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Interference of neuronal activity‐mediated gene expression through serum response factor deletion enhances mortality and hyperactivity after traumatic brain injury

Traumatic brain injury (TBI) is one of the most frequent causes of brain injury and mortality in young adults with detrimental sequelae such as cognitive impairments, epilepsy, and attention‐deficit hyperactivity disorder. TBI modulates the neuronal excitability resulting in propagation of a neuronal activity‐driven gene expression program. However, the impact of such neuronal activity mediated gene expression in TBI has been poorly studied. In this study we analyzed mouse mutants of the prototypical neuronal activity‐dependent transcription factor SRF (serum response factor) in a weight‐drop TBI model. Neuron‐restricted SRF deletion elevated TBI inflicted mortality suggesting a neuroprotective SRF function during TBI. Behavioral inspection uncovered elevated locomotor activity in Srf mutant mice after TBI in contrast to hypoactivity observed in wild‐type littermates. This indicates an SRF role in modulation of TBI‐associated alterations in locomotor activity. Finally, induction of a neuronal activity induced gene expression program composed of immediate early genes (IEGs) such as Egr1, Egr2, Egr3, Npas4, Atf3, Arc, Ptgs2, and neuronal pentraxins (Nptx2) was compromised upon SRF depletion. Overall, our data show a role of neuronal activity‐mediated gene transcription during TBI and suggest a molecular link between TBI and such post‐TBI neurological comorbidities involving hyperactivity phenotypes.


| INTRODUCTION
Traumatic brain injury (TBI) is one of the most frequent CNS injuries accounting for high accident-related mortality rates in young adults. 1,2 TBI patients, including many contact-sport related injuries, 3 suffer from cognitive decline and frequently further neurological impairments including epilepsy, neurodegenerative diseases, 4,5 and attention-deficit hyperactivity disorder (ADHD). 6 Immediately after impact, TBI induces a strong glutamate efflux that may contribute to the acute neuronal network dysfunction. 7 Particularly within the first hours after TBI, an increased neuronal excitability and reduced GABAergic inhibitory transmission were reported. [8][9][10][11][12] Such early neuronal hyperexcitability involves NMDA receptor subunit upregulation 7 and a function of, for example, GluN2B as injury mechanoreceptors. 9 Taken together, previous reports describe enhanced neuronal activity at early stages after TBI.
Enhanced neuronal transmission results in nuclear activation of activity-driven transcription factors (TFs) triggering neuronal activity directed gene expression programs. 13,14 So far, CREB (cAMP response element-binding protein) was considered one of the major TFs involved in propagation of neuronal activity-mediated gene transcription. 13,14 In TBI, CREB phosphorylation, indicative of activation, is both down 15,16 and upregulated 17,18 which may depend on the brain area and timepoint. However, up until now, no functional role for CREB in TBI has been reported in neurons, for example, using mouse mutagenesis. In contrast, in astrocytes, enhanced CREB activation mediates neuroprotective TBI functions. 19 Thus, the connection between TBI and neuronal activity directed gene transcription has currently not been investigated in great detail.
In this study, we aim at identification and functional characterization of such a neuronal activity dependent TF during TBI. For this, we analyzed a further prototypical neuronal activity-driven TF, the serum response factor (SRF). 20,21 SRF responds to neuronal activity, for instance through calcium influx via NMDA receptors. 22 After activation, SRF propagates in neurons an immediate early gene (IEG) response resulting in rapid (within minutes) but transient upregulation of target genes including cFos, Egr1, Egr2, Egr3, and, for example, Arc. 20,21 Since many SRF effector genes encode TFs, this results in a second gene expression wave involved in modulation of learning and memory, synaptic plasticity as well as neuro-behavioral changes such as hyperactivity and modulated anxiety. [23][24][25] So far, selected IEGs such as cFos, cJun, and Egr1 were found upregulated by TBI in humans 26,27 and in rodent TBI models. [28][29][30][31][32] However, a functional role of SRF or one of these IEGs in TBI has not been described. In this study we employ neuron restricted SRF deletion in mice 33,34 to show a first SRF function in a rodent TBI model. 35,36 In previous reports, such SRF deficient mice revealed impaired stress responsiveness, 33 a hyperactivity and decreased anxiety phenotype, 33,37 altered seizure propagation 34,38 and impaired long-term potentiation 39 and depression. 40 For instance, challenging mice with a novel environment, 39 acute stress, 33 electroconvulsive shocks, 39 or epileptic seizures 34,41 upregulates IEGs in wildtype (wt) mice whereas this was strongly diminished for several IEGs after SRF ablation. This suggests a pivotal role for SRF as the TF mediating a neuronal activity-driven IEG response. Furthermore, mouse injury models for axon 42,43 and myelin 44 regeneration have shown that SRF exerts a neuroprotective function in wt mice. Herein, using a mouse TBI model, we expand on such neuroprotective SRF functions. We show that SRF in mice limits TBI inflicted mortality and SRF deficiency modulates neuro-behavioral changes elicited by TBI such as a hyperactivity phenotype not observed in wt mice. Finally, SRF directs expression of a neuronal activity-associated gene set consisting of several IEGs immediately after the TBI impact.

| Srf deficient mice and housing conditions
In this study, previously described neuron specific Srf mutant mice were used. 33,34,44 We used male and female offspring derived from breeding pairs of parental mice harboring the CreERT2 allele and one "floxed" and one wild-type Srf allele (for more details see "Experimental details," below). To induce a neuron specific deletion of Srf, CreERT2 positive mice with two "floxed" Srf alleles were daily ip injected with tamoxifen (2 mg dissolved in 10% ethanol in peanut oil, Sigma) for 5 consecutive days. After tamoxifen treatment, SRF deficient animals were designated as Srf ko. As wild-type control, CreERT2 positive littermates carrying two wild-type (wt) Srf alleles were used and treated with the same tamoxifen injection schedule. Thus, all animals (wt and ko) analyzed were treated with the same tamoxifen protocol (see Figure 1A). Of note, wt mice treated with or without tamoxifen before TBI were indistinguishable with regard to open field distance and center visits, NSS score, exit circle time, and ladder walk traversing time as well as slip number (data not shown). All mice were kept under standard laboratory housing conditions (12 hours light/12 hours dark cycle, lights on at 07.00 am, 22°C, 60% humidity) and had free access to water and standard mouse diet. All animal experiments were in compliance with international regulations for the care and use of laboratory animals (ARRIVE guide-lines and EU Directive 2010/63/EU for animal experiments). Mouse experiments | 3

