Chem. Senses 25: 401-406,
2000
© Oxford University Press 2000
Regional Distribution of Protein Kinases in Normal and Odor-deprived Mouse Olfactory Bulbs
Laboratory of Molecular Neurobiology, Weill Medical College of Cornell University at The Burke Medical Research Institute, White Plains, NY 10605, USA
Correspondence to be sent to: Nian Liu, Laboratory of Molecular Neurobiology, Weill Medical College of Cornell University at The Burke Medical Research Institute, 785 Mamaroneck Avenue, White Plains, NY 10605, USA. e-mail: nliu{at}burke.org
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Unilateral naris closure produced dramatic down-regulation of tyrosine hydroxylase (TH) gene expression in periglomerular dopaminergic neurons in the olfactory bulb. To explore molecular mechanisms of TH gene regulation, the present study investigated the regional distribution of protein kinase A (PKA
), protein kinase C (PKC), and CaM kinases II (CaMKII, ß) and IV (CaMKIV) in the normal olfactory bulb and in response to odor deprivation. Strong PKA immunostaining was found in the glomerular, granule cell, external plexiform and olfactory nerve layers. PKC staining was strong in granule cell and external plexiform layers but weak in the glomerular layer. Whereas CaMKIV was primarily found in granule cells, CaMKII was present in the glomerular, external plexiform, mitral cell and granule cell layers. No change in immunoreactivities of these kinases occurred in the olfactory bulb ipsilateral to naris closure. The expression of PKA, PKC and CaMKII, but not CaMKIV, in periglomerular cells suggests that these three kinases may play a role in TH gene regulation in the olfactory bulb. The lack of change in kinase protein levels after naris closure also suggests that any involvement of these kinases in TH gene expression in the olfactory bulb must be through altered kinase activity and not protein levels. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is highly expressed in periglomerular dopaminergic neurons of the mammalian olfactory bulb. TH gene expression in the olfactory bulb is regulated by afferent innervation. For example, unilateral odor deprivation or olfactory deafferentation resulted in a dramatic reduction of TH message and protein levels in the ipsilateral olfactory bulb (Brunjes et al., 1985
; Stone et al., 1990; Baker et al., 1993; Cho et al., 1996). In vitro studies have demonstrated that activation of protein kinase A (PKA; also called cAMP-dependent protein kinase) and protein kinase C (PKC) strongly induced TH gene expression in neuroblastoma cells (Carroll et al., 1991; Kim et al., 1993a,b). Analyses of TH gene revealed that the proximal 5' upstream region of the promoter contains the cis-acting elements CRE (cAMP-response element) and AP1 (activating protein 1), which are activated by the PKA and PKC signaling pathways respectively (Harrington et al., 1987; Lewis et al., 1987; Icard-Liepkalns et al., 1992; Kim et al., 1993a). Moreover, calcium/calmodulin-dependent protein kinases, including CaM kinases II (CaMKII) and IV (CaMKIV), have been shown to regulate transcription of genes containing the CRE motif in their promoters (Sheng et al., 1990, 1991; Enslen et al., 1994; Matthews et al., 1994).
Previous reports (Ouimet et al., 1984; Erondu and Kennedy, 1985; Saito et al., 1988; Ito et al., 1990; Nakamura et al., 1995) demonstrated the presence of PKC, CaMKII and CaMKIV in the rodent olfactory bulb but did not provide a detailed delineation of their distributions in this brain region. Biochemical studies (Elkabes et al., 1993) demonstrated a reduced PKC activity in the olfactory bulb ipsilateral to naris closure. Not defined were the anatomical substrates for PKC changes in the olfactory bulb and, specifically, the relevance to TH gene expression in the glomerular layer. Any change in protein level of the kinases may have significant impact on the signal transduction in neurons. For instance, monocular deprivation resulted in up-regulation of CaMKII gene expression in ocular dominance columns of the primary visual cortex originally innervated by the deprived eye (Hendry and Kennedy, 1986; Benson et al., 1991). Dark rearing also produced an increase in CaMKII gene transcription in the visual cortex (Neve and Bear, 1989). Thus, CaMKII is thought to play an important role in maintenance of the homeostasis of visual signal transduction (Neve and Bear, 1989).
