A Drosophila Model of Closed Head Traumatic Brain Injury

A Drosophila model of closed head traumatic brain injury

Rebeccah J. Katzenbergera, Carin A. Loewenb, Douglas R. Wassarmana, Andrew J. Petersena, Barry Ganetzkyb,1, and David A. Wassarmana,1

aDepartment of Cell and Regenerative Biology, School of Medicine and Public Health, and bLaboratory of Genetics, University of Wisconsin–Madison, Madison, WI 53706

Contributed by Barry Ganetzky, September 6, 2013 (sent for review August 6, 2013) Traumatic brain injury (TBI) is a substantial health issue world- three regions, the protocerebrum, deutocerebrum, and trito- wide, yet the mechanisms responsible for its complex spectrum of cerebrum, which are homologous to the forebrain, midbrain, and pathologies remains largely unknown. To investigate the mecha- hindbrain, respectively, of humans (10). The fly brain is composed nisms underlying TBI pathologies, we developed a model of TBI in of functionally diverse neurons with all of the structural features, Drosophila melanogaster. The model allows us to take advantage neurotransmitters, and ion channels found in human neurons of the wealth of experimental tools available in flies. Closed head (11). Glial cell types and functions in flies are also similar to those TBI was inflicted with a mechanical device that subjects flies to in humans (12). For instance, surface glial cells cover brain neu- rapid acceleration and deceleration. Similar to humans with TBI, rons to form the blood–brain barrier, and glial cells are part of the flies with TBI exhibited temporary incapacitation, ataxia, activa- innate immune system (13, 14). The fly brain is encapsulated by an tion of the innate immune response, neurodegeneration, and exoskeleton, also known as the cuticle (15). Like the human cra- death. Our data indicate that TBI results in death shortly after nium, the cuticle is relatively inelastic. It provides protection from a primary injury only if the injury exceeds a certain threshold the environment, and it defines the structure of the head. The and that age and genetic background, but not sex, substantially brain is further separated from the cuticle by fluid hemolymph, affect this threshold. Furthermore, this threshold also appears to a blood-like carrier system for macrophages and nutrients (16). be dependent on the same cellular and molecular mechanisms that The topology of the fly brain relative to the cuticle is such that an control normal longevity. This study demonstrates the potential of impact to the head or body most likely causes the brain to ricochet flies for providing key insights into human TBI that may ultimately and deform against the cuticle resulting in TBI. provide unique opportunities for therapeutic intervention. Here, we describe the development and initial characterization of a fly model of TBI. Using the model, we found that concussion | insect | chronic traumatic encephalopathy fundamental characteristics of human TBI also occur in flies. We also found that primary injuries exacerbate the normal age- raumatic brain injury (TBI) is a leading cause of neurological related decline in flies. This may explain why human TBI is as- Tdeficits and mortality worldwide (1, 2). TBI outcomes result sociated with cognitive and neurodegenerative disorders that are from primary and secondary injuries that cause cell damage and typical of older individuals and why TBI outcomes are worse in death in the brain. Primary injuries occur during the initial im- older individuals. pact and are triggered by external mechanical forces that deform the brain, whereas secondary injuries are triggered by cellular Results and molecular responses that occur over time in reaction to the The HIT Device Reproducibly Inflicts Closed Head TBI in Flies. To in- primary injuries. TBI outcomes are heterogeneous in the human flict TBI in flies, we built the “high-impact trauma” (HIT) device, population owing to variation in the location and strength of which consists of a metal spring clamped at one end to a wooden primary injuries as well as genetic and environmental factors that affect the severity of primary and secondary injuries. This het- Significance erogeneity is one of the most significant barriers to the development of therapeutic interventions (3–5). Therefore, research Traumatic brain injury (TBI) occurs when a strong jolt to the aimed at determining the genetic and environmental factors that head causes damage to brain cells, resulting in immediate and affect the severity of primary and secondary injuries is essential for long-term consequences including physical, behavioral, and developing treatments for TBI. cognitive problems. Despite the importance of TBI as a major To investigate the underlying cellular and molecular basis of health issue, our understanding of the underlying cellular and TBI, we developed a Drosophila melanogaster model. Key advan- molecular mechanisms is limited. To unravel these mechanisms, tages of flies are that (i) large numbers of animals can be rapidly we have developed a model of TBI in the fruit fly, Drosophila and inexpensively analyzed to establish causality between injuries melanogaster, where we can apply many powerful experi- and outcomes, (ii) many molecular and genetic tools are available mental tools. The main features of human TBI also occur in flies, to investigate the molecules and pathways that underlie injuries, and suggesting that the underlying mechanisms are conserved. Our (iii) outcomes can readily be evaluated over the entire animal life- studies demonstrate the value of a flymodelforunderstanding span. Thus, flies provide unique opportunities to understand TBI. the consequences of TBI and may ultimately enable development It is reasonable to expect that TBI can be modeled in flies be- of therapies for their prevention and treatment. cause Drosophila has already proved to be an extremely useful model for studying other human neurodegenerative disorders (6). Author contributions: R.J.K., A.J.P., B.G., and D.A.W. designed research; R.J.K., C.A.L., In fact, research in flies has already provided novel insights into D.R.W., A.J.P., and D.A.W. performed research; D.A.W. contributed new reagents/analytic tools; R.J.K., C.A.L., B.G., and D.A.W. analyzed data; and R.J.K., C.A.L., B.G., and D.A.W. neurodegeneration, memory, and sleep, all of which are affected wrote the paper. – fl in human TBI (6 8). Additionally, y and human brains have The authors declare no conflict of interest. fl common architectural, cellular, and molecular features. The y 1To whom correspondence may be addressed. E-mail: [email protected] or dawassarman@ brain is bilaterally symmetrical and is joined to the ventral gan- wisc.edu. glion that innervates the body, similar to the way that the spinal This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cord innervates the human body (9). The fly brain is organized into 1073/pnas.1316895110/-/DCSupplemental.

