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 Público
Giuliano, Jonathan (Spring 2018)
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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