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The expression of KLF4 in zebrafish retina during optic nerve regeneration
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Abstracts / Neuroscience Research 68S (2010) e223–e334
large part of CFP positive cells were differentiated into myelinating mature oligodendrocytes. Nevertheless the number of CFP-labeled mature oligodendrocytes was not significantly increased, which would not be insufficient to repair the injury. Taken together, these results indicate that OPCs have a potential capacity as an endogenous cell source to regenerate oligodendrocytes after WMI. Development of interventions that promote this process are needed for achieving complete restoration from the injury. doi:10.1016/j.neures.2010.07.1116
P2-e04 Temporal profile of regenerative cells in the adult brain after stroke Takashi Tanaka , Wataru Yamaguti, Shinobu Inagaki Div of Health Sci, Grad Sch of Med, Univ of Osaka, Osaka Ischemic stroke is a devastating injury of neuron and glial cells, whereas cerebral ischemia also stimulates regenerative responses in the tissue adjacent and remote to the impaired area. Even in the adult brain, neural stem cells (NSCs) or neuronal progenitor cells (NPCs) can be activated by injury, however, it is not well known when and where neocortical NSCs/NPCs are generated to function in the central nervous system. In this study, we investigated the temporal expression profile of newly generated progenitor cells and their fates in the adult mouse cortex.Unilateral middle cerebral artery occlusion is induced in adult C.B17/Icr mice. Animals were injected with bromodeoxyuridine (BrdU) respectively at 3, 7, 14, 30 and 60 days after ischemia, and were sacrificed 6 h after the injection. BrdU-positive cells in the periinfarct cortex were remarkably increased in number at day 3 post-stroke with a peak at 7, while only a very few BrdU-positive cells were observed in the intact neocortex and the remote area. After day 7 post-stroke, the increase of BrdU-positive cells became less remarkable and a few BrdU-positive cells were still found at day 60. To investigate the fates of newly generated cells, the other mice injected with BrdU at day 7 post-stroke were sacrificed at day 30 and 60. Newly generated cells in post-stroke cortex were evaluated by immunohistochemical staining using various cell markers such as Nestin, NeuN, NG2, MAG, S100 and Iba1. doi:10.1016/j.neures.2010.07.1117
P2-e05 The expression of KLF4 in zebrafish retina during optic nerve regeneration Maki Nishitani 1 , Mikiko Nagashima 1,2 , Toru Matsukawa 2 , Kazuhiro Mawatari 1 , Satoru Kato 2 1
Div. Health Sci., Grad. Sch. Med., Kanazawa University, Kanazawa, Japan Department Mol. Neurobiol., Grad. Sch. Med., Kanazawa University, Kanazawa, Japan 2
Unlike mammals, fish can regenerate their axons following nerve injury. Regenerative capacity of fish CNS is their competence to induce regenerationassociated genes. Krüppel-like factor 4 (KLF4) is one of the transcription factor known to produce iPS cells. In this study, we investigated the expression of KLF4 mRNA in zebrafish retina during regeneration after nerve injury. To clone zebrafish KLF4 cDNA, we performed RT-PCR using unlesioned retinal total RNA. We obtained two different mRNAs in length, shorter (KLF4-S) and longer (KLF4-L) cDNAs. Sequencing results for these cDNAs revealed that KLF4-S was homologous sequence for registered KLF-4 cDNA. In contrast, KLF4-L was 111 bp longer than that of registered cDNA. The 111 bp inserted sequences just matched with intronic sequence (intron2) of the gene and contained a stop codon for termination of protein translation. KLF4 protein is composed with activation domain, repression domain, nuclear transport signal domain and DNA binding domain (DBD). Our sequencing results strongly indicate that the protein translated from KLF4-L might lack C-terminal which contains DBD and act as a regulator for KLF4-L protein. To study the expression changes of two KLF4 mRNAs during zebrafish optic nerve regeneration, we performed time-course analysis using RT-PCR at 0, 3, 5, 10 days after nerve injury. KLF4-S mRNA levels significantly increased 3–10 days after injury. On the contrary, KLF4-L mRNA levels decreased during this period. These changes of the KLF4-S and KLF4-L mRNAs were mainly localized in the retinal ganglion cell layer revealing by in situ hybridization. These results strongly suggest that transcriptional activity of KLF4 might dynamically change during zebrafish optic nerve regeneration. The levels of two KLF4 proteins are now in progress. doi:10.1016/j.neures.2010.07.1118
