CNR_CONTENT C ur re nt N eu ro va sc ul ar R es ea rc h 7���+��:=����+ ���:������:=?@ 898? �������:;@= =@A> � � � � � � � ���� � ������ ���� � � ��� Send Orders for Reprints to reprints@benthamscience.ae Current Neurovascular Research, 2018, 15, 3-9 3 RESEARCH ARTICLE Temporal Expression of Mutant TDP-43 Correlates with Early Amyotro- phic Lateral Sclerosis Phenotype and Motor Weakness � Qihua Chen1, Jinxia Zhou1, Cao Huang2, Bo Huang2, Fangfang Bi1, Hongxia Zhou2,* and Bo Xiao1,* 1Department of Neurology, Xiangya Hospital of Central South University, Changsha 410008, P.R. China; 2Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107, USA � A R T I C L E H I S T O R Y� Received: October 10, 2017 Revised: December 21, 2017 Accepted: December 29, 2017 DOI: 10.2174/1567202615666180109161541� Abstract: Background: Mutant transactive response DNA-binding protein (TDP-43) is closely correlated to the inherited form of amyotrophic lateral sclerosis (ALS). TDP-43 transgenic rats can reproduce the core phenotype of ALS and constitutive expression of TDP-43 caused postnatal death. Objective: The study aimed to understand whether neurologic deficiency caused by mutant TDP- 43 is dependent on its temporal expression. Method: Transgenic rats were established that express mutant human TDP-43 (M337V substitu- tion) in neurons, then a Tet-off system was used to regulate its expression. Results: TDP-43 mutant transgenic rats developed significant weakness after the transgene was activated. Rats with expression of mutant TDP-43 at 30 days showed a more aggressive pheno- type. More severe pathological changes in neurogenic atrophy were observed in these rats. Conclusion: Temporal expression of mutant TDP-43 in neurons promoted serious phenotype in rats. The dysfunction of TDP-43 had a profound impact on the development of motor neurons and skeletal muscles. Keywords: Amyotrophic Lateral Sclerosis (ALS), TAR DNA-binding protein 43, motor neurons, transgenic rats, CAG, Tet- responsive transactivator. 1. INTRODUCTION Amyotrophic Lateral Sclerosis (ALS) is the most fre- quent form of adult-onset motor neuron disease, in which loss of motor neurons leads to weakness and denervation atrophy of voluntary muscles. The disease often develops around middle life and inexorably progresses to paralysis [1, 2]. Although most ALS cases are sporadic, Familiar ALS (FALS) accounts for 5 to 10% of cases with an autosomal dominant pattern of inheritance. Transactive response DNA-binding Protein (TDP-43) is a heterogeneous nuclear RNA binding protein encoded by TDP-43 gene. TDP-43 plays important roles in multipro- tein/RNA complex and regulating gene expression [3]. TDP- 43 has been shown to promote neurodegeneration by impair- ing chromatin remodeling, accelerate age-dependent degen *Address correspondence to these authors at the Thomas Jefferson Univer- sity, 508 JAH, 1020 Locust Avenue, Philadelphia, PA19107, USA; Xiangya Hospital of Central South University, 87 Xiangya Road, Changsha, 410008, P.R. China; Tel: 086-13908487631; Fax: 086-0731-84327401; E-mail: 119072898@qq.com; xiaobo_xy@126.com eration of interneurons, and cause defects in dendritic growth [4-6]. Accumulating evidence have implicated that the pathogenic mutation of TDP-43 causes an inherited form of ALS [7]. Mutation of TDP-43 gene has been identified re- sponsible for some familiar ALS. Including M337V muta- tion, no less than 30 pathogenic TDP-43 mutants have been reported so far [8]. Using a Tet-off system, inducible expression of human TDP-43 with a pathogenic mutation (M337V substitution) successfully induced progressive paralysis in transgenic rats [9]. The removal of mutant TDP-43 partly restored motor function in ALS rats expressing the causative gene in neu- rons and muscles, indicating that the motor neuron degenera- tion is partially reversible [10]. However, it is unclear whether neurologic deficiency caused by mutant TDP-43 is correlated with the time of its expression. To address this question, we used Tet-off system to produce inducible gene expression in transgenic