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

Traumatic brain injury (TBI) is a common neurological disorder whose outcomes vary widely depending on a variety of environmental factors, including diet. Using a Drosophila melanogaster TBI model that reproduces key aspects of TBI in humans, we previously found that the diet consumed immediately following a primary brain injury has a substantial effect on the incidence of mortality within 24 h (early mortality). Flies that receive equivalent primary injuries have a higher incidence of early mortality when fed high-carbohydrate diets versus water. Here, we report that flies fed high-fat ketogenic diet (KD) following TBI exhibited early mortality that was equivalent to that of flies fed water and that flies protected from early mortality by KD continued to show survival benefits weeks later. KD also has beneficial effects in mammalian TBI models, indicating that the mechanism of action of KD is evolutionarily conserved. To probe the mechanism, we examined the effect of KD in flies mutant for Eip75B, an ortholog of the transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) that contributes to the mechanism of action of KD and has neuroprotective effects in mammalian TBI models. We found that the incidence of early mortality of Eip75B mutant flies was higher when they were fed KD than when they were fed water following TBI. These data indicate that Eip75B/PPARγ is necessary for the beneficial effects of KD following TBI. In summary, this work provides the first evidence that KD activates PPARγ to reduce deleterious outcomes of TBI and it demonstrates the utility of the fly TBI model for dissecting molecular pathways that contribute to heterogeneity in TBI outcomes.

Introduction

Traumatic brain injury (TBI) is a major health issue worldwide [1]. It is a leading cause of disability and death, and its clinical management is challenging because the physical, behavioral, cognitive, and emotional sequelae are highly variable. Variation in sequelae among TBI patients stems from heterogeneity of primary injuries to the brain as well as heterogeneity of genetic and environmental factors such as physical activity, sleep, and diet that promote tissue repair or exacerbate tissue damage through secondary injury mechanisms [2, 3]. Cellular and molecular mechanisms associated with secondary injuries include ionic imbalance, excitotoxicity, oxidative stress, inflammation, and mitochondrial dysfunction that disrupt cellular metabolism leading to neuronal dysfunction and cell death [4].

Glucose is the main energy source for the brain, but following TBI, glucose uptake and use by the brain is progressively reduced [5, 6]. Under these circumstances, ketone bodies, derived from fatty acid oxidation in the liver, become the main energy source for the brain [7, 8]. Ketone bodies such as β-hydroxybutyrate, acetone, and acetoacetate improve mitochondrial metabolism, reduce production of reactive oxygen species and proinflammatory proteins, and have broad neuroprotective effects [8, 9]. Elevated levels of ketone bodies in the blood, a state known as ketosis, can be induced by fasting and by high-fat, low-carbohydrate, low-protein ketogenic diet (KD). KD reduces seizures in refractory childhood epilepsy and ameliorates detrimental outcomes in mammalian models of neurological disorders, including TBI [10, 11]. In rat TBI models, KD reduces apoptosis, contusion volumes, and anxiety- and depressive-like behaviors and improves motor and cognitive performance [12-18]. However, much remains to be learned about the influence of KD in TBI, including the extent to which genetic background modulates its beneficial effects.

The ligand-dependent transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) contributes to the mechanism of action of KD and has anti-inflammatory and neuroprotective effects in mammalian models of neurological disorders, including TBI [19, 20]. Activation of PPARγ by fatty acids inhibits inflammation by a variety of mechanisms, including by reducing the activity of nuclear factor-kappa B (NF-κB) transcription factors that promote expression of inflammatory genes encoding cytokines, chemokines, and adhesion molecules [21]. In rodent TBI models, the PPARγ agonist pioglitazone is protective against mitochondrial dysfunction, cognitive impairment, cortical tissue loss, inflammation, dendritic morphological changes, and long-term memory loss [22-25]. However, it is not yet known if PPARγ mediates the beneficial effects of KD in TBI.

To investigate the role of genetic and environmental factors in TBI outcomes, we developed a Drosophila melanogaster model of TBI [26, 27]. The fly TBI model uses a High-Impact Trauma (HIT) device to deliver blunt force injuries to the head and body of unanesthetized flies. 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 (MI24)—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 MI24 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 MI24 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. The MI24 of flies injured at a younger age is lower than at an older age, and the MI24 is lower for flies fed water versus high-carbohydrate diets during the 24 h following primary injuries [28, 29]. Furthermore, using the HIT device, Lee et al. (2019) demonstrated that β-hydroxybutyrate, a metabolite of KD, reduces TBI-induced aggression in flies [35]. Thus, to further explore the utility of the fly TBI model, we examined the effect of KD on the MI24 and lifespan following TBI. We found that, relative to high-carbohydrate diets, high-fat KD reduced the MI24 and increased lifespan following TBI and that Eip75B, an ortholog of PPARγ, was necessary to mediate the beneficial effect of KD on the MI24.

Materials and methods

Fly lines and culturing

Flies were maintained in humidified incubators at 25°C on solid CMYD. DGRP lines and Eip75B mutant fly lines were obtained from the Bloomington Stock Center (Indiana University).

