Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Mar 1.
Published in final edited form as: Exp Neurol. 2023 Dec 12;373:114650. doi: 10.1016/j.expneurol.2023.114650

Systemic treatment with ubiquitin carboxy terminal hydrolase L1 TAT protein ameliorates axonal injury and reduces functional deficits after traumatic brain injury in mice

Zhiping Mi 1,2, Jie Ma 1,2, Dennis Zeh 1,2, Marie E Rose 1,2, Jeremy J Henchir 1,3,4, Hao Liu 1,5, Michelle Ma 3,4, Guodong Cao 1,2, C Edward Dixon 1,3,4, Steven H Graham 1,2
PMCID: PMC10939891  NIHMSID: NIHMS1953315  PMID: 38092186

Abstract

Traumatic brain injury (TBI) is often associated with axonal injury that leads to significant motor and cognitive deficits. Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is highly expressed in neurons and loss of its activity plays an important role in the pathogenesis of TBI. Fusion protein was constructed containing wild type (WT) UCHL1 and the HIV trans-activator of transcription capsid protein transduction domain (TAT-UCHL1) that facilitates transport of the protein into neurons after systemic administration. Additional mutant proteins bearing cysteine to alanine UCHL1 mutations at cysteine 152 (C152A TAT-UCHL1) that prevents nitric oxide and reactive lipid binding of C152, and at cysteine 220 (C220A TAT-UCHL1) that inhibits farnesylation of the C220 site were also constructed. WT, C152A, and C220A TAT-UCHL1 proteins administered to mice systemically after controlled cortical impact (CCI) were detectable in brain at 1 h, 4 h and 24 h after CCI by immunoblot. Mice treated with C152A or WT TAT-UCHL1 decreased axonal injury detected by NF200 immunohistochemistry 24 h after CCI, but C220A TAT-UCHL1 treatment had no significant effect. Further study indicated that WT TAT-UCHL1 treatment administered 24 h after CCI alleviated axonal injury as detected by SMI32 immunoreactivity 7 d after CCI, improved motor and cognitive deficits, reduced accumulation of total and K48-linked poly-Ub proteins, and attenuated the increase of the autophagy marker Beclin-1. These results suggest that UCHL1 activity contributes to the pathogenesis of white matter injury, and that restoration of UCHL1 activity by systemic treatment with WT TAT-UCHL1 after CCI may improve motor and cognitive deficits. These results also suggest that farnesylation of the C220 site may be required for the protective effects of UCHL1.

Keywords: Ubiquitin carboxy terminal hydrolase L1, TAT fusion protein, traumatic brain injury, ubiquitin proteasome pathway, autophagy, axonal injury

1. Introduction

Ubiquitin carboxy terminal hydrolase L1 (UCHL1) is highly expressed in brain, constituting 1% of brain protein (Day and Thompson, 2010). UCHL1 is selectively expressed in neurons, suggesting that it is important in neuronal function (Day and Thompson, 2010; Wilson et al., 1988). It also closely interacts with proteins of the neuronal cytoskeleton, and mice that do not express UCHL1 exhibit abnormalities in axonal structure and motor function (Bheda et al., 2010; Kabuta et al., 2008). Thus, UCHL1 may play an important role in axonal integrity and function.

Traumatic brain injury (TBI) is often associated with axonal pathology leading to significant motor and cognitive deficits (Adams et al., 1984; Frati et al., 2017). Although mechanical forces can shear and break axons, intact axons may also be injured, resulting in impaired axonal transport (Buki et al., 1999). Injured axons develop swellings and varicosities within hours after TBI, indicative of injury that is potentially salvageable (Johnson et al., 2013; Weber et al., 2019).

TBI is also companied by the generation of reactive lipids and nitric oxide (NO), which may covalently modify UCHL1 by binding to cysteine 152 and impair its function, resulting in the accumulation of misfolded proteins and reduced cellular solubility (Koharudin et al., 2010; Nakamura et al., 2021). We have previously shown that knockin mice expressing the UCHL1 C152A mutation exhibit significantly attenuated gray and white matter injury and significantly improved sensorimotor recovery after TBI compared to their wild type controls (Mi et al., 2021a). UCHL1’s cysteine 220 may be farnesylated resulting in translocation of UCHL1 to the membrane and increased neurotoxicity of alpha -synuclein (Liu et al., 2009). These data suggest that posttranslational modification of UCHL1 could be important in the pathogenesis of TBI.

We and others have shown that the protein transduction domain of the human immunodeficiency virus trans-activator of transcription capsid protein (TAT) allows proteins to readily transduce neurons in vitro and in vivo (Cao et al., 2002; Gong et al., 2006; Liu et al., 2017). In a prior study, we found that acute systemic administration of a TAT-UCHL1 fusion protein readily transduces neurons after the CCI model of TBI in mice and improves ubiquitin proteasome pathway (UPP) function and histological outcomes (Liu et al., 2017). The current study aims to extend these results by determining whether restoring activity of UCHL1 by TAT-UCHL1 treatment up to 24 h after CCI ameliorates UPP function, axonal injury and improves behavioral outcome after CCI. In addition, the study examines the effect of mutations that prevent the posttranslational modification of UCHL1 at cysteines 152 and 220 upon TAT-UCHL1’s amelioration of axonal injury after CCI. These studies address whether UCHL1 is an important determinate of alteration of protein degradation, axonal injury and functional recovery after TBI and whether TAT-UCHL1 has potential as a novel therapeutic strategy in TBI.

2. Material and methods

This study was carried out in accordance with recommendations from the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was 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 suffering. Animals were housed in a temperature- and humidity-controlled environment with 12 h light cycles and free access to food and water.

2.1. Reagents and antibodies

Table 1 lists the sources, catalog numbers and dilution factors for primary and secondary antibodies used in the study.

Table. 1.

