Abstract
Neuronal degeneration following traumatic brain injury (TBI) leads to intracellular accumulation of dysfunctional proteins and organelles. Autophagy may serve to facilitate degradation to overcome protein debris load and therefore be an important pro-survival factor. On the contrary, clearing may serve as pro-death factor by removal of essential or required proteins involved in pro-survival cascades. Sequestosome 1 (SQSTM1/p62) is a main regulator of the autophagic pathway that directs ubiquinated cargoes to autophagosomes for degradation. We show that SQSTM1 protein levels are suppressed 24 h and by trend 5 days after trauma. In line with these data the expression of Sqstm1 mRNA is reduced by 30% at day 3 after and stays depressed until day 5 after injury, indicating an impaired autophagy post controlled cortical impact (CCI). To determine the potential role of SQSTM1-dependent autophagy after TBI, mice lacking SQSTM1 (SQSTM1-KO) and littermates (WT) were subjected to CCI and brain lesion volume was determined 24 h and 5 days after insult. Lesion volume is 17% smaller at 24 h and immunoblotting reveals a reduction by trend of cell death marker αII-spectrin cleavage. But there is no effect on brain damage and cell death markers 5 days after trauma in SQSTM1-KO compared with WT. In line with these data neurofunctional testing does not reveal any differences. Additionally, gene expression of inflammatory (Tnf-α, iNos, Il-6, and Il-1β) and protein degradation markers (Bag1 and Bag3) were quantified by real-time PCR. Protein levels of LC3, BAG1, and BAG3 were analyzed by immunoblotting. Real-time PCR reveals minor changes in inflammatory marker gene expression and reduced Bag3 mRNA levels 5 days after trauma. Immunoblotting of autophagy markers LC3, BAG1, and BAG3 does not show any difference between KO and WT 24 h and 5 days after TBI. In conclusion, genetic ablation of SQSTM1-dependent autophagy leads to a delay but shows no persistent effect on post-traumatic brain damage formation. SQSTM1 therefore only plays a minor role for secondary brain damage formation and autophagic clearance of debris after TBI.
Introduction
Immediately after impact, a complex series of biochemical events is initiated by traumatic brain injury (TBI) resulting in expansion of the primary lesion to cause secondary injury (Giza and Hovda, ). Destruction of brain tissue leads to an accumulation of dysfunctional organelles and protein debris in the lesion core and perilesional area, which alters functional integrity and triggers cerebral inflammation (Chen et al., ). Clearance of protein debris and restoration of homeostasis is of central importance to maintain normal integrity and function of the brain (Mochida et al., ). Autophagy (macroautophagy) is a sequestration system which facilitates fast identification and degradation of dysfunctional protein aggregates (Levine and Kroemer, ) and takes part in multiple physiological functions (e.g., programmed cell death and repair mechanisms) (Kuusisto et al., ; Mizushima et al., ; Weidberg et al., ). Degraded protein complexes are identified by receptors (co-chaperons) and are encapsulated in double membrane vesicles (autophagosomes). Autophagosomes fuse with lysosomes forming the autophagolysosomes for final degradation.
Sequestosome 1 (SQSTM1), also known as p62, is an ubiquitin-binding scaffold protein, which recognizes ubiqinated degradation prone substrates. It is one of the specific substrates degraded by the autophagic pathway. During autophagy, SQSTM1 localizes to autophagosomes by binding to microtubule-associated light chain 3 (LC3) and promoting selective autophagy of proteins in e.g., aged and acutely stressed neurons (Ichimura et al., ; Gamerdinger et al., ; Lamark et al., ; Rusten and Stenmark, ). After formation of the autolysosome, LC3 disassociates, whereas SQSTM1 and attached ubiquitin shuttles are degraded by lysosomal hydrolases (Klionsky and Emr, ). Importantly, SQSTM1 is a known marker for autophagic flux activity. In pathological settings like aging and disease, SQSTM1 accumulates as result of failed autophagic clearance (Moscat et al., ; Evans et al., ). Stimulation of autophagy flux causes degradation of SQSTM1 (Sarkar et al., ). In posttraumatic tissue signs of impaired autophagy have been observed, but molecular mechanism and consequences for brain damage formation remain controversial (Luo et al., ; Lipinski et al., ). Autophagy may be an important pro-survival factor by enabling brain tissue to overcome protein debris load. On the other hand, autophagy may serve as pro-death factor by clearing essential or required proteins involved in pro-survival cascades.
