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P7C3 Neuroprotective Chemicals Block Axonal Degeneration and Preserve Function after Traumatic Brain Injury

Graphical Abstract

Highlights Treatment with (!)-P7C3-S243 after blast injury blocks wide- spread axonal degeneration

Treatment with (!)-P7C3-S243 after blast injury preserves learning and memory

Treatment with (!)-P7C3-S243 after blast injury preserves motor coordination

Without treatment, neuropsychiatric deficits persist chronically

after blast injury

Authors Terry C. Yin, Jeremiah K. Britt, ...,

Joseph M. Ready, Andrew A. Pieper

Correspondence [email protected]

In Brief E-TOC: Blast exposure from explosive

devices elicits traumatic brain injury

associated with neurodegeneration,

cognitive and motor decline, and psychi-

atric symptoms. Yin et al. now show that

treatment with the neuroprotective agent

(!)-P7C3-S243 24 hr after blast injury blocks axonal degeneration in the brain

and preserves normal learning, memory,

and motor coordination. Neuropsychi-

atric symptoms persist chronically in un-

treated mice. Optimized P7C3 variants

offer hope for identifying neuroprotective

agents for conditions involving axonal

damage, neuronal cell death, or both.

Yin et al., 2014, Cell Reports 8, 1731–1740 September 25, 2014 ª2014 The Authors http://dx.doi.org/10.1016/j.celrep.2014.08.030

Cell Reports

Report

P7C3 Neuroprotective Chemicals Block Axonal Degeneration and Preserve Function after Traumatic Brain Injury Terry C. Yin,1,10 Jeremiah K. Britt,1,10 Héctor De Jesús-Cortés,1,9,10 Yuan Lu,1 Rachel M. Genova,1 Michael Z. Khan,1

Jaymie R. Voorhees,1,5,7 Jianqiang Shao,6 Aaron C. Katzman,1 Paula J. Huntington,8 Cassie Wassink,1 Latisha McDaniel,1

Elizabeth A. Newell,2 Laura M. Dutca,7 Jacinth Naidoo,8 Huxing Cui,1 Alexander G. Bassuk,2,3,4 Matthew M. Harper,7

Steven L. McKnight,8 Joseph M. Ready,8 and Andrew A. Pieper1,3,5,7,* 1Department of Psychiatry 2Department of Pediatrics 3Department of Neurology 4Department of Pediatric Neurology 5Interdisciplinary Graduate Program in Human Toxicology 6Central Microscopy Facility 7Department of Veterans Affairs Center for the Prevention and Treatment of Visual Loss, Department of Ophthalmology and Visual Sciences University of Iowa Carver College of Medicine, 200 Hawkins Drive, Iowa City, IA 52242, USA 8Department of Biochemistry 9Graduate Program of Neuroscience UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA 10Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2014.08.030 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

SUMMARY

The P7C3 class of neuroprotective aminopropyl car- bazoles has been shown to block neuronal cell death in models of neurodegeneration. We now show that P7C3 molecules additionally preserve axonal integrity after injury, before neuronal cell death oc- curs, in a rodent model of blast-mediated traumatic brain injury (TBI). This protective quality may be linked to the ability of P7C3 molecules to activate nicotinamide phosphoribosyltransferase, the rate- limiting enzyme in nicotinamide adenine dinucleotide salvage. Initiation of daily treatment with our recently reported lead agent, P7C3-S243, 1 day after blast- mediated TBI blocks axonal degeneration and pre- serves normal synaptic activity, learning and mem- ory, and motor coordination in mice. We additionally report persistent neurologic deficits and acquisition of an anxiety-like phenotype in untreated animals 8 months after blast exposure. Optimized variants of P7C3 thus offer hope for identifying neuroprotec- tive agents for conditions involving axonal damage, neuronal cell death, or both, such as occurs in TBI.

INTRODUCTION

Traumatic brain injury (TBI) has emerged as the signature injury of military conflict, estimated to affect 20% of the 2.3 million servicemen and women deployed since 2001. Blast exposure

from explosive devices affects soldiers and civilians around the globe, placing them at increased risk for TBI associated with long-term neurologic complications, including cognitive and motor decline, acquisition of psychiatric symptoms, and neuropathological features similar to Alzheimer’s disease (Hoge et al., 2008; Wolf et al., 2009; Shively et al., 2012; Gold- stein et al., 2012). Whereas the mechanisms of injury from exposure to the blast-generated shockwave are incompletely understood, the associated sheer forces are known to lead to widespread, diffuse, and progressive axonal injury (Nakagawa et al., 2011; Magnuson et al., 2012). Unfortunately, just as there are currently no pharmacologic agents that arrest neuron cell death in any of the wide spectrum of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis, there are also currently no treatment options for patients with TBI beyond supportive and rehabilitative care. The lack of pharmacologic strategies to block neuron cell

death relates to the failure of target-directed drug discovery programs to develop neuroprotective therapeutics. Phenotypic screening, by contrast, offers the opportunity for discovery of new compounds with a desired biologic effect without bias con- cerning mechanism (Pieper et al., 2014). With this in mind, we previously implemented an in vivo phenotypic screen in living mice to identify small drug-like molecules that safely increased the net magnitude of postnatal hippocampal neurogenesis. We designed the screen to capture agents that would either enhance proliferation or block death of newborn hippocampal neural pre- cursor cells (Pieper et al., 2010) and identified an aminopropyl carbazole, named ‘‘P7C3,’’ that was fortuitously endowed with favorable pharmacokinetic properties (Pieper et al., 2010). It

Cell Reports 8, 1731–1740, September 25, 2014 ª2014 The Authors 1731

was subsequently demonstrated that P7C3 achieved its pro- neurogenic effect by virtue of blocking death of neural precursor cells. We later demonstrated additional protective benefit of P7C3 molecules, specifically the highly active analog P7C3- A20, in blocking neuronal cell death and improving neurologic outcomes in a variety of rodent models of neurodegenerative disease and injury, including those involving death of mature neurons in other regions of the CNS (MacMillan et al., 2011; Nai- doo et al., 2013; Walker et al., 2014; De Jesús-Cortés et al., 2012; Tesla et al., 2012; Blaya et al., 2014).

Recently, we reported the synthesis and characterization of an optimized member of the P7C3 series, (!)-P7C3-S243, which exhibits improved polarity and lacks the aniline moiety present in other members of the P7C3 series. This analog is readily available as a single enantiomer with selective neuro- protective activity (Naidoo et al., 2014). Because blast expo- sure in rodents results in a phenotype that recapitulates the consequences of blast-mediated TBI experienced by humans (Goldstein et al., 2012; Mohan et al., 2013), this animal model is suitable for evaluating the efficacy of neuroprotective agents. Here, we describe protective efficacy of P7C3-S243 in the mouse model of blast-induced TBI, which has also led to the discovery of a protective quality of the P7C3-class— the ability to block axonal degeneration preceding neuronal cell death.