E4152–E4159 | PNAS | Published online October 14, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1316895110 Downloaded by guest on September 27, 2021 the percentage of flies that died within 24 h. We define the PNAS PLUS percentage of flies that died within 24 h after injury as the mortality index at 24 h (MI24). To minimize any variation in outcome associated with differences in age, genotype, or sex, we used 0- to 3-d-old white (w1118) flies in every experiment, with an approximately equal number of males and females. Flies received 0–10 strikes with the spring deflected to 90° and with 5 min recovery periods between strikes. After a single strike, the MI24 was 4.5 ± 1.2 (Fig. 2A). Additional strikes resulted in an increase in the MI24; however, additional strikes did not significantly increase the MI24 per strike (Fig. 2B). The fact that not all flies died after a single strike indicates that primary injuries cause death within 24 h only if the injuries exceed a specific threshold, where threshold is presumably a composite measure of impact location and strength. Furthermore, the fact that the number of strikes did not affect the MI24 per strike indicates that flies subjected to primary injuries below the threshold do not have increased susceptibility for mortality from subsequent primary injuries. We also examined whether varying the recovery time between repeated strikes had any effect on the MI24. The expectation was that if the time between primary injuries was increased, then secondary injuries or injury repair mechanisms would have more Fig. 1. The HIT device was used to inflict TBI in 0- to 4-d-old w1118 flies. time to occur, thereby altering the threshold for death caused by fl Images show the HIT device and ies before and after a strike. Shown are (A) subsequent primary injuries. Thus, the MI24 would change as the the HIT device deflected to 90° before a strike, (B) the HIT device immedi- time between strikes changed. To test this hypothesis, flies re- ately after a strike, (C)60flies in a vial before a strike, and (D) after a strike. ceived four strikes with time intervals from 1 to 60 min between successive strikes. The temporal spacing of strikes did not significantly affect the MI24 (Fig. S3A). Therefore, over the times board with the free end positioned over a polyurethane pad (Fig. tested, the time between successive strikes neither exacerbates 1A and Fig. S1). A standard plastic vial containing unanesthetized fl fi nor ameliorates the consequences of multiple primary injuries. ies that are con ned to the bottom quarter of the vial by a sta- The fact that the time between strikes did not affect the MI tionary cotton ball is connected to the free end of the spring. 24 fl indicates that secondary injuries and injury repair mechanisms When the spring is de ected and released, the vial rapidly contacts either neutralize one another or do not contribute to the primary the polyurethane pad, and a mechanical force is delivered to injury threshold for death within 24 h.