The present study investigates if an uncoupling of the SQSTM1 dependent drive of autophagy by use of SQSTM1 deficient animals is important for the formation of brain damage and impairment of neurological function after experimental TBI.
Materials and methods
Experimental animals
After approval by the Landesuntersuchungsamt Rheinland-Pfalz (protocol number: G14-1-037 and G12-1-010) experiments were performed in compliance with the institutional guidelines of the Johannes Gutenberg University, Mainz and in accordance with the German law for animal protection. Homozygous p62/SQSTM1 (Sqstm1tm1a(EUCOMM)Wtsi) mutant mice (SQSTM1−/−) with a neomycin cassette insertion on chromosome 11 on a C57/BL6 background and corresponding wildtype littermates (SQSTM+/+) were investigated (Komatsu et al., ). Animals were kept under standard conditions of temperature and light with free access to water and food. The food that the animals received was a standard commercial regular rodent diet.
Experimental TBI
Under anesthesia with isoflurane (induction: 4 Vol%, maintenance: 2 Vol%) in an air mixture (40% O2 and 60% N2) via face mask, moderate focal mechanical TBI was performed by controlled cortical impact (CCI) on the right brain cortex as described previously (Sebastiani et al., ). Following placement into a stereotactic frame a craniotomy was performed to remove a part of the scull. A localized contusive impact was delivered to the exposed dura by a custom fabricated pneumatic controlled impactor (L. Kopacz, Mainz, Germany) which was placed perpendicular to the surface of the brain. The following variables were applied: tip diameter of 3 mm, brain penetration of 1.0 mm, impact duration of 150 ms, and impact velocity of 8 m/s. After trauma, the craniotomy was immediately sealed and the wounds were sutured closed. During surgical preparation, body temperature was maintained at 37°C by a heating plate, autoregulated, and monitored by a rectal thermocouple probe (Hugo Sachs, March-Hugstetten, Germany). Animals were returned to their own cages and placed in an incubator (33°C, 35% humidity; IC8000, Draeger, Germany) for 2 h.
Histological evaluation of brain damage
Animals were euthanized in deep isoflurane anesthesia (4 Vol% for 1 min). Brains were quickly removed, frozen in powdered dried ice, and stored at −20°C. Each brain was cut in the coronal plane using a cryostat (HM 560 Cryo Star, Thermo Fisher Scientific, Walldorf, Germany). Sections (10 μm) were serially collected at 500 μm intervals and stained with cresyl violet according to the manufacturer's instruction. Areas of both hemispheres and the injured brain tissue were measured using a computerized image system (Delta Pix Insight, Delta Pix, Maalov, Denmark) by an investigator blinded to the randomization. Lesion volumes were calculated by multiplying contusion areas obtained from 16 consecutive sections with the distance-interval of 500 μm (0.5 * [A1 +A2 +A3 +…+ An]).
Motor function
Motor function was analyzed by the rotarod test as described previously by an investigator blinded to the group allocation (Onyszchuk et al., ; Sebastiani et al., ). Mice were tested before, 1 and 5 days after TBI. The latency to balance until fall from the rod was recorded using a five-lane rotarod device (Panlab Rota Rod, Harvard Apparatus, Holliston, MA). For the acceleration, the speed was linearly increased from 4 to 40 rpm over 5 min. The investigation ended when the mice fell off the rod. Four rotarod tests were performed before TBI to score the baseline latencies for each animal. The average of these trials was taken as the baseline. After injury, animals were tested in two averaged trials per investigated time point.
