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Changes in the expression of novel Cdk5 activator messenger RNA (p39nck5ai mRNA) during rat brain development
Neuroscience Research 28 (1997) 355 – 360
Changes in the expression of novel Cdk5 activator messenger RNA (p39nck5ai mRNA) during rat brain development Xiao-Hui Cai a,b, Kazuhito Tomizawa c, Damu Tang d, Yun-Fei Lu c, Akiyoshi Moriwaki c, Masaaki Tokuda a, Shunichiro Nagahata b, Osamu Hatase a, Hideki Matsui c,* b
a Department of Physiology, Faculty of Medicine, Kagawa Medical Uni6ersity, 1750 -1 Ikenobe, Miki, Kagawa 761 -07, Japan Oral and Maxillo-facial Surgery, Faculty of Medicine, Kagawa Medical Uni6ersity, 1750 -1 Ikenobe, Miki, Kagawa 761 -07, Japan c The First Department of Physiology, Okayama Uni6ersity Medical School, 2 -5 -1 Shikata-cho, Okayama 700, Japan d Department of Biochemistry, The Hong Kong Uni6ersity of Science and Technology, Clear WaterBay, Kowloon, Hong Kong
Received 19 February 1997; accepted 20 May 1997
Abstract We previously reported that a neuron-specific Cdk5 activator, p35nck5a, was most prominent in the newborn rat brain. In the adult brain, the expression decreased in most regions except hippocampus and primary olfactory cortex. A novel neuron-specific Cdk5 activator, p39nck5ai, has been recently cloned. To clarify whether two activators were differentially distributed throughout brain development, in this study, we examined the spatial and temporal expression of p39nck5ai in the development rat brain. Northern blot analysis showed that p39nck5ai expression was low in 15-day old fetuses and newborn, and was most prominent in the 1–3 week-old rat brains. In the adult rat brain, expression declined to the same level as in newborn rat brain. In situ hybridization showed that p39nck5ai mRNA was weakly expressed in all neurons of all regions in the newborn rat brain and the transcriptional level was highest in all regions in the 3 week-old rat brain. In the adult, expression was decreased in most neurons except Purkinje and granule cells in the cerebellum which retained high levels. These results suggest that p35nck5a and p39nck5ai may have different functional roles in distinct brain regions during different states of the rat brain development. © 1997 Elsevier Science Ireland Ltd. Keywords: Cyclin-dependent kinase; Cdk5; p39nck5ai; Development; Neuron; In situ hybridization; Cerebellum
1. Introduction Cdc2-related proteins are well known cyclin-dependent kinases (Cdks). They are serine/threonine protein kinases that play a critical role in the regulation of the cell cycle in eukaryotic cells (Hartwell, 1973; Nurse, 1975). Members of this kinase family are heterodimeric proteins comprised of a Cdc2-related protein and a regulatory subunit, a member of the cyclin family (Ghiara et al., 1991). Both Cdc2-related proteins and cyclins exist as extended families. By combining members of these two protein families, a large number of * Corresponding author. Tel.: +81 86 2357104; fax: + 81 86 2235699; e-mail: [email protected]
heterodimetric protein kinases that are called Cdc2-like protein kinases have been obtained (Xiong et al., 1991; Sherr, 1993; Hunter and Pine, 1994). While most of the Cdc2-like kinases are cell cycle regulators, a concept has recently emerged that some of Cdc2-like kinases may have major functions unrelated to cell division (Hellmich et al., 1992). A Cdc2-like kinase has recently been purified from mammalian brains, and extensively characterized (Lew et al., 1992a,b; Beaudette et al., 1993). The purified kinase has been shown to be a heterodimer of Cdk5 and a 25-kDa regulatory subunit which is derived from a 35 kDa protein, p35nck5a (Lew et al., 1994; Tsai et al., 1994). The holoenzyme has been designated neuronal Cdc2like kinase.
0168-0102/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 0 1 0 2 ( 9 7 ) 0 0 0 6 3 - 1
X.-H. Cui et al.
