Protective Effect Of Dimethoxy Curcumin On Mitochondrial Dysfunction In Mutated Human TDP-43 Transfected NSC34 Cell Line

Objectives: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Most of the cases are sporadic (sALS), but 10% are familial (fALS) forms inherited in a dominant manner. Approximately 20% of familial cases are caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene, and the development of transgenic mouse models of human SOD1 mutations opened an area of intense investigation into the pathogenesis of familial ALS. However, the role of biochemically altered SOD1 in sporadic ALS is highly speculative and not supported by strong human or animal data. Most recently, mutations of the RNA metabolizing protein, the 43-kDa TAR–DNA-binding protein (TDP-43), have been uncovered by multiple groups worldwide in several rare autosomal dominant familial cases of ALS and a pathological alteration of this protein is seen in most sporadic ALS cases, potentially making TDP-43 enormously significant in understanding sporadic and familial ALS. Currently more than thirty mutations in TDP-43 have been identified in familial and sporadic ALS. TDP-43 is a 414 amino acid nuclear protein encoded by the TARDBP gene on chromosome 1. TDP-43 is an mRNA binding protein that plays important functions in regulating mRNA metabolism involved in several functions, including transcriptional repression, exon skipping and RNA splicing. TDP-43 predominantly expresses in the nucleus where it exerts its biological functions, but in pathological tissue TDP-43 is aberrantly processed and forms inclusions in the cytoplasm.Mitochondria have increasingly taken a center stage in various models of neurodegeneration. Several studies have reported mitochondrial morphological changes and mitochondrial dysfunction in ALS patients and in a variety of experimental models of SOD1-linked fALS. Mitochondrial swelling and vacuolization are apparent at early, presymptomatic stages in SOD1G37R mice. At the onset of motor symptoms there is a massive degeneration of mitochondria in motor neurons which occurs long before the motor neurons die. Moreover, deficits in the activities of mitochondrial respiratory chain complex I and complex IV have been identified in the skeletal muscle and in the spinal cord of sALS patients. However most of the work on the mitochondrial dysfunction in ALS has been done in the SOD1 model, it is important to extend these studies to other models to determine if mitochondrial dysfunction is a common outcome of the disease. To further elucidate contribution of mitochondrial dysfunction to the pathogenesis of TDP-43 linked ALS, as well as to identify therapeutic approaches that target mitochondrial dysfunction and its consequences. We measured some mitochondrial parameters from NSC-34 cells transfected human normal and mutated TDP-43, meanwhile observed therapeutic effect of dimethoxy Curcumin (DMC), an analog of curcumin obtained by methylation of both free phenolic groups in the parent compound.Methods:1 Cell culture and generation of NSC-34 cell lines stably transfected with human TDP-43NSC-34 is a hybrid cell line that expresses many properties of motor neurons. NSC-34 was routinely maintained in the high glucose formulation of DMEM supplemented with 10% FBS and antibiotics at 37°C and 5% CO2 atmosphere. NSC-34 cells were transfected with the empty pCI-neo vector or the vector cloned with wild-type (WT), M337V and Q331K mutant human TDP-43 cDNAs using LipofectamineTM 2000 transfection reagent, following the manufacturer's protocol. Individual G418-resistant clones were isolated, grown, and tested for expression of human TDP-43 protein by Western blotting. The NSC-34 cell subclones transfected with empty, WT, Q331K or M337V mutant TDP-43 were maintained in selective medium (0.5 mg/ml of G-418) containing 10% FBS for passages.2 Therapeutic agent and administration protocol Dimethoxy Curcumin was synthesized as described previously. We dissolved it in dimethyl formamide (DMF) and treated NSC-34 cells transfected with Q331K or M337V mutant TDP-43 with 15μM DMC for 3 days, control cells received DMF only.3 Western blotCells were harvested and protein was extracted using a total protein extraction kit following the manufacturer's instruction. Protein extracts were quantified using the BCA protein measure method. Fifty micrograms of protein from each sample was run on SDS–PAGE gels and blotted onto PVDF membranes. The membrane was