Developmental expression of neuronal calmodulin-binding proteins in rat brain

62

Developmental Brain ResearOz. 53 (1990) 02--70 Elsevier

BRESD 51057

Developmental expression of neuronal calmodulin-binding proteins in rat brain Joseph W. Polli, Christine M. Patanow and Melvin L. Billingsley Department of Pharmacology and Center for Cell and Molecular Biology, The Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, PA (U.S.A.)

(Accepted 14 November 1989) Key words: Calmodulin-binding protein; Biotinylated calmodulin; Neuronal development; Calcineurin; Neuromodulin/growth-associated protein 43; Multifunctional CaM-kinase

The developmental patterns of calmodulin-binding proteins (CaM-BPS) in rat brain were examined using biotinylated calmodulin overlays of one- and two-dimensional gels. Hippocampus showed the earliest onset of CaM-BP expression (postnatal day 5; PND5), followed by cerebral cortex and striatum, both of which had detectable levels of CaM-BPs by PND7. Cerebellum had the latest onset of CaM-BP expression, CaM-BPs were not detectable until PND9. Very few CaM-BPs were present in brain before PND5 and all regions reached near adult levels by PND20. However, several unique CaM-BPs were seen in embryonic brain and these proteins may have an important role in developing neurons. These data suggest an orderly, complex expression of CaM-BPs which increases during times of synaptogenesis and synaptic maturation. INTRODUCTION Development of the central nervous system is a highly complex and orchestrated event. The underlying processes which control neuronal cell division, migration, differentiation and synaptic organization remain to be elucidated. Calmodulin (CAM) is a major calciumbinding protein thought to have a pivotal role in several neuronal processes such as neurotransmitter release 11, cyclic nucleotide metabolism 2° and signal transduction 3°. CaM has been detected concurrent with the appearance of neurofilament proteins in the developing nervous system of mice by gestational day 9.59; therefore, it has been suggested that CaM may have a role in elongation of axons 9 or dendrites 28. CaM is localized within fetal growth cones, providing further support to the hypothesis that CaM may regulate several aspects of neuronal migration during CNS development 15. A second major component of axonal growth cones is neuromodulin (Growth-associated protein (GAP)-43, B50, p57, F1, pp43) 22. Neuromodulin is a unique calmodulin-binding protein (CaM-BP) which displays the property of binding to CaM with high affinity in the absence of calcium. Models have been proposed suggesting that neuromodulin acts to localize CaM within the discrete areas of the neuron when calcium levels are low 25.

Neuromodulin is phosphorylated by protein k_inase C and phosphorylation decreases the affinity for CAM1. Recently, phosphorylated neuromodulin was shown to be dephosphorylated by calcineurin (CN), a CaM-dependent protein phosphatase, suggesting that CN and protein kinase C regulate CaM-binding to neuromodulin 21. Several proteins isolated from fetal growth cones have been shown to bind [125I]CAM in nitrocellulose overlay assays15; a series of growth cone proteins may also be phosphorylated in a calcium/CaM-dependent manner 16. Two growth cone CaM-binding proteins were identified as possible subunits of the multifunctional CaM kinase; however, other CaM-dependent enzymes such as calcineurin (CN) and CaM-dependent 3',5'-cyclic nucteotide phosphodiesterase (CaM-PDE) were not detected. The developmental pattern of CaM-binding proteins has not been fully explored. Establishing the presence and time course of CaM-dependent enzyme expression in the developing nervous system is important because: (1) the CaM-dependent multifunctional protein kinase (CaM-kinase II) is the major postsynaptic density protein in the adult nervous system 14'17 and it has been postulated that this enzyme plays a critical role in neuronal development and synapse formation33; (2) CaM and its target enzymes may alter protein phosphorylation 2j and cyclic nucleotide metabolism in maturing neurons 12 and

Correspondence: M.L. Billingsley, Department of Pharmacology, P.O. Box 850, Milton S. Hershey Medical Center, Hershey, PA 17033, U.S.A.