Antibodies used in the study

Primary Antibody (host) Catalog number Dilution Source
Beclin-1 (mouse) MAB5295 1:2000 R&D Systems
GAPDH (mouse) AM4300 1:8000 Thermo Fisher
K48 (rabbit) 8081S 1:1000 Cell Signaling Technology
K63 (mouse) BML-PW0600-0100 1:500 Enzo Life Sciences
NF200 (rabbit) ab8135 1:500 Abcam
Poly-Ub (rabbit) PA1-187 1:500 Invitrogen
SMI32 (rabbit) 559844 1:500 Millipore
Secondary Antibody
IRDye® 680RD Goat anti-Mouse IgG 926-68070 1:10000 LICOR Biosciences
IRDye® 800CW Goat anti-Rabbit IgG 926-32211 1:10000 LICOR Biosciences
Goat anti-Rabbit IgG, Cy3 A-10520 1:750 Thermo Fisher
Open in a new tab

2.2. Generation of trans-activator of transcription (TAT)-UCHL1 fusion proteins

Generation and characterization of TAT-UCHL1 fusion proteins has been described in detail previously (Cao et al., 2002; Liu et al., 2017). The DNA sequence encoding the full-length mouse WT UCHL1 was amplified by PCR using primers 5’-GCTCGATATCATGCAGCTGAAACCGATG-3’ and 5’-ACTTTCTCGAGTTAGGCTGCTTTGCAGAGAGC-3’. Two UCHL1 point mutations substituting alanine for cysteine (C152A and C220A) were introduced using site-directed mutagenesis. The WT UCHL1, and mutant UCHL1 C152A and C220A sequences were cloned into a modified pET30a vector containing the HAT, 6X His and HA sequences using ECoRV and XhoI restriction sites. Constructs were confirmed by sequencing. Plasmids were introduced into Escherichia (E.) coli Rosetta 2 (DE3) strains (Novagen, San Diego, CA) producing 6X His-tagged WT TAT-UCHL1 or its mutant proteins. Generation of WT and mutant TAT-UCHL1 proteins used in the efficacy comparison study were induced by shaking the E. coli Rosetta cells in medium containing 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 35°C for 4h before harvest by centrifugation. Cell pellets were then lysed and purified with a QIAexpress Ni-NTA Fast start kit (Qiagen, Hilden, Germany). Purified protein was dialyzed against 1XPBS buffer and protein concentration was determined by bicinchoninic acid (BCA) protein assay (Pierce). Protein purity was assessed by SDS-PAGE and Coomassie blue staining. Large scale WT TAT-UCHL1 protein was manufactured by Genscript Biotech (Piscataway, NJ) using a protocol similar to that described above.

2.3. Induction of TBI using controlled cortical impact (CCI) surgery and TAT-UCHL1 protein administration

Male mice (C57BL/6J, aged 10–12 weeks) obtained from The Jackson Laboratory (Bar Harbor, ME), were housed in a humidity- and temperature-controlled environment with free access to food and water and 12 h light cycles. General CCI procedures, and postsurgical care were described in detail previously, but with a more severe injury (Dixon et al., 1991). Prior to surgery, mice were randomly assigned to either sham or CCI groups. Mice were anesthetized using 5% isoflurane, placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) and fitted with a nosecone. A midline incision was made, the skin retracted, and a 5 mm craniectomy was performed over the right parietotemporal cortex. The impactor tip (3 mm) was zeroed to the surface of the brain prior to inducing injury (impact speed: 6 m/sec, injury depth: 2.0 mm, dwell time: 50 msec, angle of impact 20°). Sham-operated mice underwent identical surgical procedures with the impactor fired adjacent to the mouse. The incision, but not the craniotomy, was closed. Anesthesia was discontinued after CCI, followed by application of local analgesia (Emla cream and Sensorcaine) for pain relief. Emla cream was used once daily for three days. After randomization, mice were administered 60 mg/kg WT, C152A or C220A TAT-UCHL1, or saline as vehicle control in blinded fashion, via intraperitoneal injection 15 min or 24 h post CCI or sham surgery and monitored daily. The purity of WT TAT-UCHL1 used in the in vivo study was > 90% and endotoxin level was ≤ 0.2 EU /mg as reported by the manufacturer. Mice showing signs of pain (freezing, hunched posture, or vocalization) or infection (swelling, redness, or discharge) were removed from the study and euthanized by carbon dioxide inhalation.

2.4. Beam balance task

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

2.5. Morris water maze

Analysis of spatial learning and memory was performed using Morris water maze testing (MWM) as previously described with minor modifications (Adelson et al., 2013; Whalen et al., 1999). To ensure recovery from surgical-induced 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 interval by an observer blinded to the treatment identity. 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.

2.6. Total protein extraction and western blotting

Mouse cortices and hippocampi were dissected 4 h or 48 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 BCA assay. Total protein lysates from TAT-UCHL1- or vehicle-treated mice having undergone sham or CCI surgery were resolved on 4–20% Mini-PROTEAN® TGX precast protein gels (BIO-RAD) and transferred onto Odyssey® Nitrocellulose Membranes (LICOR Biosciences, Lincoln, NE) or polyvinylidene difluoride (PVDF) membranes (BioRad, Hercules, CA). The membranes were airdried, blocked in either LICOR Intercept® blocking buffer or 5% non-fat milk in Tris-buffered saline (TBS) /Tween-20 for one hour respectively and probed with the indicated markers and appropriate secondary antibodies following immunoblotting protocols described in detail previously (Mi et al., 2021a; Mi et al., 2021b). Immunoreactive bands were visualized using an Odyssey CLx Imaging System (LICOR) or with ECL (Enhanced Chemiluminescence) reagents (Pierce, Fisher). GAPDH was used as loading control. Densitometric analysis was performed using Image Studio Lite Ver. 5.2 (LICOR) or ImageJ 1.50i software (Schneider et al., 2012). Results were normalized to the corresponding contralateral band for all markers analyzed.

2.7. NF200 and SMI32 immunohistochemistry and quantification

Anti-Neurofilament H antibody NF200 and anti-Neurofilament H Non-Phosphorylated antibody SMI32 immunohistochemistry were performed as previously described (Marmarou and Povlishock, 2006; Mi et al., 2021b). Mice were sacrificed at 24 h (for NF200) after injection of TAT-UCHL1 fusion proteins or 7 d (for SMI32) after CCI, and brain sections were incubated with anti-NF200 or anti-SMI32 antibodies then incubated with Cy3 goat anti-rabbit secondary antibody. To measure NF200 protein levels, a fixed size region of interest was sampled. Thresholding was used to select positive pixels; these pixels were counted using ImageJ software. SMI32 protein levels were assessed by measuring fluorescence intensity using ImageJ software after normalization of background. Brain sections incubated without application of primary antibody served as negative controls.

