Increased tolerance to ischemic neuronal damage by knockdown of Na+-Ca2+ exchanger isoform 1.
We hypothesize that stimulation of Na+-K+-Cl+ cotransporter (NKCC1) causes Na+ overload that may lead to reversal of Na+-Ca2+ exchanger isoform 1 (NCX1) and ischemic neuronal damage. NCX1 protein expression and Ca2+ influx via reversal of NCX were decreased by approximately 70% in NCX1+/- neurons. Compared to NCX1+/+ neurons, NCX1+/- neurons exhibited significantly less cell death (approximately 30%) after 3 h oxygen and glucose deprivation (OGD) and 21 h reoxygenation. Additional neuroprotection was found in NCX1+/- neurons treated with NCX inhibitor KB-R7943. Moreover, expression of NCX1 protein was approximately 40% lower in NCX1+/- brains than in NCX1+/+ brains. However, there was no significant reduction in cerebral infarction in NCX1+/- mice following middle cerebral artery occlusion (MCAO). These data suggest that moderate reduction of NCX1 protein may be not enough to exert protection. We used small RNA-interference (siRNA) approach to further elucidate the role of NCX1 in ischemic cell damage. Efficacy of anti-NCX1 siRNA was tested in astrocytes and approximately 50% knockdown of NCX1 protein expression was achieved after 24-72 h transfection. Reduction in NCX1 protein expression was also found in brains of NCX1+/- mice after the siRNA injection. NCX1+/- mice treated with siRNA showed approximately 20% less MCAO-induced infarction, compared to NCX1+/- mice. Approximately 50% neuroprotection was detected in NKCC1+/-/NCX1+/- mice following MCAO. In conclusion, these data suggest that NCX1 plays an important role in ischemia/reperfusion-induced neuronal injury.
Luo
Jing
J
Department of Neurological Surgery, University of Wisconsin Medical School, H4/332 Clinical Sciences Center, 600 Highland Ave., Madison, WI 53792, USA.
Wang
Yanping
Y
Chen
Xinzhi
X
Chen
Hai
H
Kintner
Douglas B
DB
Shull
Gary E
GE
Philipson
Kenneth D
KD
Sun
Dandan
D
D000222:Adaptation, Physiological; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D051379:Mice; D008822:Mice, Transgenic; D020033:Protein Isoforms / Q000502:physiology*; D034741:RNA, Small Interfering; D019831:Sodium-Calcium Exchanger / Q000502:physiology*
C499849:NCX1 protein, mouse; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D019831:Sodium-Calcium Exchanger