Identification of the role of C/EBP in neurite regeneration following microarray analysis of a L. stagnalis CNS injury model.
BACKGROUND
Neuronal regeneration in the adult mammalian central nervous system (CNS) is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. In contrast, the CNS of adult Lymnaea stagnalis (L. stagnalis), a freshwater pond snail, is capable of spontaneous regeneration following neuronal injury. Thus, L. stagnalis has served as an animal model to study the cellular mechanisms underlying neuronal regeneration. However, the usage of this model has been limited due to insufficient molecular tools. We have recently conducted a partial neuronal transcriptome sequencing project and reported over 10,000 EST sequences which allowed us to develop and perform a large-scale high throughput microarray analysis.


RESULTS
To identify genes that are involved in the robust regenerative capacity observed in L. stagnalis, we designed the first gene chip covering ~15, 000 L. stagnalis CNS EST sequences. We conducted microarray analysis to compare the gene expression profiles of sham-operated (control) and crush-operated (regenerative model) central ganglia of adult L. stagnalis. The expression levels of 348 genes were found to be significantly altered (p < 0.05) following nerve injury. From this pool, 67 sequences showed a greater than 2-fold change: 42 of which were up-regulated and 25 down-regulated. Our qPCR analysis confirmed that CCAAT enhancer binding protein (C/EBP) was up-regulated following nerve injury in a time-dependent manner. In order to test the role of C/EBP in regeneration, C/EBP siRNA was applied following axotomy of cultured Lymnaea PeA neurons. Knockdown of C/EBP following axotomy prevented extension of the distal, proximal and intact neurites. In vivo knockdown of C/EBP postponed recovery of locomotory activity following nerve crush. Taken together, our data suggest both somatic and local effects of C/EBP are involved in neuronal regeneration.


CONCLUSIONS
This is the first high-throughput microarray study in L. stagnalis, a model of axonal regeneration following CNS injury. We reported that 348 genes were regulated following central nerve injury in adult L. stagnalis and provided the first evidence for the involvement of local C/EBP in neuronal regeneration. Our study demonstrates the usefulness of the large-scale gene profiling approach in this invertebrate model to study the molecular mechanisms underlying the intrinsic regenerative capacity of adult CNS neurons.
Department of Physiology, Faculty of Medicine, University of Toronto, 1 King's College Circle, Toronto, Ontario, M5S 1A8, Canada.
Mila
M
Aleksic
Zhong-Ping
ZP
Feng
D000818:Animals; D019771:Axotomy; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002493:Central Nervous System Diseases / Q000473:pathology* / Q000503:physiopathology*; D004195:Disease Models, Animal; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D008195:Lymnaea; D009043:Motor Activity / Q000187:drug effects; D009416:Nerve Regeneration / Q000187:drug effects / Q000502:physiology*; D016501:Neurites / Q000187:drug effects / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D016415:Sequence Alignment; D013997:Time Factors
D022762:CCAAT-Enhancer-Binding Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering