Design And Evaluation Of The Effectiveness Of Glial Glutamate Transporter-1a Antisense Oligodeoxynucleotides

In the central nervous system, glutamate is the predominant excitatory neurotransmitter and is also a potential neurotoxin. Under normal conditions, the intracellular concentration of glutamate is about 10000 times higher than the extracellular. Intracellular glutamate mainly exists in the vesicles of glutamatergic nerve endings and is released when the nerve ending is excited. Under physiological conditions, because of the presence of glutamate uptake system, the extracellular concentrations of glutamate is kept at a low level, only a little of glutamate is involved in neurotransduction as excitatory neurotransmitter.The extracellular concentration of glutamate is controlled mostly by a family of Na+-dependent'high-affinity'excitatory amino acids transporters (EAATs). Five EAATs have been identified which includes EAAT1, EAAT2, EAAT3, EAAT4 and EAAT5. Among the five EAATs, EAAT1 and EAAT2 are glial transporters and also called glutamate aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), respectively. EAAT3 is also known as excitatory amino acid carrier-1 (EAAC-1). The main function of the transporters is to uptake extracellular glutamate into cells to keep extracellular concentrations of glutamate at a low level. GLT-1 is responsible for the most of the glutamate transport activity. It has been reported that there are three C-terminal splice variants of GLT-1 including GLT-1a, GLT-1b and GLT-1c. In the hippocampus, GLT-1a represents 90%±1% of total GLT-1.Our recent study showed that GLT-1 played an important role in the neuronal protection of cerebral ischemic preconditioning (CIP) against global brain ischemic insult. However, it is remains to be clarified that if the variant of GLT-1a plays a role in the neuronal protection of CIP against global brain ischemia. It is an important strategy to inhibiting the expression and function of GLT-1a in the study of the involvement of GLT-1a in the neuronal protection of CIP. But there is no specific antagonist of GLT-1a up to now.Antisense technology is a method according to the Watson-Cricker principle of base complementary pairs to synthesize a fragment of DNA or RNA which sequence can complementary to the target gene. After hybridization to target sequences, the expression of target gene can be blocked selectively. Antisense oligodeoxynucleotides (AS-ODNs) refers to the use of a short (14 to 23 bases), single stranded, synthetic ODNs to inhibit gene expression selectively and specifically. The key of the design for AS-ODNs is how to select the best effect sites on the mRNA chain. In order to observe the effect of inhibiting GLT-1a expression and function on the neuronal protection of CIP, the present study was undertaken to design GLT-1a AS-ODNs according to C terminus specific sequences of GLT-1a mRNA, and then to evaluate the effectiveness of the designed AS-ODNs on the expression of GLT-1a using western blot analysis in vivo.1 Design of GLT-1a AS-ODNsAccording to C terminus specific sequences of GLT-1a mRNA, five GLT-1a AS-ODNs were designed using Antisense design software from IDT company. The five GLT-1a AS-ODNs chains were named as P1, P2, P3, P4 and P5, respectively. The sequences of them were as follows: P1: 5'-AGCTGACTTTCCATTGGCCGC-3'P2: 5'-GGTTCTTCCTCAACACTGCA-3'P3: 5'-CACGTTTCCAAGGTTCTTCC-3'P4: 5'-TTGGCTGAGAATCGGGTCATT-3'P5: 5'-ATTGGCTGAGAATCGGGTC-3'2 Evaluation of the effectiveness of the designed GLT-1a AS-ODNsRothstein et al reported in 2005 that Ceftriaxone (Cef) could up-regulate the expression of GLT-1 and its mRNA. In order to find an effective chain among the five GLT-1a AS-ODNs chains efficiently, we first observed the inhibition of the chains of GLT-1a AS-ODNs on the Cef-induced up-regulation in the expression of GLT-1a and GLT-1b to evaluate the effectiveness of the designed GLT-1a AS-ODNs on the expression of GLT-1a. Based above, the inhibition of the selected AS-ODNs chain on the expression of GLT-1a in normal and CIP rats were examined.2.1 Cef Up-regulates the expression of GLT-1a protein in the rat CA1 hippocampusRothstein et al