Neurological severity score
Before and after trauma neurological outcome was tested by an investigator blinded to the group allocation by a neurological severity adapted from Tsenter et al. () which consists of 10 different tasks. These tasks are evaluating the alertness, motor ability, balancing, and general behavior of mice. Healthy mice were successful in all tasks and received 0 points. For failure to successfully perform a task 1–3 points were awarded (see Table 1).
Table 1
| Task | Points | |
|---|---|---|
| Presence of a mono- or hemiparesis | 1 | |
| Failure to walk a straight line | 1 | |
| Startle behavior | 1 | |
| Seeking behavior | 1 | |
| Balance on a 1 cm-wide beam | 1 | |
| Balance on a 0.5 cm-round beam | 1 | |
| Exit a 25 cm-diameter wide circle | 30–60 s | 1 |
| 60 s–2 min | 2 | |
| >2 min | 3 | |
| Walk on a 3 cm-wide beam | >3 feet misplacement | 1 |
| Inability to move | 2 | |
| Walk on a 2 cm-wide beam | >3 feet misplacement | 1 |
| Inability to move | 2 | |
| Walk on a 1 cm-wide beam | >3 feet misplacement | 1 |
| Inability to move | 2 | |
| Maximum total | 15 |
Tasks of the adapted neurological severity score.
RNA extraction and real-time polymerase chain reaction
Tissue preparations were performed as follows: For the time series analysis, brains were removed and placed into a cooled brain matrix (Zivic Instruments, Pittsburgh, PA). Perilesional brain tissue was dissected and immediately frozen in liquid nitrogen. For the 24-h and the 5-day studies perilesional brain tissue was collected during the cryosectioning process. Tissue was stored at −80°C until RNA isolation. Samples were homogenized in QIAzol® reagent (Qiagen). RNA isolation was performed with RNeasy® Lipid Tissue Mini kit (Qiagen) according to manufacturer's instructions. Absolute copy numbers of target genes were normalized against the housekeeping gene cyclophilin A (PPIA) (Thal et al., ). For applied primer sequences see Table 2. Same amounts of cDNA were amplified in duplicates using Absolute Blue qPCR SYBR Green Mix (Thermo fisher Scientific) for Ppia, Tnf-α, inos, and Sqstm1, Maxima Probe qPCR Mastermix (Thermo Fisher Scientific) for Il-1β and Bag3, Light Cycler 480 Probes Master (Roche) for Bag1, and Quanti Nova qPCR kit (Qiagen) for IL-6 according to the manufacturer's instructions.
Table 2
| PCR assay (amplicon size, annealing temp) | Oligonucleotide Sequence (5′–3′) | Gene bank no. |
|---|---|---|
| Cyclophilin A (PPIA) (146 bp, 55°C) | Forw: 5′-GCGTCTSCTTCGAGCTGTT-3′ Rev: 5′-RAAGTCACCACCCTGGCA-3′ FL: 5′-GCTCTGAGCACTGGRGAGAAAGGA-FL Cy5: Cy5-TTGGCTATAAGGGTTCCTCCTTTCACAG-Phos | NM_008907 |
| Bag1 (306 bp, 55°C) | Forw: 5′-CACCCACAGCAATGAGAGGTAT-3′ Rev: 5′-AATTCTTGCAGGTGGTTAGCTATC-3′ FL: 5′-GGGAAAATCTCTGAAAGAAATGGAAACACC-FL Cy5: Cy5-TTGTCAGCACTTGGAATGCAAAATGGT-Phos | NM_001171739 |