Diets

Solid CMYD contains 30 g Difco granulated agar (Becton-Dickinson, Sparks, MD), 44 g YSC-1 yeast (Sigma, St. Louis, MO), 328 g cornmeal (Lab Scientific, Highlands, NJ), 400 ml unsulfured Grandma’s molasses (Lab Scientific), 3.6 L water, 40 ml propionic acid (Sigma), and tegosept (8 g Methyl 4-hydroxybenzoate in 75 ml of 95% ethanol) (Sigma). YD contains YSC-1 yeast (Sigma) in water. KD is commercial mouse Teklad ketogenic diet (TD.96355) (Envigo) that contains 173.3 g/Kg casein, 2.6 g/Kg DL-methionine, 586.4 g/Kg vegetable shortening (Crisco), 86.2 g/Kg corn oil, 88.0 g/Kg cellulose, 13.0 g/Kg vitamin mix (Teklad 40060), 2.5 g/Kg choline bitartrate, 0.1 g/Kg tertiary butylhydroquinone (TBHQ), 20.0 g/Kg mineral mix (calcium phosphate deficient), 19.3 g/Kg dibasic calcium phosphate, 8.2 g/Kg calcium carbonate, and 0.4 g/Kg magnesium oxide. KD at 0.3 cal/200 μl was prepared by adding 1.1 g of Teklad ketogenic diet to 5 ml of water and stirring the solution for 1 min at about 95°C. Table 1 provides the caloric contribution of carbohydrate, protein, and fat for each diet as well as the amount of each diet used to make 0.3 cal/200 μl solutions. Flies were fed water and diluted diets by placing 200 μl on a filter paper disc at the bottom of a vial.

Table 1

Caloric content of diets used in Figs 1–4.

Diet	Percent calories from:			cal/g	0.3 cal/200 μl
	Protein	Carbohydrate	Fat		g diet/ml water
KD	9.2	0.3	90.5	6.70	0.22
CMYD	4.9	92.7	2.4	3.31	0.45
YD	41.0	42.0	17.0	3.25	0.46
Glucose	0	100.0	0	3.74	0.40
Sucrose	0	100.0	0	3.94	0.38

KD, ketogenic diet; CMYD, cornmeal-molasses-yeast diet; YD, yeast diet.

MI24 and lifespan assays

Flies were injured using a HIT device as described by Katzenberger et al. [26, 27]. Vials containing 60 flies at 0–7 days old were injured by 4 strikes at 5 min intervals with the spring deflected to 90°. Vials with mixed sex flies had approximately 30 males and 30 females. The Mortality Index at 24 h (MI24) was calculated by subtracting the percent of uninjured flies that died from the percent of injured flies that died during the 24 h following TBI. The lifespan of adult flies that survived 24 h following TBI flies was determined using vials with 20 flies each. The number of surviving flies was counted daily until all flies had died. Flies were transferred to new vials approximately every 3 days. Flies were considered dead if they did not show obvious locomotor activity. Statistical analysis of survival by the Kaplan-Meier Fisher’s Exact Test was performed using OASIS 2 (Online Application for Survival Analysis 2) [36].

Results

Ketogenic diet following TBI reduces the incidence of early mortality

We previously found that the diet consumed directly after TBI in Drosophila substantially affects the incidence of early mortality [28, 29]. Flies fed cornmeal-molasses-yeast diet (a standard laboratory fly diet) or simple carbohydrates (i.e., sucrose, glucose, and fructose) during the 24 h following TBI have a significantly higher MI24 than flies fed water. To further explore the effect of diet on the MI24, we examined different concentrations of cornmeal-molasses-yeast diet (CMYD), yeast diet (YD, S. cerevisiae), and ketogenic diet (KD, a commercial mouse ketogenic diet). Based on caloric content, CMYD is high in carbohydrate and low in protein and fat, YD is high in carbohydrate and protein and low in fat, and KD is high in fat and low in carbohydrate and protein (Table 1). Diets were dissolved in water at 0.5 g/ml, serially diluted by 2-fold increments in water down to 0.0625 g/ml, and 200 μl was absorbed onto a filter paper disc that was placed at the bottom of a vial. 0–7 day old, mixed sex w flies cultured on solid CMYD (i.e., undiluted CMYD), were subjected to four strikes from the HIT device with 5 min between strikes and transferred to vials with diets at different concentrations.

We found that the MI24 increased to a similar extent with increasing concentrations of CMYD and YD (Fig 1A). In contrast, the MI24 was not affected by increasing concentrations of KD. We also examined the effect of CMYD, YD, and KD as well as glucose and sucrose at approximately the same caloric content (0.3 cal/200 μl) (Table 1). MI24 values were similar for flies fed CMYD, YD, glucose, or sucrose and they were significantly higher than the MI24 of flies fed water (Fig 1B). In contrast, the MI24 of flies fed KD was the same as that of flies fed water. These data support our prior finding that ingestion of carbohydrate after TBI increases the MI24 and demonstrate that ingestion of fat after TBI does not increase the MI24 compared with water.

Fig 1

Analysis of the effect of CMYD, YD, and KD on the MI24.