rats, and the consequence events including the onset age and severity of illness were assessed in transgenic models. Our findings support the hypothesis that earlier expression of mutant TDP-43 can cause a more aggressive phenotype of ALS in transgenic rats. 1875-5739/18 $58.00+.00 © 2018 Bentham Science Publishers 4 Current Neurovascular Research, 2018, Vol. 15, No. 1 Chen et al. 2. MATERIALS AND METHODS 2.1. Generation of Transgenic Rats The animal use protocol was approved by the Ethical Re- view Board from Thomas Jefferson University, PA, USA. Transgenic rats were established and maintained in a Spra- gue Dawley background. The TDP-43 gene was isolated from BAC clone (RP11-829B14), and an M337V substitu- tion was integrated into TDP gene by homologous recombi- nation in Escherichia coli [11]. Tetracycline (Tet)-inducible transgenic rats carried a mutant human TDP-43 (TDP- 43M337V) transgene at the control of a Tet-responsive Element (TRE) [12]. Selected Cytomegalovirus enhancer fused to chicken beta actin promoter (CAG) was used to drive Tet- responsive Transactivator (tTA) gene. TRE-TDP-43 M337V transgenic rats were crossed with CAG-tTA positive rats. To selectively induce human TDP-43 gene expressed in rats, breeding rats and transgenic offspring were drunk with Dox (50 ug/ml) to inhibit transgene expression during embryonic and postnatal phase. Then CAG-tTA/TRE-TDP-43M337V double transgenic rats were deprived of Dox to activate mu- tant TDP gene. The time of withdrawal of Dox (Dox-) was selected as 30-day-old and 70-day-old, to mimic the juvenile stage and adulthood of rats. 2.2. Behavioral Tests and Disease Stages The onset and progression of the ALS phenotypes in transgenic rats were detected by mobility and grip strength. Mobility was surveyed by open field test (Med Associates), which measured the total distance a rat travelled within 10 min [13]. Grip strength was measured with a grip-strength meter (Columbus Instruments). We defined the disease onset as an irreversible reduction in travelled distance. Paralysis was defined as dragging of legs or an inability to retract in- dividual legs. Disease end stage was defined as the inability to retract two or more legs, or inability to right itself when the rat was placed on its side, or as a severe weight loss. Re- lated data were compared using unpaired t-tests. P-value < 0.05 was regarded as statistically significant. 2.3. Immunofluorescence, Immunohistochemistry and Cresyl Violet Staining Coronal forebrain sections and cross-sections of spine were used for immunohistochemistry and Cresyl violet stain- ing. The colocalization of human TDP-43 with NeuN was detected by double-labelling immunofluorescence. Primary antibodies used for immunofluorescence were; anti-human TDP-43 (Abnova, clone 2E2-D3), polyclonal antibody against NeuN from rabbits immunized with a peptide (MAQPYPPAQYPPC) and affinity purified. Immunostained tissues were visualized using a Nikon microscope, and im- ages were acquired using a Nikon digital camera as previ- ously reported [14]. Motor neurons in the spinal cord were observed by Cresyl violet staining and quantified as previ- ously reported [14]. The motor neurons in a long segment of lumbar cord (L3-L5) were assessed to increase the accuracy of cell counting. The central segment of lumbar cord was cut into 30-um thick sections using a freezing microtome, and motor neurons were counted in every 10th section on both sides using a fractionator-based stereology software (Stere- ologer). Numbers of neurons were compared using unpaired t-tests. P-value < 0.05 was regarded as statistically signifi- cant. 2.4. Histology and Histochemistry in Skeletal Muscles Fresh gastrocnemius muscles were quick-frozen in isopentane/liquid nitrogen, then cut into cross sections (10 �m) on a Cryostat Microtome. The structures of muscle were assessed by H&E staining, histochemistry for nonspecific esterase and ATPase. Nonspecific esterase activity was de- tected using a-napthyl acetate protocol. Myosin ATPase staining (pH 4.5) was used to discriminate three types of skeletal muscle fibers. 