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. Author manuscript; available in PMC: 2022 Nov 1.
Published in final edited form as: Neuroscience. 2021 Sep 8;475:127–136. doi: 10.1016/j.neuroscience.2021.09.001

Mutation of a ubiquitin carboxy terminal hydrolase L1 lipid binding site alleviates cell death, axonal injury, and behavioral deficits after traumatic brain injury in mice

Zhiping Mi 1,2, Hao Liu 1,2, Marie E Rose 1,2, Jie Ma 1,2, Daniel P Reay 1,2, Xiecheng Ma 1,3,4, Jeremy J Henchir 1,3,4, C Edward Dixon 1,3,4, Steven H Graham 1,2
PMCID: PMC8530956  NIHMSID: NIHMS1743327  PMID: 34508847

Abstract

Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is a protein highly expressed in neurons that may play important roles in the ubiquitin proteasome pathway in neurons, axonal integrity, and motor function after traumatic brain injury (TBI). Binding of reactive lipid species to cysteine 152 of UCHL1 results in unfolding, aggregation, and inactivation of the enzyme. To test the role of this mechanism in TBI, mice bearing a cysteine to alanine mutation at site 152 (C152A mice) that renders UCHL1 resistant to inactivation by reactive lipids were subjected to the controlled cortical impact model (CCI) of TBI and compared to wild type (WT) controls. Alterations in protein ubiquitination and activation of autophagy pathway markers in traumatized brain were detected by immunoblotting. Cell death and axonal injury were determined by histological assessment and anti-amyloid precursor protein (APP) immunohistochemistry. Behavioral outcomes were determined using the beam balance and Morris water maze tests. C152A mice had reduced accumulation of ubiquitinated proteins, decreased activation of the autophagy markers Beclin-1 and LC3B, a decreased number of abnormal axons, decreased CA1 cell death, and improved motor and cognitive function compared to WT controls after CCI; no significant change in spared tissue volume was observed. These results suggest that binding of lipid substrates to cysteine 152 of UCHL1 is important in the pathogenesis of injury and recovery after TBI and may be a novel target for future therapeutic approaches.

Keywords: deubiquitinating enzymes, protein ubiquitination, controlled cortical impact, ubiquitin proteasome pathway, axonal injury

INTRODUCTION

UCHL1 belongs to the UCH family of deubiquitinating enzymes that are expressed at high levels in neurons throughout the brain (Larsen et al., 1996). UCHL1 may be involved in repair of axons and neurons after brain injury by removal of abnormal proteins through the ubiquitin proteasome pathway (UPP) and autophagy (Bilguvar et al., 2013; Chen et al., 2010; Graham and Liu, 2017; Kabuta and Wada, 2008; Kim et al., 2019; Liu et al., 2002). In addition, UCHL1 closely interacts with proteins of the neuronal cytoskeleton and may affect axonal transport and maintaining axonal integrity (Bheda et al., 2010; Gong et al., 2006; Liu et al., 2015; Pukass and Richter-Landsberg, 2015; Sakurai et al., 2008). UCHL1 also regulates synaptic function and long-term potentiation (LTP) and may be involved in memory function (Gong, et al., 2006; Sakurai, et al., 2008). Multiple models of UCHL1-deficient mutant mice exhibit axonal degeneration and extensive deficits in motor function (Chen, et al., 2010; Coulombe et al., 2014; Reinicke et al., 2019; Saigoh et al., 1999; Walters et al., 2008; Yamazaki et al., 1988). Thus, there is evidence that UCHL1 may play an essential role in axonal integrity in the motor system.