To establish a role for specific protein kinases in signal processing and gene regulation in the mammalian olfactory bulb, the present study investigated the laminar and cellular distribution of PKA, PKC, CaMKII, CAMKIIß and CaMKIV in the adult mouse olfactory bulb. Specifically, the present study sought to determine the correlation between these major kinases and TH expression in periglomerular dopaminergic neurons. Also studied were changes in expression of the kinases in response to naris closure, a treatment that reduces odorant stimulation of the olfactory system and results in down-regulation of TH expression.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Animals
Adult male CD-1 mice were purchased from Charles River Breeding Laboratory (Kingston, NY) and housed under a 12:12 h light:dark cycle. Under pentobarbital anesthesia (30 mg/kg Nembutal), animals were subjected to unilateral naris closure by means of a spark-gap electrocautery. Closure was confirmed visually at the time of sacrifice and studies were carried out at least 2 months post-closure. All procedures were performed under protocols approved by the Cornell University Institutional Animal Care and Use Committee and conformed to NIH guidelines.
Materials
Rabbit anti-PKA catalytic subunit (PKA; cat. sc-903, 1:5000 dilution), rabbit anti-PKC isoform (PKC; cat. sc-208, 1:10 000), and goat anti-CaMKIV (cat. sc-1546, 1:1000) were purchased from Santa Cruz Biotechniques (Santa Cruz, CA). Mouse monoclonal anti-CaMKII subunit (CaMKII; cat. 1481703, 1:10 000) was purchased from Boehringer Mannheim (Indianapolis, IN). Mouse monoclonal anti-CaMKII ß subunit (CaMKIIß; cat. 13232012, 1:5000) was purchased from Gibco-BRL (Gaithersburg, MD). Polyclonal TH antisera (1:25 000 dilution) were prepared in the laboratory (Joh et al., 1973).
Immunocytochemistry
Mice were perfused under deep pentobarbital anesthesia with saline containing 0.5% sodium nitrite and 10 units/ml of Heparin followed by 4% buffered (0.1 M sodium phosphate, pH 7.2) formaldehyde. Brains were removed, post-fixed for 1 h and then infiltrated overnight with 30% sucrose. Frozen sections (40 µm) were obtained on a sliding microtome and incubated with specific antibodies overnight at room temperature followed by incubation with the appropriate biotinylated secondary antisera, the Vector Elite kit (Vector Laboratories; Burlingame, CA) and with 3,3'-diaminobenzidine–HCl (0.05%) and hydrogen peroxide (0.003%) as chromogen (Cho et al., 1996).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
TH immunoreactivity was localized to the glomerular layer of the mouse olfactory bulb (Figure 1A,A'). TH staining was strong in both cell bodies and processes of periglomerular neurons in the olfactory bulb with normal afferent innervation (Figure 1A'). Unilateral naris closure dramatically decreased TH immunoreactivity in the olfactory bulb ipsilateral to the closure. PKA
immunoreactivity was found in all layers of the olfactory bulb (Figure 1B,B'). Strong, often punctate, PKA staining occurred in granule and periglomerular cell somata as well as in processes within the inner part of the external plexiform layer (Figure 1B'). Mitral cell staining was weak. Ascending dendrites of granule cells were also stained (Figure 1B', inset). In contrast to TH, odor deprivation had no effect on PKA immunoreactivity in any layer of the olfactory bulb ipsilateral to naris closure.
|
Strong PKC
staining was found in the plasma membrane of granule cells and processes within the external plexiform layer (Figure 2A,A'). PKC immunoreactivity was weak in scattered periglomerular neurons and the glomerular neuropil, and absent from the mitral cell bodies as well as the olfactory nerve layer. Strong CaMKII staining occurred in the somata of periglomerular, tufted, mitral and granule cells (Figure 2B,B'). CaMKII staining was modest in the external plexiform layer, in which the apical and lateral dendrites of mitral cells were clearly seen (Figure 2B'). In contrast, CaMKII staining was very weak within the glomerular neuropil, suggesting that the processes of periglomerular cells and the apical tufts of mitral cells were weakly stained (Figure 2B'). Unilateral naris closure did not affect PKC or CaMKII immunoreactivity in the olfactory bulb (Figure 2A,B). CaMKIIß and CaMKII exhibited a similar distribution pattern in the olfactory bulb. Naris closure did not affect CaMKIIß immunoreactivity in any layer (data not shown).