RESULTS

To test the neuroprotective efficacy of P7C3-A20 and P7C3- S243, we employed a model of blast-induced TBI in which the blast wave is propagated following rupture of a mylar membrane (Mohan et al., 2013). Briefly, anesthetized mice are placed in an enclosed blast chamber constructed from an air tank partitioned into two sides. A sealed mylar membrane covers a small port be- tween the two halves of the tank. When the pressure is increased in the side without the mouse, the membrane ruptures at z22 kilopascal (kPa), generating a blast wave that travels through the mouse’s head. The head of the mouse is untethered and located in a padded holder, whereas the body is shielded from the blast by a metal tube. As previously reported (Mohan et al., 2013), the intensity of the blast wave in our system is 149.8 ± 2.09 kPa, and the duration of the total pressure (blast wave + wind gust) is z10–15 ms.

We first evaluated hippocampal-dependent memory in the Barnes maze task after blast injury, with immediate administra- tion of test compounds. The Barnes maze consists of a circular table with holes equally spaced around the perimeter. One of these holes contains an escape cup, such that the mouse can enter the hole and hide in the cup to avoid exposure on the table. Testing in the Barnes maze was initiated 7 days after blast injury. To begin, mice underwent 4 days of training, during which time they were allowed to find and enter the escape hole and rest in the protective cup. By the 4th day of training, mice typically learn how to rapidly locate the escape hole, based on visual cues permanently positioned around the table throughout training. Af- ter 4 days of training, the cup is removed and the ability of the mouse to remember where the cup was previously located is as- sessed in the ‘‘probe test’’. Memory is reflected by measuring

the amount of time the mouse spends in the area around where the cup was previously located. Sham-injured animals that were intraperitoneally administered daily vehicle, P7C3-A20, or P7C3- S243 spent 55%–60% of their time in the quadrant that con- tained the escape hole, known as the ‘‘escape quadrant,’’ during the probe test (Figure S1A), indicating that normal memory is not affected by P7C3 compounds in this task. By contrast, blast- injured animals administered vehicle were notably impaired, spending only z25% of their time in the escape quadrant, which would be expected by chance alone (Figure S1A). Daily treat- ment with P7C3-A20 or P7C3-S243 immediately after injury, however, restored the time spent in the escape quadrant to normal levels seen in uninjured mice (Figure S1A).

Delayed Initiation of Treatment with P7C3-S243 Preserves Learning and Memory after Blast-Mediated TBI On the basis of the promising results seen with administration of P7C3 molecules immediately after injury, we sought to deter- mine whether P7C3-S243 could offer protective efficacy when treatment was initiated later. Because TBI can disrupt the blood-brain barrier (BBB), we first examined BBB integrity over time by means of the Evans blue assay. Evans blue is an azo dye with tight affinity for albumin, such that in serum virtu- ally all Evans blue is albumin bound. Normally, serum albumin cannot cross the BBB. However, when the BBB is compro- mised, albumin and its bound Evans blue can enter the CNS. The magnitude of Evans blue accumulation in the brain can then be spectrophotometrically determined in order to monitor disruption of the BBB (Uyama et al., 1988; Hawkins and Egle- ton, 2006). With this technique, we observed significant disrup- tion of the BBB 6 hr after blast injury, with integrity of the BBB recovering to normal 18 hr later (Figure S1B). BBB integrity re- mained intact 102 hr after injury, and 3 days of daily treatment with P7C3-S243 did not compromise BBB integrity in sham- injured mice. We next tested whether P7C3-S243 could preserve function

when treatment was initiated 24 hr after blast injury, when the BBB was intact. A schematic of this experimental design is illus- trated in Figure S1C, and we have previously reported that P7C3-S243 readily enters the brains of mice with intact BBBs (Naidoo et al., 2014). We reasoned that this time point for starting treatment represented an important milestone, as most patients would be expected to access treatment within 24 hr of their injury. Every group consisted of 25 male C57/Bl6 wild-type mice, aged 12–14 weeks, and data were collected and analyzed in an automated manner blind to treatment group. The most stringent measure of performance in the Barnes maze probe test is percent time in the escape area, defined as a 5 cm radius surrounding the escape hole. Sham-injured animals treated with vehicle or 10 mg/kg/day P7C3-S243 spent z39%–43% of their time in the escape area, as opposed to only z15% for injured animals treated with vehicle (Figure 1A). Daily treatment with P7C3-S243 initiated 24 hr after injury dose-dependently pre- served performance in this measure, with 3, 10, and 30 mg/kg/ day doses showing complete protection and 1 mg/kg/day par- tial, though not statistically significant, protection (Figure 1A). Administration of the active enantiomer (!)-P7C3-S243 at

1732 Cell Reports 8, 1731–1740, September 25, 2014 ª2014 The Authors

3 mg/kg/day showed complete protection, whereas the same dose of the less-active enantiomer, (+)-P7C3-S243, showed no efficacy (Figure 1A). Next, we sought to define the time window of treatment effi-

cacy by initiating daily intraperitoneal (i.p.) administration of P7C3-S243 at later points after injury. Whereas both 3 and 30 mg/kg/day doses preserved normal function when treatment was initiated 24 hr after injury, only the 30 mg/kg/day dose was partially efficacious when initiated 36 hr after injury (Figure 1B). When initiated 48 hr after injury, no protective efficacy was

noted at any dose (Figure 1B). Thus, acute memory deficits after blast-mediated TBI can be effectively mitigated when treatment with P7C3-S243 is initiated within 36 hr. Finally, we tested whether oral administration of the highly active (!)-P7C3- S243 enantiomer could achieve protective effect. Indeed, initia- tion of daily oral administration of (!)-P7C3-S243 at 24 hr after injury preserved memory at 3, 10, and 30 mg/kg/day doses (Figure 1C). In addition to measuring time spent in the 5 cm radius around

the escape hole in the probe test, we also tested other com- monly used measures of memory. Specifically, we determined the percent of nose pokes into the correct hole, defined as ‘‘target entry,’’ and the percent of time spent in the quadrant of the maze containing the escape hole, defined as ‘‘percent quadrant time’’. Monitoring of target entry in the treatment groups showed the same protective effects as with the escape area measure (Figures S2A–S2C). Similar findings were also obtained with the metric of percent quadrant time, with the exception that 3 mg/kg/day of the less active (+)-P7C3-S243 enantiomer achieved some measure of protective efficacy at the margin of statistical significance (Figures S2D–S2F). This modest effect indicates that low-level activity may reside in (+)-P7C3-S243. As controls in the Barnes maze, we examined the ability of

animals to physically participate in the task, as well as to learn the task over the 4-day training period. Physical participation was defined as speed and distance traveled, and none of the groups differed significantly in these measures (Figures S2G– S2L). Learning was assessed as the percent latency to escape, defined as the percentage of time the mouse took to enter the escape hole on day 4 out of the time originally required on day 1. All animals were able to learn, though mice subjected to blast-mediated TBI and then treated with vehicle, 0.3 mg/kg/ day P7C3-S243, or 3 mg/kg/day of the less-active enantiomer (+)-P7C3-S243 learned significantly worse than sham-injury mice (Figure S2M). When treatment with 1, 3, 10, or 30 mg/kg/ day P7C3-S243 or 3 mg/kg/day (!)-P7C3-S243 was initiated 24 hr after injury, however, mice learned the task as well as sham-injury mice (Figure S2M). Notably, the 1 mg/kg/day dose failed to improve memory in the probe test (Figure 1A) yet still facilitated learning during the training period. Initiation of administration of 3 and 30 mg/kg/day doses of