2.8. Assessment of spared tissue volume

Spared tissue volume was calculated using the method of Swanson et al by measuring surviving brain area in ipsilateral and contralateral hemispheres of slices obtained every 0.5 mm using ImageJ 1.50i software (Swanson et al., 1990). Spared volume was determined by multiplying slice area by slice interval thickness then adding together all slices (10 per animal). Spared tissue volume is expressed as percent contralateral and is calculated as follows: ipsilateral / contralateral *100.

2.9. Protocol and Experimental groups

To detect the levels of TAT-UCHL1 fusion proteins in mouse brains at different time points, thirty-six CCI- operated mice were randomly assigned into four groups (n=3 per group) and received 60 mg/kg of the WT, C152A, C220A TAT-UCHL1 fusion proteins respectively or comparable volume of vehicle intraperitoneally 15 min after surgery. Mice were sacrificed and brains removed at 1 h, 4 h and 24 h after injection and TAT-UCHL1 fusion proteins were detected using anti-HA antibody by immunoblot.

To determine the efficacy of TAT-UCHL1 fusion proteins in reducing CCI-induced axonal injury, fifty-two mice were randomly assigned into eight groups. Four groups of CCI- operated mice (n=10 per group) received either 60 mg/kg of WT, C152A, or C220A TAT-UCHL1 fusion proteins, or a comparable volume of saline intraperitoneally 24 h after surgery; the other four groups received identical fusion protein or vehicle treatment after sham surgery (n= 3 per group). Mice were sacrificed and brains removed 24 h after injection. NF200 immunoreactive puncta were counted in cerebral peduncle.

For the effect of WT TAT-UCHL1 treatment on ubiquitinated protein accumulation and autophagy activation after CCI, twenty mice were randomly assigned into two groups. One group received 60 mg/kg of WT TAT-UCHL1 fusion protein and the other group received an equal volume of saline intraperitoneally 24 h after CCI surgery. Mice were sacrificed and brains removed 24 h after injection. Total protein lysates of ipsilateral and contralateral hippocampi were probed with antibodies detecting total poly-Ub, K48-linked (K48) and K63-linked (K63) poly-Ub proteins, and Beclin-1. The contralateral side was used as control.

To examine the effect of WT TAT-UCHL1 on axonal damage and contusion size after CCI, twenty mice were randomly assigned into two groups. One group received 60 mg/kg of WT TAT-UCHL1 fusion protein and the other group received an equal volume of vehicle intraperitoneally 24 h after CCI surgery. Mice were sacrificed and brains removed 7 d after CCI. SMI32 immunofluorescent intensity was measured in ipsilateral and contralateral cingulate bundle with the contralateral side serving as control. Spared tissue volume was calculated by measuring surviving brain tissue in ipsilateral and contralateral hemispheric areas at each slice.

For the effect of WT TAT-UCHL1 on motor and cognitive recovery after CCI, fifty-two mice were randomly assigned into four groups. Two groups of CCI-operated mice (n=16) received either 60 mg/kg of WT TAT-UCHL1 fusion proteins or saline intraperitoneally 24 h after surgery; two groups of sham-operated mice (n=10) received the same fusion protein or vehicle treatment as the CCI-operated groups. Vestibulomotor function was assessed using the beam balance test on D0 (baseline, pre-injury) and D1 – 5 after WT TAT-UCHL1 or vehicle injection. Cognitive function was assessed by Morris water maze testing on D14-D18 after WT TAT-UCHL1 or vehicle injection.

2.10. Statistical Analysis

Data sets were examined for normality and equal variance using Shapiro-Wilk and Levene tests respectively before statistical analysis. Densitometric comparison of immunoblots, SMI32 protein expression and spared tissue volume were analyzed using two sample independent samples t-test; NF200 protein expression was analyzed using the Kruskal Wallis one-way ANOVA followed by Dunn’s post hoc testing; beam latency and Morris water maze data were analyzed employing two-way repeated measures ANOVA or two-way ANOVA (probe test) followed by Tukey or LSD post hoc testing. All statistical analysis was performed using IBM SPSS Statistics 29 (IBM Corporation, Armonk, NY). Graphs were generated using Prism 7.0 (GraphPad, San Diego, CA), Adobe Illustrator 27.01(Adobe, San Jose, CA) and Microsoft Excel version 2305 (Microsoft, Redmond WA). Results were considered to be significant when p < 0.05. Sample sizes indicate number of animals reported.

3. Results

3.1. Efficacy of WT, C152A and C220A TAT-UCHL1 in reducing CCI-induced axonal injury

Fig. 1A illustrates the construction of WT, C152A, and C220A TAT-UCHL1 fusion proteins. Purity of the WT and mutant TAT- UCHL1 fusion proteins were estimated to be >90% as evaluated by densitometric analysis of the Coomassie Blue-stained SDS-PAGE gel under reducing conditions (Fig. 1B). Function of these TAT- UCHL1 proteins was verified by assessing hydrolase activity (Fig. 1C). The time course expression of WT, C152A and C220A TAT-UCHL1 in mouse cortical samples was detected at 1 h, 4 h and 24 h post injection by immunoblot analysis using anti-HA (Fig 2A). As shown in Fig 2A, all three TAT-UCHL1 fusion proteins were detectable at 1 h, 4 h and to a lesser extent, 24 h post injection, with peak TAT-UCHL1 protein levels at 4 h after injection. To determine whether TAT-UCHL1 proteins protect axons from traumatic injury, we compared axonal injury detected by NF200 24 h after treatment with WT TAT-UCHL1, C152A TAT-UCHL1, C220A TAT-UCHL1, or vehicle after sham or CCI operation. Treatment with WT and C152A TAT-UCHL1, but not C220A TAT-UCHL1 proteins significantly decreased NF200 immunoreactive pixels in cerebral peduncle (Fig. 2B). No significant difference was detected between the protective effect of WT and C152A TAT-UCHL1 protein treatment. No NF200 immunoreactive puncta were detected in sham-operated mice. Representative images of NF200 immunoreactive puncta in cerebral peduncle with different TAT-UCHL1 protein treatment after sham and CCI operations are shown in Fig. 2C. Since there was no significant difference in axonal protection between WT and C152A TAT-UCHL1-treated mice, further in vivo studies were conducted using WT TAT-UCHL1 treatment only.

