Acute phase expression pattern of autophagic/apoptotic and Zinc signal-related genes and their relatedness in rat cerebral cortex following recurrent neonatal seizures

Hong Ni

Neurology Laboratory, The Children’s Hospital Affiliated to Soochow University, Suzhou, China

It has been reported that autophagy and apoptosis both play the key roles in neuroexcitotoxicity. Recently, we have also revealed disturbed expression pattern of zinc transporter 1 (ZnT-1) and ZnT-3 following neonatal seizure-induced mossy fiber sprouting and cognitive deficits. However, the relatedness among autophagy, apoptosis and zinc signals has been never studied. The present study was sought to determine the acute phase expression pattern of autophagic maker (Beclin-1, microtubule-associated protein 1A/1B light chain 3, LC3), lysosome marker (Cathepsin-B, Cathepsin-D), apoptotic marker caspase-3, and ZnT-1～ZnT-3 expression, and the relatedness among the eight genes in rat cerebral cortex. We further investigate an autophagy inhibitor (3-methyladenine, 3-MA) on the regulation of autophagy/lysosome, apoptosis and ZnTs-related genes in cerebral cortex and on cognitive deficit following recurrent neonatal seizures. We initially examined the expression pattern at 1.5 h, 3 h, 6 h and 24 h after the last seizures using real-time RT-PCR and western blot methods, respectively. During postnatal day 31 (P31) to P37, the rats were tested for spatial learning ability with the Morris water maze task. The results are as follows: 1. real-time RT-PCR analysis: ①At 1.5 h and 3 h after the last seizures, the expression of both autophagy/lysosome and apoptosis marker genes (Beclin-1, LC3, Cathepsin-B, Cathepsin-D, caspase-3), along with ZnT-1 and ZnT-2, increased significantly in cerebral cortex of RS group compared with those at the corresponding time point in control group. Pre-treatment with 3-MA remarkably attenuated the expression increase of the above genes. At 6 h after the last seizures, there were still up-regulated expression of LC3, cathepsin-B and ZnT-2 in RS group; however, the expression of caspase-3, ZnT-1 and ZnT-3 was not significantly different among three groups. At 24 h after the last seizures, the expression of lysosome marker Cathepsin-B and Cathepsin-D elevated markedly in 3-MA group compared with those in control and RS groups; besides, ZnT-1 and ZnT-2 expression elevated and ZnT-3 decreased in RS group compared with those in control; on the other hand, the mRNA level of autophagy and apoptosis markers, Beclin-1, LC3 and caspase-3, showed no significant difference among three groups. ②Linear correlations among all the eight genes could be seen in control group (except Cathepsin-B to Caspase-3), while the linear correlations were disturbed in 3-MA and control groups. In detail, in RS group, the linear correlations were scarcely observed between Beclin-1 and LC3 (or Cathepsin-B, Cathepsin-D, Caspase-3), and between ZnT-2 with Beclin-1 (or Cathepsin-B, Cathepsin-D), or between ZnT-1 with Caspase-3, ZnT-3 with Cathepsin-D; in 3-MA group, linear correlations were mainly seen among Beclin-1 a Cathepsin-B, Cathepsin-D,Caspase-3, and between ZnT-1 with Beclin-1 (or Caspase-3, Cathepsin-D), or between ZnT-2 with Cathepsin-B. 2. Western blot analysis: Significant increases in the ratio of LC3-II/LC3-I and in beclin-1 in cerebral cortex were observed at 1.5 h, 3 h, 6 h and 24 h after the last seizures. Up-regulation of LC3 and beclin-1 was blocked by pretreatment with 3-MA. 3. Morris water maze tests showed that RS rats performed worse than the control rats in the Morris maze navigation test. These dates demonstrated, for the first time, that there exists an interaction of Zn2+ with autophagy/lososome and apoptosis signals that are immediately activated in cerebral cortex after recurrent neonatal seizures. The autophagic mechanism participates in neonatal seizure-induced brain damage through regulating the lososome/apoptotic and Zn2+–mediated excitotoxicity system, inhibition of this key system was shown to be neuroprotective.