| BackgroundL-Glutamate is the major excitatory neurotransmitter in the central nervous system. Excessive stimulation or over-activation of neurons by glutamate causes neuronal death, a pathological process referred as glutamate excitotoxicity.It has been proposed that glutamate excitotoxicity is an important pathological mechanism in a wide range of neurological disorders including cerebral ischemia, stroke, brain trauma and even neurodegenerative diseases such as amyotrophic lateral sclerosis, Parkinson’s disease and Huntington’s disease. The detailed mechanisms underlying glutamate excitotoxicity-induced neuronal death are not fully elucidated. However, mitochondrial dysfunction including mitochondrial membrane potential (MMP) reduction and ROS overproduction was reported as a primary event in glutamate induced neurotoxicity.Mitochondria are highly dynamic organelles that undergo continual fission/fusion events which plays a critical role in maintaining mitochondrial homeostasis. Unopposed fission leads to fragmentation while unopposed fusion causes elongation, both of which could impair mitochondrial function. Mitochondria fission and fusion events are tightly regulated by mitochondrial fission and fusion proteins:dynamin-like protein 1 (DLP1, also referred to as Drp1) and its recruiting factors on mitochondria such as Fisl, Mff, MiD49 and MiD51 for fission; and Mitofusin 1 (Mfh1), Mitofusin 2 (Mfn2) and optic atrophy protein 1(OPA1) for fusion. In addition to regulating mitochondrial morphology, mitochondrial fission and fusion processes are essential for the maintenance of various aspects of mitochondrial function (14). Thus, not surprisingly, emerging studies suggest the altered balance in mitochondrial fission and fusion might be a potential mechanism leading to mitochondrial dysfunction in neurological disorders.Mitochondrial dysfunction was reported as a primary event in glutamate induced neurotoxicity, and mitochondrial function is highly dependent on mitochondrial dynamics. Whether mitochondrial dysfunction plays a central role in glutamate-evoked neuronal excitotoxicity is still unclear. Here we sought to investigate whether and how mitochondrial dynamics play a role in glutamate-induced mitochondrial dysfunction and neuronal death in spinal cord motor neurons in vitro, in mice and in human.ObjectTo research mechanism of the toxic effect of glutamate in the primary neurons, glutamate infused mouse models, mfn2 overexpress mouse models and human tissue by exploring signaling pathways of the glutamate neurotoxicity,finding and confirming protein that blocking glutamate neurotoxicityMaterials & MethodsExpression vectors, antibodies, chemicals, and measurementsMitoDsRed2 (Clontech, Mountain View, CA), Casel2 construct (Evrogen, Moscow, Russia), GFP-Cre (Addgene, Cambridge MA) and GFP/Myc-tagged Mfn2 (gift of Dr. Margaret T. Fuller, Stanford University) were obtained. MitoDsRed2/Cre dual promoter regular construct was generated by replacing the neomycin/kanamycin resistance gene in MitoDsRed2 construct with sequence of recombinase Cre with nuclear localization signal (NLS-Cre from GFP-Cre construct). MitoDsRed2/Cre dual promoter lentiviral construct was generated by cloning mitoDsRed2 into pLVX-Puro (Clontech, Mountain View, CA) and replacing puromycin resistance gene with NLS-Cre, while Cre lentiviral construct was generated by cloning NLS-Cre into pLVX-Puro. Lentivirus were generated using 3rd generation packaging system (Packaging plasmid:pMDLg/pRRE and pRSV-Rev; Envelope Plasmid:pMD2.G from Addgene, Cambridge MA) as previously described (23). Primary antibodies used included mouse anti-VDACl/rabbit anti-Mff/Spectrin (Abeam, Cambridge, MA), rabbit anti-Calpain1, Calpain 2 and cleaved caspase-3 (Cell signaling, Danvers, MA), rabbit anti-Mfn1/mouse anti-Mfn2 (Santa Cruz, Dallas, Texas), mouse anti-HB9 (DSHB, Iowa City, IA), mouse anti-GFAP (Invitrogen, Grand Island, NY), rabbit anti-Iba1 (Wako, Richmond, VA) mouse anti-DLPl/OPAl/Tom20 (BD, Franklin Lakes, NJ) and mouse anti-actin/rabbit anti-MAP2 (Millipore, Billerica, MA). Glutamate (Sigma, St. Luis, MO) and MK-801/Calpeptin/BAPTA-AM/ Z-VAD-FMK (Tocris, Minneapolis, MN) were also obtained. ATP levels were measured by the ATP Colorimetric/Fluorometric Assay Kit (Biovision, Milpitas, CA).The ROS level and