A New Cellular Model Of Stably Expressed TDP-25 And The Role Of TDP-25 On Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis(ALS) is a lethal neurodegenerative disease of motoneurons that causes progressive muscle weakness, paralysis, and premature death. ALS is a multifactorial disease, with etiological heterogeneity and a high variability of clinical presentation. It is characterized by a selective degeneration and death of upper and lower motor neurons, initiating in mid-adult life and progressing to paralysis and death in 1–5 years. Recently, TAR DNA binding protein of 43 k Da(TDP-43)-positive inclusions are present in Parkinson disease, dementia with Lewy bodies, and 30% of Alzheimer disease cases. TDP-43(transactive response DNA-binding protein 43) is a conserved and ubiquitously expressed nuclear protein which is encoded by the TARDBP gene on chromosome 1. Structurally, TDP-43 has five functional regions, including two RNA recognition motifs(RRM1 and RRM2), one glycine-rich region(GRR), as well as a nuclear localization signal(NLS) and nuclear export signal(NES) that mediate nucleocytoplasmic shuttling. Functionally, TDP-43 is a nuclear protein involved in exon skipping and alternative splicing. TDP43 is a predominantly nuclear protein but translocates to the cytosol under a pathological condition, where it is ubiquitnated and/or phosphorylated and cleaved into smaller fragments. A major supplement in our understanding of ALS pathogenesis started in 2006 with the identification of the TDP-43 as the main component of ubiquitinated protein aggregates found in sporadic ALS patients and in patients with FTLD. Sporadic and familial forms of FTLD-U and ALS are characterized by cytoplasmic accumulation of insoluble, hyperphosphorylated, ubiquitinated, and proteolytically cleaved C-terminal fragments in affected brain and spinal cord regions. Notably, the ~25-k Da C-terminal fragment accumulates in affected brain regions, indicating that it may be involved in the disease pathogenesis. TDP-43 is a multifunctional nuclear factor which is involved in many crucial cell processes and neuronal development. A large number of researches indicated that overexpression of the 25-k Da C-terminal fragment was sufficient to cause the mislocalization and cytoplasmic accumulation of endogenous full-length TDP-43. However, a cellular model stably expressing TDP-25 still has not been established in ALS.Abnormal protein aggregation in motor neurons has been recognized as a pathological hallmark in ALS, moreover, the TDP-43 inclusions in the brains of ALS are enriched with TDP-43 C terminal fragments. There are two pathways for protein degradation, the ubinquitin proteasomal system(UPS) and autophagy-lysosomal system in eukaryotic cells. UPS plays important roles in maintaining the balance of cellular proteins that are involved in multiple cellular processes, such as cell cycle, differentiation and development, stress response and DNA repair. Many neurodegenerative disease proteins form ubiquitinated inclusions in diseased brains are degraded by UPS. TDP-43 is localized in ubiquitin-positive inclusions in diseased brains, suggesting that UPS may be involved in TDP-43 degradation or its pathogenesis. The proteasome inhibitors are broadly categorized as synthetic analogs and natural products due to their chemical structures. Peptide aldehyde inhibitors were the first proteasome inhibitors identified and researchers have developed a myriad of aldehyde inhibitors because of the convenience in synthesis and optimization. MG132(Z-Leu-Leu-Leu-al, also termed Cbz-LLL or z-LLL), which was developed by Rock et al.(1994) has been widely used in proteasome biology. MG132 is an effective, reversible 26 S peptide aldehyde proteasome inhibitor, which can inhibit the proteasome pathway protein degradation, thereby affecting cell proliferation and promoting apoptosis. Lactacystin is a kind of natural streptomyces metabolite existing in the soil, and also a selective 20 S inhibitor. Lactacystin and β2 lactone of its intermediates selectively and irreversibly bind to the proteasome β5 subunit, thereby inhibiting the activity of various peptidases. Autophagy-lysosomal system is involved in the clearance of disease proteins and in the removal of aggregated proteins or damaged organelles such as mitochondria. In neurodegenerative diseases, disease proteins prone to aggregation are usually resistant to proteasomal degradation and prefer to be degraded via autophagy-lysosomal system. 3MA, a widely used autophagy inhibitor, inhibits autophagy by blocking autophagosome formation via inhibition of type III phosphatidylinositol 3 kinases(PI3 kinase). Bafilomycin A1 is a vesicular proton pump inhibitor of enzyme H+-ATP, which is from the gray macrolide antibiotic streptomycin. Bafilomycin A1 can inhibit the formation of late autophagy by inhibiting the formation of vesicles. In mammalian cells, rapamycin inhibits the kinase activity of mammalian target of rapamycin(m TOR) by forming a complex with the immunophilin FK506-binding protein of 12 k Da(FKBP12) that binds to and inactivates m TOR, leading to the upregulation of autophagy. The previous studies demonstrated that both TDP-43 and TDP-25 were degraded by UPS and autophagy-lysosomal system. However, it is largely unknown whether the stably expressed TDP-25, i.e. consistently expressed TDP-25 is toxic and how the two pathways respond to the protein. Part Ⅰ A cellular model stably expressing TDP-25’s established and identifiedObjective: To establish a cellular model stably expressing TDP-25 and study the toxicity.Methods: Following the