356
Neuroscience
Unlike Cdk5, which is ubiquitously distributed in mammalian tissues (Tsai et al., 1993), ~35”‘~‘” is specifically localized in mammalian neurons (Lew et al., 1994). Therefore, Cdk5 kinase activity is only exhibited in neurons. The kinase phosphorylates neurofilament protein and tau protein in vitro (Paudel et al., 1993; Lew and Wang, 1995). The phosphorylation of these proteins plays an important role in mediating both neurofilament transport and the interaction of neurofilaments with other cytoskeletal systems (de-Waegh et al., 1992). In the juvenile brain, tau and neurofilament proteins are heavily phosphorylated in the soma in preparation for the axonal transport of these proteins (Rebhan et al., 1995; Rosner et al., 1994). We previously reported that the expression of “ck’a was most prominent from newborn to the P35 fourteenth postnatal day and significantly decreased in the adult rat brain (Tomizawa et al., 1996). Cdk5/ with the expression P35 “ck5a kinase activity correlations of p35”=k5a, but not with Cdk5, in brain development (Uchida et al., 1994; Tomizawa et al., 1996). These data suggest that Cdk5/p35”ck5” kinase activity is important for axonal transport and neuronal process growth and may play critical roles in neuronal maturation, synapse formation. and neuronal plasticity in the developing brain. Northern blot analysis has revealed two populations of mammalian brain mRNA (4.0 and 2.1 kb mRNA) that hybridize with a ~35”“~~” probe, suggesting the existence of an isoform of ~35”‘~~” (Lew et al., 1994; Tomizawa et al., 1996). We have cloned the isoform, which shares a high degree of amino acid sequence homology with ~35”“~~” (57%) (Tang et al., 1995). The protein, tentatively designated the neuronal Cdk5 activator isoform (p39”cksai), is similar to p35”“k5” in terms of displaying Cdk5 activation activity and neuron-specific expression (Tang et al., 1995). However, the difference in the functional properties between ~35”‘~~” and in the P39 “ck5a1 is not clear because the differences spatial and temporal expression of ~35”~~~” and P39 “ck5ai have not been elucidated in the developing and adult brain. Here, we present the first extensive study on the expression of ~39”‘~~“’ using in situ hybridization and Northern blot techniques.
2. Materials
and methods
2.1. Probe construction A 198-bp fragment (nucleotides 903-1101) of the full-length human ~39”“~~“’ cDNA (Tang et al., 1995) was subcloned into Bluescript KS Ml 3 ( - ) (Stratagene, San Diego, CA). This fragment exclude the region of high similarity between ~39”‘~~“’ and ~35”‘~‘“, thus minimizing the probability of cross hybridization. To
Researcl~ 28 (1997) 355-360
study in situ hybridization, the plasmid was linearized and transcribed with T3 or T7 RNA polymerase. The transcribed RNA probe was labeled with 2.0 pg/ml digoxigenin-UTP, as described previously (Tomizawa et al., 1995). To study Northern blot analysis, the probes was labeled with [x-32P]dCTP (111 TBq/mmol, NEN) using a random priming DNA Labeling Kit (United States Biochemical, Cleveland, OH) as described previously (Tomizawa et al., 1995). 2.2. Northern hybridization Total RNA from developing and adult rat brains was isolated by the guanidinium thiocyanate method. Then, the mRNA was isolated by three rounds of oligo (dT)cellulose (Boehringer Mannheim) chromatography. Messenger RNA (1 lug) was separated in a 1.5% formaldehyde-agarose gel and transferred to a nylon membranes (HYBOND-N, Amersham, Buckinghamshire, UK), which was prehybridized for more than 2 h at 42°C in 20 x SSPE, 20% deionized formamide, 100 x Denhardt’s solution, 10% SDS, and 100 fig/ml boiled fragmented salmon sperm DNA, and then hybridized overnight with radiolabeled probes in the same solution at 42°C. The membranes were washed three times, in 2 x SSC, 0.1% SDS at 42’C for 30 min. The autoradiogram was analyzed by the use of Fujix BioImage Analyzer BAS 2000. As a control, a 1.1 kb probe for human G3PDH (CLONTEC Laboratories) was also used to monitor the procedures. It was confirmed that the blotting and detection efficiency was reasonably constant among different samples and experiments under the conditions used (Safaei and Fischer, 1989). 2.3. In situ hybridization In situ hybridization was carried out at high stringency essentially as described previously (Tomizawa et al., 1995). Briefly, surgically obtained tissues were fixed in an RNase-free 4% paraformaldehyde solution overnight, sequentially dehydrated with 70, 80, 90, and 100% ethanol, embedded in paraffin, and sectioned to a thickness of 5.0 pm. Deparaffined tissue specimens were incubated for 15 min in TrissHCl (pH 7.4) containing 0.01% proteinase K (Sigma, St. Louis, MO) and acetylated for 15 min with 0.1 M triethanolamine containing 0.2”% acetic acid. After sequential dehydration through a graded alcohol series, each specimen was hybridized with digoxigenin-labeled probe. After hybridization, the slides were incubated with RNase A (10 /Lgg/ml), and washed with 2 x SSC and 0.2 x SSC. After washing, the specimens were incubated with alkaline phosphatase-conjugated anti-Dig antibodies (Boehringer Mannheim GmbH Biochemica, Mannheim, Germany) for 30 min. They were then
X.-H. Cai et al. /Neuroscience
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3. Results 3.1. Expression rat brain
2.1 Kb-
123456 Fig. I. Northern blot analysis of ~39”‘~~“’ mRNA in the developing rat brain. Isolated mRNA (I pg) was analyzed in each lane, as described in Section 2. The size of the ~39”‘~~“’ transcripts ( z 2. I kb) were determined relative to a RNA size marker (GIBCO BRL, Gaithersburg, MD). Lane 1, 15-day fetus; lane 2, adult rat brain. The lower panel shows the expression of G3PDH in each lane as a control.
