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TDP-43 proteolysis is associated with astrocyte reactivity after traumatic brain injury in rodents
Accepted Manuscript TDP-43 proteolysis is associated with astrocyte reactivity after traumatic brain injury in rodents
Chih-Yuan Huang, Yi-Che Lee, Ping-Chia Li, Po-Chou Liliang, Kang Lu, Kuo-Wei Wang, Li-Ching Chang, Li-Yen Shiu, MingFeng Chen, Yuan-Ting Sun, Hao-kuang Wang PII: DOI: Reference:
S0165-5728(17)30394-6 doi:10.1016/j.jneuroim.2017.10.011 JNI 476647
To appear in:
Journal of Neuroimmunology
Received date: Revised date: Accepted date:
30 August 2017 14 October 2017 16 October 2017
Please cite this article as: Chih-Yuan Huang, Yi-Che Lee, Ping-Chia Li, Po-Chou Liliang, Kang Lu, Kuo-Wei Wang, Li-Ching Chang, Li-Yen Shiu, Ming-Feng Chen, Yuan-Ting Sun, Hao-kuang Wang , TDP-43 proteolysis is associated with astrocyte reactivity after traumatic brain injury in rodents. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jni(2017), doi:10.1016/ j.jneuroim.2017.10.011
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Research report TDP-43 proteolysis is associated with astrocyte reactivity after traumatic brain injury in rodents
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Abbreviated title: TDP-43 proteolysis is associated with reactive astrocyte
Chih-Yuan Huang,1 Yi-Che Lee,2 Ping-Chia Li,3 Po-Chou Liliang,3,4 Kang Lu,3,4
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Kuo-Wei Wang,3,4 Li-Ching Chang,3 Li-Yen Shiu,5 Ming-Feng Chen,5 Yuan-Ting Sun,6
Neurosurgical Service, Department of Surgery, National Cheng Kung University
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1
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and Hao-kuang Wang3,4*
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Hospital, Tainan, Taiwan
Department of Nephrology, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan
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School of Medicine for International Students, I-Shou University, Kaohsiung, Taiwan.
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Department of Neurosurgery, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan
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Department of Medical Research, Cell Therapy and Research Center, E-Da Hospital,
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I-shou University, Kaohsiung, Taiwan. Department of Neurology, National Cheng Kung University Hospital, College of
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Medicine, National Cheng Kung University, Tainan, Taiwan
Address correspondence to:
Hao kuang Wang, MD, PhD Department of Neurosurgery, E-Da Hospital/ I-Shou University, Taiwan No.1, Yida Road, Jiaosu Village, Yanchao District, Kaohsiung City 82445, Taiwan
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Tel: +866-975106080 E-mail: [email protected], [email protected]
Word count for abstract: 162, Number of words: 3089
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Number of references: 27, Number of figures: 6
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Keywords: astrocyte; astrogliosis; traumatic brain injury; transactivation response
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DNA-binding protein 43.
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Abbreviations: Amyotrophic lateral sclerosis
ANOVA
A two-way analysis of variance
FTLD GFAP
Frontotemporal lobar degeneration Glial fibrillary acidic protein
TBI
Traumatic brain injury
TDP-43 TX
Transactivation response DNA-binding protein 43 High-salt Triton X-100
SARK
Sarkosyl
z-DEVD-fmk
N-benzyloxycarbonyl-Asp-Glu-Val-Asp- fluoromethyl ketone
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ALS
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Abstract The aggregation and deposition of transactivation response DNA-binding protein 43 (TDP-43) in neurons and astrocytes is characteristic in a number of neurodegenerative diseases including Alzheimer ’s disease, frontotemporal lobar degeneration, and
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amyotrophic lateral sclerosis. Nevertheless, the exact role of TDP-43 in astrocytes is unknown. Recently, TDP-43 was identified in neurons but not astrocytes after traumatic
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brain injury (TBI) in humans. In the present study, we evaluated TDP-43 expression and
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proteolysis in astrocytes in a rat model of TBI. We assessed TDP-43 fragment expression, astrocyte morphology, neuronal population numbers, and motor function
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after TBI with or without intracerebroventricular administration of a caspase-3 inhibitor.
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Motor dysfunction was observed after TBI in potential association astrocytic TDP-43 short fragment mislocalization and accumulation, astrogliosis, and neuronal loss.
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Notably, caspase-3 inhibition prevented these changes after TBI. Our findings suggest that TDP-43 proteolysis in astrocytes is related to astrogliosis and subsequent neuronal
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loss in TBI, and that TDP-43 may be an important therapeutic target for preventing
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motor dysfunction after TBI.
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1. Introduction Transactive response DNA binding protein (TDP-43) is a heterogeneous nuclear RNA-binding protein that regulates gene expression, transcription, and multiple aspects of RNA processing and functions including splicing, stability, transport, translation, and
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microRNA maturation (Sieben et al., 2012; Al-Chalabi et al., 2012; Gendron et al., 2013). Under physiological conditions, TDP-43 is enriched in the nucleus; however,
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TDP-43 can be cleaved by caspase-3 to generate short 25-kDa and 35-kDa C-terminal
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fragments, resulting in the formation of ubiquitin-positive cytoplasmic inclusions, cellular toxicity, and ultimately death (Zhang et al., 2009; Barmada et al., 2010; Che et
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al., 2011). TDP-43 was first identified in neuronal cytoplasmic inclusions in
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frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) in 2006 (Sieben et al., 2012; Al-Chalabi et al., 2012). Recently pathological TDP-43
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inclusions were also identified in Alzheimer’s disease (Fang et al., 2014). Thus, TDP-43 accumulation and deposition is presumed to play a role in the pathogenesis of
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neurodegenerative diseases.
Traumatic brain injury (TBI) is a leading cause of death and disability that has a
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limited number of clinical treatment options (Maas et al., 2008). TBI leads to the activation of multiple inflammatory pathways including astrocyte activation (Blennow et al., 2012). Whereas astrocytes play neurotrophic and supportive roles in nervous tissues under physiological conditions (Pekny and Pekna, 2014; Pekny and Wilhelmsson, 2014), astrocytes are subject to hypertrophy and proliferation after TBI that can lead to a number of positive (e.g., neural protection and repair) and negative (e.g., glial scarring and alterations in plasticity) effects; yet, the role of astrocytes after TBI is not well understood. Moreover, few studies have investigated a role for TDP-43 in TBI.
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Nuclear depletion and cytoplasmic aggregation of TDP-43 has been documented in astrocytes; however, the toxicity of TDP-43 fragments in astrocytes is different from that observed in neurons (Haidet-Phillips et al., 2013; Serio et al., 2013; Tong et al., 2013; Rojas et al., 2014). Haidet-Phillips et al. showed that TDP-43 overexpression or
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knockout in astrocytes did not produce a cytotoxic phenotype (Haidet-Phillips et al., 2013). Additionally, it is not clear as to whether pathogenic TDP-43 accumulation in
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astrocytes causes non-cell-autonomous neuronal death (Haidet-Phillips et al., 2013;
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Serio et al., 2013; Tong et al., 2013; Rojas et al., 2014); whereas some evidence suggests that astrocytes expressing mutated forms of TDP43 can cause the
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non-cell-autonomous death of motor neurons (Tong et al., 2013; Rojas et al., 2014),
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other evidence contradicts these findings (Haidet-Phillips et al., 2013; Serio et al., 2013).
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In the present study, we investigated whether TBI was associated with alterations in TDP-43 expression and cleavage in astrocytes using a rodent model. Rats were
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treated with a caspase-3 inhibitor or vehicle immediately after TBI and TDP-43 fragment expression, astrocyte morphology, neuronal population numbers, and motor
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function were examined at up to 7 days post-injury.