Fig. 1. Construction and characterization of WT, C152A and C220A mutant TAT-UCHL1 fusion proteins.

Fig. 1.

Open in a new tab

a. WT and mutant TAT-UCHL1 constructs used to generate UCHL1 fusion proteins. Arrows indicate cysteines 152 and 220 replacement by alanine. b. TAT-UCHL1 fusion protein purity shown in Coomassie blue stained SDS-PAGE gel. M: molecular marker. c. Function of WT and mutant TAT-UCHL1 fusion protein as measured by the in vitro hydrolase activity assay.

Fig. 2. WT and mutant TAT-UCHL1 efficacy comparison in mice after CCI.

Fig. 2.

Open in a new tab

a. Top: WT and mutant TAT-UCHL1 fusion proteins detected in paralesional cortex by HA immunoblot analysis 1 h, 4 h and 24 h after CCI. Bottom: Densitometric analysis of HA expression levels normalized to GAPDH grouped by post-treatment time of sacrifice. b. Protective effect of WT and C152A TAT-UCHL1 fusion proteins in cerebral peduncle as measured by NF200 immunohistochemistry 24 h after CCI (indicated by green square). c. Representative images of NF200 immunoreactive pixels (red) in cerebral peduncle with WT and mutant TAT-UCHL1 protein treatment after sham and CCI operation. Bar = 100μm. Veh: vehicle; pos: WT TAT-UCHL1 fusion protein as positive control. Data are means ± SE. * P < 0.05, ** P < 0.01 and NS = Not significant vs. vehicle control. One-way ANOVA followed by Dunnett’s post hoc testing. N = 8 –10 per group.

3.2. WT TAT-UCHL1 treatment decreases CCI-induced Ub-protein accumulation and autophagy activation

To examine the effects of WT TAT-UCHL1 treatment after CCI upon ubiquitin proteasome pathway (UPP) function and autophagy, total, K48-linked, and K63-linked poly-Ub proteins, and the autophagy marker Beclin-1, were detected in hippocampus 24 h after injection by immunoblotting. As shown in Fig. 3, WT TAT-UCHL1 treatment decreased CCI-induced accumulation of total and K48-linked poly-Ub protein levels in ipsilateral hippocampus compared to that of vehicle-treated mice, whereas no significant difference in CCI-induced accumulation of K63-linked poly-Ub protein level was detected. In addition, WT TAT-UCHL1 treatment reduced the accumulation of the autophagy activation marker Beclin-1 after CCI.

Fig. 3. WT TAT-UCHL1 attenuates CCI-induced increase in Poly-ubiquitinated (Poly-Ub) protein accumulation and autophagy activation in hippocampus 24 h post injury.

Fig. 3.

Open in a new tab

Total protein lysates were probed with antibodies detecting total Poly-Ub, K48-linked (K48), K63-linked (K63) Poly-Ub proteins and Beclin-1. Top: Densitometric analysis normalized to contralateral (Y axis indicates the ratio of ipisi / contra). Bottom: Representative immunoblots. GAPDH was used as a loading control. Data are means ± SE. Two sample t-test. *P < 0.05, **P < 0.01, N= 9–10 per group. Veh: vehicle, contra: contralateral, ipsi: ipsilateral.

3.3. WT TAT-UCHL1 treatment attenuates CCI-induced axonal injury

To examine the protective effect of WT TAT-UCHL1 treatment in reducing CCI-induced axonal injury, the accumulation of SMI32, an indicator of non-phosphorylated pathological neurofilaments in neurites after CCI, was detected using immunochemistry. SMI32 accumulation was significantly decreased in ipsilateral cingulate bundle in WT TAT-UCHL1-treated mice compared to vehicle-treated mice 7 d after CCI (Fig. 4).

Fig. 4. WT TAT- UCHL1 reduces CCI-induced axonal damage detected by SMI32 immunostaining.

Fig. 4.

Open in a new tab

Left: SMI32 fluorescence intensity was quantified in ipsilateral cingulate bundle normalized to contralateral at 7 d post injury. Right: Representative images illustrating SMI32- positive staining (at arrows). Data are means ± SE. *P < 0.05, two sample t-test, N=8 – 10 mice per group. Bar = 100 μm. TAT: WT TAT-UCHL1, Veh: vehicle, contra: contralateral, ipsi: ipsilateral.

3.4. WT TAT-UCHL1 treatment improves vestibulomotor function after CCI

The effect of WT TAT-UCHL1 treatment on vestibulomotor function was evaluated using the beam balance test. The CCI-induced deficits observed in vehicle-treated mice were abrogated in WT TAT-UCHL1-treated mice on D1–D5 post injury. No significant difference was detected between vehicle- and WT TAT-UCHL1-treated sham-operated mice (Fig. 5A).

Fig. 5. WT TAT-UCHL1 treatment improves motor function and spatial learning and memory after CCI.

Fig. 5.

Open in a new tab

Mice subjected to CCI or sham-operation were treated with WT TAT-UCHL1 or vehicle 24 h after CCI. a. Beam balance latency was measured on D1 – D5 post injury. Data are means ± SEM. b: Morris Water Maze (MWM) testing was performed on days 14 – 17 post injury. Data are means ± SE. *P < 0.05, **P < 0.01. Two-way repeated ANOVA followed by LSD post hoc analysis. N = 10–16 mice per group. Veh: vehicle, TAT: WT TAT-UCHL1, S: second.

3.5. WT TAT-UCHL1 treatment improves cognitive recovery after CCI

MWM testing was used to examine the effect of WT TAT-UCHL1 treatment on spatial memory acquisition. As shown in Fig. 5B, the swim latencies of WT TAT-UCHL1-treated mice were significantly less than vehicle-treated mice after CCI. There were no significant differences in the swim latencies between WT TAT-UCHL1- and vehicle-treated sham-operated groups. Visual platform evaluation at day 18 revealed no significant differences in swim latencies between WT TAT-UCHL- and vehicle-treated groups after CCI, indicating that there were not any nonspecific deficits contributing to the poor performance of vehicle-treated CCI-operated mice. Probe trial data was not significantly different between groups (data not shown).