washed several times, and developed by addition of the substrate for alkaline phosphatase, BCIP/NBT (5 Bromo-4-chloro-3-indolyl-phosphate/Nitro blue tetrazolium). As a control, the sense RNA probe was synthesized and used for hybridization at the same concentration and in the same way as described above.
of ~39”“~” mRNA
in the developing
Northern blot analysis revealed a single ~39”‘~~“’ mRNA transcript of approximately 2.1 kb hybridizing at all stages in the developing rat brain (Fig. 1). This result suggested that the probe used was not cross reactive with ~35”‘~‘” mRNA. Fig. 1 showed that the mRNA signal of ~39”“~‘“’ was very weak in 15-day fetuses, and then was higher in the newborn brain. The mRNA level was most prominent in the 1 week-old rats, and the level was maintained until they were 3 week-old. In the adult rat brain, the intensity of P39 nck5aimRNA signal decreased to the same levels as in the newborn rat brain. 3.2. In situ hybridization developing rat brain
0fp39”~~~~’ mRNA
in the
Fig. 2 revealed the expression of ~39”~~‘“’mRNA in sagittal sections of the newborn, 3 week-old, and adult rat brains. In the newborn brain, the mRNA transcript was expressed in the neurons of all brain regions (Fig. 2A). However, the intensity of the signal was low. The highest expression of ~39”“~~“’mRNA was reached in all regions of the brain at 3 weeks after birth (Fig. 2B). In the adult brain, the high intensity of mRNA expres-
l3
Fig. 2. In situ hybridization of ~39”‘~‘“’ mRNA in sagittal sections of newborn (A), 3 week-old (B) and adult hybridization using the digoxigenin-labeled sense probe in the newborn rat brain. Scale bar = 2.5 mm.
(C) rat brains.
(D) In situ
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was strongly expressed in the pyramidal cells in the fifth layer and weakly expressed in neurons in the other cell layers. A distinct expression pattern of p39nck5ai mRNA became obvious in the hippocampus throughout brain development (Fig. 4A, 4B and 4C). The gene transcript showed low signal intensity in the pyramidal cell layer and granule cell layer in the neonatal hippocampus (A). In the 3 week-old hippocampus, the mRNA was expressed at high levels in the pyramidal cell layer and granule cell layer (B). The expression of p39nck5ai mRNA in the adult hippocampus decreased in comparison with that in the 3 week-old rats. There was
Fig. 3. In situ hybridization of p39nck5ai in the neocortex of the newborn (A), 3-week-old (B) and adult rats (C). Scale bar = 200 mm.
sion was maintained only in the cerebellum, whereas the transcription level became low in the other parts of the brain (Fig. 2C). The gene transcript level in the adult forebrain was even lower than that in the newborn brain forebrain. Fig. 3. revealed p39nck5ai expression in the neocortex of newborn (A), 3 week-old (B), and adult rat brains (C). In the neonatal neocortex, mRNA was expressed in all neuronal cells of all cell layers. Expression was also detected in all cell layers in the 3 week-old rat neocortex. However, an intriguing expression pattern of p39nck5ai was found in the adult neocortex. The mRNA
Fig. 4. The expression of p39nck5ai mRNA in the hippocampus of the newborn (A), 3 week-old (B) and adult rats (C). Scale bar = 200 mm.
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and Purkinje cell layer (Fig. 5B). Expression was maintained in the granule cell layer and Purkinje cell layer in the adult cerebellum (Fig. 5C).
4. Discussion
Fig. 5. In situ hybridization of p39nck5ai mRNA in the cerebellum of the newborn (A), 3 week-old (B) and adult rats (C). Scale bar=300 mm.