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2. Materials and methods 2.1 Ethical statement All procedures were reviewed and approved by the Institution Animal Care and
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Use Committee(IACUC) of E-Da hospital (IACUC), and conformed to the guidelines of
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the Taiwan Council for Animal Care. The number of IACUC is 104004.
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2.2 Study design
Adult male Sprague-Dawley rats weighing 300–350 g were purchased from
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Charles River Laboratories. Then those rats were maintained at constant temperature (22 ± 2°C) and a 12 hour light/dark cycle with food and water available ad libitum for 1
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week before procedures. Details on the injury device, TBI model, and behavioral tests
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were previously reported (Wang et al., 2015). Table 1 showing the time-line diagram of TBI with experimental procedure. Finally, animals were divided into three groups to examine whether TBI induced the cleavage and aggregation of TDP-43 species in lesion
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sites and specifically in astrocytes.
2.3 Experimental animals 2.3.1 Traumatic brain injury model. TBI was induced using a weight drop device as previously established in the Feeney weight-drop model (Feeney et al., 1981, Liliang et al., 2010; Wang et al., 2015).
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Briefly, rats were anesthetized, the skull was exposed, and a left partial craniotomy was performed. A 40-g steel rod with a flat end was freely dropped from 30 cm onto strike a transducer rod (diameter, 4.5 mm) that was oriented with its tip placed directly onto the rat’s skull, posterior to bregma (Feeney et al., 1981, Liliang et al., 2010; Wang et al.,
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2015). Rats were anesthetized and underwent left parietal craniotomy without cortical contusion injury. After postoperative procedures, rats were returned to their home cages
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and monitored carefully.
2.3.2 Intracerebroventricular administration of a caspase-3 inhibitor.
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Rats were placed in a stereotaxic apparatus for intracerebroventricular
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administration. The cell-permeable and irreversible caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val- Asp- fluoromethyl ketone (EMD Millipore, Billerica, MA) was dissolved in 0.2% DMSO (500 ng in 1 μL of DMSO) (Clark et al., 2000;
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Knoblach et al., 2004). The caspase-3 inhibitor (2 μL) or vehicle (2 μL) was
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administered by intracerebroventricular injection ipsilateral to the lesion site at 10 min
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post-TBI via a micro-osmotic pump and infusion cannula.
2.4 Experimental procedures 2.4.1 Western blotting.
To evaluate protein expression levels in the cerebral cortical regions surrounding TBI lesions, protein extracts were obtained by homogenization of cortical tissues in RIPA lysis buffer (Clark et al., 2000; Knoblach et al., 2004; Wang et al., 2012), samples were electrophoresed and transferred to blotting membranes, and proteins were probed
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with the following primary antibodies: anti-TDP-43 (1:5000; Proteintech), anti-caspase-3 (1:2000; Cell Signaling), and anti-actin (1:15,000; Millipore). The anti-TDP-43 antibody used in this study recognized not only the full- length TDP-43 product but also 25–35 kDa cleavage products and phosphorylated forms of TDP-43.
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The anti-caspase-3 antibody recognized the activated form caspase-3 resulting from
expressed as the mean ± standard error of the mean.
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cleavage at Asp175. The relative intensities of bands were normalized to actin and
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For the analysis of TDP-43 in cellular inclusions, brain tissues were dissected and
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proteins were extracted with specialized buffers (Wang et al., 2012). Low-salt buffer, high-salt Triton X-100 buffer, myelin floatation buffer, and sarkosyl buffer were used to
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extract proteins from tissues at 5 mL/g (volume/weight). Sarkosyl-insoluble materials were further extracted in 0.25 mL/g urea buffer. Urea-soluble proteins were analyzed by
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Western blot analysis with the above primary antibodies.
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2.4.2 Immunohistochemistry.
For immunohistostaining, brain sections were stained with anti–TDP-43 (1:50;
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Gene Tex), glial fibrillary acidic protein (GFAP) (1:100, Abcam),anti–caspase-3 (1:100;Cell Signaling) or antineuron-specific nuclear protein (NeuN) (Millipore), and then with biotin-conjugated secondary antibodies (Millipore) followed by detection with 3,3′ diaminobenzidine (DAB) (Millipore) (Wang et al., 2012). For immunofluorescence staining, coronal brain sections were stained with anti-LC3 antibody (1:200, Novus Biologicals). DAPI staining was used to locate the nuclei. 2.4.3 Motor function assessments.
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We used the beam-walking test to evaluate the recovery of motor function after TBI (Loane et al. 2009; Wang et al. 2012). A basal level of competence (< 10 faults over 50 steps) was established before surgery. Then, the beam walk task was performed at 1, 3, and 7 days post-injury.
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The rotarod test was used to assess the motor coordination after TBI (Loane et al.,
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2009; Wang et al., 2014). Each rat received a training session on the rotarod apparatus at a constant speed of 8 rpm before surgery. Test trials were conducted at 1, 3, and 7 days
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post-injury.
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2.4.4 Flow cytometry
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For quantifying the survival of neuronal number, whole ipsilateral semi-brain images were acquired by TissueFAXS and analyzed by TissueQuest (TissueGnostics,
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Viena, Austria) as previously described (Wang et al., 2012). Stereologic evaluation of ipsilateral semi-brain was conducted on every 12th section throughout the forebrain
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with the damaged region by the Cavalieri principle. Flow cytometry- like data (dot-plot) were analyzed and organized by TissueQuest, with each dot on the plot representing
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intensity of NeuN and Dapi from a single cell. The plot accumulated the values of all cells from the semi-brain, and then the positive signal number could be identified by gating the immunoreactive intensity. Neuronal cell counts were comparable between the sham and caspase-3 inhibitor-treated TBI groups.
2.5 Housing and husbandry Rats were housed in the Animal Center of I-Shou University, Kaohsiung, Taiwan
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and maintained on a 12:12-h light/dark cycle (lights on at 07:00) in a room with controlled temperature (22 ± 2°C) and humidity (60 ± 5%), with ad libitum access to food and water. Rats were allowed to acclimate for at least 1 week before the
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experimental sessions.
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2.6 Sample size
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Adult male Sprague-Dawley rats weighing 300–350 g were purchased from
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Charles River Laboratories (Wang et al., 2015). Rats were randomly divided into 3 groups: TBI with vehicle treatment (TBI, n = 27), TBI with caspase-3 inhibitor
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treatment (TBI + CI, n = 12), and sham group (sham, n = 5) (Figure 1A). In TBI group, an equal amount of saline was injected. In sham group, no procedure was performed.
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All the experiments were conducted and analyzed in blinded manner.
2.7 Allocating animals to experimental groups
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Cage randomization was used in our study. On the day of surgery, a cage will be selected randomly from the pool of all cages containing animals eligible for inclusion in the study, regardless of arrival date. Primary randomization is conducted by an individual other than the surgeon. On the basis of their position on the rack, cages are given a temporary numerical designation for the purpose of primary randomization.
2.8 Experimental outcomes
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The primary endpoints of the present study were the cleavage and aggregation of TDP-43 species in lesion sites and specifically in astrocytes. Then we assessed TDP-43 fragment expression, astrocyte morphology, neuronal population numbers, and motor function after TBI with or without intracerebroventricular administration of a caspase-3
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inhibitor.
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2.9 Statistical methods
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Results are reported as the mean ± standard error of the mean. To examine whether the data is normally distributed, D`Agostino and Pearson omnibus normality test was
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used before single mean or multiple mean comparison analysis. All data were confirmed that were normally distributed. Statistical differences were analyzed using a 2-way
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analysis of variance (ANOVA) followed by post hoc Bonferroni tests for multiple comparisons. For 2-group comparisons, data were analyzed using unpaired t-tests.
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Differences were considered to be statistically significant when P < 0.05.