0165-3806/90/$03.50 t~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

63 binding proteins and to begin to explain the possible roles and regulation of these proteins in the developing nervous system.

(3) the patterns of CaM-BP expression may provide insights into the regulation of individual CaM-BPs during synaptogenesis. The purpose of this study was to examine the overall developmental pattern of neuronal CaM-

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Fig. 1. Developmental pattern of neuronal CaM-BPs from embryonic day 15 (El5) through adult rat cerebral cortex. Panels A and C show protein staining with Coomassie brilliant blue for synaptosomal (P2) and cytosolic ($2) fractions. Panels B and D are biotinylated CaM overlays demonstrating the developmental pattern of CaM-BP expression. Each lane contained 100 btg of protein. Panel E: densitometric analysis of 50 kDa CaM-BP expression during development.

64 MATERIALS AND METHODS

Materials Electrophoretic supplies were purchased from Biorad Labs, molecular weight standards and ampholines from Pharmacia, and biotinylation reagents from Pierce Chemical Co. Avidin was purchased from Biomedia, alkaline phosphatase and leupeptin from Boehringer-Mannheim, and Nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt (BCIP) from Amresco. Nitrocellulose (0.2 ~tm) was purchased from Schleicher and Schuell. SDS-PA GE and protein blotting procedures One- and two-dimensional SDS-PAGE was carried out as previously described24 using a mixture of 3-10 and 5-8 ampholines (1:3 v/v) for isoelectric focusing and pyronin Y as a tracking dye. Proteins were transferred to nitrocellulose by electrohlotting for 3-4 h at 50 V32 and blots were incubated in blocking solution (5% non-fat dry milk, 50 mM Tris-HCl, pH 7.4, 150 mM NaCI, 1 mM CaCI2) for 1 h. After blocking, blots were incubated in blocking buffer containingbiotinylated CaM (50/ag/10 ml blocking buffer) for 30 min as described by Billingsley et al.4'5, followed by three 10-min washes in buffer A (50 mM Tris-HCl, pH 7.4,150 mM NaCI, 1 mM CaCI~2). Biotinylated CaM (BioCaM) was detected using preformed avidin-alkalinephosphatase complexes (10/tl of 2 mg/ml avidin, 25 /al of 1 mg/ml biotinylated alkaline phosphatase) in 10 ml of buffer A (preincubated by rocking for 30 min at 25 °C). Blots were incubated in the presence of avidin-alkaline phosphatase for 30 min, washed, and CaM-BPs visualized by using the NBT/BCIP chromogen system. All blots were air-dried and photographed using Kodak Technical Pan Film at ASA 25. In several cases, CaM-BPs were quantitated using a Molecular Dynamics laser scanning densitometer coupled with detection software from Protein Data Bases. Neuronal tissue isolation Neuronal tissue was prepared from Sprague-Dawley rats over a developmental time course from embryonic day 15 (El5) through 120 day adult. Regional brain areas examined included cerebral cortex, cerebellum, hippocampus, and striatum. Age of embryonic tissue was determined by vaginal smears before and after copulation; the day when sperm was present in the smear was termed day 0 (E0). Embryonic tissue was collected by anesthetizing the dam with sodium pentobarbital (45 mg/kg i.p.), isolating the embryonic rat pups by caesarean section and removing the whole fetal brain. Immediately after isolation of brain regions, the tissue was homogenized in 25 mM HEPES, pH 7.25, 0.25 M sucrose, 1 mM EDTA, 10/tM leupeptin and 1 mM PMSE Homogenates were subjected to centrifugation (1000 g for 10 rain) to yield a crude nuclear pellet (P1) and crude cytosol ($1). The S1 fraction was centrifuged at 37,000 g for 45 rain to yield a synaptosomal/membrane (P2) fraction and cytosolic ($2) fraction. Protein concentration was determined by the method of Bradford 7. Subcellular fractionation Subcellular fractionation of cerebral cortex from adult rats was completed as described by Kelly and Cotman 18. Purified cortical synaptic junctions were a generous gift from Dr. Paul Kelly (University of Texas Medical Center, Houston, TX). Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose and CaM-BPs detected as described in SDS-PAGE and protein blotting procedures. Biotinylation of calmodulin and alkaline phosphatase CaM was purified from bovine testis as previously described x9. Calmodulin and alkaline phosphatase were biotinylated using biotinyl-e-aminocaproic aeid/N-hydroxysuceiniruldeester (Pierce) at a molar ratio of 10:1 (biotin:protein) as described4,s. This protocol yields a biologically active CaM molecule with optimal sensitivity