moderate p39nck5ai expression in the granule cell layer, whereas the gene transcript showed lower signal intensity in the pyramidal cell layer of the adult hippocampus (C). Fig. 5 showed p39nck5ai mRNA expression in the neonatal, 3 week-old, and adult cerebellum. In situ hybridization showed that mRNA was detectable in almost all the neurons in the cerebellum, however expression was very weak in the neonatal cerebellum (Fig. 5A). In the 3 week-old cerebellum, the mRNA transcript was strongly expressed in the granule cell layer
Three principle findings emerged from this study: (1) p39nck5ai mRNA levels were lower in the 15-day fetus and newborn, and were most prominent in the 1–3 week-old rat brains. In the adult rat brain, the mRNA declined to the same level as in the newborn; (2) p39nck5ai mRNA was expressed in most regions of the newborn and 3 week-old rat brains. However, the expression level was low in the newborn, whereas the mRNA was strongly expressed in the 3 week-old rat brain; (3) In the adult brain, the expression of p39nck5ai mRNA was localized in the cerebellum. p39nck5ai has been cloned as a isoform of p35nck5a from human brain library (Tang et al., 1995). The protein can activate Cdk5 activity with similar affinity and efficiency as p35nck5a in vitro. The existence of two brain- and neuron-specific Cdk5 activators has raised the question as to the physiological significance of the isoforms. We and other investigators have previously reported that the p35nck5a transcript level was specifically high from newborn until 2 week-old, and then the expression decreased in the 3 week-old rat brain (Uchida et al., 1994; Tomizawa et al., 1996). In the present study, we showed that the chronological change of p39nck5ai expression occurred one-week later than p35nck5a in the rat brain development. Moreover, spatial expression of the two are different in the adult brain. The expression of p39nck5ai mRNA was prominent in the cerebellum compared with other regions. We previously reported that both mRNA signal and immunoreactivity for p35nck5a in the hippocampus and primary olfactory cortex were stronger than those for other brain regions (Tomizawa et al., 1996). These results suggest that p35nck5a and p39nck5ai may have different functional roles in distinct brain regions. The regulation of Cdk5 activity by p35nck5a or p39nck5ai may be regionally and temporally different. The situation may be similar to the regulation of Cdc2 by phase-specific cyclins in yeast. Cdc2 associates with phase-specific cyclins, G1 or M phase cyclins, to regulate cell cycle progression at specific phases in yeast cells (Nurse, 1990). We previously reported a contradictory result that the subcellular localization of p35nck5a was different from that of Cdk5 although p35nck5a was identified as an activator for Cdk5 in brain (Tomizawa et al., 1996; Matsushita et al., 1996). Cdk5 changed its subcellular localization from soma to axon, whereas p35nck5a continuously existed in soma throughout brain development. We postulated from these data that there might
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be another activator for Cdk5 in axons and axon terminals. p39nck5ai shares many common characteristics with p35nck5a, including association with Cdk5, Cdk5 activating activity and neuron-specific expression (Tang et al., 1995). We hypothesize that p39nck5ai may be an activator for Cdk5 in axons and axonal terminals. Therefore, subsequent study of subcellular localization of p39nck5ai using specific antibodies in developing and mature brains will be very important.
Acknowledgements This study was supported by a Grant-in-Aid for Science Research from Ministry of Education, Science and Culture, Japan and a research grant from Toyota Physical and Chemical Research Institute.
References Beaudette, K.N., Lew, J., Wang, J.H., 1993. Substrate specificity characterization of a Cdc2-like protein kinase purified from bovine brain. J. Biol. Chem. 268, 20825–20830. de-Waegh, S.M., Lee, V.M., Brady, S.T., 1992. Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells. Cell 68, 451–463. Ghiara, J.B., Richardson, H.E., Sugimoto, K., Henze, M., Lew, D.J., Wittenberg, C., Reed, S.I., 1991. A cyclin B homolog in S. cere6isiae: chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis. Cell 65, 163–174. Hartwell, L.H., 1973. Three additional genes required for deoxyribonucleic acid synthsis in Saccharomyces cerevisiae. J. Bacteriol. 115, 966 – 974. Hellmich, M.R., Pant, H.C., Wada, E., Battey, J.F., 1992. Neuronal Cdk2-like kinase: a Cdk2-related protein kinase with predominantly neuronal expression. Proc. Natl. Acad. Sci. USA 89, 10867 – 10871. Hunter, T., Pine, J., 1994. Cyclins and cancer II: cyclin D and Cdk inhibitor come of age. Cell 79, 573–582. Lew, J., Beaudette, K., Litwin, C.M., Wang, J.H., 1992b. Purification and characterization of a novel proline-directed protein kinase from bovine brain. J. Biol. Chem. 267, 13383–13390. Lew, J., Wang, J.H., 1995. Neuronal cdc2-like kinase. Trends Biochem. Sci. 20, 33 –37. Lew, J., Winkfein, R.J., Huang, Q.-Q., Qi, Z., Aebersold, R., Hunt, T., Wang, L.H., 1994. Neuronal cdc2-like kinase is a complex of cylin-dependent kinase 5 and a novel brain-specific regulatory subunit. Nature 371, 423–426.
