Zinc is released from synaptic terminals and mediates synaptic plasticity in various physiological conditions. In pathological conditions, high concentration of zinc released from synaptic terminals and translocated to postsynaptic neurons could induce neuronal cell death. In this study, the molecular mechanisms underlying zinc toxicity in primary cultured cortical neurons and in glial cells were explored.
The mechanism by which neurotrophic factors potentiate zinc toxicity was investigated using primary cultured cortical neurons, While a brief treatment of low concentration of zinc [eg. 80 uM, 30 min] produced a low level [eg. 25%] of cell death, such zinc toxicity was strongly potentiated by pretreatment of various neurotrophic factors including BDNF, NT-3, NT-4, insulin and IGF-I, indicating that these neurotrophic factors would act as cytotoxic factors in certain conditions. In the current study, IGF-I was used in studying the mechanism underlying the zinc-toxicity potentiation by IGF-L Administration of cycloheximide and anisomycin during IGF-1 pretreatment inhibited the potentiation of zinc toxicity, indicating that new protein synthesis was essential in the process of potentiated cell death by IGF-I. The potentiation of zinc toxicity was completely or partly inhibited by GF109203X (a general inhibitor of PKC), LY294002 (a specific inhibitor of PI-3K), or rapamycin (a specific inhibitor of p70s6K), suggesting that PKC, PI-3K, and p70s6K signaling pathways were involved in the IGF-I mediated zinc-toxicity potentiation. In addition, the zinc-toxicity potentiation was suppressed by deferoxamine, an inhibitor of G1 to S cell cycle progression, and olomoucine, a specific inhibitor of cdk4, 6, indicating that the cell cycle regulators, cdk4 and cdk6, could be involved in the zinc-toxicity potentiation. Consistently, it was observed that the cyclin D1 expression was increased during IGF-I treatment. These results suggest that cyclin Dl may act as an endogenous factor that regulates the zinc toxicity potetiation. In primary cultured cortical neurons, the zinc-toxicity potentiation by IGF-I was completely blocked by antioxidants (eg. trolox), revealing that mechanism underlying the increased cell death involves ROS generation. In fact, the increased ROS was able to be visualized in cultured cortical neurons undergoing the potentiated cell death. The potentiated cell death was inhibited by aspirin or NS-398, inhibitors of cyclooxygenase, indicating that cyclooxygenase activity may mediate the increased ROS and cell death. The RNA level, the protein level, and the activity of cyclooxygenase were consistently increased by IGF-I treatment. In support of these results, neuronal cell death induced by arachidonic acid, a substrate of cyclooxygenase, was also potentiated by pretreatment of IGF-I. Therefore, cyclooxygenase-2 was proposed to play as another endogenous factor important for the IGF-I induced zinc toxicity potentiation.
The molecular mechanism of zinc toxicity in astroglial cell death was also investigated. Zinc induced toxicity was observed in chronic treatment of zinc (35 uM, 24 hr), but not in acute insult (500 uM, 30 min). Unlike that of primary cultured cortical neurons, zinc toxicity was not attenuated by antioxidant, trolox. Consequently, there seem to be distinct mechanisms underlying zinc induced cell death between cortical neurons and astroglia. However, strong zinc influx was observed in astroglia during the chronic zinc treatment, and zinc entrance was correlated to the severity of cell death, indicating that zinc influx appeared to be the primary cause of the zinc toxicity as was in cortical neurons. Application of glutathione or cysteine protected astroglia completely from the zinc toxicity. When DEM or DIA, thiol depleleting agents, was treated together with glutathione or oxidized glutathione, the potentiated zinc toxicity was not affected. These data suggest that reduced thiol may be responsible for the protective effect of glutathione and cysteine. Consistently, extracellular glutathione or cysteine completely suppressed the zinc influx. These results implicate that the zinc toxicity protection might involve the chelation of free zinc by reduced thiols in cells. In agreement with this notion, when intracellular glutathione was increased by treating with glutathione monoethyl ester, zinc toxicity was notably inhibited. In addition, the zinc toxicity was aggravated when intracellular glutathione was depleted by BSO, an inhibitor for T-glutamylcysteine synthetase. These results indicate that intracellular thiol homeostasis was important in zinc-toxicity potentiation in primary cultured astroglia. Indeed, the level of glutathione, the major non-protein thiol molecule in cells, was decreased in zinc treated astroglia in dose and time dependent manners, indicating that zinc influx may mediate intracellular thiol oxidation.
In conclusion, zinc induce cell death both in primary cultured cortical neurons and astroglial cells. The mechanisms underlying the zinc induced cell death in each of the cell types, are similar in that ROS generation plays a critical role. However, unlike in primary cultured cortical neurons, the antioxidant, trolox failed to block zinc toxicity in primary cultured astroglia. In astroglial cells, zinc induces cell death by depleting reduced glutathione in cells.