(nanogram detection of CaM-binding proteins). Biotinylation ol alkaline phosphatase with this protocol also yields a biologically active enzyme.

RESULTS The developmental pattern of CaM-BPs was determined in n e u r o n a l tissue isolated from embryonic days 15 ( E l 5 ) , E l 7 , E l 9 , birth, postnatal day 2 (PND2), -4, -5, -7, -9, -12, -15, -20, -60 and adult (120 day). Cerebral cortex, cerebellum, striatum, and hippocampus were isolated from PND5 through adult; whole brain samples were used for fetal and P N D 0 rats. Proteins were separated by S D S - P A G E , transferred to nitrocellulose and CaM-BPs detected using biotinylated CaM. Fig. 1 shows the developmental pattern of CaM-BP expression in cerebral cortex. Panels A (synaptosomal fraction - P2) and C (cytosolic - - $2) are a Coomassie brilliant blue stain of total protein to demonstrate that equal amounts of protein were loaded in each lane. Panels B and D show a biotinylated CaM overlay detecting CaM-BPs during n e u r o n a l development. The synaptosomal (P2) blot (panel B) shows that very low levels of CaM-BPs were present in the developing fetal brain and that low levels

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Fig. 3. Two-dimensional SDS-PAGE of cerebral cortex synaptosomes (P2) during development. Proteins (200 ,ug/tube) were subjected to isoelectric focusing, 10% SDS-PAGE, transferred to nitrocellulose and CaM-BPs detected using BioCaM. This figure shows the changes in CaM-BP expression which occur during development. Panel A: molecular weight standards stained with Coomassie brilliant blue.

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Fig. 4. Hippocampal expression of CaM-BPs during neuronal development. Synaptosomal (P2) and cytosolic (52) fractions were subjected to SDS-PAGE (100 gg/lane), transferred to nitrocellulose and CaM-BPs detected with BioCaM. Hippocampus displays the earliest onset of CaM-BPs expression; several CaM-BPs can be detected by PNDS.

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Fig. 5. Developmental expression of CaM-BP in striatum. Synaptosomal (P2) and cytosolic ($2) fractions were run on 10% SDS polyacrylamide gels (100 #g/lane), transferred to nitrocellulose and CaM-BPs detected with BioCaM. CaM-BPs in striatum are detected as early as PND7.

67 performed and each fraction examined for CaM-BPs by incubation with BioCaM (Fig. 2). Crude nuclear pellet (P1) had very few CaM-BPs; a 50 kDa band was detected. However, high-speed supernatant (100,000 g S3 fraction) contained major CaM-BPs of 36, 50 and a 60-62 kDa doublet. The synaptosomal membrane fraction (P2'INT, after incubation in idonitrotetrazolium), had CaM-BPs of 36, 40, 50, 51-53, 60-62, 68, 82, and 94 kDa. This complement of proteins was also seen in adult cerebral cortex synaptosomes (see Fig. 1, panel B). The light (L2) and heavy (L3) synaptic membrane fractions expressed similar CaM-BPs. Peptides of 72-74 and 99 kDa were identified as potential avidin-binding proteins. Purified synaptic junctions (SJ) contained CaM-BPs of 36, 50, and 60-62 kDa. Two-dimensional gel electrophoresis was done to separate CaM-BPs which comigrate on one-dimensional SDS-PAGE gels. Fig. 3 shows the developmental pattern of CaM-BPs in a synaptosomal fraction. Following two-dimensional gel electrophoresis of developing cerebral cortex (PND7 and adult) and whole brain homogenate (from E15 and PND1). On days E15 (panel B) and PND1 (panel C), only a few protein spots were detected using BioCaM; these proteins migrated at 99, 97, 94, 82, 74, and 72 kDa. We were not able to identify any of these