Diffuse axonal injury is a major component of the motor and cognitive sequelae of traumatic brain injury (Povlishock and Christman, 1995; Smith et al., 2013). There are few available approaches to address axonal injury and recovery after TBI, and no approaches have been successfully translated to human trials. TBI induces significant oxidative stress resulting in production of a variety of free radical and reactive lipids including cyclopentenone prostaglandins (CyPgs) that may exacerbate injury and impede recovery via inhibition of the UPP and other mechanisms (Figueiredo-Pereira et al., 2014; Hickey et al., 2007; Kunz et al., 2002; Rodriguez-Rodriguez et al., 2014; Solaroglu et al., 2005). CyPgs bind to UCHL1 at cysteine 152, unfold the enzyme, and inhibit its ubiquitin hydrolase activity (Koharudin et al., 2010). Mutation of cysteine 152 to alanine preserves UCHL1 hydrolase activity and ameliorates axonal damage in primary neurons treated with CyPgs (Liu, et al., 2015). Since UCHL1 may play an important role in maintaining axonal integrity and function, we hypothesized that inactivation of UCHL1 by binding of reactive lipid species to cysteine 152 may contribute to the pathogenesis of axonal injury and behavioral deficits after TBI.

To address this hypothesis, we generated a knock-in mouse bearing a C152A mutation (C152A mouse) that is resistant to inactivation of UCHL1 hydrolase activity by CyPgs (Liu, et al., 2015; Liu et al., 2019). C152A and wild type mice were subjected to TBI using the CCI model. The effect of the C152A mutation on protein ubiquitination, autophagy activation, neuronal cell death, white matter injury, and motor and cognitive function after CCI was determined.

EXPERIMENTAL PROCEDURES

Animals

Mice were housed in individually ventilated cages under standard conditions (22°C, 12h light–dark cycle) with free access to food and water. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize the number of animals used and their suffering.

Reagents and antibodies

Table 1 lists the sources, catalog numbers and dilution factors for primary and secondary antibodies used in the study. Hematoxylin and eosin reagents were purchased from Fisher Scientific (Pittsburgh, PA).

Table 1.

Antibodies used in the study

Primary Antibody (host) Catalog number Dilution Source
APP (rabbit) CT695, 51–2700 1:300 Thermo Fisher
Beclin-1 (rabbit) 3495 1:1000 Cell Signaling Technology
GAPDH (mouse) AM4300 1:8000 Thermo Fisher
K48 poly-Ub(rabbit) 05–1307 1:1000 Millipore Sigma
K63 poly-Ub (rabbit) ab179434 1:1000 Abeam
LC3B (rabbit) 2775 1:1000 Cell Signaling Technology
Poly-Ub (Mouse IgM) BML-PW8805-0500 1:500–1:1000 Enzo Life Sciences
Secondary Antibody
HRP conjugated goat anti-Mouse IgG AP127P 1:8000 Millipore Sigma
HRP conjugated goat anti-Mouse IgM 31456 1:8000 Thermo Fisher
HRP conjugated goat anti-Rabbit IgG 32260 1:8000 Thermo Fisher
Goat anti-Rabbit IgG, Alexafluor 488 A-11034 1:700 Thermo Fisher
Goat anti-Mouse IgG, Cy3 A-10521 1:700 Thermo Fisher
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Generation of UCHL1 C152A mice

The C152A mouse was constructed using the bacterial artificial chromosome (BAC) technique in collaboration with the University of Michigan Transgenic Core (Araki et al., 1997; Dymecki and Tomasiewicz, 1998; Hughes et al., 2007) as described in detail previously (Liu, et al., 2015; Liu, et al., 2019). Homologous recombination of DNA fragments was used to modify a BAC containing UCHL1 to produce a point mutation in UCHL1 that converts cysteine 152 to alanine (TGT>GCT) within the normal chromosome of the mouse (Liu, et al., 2015). The C152A heterozygous males were backbred to C57BL/6J female mice (The Jackson Laboratories, Bar Harbor, ME), and the resulting heterozygous offspring were crossbred to produce homozygous mutant C152A and WT UCHL1 lines. Mouse genotypes were verified by PCR amplification of genomic DNA with primers 5′-GGAATCTTGACAGCAGTGCC -3’ and 5′-ACAATCACCTTCACTCTAGTGCC -3’.

Induction of TBI using controlled cortical impact (CCI) surgery

CCI procedures, parameters and postsurgical care were described in detail previously (Dixon et al., 1991; Liu et al., 2017). Male WT and C152A mice, age 10 – 12 weeks old, were anesthetized using 5% isoflurane, placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) fitted with a nosecone. The incision was closed, and anesthesia discontinued after CCI, followed by application of bupivacaine for pain relief. Sham-operated mice underwent all described procedures except the cortical impact. Animals were monitored daily post injury. Mice showing signs of pain (freezing, hunched posture or vocalization) or infection (swelling, redness, or discharge) or weight loss greater than 20% of controls were removed from the study and euthanized by carbon dioxide inhalation.