(A) Dose-response analysis of the effect of CMYD, YD, and KD on the MI24. The MI24 represents the percent mortality of injured flies minus the percent mortality of uninjured flies 24 h following TBI. MI24 values were determined for 0–7 day old, mixed sex w flies fed CMYD, YD, or KD at the indicated concentrations following TBI. At least three biological replicates of 60 flies were tested for each condition. Dots indicate the average MI24, and error bars indicate the standard error of the mean (SEM). (B) MI24 values were determined for 0–7 day old, mixed sex w flies fed water or CMYD, YD, KD, glucose, or sucrose at 0.3 cal/200 μl following TBI. Dots indicate biological replicates of 60 flies, bars indicate averages, and error bars indicate the SEM. (C) MI24 values were determined for 0–7 day old, mixed sex w flies fed water, 0.3 cal/200 μl KD, or no food or water (starved) following TBI. Dots indicate biological replicates of 60 flies, bars indicate averages, and error bars indicate the SEM. (D) Percent survival was determined for uninjured 0–7 day old, mixed sex w flies fed water (n = 240) or 0.3 cal/200 μl CMYD (n = 200) or KD (n = 239) over the course of the experiment. Error bars indicate the SEM, and the horizontal line at 50% indicates the median lifespan. Significance for panels A, B, and C was determined using ordinary one-way ANOVA with Dunnett’s Multiple Comparison test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

An alternative interpretation of the data in Fig 1A and 1B is that flies did not consume KD, suggesting that starvation and water have equivalent effects on the MI24. To test this possibility, we determined the MI24 of 0–7 day old, mixed sex w flies that were starved by placing them in vials with a dry filter paper disc following TBI. In contrast with flies fed KD, the MI24 of starved flies was substantially higher than that of flies fed water (Fig 1C), demonstrating that consuming KD rather than starvation was beneficial. As an additional approach to test if flies consumed KD, we examined the lifespan of uninjured, mixed sex w flies cultured on solid CMYD to 0–7 days old and thereafter on water or 0.3 cal/200 μl CMYD or KD. The median and maximum lifespans of flies cultured on KD (14.7 ± 1.1 days and 37 days, respectively) were longer than those of flies cultured on water (10.6 ± 0.1 days and 13 days, respectively) and shorter than those of flies cultured on CMYD (23.4 ± 0.6 days and 74 days, respectively), indicating that flies examined in Fig 1A and 1B consumed KD (Fig 1D). Further support for this conclusion is provided in Fig 4B.

Ketogenic diet is similarly beneficial following TBI in females versus males and in different genetic backgrounds

To investigate whether KD has sex-specific effects on TBI outcomes, we compared effects of KD, water, and solid CMYD on the MI24 of 0–7 day old female, male, and mixed sex w flies. In each case, solid CMYD resulted in a significantly higher MI24 than both water and KD, and water and KD had equivalent MI24 values (Fig 2A). Moreover, comparisons of male, female, and mixed sex flies, revealed that KD as well as solid CMYD and water had similar effects on the MI24. Therefore, sex does not alter the effects of KD, water, and solid CMYD on secondary injury mechanisms that cause early mortality following TBI.

Fig 2

The beneficial effect of KD on early mortality after TBI is equivalent in females and males and is conserved in different genetic backgrounds.

(A) MI24 values were determined for 0–7 day old female, male, and mix sex w flies fed solid CMYD, water, or 0.3 cal/200 μl KD. The MI24 represents the percent mortality of injured flies minus the percent mortality of uninjured flies 24 h following TBI. (B) MI24 values were determined for 0–7 day old, mixed sex fly lines (RAL441, RAL116, and RAL391) from the DGRP fed 0.3 cal/200 μl CMYD or KD following TBI [34]. Dots indicate biological replicates of 60 flies, bars indicate averages, and error bars indicate the SEM. Significance was determined using ordinary one-way ANOVA with Dunnett’s Multiple Comparison test. **p<0.01 and ***p<0.001.

We previously found that the MI24 of flies fed solid CMYD varied significantly among fly lines with different genetic backgrounds, including inbred fly lines from the Drosophila Genetic Reference Panel (DGRP) [26, 28, 29, 34]. To determine the extent to which genetic background affects the MI24 of flies fed KD following TBI, we examined three lines from the DGRP that have different MI24 values when fed solid CMYD following TBI [28, 29]. We fed 0–7 day old, mixed sex flies 0.3 cal/200 μl KD or CMYD for 24 h following TBI and determined the MI24 of each line. For all three DGRP lines, the MI24 of flies fed KD was lower than the MI24 of flies fed CMYD (Fig 2B). Moreover, the w line and the DGRP lines fed KD had comparable MI24 values, whereas these values varied among the same fly lines when fed CMYD (Figs 1B and 2B). These results indicate that the beneficial effect of KD on the MI24 does not depend on the starting value of the MI24 (on CMYD) in different fly lines, leading to uniformly low MI24 values for flies fed KD. However, it does appear that the beneficial effect of KD has a limiting threshold beyond which it cannot act further, resulting in a proportionally greater rescuing effect for lines with higher MI24 values on CMYD.