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3. Results 3.1 Cleavage products of TDP-43 are increased in astrocytes after TBI Whole-cell extracts from lesion sites were immunoblotted to investigate changes in TDP-43 expression post-injury. Levels of the 25-kDa and 35-kDa fragments of TDP-43
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were increased whereas full- length TDP-43 (43 kDa) was decreased at 7 days post-injury (Figure 2A). Additionally, glial fibrillary acidic protein (GFAP) was
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co-localized with TDP-43 and its cleavage products in morphologically activated
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astrocytes at 7 days post-injury (Figure 2B). Next, we examined urea-soluble fractions of forebrain extracts to investigate the presence of cytoplasmic TDP-43 inclusions.
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Cleavage products including the 25-kDa and 35-kDa fragments of TDP-43 were
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upregulated at 7 days post-injury (Figure 2C). These data suggested that TBI induced the cleavage and aggregation of TDP-43 species in lesion sites and specifically in
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astrocytes.
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3.2 Caspase-3 is activated in astrocytes after TBI Next, we performed immunoblotting to quantify the expression levels of activated
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caspase-3 in cortical extracts post-injury. Activated caspase-3 expression was increased in injured cortex extracts at 3 and 7 days post-injury (Figure 3A). Additionally, GFAP was co-localized with activated caspase-3 in morphologically activated astrocytes at 7 days post-injury (Figure 3B). Thus, it was concluded that TBI induced caspase-3 activation in astrocytes.
3.3 Caspase-3 inhibitor treatment decreases TDP-43 cleavage and astrocyte activation after TBI
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Intracerebroventricular administration of a caspase-3 inhibitor immediately after TBI resulted in a 50% reduction in expression of the 35-kDa fragment of TDP-43 (Figure 4A) and a 30% reduction in expression of activated caspase-3 in injured cortex extracts at 7 days post-injury (Figure 4B); however, there was no obvious effect on the
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25-kDa fragment of TDP-43. Moreover, caspase-3 inhibitor treatment prevented the development of astrogliosis after TBI: non-reactive astrocyte morphology was observed
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in inhibitor-treated animals at 7 days post-injury (Figure 4C).
3.4 Motor dysfunction after TBI is sensitive to caspase-3 inhibition
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To assess motor function, rotarod testing and beam walk testing were performed at
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1, 3, and 7 days post-injury. Rotarod test latencies (Figure 5A) and beam walking latencies (Figure 5B) were significantly shorter in TBI rats compared to rats in the sham
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group or TBI rats treated with the caspase-3 inhibitor. Moreover, we analyzed foot placement accuracy (number of errors) and found that TBI rats treated with the
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caspase-3 inhibitor performed significantly better on the right (injury) side than did untreated TBI rats (Figure 5C). However, no between-group differences were observed
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for the left side. These results suggested that caspase-3 inhibition prevented the short-term impairment of motor coordination and balance after TBI.
3.5 Caspase-3 inhibition promotes neuronal survival after TBI To investigate whether neuronal death after TBI was sensitive to caspase-3 inhibition and potentially related to the presence of TDP-43 cleavage products in reactive astrocytes, we conducted immunohistochemistry staining for NeuN and Dapi and quantified the neuronal number by a flow cytometry- like analysis system
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(TissueFAX and TissueQuest) in the ipsilateral semi-brain at 7 days after TBI dmage. Neuronal cell counts were comparable between the sham and caspase-3 inhibitor-treated TBI groups by TissueQuest-based dot plot (Figure 6A). The quantified result indicated that TBI significantly leaded neuronal loss in the ipsilateral semi-brain at 7 days after
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TBI, but caspase-3 inhibitor improved the neuronal survival of TBI rats, a higher level of survived neuron were observed in caspase-3 inhibitor treated TBI rats (n = 5 rats per
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group).
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4. Discussion The present study demonstrates highlights a potential relationship between TDP-43 and astrogliosis after TBI. First, we found that TDP-43 cleavage products were increased after TBI (potentially owing to caspase-3 activation) in a manner associated
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with astrocytic hypertrophy. Second, rats developed severe, short-term impairments in motor coordination and balance after TBI. These behavioral impairments were possibly
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associated with TDP-43 short fragment mislocalization and accumulation in astrocytes.
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Third, intracerebroventricular treatment with a caspase-3 inhibitor immediately after TBI decreased the abundance of TDP-43 cleavage products, prevented astrocytic
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hypertrophy as well as motor impairments, and promoted neuronal survival. These
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results suggest that TDP-43 cleavage and short fragment mislocalization are pathogenic components of TBI, and that TDP-43 proteolysis is possibly related to astrogliosis after
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TBI.
TDP-43 plays a role in the repression of gene transcription and translation as well
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as multiple aspects of RNA processing and function. In pathological contexts, TDP-43 can be cleaved, phosphorylated, and ubiquitinated (Sieben et al., 2012; Al-Chalabi et al.,
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2012; Gendron et al., 2013; Wang et al., 2014), and finally transported outside of the nucleus to the cytoplasm (Zhang et al., 2009; Barmada et al., 2010; Che et al., 2011; Sieben et al., 2012; Al-Chalabi et al., 2012; Gendron et al., 2013). Zhang et al. showed that accumulation of the 25-kDa fragment of TDP-43 led to the formation of toxic, insoluble, and ubiquitin- and phospho-positive cytoplasmic inclusions within cells (Zhang et al., 2009). In another study, Che et al. reported that abundance of the 35-kDa fragment of TDP-43 led to cellular redistribution, inclusion body formation, and altered RNA processing (Che et al., 2011). Therefore, short C-terminal fragments of TDP-43
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produce neuronal toxicity and cell death by forming ubiquitin-positive cytoplasmic inclusions within cells (Zhang et al., 2009; Barmada et al., 2010; Che et al., 2011; Sieben et al., 2012; Al-Chalabi et al., 2012; Gendron et al., 2013). Neuronal TDP-43 aggregation is accordingly observed in a number of neurological disorders including
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FTLD and ALS (Sieben et al., 2012, Al-Chalabi et al., 2012, Gendron et al., 2013). TDP-43 aggregation and nuclear depletion also occur in astrocytes, but TDP-43 toxicity
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is not well studied in this context. In the present study, levels of the 25- and 35-kDa
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fragments of TDP-43 were increased and full-length TDP-43 (43 kDa) was decreased after TBI in a manner associated with neuronal loss and motor impairment. Moreover,
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TDP-43 cleavage products were co- localized with GFAP in reactive astrocytes after TBI.
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These findings suggest that TBI may induce TDP-43 proteolysis in astrocytes as a process related to astrocyte activation and downstream functional consequences of TBI.
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The exact role of TDP-43 in astrocytes and its relationship with neuronal death is not fully understood. Astrocytes are the most abundant cell type in the brain and serve a
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variety of supportive functions (Pekny et al., 2014). Astrogliosis or astrocyte reactivity refers to morphological and functional changes in astrocytes that occur in response to
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insult or injury. Reactive astrocytes permit the isolation and sequestration of injured tissue, limiting the lesion size and its influence on surrounding tissues; however, if left unresolved, reactive gliosis can affect neuroplasticity and CNS regeneration. Indeed, neurons cannot survive in the brain without close interaction with astrocytes (Pekny et al., 2014). Excitotoxicity is another common mechanism of secondary brain injury after TBI. Neurons are highly vulnerable to excitotoxicity after TBI. Therefore, neuronal cell counts were significantly lower in the TBI group.
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Some studies have shown that astrocytes expressing mutations in TDP43 can induce motor neuron cell death (Tong et al., 2013; Rojas et al., 2014). In contrast, other studies have reported that TDP-43 proteinopathies do not produce astrocyte non-cell-autonomous neuronal death in cell culture (Haidet-Phillips et al., 2013; Serio et
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al., 2013). In the present study, local administration of a caspase-3 inhibitor reduced TDP-43 cleavage, prevented astrocyte activation, and increased neuronal survival. The
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observed neuron-sparing effect may have been related to the preservation of healthy
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astrocyte-neuron interactions after TBI.