| Bag3 (269 bp, °C) | Forw: 5′-CACCACGACGTGGAACG-3′ Rev: 5′-GGGACCTCTGCGGAGTG-3′ FL: 5′-GCTCCGACCAGGCTACATTCCCA-FL Cy5: Cy5-CCCCGTCCTCCATGAAGGCTCCG-Phos | NM_013863 |
| Sqstm1 (292 bp, 55°C) | Forw: 5′-CTCAGCCCTCTAGGCATTGAG-3′ Rev: 5′-AAGACAAATGTGTCCAGTCATCGT-3′ FL: 5′-CCTTGGAGTCGGTGGGACAGCC-FL Cy5: Cy5-GAACAGATGGAGTCGGGAAACTGCTCA-Phos | NM_011018 |
| IL-1β (348 bp, 55°C) | Forw: 5′-59-GTGCTGTCGGACCCATATGAG-3′ Rev: 5′-CAGGAAGACAGGCTTGTGCTC-3′ FL: 5′-TAATGAAAGACGGCACACCCACCC-FL Cy5: Cy5-CAGCTGGAGAGTGTGGATCCCAAGC-Phos | NM_008361 |
| IL-6 (141 bp, 55°C) | Forw: 5′-GAGGATACCACTCCCAACAGACC-3′ Rev: 5′-AAGTGCATCATCGTTGTTCATACA | NM_031168 |
| TNF-α (212 bp, 62°C) | Forw: 5′-TCTCATCAGTTCTATGGCCC-3′ Rev: 5′-GGGAGTAGACAAGGTACAAC-3′ | NM_ 008361 |
| iNOS (NOS2) (312 bp, 55°C) | Forw: 5′-TGTGTCAGCCCTCAGAGTAC-3′ Rev: 5′-CACTGACACTYCGCACAA-3′ R640: Red-GCTCCTCCCAGGACCACACCC-Phos FL: 5′-GAAGCCCCGCTACTACTCCATC-FL | NM_010927 |
Specific primer and probes and optimized temperature conditions for real-time polymerase chain reaction (PCR, polymerase chain reaction; Forw, sense primer; Rev, antisense primer; Cy5, Cyanine 5; Phos, Phosphate; FL, fluorescein).
Immunoblotting
Perilesional brain tissue was homogenized in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.5 mM EDTA; 0.5% NP-40; protease and phosphatase inhibitors). Protein concentration was determined by Lowry Protein Assay (Promega, Madison, WI, USA) and 40–50 μg of each sample were separated in 12%-SDS polyalcryamide gel electrophoresis (SDS-PAGE), for spectrin and SQSTM1 in 4–12% NuPAGE (4–12% Bis-Tris Protein Gel, 1.5 mm; Thermofisher Scientific, Waltham, MA, USA), respectively, and transferred to a nitrocellulose membrane. After blocking with 5% skimmed milk for 1.5 h at room temperature and washing with Tris-buffered saline with 1% Tween-20 (Carl Roth, Karlsruhe, Germany) primary antibodies against SQSTM1 (1:1,000; GP62-C, Progen, Heidelberg, Germany), BAG3 (1:500, 10599-1-AP, Proteintech, Rosemont, IL, USA), BAG1 (1:500, aliquot kindly provided by Prof. Dr. Franz-Ulrich Hartl, Max-Planck-Institute of Biochemistry, Martinsried, Germany), spectrin (1:750, BML-FG6090, Enzo Life Science, Farmingdale, NY, USA), and LC3 (1:1,000, NB100-2220, Novus Biologicals, Littleton, CO, USA) or GAPDH (1:4,000, ACR001PS, Acris, Rockville, MD, USA) as loading control were applied in TBST or 2.5% skimmed milk (BAG3, Spectrin, GAPDH) and incubated overnight at 4°C, respectively, 2 h at room temperature (GAPDH). Secondary Antibodies against mouse, rabbit or guinea-pig (Li-Cor Bioscience, Lincoln, NE, USA) were applied for 1 h at room temperature. After washing protein bands were detected using Odyssey Imaging Systems and their intensities were measured with Image Studio Version 3.1 (both Li-Cor Bioscience, Lincoln, NE, USA) and normalized to the sample's GAPDH expression.