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Lew, J., Winkfein, R.J., Paudel, H.K., Wang, J.H., 1992a. Brain proline-directed protein kinase is a neurofilament kinase which displays high sequence homology to p34cdc2. J. Biol. Chem. 267, 25922 – 25926. Matsushita, M., Tomizawa, K., Lu, Y.-F., Moriwaki, A., Tokuda, M., Itano, T., Wang, J.H., Hatase, O., Matsui, H., 1996. Distinct cellular compartment of cyclin-dependent kinase 5 (Cdk5) and neuron-specific Cdk5 activator protein (p35nck5a) in the developing rat cerebellum. Brain Res. 734, 319 – 322. Nurse, P., 1975. Genetic control of cell size at cell division in yeast. Nature 256, 451 – 457. Nurse, P., 1990. Universal control mechanism regulating onset of M-phase. Nature 344, 503 – 508. Paudel, H.K., Lew, J., Ali, Z., Wang, J.H., 1993. Brain proline-directed protein kinase phosphorylates tau on sites that are abnormally phosphorylated in tau associated with Alzheimer’s paired helical filaments. J. Biol. Chem. 268, 23512 – 23518. Rebhan, M., Vacun, G., Rosner, H., 1995. Complementary distribution of tau proteins in different phosphorylation states within growing axons. Neuroreport 6, 429 – 432. Rosner, H., Rebhan, M., Vacun, G., Vanmechelen, E., 1994. Expression of a paired helical filament tau epitope in embryonic chicken central nervous system. Neuroreport 5, 1164 – 1166. Safaei, R., Fischer, I., 1989. Regulation of microtubule-associated protein 2 (MAP2) mRNA expression during rat brain development. J. Mol. Neurosci. 1, 189 – 198. Sherr, C.J., 1993. Mammalian G1 cyclins. Cell 73, 1059 – 1065. Tang, D., Yeung, J., Li, K.-Y., Matsushita, M., Tomizawa, K., Matsui, H., Hatase, O., Wang, J.H., 1995. An isoform of neuronal cyclin-dependent kinase 5 (Cdk5) activator. J. Biol. Chem. 270, 26897 – 26903. Tomizawa, K., Matsui, H., Kondo, E., Miyamoto, K., Tokuda, M., Itano, T., Nagahata, S., Akagi, T., Hatase, O., 1995. Developmental alteration and neuron-specific expression of bone morphogenetic protein-6 (BMP-6) mRNA in rodent Brain. Mol. Brain Res. 28, 122 – 128. Tomizawa, K., Matsui, H., Matsushita, M., Lew, J., Tokuda, M., Itano, T., Konishi, R., Wang, J.H., Hatase, O., 1996. Localization and developmental changes in the neuron-specific cyclin-dependent kinase 5 activator (p35nck5a) in the rat brain. Neuroscience 74, 519 – 529. Tsai, L.H., Takahashi, T., Caviness, V. Jr., Harlow, E., 1993. Activity and expression pattern of cyclin-dependent kinase 5 in the embryonic mouse nervous system. Development 119, 1029 – 1040. Tsai, L.H., Deialle, I., Caviness, V. Jr., Chae, T., Harlow, E., 1994. p35, a neuronal specific regulatory subunit of the Cdk5 kinase. Nature 371, 419 – 423. Uchida, T., Ishiguro, K., Ohnuma, J., Takamatsu, M., Yonekura, S., Imahori, K., 1994. Precursor of cdk5 activator, the 23 kDa subunit of tau protein kinase II: its sequence and developmental change in brain. FEBS lett. 355, 35 – 40. Xiong, Y., Connolly, T., Futcher, B., Beach, D., 1991. Human D-type cyclin. Cell 65, 691 – 699.
Changes in the expression of novel Cdk5 activator messenger RNA (p39nck5ai mRNA) during rat brain development Xiao-Hui Cai a,b, Kazuhito Tomizawa c, Damu Tang d, Yun-Fei Lu c, Akiyoshi Moriwaki c, Masaaki Tokuda a, Shunichiro Nagahata b, Osamu Hatase a, Hideki Matsui c,* b
a Department of Physiology, Faculty of Medicine, Kagawa Medical Uni6ersity, 1750 -1 Ikenobe, Miki, Kagawa 761 -07, Japan Oral and Maxillo-facial Surgery, Faculty of Medicine, Kagawa Medical Uni6ersity, 1750 -1 Ikenobe, Miki, Kagawa 761 -07, Japan c The First Department of Physiology, Okayama Uni6ersity Medical School, 2 -5 -1 Shikata-cho, Okayama 700, Japan d Department of Biochemistry, The Hong Kong Uni6ersity of Science and Technology, Clear WaterBay, Kowloon, Hong Kong
Received 19 February 1997; accepted 20 May 1997
Abstract We previously reported that a neuron-specific Cdk5 activator, p35nck5a, was most prominent in the newborn rat brain. In the adult brain, the expression decreased in most regions except hippocampus and primary olfactory cortex. A novel neuron-specific Cdk5 activator, p39nck5ai, has been recently cloned. To clarify whether two activators were differentially distributed throughout brain development, in this study, we examined the spatial and temporal expression of p39nck5ai in the development rat brain. Northern blot analysis showed that p39nck5ai expression was low in 15-day old fetuses and newborn, and was most prominent in the 1–3 week-old rat brains. In the adult rat brain, expression