TDP-43 has been previously implicated in TBI (Moisse et al., 2009; McKee et al.,
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2010; Johnson et al., 2011; Yang et al., 2014). Previous studies have suggested that
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TDP-43 proteinopathy associated with repetitive head trauma is similar to that found in FTLD; indeed, the increased expression of TDP-43 cleavage products, protein
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redistribution to the cytoplasm, and inclusion formation have been reported in TBI models (McKee et al., 2010, Yang et al., 2014). In contrast, a recent study found no
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association between a history of TBI and the presence of abnormally phosphorylated TDP-43 inclusions (Johnson et al., 2011). The present study findings supplement the
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gaps in these previous studies by defining a more clear relationship between TBI and TDP-43 proteolysis in astrocytes. Most TBI studies use saline injection as sham control groups. In those studies, they want to prove the effect of treatment. In our study, rats were randomly divided into 3 groups in our study: TBI with vehicle treatment (TBI), TBI with caspase-3 inhibitor treatment (TBI + CI), and sham group (sham). In our sham group, no procedure was performed. We focus on the TDP-43 proteolysis in astrocytes after TBI. Therefore, we used normal rats as sham group.
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Caspases 3 play an essential role during apoptotic cell death. Recent studies suggest that caspase-3 also functions as a regulatory molecule in neurogenesis and synaptic activity (Clark et al., 2000; D'Amelio et al., 2010). Therefore, active caspase 3 could be detect at sham group. After TBI, caspase-3 immunoreactivity represents active
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caspase-3 is increased and caspase 3 inhibitor treatment reduces active caspase-3 activity. This study demonstrates caspase-3 cleavage and bioactivity after TBI in rats.
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Local posttreatment with caspase 3 inhibitor reduces TDP-43 cleavage, caspase-3
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activity and neuron loss after injury.
Our study had several limitations. First, we did not use an in vitro model to
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confirm the influence of TDP-43 cleavage in astrocytes on astrocyte-neuron interactions.
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Thus, we cannot speculate as to the exact mechanism of neurotoxicity involved. Second, it would be useful to induce TBI in transgenic animals overexpressing wild-type human
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or mouse TDP-43. Future studies using this strategy can provide information about whether TDP-43 overexpression leads to astrocyte activation. Third, we did not
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specifically inhibit TDP-43 cleavage in our study. Currently, there is no drug available that specifically targets TDP-43 cleavage. Future molecular research is necessary to
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answer additional questions about the exact role of TDP-43 processing in neurological and neurodegenerative disease.
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5. Conclusions Our study demonstrates that short fragments of TDP-43 are generated in astrocytes local to the lesion site after TBI in rats. Moreover, our findings suggest that early management of TDP-43 proteolysis can effectively reduce astrogliosis and improve
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functional outcomes post-injury. TDP-43, its proteolysis, and its cleavage products in astrocytes may be an important therapeutic target for motor dysfunction after TBI.
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Further molecular research is necessary to identify a specific inhibitor of TDP-43
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proteolysis and confirm its utility in TBI and other brain disorders.
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ACKNOWLEDGEMENTS This study was supported in part by grants from the Ministry of Science and Technology, Taiwan (103-2314-B-214 -007 -MY2) and the E-da hospital, Kaohsiung,
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Taiwan (EDAHP-101026, EDAHP-103012, EDPJ103075 and EDPJ104072).
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DISCLOSURE
The authors report no conflict of interest concerning the materials or methods used
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in this study or the findings specified in this paper.
CONTRIBUTORSHIP STATEMENT
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Chih-Yuan Huang, Yi-Che Lee, Ping-Chia Li, Po-Chou Liliang, and Hao-kuang
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Wang designed research; Kang Lu, Kuo-Wei Wang, Li-Ching Chang, Li- Yen Shiu, Ming-Feng Chen, Yuan- Ting Sun, and Hao-kuang Wang analyzed data; H.-K. Wang
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wrote the paper.
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Figure Legends Figure 1. (A) Flowchart showing the animal model of TBI with caspase-3 inhibitor treatment. Animals were divided into three groups: sham surgery with no treatment (sham, n = 5), TBI with vehicle (0.2% DMSO) treatment (TBI, n = 27), and TBI with
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caspase-3 inhibitor treatment (TBI + CI, n = 12).
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Figure 2. TBI induces TDP-43 proteolysis in astrocyte. (A) Western blot analysis of
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TDP-43 levels in extracts of the cortex obtained from sham, TBI Day 1, Day 3 and Day 7 rats. The amounts of the 35-kDa TDP-43 fragments were increased 3 days, and 7 days
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post-TBI, and the 25- kDa TDP-43 fragments were increased 7 days post-TBI. Full-
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length TDP-43 decreased at 1, 3, and 7 days post-TBI (n = 5 rats per group). Blotting patterns are shown in the upper panel, and the statistical analysis is shown in the bottom
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panel. Results are the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.005; ***P < 0.001. (B) Representative photomicrographs showing TDP-43
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immunoreactivity in brain sections from sham, and 7 days post- TBI rats. TDP-43 (green)/ Glial fibrillary acidic protein (GFAP; red)/ 4',6-diamidino-2-phenylindole
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(DAPI; blue) triple labeling in the sham or contusion cortices. TDP-43 were co-localized with GFAP. (n = 5 rats per group). (C) Representative TDP-43 protein bands in the urea-soluble fraction of brain extracts. The arrow points to the unmodified form of TDP-43 on the gel (n = 7 rats per group).
Figure 3. TBI increases active form of caspase-3. (A) Western blot analysis of the level of the active form of caspase-3 in extracts of the cortex obtained from sham, TBI Day 1, Day 3, and D7 rats (n = 5 rats per group). *P < 0.05; **P < 0.005 (B) Representative
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photomicrographs showing TDP-43 immunoreactivity in brain sections from sham, and 7 days post- TBI rats. GFAP (red)/ Caspase-3 (green)/ 4',6-diamidino-2-phenylindole (DAPI; blue) triple labeling in the sham or contusion cortices. Caspase-3 were
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co-localized with GFAP. (n = 5 rats per group).
Figure 4. Caspase 3 inhibitor blocked cytoplasmic redistribution of TDP-43 in the
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astrocyte. (A) Representative protein bands of TDP-43, TDP-35, and TDP-25 in the
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lesion from rat brains treated with caspase-3 inhibitor or vehicle. C-terminal fragment of TDP-43 generated after TBI and was rescued by caspase-3 inhibitor treatment. (n = 5
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rats per group). *P < 0.05 (B) Western blot analysis of the level of the active form of
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caspase-3 in extracts of the cortex obtained from TBI with and without caspase-3 inhibitor (n = 5 rats per group). *P < 0.05 (C) TDP-43 (green)/ Glial fibrillary acidic
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protein (GFAP; red)/ 4',6-diamidino-2-phenylindole (DAPI; blue) triple labeling in the contusion cortices with caspase-3 inhibitor. The morphology of reactive astrocyte
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become less hypertrophy.
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Figure 5. TBI induce motor dysfunction. (A) The latencies of the rotarod test were significantly shorter in rats TBI rats. (B, C, D) Beam walking performance of sham, TBI Day 7, and TBI + CI Day 7 rats. The latencies, the total faults of the right hind legs, and the total faults of the left hind legs of the three groups are shown. *P < 0.05; **P < 0.005; ***P < 0.001.
Figure 6. Early treatment of caspase-3 inhibitor can improve neuron survival. (A) The dot plot from FACS-like analysis system-based neuronal counting in
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immunocytochemistry staining for NeuN and Dapi. (B) Quantification of survived
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neuronal number by FACS-like analysis system (n = 5 rats per group). *P < 0.05.
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Graphical abstract
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HIGHLIGHTS
TBI increased TDP-43 proteolysis in astrocyte, and induced astrocyte hypertrophy.