Statistical analysis
All experiments were randomized and performed by an investigator blinded to the group allocation. Analysis was performed by Sigma Plot 12.5 software (Systat Software Inc., San Jose, CA, USA). Exact Wilcoxon Mann–Whitney tests were used and values were adjusted for multiple comparisons with the Holm–Bonferroni method. P < 0.05 was considered to be significant. Graph bars indicate mean and standard deviation throughout the figures.
Results
SQSTM1 is reduced after experimental TBI
Time frame analysis shows that Sqstm1 mRNA expression is suppressed 3 days after trauma compared with naïve mice and stays depressed until day 5 after injury (naïve: 100.0 ± 26.8% naïve; 1 day post injury: 93.7 ± 8.9% naïve; P = 0.566 vs. naïve; 3 days post injury: 77.4 ± 7.4% naïve, P = 0.0377 vs. naïve; 5 days post injury: 71.0 ± 16.3% naïve; P = 0.0311; n = 9–10 mice/group; Figure 1A). Additionally, SQSTM1 protein levels were investigated by immunoblotting. SQSTM1 protein levels are reduced 1 day after TBI and nearly return to naïve values 5 days after trauma (naïve: 100.0 ± 17.2% naïve; 1 day post injury: 68.1 ± 9.7% naïve, P = 0.0071 vs. naïve; 5 days post injury: 85.0 ± 12.2, P = 0.042 vs. 1 day post injury; n = 5 mice/group; Figure 1B).
Figure 1
The lack of SQSTM1 delays formation of brain damage
SQSTM1 knockout is confirmed by western blot analysis in brain tissue lysates of investigated animals 24 h (P < 0.001; n = 7/group; Figure 2A) and 5 days after trauma (P = 0.008; n = 5/group; Figure 2B). Twenty four hours following trauma lesion volume is smaller in SQSTM1−/− (32.1 ± 3.5 mm3, n = 8 mice/group) compared with SQSTM1+/+ mice (38.8 ± 8.6 mm3, n = 12; P = 0.025; Figure 3A). Additionally, lesion volume was analyzed 5 days after experimental TBI. Lesion volumes in SQSTM1−/− and wildtype littermates are indistinguishable (28.8 ± 5.8 vs. 28.3 ± 8.0 mm3; P = 0.937; n = 6 mice/group; Figure 3B). To determine the influence of SQSTM1 absence on neuronal cell death, perilesional brain samples of SQSTM1−/− and SQSTM1+/+ mice were investigated 24 h and 5 days after trauma. Western blot analysis of caspase-dependent 120 kDa αII-spectrin fragment and calpain-dependent 145 kDa αII-spectrin was performed. Calpain-dependent cell death is reduced by trend in SQSTM1−/− mice compared with SQSTM1+/+ littermates 24 h after injury (P = 0.0797; n = 7 mice/group; Figure 3C). In line with the data investigating brain damage formation, there is no difference in 145 kDa αII-spectrin fragment levels 5 days after trauma between SQSTM1−/− and SQSTM1+/+ animals (P = 0.6633; n = 5 mice/group, Figure 3D). SQSTM1 absence does not influence caspase-dependent αII-spectrin cleavage 24 h (P = 0.507; n = 7 mice/group, Figure 3E) or 5 days after TBI (P = 0.9339; n = 5 mice/group, Figure 3F).
Figure 2
Figure 3
Neurological function is not affected by SQSTM1 mutation
Motor coordination was analyzed by rotarod before, 24 h and 5 days after trauma in SQSTM1−/− and corresponding SQSTM1+/+ littermates. SQSTM1 deficiency does not influence motor coordination compared with littermate animals 24 h (P = 0.065) or 5 days (P = 0.818) after TBI (n = 6 mice/group; Figure 4A). Neurological Severity Score does not reveal any differences between SQSTM1−/− and SQSTM1+/+ 24 h (P = 0.699) or 5 days (P = 0.937) after trauma (SQSTM1+/+, 24 h: 8.2 ± 3.0 points, 5 days: 5.3 ± 3.1 points; SQSTM1−/−, 24 h: 7.2 ± 1.7 points, 5 days: 5.5 ± 3.4 points, n = 6 mice/group; Figure 4B).