아연은 생리적조건에서 시냅스말단으로부터 분비되어 시냅스활성을 조절한다. 그러나 병리적조건에서는 시냅스과립으로부터 고농도의 아연이 후시냅스 뉴런으로 이동하여 뉴런의 세포사를 유도한다. 본 연구에서는 1차 배양신경세포와 글리아세포에서 아연독성의 분자적기전을 조사하였다.
신경영양인자에 의한 아연독성 증폭기전이1차배양신경세포에서 조사되었다. BDNF, NT-3, NT-4, insulin, IGF-I 과 같은 신경연양인자의 전처리에 의해 저농도의 [80 uM, 30 min] 아연독성은 큰 폭으로 증가하였다. 본연구에서는 아연독성증폭기전을 조사하는데 IGF-I을 사용하였다. Cycloheximide, anisomycin은 IGF-I에 의한 아연독성 증폭을 억제하므로 새로운 단백질의 합성이 IGF-I에 의한 독성증폭과정에 필요하다는 것을 알 수 있었다. 아연독성증폭은 PKC 저해제인 GF109203X, PI-3K 저해제인 LY294002, p70s6k 저해제인 rapamycin에 의해 저해되므로 이들 효소에 의한 신호전달 과정이 IGF-I에 의한 아연독성증폭을 매개하는 것을 알 수 있었다. 또한 아연독성증폭은 G1-> S 세포주기 진행저해제인 deferoxamine과 cdk4,6의 저해제인 olomoucine에 의해 억제되었다. 이와 일치하게도 IGF-I에 의해 cyclin D1의 발현이 증가하였다. 이 결과는 cyclin D1이 아연독성증폭을 조절하는 세포내 인자임을 제시한다. 1차배양신경세포에서 IGF-I에 의한 아연독성증폭은 항산화제인 trolox에 의해 저해되므로 ROS 생성이 증가된 세포사를 매개하는 것을 알 수 있었다. 실지로 증폭된 세포사가 일어나는 신경세포에서 ROS가 증가하였다. 증폭된 세포사는 COX 제해제인 aspirin, NS-398에 의해 감소되므로 COX 활성이 ROS 증가 및 세포사증가를 매개함을 알 수 있었다. COX-2의 RNA 수준, 단백질 수준 및 활성이 IGF-I에 의해 증가하였다. COX의 기질인 아라키돈산에 의한 신경세포사가 증폭된다는 사실이 이 결과들을 뒷받침하였다. 따라서 COX-2는 IGF-I 에 의한 아연독성증폭을 매개하는 또 다른 인자로 제시되었다.
글리아세포에서 아연독성의 분자적기전이 조사되었다. 아연독성은 장기처리(35 uM, 24 hr)에 의해서만 그 독성이 관찰되었다. 1차배양신경세포와 달리 아연독성은 항산화제인 trolox에 의해 저해되지 않았다. 따라서 신경세포와 글리아세포에는 서로 다른 아연독성기전이 존재함을 알 수 있었다. 그러나 아연의 유입은 글리아세포에서도 관찰되고 세포사의 정도와 직접 연관성을 가지므로 신경세포에서와 같이 주요한 원인으로 작용함을 알 수 있었다. 글루타치온이나 시스테인은 아연독성으로부터 글리아세포를 보호하였다. DEM, DIA와 같은 thiol 고갈시약을 처리하거나 산화된 글루타치온을 처리하였을 때에는 아연독성이 영향을 받지않았다. 이 결과로부터 환원된 thiol이 보호효과를 담당함을 알 수 있었다. 글루타치온이나 시스테인은 세포외부에서 아연유입을 완벽히 억제하였다. 이 결과는 세포내에서 환원된 thiol이 아연이온을 chelation하여 아연독성을 억제할 수 있음을 암시한다. 이와 일치하게도 세포내 글루타치온을 증가시켰을 때 아연독성은 크게 감소하였다. 또한 BSO를 처리하여 세포내 글루타치온을 고갈시켰을 때 아연독성을 크게 증가시켰다. 이 결과는 세포내 thiol 항상성이 아연독성에 중요하다는 사실을 알려준다. 실지로 주요한 비단백 thiol인 글루타치온의 수준은 아연에 의해 농도, 시간 의존적으로 감소하였고, 이는 아연의 유입이 세포내 thiol을 산화시킨다는 사실을 말해준다.
결론적으로 아연은 일차배양신경세포 및 글리아세포에서 세포사를 유도한다. 각 세포에서 아연독성에 의한 세포사기전은 ROS 생성이 중요한 역할을 한다는 점에서 공통점을 가진다. 그러나 신경세포와 달리 글리아세포에서 trolox는 아연독성을 억제하지 못했다. 따라서 글리아세포에서 아연은 세포내 글루타치온 고갈을 통해 세포사를 유도하는 것으로 생각되어진다.