TBI induced impaired behaviors that was associated with TDP-43 and its fragments
in astrocyte. Early management of TBI is effective in reducing TDP-43-associated impaired
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behaviors
Chih-Yuan Huang, Yi-Che Lee, Ping-Chia Li, Po-Chou Liliang, Kang Lu, Kuo-Wei Wang, Li-Ching Chang, Li-Yen Shiu, MingFeng Chen, Yuan-Ting Sun, Hao-kuang Wang PII: DOI: Reference:
S0165-5728(17)30394-6 doi:10.1016/j.jneuroim.2017.10.011 JNI 476647
To appear in:
Journal of Neuroimmunology
Received date: Revised date: Accepted date:
30 August 2017 14 October 2017 16 October 2017
Please cite this article as: Chih-Yuan Huang, Yi-Che Lee, Ping-Chia Li, Po-Chou Liliang, Kang Lu, Kuo-Wei Wang, Li-Ching Chang, Li-Yen Shiu, Ming-Feng Chen, Yuan-Ting Sun, Hao-kuang Wang , TDP-43 proteolysis is associated with astrocyte reactivity after traumatic brain injury in rodents. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jni(2017), doi:10.1016/ j.jneuroim.2017.10.011
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Research report TDP-43 proteolysis is associated with astrocyte reactivity after traumatic brain injury in rodents
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Abbreviated title: TDP-43 proteolysis is associated with reactive astrocyte
Chih-Yuan Huang,1 Yi-Che Lee,2 Ping-Chia Li,3 Po-Chou Liliang,3,4 Kang Lu,3,4
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Kuo-Wei Wang,3,4 Li-Ching Chang,3 Li-Yen Shiu,5 Ming-Feng Chen,5 Yuan-Ting Sun,6
Neurosurgical Service, Department of Surgery, National Cheng Kung University
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1
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and Hao-kuang Wang3,4*
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Hospital, Tainan, Taiwan
Department of Nephrology, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan
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School of Medicine for International Students, I-Shou University, Kaohsiung, Taiwan.
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Department of Neurosurgery, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan
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Department of Medical Research, Cell Therapy and Research Center, E-Da Hospital,
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I-shou University, Kaohsiung, Taiwan. Department of Neurology, National Cheng Kung University Hospital, College of
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Medicine, National Cheng Kung University, Tainan, Taiwan
Address correspondence to:
Hao kuang Wang, MD, PhD Department of Neurosurgery, E-Da Hospital/ I-Shou University, Taiwan No.1, Yida Road, Jiaosu Village, Yanchao District, Kaohsiung City 82445, Taiwan
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Tel: +866-975106080 E-mail: [email protected], [email protected]
Word count for abstract: 162, Number of words: 3089
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Number of references: 27, Number of figures: 6
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Keywords: astrocyte; astrogliosis; traumatic brain injury; transactivation response
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DNA-binding protein 43.
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Abbreviations: Amyotrophic lateral sclerosis
ANOVA
A two-way analysis of variance
FTLD GFAP
Frontotemporal lobar degeneration Glial fibrillary acidic protein
TBI
Traumatic brain injury
TDP-43 TX
Transactivation response DNA-binding protein 43 High-salt Triton X-100
SARK
Sarkosyl
z-DEVD-fmk
N-benzyloxycarbonyl-Asp-Glu-Val-Asp- fluoromethyl ketone
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Abstract The aggregation and deposition of transactivation response DNA-binding protein 43 (TDP-43) in neurons and astrocytes is characteristic in a number of neurodegenerative diseases including Alzheimer ’s disease, frontotemporal lobar degeneration, and
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amyotrophic lateral sclerosis. Nevertheless, the exact role of TDP-43 in astrocytes is unknown. Recently, TDP-43 was identified in neurons but not astrocytes after traumatic
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brain injury (TBI) in humans. In the present study, we evaluated TDP-43 expression and
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proteolysis in astrocytes in a rat model of TBI. We assessed TDP-43 fragment expression, astrocyte morphology, neuronal population numbers, and motor function
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after TBI with or without intracerebroventricular administration of a caspase-3 inhibitor.
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Motor dysfunction was observed after TBI in potential association astrocytic TDP-43 short fragment mislocalization and accumulation, astrogliosis, and neuronal loss.
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Notably, caspase-3 inhibition prevented these changes after TBI. Our findings suggest that TDP-43 proteolysis in astrocytes is related to astrogliosis and subsequent neuronal
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loss in TBI, and that TDP-43 may be an important therapeutic target for preventing
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motor dysfunction after TBI.
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1. Introduction Transactive response DNA binding protein (TDP-43) is a heterogeneous nuclear RNA-binding protein that regulates gene expression, transcription, and multiple aspects of RNA processing and functions including splicing, stability, transport, translation, and
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microRNA maturation (Sieben et al., 2012; Al-Chalabi et al., 2012; Gendron et al., 2013). Under physiological conditions, TDP-43 is enriched in the nucleus; however,
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TDP-43 can be cleaved by caspase-3 to generate short 25-kDa and 35-kDa C-terminal
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fragments, resulting in the formation of ubiquitin-positive cytoplasmic inclusions, cellular toxicity, and ultimately death (Zhang et al., 2009; Barmada et al., 2010; Che et
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al., 2011). TDP-43 was first identified in neuronal cytoplasmic inclusions in
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frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) in 2006 (Sieben et al., 2012; Al-Chalabi et al., 2012). Recently pathological TDP-43
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inclusions were also identified in Alzheimer’s disease (Fang et al., 2014). Thus, TDP-43 accumulation and deposition is presumed to play a role in the pathogenesis of
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neurodegenerative diseases.
Traumatic brain injury (TBI) is a leading cause of death and disability that has a
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limited number of clinical treatment options (Maas et al., 2008). TBI leads to the activation of multiple inflammatory pathways including astrocyte activation (Blennow et al., 2012). Whereas astrocytes play neurotrophic and supportive roles in nervous tissues under physiological conditions (Pekny and Pekna, 2014; Pekny and Wilhelmsson, 2014), astrocytes are subject to hypertrophy and proliferation after TBI that can lead to a number of positive (e.g., neural protection and repair) and negative (e.g., glial scarring and alterations in plasticity) effects; yet, the role of astrocytes after TBI is not well understood. Moreover, few studies have investigated a role for TDP-43 in TBI.
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Nuclear depletion and cytoplasmic aggregation of TDP-43 has been documented in astrocytes; however, the toxicity of TDP-43 fragments in astrocytes is different from that observed in neurons (Haidet-Phillips et al., 2013; Serio et al., 2013; Tong et al., 2013; Rojas et al., 2014). Haidet-Phillips et al. showed that TDP-43 overexpression or
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knockout in astrocytes did not produce a cytotoxic phenotype (Haidet-Phillips et al., 2013). Additionally, it is not clear as to whether pathogenic TDP-43 accumulation in
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astrocytes causes non-cell-autonomous neuronal death (Haidet-Phillips et al., 2013;
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Serio et al., 2013; Tong et al., 2013; Rojas et al., 2014); whereas some evidence suggests that astrocytes expressing mutated forms of TDP43 can cause the
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non-cell-autonomous death of motor neurons (Tong et al., 2013; Rojas et al., 2014),
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other evidence contradicts these findings (Haidet-Phillips et al., 2013; Serio et al., 2013).
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In the present study, we investigated whether TBI was associated with alterations in TDP-43 expression and cleavage in astrocytes using a rodent model. Rats were
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treated with a caspase-3 inhibitor or vehicle immediately after TBI and TDP-43 fragment expression, astrocyte morphology, neuronal population numbers, and motor
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function were examined at up to 7 days post-injury.
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2. Materials and methods 2.1 Ethical statement All procedures were reviewed and approved by the Institution Animal Care and
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Use Committee(IACUC) of E-Da hospital (IACUC), and conformed to the guidelines of
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the Taiwan Council for Animal Care. The number of IACUC is 104004.