of CaM-BPs persist through PNDS. The protein doublet at 72-74 kDa was determined to be avidin-binding proteins of unknown function; these proteins were identified by incubating an identical blot with BioCaM in the presence of EGTA (data not shown). It was not until PND7-9 that detectable levels of CaM-BPs began to appear. Synaptosomes from adult cerebral cortex had CaM-BPs of 36, 42, 50, 60-62, 68, 82, and 94 kDa. The peak expression of these proteins occurred between PND7 and -20; near adult levels were reached by PND20. The development of CaM-BPs in the cerebral cortex cytosol ($2) is shown in panel D. As in the synaptosomal fraction, the 50 and 60-62 kDa proteins were expressed between PND7 and -20; these proteins were first detected at PND4 in the cytosolic fraction. Expression of a 36 kDa protein also increased during development in this cellular fraction. Panel E shows a densitometric analysis of the developmental expression of the major 50 kDa protein of synaptosomes and cytosol. The integrated optical density increased approximately 4-fold in synaptosomes between PND5-20; the cytosolic fraction increased approximately 2-fold during this same time. Thus, most of the increase in the major 50 kDa CaM-BP occurred during times of peak synapse formation in neocortex. Subcellular fractionation of adult cerebral cortex was

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Fig. 6. CaM-BP expression in the developing cerebellum. Synaptosomal (P2) and cytosolic ($2) fractions were subjected to SDS-PAGE (100 #g/lane), transferred to nitrocellulose and CaM-BPs detected with BioCaM. Cerebellum had the latest onset of CaM-BP expression in the rat brain. Very few CaM-BPs could be detected before PND9; adult levels were reached by PND20.

68 spots as CN or CaM-PDE using afffinity-purified antibodies (data not shown). The large spots in the El5 (panel B) migrating at 99 and 72 kDa were identified as avidin-binding proteins (ABP). Two CaM-BPs of 94 and 82 kDa, seen on El5 (panel B), were lost during maturation of the nervous system; therefore, they may be developmentally regulated CaM-BPs. As the cerebral cortex developed, several dramatic changes in the pattern and intensity of expression of CaM-BPs occurred between El5 and adult. By PND7 (panel D), a more basic 72 kDa and 3 additional CaM-BPs appeared. The basic 72 kDa protein was identified as an avidin-binding protein. Spots migrating at 50 and 53 kDa were identified as CaM-BPs. As the brain developed to maturity, the expression of at least a dozen new CaM-binding spots emerged (panel E). A high molecular weight protein of 235 kDa was detected in adult synaptosomes; half a dozen high molecular weight spots (>94 kDa) and several CaM-BPs which migrated between 30 and 67 kDa were also detected. When other areas of the brain were examined, similar developmental patterns for CaM-BPs were seen. Fig. 4 shows the developmental CaM-BP pattern in the hippocampus. CaM-BP expression can be seen by PND5 and adult levels were reached by PND20. A predominant 50 kDa band was seen, as well as a protein doublet at 60-62 kDa. ABPs were identified at 67-70 and at 99 kDa. Striatum (Fig. 5) showed CaM-BP expression at slightly later times than that of hippocampus. In striatal homogenates, the 50 and 60-62 kDa proteins were detectable by PND7. The largest increase in enzyme expression occurred between PND9 and -20, a pattern similar to that of other brain regions, Cerebellum, a region that undergoes significant postnatal development in the rat, had a different pattern of CaM-BP expression. Expression of CaM-dependent proteins was not seen until PND9 (Fig. 6); adult levels were reached by PND20, characteristic of the other brain regions. A major difference in cerebellar CaM-BP pattern was the lower levels of the 50 kDa CaM-BP relative to other brain regions. The 60 kDa CaM-BP was seen by PND9 and its expression increased as the cerebellum matured. Fig. 6 also shows the presence of 58 and 62 kDa CaM-BPs in PND20, PND60 and adult cytosolic fraction ($2). DISCUSSION The developmental patterns of neuronal CaM-BPs were studied using a biotinylated derivative of CaM in combination with one- and two-dimensional gel electrophoresis. The greatest increase in CaM-BP expression occurred between PND7 and PND20; one exception was