declined to the same level as in newborn rat brain. In situ hybridization showed that p39nck5ai mRNA was weakly expressed in all neurons of all regions in the newborn rat brain and the transcriptional level was highest in all regions in the 3 week-old rat brain. In the adult, expression was decreased in most neurons except Purkinje and granule cells in the cerebellum which retained high levels. These results suggest that p35nck5a and p39nck5ai may have different functional roles in distinct brain regions during different states of the rat brain development. © 1997 Elsevier Science Ireland Ltd. Keywords: Cyclin-dependent kinase; Cdk5; p39nck5ai; Development; Neuron; In situ hybridization; Cerebellum
1. Introduction Cdc2-related proteins are well known cyclin-dependent kinases (Cdks). They are serine/threonine protein kinases that play a critical role in the regulation of the cell cycle in eukaryotic cells (Hartwell, 1973; Nurse, 1975). Members of this kinase family are heterodimeric proteins comprised of a Cdc2-related protein and a regulatory subunit, a member of the cyclin family (Ghiara et al., 1991). Both Cdc2-related proteins and cyclins exist as extended families. By combining members of these two protein families, a large number of * Corresponding author. Tel.: +81 86 2357104; fax: + 81 86 2235699; e-mail: [email protected]
heterodimetric protein kinases that are called Cdc2-like protein kinases have been obtained (Xiong et al., 1991; Sherr, 1993; Hunter and Pine, 1994). While most of the Cdc2-like kinases are cell cycle regulators, a concept has recently emerged that some of Cdc2-like kinases may have major functions unrelated to cell division (Hellmich et al., 1992). A Cdc2-like kinase has recently been purified from mammalian brains, and extensively characterized (Lew et al., 1992a,b; Beaudette et al., 1993). The purified kinase has been shown to be a heterodimer of Cdk5 and a 25-kDa regulatory subunit which is derived from a 35 kDa protein, p35nck5a (Lew et al., 1994; Tsai et al., 1994). The holoenzyme has been designated neuronal Cdc2like kinase.
0168-0102/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 0 1 0 2 ( 9 7 ) 0 0 0 6 3 - 1
X.-H. Cui et al.
356
Neuroscience
Unlike Cdk5, which is ubiquitously distributed in mammalian tissues (Tsai et al., 1993), ~35”‘~‘” is specifically localized in mammalian neurons (Lew et al., 1994). Therefore, Cdk5 kinase activity is only exhibited in neurons. The kinase phosphorylates neurofilament protein and tau protein in vitro (Paudel et al., 1993; Lew and Wang, 1995). The phosphorylation of these proteins plays an important role in mediating both neurofilament transport and the interaction of neurofilaments with other cytoskeletal systems (de-Waegh et al., 1992). In the juvenile brain, tau and neurofilament proteins are heavily phosphorylated in the soma in preparation for the axonal transport of these proteins (Rebhan et al., 1995; Rosner et al., 1994). We previously reported that the expression of “ck’a was most prominent from newborn to the P35 fourteenth postnatal day and significantly decreased in the adult rat brain (Tomizawa et al., 1996). Cdk5/ with the expression P35 “ck5a kinase activity correlations of p35”=k5a, but not with Cdk5, in brain development (Uchida et al., 1994; Tomizawa et al., 1996). These data suggest that Cdk5/p35”ck5” kinase activity is important for axonal transport and neuronal process growth and may play critical roles in neuronal maturation, synapse formation. and neuronal plasticity in the developing brain. Northern blot analysis has revealed two populations of mammalian brain mRNA (4.0 and 2.1 kb mRNA) that hybridize with a ~35”“~~” probe, suggesting the existence of an isoform of ~35”‘~~” (Lew et al., 1994; Tomizawa et al., 1996). We have cloned the isoform, which shares a high degree of amino acid sequence homology with ~35”“~~” (57%) (Tang et al., 1995). The protein, tentatively designated the neuronal Cdk5 activator isoform (p39”cksai), is similar to p35”“k5” in terms of displaying Cdk5 activation activity and neuron-specific expression (Tang et al., 1995). However, the difference in the functional properties between ~35”‘~~” and in the P39 “ck5a1 is not clear because the differences spatial and temporal expression of ~35”~~~” and P39 “ck5ai have not been elucidated in the developing and adult brain. Here, we present the first extensive study on the expression of ~39”‘~~“’ using in situ hybridization and Northern blot techniques.