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2.2 Study design
Adult male Sprague-Dawley rats weighing 300–350 g were purchased from
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Charles River Laboratories. Then those rats were maintained at constant temperature (22 ± 2°C) and a 12 hour light/dark cycle with food and water available ad libitum for 1
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week before procedures. Details on the injury device, TBI model, and behavioral tests
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were previously reported (Wang et al., 2015). Table 1 showing the time-line diagram of TBI with experimental procedure. Finally, animals were divided into three groups to examine whether TBI induced the cleavage and aggregation of TDP-43 species in lesion
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sites and specifically in astrocytes.
2.3 Experimental animals 2.3.1 Traumatic brain injury model. TBI was induced using a weight drop device as previously established in the Feeney weight-drop model (Feeney et al., 1981, Liliang et al., 2010; Wang et al., 2015).
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Briefly, rats were anesthetized, the skull was exposed, and a left partial craniotomy was performed. A 40-g steel rod with a flat end was freely dropped from 30 cm onto strike a transducer rod (diameter, 4.5 mm) that was oriented with its tip placed directly onto the rat’s skull, posterior to bregma (Feeney et al., 1981, Liliang et al., 2010; Wang et al.,
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2015). Rats were anesthetized and underwent left parietal craniotomy without cortical contusion injury. After postoperative procedures, rats were returned to their home cages
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and monitored carefully.
2.3.2 Intracerebroventricular administration of a caspase-3 inhibitor.
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Rats were placed in a stereotaxic apparatus for intracerebroventricular
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administration. The cell-permeable and irreversible caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val- Asp- fluoromethyl ketone (EMD Millipore, Billerica, MA) was dissolved in 0.2% DMSO (500 ng in 1 μL of DMSO) (Clark et al., 2000;
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Knoblach et al., 2004). The caspase-3 inhibitor (2 μL) or vehicle (2 μL) was
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administered by intracerebroventricular injection ipsilateral to the lesion site at 10 min
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post-TBI via a micro-osmotic pump and infusion cannula.
2.4 Experimental procedures 2.4.1 Western blotting.
To evaluate protein expression levels in the cerebral cortical regions surrounding TBI lesions, protein extracts were obtained by homogenization of cortical tissues in RIPA lysis buffer (Clark et al., 2000; Knoblach et al., 2004; Wang et al., 2012), samples were electrophoresed and transferred to blotting membranes, and proteins were probed
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with the following primary antibodies: anti-TDP-43 (1:5000; Proteintech), anti-caspase-3 (1:2000; Cell Signaling), and anti-actin (1:15,000; Millipore). The anti-TDP-43 antibody used in this study recognized not only the full- length TDP-43 product but also 25–35 kDa cleavage products and phosphorylated forms of TDP-43.
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The anti-caspase-3 antibody recognized the activated form caspase-3 resulting from
expressed as the mean ± standard error of the mean.
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cleavage at Asp175. The relative intensities of bands were normalized to actin and
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For the analysis of TDP-43 in cellular inclusions, brain tissues were dissected and
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proteins were extracted with specialized buffers (Wang et al., 2012). Low-salt buffer, high-salt Triton X-100 buffer, myelin floatation buffer, and sarkosyl buffer were used to
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extract proteins from tissues at 5 mL/g (volume/weight). Sarkosyl-insoluble materials were further extracted in 0.25 mL/g urea buffer. Urea-soluble proteins were analyzed by
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Western blot analysis with the above primary antibodies.
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2.4.2 Immunohistochemistry.
For immunohistostaining, brain sections were stained with anti–TDP-43 (1:50;
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Gene Tex), glial fibrillary acidic protein (GFAP) (1:100, Abcam),anti–caspase-3 (1:100;Cell Signaling) or antineuron-specific nuclear protein (NeuN) (Millipore), and then with biotin-conjugated secondary antibodies (Millipore) followed by detection with 3,3′ diaminobenzidine (DAB) (Millipore) (Wang et al., 2012). For immunofluorescence staining, coronal brain sections were stained with anti-LC3 antibody (1:200, Novus Biologicals). DAPI staining was used to locate the nuclei. 2.4.3 Motor function assessments.
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We used the beam-walking test to evaluate the recovery of motor function after TBI (Loane et al. 2009; Wang et al. 2012). A basal level of competence (< 10 faults over 50 steps) was established before surgery. Then, the beam walk task was performed at 1, 3, and 7 days post-injury.
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The rotarod test was used to assess the motor coordination after TBI (Loane et al.,
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2009; Wang et al., 2014). Each rat received a training session on the rotarod apparatus at a constant speed of 8 rpm before surgery. Test trials were conducted at 1, 3, and 7 days
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post-injury.
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2.4.4 Flow cytometry
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For quantifying the survival of neuronal number, whole ipsilateral semi-brain images were acquired by TissueFAXS and analyzed by TissueQuest (TissueGnostics,
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Viena, Austria) as previously described (Wang et al., 2012). Stereologic evaluation of ipsilateral semi-brain was conducted on every 12th section throughout the forebrain
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with the damaged region by the Cavalieri principle. Flow cytometry- like data (dot-plot) were analyzed and organized by TissueQuest, with each dot on the plot representing
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intensity of NeuN and Dapi from a single cell. The plot accumulated the values of all cells from the semi-brain, and then the positive signal number could be identified by gating the immunoreactive intensity. Neuronal cell counts were comparable between the sham and caspase-3 inhibitor-treated TBI groups.
2.5 Housing and husbandry Rats were housed in the Animal Center of I-Shou University, Kaohsiung, Taiwan
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and maintained on a 12:12-h light/dark cycle (lights on at 07:00) in a room with controlled temperature (22 ± 2°C) and humidity (60 ± 5%), with ad libitum access to food and water. Rats were allowed to acclimate for at least 1 week before the
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2.6 Sample size
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Adult male Sprague-Dawley rats weighing 300–350 g were purchased from
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Charles River Laboratories (Wang et al., 2015). Rats were randomly divided into 3 groups: TBI with vehicle treatment (TBI, n = 27), TBI with caspase-3 inhibitor
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treatment (TBI + CI, n = 12), and sham group (sham, n = 5) (Figure 1A). In TBI group, an equal amount of saline was injected. In sham group, no procedure was performed.
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All the experiments were conducted and analyzed in blinded manner.
2.7 Allocating animals to experimental groups
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Cage randomization was used in our study. On the day of surgery, a cage will be selected randomly from the pool of all cages containing animals eligible for inclusion in the study, regardless of arrival date. Primary randomization is conducted by an individual other than the surgeon. On the basis of their position on the rack, cages are given a temporary numerical designation for the purpose of primary randomization.
2.8 Experimental outcomes
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The primary endpoints of the present study were the cleavage and aggregation of TDP-43 species in lesion sites and specifically in astrocytes. Then we assessed TDP-43 fragment expression, astrocyte morphology, neuronal population numbers, and motor function after TBI with or without intracerebroventricular administration of a caspase-3
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inhibitor.
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2.9 Statistical methods
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Results are reported as the mean ± standard error of the mean. To examine whether the data is normally distributed, D`Agostino and Pearson omnibus normality test was
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used before single mean or multiple mean comparison analysis. All data were confirmed that were normally distributed. Statistical differences were analyzed using a 2-way
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analysis of variance (ANOVA) followed by post hoc Bonferroni tests for multiple comparisons. For 2-group comparisons, data were analyzed using unpaired t-tests.
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Differences were considered to be statistically significant when P < 0.05.
