Methyl Vitamin B12 but not methylfolate rescues a motor neuron-like cell line from homocysteine-mediated cell death |
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Authors: | Hemendinger Richelle A Armstrong Edward J Brooks Benjamin Rix |
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Affiliation: | Motor Neuron Cell Biology Group, Department of Neurology, Carolinas Neuromuscular/ALS-MDA Center, Carolinas Medical Center, Charlotte, NC 28203, USA |
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Abstract: | Homocysteine is an excitatory amino acid implicated in multiple diseases including amyotrophic lateral sclerosis (ALS). Information on the toxicity of homocysteine in motor neurons is limited and few studies have examined how this toxicity can be modulated. In NSC-34D cells (a hybrid cell line derived from motor neuron-neuroblastoma), homocysteine induces apoptotic cell death in the millimolar range with a TC50 (toxic concentration at which 50% of maximal cell death is achieved) of 2.2 mM, confirmed by activation of caspase 3/7. Induction of apoptosis was independent of short-term reactive oxygen species (ROS) generation. Methyl Vitamin B12 (MeCbl) and methyl tetrahydrofolate (MTHF), used clinically to treat elevated homocysteine levels, were tested for their ability to reverse homocysteine-mediated motor neuron cell death. MeCbl in the micromolar range was able to provide neuroprotection (2 h pretreatment prior to homocysteine) and neurorescue (simultaneous exposure with homocysteine) against millimolar homocysteine with an IC50 (concentration at which 50% of maximal cell death is inhibited) of 0.6 μM and 0.4 μM, respectively. In contrast, MTHF (up to 10 μM) had no effect on homocysteine-mediated cell death. MeCbl inhibited caspase 3/7 activation by homocysteine in a time- and dose-dependent manner, whereas MTHF had no effect. We conclude that MeCbl is effective against homocysteine-induced cell death in motor neurons in a ROS-independent manner, via a reduction in caspase activation and apoptosis. MeCbl decreases Hcy induced motor neuron death in vitro in a hybrid cell line derived from motor neuron-neuroblastoma and may play a role in the treatment of late stage ALS where HCy levels are increased in animal models of ALS. |
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Keywords: | AAPH, 2,2&prime -azobis(2-amidinopropane) dihydrochloride ALS, amyotrophic lateral sclerosis DCFH-DA, 2&prime ,7&prime -dichlorodihydrofluorescein diacetate ER, endoplasmic reticulum FCS, fetal calf serum GI50, concentration at which 50% of cell growth is inhibited Hcy, homocysteine IC50, concentration at which 50% of maximal cell death is inhibited MeCbl, methyl Vitamin B12 or methylcobalamin MTHF, 5-methyltetrahydrofolic acid disodium salt NCSS, Number Cruncher Statistical Software NSC-34D, differentiated form of a motor NSC-34 cell line PI, propidium iodide ROS, reactive oxygen species TC50, toxic concentration at which 50% of maximal cell death is achieved |
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