Autophagy has been shown to be involved in the pathophysiology of developmental seizure-induced brain damage. The present study aimed to examine whether E-64d, an autophagy inhibitor, was able to facilitate developmental seizure-induced hippocampal mossy fiber sprouting, in particular sprouting-associated zinc transporter signals. Recurrent seizures were induced by penicillin every other day in Sprague-Dawley rats from postnatal day 21 (P21). Rats were randomly assigned into the control group (CONT), recurrent seizure group (RS) and the seizure plus E-64d group (E64D). The expression levels of beclin-1 and B-cell lymphoma 2 were analyzed at 1.5, 3, 6 and 24 h after the last seizures using western blot analysis. At P51, mossy fiber sprouting and the mRNA expression levels of zinc transporter 2 (ZnT-2), ZnT-4, ZnT-5, ZnT-6, ZnT-7, divalent cation transporter 1, Zrt-Irt-like protein 6 (ZIP-6), ZIP-7, cathepsin D and cathepsin L in the rat hippocampus were assessed using Timm staining and reverse transcription-quantitative polymerase chain reaction analysis, respectively. Reduced hippocampal mossy fiber sprouting were detected in the E-64d-treated rats compared with the non-treated control. In parallel with these observations, there was a marked reduction in the mRNA expression levels of ZnT-4 at P51 in the E-64d-treated rat hippocampus compared with the non-treated seizure group. Linear correlation analysis showed significant inter-relationship among ZIP-7, ZnT-4, ZnT-5, ZnT-7, cathepsin D and cathepsin L. These results indicate that the ZnT-4/ZIP-7/cathepsin signaling pathway serves a crucial function in the neuroprotective effects of E-64d. Thus, E-64d may offer a novel strategy for the development of therapeutic interventions for developmental seizure-induced brain damage.
Early intervention in developmental seizure-induced brain damage is a focus area in recent pediatric clinical and animal studies. The autophagic/lysosomal pathway has been recently considered as a promising therapy for preventing and treating neurodegenerative diseases, in addition to seizure-induced brain injuries (
Developmental seizure-induced long-term adverse effects include neurodysfunction and pathological phenomena such as regenerative aberrant mossy fiber sprouting in the hippocampus, which is positively correlated with long-term neurofunctional impairment (
The aim of the present study was to characterize the long-term expression profiles of ZnT-associated genes in the hippocampus following developmental seizures. It was hypothesized that the disturbed expression of ZnTs would correlate with increases in hippocampal axon sprouting which could be inhibited by pretreatment with E-64d, another autophagy inhibitor.
A total of 130 Sprague-Dawley male rats (obtained from the Experimental Animal Center of Zhejiang University, Hangzhou, China) at postnatal day 21 (P21) were used in all experiments. Animals were treated in accordance with the guidelines set by the National Institutes of Health for the humane treatment of animals and were approved by the animal care and use committee of Soochow University. Attempts were made to minimize the number of animals used. Rats were randomly assigned to three groups: Control (CONT; n=30); penicillin-induced recurrent seizures group (RS; n=50); and the E-64d-treated seizure group (E64D; n=50). Penicillin (5.1 U/mg dissolved in saline; RS and E64D; North China Pharmaceutical Co., Ltd., Shijiazhuang, China) or saline (CONT) was administered intraperitoneally (i.p.) to induce seizure, as previously reported (
At 3, 6 and 24 h after the final seizure, rats (n=30 per group) were sacrificed by 4% chloral hydrate (1 ml/100 g i.p.; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; cat. no. 30037517) and the hippocampus tissues in every time point from each group (n=6) were rapidly removed and immediately frozen in dry ice for western blot analysis, as described previously by Luo
A subset of rats (n=6 each group) underwent Timm staining on P51 according to the method previously described (
A total of 6 rats from each group were sacrificed using 4% chloral hydrate (1 ml/100 g i.p.) at P51. Hippocampal samples from each group were subjected to RT-qPCR analysis, as described previously (
The protein and mRNA expression levels (2−ΔΔCq) were analyzed with post-hoc comparisons using analysis of variance, followed by Bonferroni test. Timm staining scores were compared using a non-parametric Kruskal-Wallis test. SAS 8.0 statistical software (SAS Institute, Cary, NC, USA) was used to perform statistical analysis. Data is presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.