P2-e06 The pancreatitis-associated protein-III (PAP-III) is polymerized into a fibrillar structure by the cleavage of its N-terminus Hiroyuki Konishi , Shinichi Kawahara, Sakiko Matsumoto, Rie Maeda, Hiroshi Kiyama Dept of Anat and Neurobiol, Osaka City Univ Grad Sch of Med, Osaka Pancreatitis-associated protein-III (PAP-III) is a C-type lectin secreted from neurons and glial cells in response to both PNS and CNS injury. In this study, we attempted to characterize consequences of PAP-III expression in response to neuronal injury. First we found that proteolytic cleavage of the N-terminus of PAP-III occurred in injured sciatic nerve. Western blot analysis showed that the immunoreactive band for the PAP-III in the sample prepared from injured nerves was slightly lower than that for the recombinant protein and the size was approximately equal to that of N-terminus-truncated PAP-III (NPAP-III) that was previously reported as the processed form by a trypsin-like protease. We then characterized the structure of N-PAP-III, and identified that the PAP-III protein became insoluble upon the trypsin digestion and were polymerized into a fibrous structure. The insolubility varied due to pH and concentration of Na+ in the solution. Ultrastructural observation of the fibrous structure of N-PAP-III by scanning electron microscopy revealed that N-PAP-III formed highly organized fibrillar structures with a diameter of 10–100 nm under neutral pH. The fibrillar N-PAP-III preferentially associated with the surface of neurites as well as cell bodies of primary cultured cortical neurons when N-PAP-III was added to the culture media. These results suggest that the fibrillar N-PAP-III may have a novel role either in neurite elongation or neuronal protection after traumatic neuronal injury. doi:10.1016/j.neures.2010.07.1119
P2-e07 FGF-2 induced fibronectin-positive cells retained the potential to repair spinal cord injury through several passages Masaki Kasai Furukawa
, Hidefumi Fukumitsu, Hitomi Soumiya, Shoei
Lab of Mol Biol, Gifu Pharm Univ, Gifu We have previously shown that administration of basic fibroblast growth factor (FGF-2) into the legion site of the spinal cord markedly improved lcomotor function after spinal cord injury (SCI). A mass composed of FGF-2-induced fibronectin-positive cells (FIFs) had appeared and filled large cavities, into which numerous regenerated axons penetrated and through which they coursed. The SCI rats which received transplantation of the cultured FIFs could extensively move their hindlimb joints. This time, we assessed the effect of culture time or passage number of FIFs on the ability to ameliorate SCI-induced locomotor dysfunction to identify the molecules expressing in FIFs responsible for the function of repair SCI. The FIFs were primarily cultured from the injured spinal cord 2 days after FGF-2 administration. First, confluent cultures inoculated on 10-cm dish were termed as the FIFs of the 1st generation (FIF1). FIFs grown to near confluence were repeatedly passaged into new 10-cm dishes every 3–4 days. The 2nd, 4th, 6th and 8th passaged FIFs (FIF2, FIF4, FIF6 and FIF8, respectively) were used for this study. Hindlimb locomotor activity of the SCI rats was significantly improved after transplantation of FIF2, FIF4 or FIF6, but not transplantation of FIF8. As it was suggested that the loss of function of FIFs occurred during cell passages, we investigated the alternation in gene expressions of N-cadherin depending on cell passages by RT-PCR. The expression level of N-cadherin mRNA in FIF8 was significantly lower than that in younger generations FIFs. Therefore, the timing of the decrease in N-cadherin in FIFs may coincide with the timing of the loss of the ability of FIFs to repair SCI. The primarily cultured FIFs retained the potential to repair SCI-induced locomotor dysfunction even after having proliferated during several passages. It is highly plausible that N-cadherin, potentially expressed in FIFs, plays a pivotal role in enhanced locomotor function after SCI. doi:10.1016/j.neures.2010.07.1120