mitochondrial membrane potential were measured as described before (24). Real-time measurement of oxygen consumption rate (OCR) in live cultured motor neurons with optimal seeding density (60,000 cells/well) was measured using the Seahorse XF24 Analyzer (Seahorse Bioscience, North Billerica, MA) according to the manufacture’s instruction. ATP synthase inhibitor oligomycin (1 μM), uncoupler FCCP (4 μM) and complex I inhibitors antimycin A(1 μM) and rotenone (1 μM) were injected at indicated times. After measurement, cells were lysed and OCR data was normalized by total protein. Cell death and viability was measured by Cytotoxicity Detection Kit (LDH; Roche, Nutley, NJ) or Cell Proliferation Kit (MTT; Roche, Nutley, NJ). Neuronal viability propidium iodide assay in positively transfected neurons was performed as we described (25). Neurons with propidium iodide-positive nuclei and/or obvious fragmented nuclear/neurites were counted as non-viable neurons, whereas neurons without propidium iodide-positive nuclei and clear nuclear contour/neurites were counted as viable neurons.Embryonic primary neurons isolationMfn2 floxed male and female mice were kind gifts of Dr. David Chan (California Institute of Technology). Mfn2 transgenic male and female mice were generated by pronuclear injection of the Thyl-Mfn2 transgene into C57BL/6N fertilized eggs using the murine Thy-1.2 genomic expression cassette (gift of Dr. Philip C. Wong, Johns Hopkins University). Timed pregnant Sprague-Dawley female rats (Harlan or Charles River) or C57BL/6N female mice were sacrificed following the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Case Western Reserve University. Primary motor neurons were isolated from E13-15 female rat/E12-14 female mice embryos as we described before (21). Primary cortical neurons were isolated from E18 female rats as previously described (22) but with some modifications.Mitochondria isolation and western blot analysisCrude and purified mitochondria were isolated as described before (21). Purified mitochondria from cells were lysed with 1×Cell Lysis Buffer (Cell Signaling, Danvers MA) plus 1 mM PMSF (Sigma, St. Louis, MO) and Protease Inhibitor Cocktail (Sigma, St.Louis, MO). Equal amounts of total protein extract were resolved by SDS-PAGE and transferred to Immobilon-P (Millipore, Billerica, MA).Following blocking with 10% nonfat dry milk, primary and secondary antibodies were applied as previously described (24) and the blots developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA).Animal surgery and glutamate infusionMice surgery/procedures were performed according to the NIH guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) at Case Western Reserve University. One day before implantation, mini-osmotic pumps (Model 2001, Alzet, Cupertino, CA; flow rate of 1 μl/hour) and brain infusion cannula attached with 2 cm catheter tubes (Brain infusion kit 3, Alzet, Cupertino, CA) were filled with artificial cerebrospinal fluid (aCSF:124 mM NaCl,25 mM NaHCO3,10 mM D-glucose,2.5 mM KCl,1 mM MgC12,2 mM CaC12 and 1 mM NaH2PO4, adjusted to pH 7.2-7.4 using NaOH) or aCSF containing 10 mM glutamate followed by pump incubation in phosphate buffered saline (PBS) at 37℃ overnight according to the manufacturer’s instructions. For stereotaxic surgery, male mice were anesthetized with avertin and placed on a stereotaxic frame. A small incision was first made to expose skull and bregma, and both the catheter and the connected mini-osmotic pump were implanted subcutaneously. A hole was drilled in the skull (relative to bregma:AP-0.2mm, ML 1mm) and the cannula was positioned 2 mm above the lateral ventricle. Another two holes were drilled by the edge of cannula and self-tapping bone screws (MD-1310, BASi, West Lafayette, IN) were screwed into the holes. The cannula and screws were finally secured by cement.7 day after surgery, male mice were deeply anesthetized with avertin and transcardially perfused with cold PBS and the spinal cord/brain were collected for further analysis.Immunocytochemistry and immunofluorescence for central nervous system of mouseTo investigate the mitochondrial morphology in motor neurons in male mice, we also developed an optimized immunofluorescence-based imaging technique that could directly visualize individual filamentous mitochondria in