3.6. WT TAT-UCHL1 treatment on spared tissue volume after CCI

There was no significant difference in spared tissue volume between vehicle and WT TAT-UCHL1-treated groups detected 7 d after CCI (Fig. 6).

Fig. 6. WT TAT-UCHL1 treatment did not increase spared tissue volume analysis after CCI.

Fig. 6.

Open in a new tab

Spared tissue volume was calculated by measuring surviving brain tissue in ipsilateral and contralateral hemispheric areas at each slice. Left: Spared tissue volume expressed as percent contralateral. Right: Representative H&E-stained Veh- and TAT-treated brain sections. N = 10 per group. Data are means +/− SE. Two sample t-test. Veh: vehicle, TAT: WT TAT-UCHL1.

4. Discussion

The major findings of the study are: 1) WT TAT-UCHL1, mutant C152A and C220A TAT-UCHL1 fusion proteins administered intraperitoneally after CCI cross the blood brain barrier and were detected in brain by immunoblot. Treatment with WT and C152A TAT-UCHL1 decreased axonal injury as detected by NF200 immunostaining, but C220A T-UCHL1 treatment had no significant effect. 2) WT TAT-UCHL1 treatment alleviated CCI-induced axonal injury as detected by SMI32 immunoreactivity 7 d after CCI. 3) Treatment with WT TAT-UCHL1 improved CCI-induced motor and cognitive deficits. 4) Less accumulation of total and K48-linked poly-Ub proteins and autophagy marker Beclin-1 was observed in WT TAT-UCHL1 treated groups compared to contralateral hemisphere after CCI.

TBI is characterized by axonal injury which may lead to devasting neurological impairment (Adams et al., 1984; Frati et al., 2017). UCHL1 is released into the cerebrospinal fluid and blood after TBI and has been employed as a biomarker in patients with suspected TBI (Papa et al., 2016; Papa et al., 2019; Wang et al., 2021). TBI is also accompanied by the generation of reactive lipids and NO, which may modify UCHL1 and impair its function (Koharudin et al., 2010; Kozlov et al., 2017)

The UPP is a key regulator of protein homeostasis and neuroinflammation and plays a central role in many of the cellular processes that are disrupted after TBI (Staal et al., 2009). Emerging evidence suggests that toxic accumulation of misfolded proteins is one of the key pathogenic hallmarks of TBI (McKee and Daneshvar, 2015; Rokad et al., 2017). Marked reduction of free ubiquitin levels and a significant increase in ubiquitin-conjugated protein aggregates in the cerebral cortex and hippocampus have been reported after TBI (Yao et al., 2008).

UCHL1 is highly expressed in neurons and may play a critical role in the neuronal UPP (Day and Thompson, 2010; Wang et al., 2017). UCHL1’s hydrolase activity cleaves Ub from poly-Ub chains allowing Ub to be recycled for additional uses including labeling misfolded or damaged proteins for transport to the proteasome (Liu et al., 2002). UCHL1 is also involved in converting ubiquitin from its pro molecule into its active form (Larsen et al., 1998). In addition, UCHL1 can bind to and stabilize monoubiquitin in neurons (Osaka et al., 2003). UCHL1 dysfunction has been associated with the accumulation of Ub-proteins (Liu et al., 2011; Liu et al., 2015). In the current study we found that treatment with WT TAT-UCHL1 significantly attenuated the levels of total and K48-linked poly-Ub-proteins, but not K63-linked poly-Ub proteins compared to the vehicle-treated group after CCI. The K48-linked poly-Ub modification tags proteins for transport to the proteasome; the K63 poly-Ub linkage is not associated with proteasome degradation but may play a role in endosome transport and other intracellular functions (Todi and Paulson, 2011). These results suggest that WT TAT-UCHL1 treatment enhances UPP function by attenuating accumulation of damaged and misfolded K48-linked poly-Ub-labeled proteins.

In addition to the UPP, clearance of cellular breakdown products and damaged proteins from neurons after TBI is also mediated by autophagy (Clark et al., 2008). Autophagy may be induced when there is failure of the UPP to remove misfolded proteins that form aggregates. Thus, the observation that Beclin-1 expression is attenuated after WT TAT-UCHL1 treatment could also be an indication of weakened activation of macroautophagy as a result of improved UPP function.

UCHL1 is necessary for the maintenance of axonal health and stability (Genc et al., 2022; Mukoyama et al., 1989). UCHL1 is expressed at very high levels in neurons and interacts with cytoskeletal proteins such as tubulin and neurofilaments, suggesting that it may have a structural role (Bheda et al., 2010). UCHL1 may also play an important role in axonal and synaptic function (Chen et al., 2010; Gong et al., 2006; Ichihara et al., 1995; Mukoyama et al., 1989). Mutations and disruption of the UCHL1 gene produce major axonal and dendritic pathology (Jara et al., 2015). The UCHL1-deficient gad mouse is characterized by ‘dying-back’-type axonal degeneration and axonal swelling and formation of spheroid bodies in nerve terminals within the brain (Goto et al., 2009; Saigoh et al., 1999). A human mutation in UCHL1 has been identified that produces extensive white matter changes and progressive motor deficits (Bilguvar et al., 2013). Thus, UCHL1 may be required for normal axonal structure and function. In the current study we found that treatment with TAT- UCHL1 decreases the amount of axonal injury detected both by NF200 and SMI32 after CCI but there was no significant effect of TAT-UCHL1 treatment on contusion volume. These data suggest that the functional improvement in TAT-UCHL1-treated mice was due primarily to effects on axonal injury rather than gray matter injury.