Beam balance task

Gross vestibulomotor function was assessed on D0 (baseline, pre-injury) and D1 – 5 after CCI injury as described previously (Dixon et al., 1987; Feeney et al., 1981). WT and C152A CCI and sham-operated mice were placed on a narrow round beam (10 mm wide) above a padded surface and latencies of up to 60 sec were recorded as the average of 3 trials daily.

Morris water maze

Analysis of spatial learning and memory was performed using standard Morris Water Maze testing (MWM) without environmental enrichment beginning at day 0 as previously described with minor modifications (Adelson et al., 2013; Whalen et al., 1999). To ensure recovery from motor deficits, testing was performed on days 14 through 18 after CCI. Cognitive performance was evaluated by placing mice in the water maze on post-injury days 14 – 17 with no prior training. Latency up to 120 s to locate the hidden platform was measured over 4 trials daily. On day 18, two probe trials were conducted and performance was assessed by measuring time spent in the “target quadrant” where the platform was previously located. Following the probe trials, visible platform trials were performed to control for nonspecific deficits. Latency to locate and climb the platform was measured in 2 trials. During all swim tests, mice were monitored for signs of distress or inability to swim; mice were warmed for 4 min between trials in a temperature-controlled incubator.

Total protein extraction and western blotting

Mouse hippocampi were dissected 24 h after injury, rapidly frozen on dry ice and stored at −80°C until homogenization with N-PER tissue protein extraction reagent supplemented with protease and phosphatase inhibitors (Pierce, Fisher). Protein concentrations were measured by bicinchoninic acid (BCA assay). Hippocampal total protein lysates from WT and C152A mice that have undergone sham or CCI surgery were resolved on 4–20% Mini-PROTEAN® TGX precast protein gels, transferred onto polyvinylidene difluoride (PVDF) membranes (BioRad, Hercules, CA) and blocked with 5% non-fat milk in TBS/Tween-20 for 1 h at room temperature before incubation with indicated primary antibodies overnight at 4°C. Blots were washed and incubated with appropriate HRP-conjugated secondary antibodies at room temperature for 1 h. Protein signal was visualized with ECL (Enhanced Chemiluminescence) reagents (Pierce, Fisher). Blots were subsequently stripped and re-probed using anti-GAPDH antibodies for verification of equal protein loading. Densitometric analysis was performed using ImageJ 1.50i software (Schneider et al., 2012) and results are normalized to the corresponding contralateral band.

Cell survival assessment

Cell survival measurement was performed as previously described (Mi et al., 2021). Twenty-one days after cortical impact or sham injury, mice were sacrificed via carbon dioxide inhalation, then perfused transcardially with 20 mL heparinized saline followed by 10 mL of 10% buffered formalin. Brains were extracted, postfixed in 10% buffered formalin, dehydrated, and embedded in paraffin. Serial sections (7 μm in thickness) were cut at 0.5 mm intervals. Hippocampi were examined after staining with hematoxylin and eosin (H&E) using 20X brightfield magnification. Neurons exhibiting visible nucleoli (Coulin et al., 2001) and with normal morphology were counted in the ipsilateral medial 1 mm of CA1 region of hippocampus and normalized to the respective contralateral region (at bregma −1.9 mm). Data are expressed as percent contralateral.

Assessment of spared tissue volume

Spared tissue volume in serial sections was calculated using the method of Swanson et al. by measuring surviving brain tissue in ipsilateral and contralateral hemispheres at each slice using ImageJ 1.50i (Schneider, et al., 2012; Swanson et al., 1990). Spared volume was then determined by multiplying slice area by slice interval thickness then adding together all slices. Spared tissue volume is expressed as percent contralateral (ipsilateral / contralateral * 100).