the flies as they contact the vial wall and rebound (Fig. 1B). In- NEUROSCIENCE fl Another parameter that could affect the MI24 is the number of dividual ies presumably contact the wall with different regions of flies per vial. To investigate this possibility, we placed 10–60 flies their head and/or body and with different forces, so primary fl per vial in increments of 10 and subjected the vials to four strikes injuries will vary among ies in the same vial. The lack of pene- with 5-min recovery periods between strikes. The number of flies trating injuries and the randomness of impact location and in a vial did not significantly affect the MI24 (Fig. S3B). Thus, in strength are features of closed head TBI in the human population. vials containing 10–60 flies, contact among flies does not appear Since closed head TBI is the most common form of TBI occurring to affect the primary injury threshold for death within 24 h. in falls, sports collisions, and automobile crashes, a fly model is of significant potential value (17). As shown in Fig. 2, the phenotypic TBI Reduces Lifespan. To investigate whether multiple TBI inci- effects on flies subjected to HIT device-induced injury are highly dents have long-term effects on survival, we determined the reproducible. lifespan of flies that survived beyond 24 h after receiving 0–10 The strength of the primary injuries inflicted by the HIT de- strikes with 5 min recovery periods between strikes. We assumed vice can be adjusted, either by varying the extent of spring de- that death from primary injuries was complete by 24 h because flection or by varying the number of strikes. Deflection of the the percent survival at 24 h was not substantially different from springto90°resultedinanimpactvelocityof∼3.0 m/s (6.7 miles/h) the percent survival at 48 h. Relative to untreated flies, flies that andanaverageforceof2.5N.Someflies subjected to a single received one strike had a substantially reduced median lifespan strike with the spring deflected to 90° became temporarily in- (48.3 ± 1.2 d vs. 38.3 ± 3.5 d) and significantly reduced maximum capacitated and fell to the bottom of the vial; however, they did not lifespan (85.7 ± 3.6 d vs. 73.0 ± 0.6 d) (Fig. 2C and Table S2). incur obvious external damage to the head, body, or appendages Additional strikes caused further reductions in the median and (Fig. 1 C and D). During the first minute after a strike, 8.8 ± 3.8% maximum lifespan, but an upper limit for median lifespan was of flies were incapacitated, lying on their back or side on the bot- reached at four strikes; additional strikes did not further reduce tom of the vial. Most of the incapacitated flies recovered locomotor the median lifespan. These results demonstrate that primary activity within 5 min (Fig. S2A). Subsequently, their mobility, as injuries that are below the threshold for death within 24 h none- measured by climbing ability, was reduced but gradually returned theless cause a reduction in lifespan that likely results from the tonormalovera2-dperiod(Fig. S2B and Table S1). The imme- long-term consequences of secondary injuries. diate loss of motor ability, followed by ataxia and gradual recovery of mobility are reminiscent of concussion in humans and are Age-Associated Processes Lower the Primary Injury Threshold. For consistent with the idea that the HIT device inflicts brain injury subsequent experiments, we developed a standard TBI protocol in flies. consisting of four strikes separated by 5 min intervals. This 1118 protocol resulted in an MI24 of 19.0 ± 4.0 for 0- to 3-d-old w Primary Injuries That Exceed a Threshold Cause Death Within 24 h. To flies (Fig. 2A). This moderate MI24 is convenient for identifying determine the effect of subjecting flies to multiple TBI incidents, genetic and environmental factors that enhance or suppress the we varied the number of strikes that flies received and measured effect of primary injuries.