|
CaMKIV was localized primarily to the nuclei of a subpopulation of granule cells in the mouse olfactory bulb (Figure 2C,C'). Ascending dendrites of granule cells were also weakly stained (Figure 2C', inset). Interestingly, CaMKIV was absent from the glomerular layer. Odor deprivation did not affect CaMKIV immunoreactivity in the olfactory bulb. CaMKIV staining in granule cells of the olfactory bulb on the naris-closed side appeared to be stronger than that on the open side. This was likely due to a higher cell-packing density in the granule cell layer. Prolonged naris closure caused shrinkage of the ipsilateral bulb, particularly the granule cell and external plexiform layers (Benson et al., 1984
). A comparison between CaMKIV and the three other kinases showed that these major protein kinases have distinct cellular and regional distribution in the mouse olfactory bulb (Table 1).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
The present study demonstrated that the investigated kinases showed distinct patterns of expression in the mouse olfactory bulb. CaMKIV immunoreactivity was limited to the granule cell layer while PKA
, PKC and CaMKII,ß exhibited a more widespread distribution. Strong PKA immunostaining occurred in the glomerular, external plexiform and granule cell layers. PKC immunoreactivity was strong in the external plexiform and granule cell layers but weak in the glomerular layer. CaMKII was highly expressed in the external plexiform, mitral and granule cell layers. CaMKII staining was strong in the somata of periglomerular cells but weak in the glomerular neuropil.
The presence of PKA, PKC and CaMKII in the periglomerular cells implied that these kinases may be involved in TH gene regulation in the olfactory bulb. This was supported by a recent report (Trocme et al., 1998) that both CRE and AP1 motifs were essential for expression of reporter genes driven by TH promoter in the adult olfactory bulb of transgenic mice. There are at least two mechanisms by which kinases may regulate gene expression. First, an increase or decrease in kinase protein levels may determine the availability of the kinases to their substrates, thus directly affecting transcription of target genes, including TH. For example, overexpression of PKA in neuroblastoma cells was shown to increase TH gene transcription (Kim et al., 1993b). Secondly, alterations in kinase activity may determine the ability of the kinases to phosphorylate substrates, thus indirectly regulating gene expression. Activation of PKA and PKC by forskolin and phorbol ester treatments, respectively, reportedly enhanced TH gene transcription in vitro (Carroll et al., 1991; Icard-Liepkalns et al., 1992). The present study demonstrated that little or no change in kinase expression occurred in the bulb ipsilateral to naris closure, suggesting that, if these kinases regulate TH expression in the olfactory bulb, it must be through altered activity and not protein levels.
CaMKII is a polymeric enzyme that is composed primarily of and ß subunits in :ß ratios of 3:1 and 1:4 in the adult rodent forebrain and cerebellum, respectively (Bennett et al., 1983; McGuinness et al., 1985). Although the two subunits differ in their molecular weights, they have similar enzyme activities and kinetic properties. CaMKII and CaMKIIß exhibited a similar distribution pattern in the olfactory bulb. CaMKIIß expression did not change in response to naris closure. A variety of isoforms also exist for PKC and PKA. The present study illustrated the regional distribution of PKA and PKC in the mouse olfactory bulb. The distribution of other isoforms of the two kinases was not examined. Therefore, the current studies do not rule out the possibility that those isoforms may be involved in the regulation of TH gene expression.
A previous report (Elkabes et al., 1993) showed that PKC activities were decreased in particulate and soluble fractions of the olfactory bulb ipsilateral to naris closure, supporting the notion that PKC is involved in down-regulation of TH gene expression in the bulb after odor deprivation. However, it was not clear if the decrease in PKC activity occurred in periglomerular cells. The present findings that PKC immunoreactivity in the periglomerular cells remained unchanged after naris closure did not rule out the possibility that a decrease in PKC activity may occur in these cells. In vitro studies (Icard-Liepkalns et al., 1992) showed that activation of PKC produced strong AP1-protein binding activity and enhanced TH gene transcription. The transcription factors c-Fos and Fos-B, which bind to the AP1 motif as Fos/Jun heterodimers, were found in periglomerular cells of the normal olfactory bulb and were significantly down-regulated by odor deprivation (Liu et al., 1999). Therefore, alterations in PKC activity in response to odorant stimulation may modulate expression of immediate early genes, thus regulating TH gene transcription through the AP1 motif.