P7C3-S243 at 36 hr after injury improved learning (Figure S2N), even though the 3 mg/kg/day dose did not improve memory in the probe test (Figure 1B). Similarly, 30 mg/kg/day P7C3-S243 restored normal learning when treatment was initiated 48 hr after injury (Figure S7C), despite having no effect on memory in the probe test (Figure 1C). Finally, oral administration of 1 mg/kg/ day (!)-P7C3-S243, which did not preserve memory in the probe test (Figure 1C), also restored normal learning (Figure S2O). Taken together, it can be concluded that treatment with P7C3- S243 mitigates acute neurocognitive deficits after blast-medi- ated TBI and improves learning during the training period of the Barnes maze at doses lower than those required for the more-challenging task of preserving memory. To ensure that the improvement observed in mice treated with

P7C3-S243 in this assay of learning and memory did not simply reflect an acceleration of what would otherwise be a normal

Figure 1. P7C3-S243 Preserves Memory after Blast-Mediated TBI (A) Daily i.p. administration of P7C3-S243 for 11 days in divided daily doses for

the total amount indicated dose-dependently preserved memory in the Barnes

maze probe test in blast-injured mice, as measured by the most stringent

measure of percent time in escape area (5 cm radius around the escape hole).

Treatment with an intermediate dose (3 mg/kg/day) of the active (!)-P7C3- S243 enantiomer preserved normal performance to the level displayed by

sham-injured mice. By contrast, mice treated with the same dose of the less-

active (+)-P7C3-S243 enantiomer showed the same deficit as injured mice that

were treated with vehicle.

(B) Daily administration of P7C3-S243 was initiated at later time periods after

injury to define the window of therapeutic efficacy. Whereas both 3 and 30 mg/

kg/day doses preserved normal function when treatment was initiated 24 hr

after injury, only the 30 mg/kg/day dose was efficacious when treatment was

initiated at 36 hr. When treatment was initiated 48 hr after injury, no protective

efficacy was noted at any dose.

(C) Oral administration of the highly active (!)-P7C3-S243 enantiomer pre- served normal hippocampal-dependent memory at 3, 10, and 30 mg/kg/day

doses. Every group shown consisted of 25 male C57/Bl6 mice, aged 12–

14 weeks, and data were collected and scored in an automated manner blind

to treatment group. Data are represented as mean ± SEM. Significance was

determined by two-way ANOVA with Bonferroni post hoc analysis. *p < 0.05,

**p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to blast-injured animals

treated with vehicle.

See also Figures S5 and S6.

Cell Reports 8, 1731–1740, September 25, 2014 ª2014 The Authors 1737

regions, axonal degeneration was blocked in the molecular layer by treatment with 3 and 30 mg/kg/day P7C3-S243, as well as 3 mg/kg/day of the highly active enantiomer (!)-P7C3-S243, but not by treatment with the same dose of the less-active enantiomer (+)-P7C3-S243 (Figures 5A and 5B). Because the cerebellum controls coordination, we next assayed balance and coordination, using standard procedures (Luong et al., 2011). Mice were trained to cross an 80 cm long beam over 2 days and then sham or blast injured on day 3. Daily oral admin- istration of (!)-P7C3-S243 was initiated 24 hr later, and animals were tested 7 and 28 days after injury. Mice were videotaped during the test and then analyzed for the number of foot slips by observers blind to treatment group. Seven days after blast injury, mice showed a trend toward increased number of foot slips, which did not reach statistical significance. By 28 days after injury, however, blast-injured mice displayed a 2-fold in- crease in number of foot slips. This behavioral deficit was normalized by daily oral treatment with (!)-P7C3-S243, initiated 24 hr after injury. Thus, protective efficacy for cerebellar axonal degeneration after injury by (!)-P7C3-S243 correlated with pres- ervation of motor coordination.

As with cognitive measures, we verified that the protective effect of treatment with P7C3-S243 did not simply reflect an ac- celeration of what might otherwise be a normal recovery process after injury. As shown in Figure S5, 8 months after a single blast injury, untreated mice continue to show a statistically significant deficit in ability to perform this task of motor coordination.

Acquisition of Anxiety-like Phenotypes Chronically after Blast-Mediated TBI We additionally analyzed mice at acute and chronic time points in the Laboras system, which allows 24 hr automated collection of normal home cage behavior using a vibration-sensitive plate to monitor motor activity (Xu et al., 2013). Here, we noted acqui- sition of anxiety-related phenotypes at the chronic time point that were not observed acutely after injury. Specifically, we observed increased grooming (Figure S6A) and increased loco- motion and rearing (Figure S6B) in untreated mice 8 months after a single blast injury. This phenotype correlates with increased susceptibility of humans to psychiatric symptoms after TBI. Future work will focus on expanding this finding, including deter- mination of whether acute or chronic treatment with P7C3 mole- cules offer protection from this aspect of TBI as well.

DISCUSSION

Here, we show that the P7C3 class of neuroprotective chemicals blocks axonal degeneration prior to neuron cell death. Briefly, administration of P7C3-S243, initiated 24 hr after blast-mediated TBI, potently preserves axonal integrity throughout the brain. This axonal rescue is associated with preservation of related measures of synaptic transmission, hippocampal-dependent learning and memory, and motor coordination. Examination of injured mice at chronic time points shows that these behavioral deficits do not resolve on their own in mice that received only vehicle and also that injured animals additionally attain behav- ioral phenotypes related to anxiety at this chronic time point after injury. We propose that P7C3-S243 serves as a chemical scaf-

fold upon which new drugs can be designed to treat patients with conditions of axonal degradation, such as occurs in TBI or other neurodegenerative diseases. Such an agent would have broad applicability, as axon degeneration proceeds through unique mechanisms distinct from cell death (Yan et al., 2010), and most forms of neurodegenerative disease involve degrada- tion of synapses and axons preceding loss of neuronal cell bodies (Li et al., 2001; Raff et al., 2002; Coleman and Yao, 2003; Fischer et al., 2004; Gunawardena and Goldstein, 2005; Luo and O’Leary, 2005). How might P7C3-S243 act to protect axons? In the accompa-

nying manuscript (Wang et al., 2014), we show that active P7C3 variants bind and enhance activity of the enzyme nicotinamide phosphoribosyltransferase (NAMPT). NAMPT synthesizes nico- tinamide mononucleotide (NMN) from nicotinamide, the rate- limiting step in nicotinamide adenine dinucleotide (NAD) salvage (Preiss and Handler, 1958), and NAD is known to play a vital role in axon degeneration. For example, the Wallerian degeneration slow strain of mice is resistant to axonal degeneration after injury (Lunn et al., 1989) by virtue of a triplicated fusion gene, resulting in overexpression of nicotinamide mononucleotide adenylyl- transferase 1 (Araki et al., 2004), the enzyme that converts NMN into NAD. Furthermore, it has also been shown that treat- ment with NAD and NAD precursors, including nicotinamide, nicotinic acid mononucleotide, and NMN, or overexpression of NAMPT achieves axonal protection in vitro (Araki et al., 2004; Sa- saki et al., 2006; Wang et al., 2005). Thus, active variants of P7C3 may protect from axonal degeneration by enhancing intracellular production of NAD through enhancing NAMPT activity. In conclusion, it is our hope that the P7C3 family of neuroprotective chemicals will form the basis for a new class of therapeutics applicable to a variety of conditions of nerve cell dysfunction currently lacking treatment options.