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in this study were approved by the local governmental authority for animal experimentation (Regierungspräsidium Tübingen, Germany).

| TBI model
A weight-drop TBI model was applied to induce TBI in mice 12 days after the last tamoxifen injection. In all experiments, mice had an age of 12-14 weeks. The TBI induction was performed as described earlier. 35,36 In brief, mice were anesthetized by 5% sevoflurane inhalation (Sevorane, AbbVie, Wiesbaden, Germany). The head was shaved, cleaned with ethanol, and a skin incision was performed with a scalpel. Afterwards, the mouse was fixed on a cushion of modeling clay and the tip (Ø of 3 mm) of the metal rod (weight: 333 g) was positioned over a rostro-lateral target area of the left cortical hemisphere. The falling height of the bar was adjusted to 2.3 cm for males or 2 cm for females and dropped on the closed skull. Directly after the hit, the bar was retracted. Until regular breathing was restored, 100% of oxygen was given for 1 to 2 minutes and the skin was sutured. For analgesia, Buprenorphin (Temgesic, Indivior, Virginia, USA; 0.03 mg/ kg) was injected s.c. shortly before the TBI induction and every 8 hours for the first 24 hours post-TBI in all mice (wt and ko, sham and TBI treated). As control, sham operated animals, treated exactly the same as TBI mice (ie, sevoflurane inhalation, skin incision, suturing, analgesia), except for the actual impact of the falling rod were included.

| Hematoma size
For the assessment of the hematoma, a picture of each freshly dissected brain was taken with a Samsung NX1000 camera. The hematoma area on the surface of the ipsilateral cortex was measured in pixel with the lasso tool of the F I G U R E 1 Neuron restricted conditional Srf mutagenesis in mice. A, Experimental outline. B, Srf mRNA levels were quantified in the hippocampus by qPCR in sham animals and 1 hour after TBI in wt and Srf mutant animals. Srf was slightly upregulated by TBI in wt animals, whereas almost no Srf mRNA was detectable in Srf mutant animals. Each dot reflects one animal. Data are depicted as mean ± SD. For statistical analysis, a one way ANOVA (P and F value in blue) was performed with TMC (P values of multiple comparison depicted by asterisks; see below). C, SRF protein levels are nearly absent in SRF deficient compared to wt animals (n = 3-4 animals/condition; see quantification in F). D and E, TBI animals were stained 7 days after TBI for SRF expression. In wt animals (D), SRF expression in neurons was comparable between the ipsilateral (hit by the weight; arrow) and contralateral cortex. After SRF depletion, SRF abundance was downregulated in both hemispheres of a TBI animal (E; see quantification in G). F and G, Quantification of SRF protein levels in immunoblotting 1 h after TBI (F) and of the dentate gyrus region in the hippocampus by immunohistochemistry at 7 days post-injury (G). Each dot reflects one animal. Data are depicted as mean ± SD. *P < .05; ***P < .001. Scale-bar (D, E) = 1 mm graphic software paint.net (dotPDN LLC, Washington State University, Washington, USA) and the size calculated as a percentage relative to the measured whole surface area of the ipsilateral cortex.

| Quantitative real-time PCR
From freshly dissected brains of TBI-treated or sham-operated mice, cortical tissue with approximately 5-6 mm in diameter was dissected from the target area of the ipsilateral cortex as well as of the equivalent area of the contralateral side with the following coordinates: from Bregma to 3.5 mm lateral and from Bregma to −4 mm posterior. In addition, the ipsilateral and contralateral hippocampi were dissected. After total RNA was isolated with the Isolate II RNA/DNA/ Protein kit (Bioline, Luckenwalde, Germany), cDNA synthesis was performed with 0.75 µg RNA, random hexamers (Biomers, Ulm, Germany), and the M-MLV reverse transcriptase (RT, Promega). RT-quantitative real-time PCR (qPCR) was performed with 2 µL of cDNA, specific primer pairs and SYBR Premix Ex Taq (Tli RNase H Plus) PCR Master Mix (TaKaRa Bio Europe, Saint-Germain-en-Laye, France) in a 10 µL reaction volume/well of a 96-well plate in a Roche Light Cycler 480 (Roche). The Ct value of a target gene was detected with the LC480 II software and the relative mRNA level of the target gene was calculated relative to the measured Ct value of the house-keeping gene Gapdh . Primer sequences were reported earlier or can be provided upon request. 35,45,46

| Biochemistry
Protein lysates were prepared with the Bioline kit and Western blots were performed as previously described. 35 The membranes were incubated with the following primary antibodies: anti-SRF (rat, 1:500; a kind gift of Prof. Dr A. Nordheim, Tübingen University, Germany) or anti-GAPDH (mouse, 1:60 000; Acris Origine Europe, Herford, Germany; ACR001P) overnight at 4°C. For detection horseradish peroxidase conjugated secondary antibodies (1:2000; Santa Cruz, Heidelberg, Germany) were applied in combination with ECL Western Blotting Substrate (Merck Millipore, Burlington, MA, USA). Band intensities were quantified with the FIJI software (Gels tool). For SRF, the combined intensity of both bands was measured and normalized on the intensity of the GAPDH signal.

| Neurological severity score with exit circle test
The Neurological severity score (NSS) was performed as described before. 36 The NSS includes the following 10 tests: (a) exit circle, (b) seeking behavior, (c) paresis, (d) straight walk, (e) startle reflex, (f) beam balancing (7 × 7 mm), (g-i) beam walk (3, 2, and 1 cm wide stick), and (j) round stick balancing (5 mm diameter). In the NSS only male mice were used. On average it took 10 minutes for one mouse to complete all 10 tests. For a failed test one point was awarded, while a passed test was counted as zero. An NSS score of "0" indicates that the mouse passed all tests whereas a score of "10" indicates maximum neurological impairment. The NSS was measured 1 day pre, as well as 1, 3, and 5 days post-TBI. The NSS includes the exit circle test where a mouse is located in the middle of a circular arena (Ø 30 cm) with a small exit whole (5 × 5 cm; see Figure 5). We measured the time to exit the | 5 FÖRSTNER aNd KNÖLL circle, which for a healthy mouse is typically under 3 minutes. 36 The NSS also includes a test for the paresis formation. Here, the mouse was lifted by the tail and the mouse's capability to hold on to a pole was tested. Paresis formation was additionally analyzed at 4 hours after injury.