Katzenberger et al. PNAS | Published online October 14, 2013 | E4153 Downloaded by guest on September 27, 2021 1118 Fig. 2. The effect of the number of strikes on the MI24 and the lifespan of w flies. (A) The MI24 is graphed vs. the number of strikes. MI24 values were normalized to those of untreated flies. (B) Data from A are graphed as the MI24 per strike vs. the number of strikes. The MI24 per strike was not significantly affected by the number of strikes (P = 0.82, one-way ANOVA). (C) The percent survival is graphed vs. age for flies that received the indicated number of strikes. Only flies that survived 24 h after treatment were included in the analysis. The line at 50% indicates the median lifespan. Error bars indicate the SD for at least three independent trials of 60 flies each. Exact values and SD for median and maximum lifespans are provided in Table S2.

We used the standard TBI protocol to determine the effect of performed histological analysis on brains of younger (0- to 4-d- 1118 sex and age on the MI24. w male and female flies at 0–4 and old) and older (20- to 21-d-old) flies 14 d after they were sub- 20–22 d old were assayed separately. Sex did not have a signifi- jected to the standard TBI protocol. Neurodegeneration in flies cant effect on the MI24 at either age (Fig. 3A). In contrast, age is commonly manifested by the appearance of vacuolar lesions in fi did have a signi cant effect; the MI24 was approximately twofold the brain neuropil, a region enriched for axons, synaptic termi- higher for older flies compared with younger flies. nals, and glial cells (6). In contrast with untreated flies whose To more systematically examine the effect of age on the MI24, brains had a smooth and uniform appearance throughout the fl – – we examined ies in 12 age groups ranging from 0 3to28 29 d neuropil (Fig. 4A), treated flies exhibited neuropathology man- old that were treated with the standard TBI protocol. The data ifested by the appearance of numerous small vacuolar lesions revealed an increase in the MI24 as age increased (Fig. 3B). (∼1.0 μm in diameter) (Fig. 4B). We also observed large vacuolar These results suggest that cellular and molecular changes occur lesions (5.0–10.0 μm in diameter) in injured flies. The incidence during aging that lower the primary injury threshold for death of large vacuolar lesions in the central region of the brain in- within 24 h. creased with the number of times flies were subjected to the Age-Associated Processes Enhance Neurodegeneration Due to Brain standard TBI protocol (Fig. 4C). In addition, the incidence of fl Injuries. TBI in rodents and humans increases the occurrence of large vacuolar lesions was higher in older ies subjected to TBI chronic traumatic encephalopathy (CTE), a form of neuro- compared with similarly treated younger flies. These data indicate degeneration that generally appears long after (years to decades that brain injuries in flies elicit neurodegeneration as a long-term in the case of humans) the primary injury (18). To determine if consequence and that the extent of neurodegeneration is de- neurodegeneration is also a long-term outcome of TBI in flies termined by the severity of primary injuries and the age at the time and whether it is affected by age at the time of primary injury, we of primary injuries.

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1118 Fig. 3. The MI24 of w flies subjected to the standard TBI protocol is not affected by sex but is affected by age at the time of injury. (A) For flies treated with the standard TBI protocol, the MI24 is graphed vs. sex. The experiment was performed with 0- to 4-d-old and 20- to 22-d-old flies. MI24 values were normalized to those of untreated flies. Sex did not have a significant effect on the MI24 for either 0- to 4-d-old flies (P = 0.27, one-tailed t test) or 20- to 22-d- old flies (P = 0.34, one-tailed t test). Age at the time of injury did have a significant effect on the MI24 for both males (P = 0.004, one-tailed t test) and females (P = 0.002, one-tailed t test). (B) The MI24 is graphed vs. age at the time of treatment with the standard TBI protocol. MI24 values were normalized to those of untreated flies. Error bars indicate the SD for at least three independent trials of 60 flies each.