Abstracts / Neuroscience Research 68S (2010) e223–e334
large part of CFP positive cells were differentiated into myelinating mature oligodendrocytes. Nevertheless the number of CFP-labeled mature oligodendrocytes was not significantly increased, which would not be insufficient to repair the injury. Taken together, these results indicate that OPCs have a potential capacity as an endogenous cell source to regenerate oligodendrocytes after WMI. Development of interventions that promote this process are needed for achieving complete restoration from the injury. doi:10.1016/j.neures.2010.07.1116
P2-e04 Temporal profile of regenerative cells in the adult brain after stroke Takashi Tanaka , Wataru Yamaguti, Shinobu Inagaki Div of Health Sci, Grad Sch of Med, Univ of Osaka, Osaka Ischemic stroke is a devastating injury of neuron and glial cells, whereas cerebral ischemia also stimulates regenerative responses in the tissue adjacent and remote to the impaired area. Even in the adult brain, neural stem cells (NSCs) or neuronal progenitor cells (NPCs) can be activated by injury, however, it is not well known when and where neocortical NSCs/NPCs are generated to function in the central nervous system. In this study, we investigated the temporal expression profile of newly generated progenitor cells and their fates in the adult mouse cortex.Unilateral middle cerebral artery occlusion is induced in adult C.B17/Icr mice. Animals were injected with bromodeoxyuridine (BrdU) respectively at 3, 7, 14, 30 and 60 days after ischemia, and were sacrificed 6 h after the injection. BrdU-positive cells in the periinfarct cortex were remarkably increased in number at day 3 post-stroke with a peak at 7, while only a very few BrdU-positive cells were observed in the intact neocortex and the remote area. After day 7 post-stroke, the increase of BrdU-positive cells became less remarkable and a few BrdU-positive cells were still found at day 60. To investigate the fates of newly generated cells, the other mice injected with BrdU at day 7 post-stroke were sacrificed at day 30 and 60. Newly generated cells in post-stroke cortex were evaluated by immunohistochemical staining using various cell markers such as Nestin, NeuN, NG2, MAG, S100 and Iba1. doi:10.1016/j.neures.2010.07.1117
P2-e05 The expression of KLF4 in zebrafish retina during optic nerve regeneration Maki Nishitani 1 , Mikiko Nagashima 1,2 , Toru Matsukawa 2 , Kazuhiro Mawatari 1 , Satoru Kato 2 1
Div. Health Sci., Grad. Sch. Med., Kanazawa University, Kanazawa, Japan Department Mol. Neurobiol., Grad. Sch. Med., Kanazawa University, Kanazawa, Japan 2
Unlike mammals, fish can regenerate their axons following nerve injury. Regenerative capacity of fish CNS is their competence to induce regenerationassociated genes. Krüppel-like factor 4 (KLF4) is one of the transcription factor known to produce iPS cells. In this study, we investigated the expression of KLF4 mRNA in zebrafish retina during regeneration after nerve injury. To clone zebrafish KLF4 cDNA, we performed RT-PCR using unlesioned retinal total RNA. We obtained two different mRNAs in length, shorter (KLF4-S) and longer (KLF4-L) cDNAs. Sequencing results for these cDNAs revealed that KLF4-S was homologous sequence for registered KLF-4 cDNA. In contrast, KLF4-L was 111 bp longer than that of registered cDNA. The 111 bp inserted sequences just matched with intronic sequence (intron2) of the gene and contained a stop codon for termination of protein translation. KLF4 protein is composed with activation domain, repression domain, nuclear transport signal domain and DNA binding domain (DBD). Our sequencing results strongly indicate that the protein translated from KLF4-L might lack C-terminal which contains DBD and act as a regulator for KLF4-L protein. To study the expression changes of two KLF4 mRNAs during zebrafish optic nerve regeneration, we performed time-course analysis using RT-PCR at 0, 3, 5, 10 days after nerve injury. KLF4-S mRNA levels significantly increased 3–10 days after injury. On the contrary, KLF4-L mRNA levels decreased during this period. These changes of the KLF4-S and KLF4-L mRNAs were mainly localized in the retinal ganglion cell layer revealing by in situ hybridization. These results strongly suggest that transcriptional activity of KLF4 might dynamically change during zebrafish optic nerve regeneration. The levels of two KLF4 proteins are now in progress. doi:10.1016/j.neures.2010.07.1118