paraffin-embedded buffered formalin fixed spinal cord tissues using specific antibodies against mitochondrial protein Tom20 or VDAC1. Taken briefly, deparaffinized and re-hydrated tissue sections were washed briefly three times with distilled H2O and placed in 1X antigen decloaker (Biocare, Concord, CA). The sections were then subject to antigen retrieval under pressure using Biocare’s Decloaking Chamber by heating to125℃ for 10 sec and cooling to 90℃ for 30 sec followed by heating to 22 psi at 128℃, and cooling to 0 psi at 94℃. After temperature decreased to 30℃, the sections were gradually rinsed with distilled H2O for five times. The sections were then blocked with 10% normal goat serum (NGS, St. Louis, MO) for 30 min at RT, and incubated with primary antibodies in PBS containing 1% NGS overnight at 4℃. After 3 washes with PBS, the sections were incubated in 10% NGS for lOmin, and then with Alexa Fluor conjugated secondary antibody (Invitrogen, Grand Island, NY) (1:300) for 2 h at room temperature in dark. Finally, the sections were rinsed three times with PBS, stained with DAPI, washed again with PBS for three times, and mounted with Fluoromount-G mounting medium (Southern Biotech, Birmingham, AL).Electron microscopy for central nervous system of mouseMale mice were transcardially perfused with EM fixative (the quarter strength Karnovsky-1.25% DMSO mixture) for 5 minutes and then spinal cords were removed quickly and placed in EM fixative solution for additional 15 min at RT followed by additional fixation in fresh EM fixative for another 2 hours at RT. After washing, tissue blocks were postfixed in 1% osmium-1.25% ferrocyanide mixture for two changes of solution of one hour each (total postfixation time is two hours) at RT. Then the specimens were rinsed, and soaked overnight in acidified 0.5% uranylacetate. After another wash, the blocks were dehydrated in ascending concentrations of ethanol, passed through propylene oxide, and embedded in Poly/Bed 812 embedding resin (Polysciences, Warrington, PA). Thin sections were sequentially stained with 2% acidified uranyl acetate followed by Sato’s triple lead staining as modified by Hanaichi et al. (26) and examined in an FEI Tecnai T12 electron microscope equipped with a Gatan single tilt holder and a Gatan US4000 4kx4k CCD camera (Gatan, Pleasanton, CA).In vitro calpain cleavage assay100ug purified mitochondria or lug recombinant Mfn2 protein (Origene, Rockville, MD) were incubated with purified human erythrocyte μ-calpain (calpain-I) (Biovision, Milpitas, CA) at 30℃ for 30 min according to manufacturer’s instruction. For inhibition, calpain-I was pre-incubated with PMSF or calpeptin for 5 min at 30℃. The reaction was stopped by the addition of SDS sample buffer containing 62.5 mM Tris-HCl,4% SDS,10% glycerol,50 mM DTT and 0.1% bromophenol blue (pH 6.8). The samples were boiled at 100℃ for 10 min.1/6 of reaction volume was loaded into SDS-PAGE gel for western blot.Results1. Glutamate induces mitochondrial fragmentation in the neurons2. Mfn2 deficiency elicits mitochondrial and neuronal toxicity in the neurons3.Mfn2 overexpression prevents glutamate-induced mitochondrial dysfunction and neuron death in vitro4. Mfn2 overexpression protects mitochondria and motor neurons from glutamate excitotoxicity in vivo5. Calpain cleaves Mfn2 in the neurons following glutamate exposure6. Recapitulated Calpain activation, Mfn2 reduction and mitochondrial fragmentation in sALS patientsConclusion:Calpain-mediated degradation of Mfn2 is responsible for glutamate induced mitochondrial dysfunction and neuronal death in neurons. Calpain-mediated Mfn2 degradation is a novel mechanism regulating mitochondrial fusion during glutamate excitotoxicity.SignificanceNeurotoxicity effect of glutamate widely exist in the nervous system diseases including cerebral hemorrhage, stroke, brain tumors, central nervous system infection. It has close relationship with some other diseases. This experiment confirmed the mechanism of the toxic effect of glutamate on injured neurons, animal models and the human body models. It has a significant meaning in the researches of nervous system diseases. Studies of this signaling pathway will likely become hot topics in related research areas in the future... |
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