Figure 4
SQSTM1 deficiency influences Tnf-α and Il-6 mRNA expression after TBI
Gene expression levels of the pro-inflammatory markers iNos, Tnf-α, Il-6, and Il-1β were investigated 24 h and 5 days after trauma by real-time PCR. SQSTM1 deficiency increases inflammatory marker Tnf-α expression 24 h after head trauma (SQSTM1−/−: 4,378 ± 1,424% control; SQSTM1+/+: 6,820 ± 2,186% control, P = 0.005; n = 10–12 mice/group; Figure 5A). Five days after head trauma Tnf-α levels are lower in SQSTM1 deficient animals (SQSTM1−/−: 5,976 ± 1,501% control; SQSTM1+/+: 8,837 ± 1,643% control, P = 0.026; n = 6 mice/group; Figure 5B), whereas Il-6 expression levels are significantly higher in SQSTM1−/− animals at 5 days after insult (SQSTM1−/−: 179 ± 46% control; SQSTM1+/+: 115 ± 29% control, P = 0.015; n = 6 mice/group, Figure 5B). There is no difference between SQSTM1−/− and corresponding SQSTM1+/+ in the expression levels of other investigated markers such as iNos (24 h: SQSTM1−/−: 323 ± 82% control; SQSTM1+/+: 281 ± 75% control, P = 0.221; n = 10–12 mice/group; 5 days: SQSTM1−/−: 271 ± 37% control; SQSTM1+/+: 284 ± 23% control, P = 0.310; n = 6 mice/group), Il-6 (24 h: SQSTM1−/−: 6,250 ± 3,090% control; SQSTM1+/+: 5,108 ± 1,921% control, P = 0.646; n = 10–12 mice/group), and Il-1β (24 h: SQSTM1−/−: 3,075 ± 1,075% control; SQSTM1+/+: 2,282 ± 909% control, P = 0.061; n = 10–12 mice/group; 5 days: SQSTM1−/−: 249 ± 114% control; SQSTM1+/+: 368 ± 69 % control, P = 0.093; n = 6 mice/group; Figures 5A,B).
Figure 5
SQSTM1 deficient animals show lower Bag3, but unchanged Bag1 mRNA expression levels
In order to investigate if the potential mechanism of the early effect on lesion volume in by SQSTM1−/− is due to changes in key regulators of protein degradation, gene expression levels for Bag1 (marker of proteasomal activity) and Bag3 (mediator of the BAG3-mediated selective macro-autophagy pathway; Gamerdinger et al., ) were analyzed by real-time PCR. In SQSTM1−/− mice Bag3 expression levels are significantly lower 5 days after TBI (24 h: SQSTM1−/−: 102 ± 5% control; SQSTM1+/+: 100 ± 11% control, P = 0.589; 5 days: SQSTM1−/−: 234 ± 33% control; SQSTM1+/+: 336 ± 36% control, P = 0.002, Figures 5C,D). Bag1 expression levels are not changed by SQSTM1 deficiency (24 h: SQSTM1−/−: 248 ± 52% control; SQSTM1+/+: 225 ± 68% control, P =; 5 days: SQSTM1−/−: 113 ± 10% control; SQSTM1+/+: 119 ± 9% control, P = 0.485; Figures 5C,D).