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3. Results 3.1 Cleavage products of TDP-43 are increased in astrocytes after TBI Whole-cell extracts from lesion sites were immunoblotted to investigate changes in TDP-43 expression post-injury. Levels of the 25-kDa and 35-kDa fragments of TDP-43
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were increased whereas full- length TDP-43 (43 kDa) was decreased at 7 days post-injury (Figure 2A). Additionally, glial fibrillary acidic protein (GFAP) was
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co-localized with TDP-43 and its cleavage products in morphologically activated
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astrocytes at 7 days post-injury (Figure 2B). Next, we examined urea-soluble fractions of forebrain extracts to investigate the presence of cytoplasmic TDP-43 inclusions.
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Cleavage products including the 25-kDa and 35-kDa fragments of TDP-43 were
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upregulated at 7 days post-injury (Figure 2C). These data suggested that TBI induced the cleavage and aggregation of TDP-43 species in lesion sites and specifically in
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astrocytes.
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3.2 Caspase-3 is activated in astrocytes after TBI Next, we performed immunoblotting to quantify the expression levels of activated
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caspase-3 in cortical extracts post-injury. Activated caspase-3 expression was increased in injured cortex extracts at 3 and 7 days post-injury (Figure 3A). Additionally, GFAP was co-localized with activated caspase-3 in morphologically activated astrocytes at 7 days post-injury (Figure 3B). Thus, it was concluded that TBI induced caspase-3 activation in astrocytes.
3.3 Caspase-3 inhibitor treatment decreases TDP-43 cleavage and astrocyte activation after TBI
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Intracerebroventricular administration of a caspase-3 inhibitor immediately after TBI resulted in a 50% reduction in expression of the 35-kDa fragment of TDP-43 (Figure 4A) and a 30% reduction in expression of activated caspase-3 in injured cortex extracts at 7 days post-injury (Figure 4B); however, there was no obvious effect on the
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25-kDa fragment of TDP-43. Moreover, caspase-3 inhibitor treatment prevented the development of astrogliosis after TBI: non-reactive astrocyte morphology was observed
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in inhibitor-treated animals at 7 days post-injury (Figure 4C).
3.4 Motor dysfunction after TBI is sensitive to caspase-3 inhibition
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To assess motor function, rotarod testing and beam walk testing were performed at
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1, 3, and 7 days post-injury. Rotarod test latencies (Figure 5A) and beam walking latencies (Figure 5B) were significantly shorter in TBI rats compared to rats in the sham
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group or TBI rats treated with the caspase-3 inhibitor. Moreover, we analyzed foot placement accuracy (number of errors) and found that TBI rats treated with the
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caspase-3 inhibitor performed significantly better on the right (injury) side than did untreated TBI rats (Figure 5C). However, no between-group differences were observed
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for the left side. These results suggested that caspase-3 inhibition prevented the short-term impairment of motor coordination and balance after TBI.
3.5 Caspase-3 inhibition promotes neuronal survival after TBI To investigate whether neuronal death after TBI was sensitive to caspase-3 inhibition and potentially related to the presence of TDP-43 cleavage products in reactive astrocytes, we conducted immunohistochemistry staining for NeuN and Dapi and quantified the neuronal number by a flow cytometry- like analysis system
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(TissueFAX and TissueQuest) in the ipsilateral semi-brain at 7 days after TBI dmage. Neuronal cell counts were comparable between the sham and caspase-3 inhibitor-treated TBI groups by TissueQuest-based dot plot (Figure 6A). The quantified result indicated that TBI significantly leaded neuronal loss in the ipsilateral semi-brain at 7 days after
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TBI, but caspase-3 inhibitor improved the neuronal survival of TBI rats, a higher level of survived neuron were observed in caspase-3 inhibitor treated TBI rats (n = 5 rats per
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group).
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4. Discussion The present study demonstrates highlights a potential relationship between TDP-43 and astrogliosis after TBI. First, we found that TDP-43 cleavage products were increased after TBI (potentially owing to caspase-3 activation) in a manner associated
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with astrocytic hypertrophy. Second, rats developed severe, short-term impairments in motor coordination and balance after TBI. These behavioral impairments were possibly
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associated with TDP-43 short fragment mislocalization and accumulation in astrocytes.
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Third, intracerebroventricular treatment with a caspase-3 inhibitor immediately after TBI decreased the abundance of TDP-43 cleavage products, prevented astrocytic
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hypertrophy as well as motor impairments, and promoted neuronal survival. These
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results suggest that TDP-43 cleavage and short fragment mislocalization are pathogenic components of TBI, and that TDP-43 proteolysis is possibly related to astrogliosis after
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TBI.
TDP-43 plays a role in the repression of gene transcription and translation as well
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as multiple aspects of RNA processing and function. In pathological contexts, TDP-43 can be cleaved, phosphorylated, and ubiquitinated (Sieben et al., 2012; Al-Chalabi et al.,
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2012; Gendron et al., 2013; Wang et al., 2014), and finally transported outside of the nucleus to the cytoplasm (Zhang et al., 2009; Barmada et al., 2010; Che et al., 2011; Sieben et al., 2012; Al-Chalabi et al., 2012; Gendron et al., 2013). Zhang et al. showed that accumulation of the 25-kDa fragment of TDP-43 led to the formation of toxic, insoluble, and ubiquitin- and phospho-positive cytoplasmic inclusions within cells (Zhang et al., 2009). In another study, Che et al. reported that abundance of the 35-kDa fragment of TDP-43 led to cellular redistribution, inclusion body formation, and altered RNA processing (Che et al., 2011). Therefore, short C-terminal fragments of TDP-43
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produce neuronal toxicity and cell death by forming ubiquitin-positive cytoplasmic inclusions within cells (Zhang et al., 2009; Barmada et al., 2010; Che et al., 2011; Sieben et al., 2012; Al-Chalabi et al., 2012; Gendron et al., 2013). Neuronal TDP-43 aggregation is accordingly observed in a number of neurological disorders including
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FTLD and ALS (Sieben et al., 2012, Al-Chalabi et al., 2012, Gendron et al., 2013). TDP-43 aggregation and nuclear depletion also occur in astrocytes, but TDP-43 toxicity
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is not well studied in this context. In the present study, levels of the 25- and 35-kDa
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fragments of TDP-43 were increased and full-length TDP-43 (43 kDa) was decreased after TBI in a manner associated with neuronal loss and motor impairment. Moreover,
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TDP-43 cleavage products were co- localized with GFAP in reactive astrocytes after TBI.
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These findings suggest that TBI may induce TDP-43 proteolysis in astrocytes as a process related to astrocyte activation and downstream functional consequences of TBI.
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The exact role of TDP-43 in astrocytes and its relationship with neuronal death is not fully understood. Astrocytes are the most abundant cell type in the brain and serve a
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variety of supportive functions (Pekny et al., 2014). Astrogliosis or astrocyte reactivity refers to morphological and functional changes in astrocytes that occur in response to
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insult or injury. Reactive astrocytes permit the isolation and sequestration of injured tissue, limiting the lesion size and its influence on surrounding tissues; however, if left unresolved, reactive gliosis can affect neuroplasticity and CNS regeneration. Indeed, neurons cannot survive in the brain without close interaction with astrocytes (Pekny et al., 2014). Excitotoxicity is another common mechanism of secondary brain injury after TBI. Neurons are highly vulnerable to excitotoxicity after TBI. Therefore, neuronal cell counts were significantly lower in the TBI group.
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Some studies have shown that astrocytes expressing mutations in TDP43 can induce motor neuron cell death (Tong et al., 2013; Rojas et al., 2014). In contrast, other studies have reported that TDP-43 proteinopathies do not produce astrocyte non-cell-autonomous neuronal death in cell culture (Haidet-Phillips et al., 2013; Serio et
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al., 2013). In the present study, local administration of a caspase-3 inhibitor reduced TDP-43 cleavage, prevented astrocyte activation, and increased neuronal survival. The
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observed neuron-sparing effect may have been related to the preservation of healthy
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astrocyte-neuron interactions after TBI.