| Open field test
To determine the locomotor activity, anxiety, and exploratory behavior, the Open field (OF) was performed 2 days pre-TBI, 6 hours post-TBI, and then daily until 7 days post-TBI. In the OF only male mice were used. To measure the locomotor activity, mice were placed in the middle of a square shaped OF arena (50 × 50 cm) and tracked for 15 minutes with a video camera. In the arena center, the light intensity was 30 lux. The Viewer III software (Biobserve, Bonn, Germany) determined the overall track-length in the 15 minutes trial as read-out for the locomotor activity as reported before. 47 To analyze anxiety, the number and time span of visits within 15 minutes in a virtual square (20 × 20 cm) predefined by the Viewer III software in the center of the OF arena was determined by the Viewer III software.

| Ladder walk
To assess the posttraumatic motor coordination, a ladder walk test was performed 2 days pre-TBI, 4 hours and then daily until 5 days post-TBI. For this test only male mice were used. In the ladder walk, mice had to walk over a horizontal ladder to reach their home cage. The ladder was built of two transparent plates (square: 69.5 × 15 cm) and cylindrical ladder rungs (8 cm long, Ø of 2 mm). The ladder walk had a length of 60 cm with a space of 1 cm between rungs (see Figure 5). Before the first ladder walk test, each mouse accomplished one test run over the ladder. At each timepoint each mouse had to cross the ladder three times, while they were recorded with a Samsung NX1000 camera. The recorded videos were analyzed at ¼ of the original speed to count slips and time needed to cross the ladder for each run. The average slip number or time out of the three runs is depicted. Typically, this test was completed in 5 minutes by each mouse.

| Acquisition of mouse body weight
The body weight was measured 30-60 minutes before TBI and 6 hours after TBI followed by a daily measurement between 2 and 5 pm until day seven post-TBI. The weight loss of the mice after TBI was calculated as a percentage relative to the measured body weight before TBI.

| Microscopy and image analysis
Images of the stained brain slices were acquired with a BIOREVO BZ-9000 microscope (KEYENCE, Neu-Isenburg, Germany) applying a 4x or 10x objective lense (Plan APO, Nikon) and merged with the software BZ-II Analyzer (version 2.2, Keyence). For image analysis for each staining one stained tissue slice was analyzed per mouse. The SRF positive area ( Figure 1) was measured in a squared area (300 × 200 µm) of the upper leaf of the granule cell layer of the dentate gyrus with the hybrid cell count tool of the BZ-II Analyzer software. The positive area of EGR1, ATF3, or cFos ( Figure 7) was measured in a squared area (300 × 100 µm) of the upper leaf of the granule cell layer of the dentate gyrus with the hybrid cell count tool of the BZ-II Analyzer software. Thereby, for all stainings the same threshold was used for all samples to distinguish the specific signal from background. The lesion area was measured in brain slices stained with anti-NeuN with the FIJI software (ImageJ 1.51s, National Institutes of Health, NIH, USA). The BZ-II Analyzer software was used to manually count the NeuN positive cells or Iba1 positive cells. NeuN positive cells were counted in a circular area (Ø 300 µm) positioned next to the lesion area in the cortical layers 2/3. Iba1 positive cells were counted in three circles (Ø 200 µm) positioned in one line next to the lesion area in cortical layers 2/3 ( Figure  3C). The GFAP positive area was measured in the same three circles with the FIJI software. The threshold to distinguish positive area from background was set for each stained cohort (including TBI treated or sham operated wt and Srf ko mice) to 50% of the measured intensity of strongly GFAP positive cells from TBI-treated wt and Srf ko mice. The intensity of P-cofilin in the cerebral peduncles was measured with the FIJI software. For the quantification of Smi32 positive signals in the cerebral peduncles the hybrid cell count tool of the BZ-II Analyzer software was applied. Here, the same threshold was used for all stained slices.

| Experimental design and statistical analysis
Wt and Srf ko mice were randomly distributed in three groups. Induction of TBI was always performed between 9.00 and 11.00 am. All behavioral testing was conducted between 1.00 and 7.00 pm. Animals performed two (ladder walk and open field) or three (ladder walk, open field, NSS) tests at the same day. Mice were allowed to move freely in their home cages for 1 hour between two tests. For mortality quantification ( Figure 2), all TBI-treated mice with a skull fracture (31 wt mice: 25 males, 6 females; 36 Srf ko: 29 males, 7 females) were included. For statistical analysis a Fisher's exact test was applied.

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FÖRSTNER aNd KNÖLL F I G U R E 2 SRF deletion enhances TBI-induced mortality in mice. A-C, Mortality was quantified irrespective of body-weight (A) and two different weight cohorts for males (B) and females (C). TBI inflicted mortality was elevated in SRF deficient mice for both males and females (A-C) as well as when accounting for weight differences (B, C). D, At 4 h post-TBI, SRF deficient mice had a slightly higher paresis incidence at three of the four extremities. E and F, Wt and Srf mutant mice exhibited comparable extent of hematoma formation on the ipsilateral cortex (arrows in E). Each dot in (F) depicts one animal. Black dots represent male whereas grey filled dots depict female animals. Data are depicted as mean ± SD. G, Weight loss after TBI was comparable for wt and Srf mutant animals. Data are depicted as mean ± SEM. # P < .05 for TBI ko versus Sham ko; ## P < .01 for TBI ko versus sham ko; ++ P < .01 for TBI wt versus sham wt. H, The overall NSS score was comparable between wt and Srf mutant TBI animals. A score of "0" signifies no impairments whereas a score of "10" reflects maximally impaired animals. I, The 10 individual tests comprising the NSS are depicted with the percentage of mice failing to complete a test. Data are depicted as mean ± SEM. # P < .05 for TBI ko versus sham ko; + P < .01 for TBI wt versus sham wt (G, H). Scale-bar (E) = 0.5 cm | 7