Autophagy and proteasomal degradation is not influenced by SQSTM1 deficiency
In order to confirm the results of mRNA analysis, the impact of SQSTM1 deficiency on protein degradation marker levels was investigated by western blot of perilesional brain tissue lysates in SQSTM1−/− and SQSTM1+/+ littermates 24 h and 5 days after trauma. Autophagy markers LC3 (24 h: P = 0.677; n = 8 mice/group; 5 days: P = 0.771, n = 6 mice/group; Figures 6A,B) and BAG3 (24 h: P = 0.610; n = 8 mice/group; 5 days: P = 0.561, n = 6 mice/group; Figures 6E,F) and proteasomal activity marker BAG1 (24 h: P = 0.354; n = 8 mice/group; 5 days: P = 0.230, n = 6 mice/group; Figures 6C,D) protein content is unchanged in the absence of SQSTM1.
Figure 6
Discussion
Several studies have identified the importance of dysregulated autophagy in different human diseases. The ubiquitin-binding protein SQSTM1 was identified as key component of the selectivity of the autophagy network. The present study investigates the role of SQSTM1-mediated autophagy for brain damage formation after experimental TBI. Our study demonstrates (I) reduced Sqstm1 mRNA expression and SQSTM1 protein levels indicating increased autophagy flux after TBI and (II) that absence of SQSTM1 leads to delayed but no long term effect on brain damage formation after TBI.
Depending on the situation autophagy may serve as pro-survival or pro-death mechanism. Autophagy is constitutively present at low levels in all healthy neurons (Rubinsztein et al., ). A dysregulation of this degradation pathway has been reported to occur in many neurodegenerative processes like post-traumatic stress disorder (Zheng et al., ), cerebral ischemia (Xu et al., ), Parkinson's disease (Wang et al., ), or Alzheimer's disease (Kuusisto et al., ).
The ubiquitin receptor SQSTM1 is an important player of the autophagic pathway. Recent findings suggest that SQSTM1-dependent autophagy negatively regulates important signaling pathways (Niida et al., ; Sandilands et al., ), key downstream components and transcription factors (Wang et al., ) changes in stress conditions (Belaid et al., ). It directly interacts with LC3 and is incorporated and sequestered within an autolysosome for degradation and thereby acts an autophagy substrate (Pankiv et al., ). Thus, SQSTM1 is used to evaluate the autophagic flux (Sahani et al., ; Geng and Klionsky, ). Vice versa, autophagy is responsible for the degradation of SQSTM1. Hence, an impairment of autophagy is usually accompanied by an accumulation of SQSTM1 protein (Komatsu and Ichimura, ; Katsuragi et al., ). In the present study Sqstm1 mRNA expression and SQSTM1 protein levels decrease after trauma compared with the naïve group indicating an induction of autophagy flux (Ichimura et al., ). Decreased Sqstm1 mRNA expression levels following insult have also been shown for other brain pathologies like hypoxic-ischemic brain injury (Xu et al., ). In contrast to the present data, other studies have shown accumulation of SQSTM1 following experimental TBI compared with sham animals (Sarkar et al., ). Consistently, in a study investigating the cerebrovascular fluid of 30 children with severe TBI, increased SQSTM1 protein levels where detected in the cerebrovascular fluid after TBI compared to a control group. In this study, peak levels of SQSTM1 were higher in patients with unfavorable outcome (Au et al., ).
These data suggest that modulation of the autophagy flux in SQSTM1−/− mice should have an influence on pathophysiological mechanisms after acute brain trauma. Unfortunately, our results did not confirm this hypothesis and failed to show a significant influence on brain damage 5 days after trauma. At a very early time point after insult SQSTM1−/− mice demonstrated a significant lower lesion volume at 24 h and by trend decreased calpain-dependent cell death after TBI compared with wildtype littermates. In line with the histological data, neurofunctional data was not significantly different between groups. Deficiency of SQSTM1 therefore delays, but does not prevent secondary brain damage. A possible explanation for this effect might be that SQSTM1 severely impairs mitochondrial function and, thereby enhances neurodegeneration (Jackson et al., ). SQSTM1 deficiency results in inhibition of SQSTM1-dependent autophagy and will result in accumulation of latter protein substrates. The data suggest that prevention of degradation may be a physiological reaction, as indicated by mRNA levels. Further inhibition of the pathways was able to further delay but not prevent damage evolution.