UCHL1 has a number of posttranslational modification sites that may be important in its function (Castegna et al., 2002; Clarke and Tamanoi, 2004; Koharudin et al., 2010; Nakamura et al., 2021). The C152 of UCHL1 is modified by nitric oxide and reactive lipids such as cyclopentenone prostaglandins resulting in a structure change, partial loss of activity, and aggregation (Koharudin et al., 2010; Nakamura et al., 2021). Since reactive lipids and NO concentrations increase after TBI (Kunz et al., 2002; Villalba et al., 2014), ligation by these substrates at the C152 cite may result in decreased UCHL1 activity and aggregation of the protein after TBI. C152A TAT-UCHL1 maintains normal hydrolase activity but is resistant to ligation by reactive lipids or NO; thus it may be resistant to aggregation and loss of activity after TBI. In addition, mice bearing the UCHL1 C152A mutation have less axonal injury after stroke and TBI suggesting that this modification may increase the potency of wild type TAT-UCHL1 (Liu et al., 2019; Mi et al., 2021a). Thus, we expected that treatment with the C152A TAT-UCHL1 would protect axons against injury but might have more potent effects than treatment with WT TAT-UCHL1. Both WT TAT-UCHL1 and C152A TAT-UCHL1 are detectable in brain up to 24 h after CCI, so the C152A modification does not appear to significant delay the metabolism of TAT-UCHL1 in brain. In addition, there was no significant difference between the efficacy of wild type TAT- and C152A TAT-UCHL1 in reducing axonal injury. The C152A modification does not appear to increase the potency of TAT-UCHL1 in reducing axonal injury after CCI.

The cysteine 220 of UCHL1 may be farnesylated resulting in translocation of UCHL1 to the membrane (Liu et al., 2009). This modification potentiates α-synuclein toxicity but the role of farnesylation at this site in normal and pathological conditions is not well understood. Since the farnesylation of cysteine 220 may potentiate α-synuclein toxicity we surmised that this mechanism could limit the protective effects of TAT-UCHL1 treatment and thus the C220A modification might increase the protective potency of wild type TAT-UCHL1. However, C220A TAT-UCHL1 treatment did not significantly reduce axonal injury after CCI as detected by NF200 immunocytochemistry compared to vehicle controls. Thus, these data suggest that the cysteine 220 cite may be necessary for the protective effects of TAT-UCHL1 treatment rather than mediating toxicity. Mice bearing a mutation in the C90 hydrolase activity site do not develop the progressive motor impairment and premature death that occur in UCHL1 null mice (Mi et al., 2021a). The C220A AT-UCHL1 fusion protein maintains its hydrolase activity (Fig 1C); thus, the cysteine 220 site may be required for neuroprotective mechanisms that are independent from UCHL1’s hydrolase activity.

In conclusion, treatment with WT TAT-UCHL1 fusion protein up to 24 h after CCI in mice reduces white matter injury and improves both motor and cognitive functional outcomes. Treatment with WT or C152A TAT-UCHL1 fusion proteins were both effective in reducing axonal injury after CCI, but C220A TAT-UCHL1 was not. Additional studies are needed to further investigate delayed treatment with TAT-UCHL1 fusion protein and to investigate the mechanisms by which TAT-UCHL1 decreases white matter injury after TBI.

  • UCHL1 fusion proteins administered systemically are detectable in mouse brain

  • WT and C152A but not C220A TAT-UCHL1 decreased CCI-induced axonal injury

  • WT TAT-UCHL1 improved CCI-induced motor and cognitive deficits

  • WT TAT-UCHL1 reduced CCI-induced accumulation of total and K48 poly-Ub proteins

  • WT TAT-UCHL1 attenuated the increase of the autophagy marker Beclin-1 after CCI

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

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Declaration 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.