EXPERIMENTAL PROCEDURES

Animals Approval for animal experiments was obtained from the University of Iowa

Institutional Animal Care and Use Committee. Mice were singly housed in

the University of Iowa vivarium in a temperature-controlled environment with

lights on 0600–1800. Mice had ad libitum access to water and standard

chow. Eight-week-old C57BL6 wild-type mice were obtained from Jackson

Laboratories.

Blast-Mediated Traumatic Brain Injury Mice were anesthetized with ketamine/xylazine (1 mg/kg and 0.1 mg/kg,

respectively) and placed in an enclosed blast chamber (50 cm long and

33 cm wide) constructed from an air tank partitioned into two sides. One

side was pressurized with a 13 cm opening between the partitions and covered

with a mylar membrane. The unpressurized partition contained a restraint

10 cm from the mylar membrane, into which the mouse was placed. The

head was freely moving whereas a metal tube shielded the body. Compressed

air was forced into the pressurized partition until the mylar membrane burst at

22 kPa. The blast wave impacted the test animal inside a foam-lined restraint

to reduce blunt impact trauma of the head against the metal tube. The left side

of the head was closest the origin of the blast wave. Sham-injured animals

were anesthetized in the same way and not subjected to the blast.

P7C3 Series of Compounds Formulation Compound formulation was conducted using previously described methods

(Pieper et al., 2010, Naidoo et al., 2014).

1738 Cell Reports 8, 1731–1740, September 25, 2014 ª2014 The Authors

Statistics GraphPad Prism 6 software was used to perform all statistical analyses.

For detailed methodology of blood-brain barrier studies, Barnes maze,

immunohistochemistry, electron microscopy, and electrophysiology studies,

IL-1 pathway analysis, foot slip assay, and LABORAS assay, see Supple-

mental Experimental Procedures.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

six figures, and two tables and can be found with this article online at http://

dx.doi.org/10.1016/j.celrep.2014.08.030.

AUTHOR CONTRIBUTIONS

T.C.Y., J.K.B., and H.D.J.-C. were equally instrumental in design of the study,

conducted experiments and data analysis, and contributed to writing the

manuscript and preparation of figures. Specifically, T.C.Y. and J.K.B. led

behavioral studies of learning and memory, coordination, and anxiety.

H.D.J.-C. and T.C.Y. led ultrastructural electron microscopy hippocampal

studies. All three investigators conducted key aspects of histochemical

analysis.

ACKNOWLEDGMENTS

We thank Mike Welsh and Randy Kardon for advice and encouragement and

Noelle Williams for pharmacologic studies. Medical illustration was provided

by Teresa A. Ruggle (Department of Internal Medicine Design Center, Univer-

sity of Iowa Hospitals and Clinics) and Victor Powell (Medical Media, Iowa City

VA Health Care System). The work was funded by a grant awarded to A.A.P.

and S.L.M. from the NIMH (5-RO1-MH087986); grant awards from the Welch

Foundation (I-1612) and the Edward N. and Della C. Thome Memorial Founda-

tion to J.M.R.; unrestricted funds provided to S.L.M. by an anonymous donor;

NIH grants 1R21MH100086-01 and 5R01NS064159-05 to A.G.B.; the Depart-

ment of Veterans Affairs, Veterans Health Administration, Rehabilitation

Research and Development Service (Center for the Treatment and Prevention

of Visual Loss, RR&D Career Development Award [to M.M.H.] and Merit Award

RX000427); and a National Science Foundation Graduate Research Fellow-

ship to H.D.J.-C.

Received: May 17, 2014

Revised: July 7, 2014

Accepted: August 15, 2014

Published: September 11, 2014

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Cell Reports, Volume 8 Supplemental Information

P7C3 Neuroprotective Chemicals Block Axonal Degeneration and Preserve Function after Traumatic Brain Injury Terry C. Yin, Jeremiah K. Britt, Héctor De Jesús-Cortés, Yuan Lu, Rachel M. Genova, Michael Z. Khan, Jaymie R. Voorhees, Jianqiang Shao, Aaron C. Katzman, Paula J. Huntington, Cassie Wassink, Latisha McDaniel, Elizabeth A. Newell, Laura M. Dutca, Jacinth Naidoo, Huxing Cui, Alexander G. Bassuk, Matthew M. Harper, Steven L. McKnight, Joseph M. Ready, and Andrew A. Pieper

Supplemental Methods Blood brain barrier (BBB) permeability assay. To analyze permeability of the BBB, we used an Evan’s blue dye (EBD) method modified from Uyama et al. (1988). EBD, which binds albumin, is incapable of crossing an intact blood-brain barrier. One hour prior to sacrifice, freshly prepared 2% EBD (Sigma) in phosphate-buffered saline (PBS) was administered at a 4 ml/kg dose via the retro-orbital venous sinus. Animals were sacrificed and perfused 6 hours, 24 hours, and 102 hours following blast-injury with cold PBS. Brains were frozen at -20 °C until all time points were complete. To quantify the extravasation of albumin-bound EBD in each brain, tissue was weighed and incubated for 30 minutes at room temperature in 50% ��ȋ��Ȍ��͵ǣͳ�Ɋ�Ȁ��tio. Samples were sonicated at 40% amplitude and centrifuged for 20 minutes at 10,000 rpm. Supernatants were transferred to new tubes and 100% ethanol was added in a 1:3 ratio. Fluorescence was measured using a SpectraMax M2e (Molecular Devices) in 100 Ɋ�� plated on a 96-well plate at excitation wavelength of 620 nm and an emission wavelength of 680 nm. Barnes Maze. The Barnes maze test assesses spatial learning and memory, and was conducted on a gray circular surface 91 cm in diameter, raised to a height of 90 cm, with 20 holes 5 cm in diameter equally spaced around the perimeter (Stoelting Co.). The surface was brightly lit and open in order to motivate the test animal to learn the location of a dark escape chamber recessed under one of the 20 holes, which was designated randomly. The maze was surrounded by a black circular curtain upon which were hung four different and equally spaced visual cues (with different shapes and colors), for orientation to the designated location of the escape chamber. Each animal was subjected to four days of training comprised of four trials per day. An area extending 4 cm from the escape hole in all directions was used as the target area for measurements (percent time in escape area, percent latency to escape and nose pokes). A probe trial was conducted on the subsequent day, during which time the escape chamber was removed and measurements were made to confirm the animal’s memory based upon spatial cues. Measurements were acquired with Anymaze video tracking software (Stoelting Co.), and analysis was conducted blind to treatment group. Immunohistochemistry. Mice were killed by transcardial perfusion with 4% paraformaldehyde at pH 7.4, and dissected brains were immersed in 4% paraformaldehyde overnight at 4°C and then cryoprotected in sucrose for 72 hours. Brains were then rapidly frozen in isopentane pre-cooled to -70ºC with dry ice. All brains were stored in a freezer at -80°C before sectioning. Serial sections (40µm) were cut coronally through the cerebrum, approximately from bregma 3.20 mm to bregma -5.02 mm and the brainstem and cerebellum, approximately from bregma - 5.52 mm to bregma -6.96 mm (Mouse Brain in Stereotaxic Coordinates by Paxinos & Franklin, 1997). Every section in a series of 12 sections (interval: 480 µm) was collected separately. All sections were stored free-floating in FD sections storage solution (FD Neurotechnologies, Columbia, MD) at -20ºC before further processing.