cerebellum, where CaM-BP levels increased between PND9 and PND20. Hippocampus had the earliest onset of CaM-BPs expression, with the first significant levels of CaM-dependent proteins seen at PND5. Cerebral cortical and striatal CaM-BP expression began on PND7 and cerebellar CaM-BPs were expressed beginning at PND9. The protein at 50 kDa was tentatively identified as the a-subunit of the multifunctional CaM-kinase while the doublet at 60-62 kDa corresponded to the fl-subunit of the multifunctional CaM-kinase and the a-subunit of the CaM-dependent phosphatase, calcineurin. The identity of the 50-62 kDa CaM-BPs as CaM-kinase 1I subunits and CN catalytic subunit was supported by results of subcellular fractionation studies (Fig. 2), CaM-dependent phosphorylation assays and immunoblot analysis (Billingsley et al., submitted). Based on previous studies, 36 kDa CaM-BP in P2 fractions may be cytosynalin, a protein which has been demonstrated to be associated with synaptic vesicles29. It is also possible that this 36 kDa CaM-BP is CaM-kinase I, a protein kinase which phosphorylates site I of synapsin I23; however, this protein has not been reported to be enriched in synaptosomes. Electrophoretic mobilities and isoelectric points were used to tentatively identify the 235 and 97 kDa spots seen on the two-dimensional SDS-PAGE gels as spectrin and caldesmon, respectively, both cytoskeletal proteins. The 94, 82 and 53 kDa spots remain unidentified at this time. Affinity-purified antibodies against CN detected 4 protein spots differing in charge in adult cerebral cortex and hippocampus, suggesting that several isoforms of CN exist in the mature nervous system 6. CaM-BPs found at high levels in mature neurons, such as CN and the multifunctional CaM-kinase, were not detectable early in development. Thus, these CaM-dependent enzymes may not play important roles in early brain development. However, it is possible that developmental isoforms of CaM-BPs (panel B - - 94 and 82 kDa) are expressed only in fetal tissue, and may be important regulators of CaM-related processes in early brain development. Several CaM-BPs have been detected in the isolated growth cones, but were low in abundance when compared to adult synaptosomal CaM-BPs 15. The [1251]CAM overlay pattern of growth cones was similar to the pattern from synaptosomes isolated from adult brain and several CaM-BPs were identified as the multifunctional CaMkinase and CaM-PDE; however, immunoblot analysis for CN indicated that this protein was below detectable limits. Earlier work by Tallant et al. 31 established that CN enzymatic activity was not measurable before PND5. CN activity tripled between PND8 and -20. Our work supports the results of Tallant et al. in that CN levels were low in the fetal and early postnatal brain, and increased between PND9-20. We therefore hypoth-