In the olfactory bulb, CaMKIV was found only in granule cells, indicating that this kinase is unlikely to regulate TH gene expression in periglomerular dopaminergic neurons. Because CaMKIV was found to phosphorylate CRE-binding protein upon activation and enhance gene transcription (Enslen et al., 1994; Matthews et al., 1994), it is possible that CaMKIV regulates TH gene expression in other brain regions. Indeed, CaMKIV immunoreactivity has been found in noradrenergic neurons in the locus ceruleus, but not in dopaminergic neurons in the substantia nigra (unpublished data). Further studies will determine if CaMKIV plays a major role in the regulation of TH expression in the locus ceruleus.
|
Conclusions |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
The variations in laminar and cellular distribution of the protein kinases in the mouse olfactory bulb suggest that they participate in different aspects of olfactory signal processing and gene regulation. The expression of PKA
, PKC and CaMKII,ß, but not CaMKIV, in the periglomerular cells suggests that these three major kinases might play a role in regulating TH gene expression in the olfactory bulb in response to odorant stimulation. However, the lack of change in protein levels of the kinases in the glomerular layer of the olfactory bulb ipsilateral to naris closure indicates that odor-induced neuronal activity does not modulate the kinase gene expression and that any role in TH gene regulation may be indirect through changes in kinase activity.
|
Acknowledgments |
|---|
This research was supported by NIH Grant AG09686. The author thanks Dr Harriet Baker for her critical reading of the manuscript.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Baker, H., Morel, K., Stone, D.M. and Maruniak, J.A. (1993) Adult naris closure profoundly reduces tyrosine hydroxylase expression in mouse olfactory bulb. Brain Res., 614, 109–116.[Web of Science][Medline]
Bennett, M.K., Erondu, N.E. and Kennedy, M.B. (1983) Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain. J. Biol. Chem., 258, 12735–12744.
Benson, D.L., Isackson, P.J., Gall, C.M. and Jones, E.G. (1991) Differential effects of monocular deprivation on glutamic acid decarboxylase and type II calcium–calmodulin-dependent protein kinase gene expression in the adult monkey visual cortex. J. Neurosci., 11, 31–47.[Abstract]
Benson, T.E., Ryugo, D.K. and Hinds, J.W. (1984) Effects of sensory deprivation on the developing mouse olfactory system: a light and electron microscopic, morphometric analysis. J. Neurosci., 4, 638–653.[Abstract]
Brunjes, P.C., Smith-Crafts, L.K. and McCarty, R. (1985) Unilateral odor deprivation: effects on the development of olfactory bulb catecholamines and behavior. Devl Brain Res., 22, 1–6.
Carroll, J.M., Kim, K.S., Kim, K.T., Goodman, H.M. and Joh, T.H. (1991) Effects of second messenger system activation on functional expression of tyrosine hydroxylase fusion gene constructs in neuronal and nonneuronal cells. J. Mol. Neurosci., 3, 65–74.[Web of Science][Medline]
Cho, J.Y., Min, N., Franzen, L. and Baker, H. (1996) Rapid down-regulation of tyrosine hydroxylase expression in the olfactory bulb of naris-occluded adult rats. J. Comp. Neurol., 369, 264–276.[Web of Science][Medline]
Elkabes, S., Cherry, J.A., Schoups, A.A. and Black, I.B. (1993) Regulation of protein kinase C activity by sensory deprivation in the olfactory and visual systems. J. Neurochem., 60, 1835–1842.[Web of Science][Medline]
Enslen, H., Sun, P., Brickey, D., Soderling, S.H., Klamo, E. and Soderling, T.R. (1994) Characterization of Ca2+/calmodulin-dependent protein kinase IV. Role in transcriptional regulation. J. Biol. Chem., 269, 15520–15527.
Erondu, N.E. and Kennedy, M.B. (1985) Regional distribution of type II Ca2+/calmodulin-dependent protein kinase in rat brain. J. Neurosci., 5, 3270–3277.[Abstract]
Harrington, C.A., Lewis, E.J., Krzemien, D. and Chikaraishi, D.M. (1987) Identification and cell type specificity of tyrosine hydroxylase gene promoter. Nucleic Acids Res., 15, 2363–2384.
Hendry, S.H.C. and Kennedy, M.B. (1986) Immunoreactivity for a calmodulin-dependent protein kinase is selectively increased in macaque striate cortex after monocular deprivation. Proc. Natl Acad. Sci. USA, 83, 1536–1540.
