Abstract
Homocysteine (HCY) is a pro-inflammatory sulphur-containing redox active endogenous amino acid, which concentration increases in neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). A widely held view suggests that HCY could contribute to neurodegeneration via promotion of oxidative stress. However, the action of HCY on motor nerve terminals has not been investigated so far. We previously reported that oxidative stress inhibited synaptic transmission at the neuromuscular junction, targeting primarily the motor nerve terminals. In the current study, we investigated the effect of HCY on oxidative stress-induced impairment of transmitter release at the mouse diaphragm muscle. The mild oxidant H2O2 decreased the intensity of spontaneous quantum release from nerve terminals (measured as the frequency of miniature endplate potentials, MEPPs) without changes in the amplitude of MEPPs, indicating a presynaptic effect. Pre-treatment with HCY for 2 h only slightly affected both amplitude and frequency of MEPPs but increased the inhibitory potency of H2O2 almost two fold. As HCY can activate certain subtypes of glutamate N-methyl D-aspartate (NMDA) receptors we tested the role of NMDA receptors in the sensitizing action of HCY. Remarkably, the selective blocker of NMDA receptors, AP-5 completely removed the sensitizing effect of HCY on the H2O2-induced presynaptic depressant effect. Thus, at the mammalian neuromuscular junction HCY largely increases the inhibitory effect of oxidative stress on transmitter release, via NMDA receptors activation. This combined effect of HCY and local oxidative stress can specifically contribute to the damage of presynaptic terminals in neurodegenerative motoneuron diseases, including ALS.
Highlights
Homocysteine (HCY, 2-amino-4-sulfanylbutanoic acid) is a sulphur-containing endogenous amino acid, which is produced in the methylation cycle of protein metabolism and involved in maintaining the cells redox balance
Miniature end-plate potentials (MEPPs) representing spontaneous release of ACh quanta from nerve terminals occurred at a mean frequency of 0.60 ± 0.05 s−1 and an amplitude of 0.52 ± 0.02 mV (n = 46 synapses/6 mice)
After 30–35 min exposure to H2O2 the mean frequency of MEPPs decreased by 32.3 ± 8.6% (n = 47 synapses/6 mice, p < 0.05 by MannWhitney test) regarding control untreated preparation
Summary
Homocysteine (HCY, 2-amino-4-sulfanylbutanoic acid) is a sulphur-containing endogenous amino acid, which is produced in the methylation cycle of protein metabolism and involved in maintaining the cells redox balance. An elevated plasma level of HCY (termed hyperhomocysteinemia, hHCY) markedly decreases cell viability (Kolling et al, 2013). According to a number of studies, abnormally elevated levels of HCY in plasma and cerebrospinal fluids correlate with amyotrophic lateral sclerosis (ALS), a motor neuronal disease that causes muscle degeneration (Zoccolella et al, 2008, 2010; Levin et al, 2010; Valentino et al, 2010; Veeranki and Tyagi, 2013). The variant MTHFR C677T, known to be associated with increased levels of HCY (Kang et al, 1988; Li et al, 2003) is more frequent among ALS patients (Kühnlein et al, 2011), an Italian population study found no association (Ricci et al, 2012). Apart from a direct link between MTHFR and ALS, there are other possible mechanisms how the increasing level of HCY due to aging or improper diet (Zhang et al, 2008; Song et al, 2013) can speed up the development of disease in patients with ALS
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