For hematoxilin & eosin (H&E) staining, sections were mounted on 1”x3” Superfrost Plus microscope slides and stained with FD hematoxylin & eosin (FD Neurotechnologies). For silver staining, sections were collected in 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde and fixed for 5 days at 4°C. Sections were then processed for the detection of neurodegeneration with FD NeuroSilver Kit II (FD Neurotechnologies) according to the manufacturer’s instructions (manual of PK301, available at www.fdneurotech.com). Sections were subsequently mounted on slides, dehydrated in ethanol, cleared in xylene, and coverslipped with Permount (Fisher Scientific, Fair Lawn, NJ). For NeuN- and GFAP- immunoreactivity, endogenous peroxidase activity was inactivated with 0.6% hydrogen peroxidase, and free-floating sections were then incubated for 43 hours at 4 °C in 0.01 M phosphate-buffered saline (PBS, pH 7.4) containing 1% normal blocking serum, 0.3% Triton X-100 (Sigma, St. Louis, MO), and either biotin- conjugated monoclonal mouse anti-NeuN IgG (1:600; Millipore, Billerica, MA) or rat anti-GFAP IgG (1:10,000; Invitrogen, Carlsbad, CA). The immunoreaction product was visualized according to the avidin-biotin complex method with the Vectastin elite ABC kit (Vector Lab., Burlingame, CA), and 3’,3’-diaminobenzidine (Sigma) was used as chromagen. All images were taken with an Aperio ScanScope (Leica biosystems) Quantification of immunohistochemistry: Optical densitometry for quantification of immunohistochemical signal was modified from published methodology (Baldock and Poole et al. 1993). Images were captured with an upright microscope (Zeiss Axiolmager.M2) equipped with a monochromatic digital camera (Zeiss AxioCam MRm Rev.3), and processed with the Zen imaging software (Zeiss 2012, Blue edition). The microscope light intensity and camera exposure were held constant. The operator outlined areas of interest around specific brain regions and recorded the intensity of light passing through the slide. Degenerating axons allowed less light to pass through the section due to their uptake of silver stain, so lower light intensity correlated with increased degeneration. The operator performing quantification was blinded to condition and treatment. This technique was verified to correlate with traditional measure of optical density of scanned tissues using NIH Image J, as described in the text. Toluidine blue staining and transmission electron microscopy (TEM). Mice were transcardially perfused with Karnovsky’s fixative solution (2% formaldehyde, 2.5% glutaraldehyde, 0.2M sodium cacodylate buffer, 1mM CaCl2, 2mM MgCl2, and 42.8mM NaCl, pH 7.4) two weeks after either sham- or blast-injury and with or without specified compound treatment. Harvested brains were incubated in Karnovsky’s fixative solution overnight at 4 °C. Whole brains were cut ��ȋͳͲͲɊ�Ȍ��ȋ��ͳͷͲͲȌǤ�� contained the hippocampus were selected, washed with 0.1M sodium cacodylate buffer, and then post fixed with 1% osmium fixative for 1hr. Sections were then dehydrated in a series of ethanol (50%, 75%, 95% and 100% ethanol) followed by embedding in EPON resin overnight at 65 °C. For toluidine blue staining, semithin

��ȋͳɊ�Ȍ��ȋ��͸Ȍ�� toluidine blue. Pictures were taken using an upright microscope (Zeiss Axio Imager.M2) with a color camera (AxioCam ICc5). For TEM, ultrathin sections (60nm) adjacent to semithin sections were cut with an ultramicrotome, loaded onto a Formvar 200-mesh Ni grid, and counterstained with uranyl acetate and lead citrate. Specimens were examined using a JEOL JEM 1230 electron microscope with a Gatan UltraScan 1000 2k x 2k CCD camera. See also Figure S4. Electrophysiology. Single-housed, naïve 7- to 9-week-old male C57BL/6J mice received intraperitoneal injections of P7C3-S243 (0.3, 3, or 30 mg/kg/day) or vehicle 24h following a single sham- or blast-injury. On the tenth day after injury, ��ȋͶͲͲ�Ɋ�) were prepared, in accordance with University of Iowa Carver College of Medicine guidelines. Briefly, hippocampal slices were cut using a Vibratome 1000 Plus (Vibratome, St. Louis, MO) in ice-cold slicing buffer (in mM: 127 NaCl, 26 NaHCO3, 1.2 KH2PO4, 1.9 KCl, 1.1 CaCl2, 2 MgSO4, 10 D-Glucose) bubbled with 95% O2 and 5% CO2. Slices were then transferred to a holding chamber containing oxygenated artificial cerebrospinal fluid (aCSF; in mM: 127 NaCl, 26 NaHCO3, 1.2 KH2PO4, 1.9 KCl, 2.2 CaCl2, 1 MgSO4, 10 D-Glucose) for 30 min at 34 qC and then for another 30 min at 22 qC for recovery, and subsequently transferred to a submersion recording chamber continually perfused with 32 qC oxygenated aCSF (rate: 2 ml/min). Slices were equilibrated for at least 15 min before each recording. aCSF-filled glass electrodes (resistance <1 M:) were positioned in the stratum radiatum of area CA1 for extracellular recording. Synaptic responses were evoked by stimulating Schaffer collaterals with 0.2 ms pulses once every 15 s. The stimulation intensity was systematically increased to determine the maximal field excitatory post-synaptic potential (fEPSP) slope, and then adjusted to yield 40-60% of the maximal (fEPSP) slope. Experiments with maximal fEPSPs of less than 0.5 mV, with large fiber volleys, or with substantial changes in the fiber volley during recording were rejected. LTP was induced by 12TBS (12 bursts, each of 4 pulses at 100 Hz). Field EPSPs were recorded (AxoClamp 900A amplifier, Axon Instruments, Foster City, CA), filtered at 1 kHz, digitized at 10 kHz (Axon Digidata 1440), and stored for off-line analysis (Clampfit 10). Initial slopes of fEPSPs were expressed as percentages of baseline averages. In summary graphs, each point represents the average of 4 consecutive responses. Time-matched, normalized data were averaged across experiments. IL-1 pathway examination with RT² Profiler PCR Array. 1Pg total RNA (from whole hippocampus) was used for cDNA synthesis from tissue dissected 2 hours post TBI, via the RT2 first strand kit (SAbioscience 330401). 2ug total RNA (from whole hippocampus) was used for subsequent cDNA synthesis. RT2 sybr green mastermix (SAbioscience 330522) and RT2 profiler PCR array (Inflammatory Response & Autoimmunity PCR Array, Cat. no. PAMM-077Z, Qiagen) were used for real time PCR following manufacturer’s instruction. Housekeeping genes used as endogenous controls included ACTB, B2M, GAPDH, GUSB, HSP90AB1.