APP immunofluorescent staining and quantification

Axonal injury was determined by APP immunohistochemistry (Gentleman et al., 1993; Hayashi et al., 2015; Li et al., 2013). APP immunofluorescent staining was performed as previously described (Marmarou and Povlishock, 2006). C152A and WT mice were sacrificed 24 h post CCI and brain sections were incubated with anti-APP antibody overnight at 4°C, followed by AlexaFluor 488-conjugated goat anti-rabbit antibody. APP stained sections were photographed using an EVOS Imaging system (ThermoFisher, Pittsburgh, PA). APP positive spots were counted in 1400 μm2 fields from 20X fluoromicrographs located in thalamus (Xia et al., 2018)(Bregma −2.2 mm). N = 5 per group. Brain sections incubated without application of primary antibody served as controls for anti-rabbit secondary antibody.

Statistical Analysis

Densitometric comparison of immunoblots, APP staining, cell counting, and spared tissue volumes were analyzed using two-way ANOVA followed by Tukey or LSD post hoc testing; beam latency and Morris water maze data were analyzed using two-way repeated measures ANOVA (for hidden and visible platform test) or two-way ANOVA (probe test) followed by Tukey or bonferroni post hoc testing. All statistical analysis was performed using IBM SPSS Statistics 25 (IBM Corporation, Armonk, NY). Graphs were generated using Prism 7.0 (GraphPad, San Diego, CA) and Microsoft Excel. Box-and-whisker plots displaying all data points were used to graphically depict the data in Figs. 1–3. The boxes represent 25–75% of the range, the whiskers indicate minimum and maximum values, and the median is shown by a horizontal line inside the box. Results were considered to be significant when p < 0.05. Sample sizes indicate number of animals used.

Fig. 1. The UCHL1 C152A mutation attenuates CCI-induced increase in poly-ubiquitinated (poly-Ub) protein accumulation and autophagy activation in mouse hippocampus 24 h post injury.

Fig. 1

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a. Total poly-Ub protein, K48-linked (K48) and K63-linked (K63) poly-Ub protein expression levels in hippocampus. b. Beclin-1 and LC3B expression levels in hippocampus. GAPDH was used as a loading control. Left: representative immunoblots; right: densitometric analysis of immunoblots expressed as ipsilateral/contralateral. Circles indicate sham surgery and triangles indicate CCI surgery. *P < 0.05, **P < 0.01. N=8 −10 mice per group

Fig. 3. The UCHL1 C152A mutation confers increased neuronal viability in CA1 after CCI.

Fig. 3

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WT and C152A mice underwent CCI or sham surgery and were sacrificed 21d post injury. a. Surviving neurons in ipsilateral medial 1 mm of CA1 normalized to the respective contralateral region (left). Representative H&E sections (right). Arrows: examples of neurons with normal morphology and intact nucleoli. b. Spared tissue volume and representative slices at bregma - 1.9 mm. a-b: * P < 0.05, *** P < 0.001, NS: not significant. N = 9 – 10 mice per group

RESULTS

The UCHL1 C152A mutation attenuates ubiquitinated protein accumulation and autophagy activation after CCI

To determine the effects of the C152A mutation on CCI-induced UPP function, poly-ubiquitinated (poly-Ub) proteins were detected in hippocampus 24 h after CCI by immunoblotting. While there were increases in K63-linked UPP-independent and total poly-Ub proteins in WT CCI-operated mice compared to sham-operated counterparts, these increases were not observed in CCI-operated C152A mice (Fig. 1A) (Two-way ANOVA: F (1,28) injury = 14.301, p < 0.001 for Poly-Ub; F (1, 32) injury = 6.26, p < 0.05 for K63 poly-Ub). In addition, the expression of K48-linked poly-Ub proteins was significantly higher in WT mice than that of C152A mice after CCI (Fig. 1A) (Two-way ANOVA: F (1,31) genotype = 5.066; p < 0.05). To determine the effects of the C152A mutation on CCI-induced autophagy, autophagy markers Beclin-1 and LC3B were detected in hippocampus 24 h after CCI by immunoblotting. Significantly higher levels of Beclin-1 and LC3B II/I were observed in WT mice compared to C152A mice after CCI (Fig. 1B) (Two-way ANOVA: F (1,31) genotype = 11.507, p < 0.01 for Beclin 1; F (1,36) genotype = 4.262, p < 0.05). And, in a pattern like that of the poly-Ub proteins, both Beclin-1 expression and LC3B II/I ratio were significantly increased in WT mice subjected to CCI compared to sham-operated controls (Two-way ANOVA, F (1,31) injury = 8.595, p < 0.01 for Beclin 1; F (1,36) injury = 0.704, p > 0.05 for LC3B). There was no difference between WT and C152A sham-operated mice in all markers examined.