manufacturer’ protocol, NSC-34 cells were transfected with the empty p CI-neo vector or the vector cloned with the TDP-25 c DNAs by using LipofectamineTM 2000 transfection reagent. After transfection for 48 h, cells were diluted to generate polyclonal cell line stably expressing vectors in the presence of G-418 for 3 weeks to select resistant clones. The purified Green fluorescent protein(GFP)-positive cells were obtained by cell sorting using a FACSAria(BD). The polyclonal cell line stably expressing TDP-25 was grown, and tested for expression of human TDP-25 protein by Western blotting. We observed the subcellular localization of TDP-25 by confocal microscopy and inverted fluorescence microscopy. Then the morphology of the aggregates in TDP-25 expressing cells was evaluated by transmission electric microscopy, and further the MDA level and cell viability was measured in the TDP-25 cells. ROS was evaluated by Mito Tracker® Red CM-H2 Xros. Finally the toxicity of stable expressed TDP-25 on the motor neuron was evaluated by apoptotic marker.Results:(1) We established the stable cell lines expressing TDP-25 and the empty p CI-neo vector sucessfully by the cell sorting technique. Western blot data showed the expression levels of TDP-25 were almost 47 percent of endogenous TDP-43.(2) TDP-25 inclusions are not restricted to the nucleus, but localized within neuronal cytoplasmic in the stably expressing TDP-25 cell line. In the stable cell line, we also found that the stably expressed TDP-25 formed some small aggregates just like dots near the nuclear membrane. However, after the cells been treated by MG132, the TDP-25 formed bigger aggregates located in the cytoplasm and nucleus. Furthermore, the aggregates were also evaluated by TEM and were characterized by filamentous bundles and electron dense granular material.(3) Stably expressed TDP-25 induced oxidative stress, lipoperoxidation and apoptosis.(4) We also investigated aggregates by confocal microscopy, and found that the TDP-25 aggregates colocalized with ubiquitin and p62 in the stable cell line.Conclusion: We established the stable cell lines expressing TDP-25 and the empty p CI-neo vector sucessfully by the cell sorting technique. Compared with control cells, stably expressed TDP-25 induced oxidative stress, lipoperoxidation and apoptosis.Part Ⅱ The effects of stably expressed TDP-25 on the ubinquitinproteasomal systemObjective: To investigate whether stably expressed TDP-25 was degraded by the proteasome pathway and the toxicity of the TDP-25.Methods: To study whether the stably expressed TDP-25 was degraded by proteasome, cells expressing TDP-25 were treated with MG132 or lactacystin for 3, 6, 12 and 24 h respectively. The fluorescence number of TDP-25 and GFP of the empty vector was evaluated by the inverted fluorescence microscopy. Then we examined the protein level of human TDP-25 in the stably expressing TDP-25 cell line by western blot, observed characters of mitochondria by transmission electron microscope.Results:(1) Our results showed that the expression of TDP-25 increased gradually and reached the highest level at 12 h. However, no change was observed in the endogenous TDP-43 and GFP of the empty vector.(2) We found that the fluorescence of TDP-25 increased in a time-dependent manner and the amount of the fluorescence of TDP-25 reached the maximum in 24 h. However, no change was observed in the GFP of the empty vector.(3) The TDP-25 had a property that it is easy to form aggregation when it was overexpressed. In the stable cell line, we also found that the stably expressed TDP-25 formed some small aggregates just like dots near the nuclear membrane. However, after the cells were treated by MG132, the TDP-25 formed bigger aggregates located in the cytoplasm and nucleus.(4) Our results showed mitochondrial swelling and cristae dilation in the TDP-25-expressing stable cells by transmission electron microscope. Furthermore, the mitochondria became more swollen after application of the proteasome inhibitor MG132.Conclusion: The stably expressed TDP-25 could mainly be degraded by the proteasome pathway. The mitochondria are swelling in the TDP-25-expressing stable cells. The toxicity of TDP-25 depends on the proteasome activity. Part Ⅲ The effects of stably expressed TDP-25 on theautophagy-lysosomal systemObjective: To investigate whether stably expressed TDP-25 was degraded by the autophagy-lysosomal pathway.Methods: To study whether the stably expressed TDP-25 was degraded by autophagy, cells expressing TDP-25 were treated with 3-methyladenine(3MA) and bafilomycin A1 which were known as inhibitors of autophagy or an autophagy activator rapamycin at different doses. The fluorescence number of TDP-25 and GFP of the empty vector was evaluated by the inverted fluorescence microscopy. Then we examined the protein level of human TDP-25 in the stably expressing TDP-25 cell line by western blot.Results:(1) The stable cells were treated with 3MA or bafilomycin A1 for 24 h, which are inhibitors of autophagy, at different doses. Then, the TDP-25 expression was evaluated by Western blot and the inverted fluorescence microscope. There was no significant change in EGFP and TDP-25 expression in all the study groups.(2) The stable cells were treated with Rapamycin for 24 h, which is an inhibitor of FRAP/m TOR, at different doses. Then, the TDP-25 expression was evaluated by Western blot and the inverted fluorescence microscope. There was no significant change in EGFP and TDP-25 expression in all the study groups.Conclusion: Therefore, the stably expressed TDP-25 was mainly not degraded by the autophagy pathway.