Ketogenic diet following TBI has beneficial long-term effects on lifespan

For both humans and flies, individuals that survive TBI manifest a variety of long-term consequences, including reduced lifespan, as a result of secondary injuries triggered by primary injuries to the brain. The exact connection between primary injuries and secondary injuries is complex, and the details are still poorly understood. The fact that the MI24 is reduced in flies fed KD immediately after TBI, raises the question of whether the beneficial effects of KD extend to longer-term pathological consequences of TBI. We examined this possibility using lifespan as a readout. Lifespan was determined for 0–7 day old, mixed sex w flies fed 0.3 cal/200 μl KD or CMYD for 24 h following TBI with surviving flies subsequently cultured on solid CMYD. As we reported previously, flies fed CMYD for 24 h after injury had a reduced lifespan relative to uninjured controls (Kaplan-Meier Fisher’s Exact Test, p = 4.1X10-9 at 50%) (Fig 3) [26, 29]. The same was true for flies fed KD (Kaplan-Meier Fisher’s Exact Test, p = 1.7X10-7 at 50%). However, notably, injured flies fed KD rather than CMYD for 24 h after injury had a significantly longer median lifespan (40.3 ± 0.2 days vs. 37.8 ± 0.28 days, Kaplan-Meier Fisher’s Exact Test, p = 1.0X10-4 at 50%). Moreover, the difference in median lifespan between injured flies and uninjured controls was much narrower for KD-fed than for CMYD-fed flies (40.3 ± 0.2 days vs. 44.2 ± 0.8 days for KD; 37.8 ± 0.28 days vs. 48.2 ± 1.0 days for CMYD). Thus, flies that avoid early mortality following TBI because of the beneficial effects of KD during a 24 h window after primary injuries continue to manifest long-term benefits of this diet weeks later.

Fig 3

KD has a long-term beneficial effect on lifespan following TBI.

Percent survival was determined for uninjured and injured 0–7 day old, mixed sex w flies fed 0.3 cal/200 μl CMYD or KD for 24 h following TBI and solid CMYD thereafter, that is, flies in the experiment that survived 24 h feeding on 0.3 cal/200 μl CMYD or KD were fed solid CMYD throughout the rest of their lifespan. At least 230 flies were examined for each condition. Error bars indicate the SEM, and the horizontal line at 50% indicates the median lifespan.

Beneficial effects of KD on early mortality are mediated by the PPARγ ortholog Eip75B

In mammals, the mechanism of action of KD is mediated by the transcription factor PPARγ, which has neuroprotective effects in a number of progressive neurological disorders, including TBI [19, 20]. Because KD exerts a protective effect following TBI in flies as well as mammals, we hypothesized that the underlying mechanism is conserved as well. If so, the protective effect in flies should depend on the transcription factor Eip75B (ecdysone-induced protein 75B), the Drosophila ortholog of PPARγ. The orthologous relationship is inferred both from amino acid sequence identity (i.e., Eip75B is the most significant match to human PPARγ in a BLAST search of the Drosophila proteome) and from common activation by the PPARγ agonist pioglitazone [37-39]. Under this hypothesis, mutational loss of Eip75B should result in loss of the beneficial effect of KD. Thus, we examined the effect of water and 0.3 cal/200 μl KD on the MI24 of 0–7 day old, mixed sex Eip75B mutant flies. Three hypomorphic alleles of Eip75B (Eip75B, Eip75B, and Eip75B) containing transposon insertions within the transcribed region were examined (Fig 4A). As in Fig 1, the MI24 was comparably low in control w flies fed either water or KD (ordinary one-way ANOVA with Dunnett’s Multiple Comparison test, p = 0.835) (Fig 4B). In contrast, although water-fed Eip75B, Eip75B, and Eip75B /Eip75B flies still had low MI24 values comparable to that of water-fed w controls, MI24 values were higher in KD-fed Eip75B mutants (ordinary one-way ANOVA with Dunnett’s Multiple Comparison test, p = 0.078, p = 0.033, and p = 0.006, respectively), indicating that the beneficial effect of KD was impaired in these mutants. Furthermore, higher MI24 values in KD-fed versus water-fed mutants provides further evidence that flies consumed KD. The beneficial effect of KD was, however, retained in Eip75B homozygotes (ordinary one-way ANOVA with Dunnett’s Multiple Comparison test, p = 0.999), which we attribute to a presumptive weaker loss of function of Eip75B caused by this mutation. Eip75B only disrupts three of the seven Eip75B pre-mRNA isoforms, whereas Eip75B and Eip75B disrupt four and five isoforms, respectively (Fig 4A). Thus, while it remains possible that differences in genetic background underlie differences in MI24 values for Eip75B mutant flies fed water versus KD, the data support the conclusion that activation of Eip75B/PPARγ by KD triggers mechanisms that reduce early mortality following TBI.

Fig 4

The beneficial effect of KD on early mortality following TBI requires Eip75B.