2. Materials
and methods
2.1. Probe construction A 198-bp fragment (nucleotides 903-1101) of the full-length human ~39”“~~“’ cDNA (Tang et al., 1995) was subcloned into Bluescript KS Ml 3 ( - ) (Stratagene, San Diego, CA). This fragment exclude the region of high similarity between ~39”‘~~“’ and ~35”‘~‘“, thus minimizing the probability of cross hybridization. To
Researcl~ 28 (1997) 355-360
study in situ hybridization, the plasmid was linearized and transcribed with T3 or T7 RNA polymerase. The transcribed RNA probe was labeled with 2.0 pg/ml digoxigenin-UTP, as described previously (Tomizawa et al., 1995). To study Northern blot analysis, the probes was labeled with [x-32P]dCTP (111 TBq/mmol, NEN) using a random priming DNA Labeling Kit (United States Biochemical, Cleveland, OH) as described previously (Tomizawa et al., 1995). 2.2. Northern hybridization Total RNA from developing and adult rat brains was isolated by the guanidinium thiocyanate method. Then, the mRNA was isolated by three rounds of oligo (dT)cellulose (Boehringer Mannheim) chromatography. Messenger RNA (1 lug) was separated in a 1.5% formaldehyde-agarose gel and transferred to a nylon membranes (HYBOND-N, Amersham, Buckinghamshire, UK), which was prehybridized for more than 2 h at 42°C in 20 x SSPE, 20% deionized formamide, 100 x Denhardt’s solution, 10% SDS, and 100 fig/ml boiled fragmented salmon sperm DNA, and then hybridized overnight with radiolabeled probes in the same solution at 42°C. The membranes were washed three times, in 2 x SSC, 0.1% SDS at 42’C for 30 min. The autoradiogram was analyzed by the use of Fujix BioImage Analyzer BAS 2000. As a control, a 1.1 kb probe for human G3PDH (CLONTEC Laboratories) was also used to monitor the procedures. It was confirmed that the blotting and detection efficiency was reasonably constant among different samples and experiments under the conditions used (Safaei and Fischer, 1989). 2.3. In situ hybridization In situ hybridization was carried out at high stringency essentially as described previously (Tomizawa et al., 1995). Briefly, surgically obtained tissues were fixed in an RNase-free 4% paraformaldehyde solution overnight, sequentially dehydrated with 70, 80, 90, and 100% ethanol, embedded in paraffin, and sectioned to a thickness of 5.0 pm. Deparaffined tissue specimens were incubated for 15 min in TrissHCl (pH 7.4) containing 0.01% proteinase K (Sigma, St. Louis, MO) and acetylated for 15 min with 0.1 M triethanolamine containing 0.2”% acetic acid. After sequential dehydration through a graded alcohol series, each specimen was hybridized with digoxigenin-labeled probe. After hybridization, the slides were incubated with RNase A (10 /Lgg/ml), and washed with 2 x SSC and 0.2 x SSC. After washing, the specimens were incubated with alkaline phosphatase-conjugated anti-Dig antibodies (Boehringer Mannheim GmbH Biochemica, Mannheim, Germany) for 30 min. They were then
X.-H. Cai et al. /Neuroscience
Research 28 (1997) 355-360
357
3. Results 3.1. Expression rat brain
2.1 Kb-
123456 Fig. I. Northern blot analysis of ~39”‘~~“’ mRNA in the developing rat brain. Isolated mRNA (I pg) was analyzed in each lane, as described in Section 2. The size of the ~39”‘~~“’ transcripts ( z 2. I kb) were determined relative to a RNA size marker (GIBCO BRL, Gaithersburg, MD). Lane 1, 15-day fetus; lane 2, adult rat brain. The lower panel shows the expression of G3PDH in each lane as a control.
washed several times, and developed by addition of the substrate for alkaline phosphatase, BCIP/NBT (5 Bromo-4-chloro-3-indolyl-phosphate/Nitro blue tetrazolium). As a control, the sense RNA probe was synthesized and used for hybridization at the same concentration and in the same way as described above.
of ~39”“~” mRNA
in the developing
Northern blot analysis revealed a single ~39”‘~~“’ mRNA transcript of approximately 2.1 kb hybridizing at all stages in the developing rat brain (Fig. 1). This result suggested that the probe used was not cross reactive with ~35”‘~‘” mRNA. Fig. 1 showed that the mRNA signal of ~39”“~‘“’ was very weak in 15-day fetuses, and then was higher in the newborn brain. The mRNA level was most prominent in the 1 week-old rats, and the level was maintained until they were 3 week-old. In the adult rat brain, the intensity of P39 nck5aimRNA signal decreased to the same levels as in the newborn rat brain. 3.2. In situ hybridization developing rat brain
0fp39”~~~~’ mRNA
in the
Fig. 2 revealed the expression of ~39”~~‘“’mRNA in sagittal sections of the newborn, 3 week-old, and adult rat brains. In the newborn brain, the mRNA transcript was expressed in the neurons of all brain regions (Fig. 2A). However, the intensity of the signal was low. The highest expression of ~39”“~~“’mRNA was reached in all regions of the brain at 3 weeks after birth (Fig. 2B). In the adult brain, the high intensity of mRNA expres-
l3
Fig. 2. In situ hybridization of ~39”‘~‘“’ mRNA in sagittal sections of newborn (A), 3 week-old (B) and adult hybridization using the digoxigenin-labeled sense probe in the newborn rat brain. Scale bar = 2.5 mm.
(C) rat brains.