P2-e06 The pancreatitis-associated protein-III (PAP-III) is polymerized into a fibrillar structure by the cleavage of its N-terminus Hiroyuki Konishi , Shinichi Kawahara, Sakiko Matsumoto, Rie Maeda, Hiroshi Kiyama Dept of Anat and Neurobiol, Osaka City Univ Grad Sch of Med, Osaka Pancreatitis-associated protein-III (PAP-III) is a C-type lectin secreted from neurons and glial cells in response to both PNS and CNS injury. In this study, we attempted to characterize consequences of PAP-III expression in response to neuronal injury. First we found that proteolytic cleavage of the N-terminus of PAP-III occurred in injured sciatic nerve. Western blot analysis showed that the immunoreactive band for the PAP-III in the sample prepared from injured nerves was slightly lower than that for the recombinant protein and the size was approximately equal to that of N-terminus-truncated PAP-III (NPAP-III) that was previously reported as the processed form by a trypsin-like protease. We then characterized the structure of N-PAP-III, and identified that the PAP-III protein became insoluble upon the trypsin digestion and were polymerized into a fibrous structure. The insolubility varied due to pH and concentration of Na+ in the solution. Ultrastructural observation of the fibrous structure of N-PAP-III by scanning electron microscopy revealed that N-PAP-III formed highly organized fibrillar structures with a diameter of 10–100 nm under neutral pH. The fibrillar N-PAP-III preferentially associated with the surface of neurites as well as cell bodies of primary cultured cortical neurons when N-PAP-III was added to the culture media. These results suggest that the fibrillar N-PAP-III may have a novel role either in neurite elongation or neuronal protection after traumatic neuronal injury. doi:10.1016/j.neures.2010.07.1119
P2-e07 FGF-2 induced fibronectin-positive cells retained the potential to repair spinal cord injury through several passages Masaki Kasai Furukawa
, Hidefumi Fukumitsu, Hitomi Soumiya, Shoei
Lab of Mol Biol, Gifu Pharm Univ, Gifu We have previously shown that administration of basic fibroblast growth factor (FGF-2) into the legion site of the spinal cord markedly improved lcomotor function after spinal cord injury (SCI). A mass composed of FGF-2-induced fibronectin-positive cells (FIFs) had appeared and filled large cavities, into which numerous regenerated axons penetrated and through which they coursed. The SCI rats which received transplantation of the cultured FIFs could extensively move their hindlimb joints. This time, we assessed the effect of culture time or passage number of FIFs on the ability to ameliorate SCI-induced locomotor dysfunction to identify the molecules expressing in FIFs responsible for the function of repair SCI. The FIFs were primarily cultured from the injured spinal cord 2 days after FGF-2 administration. First, confluent cultures inoculated on 10-cm dish were termed as the FIFs of the 1st generation (FIF1). FIFs grown to near confluence were repeatedly passaged into new 10-cm dishes every 3–4 days. The 2nd, 4th, 6th and 8th passaged FIFs (FIF2, FIF4, FIF6 and FIF8, respectively) were used for this study. Hindlimb locomotor activity of the SCI rats was significantly improved after transplantation of FIF2, FIF4 or FIF6, but not transplantation of FIF8. As it was suggested that the loss of function of FIFs occurred during cell passages, we investigated the alternation in gene expressions of N-cadherin depending on cell passages by RT-PCR. The expression level of N-cadherin mRNA in FIF8 was significantly lower than that in younger generations FIFs. Therefore, the timing of the decrease in N-cadherin in FIFs may coincide with the timing of the loss of the ability of FIFs to repair SCI. The primarily cultured FIFs retained the potential to repair SCI-induced locomotor dysfunction even after having proliferated during several passages. It is highly plausible that N-cadherin, potentially expressed in FIFs, plays a pivotal role in enhanced locomotor function after SCI. doi:10.1016/j.neures.2010.07.1120