The UCHL1 C152A mutation attenuates CCI-induced white matter injury

White matter injury in C152A and WT mice was determined in thalamus 24 h and 7 d post CCI by APP immunostaining. There were significantly fewer APP immuno-positive neurites in thalamus ipsilateral to CCI in C152A mice compared to WT mice at both 24 h and 7 d after injury (Fig. 2) (Two-way ANOVA: F (1,16) genotype = 3.221, p > 0.05 for 24 h; F (1,16) genotype = 9.68, p < 0.01 for 7 d).

Fig. 2. The UCHL1 C152A mutation alleviates CCI-induced axonal damage in thalamus.

Fig. 2

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WT and C152A mice underwent CCI surgery and were sacrificed 24 h or 7 d post injury. Brains were embedded in paraffin, cut in 7 μm sections, and immunostained using anti-APP antibody (green). Left: APP positive spots per field in ipsilateral (ipsi) and contralateral (contra) thalamus; right: representative 20X photos. Arrows indicate examples of APP positivity. Bar = 25 μm. * P < 0.05, ** P < 0.01. N = 5 mice per group

The UCHL1 C152A mutation increases neuronal viability after CCI

To examine whether the UCHL1 C152A mutation affects cortical tissue loss and cell viability post injury, histological analysis was performed 21 days post injury on WT and C152A mice. Although cell survival in CA1 hippocampus was significantly decreased in both WT and C152A groups after CCI, this reduction was significantly less in C152A mice compared to WT control (Fig. 3A) (Two-way ANOVA: F (1,34) injury = 20.177, p < 0.001; F (1,34) genotype = 2.575, p > 0.05). Spared hemispheric volume in both WT and C152A mice was significantly smaller after CCI compared to their sham-operated counterparts (Fig. 3B) (Two-way ANOVA: F (1,36) injury = 283.486, p < 0.001). There was no significant difference between either sham or CCI-operated WT and C152A mice (Fig. 3B). Collectively, the above data indicate that the C152A mutation ameliorates CCI-induced neuronal injury on hippocampal cell loss but has no effect on post-injury spared tissue volume.

UCHL1 C152A mice exhibited improved motor performance recovery after CCI

Vestibulomotor function was measured by placing mice on a 10mm round beam and measuring latency up to 60 sec in 3 trials daily on D0 (baseline, pre-injury) and D1 – 5 post injury. Motor performance deficits observed in WT mice were abrogated in C152A mice on D2 – D5 post injury. No significant difference was detected between sham-operated WT and C152A mice (Fig. 4) (Two-way ANOVA repeated measures from D1-D5: F (1, 36) injury = 8.923, p < 0.01); F (1, 36) genotype = 12.037, p < 0.01).

Fig. 4. The UCHL1 C152A mutation abrogates delayed motor function recovery in mice after CCI.

Fig. 4

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WT and C152A mice underwent the CCI or sham surgery on D0. Beam balance was measured on D1 – D5 post injury (three trials daily). Data are means +/− SE. *** P <0.001 between C152A and WT CCI groups. N = 10 mice per group

The UCHL1 C152A mutation mitigates CCI-induced spatial memory acquisition deficits

MWM testing was used to measure spatial memory acquisition in CCI and sham-operated mice. Injured WT mouse swim latencies were significantly longer than WT sham-operated controls; there were no significant differences in swim latencies between any other groups (Fig 5) (Two-way ANOVA repeated measures from D14-D17: F (1, 51) injury = 2.996, p > 0.05). These results demonstrate a significant impairment in spatial memory acquisition in WT mice after CCI, but not in C152A mice. Visual platform evaluation at day 18 revealed no significant differences in swim latency indicating that there were no nonspecific deficits contributing to the poor performance of injured WT mice. Probe trial data was not significantly different between groups.

