The Protective Effects Of Curcumin And Methoxy Derivative ASC-J9and The Underlying Mechanism In TDP-43Cell Lines

Oxidative damage plays a critical role in many neurodegenerative diseases. Astrocytes are involved in supporting the survival and protection of neurons against oxidative damage. The dysfunction of antioxidant in astrocytes has been implicated in a variety of neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), spinalmuscularatrophy (SMA). The loss of motor neuron in spinal cord has been attributed to deterioration of astrocytes. The activation of antioxidantive function in astrocytes may serve as a therapeutic strategy for neurodegenerative diseases. Nuclear factor erythroid2-related factor2(Nrf2) is a master transcriptional regulator of phase Ⅱ antioxidantive genes. TAR DNA-binding protein-43(TDP-43) has been identified as the pathological signature protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Pathological TDP-43is abnormally ubiquitinated, generates C-terminal fragments (CTFs) and is redistributed from nuclear localization to cytoplasmic insoluble aggregates. The fragments are toxic to neurons and induced cell death through a toxic gain-of-function. Macroautophagy (called autophagy hereafter) has been predominantly studied as a nonselective self-digestion process. Recent evidence suggests that one form of autophagy, termed quality control (QC) autophagy, selectively disposes of aberrant proteins and damaged organelles via ubiquination of abnormal proteins. Axons of the adult central nervous system (CNS) have very limited ability to regenerate after injury. This inhibitory effect is associated with inhibitory factors that create a nonpermissive environment for regrowth after injuries. Recent evidence suggests that these factors inhibit neurite outgrowth by abnormal activation of the small GTPase RhoA. Neuroregenerative agent that can limit nerve injury and promote neurite outgrowth/axon regeneration after injury in CNS is extremely desirable.Part I Activation of Nuclear Factor Erythroid2-Related Factor2Cytoprotective Signaling by Curcumin Protect Primary Spinal Cord Astrocytes against Oxidative ToxicityObjective:The aim of this study was to evaluate the protective effects of curcumin against oxidative damage and the relationship between detoxification and Nrf2-ARE pathway in primary astrocytes.Methods:We used transgenic animals (Nrf2+/+and Nrf2-/-). Genotypes (Nrf2+/+and Nrf2-/-) of the animals were determined by polymerase chain reaction (PCR) amplification of genomic DNA from tails. This study also used primary astrocyte culture, evaluation of intracellular ROS production and viability assay and detection of cytotoxicity by measuring LDH release and flow cytometry.Results:We report herein that Nrf2-ARE signaling pathway responsive in astrocytes but also it can be significantly activated by curcumin. Addition of curcumin in Nrf2+/+astrocytes, ROS production decreased significantly. In contrast, no significant alterations were observed in Nrf2-/-cells. Curcumin efficiently prevented the loss of mitochondrial trans-membrane potential in Nrf2+/+astrocytes, while failed to do so in Nrf2-/-astrocytes. Pretreatment of Nrf2+/+astrocytes with curcumin led to a significant protection against and decreased the sensitivity to H2O2-induced oxidative stress. In contrast, the same curcumin pretreatment in Nrf2-/-cells resulted in either none or only a slight cytoprotection against the cell injury and led to no significant alteration in the sensitivity to H2O2-induced oxidative stress.Conclusions:curcumin significantly activates Nrf2target genes in primary spinal cord astrocytes, decreases the level of intracellular reactive oxygen species (ROS), and attenuates oxidative damage and mitochondrial dysfunction. Part Ⅱ The neuroprotection effects of ASC-J9and the underlying mechanism in amyotrophic lateral sclerosis cell modelsObjective:This study is to evaluate the degradative effect ASC-J9on mutant TDP-43protein, C-terminal fragments (CTFs) and its cytoplasmic insoluble aggregates, soluble TDP-43protein and the underlying mechanism in this degradative progress.Methods:To examine the solubility profile of TDP-43, sequential extractions were performed. We used the method of reverse transcription-polymerase chain reaction, small interfering RNA, immunofluorescence methods, westernblotting and quantitative assessment of neurite outgrowth.Results:Here we