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In the first experimental group, only male mice were used. The following number of male mice was used to assess paresis, hematoma, weight loss, NSS ( Figure 2D Here, mice without a cortical lesion were excluded to analyze animals with a comparable TBI impact only. Weight loss ( Figure 2G) and behavior data ( Figures 2H and 5B,D,F-I) are presented as mean ± SEM and data were analyzed with a two-way analysis of variance (ANOVA) with Tukey's multiple comparisons test (TMC; α = 0.05). The statistical power for these data sets was 99.9%. In Figure 5B we additionally performed a two-way ANOVA with Dunnett's multiple comparisons (DuMC) test to compare each timepoint with the pre-TBI baseline for each experimental group. OF data pre-TBI ( Figure 5A,C,E) are depicted as mean ± SD with a Mann-Whitney test ( Figure 5A) and an unpaired t-test ( Figure 5C). Histology data ( Figures 1G, 3E,F, and 4M-P) are presented as mean ± SD with a Kruskal-Wallis test and Dunn's multiple comparisons test (DMC; α = 0.05). For Smi32 and P-cofilin staining (Figure 4), the number of Srf ko TBI mice was seven instead of eight. F I G U R E 3 TBI-associated neuroinflammation is not altered by SRF ablation. A-D, Cortical sections of sham animals and animals after 7 days of TBI were stained for Iba1 (red), GFAP (green), and nuclei (blue). TBI induces a comparable microglia and astrocyte response in wt (C) and Srf mutant (D) animals compared to respective sham animals (A, B). Dotted circles in (C) indicate positions used for quantification in (E, F). A′-D′, are higher magnifications of boxed areas in (A-D). E, F, Quantification of microglia (E) and astrocyte (F) immunoreactivity for all four cohorts and the three areas depicted in (C). G-I, qPCR analysis of the inflammatory genes Ccl2 (G), Ccl3 (H), and Il1b (I) 1 h after TBI in the cortex. The mRNA levels were similarly upregulated by TBI in wt and Srf mutant animals. For statistical analysis, a one-way ANOVA (P and F value in blue) was performed with TMC (P values of multiple comparison depicted by asterisks; see below). In (E-I), each dot depicts one animal. Data are depicted as mean ± SD. *P < .05 for TBI versus respective sham; **P < .01 for TBI versus respective sham. Scale-bar (A-D) = 500 μm; (A′-D′) = 100 μm 8 |

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The animals of the second experimental group were sacrificed 1 hour after TBI to measure the hematoma size ( Figure  2E,F), gene expression ( Figures 1B, 3G-I, and 6). This group included four wt sham (two males and two females), four Srf ko sham (two males and two females), seven wt TBI (five males and two females), and six Srf ko TBI (four males, two Black symbols depict twoway ANOVA with TMC *P < .05 for TBI wt versus TBI ko; **P < .01 for TBI wt versus TBI ko; ***P < .001 for TBI wt versus TBI ko; # P < .05 for TBI ko versus sham ko; & P < .05 for wt sham versus ko sham (B, D, F). Blue and red symbols depict two-way ANOVA with DuMC for wt TBI and ko TBI in relation to the pre-TBI data, respectively with *P < .05 and **P < .01 (B) are shown as mean ± SD. For statistical analysis of data, a one-way ANOVA with TMC (α = 0.05; Figures 1B, 3G-I, and 6A-L), a Kruskal-Wallis test with DMC ( Figure 1F), Mann-Whitney test ( Figure 6M), or unpaired t-test ( Figure  2F) was used. The statistical power for the gene expression data was 99.9%.

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Animals of the third experimental group were killed 2 hours after TBI for immunohistochemistry (Figure 7). This group included two wt sham (one male, one female), five wt TBI (two males, three females), and three Srf ko TBI (one male, two females) animals.
Statistical analysis was performed with the Prism7 software (Graph Pad Software, San Diego, CA, USA).
Since SRF can regulate its own expression 48 we analyzed whether TBI upregulates Srf mRNA levels 1 hour after TBI in the hippocampus ( Figure 1B). As control, sham operated mice ("sham"), that is, receiving anesthesia, analgesia, and suturing were analyzed. Further, we employed both the ipsilateral ("TBI ipsi") and contralateral brain hemisphere ("TBI contra"), not directly hit by the TBI impact. Srf mRNA abundance was significantly elevated by TBI compared to sham-treated animals and the contralateral hemisphere (wt TBI ipsi vs wt sham: P = .0061, Mann-Whitney test; wt TBI ipsi vs wt TBI contra: P = .0006, Mann-Whitney test; Figure  1B). At this 1 hour post-TBI timepoint, SRF protein abundance was also slightly elevated by TBI ( Figure 1C), however statistical significance was not fully reached (P = .057; Figure 1F).
After 7 days of TBI, SRF protein was uniformly expressed in both the ipsi (arrow in Figure 1D) and contralateral hemisphere of wt mice (quantified in Figure 1G). In SRF deficient mice, SRF protein abundance was clearly diminished ( Figure  1E-G).
In summary, at an early timepoint after TBI (ie, 1 h), SRF abundance increased after TBI whereas at a later timepoint (ie, 1 week post-TBI) this was not observed anymore.