(A) Transposon insertion locations (arrowheads) relative to the seven Eip75B transcripts drawn 5’ to 3’ (http://flybase.org). Gray boxes, black boxes, and lines indicate 5’ and 3’ untranslated regions, exons, and introns, respectively. (B) MI24 values were determined for 0–7 day old, mixed sex w and Eip75B mutant flies fed water or 0.3 cal/200 μl KD following TBI. The MI24 represents the percent mortality of injured flies minus the percent mortality of uninjured flies 24 h following TBI. Dots indicate biological replicates of 60 flies, bars indicate averages, and error bars indicate the SEM. Significance was determined using ordinary one-way ANOVA with Dunnett’s Multiple Comparison test.

Discussion

TBI patients face a spectrum of neurobehavioral sequelae initiated by primary injuries to the brain and mediated by the interplay of genetic and environmental factors that control pathophysiological cascades. Here, we discovered that an interaction between the genetic factor Eip75B/PPARγ and the environmental factor KD affects early mortality in a fly TBI model. In particular, whereas flies fed high-carbohydrate CMYD or YD exhibited a dose-dependent increase in early mortality compared with flies fed water, flies fed high-fat KD showed no increase in early mortality (Fig 1A and 1B). The beneficial effect of KD on early mortality was equivalent in males and females, conserved in different genetic backgrounds, and had long-term beneficial effects on lifespan as well (Figs 2 and 3). However, the beneficial effect of KD on early mortality was diminished in flies mutant for Eip75B, a transcription factor orthologous to mammalian PPARγ, suggesting that KD exerts its effect through Eip75B (Fig 4B). These data provide a mechanistic link between KD and PPARγ in modifying TBI outcomes and demonstrate the utility of the fly TBI model for dissecting interactions between genetic and environmental factors that affect TBI outcomes.

The KD-Eip75B/PPARγ pathway may reduce early mortality following TBI by inhibiting Relish/NF-κB

Our data suggest that KD reduces early mortality following TBI by activating Eip75B/PPARγ. However, it remains to be determined what occurs downstream of Eip75B/PPARγ to exert this effect. One possibility is that Eip75B/PPARγ controls expression of genes involved in inflammation. In mammals, activation of PPARγ by dietary fatty acids mitigates neuroinflammation by inhibiting NF-κB, a transcriptional activator of cytokine, chemokine, and adhesion genes downstream of Toll-like receptor (TLR)/Interleukin-1 receptor (IL-1R) and Tumor necrosis factor-α receptor (TNFR) innate immune response signaling pathways [19]. PPARγ inhibits NF-κB by a variety of mechanisms, including ubiquitination and degradation, export from the nucleus, competition for cofactors, and steric inhibition of DNA binding [40]. 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. Relish functions in the Immune-deficiency (Imd) pathway that is homologous to the TNFR pathway in mammals and controls transcription of numerous antimicrobial peptide genes (AMPs) that produce resistance to infection [47, 48]. A confirmed transcriptional target of Relish in TBI is the AMP gene Metchnikowin (Mtk), which when mutated reduces early mortality and increases lifespan following TBI [32]. Thus, KD-mediated activation of Eip75B/PPARγ may reduce early mortality following TBI by inhibiting Relish/NF-κB. This could be tested in the fly TBI model by examining effects of KD and pioglitazone on the MI24, lifespan, and expression of AMP genes in wild type as well as Relish and Mtk mutant flies.

KD and water appear to reduce early mortality following TBI by different mechanisms

Genetically diverse fly lines fed KD or water following TBI consistently had a lower incidence of early mortality relative to flies fed high-carbohydrate diets (Figs 1A and 1B and 2) [29]. These data suggest that KD and water might activate the same protective pathways. Water is a fasting condition where the amount of available carbohydrate is decreased, forcing a switch to the use of fatty acids as a nutrient supply through beta-oxidation and ketogenesis [49]. Ketogenesis converts acetyl-CoA to ketone bodies (e.g., β-hydroxybutyrate, acetone, and acetoacetate) that are used by the brain and other tissues to produce energy. Flies with impaired mitochondrial ATP synthase activity produce elevated amounts of β-hydroxybutyrate, indicating that ketogenesis operates in flies [50]. Additionally, aggressive behaviors and early mortality induced by TBI in flies are reduced when flies are raised on high-carbohydrate diet supplemented with β-hydroxybutyrate relative to high-carbohydrate diet alone, indicating that β-hydroxybutyrate operates in the fly TBI model [35]. Nonetheless, several lines of evidence argue that KD and water act by distinct mechanisms to reduce early mortality following TBI. First, the MI24 of Eip75B mutants differed depending on whether they were fed KD or water, indicating that water acts independently of Eip75B/PPARγ (Fig 4B). 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].

In conclusion, our observations indicate that KD signals through PPARγ to improve TBI outcomes in flies. Thus, the fly TBI model offers considerable potential for understanding the cellular and molecular mechanisms that underlie the beneficial effects of KD and may ultimately facilitate development of therapeutic intervention for TBI in humans.