Foot slip assay. We used standard procedures described by Luong et. al. (2011) to measure motor balance coordination. During the training period, mice were trained to cross the 80cm beam to enter a black box with nesting material 3 times a day over 2 consecutive days. Mice were then sham- or blast-injured on the next day. 24hrs after injury, daily administration with (-)-P7C3-S243 or vehicle was initiated. Animals were then tested 7 and 28 days after injury. Behavior was videotaped during the test and foot slips were analyzed by an observer blind to condition and treatment group. LABORAS assay. LABORAS (Metris) is a system that uses a carbon fiber plate to detect behavior-specific vibration patterns created by animals. Data were collected uninterrupted over a 24-h period, enabling comprehensive quantification of basal grooming time and locomotor activity in the home cage environment throughout the light–dark cycle. Before data collection, test animals were acclimated in the test room for 1 wk. Then, test animals were placed in a standard cage atop the carbon fiber platforms. Vibrations were recorded for 24 h, and data was processed via LABORAS 2 software. References: Baldock, R.A., and Poole, I. (1993). Video camera calibration for optical densitometry. J Microsc 172, 49-54.

Figure S1. Protective Efficacy of Immediate Administration of P7C3-S243, Dynamics of Blood-Brain-Barrier Integrity after Blast-Injury, and Experimental Design of Subsequent Experiments, Related to Figure 1. (A) Administration of P7C3-A20 or P7C3- S243 (10 mg/kg/d administered IP in divided daily doses for 11 days), within 30-60 seconds after blast-mediated TBI, preserves hippocampal-dependent spatial memory in the Barnes maze 11 days after injury. Animals subjected to sham-injury and administered vehicle, or sham-injured animals that received the same doses of P7C3- A20 or P7C3-^Ϯϰϯ͕�ƐƉĞŶƚ�уϲϬй�ŽĨ�ƚŚĞŝƌ�time in the escape quadrant, in contrast to blast- injured vehicůĞ�ĐŽŶƚƌŽůƐ͕�ǁŚŝĐŚ�ƐƉĞŶƚ�уϮϬй�ŽĨ�ƚŚĞŝƌ�ƚŝŵĞ�ŝŶ�the escape quadrant. This same treatment with P7C3-A20 or P7C3-S243 immediately after blast-injury rescued memory to normal levels in sham-injured mice. In both blast-injured and sham-injured groups, treatment with (-)-P7C3-S243 showed a nonsignificant trend in increasing time spent in the escape quadrant. 12 male C57/Bl6 mice aged 12-14 weeks were tested per group, and data was collected and scored in an automated manner blind to treatment group. Significance was determined by 2 way ANOVA with Bonferroni post-hoc analysis. p-value labeled as *<0.05, **<0.01, ***<0.001, compared to blast-injured animals treated with vehicle. (B) Blood-brain-barrier permeability is compromised at 6 hours after blast injury, and returns to normal 24 hours after injury. Daily treatment with P7C3-S243 (10 mg/kg/d) for four days does not affect blood brain barrier permeability. 5 male C57/Bl6 mice aged 12-14 weeks were tested per group. Significance was measured using two way ANOVA. Data are represented as mean ± SEM. p-value labeled as ****<0.0001, compared to uninjured animals treated with vehicle. (C) Schematic of experimental design of subsequent experiments. Figure S2. Target Entry, Quadrant Time, Speed, Distance Traveled, and Learning in Veh and P7C3-S243 Treated Mice After Blast-Injury, and Persistent Deficits in Learning and Memory in Untreated Mice After Blast-Injury in the Barnes Maze, Related to Figure 1. (A) Daily IP administration of P7C3-S243 for 11 days in divided daily doses as indicated dose-dependently preserved performance in the probe test of the Barnes maze in blast- injured mice, as measured by the percent target entry. This metric is defined as the number of times a mouse pokes its nose into the correct hole out of the total number of times it pokes its nose into any hole. Treatment with an intermediate dose (3 mg/kg/d) of the active (-)-P7C3-S243 enantiomer preserved normal performance in this measure to the level displayed by sham-injured mice. By contrast, mice treated with the less active (+)-P7C3-S243 enantiomer showed the same deficit as blast-injured mice treated with vehicle. (B) Daily administration of P7C3-S243 was initiated at progressively later time periods after injury, in order to define the window of therapeutic efficacy. Whereas both 3 and 30 mg/kg/d doses preserved a normal percent target entry when treatment was initiated 24 hours after injury, only the higher dose was significantly protective when daily treatment was initiated at 36 hours. When treatment was initiated 48 hours after injury, no protective efficacy was noted at any dose. (C) Oral (PO) administration of the highly active (-)-P7C3-S243 enantiomer showed potent preservation of percent

