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 substitution) 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 phenotype. 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 frequent 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 multiprotein/RNA complex and regulating gene expression [3]. TDP-43 has been shown to promote neurodegeneration by impairing chromatin remodeling, accelerate age-dependent degen 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 responsible for some familiar ALS. Including M337V mutation, 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 neurons and muscles, indicating that the motor neuron degeneration 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.
2. MATERIALS AND METHODS
2.1. Generation of Transgenic Rats
The animal use protocol was approved by the Ethical Review Board from Thomas Jefferson University, PA, USA. Transgenic rats were established and maintained in a Sprague Dawley background. The TDP-43 gene was isolated from BAC clone (RP11-829B14), and an M337V substitution was integrated into TDP gene by homologous recombination 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 mutant 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 individual 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. Related 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 staining. 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 images were acquired using a Nikon digital camera as previously reported [14]. Motor neurons in the spinal cord were observed by Cresyl violet staining and quantified as previously 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 (Stereologer). Numbers of neurons were compared using unpaired t-tests. P-value < 0.05 was regarded as statistically significant.
2.4. Histology and Histochemistry in Skeletal Muscles
Fresh gastrocnemius muscles were quick-frozen in isopentane/liquid nitrogen, then cut into cross sections (10 µm) on a Cryostat Microtome. The structures of muscle were assessed by H&E staining, histochemistry for nonspecific esterase and ATPase. Nonspecific esterase activity was detected 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 neurons could induce ALS-like phenotypes, we chose the regulatory promoter of CAG gene to control the activation of transgene and established CAG-tTA/TRE-TDP-43M337V double 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 expression of mutant TDP-43 in neurons. Dox was used to inhibit 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 suffered 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 fluorescence 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 expression 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 unbiased 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 identified 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 examined 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-
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 expressed 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 administration 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 significantly 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 phenotypes as (a) the interval between Dox- and disease onset was shorter, (b) there was an expedited proceeding to terminal stage, (c) their limb asthenia and muscle degeneration were more remarkable. It is unquestionable that the progressive 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 aggregation, 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 different 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 muscles.
Increasing evidences suggested that the loss of TDP-43 function is involved in neurodegeneration and ALS symptoms [20, 21]. A motor neuron-specific TDP-43 knockout mice model demonstrated age-dependent motor impairment and morphological abnormalities in the motor neuron system, 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 neuroepithelium that contains neural progenitors [25]. Given that interneuron subtypes and motor neurons were derived of neural 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 function, it is possible that earlier disturbance to TDP-43 expression 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 progression of TDP-43 induced ALS pathological changes, providing new strategy for ALS treatment.
CONCLUSION
In summary, our study suggests that mutant TDP-43 expressed at juvenile stage of the rats can cause a more aggressive phenotype than mutant TDP-43 expressed at an adult stage of the rats. The normal function of TDP-43 is important to promote the development of motor neurons and skeletal muscles.
Ethics Approval and Consent to Participate
The animal use protocol was approved by the Ethical Review Board from Thomas Jefferson University, PA, USA.
Human and Animal Rights
No Humans were used for studies that are base of this research. 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.
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 Xiangya Hospital of China (No. 2013Q09).
Conflict of Interest
The authors declare no conflict of interest, financial or otherwise.
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