Fig. 5. Spatial learning and memory in UCHL1 C152A knock-in mice after CCI as measured by Morris Water Maze (MWM).

Fig. 5

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WT and C152A mice underwent CCI or sham surgery. MWM submerged platform testing was performed on days 14 – 17 post injury; probe and visible platform tests were performed on D18. Data are means ± SE. * P < 0.05 WT CCI vs WT sham. NS: not significant. N = 15 mice per group

DISCUSSION

The major findings of the study are: 1) The C152A mutation attenuates accumulation of ubiquitinated proteins and autophagy activation in hippocampus after CCI. 2) C152A mice exhibit reduced CCI-induced white matter injury and increased neuronal cell viability. 3) C152A mice have improved motor function and cognitive functional recovery after CCI compared to WT controls.

Prostaglandins (PG) produced by the cyclooxygenase 2 pathway have been hypothesized to exacerbate neuronal injury (Figueiredo-Pereira, et al., 2014; Gong et al., 2016; Graham and Hickey, 2003; Hickey, et al., 2007; Kunz, et al., 2002; Liu et al., 2013; Nakayama et al., 1998). PGD2 is the most prevalent PG in brain (Ogorochi et al., 1984) and is nonenzymatically converted to reactive cyclopentenone prostaglandins including PGJ2 and 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) (Fukushima et al., 1982; Milne et al., 2005; Mullally et al., 2001; Ohno et al., 1992; Straus and Glass, 2001). These CyPgs are characterized by the presence of a cyclopentenone ring which can directly modify nucleophiles such as free sulfhydryls in cysteine residues of cellular proteins. 15d-PGJ2 covalently modifies UCHL1, inducing structural changes detected by nuclear magnetic resonance spectroscopy resulting in unfolding and loss of hydrolase activity (Koharudin, et al., 2010). Mutation of cysteine 152, but not the other five cysteine residues in UCHL1, prevents the unfolding of the protein and preserves UCHL1 hydrolase activity after incubation of UCHL1 recombinant protein with CyPgs (Koharudin, et al., 2010). Incubation with 15d-PGJ2 induces cell death in primary neurons, associated with accumulation of ubiquitinated proteins. Cell death induced by hypoxia is exacerbated by inhibition of UCHL1 and rescued when treated with a UCHL1 protein fused to the transduction domain of HIV trans-activator protein (TAT-UCHL1) (Liu et al., 2011). 15d-PGJ2 treatment also induces axonal damage in primary neurons, but neurons derived from mice containing the UCHL1 C152A mutation are resistant to 15d-PGJ2-induced injury suggesting that modification of the C152 site of UCHL1 by CyPgs may induce axonal injury (Liu, et al., 2015; Liu, et al., 2019). These studies demonstrate that UCHL1 activity and binding of CyPgs to cysteine 152 of UCHL1 may be an important component of axonal injury and neuronal cell death.

The UPP is a crucial mechanism by which abnormal protein accumulations within neurons are degraded. UCHL1 may be an important component of the UPP in neurons and may play a necessary role in removing abnormal protein aggregations within neurons in pathological states (Graham and Liu, 2017; Liu, et al., 2011; Shen et al., 2006). Increased ubiquitinated proteins and decreased free ubiquitin have been detected in brain after TBI and these alterations in ubiquitination may contribute to cell death and dysfunction (Sakai et al., 2014; Yao et al., 2008). Abolishing UCHL1’s hydrolase activity by mutation of cysteine 90 to alanine resulted in increased accumulation of ubiquitinated proteins and exacerbated cell death and dysfunction in mice after TBI (Mi, et al., 2021). In the present study, there was significantly less accumulation of K-48–linked poly-Ub proteins in C152A mice compared with WT mice after CCI. In addition, there was significantly increased total poly-Ub and K63-linked poly-Ub proteins detected in WT mouse hippocampus after CCI, but no significant difference was detected between sham and injured C152A mice. In addition to the UPP, clearance of misfolded and damaged proteins from neurons is also mediated by autophagy (Frake et al., 2015). The current study indicates there was a significant increase in the Bcl-2 associated protein Beclin-1 and the ratio of LC3BII to LC3BI autophagy markers in the hippocampus of WT mice post CCI, but this increase was not observed in C152A mice. These results suggest that the UCHL1 C152A mutation preserves UCHL1 activity and improves function of the UPP, thus eliminating the need for activation of autophagy pathways.