References

  1. Adams JH, Doyle D, Graham DI, Lawrence AE, McLellan DR, 1984. Diffuse axonal injury in head injuries caused by a fall. Lancet 2, 1420–1422. [DOI] [PubMed] [Google Scholar]
  2. Adelson PD, Fellows-Mayle W, Kochanek PM, Dixon CE, 2013. Morris water maze function and histologic characterization of two age-at-injury experimental models of controlled cortical impact in the immature rat. Childs Nerv Syst 29, 43–53. [DOI] [PubMed] [Google Scholar]
  3. Bheda A, Gullapalli A, Caplow M, Pagano JS, Shackelford J, 2010. Ubiquitin editing enzyme UCH L1 and microtubule dynamics: implication in mitosis. Cell cycle (Georgetown, Tex 9, 980–994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bilguvar K, Tyagi NK, Ozkara C, Tuysuz B, Bakircioglu M, Choi M, Delil S, Caglayan AO, Baranoski JF, Erturk O, Yalcinkaya C, Karacorlu M, Dincer A, Johnson MH, Mane S, Chandra SS, Louvi A, Boggon TJ, Lifton RP, Horwich AL, Gunel M, 2013. Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration. Proc Natl Acad Sci U S A 110, 3489–3494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buki A, Koizumi H, Povlishock JT, 1999. Moderate posttraumatic hypothermia decreases early calpain-mediated proteolysis and concomitant cytoskeletal compromise in traumatic axonal injury. Exp Neurol 159, 319–328. [DOI] [PubMed] [Google Scholar]
  6. Cao G, Pei W, Ge H, Liang Q, Luo Y, Sharp FR, Lu A, Ran R, Graham SH, Chen J, 2002. In Vivo Delivery of a Bcl-xL Fusion Protein Containing the TAT Protein Transduction Domain Protects against Ischemic Brain Injury and Neuronal Apoptosis. J Neurosci 22, 5423–5431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Castegna A, Aksenov M, Aksenova M, Thongboonkerd V, Klein JB, Pierce WM, Booze R, Markesbery WR, Butterfield DA, 2002. Proteomic identification of oxidatively modified proteins in Alzheimer’s disease brain. Part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. Free Radic Biol Med 33, 562–571. [DOI] [PubMed] [Google Scholar]
  8. Chen F, Sugiura Y, Myers KG, Liu Y, Lin W, 2010. Ubiquitin carboxyl-terminal hydrolase L1 is required for maintaining the structure and function of the neuromuscular junction. Proc Natl Acad Sci U S A 107, 1636–1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clark RS, Bayir H, Chu CT, Alber SM, Kochanek PM, Watkins SC, 2008. Autophagy is increased in mice after traumatic brain injury and is detectable in human brain after trauma and critical illness. Autophagy 4, 88–90. [DOI] [PubMed] [Google Scholar]
  10. Clarke S, Tamanoi F, 2004. Fighting cancer by disrupting C-terminal methylation of signaling proteins. J Clin Invest 113, 513–515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Day IN, Thompson RJ, 2010. UCHL1 (PGP 9.5): neuronal biomarker and ubiquitin system protein. Prog Neurobiol 90, 327–362. [DOI] [PubMed] [Google Scholar]
  12. Dixon CE, Clifton GL, Lighthall JW, Yaghmai AA, Hayes RL, 1991. A controlled cortical impact model of traumatic brain injury in the rat. J Neurosci Methods 39, 253–262. [DOI] [PubMed] [Google Scholar]
  13. Dixon CE, Lyeth BG, Povlishock JT, Findling RL, Hamm RJ, Marmarou A, Young HF, Hayes RL, 1987. A fluid percussion model of experimental brain injury in the rat. J Neurosurg 67, 110–119. [DOI] [PubMed] [Google Scholar]
  14. Feeney DM, Boyeson MG, Linn RT, Murray HM, Dail WG, 1981. Responses to cortical injury: I. Methodology and local effects of contusions in the rat. Brain Res 211, 67–77. [DOI] [PubMed] [Google Scholar]
  15. Frati A, Cerretani D, Fiaschi AI, Frati P, Gatto V, La Russa R, Pesce A, Pinchi E, Santurro A, Fraschetti F, Fineschi V, 2017. Diffuse Axonal Injury and Oxidative Stress: A Comprehensive Review. Int J Mol Sci 18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Genc B, Jara JH, Sanchez SS, Lagrimas AKB, Gozutok O, Kocak N, Zhu Y, Hande Ozdinler P, 2022. Upper motor neurons are a target for gene therapy and UCHL1 is necessary and sufficient to improve cellular integrity of diseased upper motor neurons. Gene Ther 29, 178–192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gong B, Cao Z, Zheng P, Vitolo OV, Liu S, Staniszewski A, Moolman D, Zhang H, Shelanski M, Arancio O, 2006. Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory. Cell 126, 775–788. [DOI] [PubMed] [Google Scholar]
  18. Goto A, Wang YL, Kabuta T, Setsuie R, Osaka H, Sawa A, Ishiura S, Wada K, 2009. Proteomic and histochemical analysis of proteins involved in the dying-back-type of axonal degeneration in the gracile axonal dystrophy (gad) mouse. Neurochem Int 54, 330–338. [DOI] [PubMed] [Google Scholar]
  19. Ichihara N, Wu J, Chui DH, Yamazaki K, Wakabayashi T, Kikuchi T, 1995. Axonal degeneration promotes abnormal accumulation of amyloid beta-protein in ascending gracile tract of gracile axonal dystrophy (GAD) mouse. Brain Res 695, 173–178. [DOI] [PubMed] [Google Scholar]
  20. Jara JH, Genc B, Cox GA, Bohn MC, Roos RP, Macklis JD, Ulupinar E, Ozdinler PH, 2015. Corticospinal Motor Neurons Are Susceptible to Increased ER Stress and Display Profound Degeneration in the Absence of UCHL1 Function. Cerebral cortex 25, 4259–4272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Johnson VE, Stewart W, Smith DH, 2013. Axonal pathology in traumatic brain injury. Exp Neurol 246, 35–43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kabuta T, Setsuie R, Mitsui T, Kinugawa A, Sakurai M, Aoki S, Uchida K, Wada K, 2008. Aberrant molecular properties shared by familial Parkinson’s disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1. Hum Mol Genet 17, 1482–1496. [DOI] [PubMed] [Google Scholar]
  23. Koharudin LM, Liu H, Di Maio R, Kodali RB, Graham SH, Gronenborn AM, 2010. Cyclopentenone prostaglandin-induced unfolding and aggregation of the Parkinson disease-associated UCH-L1. Proc Natl Acad Sci U S A 107, 6835–6840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kozlov AV, Bahrami S, Redl H, Szabo C, 2017. Alterations in nitric oxide homeostasis during traumatic brain injury. Biochim Biophys Acta Mol Basis Dis 1863, 2627–2632. [DOI] [PubMed] [Google Scholar]
  25. Kunz T, Marklund N, Hillered L, Oliw EH, 2002. Cyclooxygenase-2, prostaglandin synthases, and prostaglandin H2 metabolism in traumatic brain injury in the rat. J Neurotrauma 19, 1051–1064. [DOI] [PubMed] [Google Scholar]
  26. Larsen CN, Krantz BA, Wilkinson KD, 1998. Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases. Biochemistry 37, 3358–3368. [DOI] [PubMed] [Google Scholar]