Icard-Liepkalns, C., Biguet, N.F., Vyas, S., Robert, J.J., Sassone-Corsi, P. and Mallet, J. (1992) AP-1 complex and c-fos transcription are involved in TPA provoked and trans-synaptic inductions of the tyrosine hydroxylase gene: insights into long-term regulatory mechanisms. J. Neurosci. Res., 32, 290–298.[Web of Science][Medline]
Ito, A., Saito, N., Hirata, M., Kose, A., Tsujino, T., Yoshihara, C., Ogita, K., Kishimoto, A., Nishizuka, Y. and Tanaka, C. (1990) Immunocytochemical localization of the alpha subspecies of protein kinase C in rat brain. Proc. Natl Acad. Sci. USA, 87, 3195–3199.
Joh, T.H., Geghman, C. and Reis, D.J. (1973) Immunochemical demonstration of increased accumulation of TH protein in sympathetic ganglia and adrenal medulla elicited by reserpine. J. Neurochem., 39, 342–348.[Web of Science][Medline]
Kim, K.-S., Lee, M.K., Carroll, J. and Joh, T.H. (1993a) Both the basal and inducible transcription of the tyrosine hydroxylase gene are dependent upon a cAMP response element. J . Biol. Chem., 268, 15689–15695.
Kim, K.-S., Park, D.H., Wessel, T.C., Song, B., Wagner, J.A. and Joh, T.H. (1993b) A dual role for the cAMP-dependent protein kinase in tyrosine hydroxylase gene expression. Proc. Natl Acad. Sci. USA, 90, 3471–3475.
Lewis, E.J., Harrington, C.A. and Chikaraishi, D.M. (1987) Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP. Proc. Natl Acad. Sci. USA, 84, 3550–3554.
Liu, N., Cigola, E., Tinti, C., Jin, B.K., Conti, B., Volpe, B.T. and Baker, H. (1999) Unique regulation of immediate early gene and tyrosine hydroxylase expression in the odor-deprived mouse olfactory bulb. J. Biol. Chem., 274, 3042–3047.
Matthews, R.P., Guthrie, C.R., Wailes, L.M., Zhao, X., Means, A.R. and McKnight, G.S. (1994) Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression. Mol. Cell. Biol., 14, 6107–6116.
McGuinness, T.L., Lai, Y. and Greengard, P. (1985) Ca2+/calmodulin-dependent protein kinase II. Isozymic forms from rat forebrain and cerebellum. J. Biol. Chem., 260, 1696–1704.
Nakamura, Y., Okuno, S., Sato, F. and Fujisawa, H. (1995) An immunohistochemical study of Ca2+/calmodulin-dependent protein kinase IV in the rat central nervous system: light and electron microscopic observations. Neuroscience, 68, 181–194.[Web of Science][Medline]
Neve, R.L. and Bear, M.F. (1989) Visual experience regulates gene expression in the developing striate cortex. Proc. Natl Acad. Sci. USA, 86, 4781–4784.
Ouimet, C.C., McGuinness, T.L. and Greengard, P. (1984) Immunocytochemical localization of calcium/calmodulin-dependent protein kinase II in rat brain. Proc. Natl Acad. Sci. USA, 81, 5604–5608.
Saito, N., Kikkawa, U., Nishizuka, Y. and Tanaka, C. (1988) Distribution of protein kinase C-like immunoreactive neurons in rat brain. J. Neurosci., 8, 369–382.[Abstract]
Sheng, M., McFadden, G. and Greenberg, M.E. (1990) Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB. Neuron, 4, 571–582.[Web of Science][Medline]
Sheng, M., Thompson, M.A. and Greenberg, M.E. (1991) CREB: a Ca2+-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science, 252, 1427–1430.
Stone, D.M., Wessel, T., Joh, T.H. and Baker, H. (1990) Decrease in tyrosine hydroxylase, but not aromatic L-amino acid decarboxylase, messenger RNA in rat olfactory bulb following neonatal, unilateral odor deprivation. Mol. Brain Res., 8, 291–300.[Medline]
Trocme, C., Sarkis, C., Hermel, J.-M., Duchateau, R., Harrison, S., Simonneau, M., Al-Shawi, R. and Mallet, J. (1998) CRE and TRE sequences of the rat tyrosine hydroxylase promoter are required for TH basal expression in adult mice but not in the embryo. Eur. J. Neurosci., 10, 508–521.[Web of Science][Medline]
Accepted February 14, 2000
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Baker, N. Liu, H. S. Chun, S. Saino, R. Berlin, B. Volpe, and J. H. Son Phenotypic Differentiation during Migration of Dopaminergic Progenitor Cells to the Olfactory Bulb J. Neurosci., November 1, 2001; 21(21): 8505 - 8513. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||