3. RESULTS 3.1. TDP-43 Transgenic Rats had Worse Phenotype To examine whether mutant TDP-43 expressed in neu- rons could induce ALS-like phenotypes, we chose the regu- latory promoter of CAG gene to control the activation of transgene and established CAG-tTA/TRE-TDP-43M337V dou- ble transgenic rats, with their mutant human TDP-43 gene under the control of a Tetracycline (Tet)-responsive Element (TRE). Then, we used the Tet-off system to regulate the ex- pression of mutant TDP-43 in neurons. Dox was used to in- hibit transgene expression during embryonic and postnatal phase. To mimic mutant TDP-43 expression in juvenile stage or adulthood, transgenic rats were deprived Dox (Dox-) at the age of 30 days or 70 days, respectively. Both the groups of CAG-tTA/TRE-TDP-43M337V double transgenic rats suf- fered from progressive weakness after Dox (Fig. 1A, B). In the group of Dox- at the age of 30 days (Dox- 30 days), transgenic rats began losing grip strength and mobility as early as 16 days after Dox- (26.33±3.575 days after Dox-), while the counterpart rats with Dox- at the age of 70 days (Dox- 70days) developed early signs of paralysis (disease onset) not until 26 days after Dox- (48.17±6.39 days after Dox-) (Fig. 1C-F). There were significant differences in the time of onset between the two groups (Fig. 2A, B). 3.2. Severe Loss of Neurons in Transgenic Rats In the cortex and dentate gyrus, double-label fluores- cence staining demonstrated that mutant human TDP-43 was colocalized with the neurons (Fig. 3A, B). CAG-tTA/TRE- TDP-43M337V double transgenic rats showed apparent expres- sion of TDP-43 at the disease end stage, while TDP-43 was not detected in normal rats with matched age (Fig. 3C). To accurately estimate motor neuron death in rats, we used un- biased stereological cell counting to count the number of motor neurons in a long segment of spinal cord (L3-L5). Compared with normal rats, stereological cell counting iden- tified more loss of neurons in transgenic rats with Dox- at the age of 30 days than in rats with Dox- at the age of 70 days (Fig. 4). 3.3. Neurogenic Damages in Transgenic Rats As an outcome of motor neuron degeneration, we exam- ined skeletal muscle atrophy by H&E staining (Fig. 5A1, B1), and performed immunohistochemistry for nonspecific esterase (Fig. 5A2, B2) and ATPase (Fig. 5C1, C2) in mut- ant TDP-43 transgenic rats at end stage. In addition, CAG- TDP-43 Worsen ALS-like Phenotype Current Neurovascular Research, 2018, Vol. 15, No. 1 5 Fig. (1). Mutant human TDP-43 expressed on 30 days causes shorter onset and induces more severe paralysis in transgenic rats. Double transgenic rats developed paralysis (A): Dox- at the age of 30 days, (B): Dox- at the age of 70 days). Compared to control group, open-field assay (C) and the measured grip strength of paws (E) indicated an irreversible reduction of mobility in two groups of double-transgenic rats (*P<0.05). Compared with the rats deprived of Dox at the age of 70 days, the rats deprived of Dox at the age of 30 days developed more seri- ous paralysis (D and F). Data are means ± SD (n=6). *P<0.05. Stage 0: the time of Dox-; stage 1: onset time; stage 2: middle stage; stage 3: end stage. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper). Fig. (2). The survival of transgenic rats. (A). The disease onset in two groups of rats deprived of Dox at the age of 30 and 70 days, respec- tively. (B). The survival in two groups of rats deprived of Dox at the age of 30 and 70 days, respectively. Data are means ± SD (n=6). tTA/TRE-TDP-43M337V double transgenic rats with Dox- at the age of 30 days revealed a worse pathologic change than the rats of Dox- 70 days. In contrast, rats in control group with matched age had no sign of amyotrophy (Fig. 5C). We also counted the cross areas of myofibers in all groups of rats on cross sections of gastrocnemius stained by ATPase 4.5, and found that the size of fibers was remarkably reduced in TDP-43 rats (Fig. 5D). 