2 Aug 2021

PONE-D-21-21417

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

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript the authors test the impact of feeding flies a ketogenic (KD) vs high carbohydrate diet (CMYD) in the 24 hrs after TBI. The system they use is the so-called HIT device to administer TBI. They find that animals on a the CMYD diet or a yeast diet have standard high mortality after 24 hrs, whereas animals on a KD have low mortality similar to feeding animals water. They provide evidence that the animals are eating because starved animals have a high mortality. They show that the protective effect of a KD is conferred upon a range of genetic backgrounds, and then provide molecular insight by showing that the effect is dependent on Eip75B gene function, which is the homolog of PPAR-gamma. These findings suggest that KD, by activating Eip75B activity, mitigates the deleterious outcomes of TBI. These data are consistent with some mammalian findings.

Overall this is an interesting and appropriate manuscript. There are just a few points that need to be addressed..

In Figure 2, are the lifespans of the different strains on KD significantly different? The RAL441 and RAL391 look like they might be. Thus, whereas on a CMYD diet, the mortality is marked different, on a KD diet it may still be different but overall dramatically better.

In figure 3, at first it is rather confusing why these lifespans are so dramatically different from those in figure 1. They could clarify this by making the point more clearly that the flies are cultured differently. That is…

”Lifespan was determined…for 24 h following TBI but then surviving flies were cultured on solid CMYD.”

Also, some of their arguments about lifespan (median lifespan between injured and uninjured animals; Fig 3) seem sketchy because the uninjured CMYD animals have a better median lifespan than KD animals. So the animals survive better after TBI if on a KD for 24 hrs, but after that, the KD lifespan is compromised. So it seems like there are pros and cons. How do they explain or interpret this.

Since only 1 of two Eip75B alleles showed loss of the KD benefit, the significance of Eip75B function seems questionable also. Maybe those results are due to genetic background. Can they show somehow that the alleles are of a strength consistent with their interpretation that the MI04895 allele is less severe? This needs another allele, and/or controls for background, or some other way of validating the findings to be properly interpreted. Perhaps they could measure activation of PPAR-gamma to at least show correlation. In any case, this point needs to be strengthened.

Reviewer #2: This is an interesting paper by Blommer et al that provides novel experimental data indicating that a ketogenic diet can mediate beneficial effects in a fly model of traumatic brain injury. My comments are as followed:

1. Please make sure that gene names are consistently italicized.

2. According to the Alliance of Genome Resources, Eip75B is an ortholog of the following human genes: NR1D2, PPARA, PPARG, NR1D1, and PPARD. It may be worthwhile to mention that PPARG is not the sole ortholog of this fly gene.

3. In the Introduction, I would mention that traumatic brain injury is a known risk factor dementia. This will help highlight the broader importance of your research. There are multiple papers one could cite for this, such as this recent systematic review and meta-analysis (PMID: 33044182).

4. In the Introduction, it may be useful to mention that there is a growing interest in the interplay between lipid metabolism and aging. Dr. Anne Brunet has published excellent reviews on this topic.

5. It would be helpful if the authors can explicitly list the increase in median and mean lifespan in response to a ketogenic diet. For those looking to do systematic reviews and/or meta-analyses in the future, this information could be useful. It may be simplest to do this in the form of a table that summarizes all of the lifespan results (i.e., p-value, median increase, mean increase).

6. While there is evidence that a ketogenic diet may be beneficial in specific circumstances, it may not be as impactful as other dietary interventions (e.g., caloric restriction, intermittent fasting, Mediterranean diet, plant-based diet). In the Discussion, can the authors comment on where a ketogenic diet will be specifically beneficial vs. other dietary alterations and why they think this is the case?

7. Major sections vs. sub-sections need to be demarcated more clearly. I recommend making major sections entirely capitalized (e.g., DISCUSSION) while keeping sub-sections as sentence case (e.g., KD and water appear to reduce early mortality following TBI by different mechanisms).

8. Unless the journal handles this separately, please add a Conflict of Interest section to your manuscript.

**********

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Reviewer #1: No

Reviewer #2: No

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9 Sep 2021

Responses to reviewer’s criticisms (in bold font)

Reviewer #1: In this manuscript the authors test the impact of feeding flies a ketogenic (KD) vs high carbohydrate diet (CMYD) in the 24 hrs after TBI. The system they use is the so-called HIT device to administer TBI. They find that animals on a the CMYD diet or a yeast diet have standard high mortality after 24 hrs, whereas animals on a KD have low mortality similar to feeding animals water. They provide evidence that the animals are eating because starved animals have a high mortality. They show that the protective effect of a KD is conferred upon a range of genetic backgrounds, and then provide molecular insight by showing that the effect is dependent on Eip75B gene function, which is the homolog of PPAR-gamma. These findings suggest that KD, by activating Eip75B activity, mitigates the deleterious outcomes of TBI. These data are consistent with some mammalian findings.

Overall this is an interesting and appropriate manuscript. There are just a few points that need to be addressed.

In Figure 2, are the lifespans of the different strains on KD significantly different? The RAL441 and RAL391 look like they might be. Thus, whereas on a CMYD diet, the mortality is marked different, on a KD diet it may still be different but overall dramatically better.