The current study determined that there was decreased neuronal cell death and white matter injury in C152A mice suggesting that UCHL1 plays an important role in the pathogenesis of injury and recovery after TBI. The UPP removes excessive amounts of ubiquitinated proteins that form aggregates within neurons. These protein aggregates have been hypothesized to result in the unfolded protein response and to exacerbate neuronal cell death after TBI, ischemia and neurodegenerative diseases (Ding et al., 2017; Hu et al., 2000; Jara et al., 2013; Kabuta and Wada, 2008; Liu, et al., 2002; Proctor et al., 2010). Thus, increased cell viability in the hippocampal CA1 region of C152A mice compared to WT counterparts after CCI may be a direct result of better preserved UCHL1 hydrolase activity and UPP function in C152A mouse hippocampal neurons. UCHL1 may also exert its protective effects through other mechanisms. UCHL1 may post-translationally modify proteins that are important in signal transduction pathways by ligation of ubiquitin and modify protein function via protein-protein interactions. Examples of UCHL1-modified molecules include hypoxia-inducible factor-1α, C-Jun activation domain-binding protein-1 and lysosome receptors for chaperone-mediated autophagy (Caballero et al., 2002; Goto et al., 2015; Kabuta and Wada, 2008). These proteins may modulate a number of cellular responses including oxidative stress, autophagy, and axonal and synaptosomal transport. Thus, UCHL1 activity may have multiple molecular targets in neurons.

UCHL1’s effects on signal transduction pathways may have multiple effects on neuronal function. UCHL1 regulates LTP and synaptic function under physiological and pathological conditions and therefore may be involved in memory (Gong, et al., 2006). UCHL1-deficient mice display agglomeration of tubulovesicular profiles at the presynaptic axon terminals and impaired neuromuscular transmission (Bilguvar, et al., 2013; Chen, et al., 2010). UCHL1-null mice manifest neurodegeneration with progressive motor impairment as well as pathology in the dendritic spines of corticospinal neurons (Goto et al., 2009; Miura et al., 1993; Saigoh, et al., 1999). These and other findings indicate that UCHL1 plays an essential role in maintaining synaptic function and axonal transport, and is critical in maintaining cognitive and motor function (Genc et al., 2016; Gong, et al., 2006; Jara et al., 2015). Thus, preserving UCHL1 activity after TBI may result in ameliorating neuronal cell death and axonal injury and promoting cognitive and motor recovery after TBI.

In conclusion, mutation of cysteine 152 of UCHL1 decreases accumulation of ubiquitinated proteins and activation of autophagy, ameliorates cell death and white matter injury, and improves motor function after CCI in mice. These results suggest unfolding and inactivation of UCHL1 by covalent modification of UCHL1 by CyPgs and other reactive lipids may be a significant component of injury and motor impairment after TBI. Development of agents that inhibit binding of reactive lipids at the C152 site of UCHL1 or restoration of UCHL1 activity with TAT-UCHL1 proteins could be useful therapeutic strategies to improve outcome after TBI (Gong, et al., 2006; Liu, et al., 2017). Further studies are needed to address these hypotheses.

  • Mutation of UCHL1’s C152 lipid binding site alleviated TBI-induced cell death

  • C152 mutation decreased ubiquitinated proteins, activation of autophagy after TBI

  • C152 mutation decreased white and gray matter injury after TBI

  • C152 mutation improved motor and cognitive function after TBI

FUNDING

This work was supported by the National Institutes of Health/ National Institute of Neurological Disorders and Stroke Grant R01NS102195 (to S.H.G. and C.E.D.). The funding source had no role in study design, collection, analysis, and interpretation of data or in the writing of the report and the decision to submit the article for publication.

Footnotes

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CONFLICTS OF INTEREST

The authors declare no conflict of interest. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.

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