(D) In situ
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was strongly expressed in the pyramidal cells in the fifth layer and weakly expressed in neurons in the other cell layers. A distinct expression pattern of p39nck5ai mRNA became obvious in the hippocampus throughout brain development (Fig. 4A, 4B and 4C). The gene transcript showed low signal intensity in the pyramidal cell layer and granule cell layer in the neonatal hippocampus (A). In the 3 week-old hippocampus, the mRNA was expressed at high levels in the pyramidal cell layer and granule cell layer (B). The expression of p39nck5ai mRNA in the adult hippocampus decreased in comparison with that in the 3 week-old rats. There was
Fig. 3. In situ hybridization of p39nck5ai in the neocortex of the newborn (A), 3-week-old (B) and adult rats (C). Scale bar = 200 mm.
sion was maintained only in the cerebellum, whereas the transcription level became low in the other parts of the brain (Fig. 2C). The gene transcript level in the adult forebrain was even lower than that in the newborn brain forebrain. Fig. 3. revealed p39nck5ai expression in the neocortex of newborn (A), 3 week-old (B), and adult rat brains (C). In the neonatal neocortex, mRNA was expressed in all neuronal cells of all cell layers. Expression was also detected in all cell layers in the 3 week-old rat neocortex. However, an intriguing expression pattern of p39nck5ai was found in the adult neocortex. The mRNA
Fig. 4. The expression of p39nck5ai mRNA in the hippocampus of the newborn (A), 3 week-old (B) and adult rats (C). Scale bar = 200 mm.
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and Purkinje cell layer (Fig. 5B). Expression was maintained in the granule cell layer and Purkinje cell layer in the adult cerebellum (Fig. 5C).
4. Discussion
Fig. 5. In situ hybridization of p39nck5ai mRNA in the cerebellum of the newborn (A), 3 week-old (B) and adult rats (C). Scale bar=300 mm.
moderate p39nck5ai expression in the granule cell layer, whereas the gene transcript showed lower signal intensity in the pyramidal cell layer of the adult hippocampus (C). Fig. 5 showed p39nck5ai mRNA expression in the neonatal, 3 week-old, and adult cerebellum. In situ hybridization showed that mRNA was detectable in almost all the neurons in the cerebellum, however expression was very weak in the neonatal cerebellum (Fig. 5A). In the 3 week-old cerebellum, the mRNA transcript was strongly expressed in the granule cell layer
Three principle findings emerged from this study: (1) p39nck5ai mRNA levels were lower in the 15-day fetus and newborn, and were most prominent in the 1–3 week-old rat brains. In the adult rat brain, the mRNA declined to the same level as in the newborn; (2) p39nck5ai mRNA was expressed in most regions of the newborn and 3 week-old rat brains. However, the expression level was low in the newborn, whereas the mRNA was strongly expressed in the 3 week-old rat brain; (3) In the adult brain, the expression of p39nck5ai mRNA was localized in the cerebellum. p39nck5ai has been cloned as a isoform of p35nck5a from human brain library (Tang et al., 1995). The protein can activate Cdk5 activity with similar affinity and efficiency as p35nck5a in vitro. The existence of two brain- and neuron-specific Cdk5 activators has raised the question as to the physiological significance of the isoforms. We and other investigators have previously reported that the p35nck5a transcript level was specifically high from newborn until 2 week-old, and then the expression decreased in the 3 week-old rat brain (Uchida et al., 1994; Tomizawa et al., 1996). In the present study, we showed that the chronological change of p39nck5ai expression occurred one-week later than p35nck5a in the rat brain development. Moreover, spatial expression of the two are different in the adult brain. The expression of p39nck5ai mRNA was prominent in the cerebellum compared with other regions. We previously reported that both mRNA signal and immunoreactivity for p35nck5a in the hippocampus and primary olfactory cortex were stronger than those for other brain regions (Tomizawa et al., 1996). These results suggest that p35nck5a and p39nck5ai may have different functional roles in distinct brain regions. The regulation of Cdk5 activity by p35nck5a or p39nck5ai may be regionally and temporally different. The situation may be similar to the regulation of Cdc2 by phase-specific cyclins in yeast. Cdc2 associates with phase-specific cyclins, G1 or M phase cyclins, to regulate cell cycle progression at specific phases in yeast cells (Nurse, 1990). We previously reported a contradictory result that the subcellular localization of p35nck5a was different from that of Cdk5 although p35nck5a was identified as an activator for Cdk5 in brain (Tomizawa et al., 1996; Matsushita et al., 1996). Cdk5 changed its subcellular localization from soma to axon, whereas p35nck5a continuously existed in soma throughout brain development. We postulated from these data that there might
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be another activator for Cdk5 in axons and axon terminals. p39nck5ai shares many common characteristics with p35nck5a, including association with Cdk5, Cdk5 activating activity and neuron-specific expression (Tang et al., 1995). We hypothesize that p39nck5ai may be an activator for Cdk5 in axons and axonal terminals. Therefore, subsequent study of subcellular localization of p39nck5ai using specific antibodies in developing and mature brains will be very important.
Acknowledgements This study was supported by a Grant-in-Aid for Science Research from Ministry of Education, Science and Culture, Japan and a research grant from Toyota Physical and Chemical Research Institute.
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