Significant increases in the hippocampal protein expression levels of beclin-1 (
As shown in
RT-qPCR was employed to evaluate the relative mRNA expression levels of ZnT-2, ZnT-4, ZnT-5, ZnT-6, ZnT-7, DCT-1, ZIP-6, ZIP-7, cathepsin D and cathepsin L in the hippocampus at P51. As shown in
Linear correlation analysis showed a total of 45 Pearson correlation coefficients among the ten genes. As shown in
E-64d is an autophagy inhibitor that has been shown to perform a neuroprotective function in spinal cord injury, hypoxic-ischemic brain injury, acute optic neuritis (
In accordance with previous results (
Previous results showed that CBI, an autophagy inhibitor, could improve functional deficits and inhibit hippocampal sprouting by modulating long-term PRG-1 expression in the hippocampus and cerebral cortex (
The divalent cation Zn2+, an essential trace element that performs structural and cofactor functions, is concentrated in synaptic vesicles, particularly in the hippocampal zinc-rich mossy fiber pathway, which has the highest quantity of zinc in brain (
Apart from ZnTs, the regulation of cellular zinc homeostasis is controlled closely by a number of different mechanisms. Hence, we further investigated the expression of ZIP-6, ZIP-7 and DCT-1 in hippocampus. ZIP is responsible for the movement of zinc into a cell from the extracellular space, and transports zinc into the cytoplasm from organelles that contain zinc (
A previous study by Luo
In conclusion, the present study provides insights into the abnormalities in signal transduction of zinc, reflected by ZnTs and ZIP. These results suggest that the E-64d attenuates hippocampal mossy fiber sprouting, at least in part, via the modulation of the ZnT-4/ZIP-7/cathepsin signaling pathway. These results may offer a novel strategy for the development of therapeutic interventions for treatment of developmental seizure-induced brain damage.
This study was supported by the National Natural Science Foundation of China (grant nos. 81271458 and 81471337) and the Jiangsu Province's Key Provincial Talents Program (grant no. RC2011113), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
Western blot analysis of (A) beclin-1 and (B) Bcl-2 protein expression levels in the rat hippocampus at 3, 12 and 24 h after the last seizures. Data are expressed as the mean ± standard deviation. Data were analyzed with post-hoc comparisons using a Bonferroni test after analysis of variance.*P<0.05 and **P<0.01, vs. CONT and E64D groups. CONT, control group; RS, recurrent seizure group; E64D, E-64d-treated group; Bcl-2, B-cell lymphoma 2.
Representative images of mossy fiber sprouting by Timm staining in the (A) CONT, (B) RS and (C) E64D groups. Parts labeled as (a) represent panoramic view of hippocampus from CONT to E4D; (b) represent CA3 subfield from CONT to E64D; similarly, parts labeled as (c) represent dentate gyrus subfield. Excessive quantities of Timm staining are observed in the stratum pyramidale of CA3 subfield (as shown in B-b) and in the inner molecular layer of the granule cells (as shown in B-b) in the RS group (arrows). Mild quantities of Timm staining are observed in the stratum pyramidale of CA3 subfield (as shown in C-b) in the E64D group (arrow). Data are expressed as the mean ± standard deviation. (D) Timm staining scores compared using a non-parametric Kruskal-Wallis test. Magnification: (a) ×40; (b) and (c) ×100. *P<0.05 vs. CONT group; #P<0.05 vs. RS group. CONT, control group; RS, recurrent seizure group; E64D, E-64d-treated group.
mRNA expression levels as identified using reverse transcription-quantitative polymerase chain reaction analysis. (A) Expression of ZnT-2, ZnT-4, ZnT-5, ZnT-6, ZnT-7, DCT-1, ZIP-6, ZIP-7, cathepsin D and cathepsin L in the rat hippocampus after behavioral analysis. (B) Pearson's linear correlation analyses of the ten genes. Data are expressed as the mean ± standard deviation. Data was analyzed with post hoc comparisons using a Bonferroni test after analysis of variance. *P<0.05 vs. CONT group; #P<0.05 vs. RS group. CONT, control group; RS, recurrent seizure group; E64D, E-64d-treated group; Znt, zinc transporter; DCT-1, divalent cation transporter 1; ZIP, Zrt-Irt-like protein.
Oligonucleotide primers for reverse transcription-quantitative polymerase chain reaction analysis.