4. DISCUSSION As a highly conserved ribonucleoprotein, TDP-43 plays an important role in gene expression. The level of TDP-43 is remarkably elevated in the brain and spine of patients in late stage ALS [15-17]. Moreover, overexpression of either wild- type or mutant TDP-43 could cause cell death, suggesting the gain-of-function of TDP-43 in disease progress [18, 19]. The previous study confirmed that mutant TDP-43 gene ex- 6 Current Neurovascular Research, 2018, Vol. 15, No. 1 Chen et al. Fig. (3). Overexpression of mutant human TDP-43 (hTDP-43) in neurons of transgenic rats. Immunofluorescence showed that mutant TDP- 43 was expressed in the cortex (A) and dentate gyrus (B) of CAG-tTA/TRE-TDP43M337V double-transgenic rats after Dox was deprived (Dox-) at the age of 30 days and 70 days, respectively (green, A1, B1, A4, B4), but was not detected in the normal group (C1, C4). Neuronal marker NeuN was stained in the cortex and dentate gyrus in transgenic groups and control group (red, A2, B2, C2, A5, B5, C5). Double-label fluorescence staining demonstrated that mutant human TDP-43 was colocalized with NeuN in the cortex and dentate gyrus in transgenic rats (A3, B3, A6, B6), but not in control group (C3, C6). Scale bar: 100 �m. (C). Immunohistochemistry showed overexpression of TDP-43 in the cortex (A1, B1, C1) and dentate gyrus (A2, B2, C2). Scale bar: 100 �m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper). Fig. (4). Expression of mutant TDP-43 causes motor neuron death. (A-C) Representative images of the lumbar cord of transgenic rats at end stage (A): Dox- at the age of 30 days, (B): Dox- at the age of 70 days) and normal control subjects at matched ages (C). Scale bar: 200 �m (A1, B1, C1) and 50 �m (A2, B2, C2). (D) Stereological cell counting demonstrated a significant loss of motor neurons (>25 �m in diameter) in the L3-L5 cords of CAG-tTA/TRE-TDP43M337V double-transgenic rats compared with normal rats. Data are means ± SD (n=6). *P<0.05. TDP-43 Worsen ALS-like Phenotype Current Neurovascular Research, 2018, Vol. 15, No. 1 7 Fig. (5). Overexpression of mutant human TDP-43 in early stage results in more severe muscular atrophy. (A-C) H & E staining (A1, B1), immunohistochemistry for nonspecific esterase (A2, B2) and ATPase 4.5 (A3, B3) indicated substantial atrophy of skeletal muscle in two group of double transgenic rats at the end stage, while the normal rats with matched ages showed no sign of amyotrophy (C1, C2, C3). Scale bar: 30 �m. (D) The cross areas of myofibers in all groups of rats were measured on cross sections of gastrocnemius stained by ATPase 4.5. Data are means ± SD (n=6). pressed in motor neurons is sufficient to induce ALS-like symptoms [8]. In this study, we used CAG-tTA/TRE-TDP- 43M337V double transgenic rats that express mutant human TDP-43 in neurons. Dependent on Tet-responsive element, we regulated the expression of mutant TDP-43 by admini- stration or withdrawal of Dox. We validated that CAG- tTA/TRE-TDP-43M337V double transgenic rats developed progressive severe weakness, atrophy and weight loss. In the end stage, transgenic rat models with Dox- showed signifi- cantly reduced motor neurons in spinal cord and apparent neurogenic atrophy, consistent with the pathologic changes in ALS. Notably, transgenic rats that expressed mutant human TDP-43 at 30 days exhibited more devastating clinical phe- notypes as (a) the interval between Dox- and disease onset was shorter, (b) there was an expedited proceeding to termi- nal stage, (c) their limb asthenia and muscle degeneration were more remarkable. It is unquestionable that the progres- sive loss of motor neurons plays a critical role in ALS, and significant decreases in motor neurons in spinal cord are observed in ALS transgenic rats. However, there was no significant difference between double transgenic rats. Small sample size may explain the results because there were less motor units in transgenic rats with Dox- 30 days. It has been well established that the pathogenic mutation and elevated expression of TDP-43 are neurotoxic to motor neurons. Although the detailed mechanisms of mutant TDP- 43 induced motor neuron death are