI and CTE literature, examine the role of RING-type zinc-finger protein 114 (RNF114) and nuclear factor κB (NF-κB) in the inflammatory cascade and their possible link to CTE disease pathogenesis, and test a model for the induction of trauma in cell culture.

Methods: Using immunohistochemistry, tissue sections from the frontal cortices of CTE and Alzheimer’s disease (AD) patients were stained for RNF114. Expression of RNF114 and NF-κB in HEK 293T and BV2 cell culture was modified through transfection of plasmids and small interfering RNA (siRNA), and changes in expression were measured through western blotting and quantitative real-time PCR (qPCR). Additionally, the effect of RNF114 up-regulation on tau aggregation was determined through transfection of an RNF114 plasmid in a tau biosensor line and fluorescence resonance energy transfer (FRET) analysis. Finally, a model for the mechanical induction of chronic inflammation in BV2 cell culture was tested, and the long-term effect of agitation of cell culture on expression of RNF114 were measured through western blotting.

Results: Expression of RNF114 in CTE post-mortem brain tissue was significantly depressed, validating the reduction of RNF114 expression previously identified through quantitative proteomics. RNF114 and NF-κB overexpression in BV2 cells found that RNF114 serves as negative regulator of NF-κB. siRNA transfection did not yield a significant depression in RNF114 expression, and as such did not provide insight into the effect of RNF114 knockdown on the inflammatory cascade. Additionally, RNF114 overexpression in a tau biosensor line was not sufficient to promote the aggregation of tau. Finally, we demonstrated a significant increase in expression of RNF114 after agitation of cells in a vortex machine at four and eight days after intervention.

Conclusion: These findings suggest that RNF114 and NF-κB may play a role in the regulation of the neuroinflammatory cascade in CTE and contribute to disease pathogenesis. The results of the mechanical trauma experiments provide a foundation for future studies of cellular rmTBI models.    

Table of Contents

Introduction and Background

1

1. Overview

1

2. History of CTE

2

3. rmTBI

4

3.1. Overview

4

3.2. Mechanisms of rmTBI

5

3.2.1. Biophysics

5

3.2.2. Axonal Injury

6

3.2.3. Neuroinflammation and Microglial Activation

6

3.2.4. Other Contributors to Neuropathology

8

4. CTE

9

4.1. Diagnosis and Prevalence

9

4.2. Stages of CTE

9

4.3. Clinical Presentation

10

4.3.1. Overview

10

4.3.2. Cognitive and Behavioral Changes

11

4.4. Neuropathology

12

4.4.1. Overview

12

4.4.2. Microscopic Neuropathology

13

4.4.2.1. Tau

13

4.4.2.2. TDP-43

14

4.4.2.3. Amyloid-Beta

15

4.4.3. Gross Neuropathology

15

4.5. Risk Factors

16

5. Regulation of the Neuroinflammatory Cascade

17

5.1. NF-κB

19

5.2 Regulation of NF-κB

20

5.3. RNF114

20

6. Models of rmTBI and CTE

21

7. Rationale and Hypotheses

22

Materials and Methods

23

1. Cell Lines

23

1.1. BV2

23

1.2. HEK 293T and YFP/CFP

23

2. Protocols

24

2.1. Transfection

24

2.1.1. Plasmid

25

2.1.2. RNAi

26

2.2. Western Blot

27

2.3. Immunohistochemistry (IHC)

29

2.4. Immunocytochemistry (ICC)

30

2.5. Fluorescence resonance energy transfer (FRET)

31

2.6. qPCR

32

2.7. Mechanical Trauma

34

Experiments and Results

35

1. Validation of Data from the CTE Insoluble Proteome

35

2. Transfection of Plasmid Over-Expression Constructs

38

3. Transfection of siRNA Duplexes

42

4. Modeling Mechanical Trauma in BV2 Cell Culture

45

Discussion and Future Directions

52

1. Overview

52

2. Validation of Data from the CTE Insoluble Proteome

52

3. Transfection of Plasmid Over-Expression Constructs

54

4. Transfection of siRNA Duplexes

55

5. Modeling Mechanical Trauma in BV2 Cell Culture

57

Conclusion

59

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