target entry at 3, 10 and 30 mg/kg/day doses. (D) Daily IP administration of racemic P7C3-S243 for 11 days in divided daily doses dose-dependently preserved performance in the probe test of the Barnes maze in blast-injured mice, as measured by the percent quadrant time. This metric is defined as the percentage of time the mouse spends in the quadrant containing the escape hole. Treatment with an intermediate dose (3 mg/kg/d) of the active (-)-P7C3-S243 enantiomer completely preserved normal performance in this assay to the level displayed by sham-injured mice. Mice treated with the less active (+)-P7C3-S243 enantiomer showed some protection at the margin of statistical significance, but not to the degree effected by equivalent doses of racemic P7C3-S243 or (-)-P7C3-S243. (E) Daily administration of racemic P7C3-S243 was initiated at progressively later time periods after injury, in order to define a window of therapeutic efficacy. Whereas 3 and 30 mg/kg/d doses preserved a normal percent quadrant time when treatment was initiated 24 hours after injury, only the higher dose was significantly protective when daily treatment was initiated 36 hours after injury. When treatment was initiated 48 hours after injury, no protective efficacy in this assay was noted at any dose of racemic P7C3-S243. (F) Oral (PO) administration of the highly active (-)-P7C3-S243 enantiomer showed potent preservation of percent quadrant time at 3, 10 and 30 mg/kg/day doses. (G) Speed did not differ as a function of blast-injury or treatment initiated 24 hours after injury, (H) of compound administered (IP) 36 or 48 hours after injury, or (I) of oral (PO) administration of compound to blast-injured animals. (J) Distance traveled did not differ as a function of blast-injury or treatment (IP) initiated 24 hours after injury, (K) of compound administered (IP) 36 or 48 hours after injury, or (L) of oral (PO) administration of compound to blast-injured animals. (M) Learning was assessed as the percent latency to escape, defined as the percentage of time the mouse took to enter the escape hole on day 4 out of the time that it required on day 1. Mice subjected to blast-mediated TBI and then treated with vehicle, 0.3 mg/kg/day P7C3-S243, or 3 mg/kg/day (+)-P7C3-S243 learned significantly more poorly than did sham-injured mice. When treatment with 1, 3, 10 or 30 mg/kg/day P7C3-S243 (IP), or 3 mg/kg/day (-)-P7C3-S243 (IP), was initiated 24 hours after injury, however, all groups learned the task equally well. (N) Daily administration of P7C3-S243 was initiated at progressively later time periods after injury, in order to define a window of therapeutic efficacy. Whereas 3 and 30 mg/kg/day doses preserved a normal percent latency to escape when treatment was initiated 24 hours after injury, only the higher dose was protective when treatment was initiated at 36 hours. However, some protective efficacy was also seen at the lower dose of 3 mg/kg/day (IP) when treatment was initiated at this time point. When treatment was initiated 48 hours after injury, protective efficacy was noted at 30 mg/kg/day (IP) P7C3-S243. (O) Oral (PO) administration of the highly active (-)-P7C3-S243 enantiomer preserved normal percent latency to escape at 1, 3, 10 and 30 mg/kg/day doses. For (A–O), every group shown consisted of 25 male C57/Bl6 mice, aged 12-14 weeks, and data was collected and scored in an automated manner blind to treatment group. Data are represented as mean ± SEM. Significance was determined by two-way ANOVA with Bonferroni post-hoc analysis. p-value labeled as *<0.05, **<0.01, ***<0.001, and ****<0.0001 compared to blast-injured animals treated with vehicle. (P) After blast-injury, both sham groups

ƐƉĞŶƚ�ΕϰϬй�ŽĨ�ƚŽƚĂů�ƉƌŽďĞ�ƚƌŝĂl time in the target area, regardless of the amount of time after injury. Blast injured groups, by contrast spent less than 20й�ƚŽƚĂů�ƚŝŵĞ�ŝŶ�ƚĂƌŐĞƚ� area. Blast-injury causes a similar decrement in performance in the (Q) й�ƚĂƌŐĞƚ�ĞŶƚƌǇ� and (R) й�time spent in the escape quadrant at both acute and chronic time points after injury. (S) Speed during the probe trial was consistent between sham and blast-injured mice in both acute and chronic groups. (T) Total distance traveled during the probe trial was no different when mice were tested at 2 months of age. However, when mice were tested at the chronic time point of eight months after injury, the blast-injured group traveled a greater distance than sham animals. (U) Sham-injured animals had a decreased latency to escape compared to blast-injured animals at both 2 and 10 month of age, although older sham mice in the chronic group learned more slowly than the corresponding younger mice in the acute sham-injury group. The acute group consisted of 25 male C57/Bl6 mice in both sham- and blast-injury groups, and the chronic group consisted of 22 sham C57/Bl6 mice and 21 blast-injured C57/Bl6 mice. Data was collected and scored in an automated manner blind to treatment group. Significance was determined by two-tailed students’ T-Test. p-value labeled as *<0.05, **<0.01, ***<0.001, and ****<0.0001 compared to blast-injured animals. Figure S3. Histologic quantification of Tissue Damage After Blast-Injury, Related to Figure 3. (A) Optical densitometry of silver-stained CA1 stratum radiatum by NIH Image J from all animals in each group was used to quantify the protective effect by identifying degenerating axons in brain tissue. Every group shown consisted of the identical sections analyzed in Figure 3, from 5 male C57/Bl6 mice, aged 12-14 weeks. Data was collected and scored blind to treatment group. Significance was determined by 1 way ANOVA with Bonferroni post-hoc analysis. p-value labeled as *<0.05, **<0.01, ***<0.001, and ****<0.0001 compared to blast-injured animals treated with vehicle. (B) Shown at 80X magnification for clarity of morphology (scale bar = 5PM) is prominent silver staining of degenerating axons in CA1, corpus callosum, thalamus, cortex, olfactory bulb, striatum, dentate gyrus and cerebellum of blast-injured animals treated with vehicle, low dose (0.3 mg/kg/day) P7C3-S243, or intermediate dose (3 mg/kg/day) of the less active enantiomer (+)-P7C3-S243. Silver staining shows no evidence of axonal degeneration in sham-injured mice treated with vehicle, or in blast-injured mice treated with 3 or 30 mg/kg/day doses of P7C3-S243. Blast-injured mice were also protected from axonal degeneration by treatment with 3 mg/kg/day dose of the highly active enantiomer (-)-P7C3-S243. No axonal degeneration was observed in the hypothalamus as a result of blast-injury. Images shown are representative of brain slices from 5 animals in each group. (C) Same as (B), with lower power (40X, scale bar = 2.5PM) images showing breadth of axonal staining. (D) Optical densitometry of light transmitted through the indicated silver stained regions from all animals in each group was used to quantify the protective effect. The specific tissue area was manually delineated, and signal was quantified for 18 sections for each of the 5 animals, spaced 480 PM apart. Here, a greater value indicates that more light was able to pass unimpeded through the section by virtue of less silver staining, which indicates less axonal degeneration. Data are represented as mean ± SEM. P-value *<0.05, *<0.01, **<0.001 determined by-two