69 esize that CN (61 kDa) is not detectable in embryonic brain, suggesting that other phosphatases may regulate dephosphorylation in embryonic brain. The multifunctional CaM-kinase (CaM-kinase II), identified as the major postsynaptic density protein 14, had a developmental pattern similar to that of CN in all tissues examined. Very little of this CaM-dependent kinase could be detected before PND5, and the peak expression occurred between PND5 and PND20. Kelly et al. 17 have reported a similar pattern of development for the multifunctional CaM-kinase in isolated synaptic junctions from developing nervous tissue. They recently demonstrated that the mRNA for the a- and fl-subunit of the multifunctional CaM-kinase could be detected as early as PND4, a time when virtually no kinase activity is present 8. The results of Hyman et al. 15 suggest that during development, the multifunctionai CaM-kinase may be concentrated in growth cones; by using total cellular homogenates for analysis, this protein may be diluted to below detectable levels. Developmental changes in holoenzyme composition for the multifunctional CaM-kinase have been well documented 13A7'26"27. Early in development (PND5) the holoenzyme was composed of a 3:1 ratio of fl:a subunits whereas, by PND18 the ratio became 1:1. Weinberger et al. 35 have postulated that the multifunctional CaM-kinase plays a role in synaptic maturation and that this protein may help to establish the final morphology of the synaptic junction. Development of the multifunctional calmodulin-dependent protein kinase in the cerebellum differs from other regions studied. The cerebellar holoenzyme is primarily composed of the 60 kDa fl-subunit with low levels of the ct-subunit (50 kDa) expressed 13. The BioCaM overlay of cerebellar homogenate (Fig. 5) demonstrated the lower expression of the 50 kDa a-subunit of the kinase. The 58 kDa protein seen in the PND20, PND60 and adult fractions of the cerebellum blot (Fig. 5) has been tentatively identified as the fl'-form of the multifunctional kinase described by Bennett et al. 3. The CaM-BP neuromodulin is highly concentrated in developing growth cones 22. This CaM-BP is unusual in that it has a higher affinity for CaM in the absence of calcium. Neuromodulin levels begin to decline 2 days after birth but this protein is still expressed in discrete areas of the adult nervous system; following neuronal damage, neuromodulin expression is greatly increased 1°. The role of this unique CaM-BP is unknown. It has been suggested that this protein localizes CaM within the axon and that phosphorylation decreases its affinity for CaM t. Although CN dephosphorylates neuromodulin in vitro 2~, it is unlikely, due to the low expression of CN in the embryonic and newborn brain, that CN plays an impor-

tant role in the developing growth cone. This does not exclude the possibility that in the mature nervous system, dephosphorylation of neuromodulin by CN may have a physiological role. One conclusion of this study is that the CaM-dependent enzymes in brain have a similar pattern and time course of expression. This may suggest that common regulatory elements control the expression of some of these enzymes during development. Several CaM-BPs have been cloned; analysis of genomic clones may give further insight into the regulation of these enzymes. One level of gene regulation, demonstrated for CaM-PDE and the multifunctional CaM-kinase, is the influence of transsynaptic input on the expression of these two enzymes TM. Balaban et al. have shown that destruction of the inferior olive and the climbing fibers in the cerebellum with 3-acetylpyridine selectively reduced CaM-PDE of Purkinje cells2. Wu and Black 34 provided evidence that if synaptic input into the developing dorsal root ganglia was destroyed, levels of CaM-kinase decreased. These experiments suggest that transsynaptic input is important to maintain expression of these two CaM-BPs. One model for CaM-BP expression could be related to a complex interaction between transsynaptic regulation and pre-programmed genetic expression. When little transsynaptic interaction is occurring, such as in early development, CaM-dependent enzymes may be expressed at a very low level. Upon completion of neuronal division and migration, the neuron identifies its final target. Coincident with synaptogenesis, specific neurons may begin to express CaM-BPs. This initial synapse formation may stabilize CaM-BP expression. CaM-BPs, particularly the multifunctional CaM-kinase, may then participate in the synaptic maturation phase to help define and stabilize the final morphology of the synaptic connection as proposed by Weinberger et al. 34. Our current working hypothesis is that transsynaptic input may be a critical component in the regulation of CaM-BP expression. Experiments to test this hypothesis are presently being completed. Also, we are examining the potential role of calcium/CaM as regulators of CaM-target proteins during development. More studies are needed to elucidate the interactions between synaptogenesis and programmed expression of CaM-BPs in neuronal development.

Acknowledgements. We thank Drs. Randall Kincaid, George Oyler and Kyle Krady for their help and suggestions during this work. This work was supported by a Pharmaceutical Manufacturers Association Predoctoral Fellowship to J.W.P., and research grants from the International Life Sciences Institute Research Foundation, PHS RO-AG06377, and ACS Grant IN-109J to M.L.B.

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