Previously, we found that fly lifespan affects the MI24, that is, at a given age, flies with a short lifespan have a higher MI24 than flies with a long lifespan (Katzenberger et al. (2013) PNAS). Thus, it is likely that RAL441 flies have a longer lifespan than RAL391 flies when fed solid CMYD. We agree that it would be interesting to determine if the lifespan of these lines differs when fed KD, but we believe that this is beyond the scope of the manuscript.

In figure 3, at first it is rather confusing why these lifespans are so dramatically different from those in figure 1. They could clarify this by making the point more clearly that the flies are cultured differently. That is…

”Lifespan was determined…for 24 h following TBI but then surviving flies were cultured on solid CMYD.”

We clarified this point in several ways. In the legend for Figure 1, we added the phrase “over the course of the experiment” to the sentence “Percent survival was determined for uninjured 0-7 day old, mixed sex w1118 flies fed water (n=240) or 0.3 cal/200 �l CMYD (n=200) or KD (n=239) over the course of the experiment.” Also, in the legend for Figure 3, we added the phrase “that is, flies in the experiment that survived 24 h feeding on 0.3 cal/200 �l CMYD or KD were fed solid CMYD throughout the rest of their lifespan” to the sentence “Percent survival was determined for uninjured and injured 0-7 day old, mixed sex w1118 flies fed 0.3 cal/200 �l CMYD or KD for 24 h following TBI and solid CMYD thereafter, that is, flies in the experiment that survived 24 h feeding on 0.3 cal/200 �l CMYD or KD were fed solid CMYD throughout the rest of their lifespan.”

Also, some of their arguments about lifespan (median lifespan between injured and uninjured animals; Fig 3) seem sketchy because the uninjured CMYD animals have a better median lifespan than KD animals. So the animals survive better after TBI if on a KD for 24 hrs, but after that, the KD lifespan is compromised. So it seems like there are pros and cons. How do they explain or interpret this.

We see the reviewer’s point that the data are not black and white. But we think that our main conclusion is supported by the data. Figure 3 shows that the survival curve of injured KD flies more nearly approximates that of uninjured KD flies than does that of injured CYMD flies to their control. Thus, relative to the appropriate control, the ketogenic diet is beneficial both at 24 hours and throughout the course of the lifespan for flies that survived mortality at 24 hours. Accordingly, in the Abstract we conclude “flies protected from early mortality by KD continued to show survival benefits weeks later” and in the section about Figure 3, we similarly conclude “Thus, flies that avoid mortality following TBI because of the beneficial effects of KD during a 24 h window after primary injuries continue to manifest long-term benefits of this diet weeks later.”

Since only 1 of two Eip75B alleles showed loss of the KD benefit, the significance of Eip75B function seems questionable also. Maybe those results are due to genetic background. Can they show somehow that the alleles are of a strength consistent with their interpretation that the MI04895 allele is less severe? This needs another allele, and/or controls for background, or some other way of validating the findings to be properly interpreted. Perhaps they could measure activation of PPAR-gamma to at least show correlation. In any case, this point needs to be strengthened.

To address this point, we examined another Eip75B allele (Eip75BKG04491) and included these data in Figure 4. Thus, there are now three Eip75B alleles or allelic combinations in which the MI24 is higher for KD than water. We also added a figure (Figure 4A) that supports the conclusion that Eip75BMI04895 is a weaker allele than Eip75BKG04491 and Eip75BBG02576. Lastly, we added analyses of male and female flies to Figure 2 (new panel 2A) that reinforce the finding that water and KD have similar effects on the MI24 of flies that are wild type for Eip75B. Based on these data, we have concluded that “The beneficial effect of KD was, however, retained in Eip75BMI04895 homozygotes (ordinary one-way ANOVA with Dunnett’s Multiple Comparison test, p=0.999), which we attribute to a presumptive weaker loss of function of Eip75B caused by this mutation. Eip75BMI04895 only disrupts three of the seven Eip75B pre-mRNA isoforms, whereas Eip75BKG04491 and Eip75BBG02576 disrupt four and five isoforms, respectively (Fig. 4A). Thus, while it remains possible that differences in genetic background underlie differences in MI24 values for Eip75B mutant flies fed water versus KD, the data support the conclusion that activation of Eip75B/PPAR� by KD triggers mechanisms that reduce early mortality following TBI.”

Reviewer #2: This is an interesting paper by Blommer et al that provides novel experimental data indicating that a ketogenic diet can mediate beneficial effects in a fly model of traumatic brain injury. My comments are as followed:

1. Please make sure that gene names are consistently italicized.

We believe that all of the gene names are italicized. In accord with Drosophila convention, genes and RNAs are italicized, and proteins are not italicized.

2. According to the Alliance of Genome Resources, Eip75B is an ortholog of the following human genes: NR1D2, PPARA, PPARG, NR1D1, and PPARD. It may be worthwhile to mention that PPARG is not the sole ortholog of this fly gene.