unclear, ubiquitin aggre- gation, gliosis and cytoplasmic accumulation of TDP-43 are all correlated to motor neuron degeneration [10]. However, whether the adverse effect of mutant TDP-43 varied at dif- ferent periods in developmental process of motor system remains a conundrum. Since the copies of mutant TDP-43 transgene are equal in both groups of transgenic rats, we speculate that normal function of TDP-43 is more crucial to the early development of motor neurons and skeletal mus- cles. Increasing evidences suggested that the loss of TDP-43 function is involved in neurodegeneration and ALS symp- 8 Current Neurovascular Research, 2018, Vol. 15, No. 1 Chen et al. toms [20, 21]. A motor neuron-specific TDP-43 knockout mice model demonstrated age-dependent motor impairment and morphological abnormalities in the motor neuron sys- tem, and the loss of TDP-43 function led to the pathogenesis of ALS [22]. In addition, TDP-43 gene deletion resulted in early embryonic lethality in mammals [23, 24]. Embryonic TDP-43 expression was colocalized with the neuroepithe- lium that contains neural progenitors [25]. Given that in- terneuron subtypes and motor neurons were derived of neu- ral progenitors, it can be speculated that mutations in TDP- 43 regulation may affect the development central nervous system, including the growth of motor neurons. Since the early development of motor neurons is crucial to motor func- tion, it is possible that earlier disturbance to TDP-43 expres- sion gives rise to developmental anomalies of motor systems and causes more severe ALS-like pathological changes. Therefore, our findings suggest that it is very important to perform early intervention to prevent or delay the progres- sion of TDP-43 induced ALS pathological changes, provid- ing new strategy for ALS treatment. CONCLUSION In summary, our study suggests that mutant TDP-43 ex- pressed at juvenile stage of the rats can cause a more aggres- sive phenotype than mutant TDP-43 expressed at an adult stage of the rats. The normal function of TDP-43 is impor- tant to promote the development of motor neurons and skele- tal muscles. ETHICS APPROVAL AND CONSENT TO PARTICI- PATE The animal use protocol was approved by the Ethical Re- view Board from Thomas Jefferson University, PA, USA. HUMAN AND ANIMAL RIGHTS No Humans were used for studies that are base of this re- search. All the animals used were in accordance with the standards set forth in the 8th Edition of Guide for the Care and Use of Laboratory Animals (http:// grants.nih.gov/ grants/olaw/Guide-for-thecare-and-use-of-laboratory-ani- mals.pdf) published by the National Academy of Sciences, The National Academies Press, Washington DC, United States of America. CONSENT FOR PUBLICATION Not applicable. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS We would like to thank all participants in this study and Dr. Xugang Xia from the Thomas Jefferson University for laboratory assistance. This work was supported by Natural Science Foundation of China (No. 81401065, 81760238, 81571256) and science fund for young scientists from Xian- gya Hospital of China (No. 2013Q09). REFERENCES [1] Boillee S, Yamanaka K, Lobsiger CS, et al. Onset and progression in inherited ALS determined by motor neurons and microglia. Sci- ence 2006; 312(5778): 1389-92. 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[24] Wu LS, Cheng WC, Hou SC, Yan YT, Jiang ST, Shen CK. TDP- 43: A neuro-pathosignature factor, is essential for early mouse em- bryogenesis. Genesis 2010; 48(1): 56-62. [25] Rowitch DH. Glial specification in the vertebrate neural tube. Nat Rev Neurosci 2004; 5(5): 409-19. � � Temporal Expression of Mutant TDP-43 Correlates with Early AmyotrophicLateral Sclerosis Phenotype and Motor Weakness Abstract: Background: Objective: Method: Results: Conclusion: Keywords: INTRODUCTION MATERIALS AND METHODS RESULTS Fig. (1). Fig. (2). DISCUSSION Fig. (3). Fig. (4). Fig. (5). CONCLUSION ETHICS APPROVAL AND CONSENT TO PARTICIPATE HUMAN AND ANIMAL RIGHTS CONSENT FOR PUBLICATION CONFLICT OF INTEREST ACKNOWLEDGEMENTS REFERENCES