  27. Liu H, Li W, Ahmad M, Miller TM, Rose ME, Poloyac SM, Uechi G, Balasubramani M, Hickey RW, Graham SH, 2011. Modification of ubiquitin-C-terminal hydrolase-L1 by cyclopentenone prostaglandins exacerbates hypoxic injury. Neurobiol Dis 41, 318–328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liu H, Li W, Rose ME, Hickey RW, Chen J, Uechi GT, Balasubramani M, Day BW, Patel KV, Graham SH, 2015. The point mutation UCH-L1 C152A protects primary neurons against cyclopentenone prostaglandin-induced cytotoxicity: implications for post-ischemic neuronal injury. Cell death & disease 6, e1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liu H, Povysheva N, Rose ME, Mi Z, Banton JS, Li W, Chen F, Reay DP, Barrionuevo G, Zhang F, Graham SH, 2019. Role of UCHL1 in axonal injury and functional recovery after cerebral ischemia. Proc Natl Acad Sci U S A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liu H, Rose ME, Ma X, Culver S, Dixon CE, Graham SH, 2017. In vivo transduction of neurons with TAT-UCH-L1 protects brain against controlled cortical impact injury. PloS one 12, e0178049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu Y, Fallon L, Lashuel HA, Liu Z, Lansbury PT Jr., 2002. The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson’s disease susceptibility. Cell 111, 209–218. [DOI] [PubMed] [Google Scholar]
  32. Liu Z, Meray RK, Grammatopoulos TN, Fredenburg RA, Cookson MR, Liu Y, Logan T, Lansbury PT Jr., 2009. Membrane-associated farnesylated UCH-L1 promotes alpha-synuclein neurotoxicity and is a therapeutic target for Parkinson’s disease. Proc Natl Acad Sci U S A 106, 4635–4640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Marmarou CR, Povlishock JT, 2006. Administration of the immunophilin ligand FK506 differentially attenuates neurofilament compaction and impaired axonal transport in injured axons following diffuse traumatic brain injury. Exp Neurol 197, 353–362. [DOI] [PubMed] [Google Scholar]
  34. McKee AC, Daneshvar DH, 2015. The neuropathology of traumatic brain injury. Handb Clin Neurol 127, 45–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mi Z, Liu H, Rose ME, Ma J, Reay DP, Ma X, Henchir JJ, Dixon CE, Graham SH, 2021a. 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. Neuroscience 475, 127–136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mi Z, Liu H, Rose ME, Ma X, Reay DP, Ma J, Henchir J, Dixon CE, Graham SH, 2021b. Abolishing UCHL1’s hydrolase activity exacerbates TBI-induced axonal injury and neuronal death in mice. Exp Neurol 336, 113524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mukoyama M, Yamazaki K, Kikuchi T, Tomita T, 1989. Neuropathology of gracile axonal dystrophy (GAD) mouse. An animal model of central distal axonopathy in primary sensory neurons. Acta Neuropathol 79, 294–299. [DOI] [PubMed] [Google Scholar]
  38. Nakamura T, Oh CK, Liao L, Zhang X, Lopez KM, Gibbs D, Deal AK, Scott HR, Spencer B, Masliah E, Rissman RA, Yates JR 3rd, Lipton SA, 2021. Noncanonical transnitrosylation network contributes to synapse loss in Alzheimer’s disease. Science 371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Osaka H, Wang YL, Takada K, Takizawa S, Setsuie R, Li H, Sato Y, Nishikawa K, Sun YJ, Sakurai M, Harada T, Hara Y, Kimura I, Chiba S, Namikawa K, Kiyama H, Noda M, Aoki S, Wada K, 2003. Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron. Hum Mol Genet 12, 1945–1958. [DOI] [PubMed] [Google Scholar]
  40. Papa L, Brophy GM, Welch RD, Lewis LM, Braga CF, Tan CN, Ameli NJ, Lopez MA, Haeussler CA, Mendez Giordano DI, Silvestri S, Giordano P, Weber KD, Hill-Pryor C, Hack DC, 2016. Time Course and Diagnostic Accuracy of Glial and Neuronal Blood Biomarkers GFAP and UCH-L1 in a Large Cohort of Trauma Patients With and Without Mild Traumatic Brain Injury. JAMA Neurol 73, 551–560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Papa L, Zonfrillo MR, Welch RD, Lewis LM, Braga CF, Tan CN, Ameli NJ, Lopez MA, Haeussler CA, Mendez Giordano D, Giordano PA, Ramirez J, Mittal MK, 2019. Evaluating glial and neuronal blood biomarkers GFAP and UCH-L1 as gradients of brain injury in concussive, subconcussive and non-concussive trauma: a prospective cohort study. BMJ Paediatr Open 3, e000473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rokad D, Ghaisas S, Harischandra DS, Jin H, Anantharam V, Kanthasamy A, Kanthasamy AG, 2017. Role of neurotoxicants and traumatic brain injury in alpha-synuclein protein misfolding and aggregation. Brain Res Bull 133, 60–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Saigoh K, Wang YL, Suh JG, Yamanishi T, Sakai Y, Kiyosawa H, Harada T, Ichihara N, Wakana S, Kikuchi T, Wada K, 1999. Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice. Nat Genet 23, 47–51. [DOI] [PubMed] [Google Scholar]
  44. Schneider CA, Rasband WS, Eliceiri KW, 2012. NIH Image to ImageJ: 25 years of image analysis. Nature methods 9, 671–675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Staal JA, Dickson TC, Chung RS, Vickers JC, 2009. Disruption of the ubiquitin proteasome system following axonal stretch injury accelerates progression to secondary axotomy. J Neurotrauma 26, 781–788. [DOI] [PubMed] [Google Scholar]
  46. Swanson RA, Shiraishi K, Morton MT, Sharp FR, 1990. Methionine sulfoximine reduces cortical infarct size in rats after middle cerebral artery occlusion. Stroke 21, 322–327. [DOI] [PubMed] [Google Scholar]
  47. Todi SV, Paulson HL, 2011. Balancing act: deubiquitinating enzymes in the nervous system. Trends Neurosci 34, 370–382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Villalba N, Sonkusare SK, Longden TA, Tran TL, Sackheim AM, Nelson MT, Wellman GC, Freeman K, 2014. Traumatic brain injury disrupts cerebrovascular tone through endothelial inducible nitric oxide synthase expression and nitric oxide gain of function. J Am Heart Assoc 3, e001474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang KK, Yang Z, Sarkis G, Torres I, Raghavan V, 2017. Ubiquitin C-terminal hydrolase-L1 (UCH-L1) as a therapeutic and diagnostic target in neurodegeneration, neurotrauma and neuro-injuries. Expert Opin Ther Targets 21, 627–638. [DOI] [PubMed] [Google Scholar]
  50. Wang KKW, Kobeissy FH, Shakkour Z, Tyndall JA, 2021. Thorough overview of ubiquitin C-terminal hydrolase-L1 and glial fibrillary acidic protein as tandem biomarkers recently cleared by US Food and Drug Administration for the evaluation of intracranial injuries among patients with traumatic brain injury. Acute Med Surg 8, e622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Weber MT, Arena JD, Xiao R, Wolf JA, Johnson VE, 2019. CLARITY reveals a more protracted temporal course of axon swelling and disconnection than previously described following traumatic brain injury. Brain Pathol 29, 437–450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Whalen MJ, Clark RS, Dixon CE, Robichaud P, Marion DW, Vagni V, Graham SH, Virag L, Hasko G, Stachlewitz R, Szabo C, Kochanek PM, 1999. Reduction of cognitive and motor deficits after traumatic brain injury in mice deficient in poly(ADP-ribose) polymerase. J Cereb Blood Flow Metab 19, 835–842. [DOI] [PubMed] [Google Scholar]
  53. Wilson PO, Barber PC, Hamid QA, Power BF, Dhillon AP, Rode J, Day IN, Thompson RJ, Polak JM, 1988. The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br J Exp Pathol 69, 91–104. [PMC free article] [PubMed] [Google Scholar]
  54. Yao X, Liu J, McCabe JT, 2008. Alterations of cerebral cortex and hippocampal proteasome subunit expression and function in a traumatic brain injury rat model. J Neurochem 104, 353–363. [DOI] [PubMed] [Google Scholar]

RESOURCES