reported in 2005 that Cef could up-regulate the expression of GLT-1 and GLT-1b in the hippocampus of rats. However, it has not specifically pointed out that the Cef can up-regulate the expression of GLT-1a. So the up-regulating effect of Cef on the expression of GLT-1a was observed firstly.Methods: Ten male Wistar rats (280-300 g) were randomly assigned to sham (n=5) and Cef (n=5) groups. The rats in sham group were intraperitoneally injected with normal saline (NS) (0.8 ml/kg) once a day for 5 days, while the rats in Cef group were given with Cef (200 mg/kg) by the same way. The animals were sacrificed by decapitation 2 days after the last intraperitoneally injection. The expression of GLT-1a in the CA1 hippocampus was assayed using Western blot analysis. The relative expression of GLT-1a was represented with the ratio of integral optical density (IOD) of GLT-1a immunoblot band to that ofβ-actin. The more the ratio is, the more the expression of GLT-1a is.Results: Basal expression of GLT-1a in the CA1 hippocampus could be observed in sham group. Compared with sham group, the expression of GLT-1a in the area was significantly increased in Cef group (P<0.05) . The results indicated that the expression of GLT-1a in the CA1 hippocampus can be up-regulated by intraperitoneal administration of Cef.2.2 The effect of each chain (P1-P5) of GLT-1a AS-ODNs on the Cef-induced GLT-1a up-regulation in the rat CA1 hippocampusMethods: Thirty male Wistar rats (280-300 g) were used. A guide canula was first imbedded into the right lateral ventricle of the rats for administration of drugs. After a recovery of five days, the rats were randomly assigned to sham (n=5) and AS-ODNs (n=5) groups. The rats in sham group were intraperitoneally injected with Cef (200 mg/kg) once a day for 5 days, and were injected with 10μl distill water (DW) , the solvent of GLT-1a AS-ODNs, into the right lateral ventricle at 36 h, 72 h and 108 h after the first time of Cef injection. AS-ODNs group was further divided into five sub-groups of P1, P2, P3, P4, P5 according to the chain of GLT-1a AS-ODNs used. Rats in each subgroup were injected with 10μl solution (18 nmol) of corresponding AS-ODNs chain in the same way to that in sham group. Other procedures were the same as those in sham group. The animals were sacrificed by decapitation 2 days after the last time of the intraperitoneal injection. The expression of GLT-1a in the CA1 hippocampus of rats was assayed using Western blot analysis.Results: Strong expression of GLT-1a in the CA1 hippocampus could be observed in rats of sham group. Compared with sham group, the expression of GLT-1a was significantly decreased in subgroup of P2 chain of AS-ODNs (P<0.05) . While other chains of AS-ODNs used had no effect on the Cef-induced up-regulation in the expression of GLT-1a (P >0.05) .The results indicated that different from other 4 chains of AS-ODNs designed, P2 chain of the AS-ODNs could significantly inhibit the expression of GLT-1a.2.3 Evaluation on the specificity of the inhibition effect of GLT-1a AS-ODNs (P2, the following is the same) on the expression of GLT-1a in the rat CA1 hippocampusMethods: In order to determine the specificity of GLT-1a AS-ODNs, the inhibition of GLT-1a AS-ODNs on the expression of GLT-1a and GLT-1b was compared. Ten male Wistar rats (280-300 g) were randomly assigned to sham (n=5) and AS-ODNs (n=5) group five days after a guide canula was imbedded into the right lateral ventricle of the rats for administration of drugs. The rats in sham group were intraperitoneally injected with Cef(200 mg/kg)once a day for 6 days, and were injected with 10μl distill water (DW), the solvent of GLT1a AS-ODNs, into the right lateral ventricle immediately after each time of Cef injection. The rats in AS-ODNs group were injected with 10μl AS-ODNs solution (15 nmol) in the same way to that in sham group. Other procedures were the same as those in sham group. The animals were sacrificed by decapitation 2 days after the last intraperitoneally injection. The expression of GLT-1a and GLT-1b in the CA1 hippocampus of rats was assayed using Western blot analysis.Results: Strong expression of GLT-1a and a certain