then incubated overnight at 4°C with the following primary antibodies: rabbit anti-HA, rabbit anti-UCP2, mouse anti-NDUFB8, mouse anti-β-actin. Membranes were then incubated with a fluorescence-conjugated secondary antibody. The bands of interest on the membrane were detected using an Odyssey Infrared Imaging System. The original green or red color of a band was converted to black and white colors for data presentation.4 Electron microscopyCells grown on corning flasks were fixed in 4% glutaraldehyde in 0.1M phosphate buffered saline (PBS), postfixed with 1% OsO4 in 0.1M PBS. Samples were stained with uranyl acetate and lead citrate, dehydrated in ethanol and embedded in EPON 812 following standard procedures. Ultrathin sections were examined using a transmission electron microscope.5 Mitochodrial membranes potentialMitochondrial transmembrane potential (△Ψm) was measured using flow cytometry. NSC34 cells transfected with empty, WT, M337V or Q331K mutant TDP-43 were harvested into phosphate buffered saline (PBS) and cells was incubated with rhodamine 123 at 37°C for 30 min. The cells were then washed twice with PBS and were immediately analyzed by flow cytometry to determine fluorescence intensity. The cell line expressed the empty vector was used as the normal control. At least 10,000 cells per sample were analyzed.6 Isolation of mitochondria from cultured cells To isolate mitochondria, we used a modification of the nitrogen cavitation method. The different cell lines were harvested at the same passage number to ensure fair comparison. The cells were washed once with isolation medium, then collected into a pre-cooled cavitation chamber. The cell suspension under stirring was subjected to 1500 psi for 15 min. At the end of the 15 min period, the pressure in the chamber fell to approximately 800 psi. The cell suspension is then released through outflow tubing. After collecting the cell suspension from the cavitation chamber, it was centrifuged at 600×g for 10 min to pellet the cell debris (heavy particles, or fractions of cells). The supernatant was collected and centrifuged at 17,000×g for 10 min. This pellet (crude mitochondrial fraction) was washed and finally resuspended in buffer, as above, before snap freezing in 100μl aliquots and storing at -80°C until use.7 Measurement of mitochondrial malondialdehyde (MDA)Lipid peroxidation was determined by the measurement of MDA in the isolated mitochondria with the thiobarbituric acid reaction substances (TBARS). Following the manufacturer's instructions, mitochondria samples were mixed with the reagents at room temperature. Reaction mixtures were incubated at 95°C for 40min and then allowed to cool. Absorbance of samples was read at 532 nm. The amount of MDA was expressed as nmole of TBARS/mg protein.8 Mitochondrial respiratory chain enzyme activitiesSpectrophotometric assays were used to measure the activity of each of the components of the electron transfer chain. Activity of complex I of ETC was estimated by a modification of the method of Janssen et al. We think it is imperative to add rotenone in a separate cuvette before the start of the measurement to allow rotenone to accumulate to its binding site. Complex II activity was estimated by the method of Janssen et al. The reaction was initiated with the addition of succinate and was monitored at 600 nm at 30°C temperature. The activity was calculated using the extension coefficient of DCIP. Complex III activity was estimated by the method from book of mitochondrion. Reduction of cytochrome c (oxidized) is coupled to oxidation of decylbenzylquinol (DB.H2), and the increase in absorbance at 550 nm was followed. The reaction was initiated with the addition of cytochrome c (oxidized). Activity of Complex III was expressed as an apparent first-order rate constant. Complex IV activity was determined by the method from book of mitochondrion. Oxidation of reduced cytochrome c by complex IV was followed by a decrease in absorbance at 550 nm. The reaction was initiated with the addition of mitochondria. Activity of COX was expressed as an first-order rate constant.Results:1 Generation of stably transfected NSC-34 cellsStable transfections with the different plasmids were checked by Western blots. The expression of human TDP-43 was measured with an anti-HA antibody. In SDS-PAGE human TDP-43 were detected in cells transfected WT, Q331K or M337V mutant TDP-43. No human TDP-43 expression is seen in cells transfected with empty