Primary Injuries Activate the Innate Immune Response. One impor- at least somewhat specific because the expression of other genes tant cause of secondary injuries in TBI is activation of the innate such as TAF1 was not similarly increased. Thus, TBI in flies, as immune response (19). The innate immune response is triggered in mammals, elicits activation of an innate immune response by pathogen-derived molecules and by endogenous molecules pathway. generated by stressed and injured cells (20). A component of this response is the production of proinflammatory cytokines, such as Genetic Background Affects the Primary Injury Threshold. An in- NEUROSCIENCE tumor necrosis factor (TNF), by microglial cells and astrocytes. creasing body of evidence implicates genetic factors in the vari- In some studies, elevated levels of TNF in the cerebrospinal fluid able clinical outcomes of TBI in humans (26). To investigate the of TBI patients are correlated with unfavorable clinical out- effect of genetic background on the primary injury threshold in fl fl – comes, providing evidence of the injurious role of cytokines (21). ies, we determined the MI24 for 42 y lines (aged 0 7 d) that Likewise, inhibition of TNF shortly after primary injuries in were subjected to the standard TBI protocol. Five of these lines rodents reduces the severity of some deleterious outcomes, such are commonly used as wild-type controls in various Drosophila as tissue loss (22). On the other hand, long-term recovery of experiments; the remaining 37 lines contain mutations in genes neurological damage in TBI is impaired in TNF-deficient mice, that encode components of the Imd or Toll pathways (24). fi The MI24 showed wide variation among the lines, ranging providing evidence of a bene cial role of cytokines (23). Therefore, ± ± the innate immune response plays a critical but not yet fully un- from 14.9 1.2 to 82.5 5.0 (Fig. 6A). Variability was even derstood role in TBI. Flies provide an opportunity to advance our observed among wild-type controls, for which the MI24 ranged from 20.3 ± 1.4 to 39.9 ± 3.8. Among the mutant lines there were understanding of this role because innate immune response striking differences for different alleles of the same gene. For pathways are highly conserved between flies and humans (24). example, the MI for ImdSDK and Imd10191 was 21.0 ± 3.3 and The Toll pathway in flies is analogous with mammalian Toll-like 24 40.9 ± 4.8, respectively. Additionally, the MI did not correlate receptor (TLR) pathways, and the Immune deficiency (Imd) 24 with either the Imd or the Toll pathway. For example, mutations pathway is analogous with the mammalian TNF pathway. Among in Relish (Rel), which encodes the NF-κB transcription factor in the key functions of both the Toll and Imd pathways is the tran- the Imd pathway, had a significantly different effect on the MI scriptional activation of antimicrobial peptide (AMP) genes (24). 24 fl than mutations in kenny (key), which encodes an activator of Rel To assess activation of the innate immune response in ies (RelE20, 47.2 ± 2.3; RelE38, 41.7 ± 3.3; keyf05097, 8.5 ± 1.2; and following primary injuries, we used quantitative real-time reverse c02831 ± – key , 7.9 1.9) (24). The wide range of effects for wild-type transcription PCR (qRT-PCR) to determine AMP gene mRNA lines and the lack of coherent effects for Imd and Toll pathway 1118 fl levels in heads of w ies subjected to the standard TBI lines suggests that in young flies many genes in the genetic protocol. To control for the usual increase in expression of innate background affect the primary injury threshold for death within fl immunity genes that occurs as a function of age, treated ies 24 h. We suspect that, in some cases, the genetic background fl were normalized to age-matched untreated ies (25). Flies that masks the effect of mutations in Imd or Toll pathway genes survived the standard TBI protocol exhibited an increase in making it impossible to determine from these data whether AMP gene expression within 24 h after the primary injuries (Fig. the innate immune response specifically influences the MI24. 5). Similar results were observed in 0- to 4-d-old and 20- to 21-d- old flies. Some AMP genes were activated within 1 h after the Genetic Background Affects the Age-Dependent Reduction of the primary injury and all AMP genes were activated within 24 h. Primary Injury Threshold. To investigate the effect of genetic Activation of innate immunity genes following primary injuries is background on the primary injury threshold in older flies, we