| SRF deletion enhances TBI-induced mortality in mice
TBI is one of the leading causes of mortality in young adults, particularly males. 2 Hence, molecular identification of neuroprotective factors reducing TBI-associated death is an important topic in TBI research and animal TBI models might help in this respect.
When wt and Srf mutant mice were subjected to weightdrop based TBI we noted an immediate difference in post-TBI survival between cohorts (Figure 2A-C). In wt mice we obtained a mortality rate of 6.5% (Figure 2A) with males being slightly more affected than females. Typically, animals died within 5 minutes after TBI most likely involving the respiratory failure due to compression of the brainstem by the weight impact. 36 In contrast to wt mice, more than one third of all Srf mutant animals died immediately after TBI with females (42.9%) slightly more affected than males (37.9%; Figure 2A).
Since weight differences in adult mice might account for alterations in mortality we grouped animals in several weight ranges. Still, when controlling for weight differences ( Figure  2B,C) we noted that lighter Srf mutant animals of both sexes (<28 g males, <23.5 g females) were experiencing higher mortality rates of 57.1% (males) and 50% (females) compared to weight matched wt littermates (14.3% males, 0% females; Figure 2B,C). The same holds true for animals with more than 23.5 g (females) or 28 g (males) bodyweight ( Figure 2B,C). Also, the changes in mortality are most likely not due to differences in weight or brain size before TBI since only subtle differences in body weight were observed between wt and Srf mutant animals (wt males: 29.5 ± 1.9 g; ko: 28.1 ± 2.0 g; P = .01; wt female: 24.03 ± 0.8 g; ko: 23.5 ± 1.4 g; P = .46; Figure 2E-G).
Next, we analyzed paresis prevalence at 4 hours ( Figure 2D) and at several timepoints after TBI ( Figure 2I) in those animals surviving TBI. In general, forelimbs were more frequently affected than hindlimbs in both genotypes. Also, extremities on the right side, contralateral to left brain hemisphere hit by the weight-drop, were more frequently affected than left extremities, particularly in Srf mutants ( Figure 2D). Comparing wt and Srf mutant mice, the paresis formation was slightly higher after SRF ablation at three out of the four limbs at 4 hours after TBI ( Figure 2D; for later timepoints see also I).
F I G U R E 6 Impaired neuronal activity-driven gene expression after TBI upon SRF ablation. A-L, qPCR analysis of wt and Srf mutant sham treated hippocampal samples as well as ipsi and contralateral hippocampi collected 1 h after TBI of both genotypes. In wt mice, TBI upregulated all genes tested except for β actin (I). Upon SRF ablation, induction of indicated genes was reduced for Egr1 (A), Egr2 (B), Egr3 (C), Npas4 (D), Arc (E), Atf3 (F), Ptgs2 (G), and Nptx2 (H). In contrast, TBI-associated cFos (J), ΔFosb (K), and JunB (L) upregulation was SRF independent. For statistical analysis, a one-way ANOVA (P and F value in blue) was performed with TMC (P values of multiple comparison depicted by asterisks; see below). M, qPCR analysis in the cortex of wt and Srf mutant animals 1 h after TBI. Values were normalized to respective sham values of wt or Srf mutant mice. Each dot depicts one animal. Black dots show male whereas gray filled dots represent female animals. Data are depicted as mean ± SD. *P < .05; **P < .01; ***P < .001 The weight hitting the skull resulted in hematoma formation in the ipsilateral cortical layers ( Figure 2E,F). Quantification of hematoma size on the cortical surface did not result in a statistically significant difference between cohorts (P = .97, unpaired t-test; Figure 2F). Thus, on average the hematoma size was 10% of the entire ipsilateral cortical hemisphere regardless of genotype ( Figure 2F).
Since TBI results in decreased body-weight in subsequent days, weight loss of wt and Srf mutant animals either sham or TBI-treated was quantified over 7 days post-TBI ( Figure 2G).
Sham-treated animals experienced weight loss of approximately 7%, most likely due to stress, anesthesia and performance of sham surgery with suturing ( Figure 2G). In TBI treated animals, weight loss was more pronounced and peaked at 12%-15% at 1 day after TBI. Subsequently, animals gained weight in the following days ( Figure 2G). Weight loss in TBI treated mutant compared to wt animals was slightly enhanced, however not reaching statistical significance (two-way ANOVA: P ≤ .0023; F time × group = 2.18; with TMC for eg, wt TBI vs Srf ko TBI: adjusted P = .32 (6 h), P = .51 (1d); Figure 2G). F I G U R E 7 SRF regulates EGR1 and ATF3 protein abundance after TBI but not c-Fos. A-C, EGR1 was upregulated in the hippocampus and cortex 2 h after TBI in wt (B) compared to sham-treated animals (A). In SRF deficient animals, EGR1 expression was lower after TBI (C). D-F, ATF3 was induced by TBI in wt TBI-treated animals (E) whereas only low ATF3 expression was present in sham animals (D). ATF3 expression was reduced after TBI in SRF deleted animals (F). G-I, c-Fos was upregulated to comparable extent in wt (H) and Srf mutant (I) animals after TBI. In contrast, in sham animals c-Fos abundance is low (G). A′-I′, are higher magnifications of boxed areas in (A-I). Scale-bar (A-I) = 1 mm; (A′-I′) = 50 μm | 13

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Above, we noted that wt and SRF depleted animals slightly differed in paresis prevalence early after TBI ( Figure 2D). In order to address whether both genotypes had comparable motor and neuro-behavioral outcome in the following days after TBI, the Neurological Severity Score (NSS; Figure 2H,I) was performed. The NSS consists of 10 individual tasks and failure to complete one task is given a score of "1." Thus, animals with scores of "0" or "10" have succeeded or failed in all tests, respectively. After 1 day post-TBI the average NSS score reached 2.5 irrespective of genotype indicating an overall moderate TBI severity. When taken together all 10 tests of the NSS, mice of both genotypes recovered almost identically thereafter reaching an NSS of 1.5 at 5 days post-TBI (two-way ANOVA with TMC: P ≤ .06; F time × group = 1.96; Figure 2H). However, we noted that wt and Srf mutant mice after TBI differed in individual NSS tests ( Figure 2I). For instance, wt mice after TBI more often failed to exit the circle ( Figure 2I; see also Figure 5G). In contrast, the percentage of Srf mutant mice having paresis at 3 and 5 days post-TBI and failing the round stick balancing was elevated ( Figure 2I).
Overall, our data suggest that SRF deficiency results in higher mortality immediately after TBI. However, after TBI there was no major difference with regard to weight loss and overall NSS performance.