way ANOVA with Bonferroni post-hoc analysis. (E) Immunohistochemical staining for NeuN shows no evidence of frank neuronal cell loss after blast-mediated TBI in CA1, corpus callosum, thalamus, cortex, olfactory bulb, striatum, or hypothalamus. Images shown are representative of brain slices from 5 animals in each group, directly adjacent to those shown in Figure S7. Scale bar = 5PM. (F) NeuN-stained cells were quantified for 18 sections for each of the 5 animals, spaced 480 PM apart. Data are represented as mean ± SEM. No significant differences between groups were noted by-two way ANOVA with Bonferroni post-hoc analysis. (G) Hematoxylin and eosin (H&E) staining shows no evidence of cell loss after blast-mediated TBI in CA1, corpus callosum, thalamus, cortex, olfactory bulb, striatum, or hypothalamus. Images shown are representative of brain slices from 5 animals in each group, directly adjacent to those shown in Figure S7. Scale bar = 5PM. (H) H&E staining in 18 sections for each of the 5 animals, spaced 480 PM apart, was quantified. Data are represented as mean ± SEM. No significant differences between groups were noted by-two way ANOVA with Bonferroni post-hoc analysis. (I) Immunohistochemical Staining for glial fibrillary acidic protein (GFAP) shows no elevation after blast-injury, thus providing no evidence of neuroinflammation. Images shown are representative of brain slices from 5 animals in each group, directly adjacent to those shown in (G). Figure S4. Transmission Electron Microscopy and Toluidine Blue Staining, Related to Figure 4. (A) This diagram shows the methodology of tissue processing for toluidine blue and transmission electron microscopy (TEM). Both hemispheres of the hippocampus (red rectangles) were used for evaluation of pathology. (B) Toluidine blue staining showed that blast-injury vehicle-treated mice accumulate chromatolytic and pyknotic neurons in the CA1 region, and that initiation of daily oral treatment of blast- injured mice with (-)-P7C3-S243 prevents this pathology. Pictures shown are representative of 4 animals for each condition, and are from different animals than the pictures shown in figure 4. Scale bar = 20Pm (C) TEM showing protection against myelin degeneration (red arrows) and mitochondrial swelling (blue arrows) in stratum radiatum of blast-injury mice treated with 30 mg/kg/day (-)-P7C3-S243. These pictures are from different animals than the pictures in Figure 4 (scale bar = 500nm). (D) TEM visualization of hippocampal axonal and mitochondrial structures 2 weeks after sham-injury in mice administered vehicle, showing normal myelin sheath (red arrows), along with intact outer membrane and internal cristae structures within mitochondria (blue arrows) in the hippocampus stratum radiatum (scale bars = 500nm). (E) TEM visualization of hippocampal axonal and mitochondrial structures 2 weeks after sham-injury in mice treated orally with 30 mg/kg/day of (-)-P7C3-S243, showing normal myelin sheath (red arrows), along with intact outer membrane and internal cristae structures within mitochondria (blue arrows) in the hippocampus stratum radiatum (scale bars = 500nm). (F) TEM visualization of hippocampal axonal and mitochondrial pathology 2 weeks after blast-injury in mice receiving vehicle, showing degeneration of myelin sheath (red arrows), along with abnormal outer membrane and internal cristae structures within mitochondria (blue arrows) in the hippocampus stratum radiatum (scale bars, 500nm). (G) TEM visualization of hippocampal axonal and mitochondrial structures 2 weeks after

blast-injury in mice treated orally with 0.3 mg/kg/day of (-)-P7C3-S243, showing no preservation of myelin sheath (red arrows), along with abnormal outer membrane and internal cristae structures within mitochondria (blue arrows) in the hippocampus stratum radiatum (scale bars, 500nm). (H) TEM visualization of hippocampal axonal and mitochondrial structures 2 weeks after blast-injury in mice treated orally with 3 mg/kg/day of (-)-P7C3-S243, showing preservation of myelin sheath (red arrows), along with very minimal outer membrane and internal cristae structures within mitochondria (blue arrows) in the hippocampus stratum radiatum (scale bars = 500nm). (I) TEM visualization of hippocampal axonal and mitochondrial structures 2 weeks after blast- injury in mice treated orally with 30 mg/kg/day of (-)-P7C3-S243, showing preservation of myelin sheath (red arrows), along with normal outer membrane and internal cristae structures within mitochondria (blue arrows) in the hippocampus stratum radiatum (scale bars = 500nm). Figure S5. Chronic Motor Deficits after Blast Injury, related to Figure 5. Eight months after a single blast-injury, mice display twice as many foot slips as sham-injury control mice. Sham animals consisted of 22 male C57/Bl6 mice, and blast-injured animals consisted of 21 male C57Bl6 mice. Data was collected and scored by an observer blinded to identity of the group. Significance was determined by two-tailed students’ T-Test. p- value labeled as *<0.05 compared to blast-injured animals. Figure S6. Acquisition of Chronic Neuropsychiatric Deficits Related to Anxiety in Untreated Mice after Blast-Injury, related to Figure 5. (A) Basal grooming is elevated chronically after a single blast-injury. Grooming behavior was quantified with LABORAS® equipment. At the acute time point, there was no difference in 24 hour grooming time between blast-injury and sham-injury groups. Eight months after blast- injury, however, mice showed acquisition of elevated grooming time, relative to sham- injury mice. Both blast-injury and sham-injury acute groups consisted of 25 male C57/Bl6 mice, and the chronic groups consisted of 22 sham C57/Bl6 mice and 21 blast- injured C57/Bl6 mice. Data was collected and scored in an automated manner blind to treatment group. Significance was determined by two-tailed students’ T-Test. p-value labeled as *<0.05 compared to blast-injured animals. (B) Hyperactivity is acquired chronically after a single blast-injury. 24 hr activity was monitored using LABORAS® equipment. 12 days after blast-injury, mice did not exhibit any difference in locomotion time. However, eight months after injury, locomotion was increased by 2.5 fold in the blast injured group relative to sham controls. 12 days after blast-injury, distance traveled over the course of 24hrs was not different between sham and blast-injured groups. Eight months after injury, however, blast-injured mice exhibited a two-fold increase in distance traveled compared to sham controls. 12 days after blast-injury, there was no difference in rearing activity between blast-injured and sham-injured groups. However, eight months after injury, blast-injured mice exhibited ~3 fold increase in rearing time over sham-injury controls. 12 days after blast-injury, the time spent immobile was not different between blast-injured and sham-injured groups. However, eight months after blast injury, the blast-injured group exhibited Ă�Ϯϱй�

decrease in immobile time relative to sham-injury controls. Both acute groups consisted of 25 male C57/Bl6 mice, and the chronic groups consisted of 22 sham C57/Bl6 mice and 21 blast-injured C57/Bl6 mice. Data was collected and scored in an automated manner blind to treatment group. Significance was determined by two-tailed students’ T-Test. p- value labeled as ****<0.0001 compared to blast-injured animals.

Gene Fold Change Blast/Sham

t-Test p value Average raw Ct, blast

Average raw Ct, sham

IL-1α -2.24 0.101177 31.61 29.39 IL-1β 1.17 0.166437 32.77 31.94 IL1R1 1.28 0.207558 26.53 25.83 IL1RAP 1.05 0.539049 24.62 23.64 IL1RAN 2.71 0.230179 34.40 34.79

Table S1. Change in relative gene expression in hippocampus 2 hours following blast induced brain injury. None of the examined genes of the IL-1 pathway reached a greater than 3 fold change following

blast injury. Gene expression data represent average from 3 mice per injury and sham groups.

Gene Fold Change Blast/Sham

t-Test p value Average raw Ct, blast

Average raw Ct, sham

IL-1ɲ -1.02 0.792652 29.83 29.85 IL-1ɴ -1.71 0.193353 33.06 32.33 IL1R1 1.02 0.794696 26.44 26.53 IL1RAP 1.01 0.850761 23.89 23.95 IL1RAN 1.10 0.587179 34.69 34.88

Table S2. Change in relative gene expression in hippocampus 24 hours following blast induced brain injury. None of the examined genes of the IL-1 pathway reached a greater than 3 fold change following blast injury. Gene expression data represent average from 4 mice per injury and sham groups.