Katzenberger et al. PNAS | Published online October 14, 2013 | E4155 Downloaded by guest on September 27, 2021 Fig. 4. Treatment of w1118 flies with the standard TBI protocol causes neurodegeneration. Images of sections of fly brains from 14- to 18-d-old (A, Upper and Lower) untreated and (B, Upper and Lower) treated flies are shown. The treated fly received the standard TBI protocol at 0–4 d old. The large vacuole indicated by the arrow in the magnified image (B, Lower) is 7.75 μm in diameter. (Scale bars in A, Upper and B, Upper: 100 μm. Images in Lower panels are magnified an additional 5×.) (C) The number of large, i.e., 5.0- to 10.0-μm-diameter vacuoles in the central region of the brain is graphed vs. the number of times flies were subjected to the standard TBI protocol. Brain sections chosen for analysis were at equivalent depths in the brain, as exemplified by A and B. Multiple treatments with the standard TBI protocol occurred over successive days. All flies were analyzed 14 d after the time of the first treatment, i.e., flies labeled 14–18 d were treated beginning at 0–4 d old, and flies labeled 34–35 d were treated beginning at 20–21 d old. Error bars indicate the SD for at least three heads. When treated with one standard TBI protocol, young flies (14–18 d old) developed significantly fewer large vacuoles that older flies (34–35 d old) (P = 0.003, one-tailed t test), but the difference between young and older flies was not significant for treatment with two or three standard TBI protocols (P = 0.07 and P = 0.07, one-tailed t test, respectively). Both young and older flies developed significantly more large vacuoles when treated with three vs. one standard TBI protocol (P = 0.029 for young flies and P = 0.03 for older flies, one-tailed t test).

3 reexamined the 42 fly lines at 20–27 d old. The results largely spz flies had substantially different MIs24 (22.5 ± 4.2 and 55.1 ± paralleled those observed in young flies in that wide variability 9.4, respectively) (Fig. S4). Conversely, 0- to 7-d-old necrotic (nec2) wasobservedintheMI24, and the variability did not correlate and TAK1-associated binding protein 2 (Tab2EY00380) flies had with the Imd or the Toll pathway (Fig. 6B). In addition, for every substantially different MIs24 (17.6 ± 4.1 and 44.2 ± 4.6, re- 2 EY00380 line, the MI24 for 20- to 27-d-old flies was higher than the MI24 spectively) but 20- to 27-d-old nec and Tab2 flies had fl fl for 0- to 7-d-old ies (Fig. S4). MIs24 of younger and older ies similar MIs24 (62.4 ± 5.9 and 65.1 ± 6.3, respectively). These data had a correlation coefficient (r) of 0.77. Thus, in all genetic indicate that the genes that affect the primary injury threshold for backgrounds, age appears to be a critical determinant of the death within 24 h in young flies are different from those that affect primary injury threshold for death within 24 h. However, the the age-dependent reduction in the primary injury threshold. MI24 in younger flies was not entirely predictive of the extent of increase in the MI24 in older flies. For example, 0- to 7-d-old Variation in Primary Injury Threshold Correlates with Variation in f05097 3 key and spatzle (spz ) flies had similar MIs24 (14.9 ± 1.2 Longevity. Because the primary injury threshold varies with f05097 and 15.9 ± 3.2, respectively) but 20- to 27-d-old key and age, we examined the correlation between MI24 and longevity

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Fig. 5. Treatment of w1118 flies with the standard TBI protocol activates the innate immune response. The histogram shows the fold-increase in mRNA levels in treated vs. untreated flies at the indicated timepoints after treatment of 0- to 4- or 20- to 21-d- old flies. The AMP genes examined were Attacin-C (AttC), Diptericin B (DiptB), and Metchnikowin (Mtk), and the control gene examined was TBP-associated factor 1 (TAF1). Error bars indicate the SEM for at least three independent trials.

in the absence of TBI. We analyzed 14 fly lines, including lines between the MI24 and the median lifespan was −0.67 for 0- with low, average, or high MI24 after treatment with the stan- to 7d-old flies and −0.84 for 20- to 27-d-old flies. Thus, the dard TBI protocol. We found a negative linear relationship longevity of a particular fly line and its primary injury thresh- between the MI24 and the median lifespan for both 0- to 7-d-old old for death within 24 h are largely determined by the same and 20- to 27-d-old flies (Fig. 7A). The correlation coefficient r genetic factors. NEUROSCIENCE

Fig. 6. The MI24 is strongly affected by genetic background. Histograms show the MI24 for 42 different ﬂy lines treated with the standard TBI protocol and tested at (A)0–7 d old and (B)20–27 d old.

