Survival Following Traumatic Brain Injury in Drosophila Is Increased by Heterozygosity for a Mutation of the NF-κB Innate Immune Response Transcription Factor Relish

Abstract

Traumatic brain injury (TBI) pathologies are caused by primary and secondary injuries. Primary injuries result from physical damage to the brain, and secondary injuries arise from cellular responses to primary injuries. A characteristic cellular response is sustained activation of inflammatory pathways commonly mediated by NF-κB transcription factors. Using a Drosophila melanogaster TBI model, we previously found that the main proximal transcriptional response to primary injuries is triggered by activation of Toll and Imd innate immune response pathways that engage NF-κB factors Dif and Relish (Rel), respectively. Here, we found by mass spectrometry that Rel protein level increased in fly heads at 4-8 h after TBI. To investigate the necessity of Rel for secondary injuries, we generated a null allele, Reldel, by CRISPR/Cas9 editing. When heterozygous but not homozygous, the Reldel mutation reduced mortality at 24 h after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygosity for Reldelon mortality was modulated by genetic background and diet. To identify genes that facilitate effects of Reldel on TBI outcomes, we compared genome-wide mRNA expression profiles of uninjured and injured +/+, +/Reldel, and Reldel/Reldel flies at 4 h following TBI. Only a few genes changed expression more than two-fold in +/Reldel flies relative to +/+ and Reldel/Reldel flies, and they were not canonical innate immune response genes. Therefore, Rel is necessary for TBI-induced secondary injuries but in complex ways involving Rel gene dose, genetic background, diet, and possibly small changes in expression of innate immune response genes.

Repetitive mild traumatic brain injury causes synergistic effects on mortality

“…Our data shows that mild TBI causes synergistic effects on mortality after a requisite number of injuries, possibly because cumulative cellular stress surpasses a critical threshold. We expect that many of the secondary injury pathways active in animals following our mild TBI (60°) methodology overlap with pathways previously reported (Katzenberger et al 2015; Katzenberger et al 2016; Anderson et al 2018; Saikumar et al 2020; Swanson, Rimkus, et al 2020; Swanson, Trujillo, et al 2020). However, the identity of any specific factor(s) which set or scale the sensitivity to rmTBI remain unknown.…”

Ketogenic diet reduces early mortality following traumatic brain injury in Drosophila via the PPARγ ortholog Eip75B

“…In mammalian TBI models, reduced NF-κB activity resulting from treatment with the PPARγ agonist pioglitazone or other pharmacological agents improves outcomes [ 22 – 25 , 41 – 46 ]. Reducing NF-κB activity also improves TBI outcomes in flies [ 33 ]. Heterozygosity for a null mutation of Relish , one of three NF-κB genes in Drosophila , reduces early mortality and increases lifespan following TBI.…”

“…Second, we previously found that flies fed water versus CMYD exhibited increased expression of AMP genes following TBI, suggesting that the beneficial effects of water are not mediated by inhibition of Relish, which activates the transcription of AMP genes [ 29 ]. Third, while heterozygosity for a mutation of Relish reduced the incidence of early mortality for flies fed CMYD following TBI, it did not affect the incidence of early mortality for flies fed water following TBI, suggesting that water functions either downstream or independently of Relish [ 33 ].…”

“…Behavioral outcomes of TBI shared between flies and humans include temporary incapacitation, ataxia, abnormal sleep, early mortality, and reduced lifespan [ 26 – 31 ]. Cellular and molecular outcomes are also shared, including progressive neurodegeneration, disruption of the blood-brain barrier and the intestinal barrier, transient hyperglycemia, and prolonged activation of innate immune response pathways [ 26 , 28 , 29 , 32 , 33 ]. Using the Mortality Index at 24 hours (MI 24 )—the normalized percent of flies that die within 24 h after strikes from the HIT device—as a readout, we previously found that genetic background plays a substantial role in determining TBI outcomes.…”

“…For example, the MI 24 of flies injured at 0–7 days old varies from 7 to 58 among 179 inbred lines in the Drosophila Genetic Reference Panel (DGRP) [ 28 , 34 ]. Additionally, the MI 24 is reduced by heterozygosity for a mutation of the NF-κB innate immune response transcription factor Relish [ 33 ]. Age and diet also play substantial roles in determining outcomes of TBI in flies.…”