Gene | Genbank accession no. | Primer sequence |
---|---|---|
ZnT-2 | RNU50927 | F, 5′-GGCTGGATCCTGGACTAATGTT-3′ |
R, 5′-ACACCCCAAAATCCCTTTCTG-3′ | ||
Probe, 5′-FAM-CTCACACCACAGCTGGAGAGACACTGAGG-TAMRA-3′ | ||
ZnT-4 | NM_172066.1 | F, 5′-GCTGAAGCAGAGGAAGGTGAA-3′ |
R, 5′-TCTCCGATCATGAAAAGCAAGTAG-3′ | ||
Probe, 5′-FAM-CAGGCTGACCATCGCTGCCGT-TAMRA-3′ | ||
ZnT-5 | NM_001106404.1 | F, 5′-CCAGCACATGTCTGGCCTAA-3′ |
R, 5′-TTTGCAGTACTTCATGGATTCCA-3′ | ||
Probe,5′-FAM-CACTGGCTTCCACGATGTCCTGGCTAT-TAMRA-3′ | ||
ZnT-6 | NM_001106708.1 | F, 5′-CGGCATTATCCCAGGACTCA-3′ |
R, 5′-CCAGCAAGATCGATCAGAACAA-3′ | ||
Probe, 5′-FAM-TTCTTGCCCCGCATGAACCCG-TAMRA-3′ | ||
ZnT-7 | XM_001073594.1 | F, 5′-TTGGGATCCGCGTCTGA-3′ |
R, 5′-CCCTCTAGAAGTGACTCGGTATGG-3′ | ||
Probe, 5′-FAM-TCGTCTCTGCTGTCACTGCCGCC-TAMRA-3′ | ||
DCT-1 | NM_001108855.1 | F, 5′-TGGCTGCCTCAGCATCTG-3′ |
R, 5′-CCCAGTTGTTGAGTGGTTTGG-3′ | ||
Probe, 5′-FAM-AAAACCAGAGCCCCTTCCCTACGCA-TAMRA-3′ | ||
ZIP-6 | NM_001024745.1 | F, 5′-CTCACTGCTGGCTTGTTCATG-3′ |
R, 5′-CGTGATCACTGGCATCATTGT-3′ | ||
Probe, 5′-FAM-TCGCTCTGGTCGACATGGTACCCG-TAMRA-3′ | ||
ZIP-7 | NM_001008885.1 | F, 5′-GACTTGGCACACAACTTCACAGA-3′ |
R, 5′-GGACTGTCATCGTGGTCAGGAT-3′ | ||
Probe, 5′-FAM-CTGGCCATTGGTGCTTCCTTCCG-TAMRA-3′ | ||
Cathepsin D | NM_134334 | F, 5′-CATCGCAGCCAAGTTTGATG-3′ |
R, 5′-CCGGGAGCACATTGTTAACA-3′ | ||
Probe, 5′-FAM-CATCTTGGGCATGGGCTACCCTTTTATC-TAMRA-3′ | ||
Cathepsin L | NM_013156 | F, 5′-GGGTATGGATGGCTACATCAAAA-3′ |
R, 5′-GGCTGCGGTGGCAAGTC-3′ | ||
Probe, 5′-FAM-AGCCAAAGACCGGAACAACCACTGC-TAMRA-3′ |
ZnT, zinc transporter; DCT-1, divalent cation transporter 1; ZIP, Zrt-Irt-like protein.
Linear correlation analysis among the 10 genes, showing the 14 Pearson correlation coefficients (
Associated genes | Coefficient value | P-value |
---|---|---|
ZnT-4/ZnT-5 | 0.90 | <0.0001 |
ZnT-4/ZnT-7 | 0.89 | <0.0001 |
ZnT-4/ZIP-7 | 0.64 | <0.01 |
ZnT-5/ZnT-7 | 0.90 | <0.0001 |
ZnT-5/ZIP-7 | 0.57 | <0.05 |
ZnT-6/ZIP-6 | 0.56 | <0.05 |
ZnT-7/DCT-1 | 0.64 | <0.01 |
Cathepsin D/ZnT-4 | 0.72 | <0.001 |
Cathepsin D/ZnT-5 | 0.74 | <0.001 |
Cathepsin D/ZnT-7 | 0.70 | <0.01 |
Cathepsin D/ZIP-7 | 0.62 | <0.01 |
Cathepsin L/ZnT-4 | 0.53 | <0.05 |
Cathepsin L/ZnT-5 | 0.60 | <0.01 |
Cathepsin L/ZIP-7 | 0.47 | <0.05 |
ZnT, zinc transporter; ZIP, Zrt-Irt-like protein 6; DCT, divalent cation transporter.