| Neuroinflammation and neurodegeneration after TBI is not affected by SRF deficiency
The mechanical impact during TBI induces morphological and anatomical changes to neurons, particularly in cortical layers. This is accompanied by a neuroinflammatory response resulting in astrocyte and microglia activation. 49 To find out whether SRF ablation affects those processes we performed histology of wt and Srf mutant mice at 7 days postinjury (Figures 3 and 4).
Activation of brain resident immune cells such as astrocytes and microglia was analyzed with GFAP and Iba1 directed antibodies, respectively ( Figure 3). Indeed, a statistically significant astrocyte and microglia activation in the cortex of TBI compared to sham-treated wt animals was noted (P = .012 for astrocytes in area "1"; .017 for microglia in area "1"; Kruskal-Wallis test with DMC; Figure 3E,F). Similarly to wt TBI-treated animals, both glial cell types were activated in SRF deficient animals after TBI (P = .09 for astrocytes in area "1"; .049 for microglia in area "1"; Kruskal-Wallis test with DMC; Figure 3E,F). These data suggest no overt interference of SRF with immune cell activation after 7 days of TBI.
We further corroborated these findings by quantification of chemokine and interleukin mRNA abundance 1 hour after TBI in the cortex (Figure 3G-I). TBI upregulated mRNA levels of Ccl2 ( Figure 3G), Ccl3 ( Figure 3H) and interleukin 1 beta (Il1b; Figure 3I) in wt and to a similar extent in Srf mutant animals. This further underlines no major function of SRF in regulating the TBI associated immune response within the first 7 days post-TBI.
The lesion size and neuron loss in the cortex was assessed by NeuN directed antibodies recognizing all neuronal cell bodies ( Figure 4A-D,M,N). TBI induced a cortical lesion (arrows in Figure 4C,D) of approximately 0.5-0.7 mm 2 in the cortex irrespective of the genotype ( Figure 4M). Since the actual lesion center is devoid of neurons (see Figure 4C,D) we quantified neuronal loss at 7 days post-TBI in direct vicinity to this lesion core (see dashed circle in Figure 4C). At this position there was a statistically significant reduction in NeuN positive neurons of approximately 20% in both wt and Srf mutant TBI treated animals compared to intact cortices of sham-treated animals (P = .007 for wt TBI vs wt sham and P = .049 for for Srf ko TBI vs Srf ko sham; Kruskall-Wallace with TMC; Figure 4N). Besides neuronal cell loss, mechanical injury during TBI induces axonal degeneration. 30 We used the established degeneration marker Smi32 recognizing non-phosphorylated neurofilament H that is upregulated in conditions of axonal degeneration ( Figure 4E-H,O). Here, we focused on the cerebral peduncle, an axonal trajectory connected to cortical neurons. In sham-treated animals of either genotypes, no signs of axonal degeneration were expectedly observed ( Figure 4E,F,O). In contrast, 1 week after TBI, Smi32 levels were highly upregulated in axons of wt and Srf mutant animals ( Figure 4G,H,O). As observed for neuronal loss ( Figure 4A-D,M,N) there was no difference between wt and mutant animals indicating no obvious modulation of neuronal and axonal degeneration after TBI by SRF.
Finally, we analyzed whether cofilin, an actin severing factor whose activity depends on SRF 50,51 was modulated by TBI ( Figure 4I-L,P). For this, P-cofilin abundance--indicative of inactivated cofilin-was measured in the cerebral peduncle of sham-and TBI-treated animals. In wt animals, cofilin was not phosphorylated in both sham-and TBI-treated animals suggesting no obvious regulation of this actin regulator by TBI ( Figure 4I,K,P) as also seen for acute stress exposure. 33 In opposite to this, P-cofilin levels were significantly upregulated upon SRF deletion (P = .67 for wt sham vs Srf ko sham and P = .0017 for wt TBI vs Srf ko TBI; Kruskall-Wallace with DMC; Figure 4J,L,P). This suggests cofilin inactivation by SRF ablation before and also 7 days after TBI.