show that5-hydroxy-1,7-bis(3,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one (ASC-J9) increases adaptor p62and NBRl expression, facilitates the clearance of mutant TDP-43,～25and35kDa CTFs and mutant SOD1-G93A proteins via activating QC autophagy, but has little effect on wild type TDP-43and SOD1. Knock-down of p62prevented the degradation of mutant TDP-43and its CTFs. In addition, ASC-J9decreases both soluble and insoluble mutant TDP-43. We also show that expression of mutant TDP-43results in neurite wallerian-like degeneration. Interestingly, ASC-J9significantly promotes neurite outgrowth and attenuates pathological degeneration.Conclusions:Our results suggest that ASC-J9is a promising therapeutic candidate for TDP-43proteinopathies and aggregation-related diseases.PartⅢ The role for selectively autophagy in ASC-J9-induced neurite outgrowth and growth cone extension in motor neuron-like cells through inhibition of RhoA signalingObjective:This study is to evaluated the effect of ASC-J9on neurite outgrowth and growth cone extension in motor neuron-like cells. Because of ASC-J9activated selective autophagy, we further evaluated the underlying mechanism between autophagy and RhoA inhibition.Methods:We used NSC34cell line. NSC-34is an embryonic mouse spinal motor neuron X mouse neuroblastoma hybrid neural cell line with motor neuron like properties. We also used p62siRNA, immunoblotting and quantitative assessment of neurite outgrowth. Down-regulation of p62expression in NSC-34cells was achieved using RNA interference. NSC-34cells were transfected with either p62siRNA for using Lipofectamine2000according to the manufacturer’s instructions.Results:In present study, we find that5-hydroxy-1,7-bis (3,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one (ASC-J9), significantly promote neurite outgrowth, growth cone extension and F-actin cytoskeletal remodeling in motor neuron-like cells (NSC-34cells). The ASC-J9-induced neurite outgrowth, growth cone extension and F-actin cytoskeletal remodeling can be suppressed by concomitant treatment with autophagy inhibitors or p62knock down. The neurite outgrowth promoting effect of ASC-J9is through the inhibition of RhoA pathway and also suppressed by treatment with autophagy inhibitors or p62knock down.Conclusions:Selective autophagy might play an indispensable role in the neurite outgrowth and RhoA inhibition.PartIV Dysfunction of autophagy pathway and the underlying mechanism in TDP-43mutant cellsObjective:This study is to evaluated whether the macroautophagic activity was reduced in stablely expressed mutant full length TDP-43cells and which steps were impaird and also the underlying mechanism of the reduction macroautophagic activity.Methods:We used stably expressed WT and mutant TDP-43cell line. We also used mcherry-lysosome and mcherry-GFP-LC3B plasmid transfection, immune precipitation, immunoblotting and F-actin stainning. Transent transfection was achieved using Lipofectamine2000according to the manufacturer’s instructions.Results:We found that lysosomes were distinctly enriched at one side of the nucleus of expressed wild-type full length TDP-43cells, consistent with their concentration to the microtubule organizing center. By contrast, lysosomes in expressed mutant full length TDP-43cells were more randomly distributed. These data demonstrate that some obstacles may exist in lysosome transportation and concentration in mutant TDP-43cells. We used a pH sensitive, double tagged GFP-mCherry-LC3(dtLC3) reporter to determine the fusion efficiency, found that accumulation of autophagosomes caused by a defect in lysosome-autophagosome fusion in mutant TDP-43expressed cells. We also observed aggresome-like protein aggregates are frequently surrounded by or colocalized with prominent F-actin structures in wild-type TDP-43cells. In contrast, less F-actin was found around protein aggregates in mutant TDP-43cells.The impaired lysosome-autophagosome fusion, obstacles in lysosome concentration in mutant TDP-43expressed cells were restored by re-transfection of full length HDAC6.Conclusions:We speculated that the growing tangle of endogenous nuclear TDP-43and mutant TDP-43might lead to deleterious perturbation of nuclear trafficking and/or solubility of TDP-43result in a loss of function at the nuclear level (HDAC6decreasing). The decreased HDAC6level may responsible to impaird macroautophagic activity in mutant TDP-43cells.