The MI24 vs. fly genotype is graphed. MI24 values were normalized to those of untreated flies. Geno- types are listed in Table S3. White bars indicate fly lines containing mutations in genes implicated in the Imd pathway. Gray bars indicate fly lines containing mutations in genes implicated in the Toll pathway. Black bars indicate fly lines commonly used as wild- type controls in Drosophila experiments. For a refer- ence, fly line number 7 is w1118. Error bars indicate the SD for at least three independent trials of 60 flies each.

Katzenberger et al. PNAS | Published online October 14, 2013 | E4157 Downloaded by guest on September 27, 2021 significantly associated with increasing age, 15% at <18 y, 44% at 18–59 y, and 52% at >59 y (29). These similarities between flies in our TBI model and human TBI patients strongly indicate that flies and humans incur TBI through similar cellular and molecular mechanisms.

The Fly Model of TBI Can Provide Unique Insights into Human TBI. Because the fly TBI model enables us to analyze many animals throughout their lifespan, we have found several properties of TBI that may be of clinical relevance: age at the time of primary injury as well as the number of primary TBI incidents are risk factors for neurodegeneration (Fig. 4C); the number of primary TBI incidents affects longevity (Fig. 2C and Table S2); the MI24 following TBI varies with genetic background (Fig. 6); and finally, the genes that affect the susceptibility to mortality are different at different ages (Fig. 6 and Fig. S4). Although it is difficult to obtain exactly comparable information from human TBI patients, available data are consistent with our conclusions. fl Fig. 7. The susceptibility of different fly lines to TBI-induced mortality is For example, as in ies, human studies suggest that age and the