degree of expression of GLT-1b in the CA1 hippocampus could be observed in sham group. Compared with sham group, the administration of AS-ODNs significantly down-regulated the expression of GLT-1a, while had no effect on the expression of GLT-1b (P>0.05) .The results indicated that the GLT-1a AS-ODNs could specifically inhibit GLT-1a expression.2.4 The GLT-1a AS-ODNs inhibits the expression of GLT-1a in normal rat CA1 hippocampusMethods: Ten male Wistar rats (280-300 g) were randomly assigned to sham (n=5) and AS-ODNs (n=5) groups. All procedures in each group were the same as to those in the part of 2.3, except for the rats was not injected with Cef.Results: Basal expression of GLT-1a in the CA1 hippocampus could be observed in sham group. Compared with sham group, the expression of GLT-1a was significantly decreased in AS-ODNs group (P<0.05).The results indicated that the GLT-1a AS-ODNs could also inhibit the basal expression of GLT1a in normal rats.2.5 The GLT-1a AS-ODNs inhibits the up-regulation in the expression of GLT-1a in the rat CA1 hippocampus induced by CIP.Methods: Thirty Wistar rats (280-300 g) were randomly assigned to the following groups (n=5) 5 days after the rats were implanted with stainless steel canula in the right lateral ventricle for administration of drugs:(1) Sham group: The bilateral vertebral arteries were exposed, and 48 hours later, the bilateral common carotid arteries (BCCAs) were separated, but without occluding the blood flow within the arteries.(2) Vertebral artery occluding (VAO) group: The bilateral vertebral arteries were first electrocauterized permanently. Then, the BCCAs were separated, but without occluding the blood flow 48 h after permanent occlusion of the bilateral vertebral arteries.(3) CIP group: The bilateral vertebral arteries of the rats were first electrocauterized permanently. The BCCAs were clamped to occluding the blood flow for 3 min at 48 h after the permanent occlusion of the bilateral vertebral arteries, and then the blood flow was reperfused.(4) CIP+AS-ODNs group: The bilateral vertebral arteries were first electrocauterized permanently. Ten min later, the rats were injected with GLT-1a AS-ODNs solution (15μl, 25 nmol) into the right cerebroventricle. In total, 4 times of the injection were performed in an interval of 24 h. Other procedures were the same as those in CIP group. Meanwhile, GLT-1a Sense ODNs and Missense ODNs groups were designed as control.The animals were sacrificed by decapitation 2 days after the cerebral ischemia or its sham operation. The expression of GLT-1a was assayed using Western blot analysis.Results: Basal expression of GLT-1a in the CA1 hippocampus could be observed in sham group. Compared with sham group, a certain degree of up-regulation of GLT-1a expression was observed in VAO group, while the expression of GLT-1a was further up-regulated after CIP in the CIP group (P<0.05) . Administration of GLT-1a AS-ODNs into the lateral cerebral ventricle significantly inhibited the up-regulation of GLT-1a expression induced by VAO and CIP, which represented with down-regulation of GLT-1a in the CIP+AS-ODNs group compared with the VAO and CIP groups. In addition, the expression of GLT-1a in CIP+ AS-ODNs group was further down-regulated compared with the Sham group, which indicated the GLT-1a AS-ODNs administrated could also inhibit the basal expression of GLT-1a. However, the administration of either GLT-1a Sense ODNs or Missense ODNs had no effect on the expression of GLT-1a induced by CIP, which was illustrated by the result that there were no significantly changes in the expression of GLT-1a in those groups compared with the CIP group (P>0.05) .The results indicated that the expression of GLT-1a induced by CIP can be inhibited effectively by GLT-1a AS-ODNs injected through the right lateral cerebral ventricle.Conclusion: The results indicated that among the five designed GLT-1a AS-ODNs (P1-P5), P2 chain could specificly inhibit the expression of GLT-1a. GLT-1a AS-ODNs (P2) could not only inhibit the basal expression of GLT-1a, but alsol could inhibit Cef- and CIP-induced up-regulation of GLT-1a expression in the rat CA1 hippocampus.