TDP-43 has been previously implicated in TBI (Moisse et al., 2009; McKee et al.,
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2010; Johnson et al., 2011; Yang et al., 2014). Previous studies have suggested that
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TDP-43 proteinopathy associated with repetitive head trauma is similar to that found in FTLD; indeed, the increased expression of TDP-43 cleavage products, protein
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redistribution to the cytoplasm, and inclusion formation have been reported in TBI models (McKee et al., 2010, Yang et al., 2014). In contrast, a recent study found no
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association between a history of TBI and the presence of abnormally phosphorylated TDP-43 inclusions (Johnson et al., 2011). The present study findings supplement the
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gaps in these previous studies by defining a more clear relationship between TBI and TDP-43 proteolysis in astrocytes. Most TBI studies use saline injection as sham control groups. In those studies, they want to prove the effect of treatment. In our study, rats were randomly divided into 3 groups in our study: TBI with vehicle treatment (TBI), TBI with caspase-3 inhibitor treatment (TBI + CI), and sham group (sham). In our sham group, no procedure was performed. We focus on the TDP-43 proteolysis in astrocytes after TBI. Therefore, we used normal rats as sham group.
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Caspases 3 play an essential role during apoptotic cell death. Recent studies suggest that caspase-3 also functions as a regulatory molecule in neurogenesis and synaptic activity (Clark et al., 2000; D'Amelio et al., 2010). Therefore, active caspase 3 could be detect at sham group. After TBI, caspase-3 immunoreactivity represents active
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caspase-3 is increased and caspase 3 inhibitor treatment reduces active caspase-3 activity. This study demonstrates caspase-3 cleavage and bioactivity after TBI in rats.
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Local posttreatment with caspase 3 inhibitor reduces TDP-43 cleavage, caspase-3
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activity and neuron loss after injury.
Our study had several limitations. First, we did not use an in vitro model to
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confirm the influence of TDP-43 cleavage in astrocytes on astrocyte-neuron interactions.
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Thus, we cannot speculate as to the exact mechanism of neurotoxicity involved. Second, it would be useful to induce TBI in transgenic animals overexpressing wild-type human
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or mouse TDP-43. Future studies using this strategy can provide information about whether TDP-43 overexpression leads to astrocyte activation. Third, we did not
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specifically inhibit TDP-43 cleavage in our study. Currently, there is no drug available that specifically targets TDP-43 cleavage. Future molecular research is necessary to
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answer additional questions about the exact role of TDP-43 processing in neurological and neurodegenerative disease.
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5. Conclusions Our study demonstrates that short fragments of TDP-43 are generated in astrocytes local to the lesion site after TBI in rats. Moreover, our findings suggest that early management of TDP-43 proteolysis can effectively reduce astrogliosis and improve
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functional outcomes post-injury. TDP-43, its proteolysis, and its cleavage products in astrocytes may be an important therapeutic target for motor dysfunction after TBI.
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Further molecular research is necessary to identify a specific inhibitor of TDP-43
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proteolysis and confirm its utility in TBI and other brain disorders.
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ACKNOWLEDGEMENTS This study was supported in part by grants from the Ministry of Science and Technology, Taiwan (103-2314-B-214 -007 -MY2) and the E-da hospital, Kaohsiung,
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Taiwan (EDAHP-101026, EDAHP-103012, EDPJ103075 and EDPJ104072).
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DISCLOSURE
The authors report no conflict of interest concerning the materials or methods used
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in this study or the findings specified in this paper.
CONTRIBUTORSHIP STATEMENT
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Chih-Yuan Huang, Yi-Che Lee, Ping-Chia Li, Po-Chou Liliang, and Hao-kuang
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Wang designed research; Kang Lu, Kuo-Wei Wang, Li-Ching Chang, Li- Yen Shiu, Ming-Feng Chen, Yuan- Ting Sun, and Hao-kuang Wang analyzed data; H.-K. Wang
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wrote the paper.
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Figure Legends Figure 1. (A) Flowchart showing the animal model of TBI with caspase-3 inhibitor treatment. Animals were divided into three groups: sham surgery with no treatment (sham, n = 5), TBI with vehicle (0.2% DMSO) treatment (TBI, n = 27), and TBI with
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caspase-3 inhibitor treatment (TBI + CI, n = 12).
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Figure 2. TBI induces TDP-43 proteolysis in astrocyte. (A) Western blot analysis of
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TDP-43 levels in extracts of the cortex obtained from sham, TBI Day 1, Day 3 and Day 7 rats. The amounts of the 35-kDa TDP-43 fragments were increased 3 days, and 7 days
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post-TBI, and the 25- kDa TDP-43 fragments were increased 7 days post-TBI. Full-
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length TDP-43 decreased at 1, 3, and 7 days post-TBI (n = 5 rats per group). Blotting patterns are shown in the upper panel, and the statistical analysis is shown in the bottom
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panel. Results are the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.005; ***P < 0.001. (B) Representative photomicrographs showing TDP-43
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immunoreactivity in brain sections from sham, and 7 days post- TBI rats. TDP-43 (green)/ Glial fibrillary acidic protein (GFAP; red)/ 4',6-diamidino-2-phenylindole
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(DAPI; blue) triple labeling in the sham or contusion cortices. TDP-43 were co-localized with GFAP. (n = 5 rats per group). (C) Representative TDP-43 protein bands in the urea-soluble fraction of brain extracts. The arrow points to the unmodified form of TDP-43 on the gel (n = 7 rats per group).
Figure 3. TBI increases active form of caspase-3. (A) Western blot analysis of the level of the active form of caspase-3 in extracts of the cortex obtained from sham, TBI Day 1, Day 3, and D7 rats (n = 5 rats per group). *P < 0.05; **P < 0.005 (B) Representative
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photomicrographs showing TDP-43 immunoreactivity in brain sections from sham, and 7 days post- TBI rats. GFAP (red)/ Caspase-3 (green)/ 4',6-diamidino-2-phenylindole (DAPI; blue) triple labeling in the sham or contusion cortices. Caspase-3 were
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co-localized with GFAP. (n = 5 rats per group).
Figure 4. Caspase 3 inhibitor blocked cytoplasmic redistribution of TDP-43 in the
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astrocyte. (A) Representative protein bands of TDP-43, TDP-35, and TDP-25 in the
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lesion from rat brains treated with caspase-3 inhibitor or vehicle. C-terminal fragment of TDP-43 generated after TBI and was rescued by caspase-3 inhibitor treatment. (n = 5
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rats per group). *P < 0.05 (B) Western blot analysis of the level of the active form of
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caspase-3 in extracts of the cortex obtained from TBI with and without caspase-3 inhibitor (n = 5 rats per group). *P < 0.05 (C) TDP-43 (green)/ Glial fibrillary acidic
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protein (GFAP; red)/ 4',6-diamidino-2-phenylindole (DAPI; blue) triple labeling in the contusion cortices with caspase-3 inhibitor. The morphology of reactive astrocyte
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become less hypertrophy.
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Figure 5. TBI induce motor dysfunction. (A) The latencies of the rotarod test were significantly shorter in rats TBI rats. (B, C, D) Beam walking performance of sham, TBI Day 7, and TBI + CI Day 7 rats. The latencies, the total faults of the right hind legs, and the total faults of the left hind legs of the three groups are shown. *P < 0.05; **P < 0.005; ***P < 0.001.
Figure 6. Early treatment of caspase-3 inhibitor can improve neuron survival. (A) The dot plot from FACS-like analysis system-based neuronal counting in
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immunocytochemistry staining for NeuN and Dapi. (B) Quantification of survived
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neuronal number by FACS-like analysis system (n = 5 rats per group). *P < 0.05.
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Graphical abstract
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HIGHLIGHTS
TBI increased TDP-43 proteolysis in astrocyte, and induced astrocyte hypertrophy.
TBI induced impaired behaviors that was associated with TDP-43 and its fragments
in astrocyte. Early management of TBI is effective in reducing TDP-43-associated impaired
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behaviors