inversely correlated with their respective longevity. (A) The MI24 of 20- to 27- severity of primary injury are important factors in CTE de- d-old flies is graphed vs. the median lifespan for 14 of the fly lines that were velopment (30, 31). In addition, studies in different rodent analyzed in Fig. 6. The lines that were analyzed are listed in Table S3. RelE20 strainsaswellasgeneticassociationstudiesinTBIpatients E38 fl and Rel ies had the same MI24 and median lifespan, so they appear as indicate that, as in flies, TBI outcomes depend on genetic back- 1118 fl a single open box on the graph. (B) The MI24 is graphed for w ies of the ground (26, 32). indicated age that were raised at either 18 °C or 25 °C. MI24 values were normalized to those of untreated flies. Temperature had a significant effect Finally, we found that the genetic and environmental factors that affect longevity in the absence of injury also affect the sus- on the MI24 for both 0- to 4-d-old flies (P = 0.012, one-tailed t test) and 21- to 22-d-old flies (P = 0.001, one-tailed t test). Error bars indicate the SD for at ceptibility to mortality following TBI (Fig. 7). Two lines of evi- least three independent trials of 60 flies each. dence suggest that fractional age rather than chronological age per se is a risk factor for TBI-induced mortality in humans as it is in flies, where fractional age is defined as chronological age fl To test this proposal, we extended the lifespan of ies and relative to the maximum longevity of flies of that genotype. First, examined the effect on the MI24. To extend the lifespan, we fl drugs such as rapamycin and resveratrol that alleviate TBI out- raised ies at 18 °C rather than at 25 °C, the temperature at comes in mammals also extend normal longevity (33–36). Sec- which flies were raised for all of the prior experiments. Un- ond, the gene apolipoprotein E (apoE), implicated in controlling treated w1118 flies raised at 18 °C had a significantly longer median longevity, is also found to be a risk factor for TBI outcomes in lifespan relative to flies raised at 25 °C (68.5 ± 0.8 d vs. 48.3 ± 1.2 d, P < 0.0001, one-tailed t test). After treatment with the standard mammals (37, 38). Interestingly, rapamycin, resveratrol, and 1118 fl fi apoE also appear to affect the severity of neurodegeneration in TBI protocol, w ies raised at 18 °C had a signi cantly lower ’ MI than equivalent-age flies raised at 25 °C (Fig. 7B). The neg- Alzheimer s disease, which is phenotypically similar to CTE (18, 24 39). Collectively, these findings suggest that secondary injuries in ative correlation between natural longevity and MI24 was observed for both 0- to 3-d-old and 21- to 22-d-old flies. Thus, flies of the TBI accelerate some normal aging processes in the brain. This same genotype and chronological age but different median lifespan idea may explain why the average age of onset (AAO) for CTE is differed in their primary injury threshold for death within 24 h. This substantially earlier than for Alzheimer’s disease (CTE: AAO = result indicates that environmental factors such as temperature 42.8 ± 12.7 y, range = 25–76 y; Alzheimer’s disease: AAO = 72.8 ± determine both the longevity of a particular line in the absence of 6.8 y, range = 49–97 y) (40, 41). injury and its primary injury threshold for death within 24 h. In summary, our studies demonstrate that a fly TBI model offers considerable potential for understanding the cellular Discussion and molecular mechanisms that underlie the pathological con- Flies Can Model Human TBI. Here, we have described the de- sequences of human TBI and may ultimately facilitate development velopment and initial characterization of a fly model of TBI. We of unique therapeutic strategies. found that inflicting mechanical injury on flies by rapid acceleration and deceleration produces outcomes that are similar to Materials and Methods outcomes characteristic of closed head TBI in humans (1, 2). Fly Lines and Culturing. Flies were maintained on standard molasses medium These outcomes include temporary incapacitation (Fig. S2A), at 25 °C unless otherwise stated. All fly lines were obtained from the Bloo- ataxia (Fig. S2B and Table S1), activation of the innate immune mington Stock Center except for ImdSDK and Imd10191, which were obtained response (Fig. 5), neurodegeneration (Fig. 4), and death (Fig. 2). from Mimi Shirasu-Hiza (Columbia University, New York). Genotypes of flies In addition, we found that risk factors for mortality in flies are used in Figs. 6 and 7A and Fig. S4 are described in Table S3. shared with humans. Our data indicate that the MI24 following injury does not vary with sex (Fig. 3A). Similarly, a study of 914 Assays for TBI Outcomes. Construction and use of the HIT device is described in Fig. S1. The impact velocity of the HIT device was determined by analyzing women and 916 men by Coimbra et al. concluded that sex is not images from a high-speed camera. MIs24 were determined by analyzing at a risk factor for mortality in TBI patients (27). The MI24, how- fl fl fl fl least 180 ies (three experiments of 60 ies). The percentage of ies that ever, is dependent on the age of ies at the time of injury (Figs. were incapacitated was determined by analyzing movie recordings of flies 3B and 6). Likewise, in a study of 5,612 TBI patients, Hukkelhoven after a single strike. Flies that did not show obvious locomotor activity were et al. found that the percent mortality within 6 mo of primary considered incapacitated. Before each timepoint, hitting the benchtop upon injuries increased with age, 21% at <35 y and 52% at >55 y (28). which the fly vial sat was used to stimulate locomotion. Climbing, lifespan, Also, in a study of 244 TBI patients, Dhandapani et al. found histology, and qRT-PCR assays were performed as described in Petersen et al. that the percent mortality within 1 mo of primary injuries was (14, 42).

E4158 | www.pnas.org/cgi/doi/10.1073/pnas.1316895110 Katzenberger et al. Downloaded by guest on September 27, 2021 ACKNOWLEDGMENTS. We thank Nicole Bertram, Grace Boekhoff-Falk, mem- velocity; and Satoshi Kinoshita for performing the histology. This work was PNAS PLUS bers of the D.A.W. and B.G. laboratories, and Ron Kalil for their insights that supported by National Institutes of Health Grants R01 NS059001 (to D.A.W.) greatly improved this research; Russell Little for help in determining the impact and R01 AG033620 (to B.G.).

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