Additionally, the influence of SQSTM1−/− on BAG1 and BAG3, other main participators of the protein degradation system, was investigated. Following brain trauma SQSTM1−/− mice show decreased Bag3 mRNA expression levels 5 days after trauma. Thereby, SQSTM1 deficiency might additionally counteract an autophagy overshoot by decreasing the expression of one of its main key players BAG3. In line with this, a neuroprotective effect of BAG3 depletion has been also described for neuronal hypoxia-ischemia injury. Here, BAG3 depletion prevented hippocampal neuronal death via depression of galactin 3 and filamin c (Cho et al., ). Protein degradation markers are unchanged in SQSTM1−/− after TBI indicating its minor role in early pathophysiological events after TBI.
Several other studies have demonstrated neuroprotective effects by addressing other players of autophagy. Mice deficient in Atg7, essential for autophagy induction, showed protection from hypoxia-ischemia induced caspase-3 activation and neuronal cell death (Koike et al., ). In a mouse model of frontotemporal dementia, inhibition of autophagy ameliorated neuronal cell loss. The authors suggested that an excessive accumulation of autophagosomes is detrimental for neuronal survival under some neurodegenerative conditions (Lee and Gao, ). Our present data do not support the notion that inhibition of autophagy, specifically the SQSTM1-dependent autophagy has a substantial and lasting effect on secondary brain damage.
Conclusion
The findings of the present study have important implications for a better understanding of SQSTM1-dependent autophagy and its involvement in cerebral trauma. The modulation of autophagy in SQSTM1−/− animals causes an attenuation of cell death processes, but does not prevent secondary brain damage and fails to influence neurological function after trauma. Therefore, data suggest a minor role of SQSTM1-dependent autophagy for pathophysiological mechanisms in the initial phase after head injury.
Statements
Author contributions
Conceived and designed the experiments: AS, CG, ST, and KE; Performed the experiments: AS, CG, PS, and WB. Analyzed the data: AS, CG, PS, WB, TM, CB, ST, and KE. Wrote the paper: AS, CG, PS, TM, ST, and KE.
Funding
This work was supported by the German Research Foundation (CRC1080/A9) to CB, KE, and TM (TP C07 and TP C02). Some data shown in this manuscript are part of a doctoral thesis presented by PS to the Medical Faculty, University Medical Center of the Johannes Gutenberg University Mainz, Germany. The study is content of the professorial dissertation (Habilitation) of AS presented to the Johannes Gutenberg-University Mainz, Germany.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Summary
Keywords
traumatic brain injury, protein degradation, autophagy, SQSTM1, p62, BAG3, mouse model
Citation
Sebastiani A, Gölz C, Sebastiani PG, Bobkiewicz W, Behl C, Mittmann T, Thal SC and Engelhard K (2017) Sequestosome 1 Deficiency Delays, but Does Not Prevent Brain Damage Formation Following Acute Brain Injury in Adult Mice. Front. Neurosci. 11:678. doi: 10.3389/fnins.2017.00678
Received
07 September 2017
Accepted
20 November 2017
Published
19 December 2017
Volume
11 - 2017
Edited by
Paula Ludovico, University of Minho, Portugal
Reviewed by
Cecilia Santos, Universidade da Beira Interior, Portugal; Savina Apolloni, Fondazione Santa Lucia (IRCCS), Italy
Updates
Copyright
© 2017 Sebastiani, Gölz, Sebastiani, Bobkiewicz, Behl, Mittmann, Thal and Engelhard.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Anne Sebastiani annesebastiani@web.de
This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience
†These authors have contributed equally to this work.
‡These authors have contributed equally to this work as senior authors.
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