plasmids.2 Morphologic alterations of mitochondria in NSC34 cells in the presence of mutant TDP-43 and effect of DMCThe morphology of the mitochondria in the presence of mutant TDP-43 was investigated by electron microscopy. In NSC34 cells transfected with empty vector alone, the mitochondrial cristae were clearly defined. In cells expressing M337V mutant TDP-43, the intracristal space of the mitochondria become dilated. After administration of DMC the abnormal appearance had an improvement.3 Presence of mutant TDP-43 results in a decrease of the complexⅠactivity of the mitochondrial electron transfer chain and effect of DMCThe activities of mitochondrial electron transfer chain complex in NSC-34 cell lines are measured. The presence of human normal and mutant TDP-43 in NSC34 cells result in a significant decrease in activity of complexⅠcompared with empty plasmid transfected cell line. The activity of complexⅠwas 31.79±1.03, 26.72±1.62, 13.92±1.46, 19.56±1.94μM/min/mg respectively in empty plasmid, wild type, M337V, Q331K TDP-43 transfected cell lines. The levels of ComplexⅡ,complexⅢand ComplexⅣactivity were not significantly altered by the presence of human normal or mutant TDP-43 in the cells. To determine whether the change in activity of complexⅠwas accompanied by alterations in the level of protein expression. The complexⅠsubunit NDUFB8 was detected by western blot. Results from three separate experiments indicate that there are no significant changes in protein levels of NDUFB8. Meanwhile we observed effect of DMC on complexⅠactivity in NSC34 cells transfected mutant TDP-43. DMC can significantly increased complexⅠactivity 2-fords more in both Q331K and M337V mutant TDP-43 cell lines.4 Mitochondrial member potential decreased in NSC-34 cells transfected human normal and mutated TDP-43 and the improved role of DMCMitochondrial membrane potential was measured in empty, TDP-43 wild type, Q331K or M337V transfected NSC-34 cells. The results showed that TDP-43 wild type, TDP-43 M337V, especially TDP-43 Q331K caused significant decrease of mitochondrial membrane potential. Moreover we have observed effect of DMC on mitochondrial membrane potential. The results showed that DMC significantly improved mitochondrial membrane potential approximately twice in NSC-34 cells transfected human mutated TDP-43.5 UCP2 expression in NSC34 cells in the presence of mutant TDP-43 and effect of DMCWe examined the expression of UCP2 in NSC-34 cells transfected with empty, WT, Q331K or M337V mutant TDP-43. UCP2 expression levels in the NSC-34 cells transfected with WT, Q331K or M337V mutant TDP-43 are significantly higher than that in the NSC-34 cells transfected with empty plasmids. Moreover, DMC significantly decreased UCP2 expression levels in the NSC-34 cells transfected with Q331K or M337V mutant TDP-43. 6 MDA levels in mitochondria isolated from transfected NSC-34 cellsTo detect levels of lipid peroxidation of mitochondria, we examined the MDA level in mitochondria isolated from transfected NSC-34 cells. The result showed that there were no significantly changes in mitochondrial MDA level in NSC-34 cells transfected with empty, WT, Q331K or M337V mutant TDP-43.Conclusions:1. Human mutated TDP-43 can cause mitochondrial dysfunction in NSC-34 cells. These abnormal phenomena are showed the following aspects. (1). Human mutated TDP-43 caused morphologic abnormal alterations of mitochondria in NSC34 cells. (2). Presence of mutant TDP-43 results in a decrease of the complexⅠactivity of the mitochondrial electron transfer chain. (3). Mitochondrial member potential decreased in NSC-34 cells transfected human normal and mutated TDP-43. (4). UCP2 expression levels in the NSC-34 cells transfected with WT, Q331K or M337V mutant TDP-43 are significantly higher than that in the NSC-34 cells transfected with empty plasmids.2. DMC has protective effect on mitochondrial dysfunction in mutated human TDP-43 transfected NSC34 cell line by the following roles. (1)DMC ameliorated morphologic abnormal alterations of mitochondria in NSC34 cells transfected human mutated TDP-43. (2). DMC ameliorated decrease of the complexⅠactivity of the mitochondrial electron transfer chain in NSC34 cells transfected human mutated TDP-43. (3). DMC significantly elevated mitochondrial member potential in NSC-34 cells transfected human normal and mutated TDP-43. (4).DMC decreased UCP2 expression levels in the NSC-34 cells transfected with Q331K or M337V mutant TDP-43.