Our assignment of Eip75B as the ortholog of PPAR� is based on two lines of evidence. First, BLAST search analysis of the Drosophila genome with the human PPAR��protein identified Eip75B as the most significant match. Second, data in references 38 and 39 demonstrate that Eip75B functions similarly to PPAR���Thus, we have added the sentence “The orthologous relationship is inferred both from amino acid sequence identity (i.e., Eip75B is the most significant match to human PPAR� in a BLAST search of the Drosophila proteome).” This information is provided under the section heading “Beneficial effects of KD on early mortality are mediated by the PPAR� ortholog Eip75B.” PPARA, PPARD, NR1D1, and NR1D2 may be listed as orthologs simply because they are similar in sequence to Eip75B. Often single genes in flies are represented by expanded gene families in mammals.

3. In the Introduction, I would mention that traumatic brain injury is a known risk factor dementia. This will help highlight the broader importance of your research. There are multiple papers one could cite for this, such as this recent systematic review and meta-analysis (PMID: 33044182).

We appreciate the suggestions to include dementia and the interplay between lipid metabolism and aging in the Introduction. Our rationale for not including these topics as well as many others related to TBI is to focus the Introduction on topics that are critical for the reader to understand and to appreciate the significance and implications of the data that are presented.

4. In the Introduction, it may be useful to mention that there is a growing interest in the interplay between lipid metabolism and aging. Dr. Anne Brunet has published excellent reviews on this topic.

Please see the response to point 3.

5. It would be helpful if the authors can explicitly list the increase in median and mean lifespan in response to a ketogenic diet. For those looking to do systematic reviews and/or meta-analyses in the future, this information could be useful. It may be simplest to do this in the form of a table that summarizes all of the lifespan results (i.e., p-value, median increase, mean increase).

For readability, we have provided median lifespan numbers and p-values in the text as they were discussed.

6. While there is evidence that a ketogenic diet may be beneficial in specific circumstances, it may not be as impactful as other dietary interventions (e.g., caloric restriction, intermittent fasting, Mediterranean diet, plant-based diet). In the Discussion, can the authors comment on where a ketogenic diet will be specifically beneficial vs. other dietary alterations and why they think this is the case?

Unfortunately, there are very little data in the mammalian literature and no data in the Drosophila literature on the effect of other dietary interventions on TBI. Thus, we do not feel able to add any meaningful comparisons among the various diets at this time.

7. Major sections vs. sub-sections need to be demarcated more clearly. I recommend making major sections entirely capitalized (e.g., DISCUSSION) while keeping sub-sections as sentence case (e.g., KD and water appear to reduce early mortality following TBI by different mechanisms).

The guidelines for style are set by PLoS One, which we are required to follow.

8. Unless the journal handles this separately, please add a Conflict of Interest section to your manuscript.

Please see the response to point 7.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

20 Sep 2021

PONE-D-21-21417R1Ketogenic diet reduces early mortality following traumatic brain injury in Drosophila via the PPARg ortholog Eip75BPLOS ONE

Dear Dr. Wassarman,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================the reviewers feel and I concur that the manuscript is ready for publication save for the clarifications presented below. It is essential in my opinion to clarify the MI24 measurements are indeed %.Please make these minor changes and submit ASAP.

==============================

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Reviewers' comments:

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript has been improved by the revisions suggested by the Reviewers.

One minor point is to make clear along the y-axes that MI24 is a percentage. It is unlabelled and not clarified in the legends.

Also in the text on page on page 4, "varies from 7 to 58" should be "7% to 58% among 179...."

Reviewer #2: It is surprising that the authors were unwilling to accommodate minor changes to the text that would have helped emphasize the importance of this work to a broader audience. While my recommendation is to accept the paper based on its technical soundness and its improvement from the review process, I would encourage the authors to be more flexible in the future.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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21 Sep 2021

Responses to reviewer’s criticisms

Reviewer #1: This manuscript has been improved by the revisions suggested by the Reviewers.

One minor point is to make clear along the y-axes that MI24 is a percentage. It is unlabelled and not clarified in the legends.

Also in the text on page on page 4, "varies from 7 to 58" should be "7% to 58% among 179...."

We appreciate the reviewer’s suggestion. To increase clarity, we added the following sentence to the legends for Figures 1, 2, and 4. “The MI24 represents the percent mortality of injured flies minus the percent mortality of uninjured flies 24 h following TBI.”

Adding percent or % to the y-axis label is problematic because the MI24 is defined as a percent. So, MI24 percent and MI24% are redundant and percent mortality and % mortality are incorrect. Furthermore, I would like the graphs to be consistent with our prior publications. We have published graphs in nine papers with y-axes labeled MI24, and other labs have also used this nomenclature.

Reviewer #2: It is surprising that the authors were unwilling to accommodate minor changes to the text that would have helped emphasize the importance of this work to a broader audience. While my recommendation is to accept the paper based on its technical soundness and its improvement from the review process, I would encourage the authors to be more flexible in the future.

We thank the reviewer for the suggestion.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

7 Oct 2021

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

PONE-D-21-21417R2

Dear Dr. Wassarman,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Efthimios M. C. Skoulakis, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

15 Oct 2021

PONE-D-21-21417R2

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

Dear Dr. Wassarman:

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