| SRF ablation induces a hyperactivity phenotype after TBI
Others and we previously described hyperactivity, memory impairments, and decreased anxiety in Srf mutant mice including the mouse strain used in this study. 33,37,40 Independently of these reports, experiments performed in this study confirmed hyperactivity and less anxiety in the open field (OF) test before TBI ( Figure 5A-F). Thus, before TBI SRF deficient animals almost ran twice the distance in the OF compared to wt animals (P = .0001 for; Mann-Whitney test; Figure 5A). Since TBI typically induces hypoactivity early after TBI we investigated the locomotor activity of wt and Srf mutant animals daily in the OF over 7 days post-TBI ( Figure  5B). We observed that wt TBI-treated animals reduced locomotor activity at 6 hours, 1, and 2 days after TBI to about half the activity measured pre-TBI (two-way ANOVA with TMC or DuMC: P ≤ .0001; F time × group = 2.75; Figure 5B). In part, this hypoactivity was also observed in wt sham-treated animals which particularly at later timepoints might be due to habituation to the daily encounter of the OF environment as shown before. 52 In contrast, sham-treated Srf mutant animals showed less habituation and revealed rather constant activity scores along the entire 8 days of measurement (red dashed line Figure 5B). The most striking changes were observed in TBI-treated Srf mutant animals. First of all, in the early timepoints after TBI (ie, 6 h, 1, and 2 d) they did not reveal the hypoactivity observed in wt animals. Second, at later timepoints post-TBI (ie, 4-7 days post-TBI), Srf mutant animals even further increased their initial pre-TBI hyperactivity and now reached track lengths statistically significantly exceeding those measured pre-TBI (eg, adjusted P = .011 for Srf ko TBI vs Srf ko sham at 6d; Two-Way ANOVA with TMC; eg, P = .01 for Srf ko post-TBI vs Srf ko pre-TBI at 6d; Two-Way ANOVA with DuMC; with a statistical power of 99.9%; Figure 5B). For instance, at 5 and 6 days post-TBI, the track length of Srf mutant animals was elevated by approximately 35%-40% compared to baseline levels. Thus, SRF ablation results in hyperactivity already before brain jury and this is further augmented by TBI.
Notably, before TBI, prospective sham (black dots with gray border) and TBI-treated (black dots) wt and Srf mutant animals were indistinguishable in OF activity ( Figure 5A; see also Figure 5C for center visits). Thus, the pre-TBI hyperactivity did not differ between designated sham and TBI-treated Srf mutant groups and therefore cannot account for enhanced hyperactivity in TBI-treated Srf mutant animals.
In the OF, elevated numbers of entries in the arena center zone are indicative of decreased anxiety. 52 Similar to locomotor activity ( Figure 5A,B) we observed that TBI-treated Srf mutant animals had more center visits pre ( Figure 5C) and also at various timepoints post-TBI ( Figure 5D,F) compared to the other three experimental cohorts. This was observed at early timepoints after TBI (ie, 6 h, 1, and 2 d) and also at the later timepoints, that is, 3-7 days after TBI (two-way ANOVA with TMC: P = .45; F time × group = 1.00; Figure 5D,F). However, at later timepoints mice might habituate to the OF challenge due to the daily testing and therefore interpretations regarding anxiety should be taken with some caution for these later timepoints.
The observed hyperactivity phenotype was confirmed by additional tests including open circle exit ( Figure 5G) and ladder walk ( Figure 5H,I). In the open circle, part of the NSS ( Figure 2H, I), we noticed that wt TBI-treated animals required approximately 100 seconds to exit the circle (two-way ANOVA with TMC: P = .065; F time × group = 1.92; Figure 5G). In contrast, Srf mutants needed significantly less time resulting in circle exit after approximately 10 seconds ( Figure 5G). In the ladder walk, TBI elevated the number of slips for wt and Srf mutant TBI animals almost identically ( Figure 5H). However, once again a hyperactivity phenotype became apparent when stopping the traversing time ( Figure  5I). Immediately after TBI (ie, at 4 h), animals of both cohorts needed the same time to cross the ladder ( Figure 5I). However, at later timepoints after TBI, Srf mutant animals were faster and reached levels comparable to sham-treated animals of either genotype although statistical significance was not reached ( Figure 5I).
In summary, SRF deficient mice show altered locomotor and anxiety related behavior after TBI.
For this, hippocampal ( Figure 6A-L) or cortical ( Figure  6M) tissue of wt and SRF deficient animals was subjected to qPCR at 1 hour after TBI. As control we included contralateral hemispheres of TBI animals as well as sham-treated animals ( Figure 6A-L). In general, mRNA levels of all genes tested were low in these control tissues and no obvious difference between genotypes was observed. In contrast β-actin, a known SRF target gene, was highly expressed in control and TBI treated hippocampi and mRNA levels were significantly reduced upon SRF deletion (P = .014 for Srf ko TBI vs wt TBI; Mann-Whitney test; Figure 6I).
Besides β-actin all other genes investigated were robustly upregulated by TBI in wt mice. In contrast, we noted that several neuronal activity regulated genes were significantly less induced in Srf mutant hippocampi including Egr1, Egr2, Egr3 (P = .001, .001, .008, respectively Mann-Whitney test; Figure  6A-C), Npas4, Arc and Atf3 (P = .035, .001, .05, respectively Mann-Whitney test; Figure 6D-F). We also analyzed two novel SRF target genes, neuronal pentraxin 2, 53,60 a gene involved in enhancing synaptic inhibition, 61 and cyclooxygenase 2 (Ptgs2; Figure 6G), a gene regulated by SRF in muscles to regulate inflammation. 54 Interestingly, both genes revealed a significant SRF dependency and were only weakly induced by TBI in SRF deficient animals (P = .005, .014, respectively Mann-Whitney test; Figure 6G, H). In opposite to the aforementioned genes, we noted that Fos and Jun family members were induced by TBI in an SRF independent manner ( Figure 6J-L).
We also analyzed mRNA levels in the cortex ( Figure 6M) and observed a similar tendency as described in the hippocampus above. Here, significant SRF-mediated gene induction by TBI for wt versus Srf mutant cortices was achieved for Npas4, Arc, Ptgs2, cFos, and Nptx2 (P = .008, .035, .001, .014, .035, respectively; Mann-Whitney test; Figure 6M). For other genes, that is, Egr1, Egr2, and Egr3 a similar SRF dependency was observed as in the hippocampus, however without reaching significance (P = .37, .53, .14, respectively; Mann-Whitney test; Figure 6M).
For gene expression we included male and female mice (black and gray dots, respectively; Figure 6). As with mortality (Figure 2), gender did not obviously alter the TBI outcome since TBI-induced gene transcription gave similar results in male and female animals ( Figure 6).
Finally, qPCR data were corroborated on protein level for the three potential SRF targets Egr1, ATF3 and c-Fos ( Figure 7). Indeed, we noted that 2 hours after TBI, EGR1, and ATF3 were induced in the hippocampus and cortex of wt mice ( Figure 7B,E) compared to sham animals ( Figure  7A,D). In the absence of SRF, the protein abundance of EGR1 and ATF3 was reduced after TBI ( Figure 7C,F). For example, in the upper leaf of the dentate gyrus granule cell layer 2.87 ± 1.75% of the area was EGR1 positive whereas only 0.35 ± 0.43% of the area was positive in Srf mutant animals (for ATF3: wt TBI: 4.00 ± 5.53% vs Srf ko TBI: 0.99 ± 1.45%). As seen for mRNA ( Figure 6J), c-Fos was comparably induced in both wt and SRF deficient TBI animals ( Figure 7H, I; wt TBI: 25.5 ± 6.03% vs Srf ko TBI: 19.85 ± 7.4%) compared to only weak c-Fos levels found in wt sham animals ( Figure 7G).
Taken together, we observed an SRF-dependent induction of several neuronal activity encoding genes after TBI.

| DISCUSSION
TBI triggers glutamate release resulting in neuronal dysfunction accompanied by enhanced network activity. 62,63 Here, we investigated SRF, a neuronal activity-driven TF in the context of TBI. SRF deletion affected the TBI outcome on three levels: i) mortality, ii) behavior, and iii) gene expression.

| SRF is a neuroprotective factor in TBIassociated mortality
TBI is a leading cause of mortality in young adults. 2 Herein, we observed a fourfold to sixfold elevated mortality upon SRF ablation compared to wt mice ( Figure 2). This was irrespective of gender and provides data also for female animals typically neglected in many studies mainly focusing on males only. So far, only few animal studies identified molecules affecting TBI induced mortality. Repression of neuronal NF-κB signaling increases the acute posttraumatic mortality rate to 40% 47 thus reaching similar rates as observed for Srf mutants in this study (Figure 2). In contrast, other mouse mutants for example, of Atf3, 35 p53, 64 complement receptor CR2, 65 TNF receptors, 66 Nogo receptor, 67 or neurodegeneration-inducing superoxide dismutase 68 affected mortality not at all or only by few percent. Thus, in relation to these factors, SRF emerges as strong modifier and protective factor of TBI-associated mortality in wt mice.
In our experiments, death occurred within 5 minutes after TBI. Given this short time window, it is unlikely that SRF confers protection from mortality via direct gene transcription since de novo mRNA production and subsequent protein translation might take too long to intervene in this protective mechanism. The only obvious correlation our data provide is a connection between the high mortality rate and the pre-TBI hyperactivity in Srf mutant mice ( Figure 5A). The existing literature does not provide any obvious connection between hyperactivity and TBI-associated mortality in animal models or human patients. Thus, our data on SRF provide a first precedence and, although speculative at this stage, suggest that hyperactivity most likely associated with neuronal network changes (see below) might be a confounding factor in TBIassociated mortality.

| Induction of a post-TBI hyperactivity phenotype upon SRF ablation in mice
Previous reports showed that SRF deletion in glutamatergic 33,69 and dopaminoceptive 37 neurons produces hyperactivity in mice without any injury or stimulation. In this study we observed that brain injury by TBI further augmented hyperactivity in Srf mutant whereas wt mice responded with hypoactivity ( Figure 5). Of note the "basal" hyperactivity in Srf mutant mice in the absence of any exogenous injury/stimulus ( Figure 5A) is modulated differently depending on the subsequent stimulus type: in TBI, the initial hyperactivity was further enhanced ( Figure 5) whereas after acute stress, Srf mutants reacted with hypoactivity. 33 This discrepancy might reflect differential involvement of brain regions in mechanical injury versus stress processing. In any case, this basal hyperactivity in Srf mutants ( Figure 5A) has to be taken carefully into account when interpreting data on Srf mutant mice additionally challenged with stress or TBI.
The higher locomotor activity after TBI suggests that SRF ablation might actually enhance recovery after TBI. Indeed, locomotor activity particularly of those Srf mutants starting with an immediate post-TBI paresis (Figure 2) accelerated very quickly. Thus, for selected aspects of post-TBI regeneration such as locomotor activity, SRF deletion might be beneficial. However, SRF might exert a dual function in TBI since-as discussed above-SRF dysfunction clearly raises the risk in mice to die immediately after TBI. Besides IEGs (discussed below), one downstream effector of SRF possibly mediating hyperactivity during TBI is the actin severing factor cofilin (Figure 4). Cofilin mouse mutants display a hyperactivity phenotype 70 similar to cofilin inhibition achieved in Srf mutant mice in our study ( Figure 4).
As previously discussed by others, 37 the hyperactivity observed in Srf mutants recapitulates one aspect of the wide behavioral spectrum often altered in ADHD patients. A mutual interaction between ADHD in patients and TBI is established 6 and ADHD is frequently a sequel of brain injury. 6 Clearly, ADHD patients present several behavioral alterations which most likely cannot all be recapitulated properly in animal models. Nevertheless, work in rodents by others reported induction of hyperactivity 3 weeks after a mild TBI 71 whereas at earlier timepoints post-TBI (ie, first 3-5 days) wt animals still showed hypoactivity 71 ( Figure  5B). In summary, our data suggest, although speculative currently and with the caveat that Srf mutants were hyperactive already without TBI ( Figure 5A and see above), a role of SRF as a potential molecular link between TBI and ADHD.

| SRF is a novel upstream gene regulator for TBI-associated induction of neuronal activity related genes
Neuronal activity results in IEG upregulation. 23 For instance, cFos is frequently used as surrogate markers to identify neurons activated by neurotransmitters in physiological but also pathologic conditions. 23 In this study, SRF was identified as upstream regulator of several IEGs including Egr1, Egr2, Egr3, Npas4, Arc, and Atf3 but also other target genes, Ptgs2 and Nptx2, so far not typically annotated as IEGs (Figures 6 and 7). Of note, Ptgs2 encoding a prostaglandin synthase (COX2) mediates the brain injury related neuroinflammation. 72 Ptgs2 induction was compromised in Srf mutants (Figure 6), however no obvious changes in neuroinflammation in wt versus Srf mutant TBI-affected brains were observed ( Figure 3). Interestingly, COX2 downregulation also affects locomotor activity in rodents. Thus, similar to SRF-dependent Cox2 mRNA downregulation ( Figure 5), pharmacological COX2 inhibition, correlated with increased locomotor activity in a rat posttraumatic stress disorder (PTSD) model. 73 In summary, SRF deletion resulted in failure to induce an entire gene set of neuronal activity encoding genes 1 hour after TBI. This 1 hour timepoint after TBI fits well with the proposed rise in neuronal activity triggered by TBI early after the impact. 10 These SRF regulated genes fall into two classes, i) genes encoding TFs (Egr1, Egr2, Egr3, ATF3, Npas4) thereby eliciting a second gene expression wave and ii) putative "effector" genes with direct impact on cellular processes such as Nptx2 and Arc. For the TF encoding SRF target genes we noted that Fos family members were largely SRF independently regulated during TBI (Figures 6 and 7), a finding in agreement with previous reports. 37,52 In contrast, during LTP and seizure propagation cFos was regulated in an SRF-dependent manner. 34,38,39 Thus, cFos and family members appear as facultative SRF target genes and other TFs such as CREB might have a stronger impact. 60 In contrast, Egr1, Egr2, Egr3, and also Npas4 appear to be more constitutively SRF dependent for a wide range of in vivo stimuli. 34,52 How might SRF-dependent gene expression during TBI be connected to the hyperactivity phenotype observed in Srf mutant mice? In one possible scenario, TBI results in an immediate increase in neuronal activity in wt brains. This activates SRF and in turn results in a first gene expression wave of TF encoding IEGs such as Egr1/2/3, Atf3 and Npas4 and "effector" genes such as Nptx2 and Ptges2. Npas4 was reported to trigger a delayed gene response promoting inhibition onto excitatory neurons while inducing excitation onto inhibitory neurons. 74,75 Similarly, Nptx2 is involved in enhancing feedforward inhibition on neuronal networks. 61 Overall, although not shown in this study, these SRF target genes could enhance mechanisms of neuronal inhibition during TBI and thereby limit hyperactivity related behavior. In keeping with this, mouse mutants of several of these IEGs including Egr3, Nptx2, Ptgs2, and Npas4 also induce hyperactivity 55-59 suggesting a function for the wt proteins in restricting hyperactive behavior.
Upon SRF deficiency, a "composite" IEG mouse mutant phenotype would be induced, affecting expression of several IEGs (Figures 6 and 7). Such impaired induction of, for example, Npas4 and Nptx2 would fail to elevate neuronal inhibition after TBI, thereby shifting the balance from inhibitory to excitatory neuronal transmission which might facilitate together with failure to upregulate hyperactivity repressing IEGs such as Egr3, Nptx2, Ptgs2, and Npas4 (see above) development of a hyperactive phenotype. Of note, the | 17 FÖRSTNER aNd KNÖLL hippocampal IEG response after TBI was in general stronger compared to the cortex ( Figure 6) suggesting a stronger hippocampus contribution to post-TBI behavioral phenotypes such as hyperactivity observed in this study. In support, a previous study in Srf mutant mice emphasized the importance of the hippocampus over the cortex for habituation in the OF. 40 Further studies will have to address this mechanism in more detail, but our data provide a first molecular link between TBI and hyperactivity related behaviors via a signaling cascade involving SRF and neuronal activity encoding genes.