Differential calmodulin gene expression in the rodent brain

Life Sciences 70 (2002) 2829 – 2855

Minireview

Differential calmodulin gene expression in the rodent brain Arpad Palfi, Elod Kortvely, Eva Fekete, Beatrix Kovacs, Szilvia Varszegi, Karoly Gulya* Department of Zoology and Cell Biology, University of Szeged, 2 Egyetem St., POB 659, Szeged H-6722, Hungary Received 20 December 2001; accepted 31 January 2002

Abstract Apparently redundant members of the calmodulin (CaM) gene family encode for the same amino acid sequence. CaM, a ubiquitous cytoplasmic calcium ion receptor, regulates the function of a variety of target molecules even in a single cell. Maintenance of the fidelity of the active CaM-target interactions in different compartments of the cell requires a rather complex control of the total cellular CaM pool comprising multiple levels of regulatory circuits. Among these mechanisms, it has long been proposed that a multigene family maximizes the regulatory potentials at the level of the gene expression. CaM genes are expressed at a particularly profound level in the mammalian central nervous system (CNS), especially in the highly polarized neurons. Thus, in the search for clear evidence of the suggested differential expression of the CaM genes, much of the research has been focused on the elements of the CNS. This review aims to give a comprehensive survey on the current understanding of this field at the level of the regulation of CaM mRNA transcription and distribution in the rodent brain. The results indicate that the CaM genes are indeed expressed in a gene-specific manner in the developing and adult brain under physiological conditions. To establish local CaM pools in distant intracellular compartments (dendrites and glial processes), local protein synthesis from differentially targeted mRNAs is also employed. Moreover, the CaM genes are controlled in a unique, gene-specific fashion when responding to certain external stimuli. Additionally, putative regulatory elements have been identified on the CaM genes and mRNAs. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Calmodulin; Gene expression; Rodent; Brain; mRNA

* Corresponding author. Tel./fax: +36-62-544049. E-mail address: [email protected] (K. Gulya). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 4 4 - 8

2830

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

Calmodulin is a ubiquitous intracellular signalling molecule Although calmodulin (CaM), a multifunctional, highly conserved calcium ion (Ca2+) sensor protein, exists as an identical amino acid sequence in species ranging from fish to human [1–3], it is encoded by a multigene family in vertebrates. Three non-allelic bona fide members of the CaM gene family have been described in mammals, e.g. in the mouse [4–8], rat [9–14] and human [15–20]. The three CaM genes transcribe altogether seven major mRNA species by means of alternative polyadenylation. In the rat, for example, these are as follows: three species for CaM I: 4.2 kb, 1.7 kb and 1.0 kb; one species for CaM II: 1.4 kb; and three species for CaM III: 2.3 kb, 1.9 kb and 0.9 kb [13,21,22]. At almost the theoretical limits of the degeneracy of the genetic code, the coding regions of the CaM genes are still 80–85% identical to each other in the rat or human [13,20]. On the other hand, in the non-coding regions, there are no significant sequence similarities between the three CaM genes within a species (Fig. 1A; [20]). However, a comparison of the non-coding regions across species reveals a noteworthy correspondence, implying conserved functions for them (Fig. 1B). Thus, the structural features of the CaM genes suggest that their redundancy is apparent and the members of the CaM gene family were indeed selected for and remained fixed in the vertebrate lineage [23,24]. To variable extents, CaM is expressed in all eukaryotic cells [1,3,25], participating in signalling pathways that regulate many crucial cellular processes, such as cell division or movement [1,26–32]. Lacking its own enzymatic activity, CaM functions by regulating a number of target proteins, most of which are enzymes [1,26–32]. The presence of many of these catalytic activities in the same cell, often with clearly opposing effects, obviously demands a careful cytoplasmic control and separation of the active CaM effectors. In order to maintain the fidelity of appropriate CaM-target interactions spatially and temporally, cells utilize CaM in subcellular ‘‘microdomains’’ formed by a very precise adjustment of multiple regulatory events, including transient Ca2+ signals, reversible storage of CaM (by binding to membranes or storage proteins, such as GAP-43), masking (e.g. by phosphorylation) and redistribution of CaM pools to certain intracellular sites, de novo CaM synthesis and control of target availability [24]. Thus, the regulation of CaM is more complex than that of most other proteins in the cell. It is reasonable to propose that, among the above control mechanisms, a multigene family is necessary to maximize the regulatory potentials at the level of the CaM gene expression. CaM is particularly abundant in the mammalian central nervous system (CNS; [33–35]). In many ways, the brain is exceptionally amenable to study of the different aspects relating to the expression of the CaM genes, and also the function of the protein itself, for the following reasons: 1) The actual levels of both CaM and its mRNAs are 5–15 times higher in the nervous tissue than in most other tissues [21,36]. 2) The cells in the CNS have traditionally been classified into characterized types, upon the basis of their exclusive morphology and, more recently, their physiology, biochemistry and molecular facets. 3) The highly polarized process-bearing cells of the CNS (i.e. neurons and glial cells) present an ideal model for study of the functional characteristics of subcellular compartmentalization. 4) The vast majority of CaM in the CNS is expressed by neurons [35,37–39], where, beside its fundamental

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2831

Fig. 1. Sequence comparison of the 50-UTRs of the CaM genes. A) Alignment of the cDNA sequences corresponding to the 50-UTRs of the three rat CaM genes. The 50-UTRs of the three CaM genes are not significantly conserved within a mammalian species (here in the rat). Sequence accession numbers are X13931, X13833 and X14265 for CaM I, II and III, respectively. B) Alignment of the cDNA sequences corresponding to the 50-UTRs of the CaM II genes from different mammalian species. There is a striking sequence correspondence among the 50-UTRs of the CaM II genes of the mouse, rat, dog and human. Sequence accession numbers are D12623, X13833, D12622 and U44757 for the mouse, rat, dog and human, respectively. Sequence alignment was performed by using the GCG computer program [183] and its extension package [184].

housekeeping actions, CaM is also involved in specialized neuronal functions, such as the synthesis and release of neurotransmitters, neurite extension, long-term potentiation (LTP) and axonal transport [26,40–44]. Some of these activities can be directly associated with

2832

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

individual neuronal subcellular compartments, such as the soma, dendrites, axons and axon terminals. 5) Although the glial CaM expression is more restricted and lower as compared to the neurons, particular glial cells may display significant levels of the protein under certain conditions [37,45–47]. Glial cells also elaborate discrete subcellular compartments, such as myelin sheets or astroglial processes and end feet. 6) In particular, the neurons are often organized into highly ordered layers of certain cell types (e.g. the pyramidal cell layer in the hippocampus), allowing even their gross measurement. These considerations have led to much of the CaM research, both in vivo and in vitro, being focused on the elements of the CNS. In a search for definite evidence of the differential utilization of the three CaM genes, most of the work in our laboratory has recently gone into describing the localization, distribution and regulation of the CaM mRNA content under both physiological and experimental conditions in the rat nervous tissue. This review aims to give a comprehensive survey of the current understanding, the developments and future prospects in this field.

CaM genes are heavily transcribed in the brain Under physiological conditions, wide expressions of the three CaM genes have been described by several authors in the adult rodent brain [21,38,39,48–51]. Strong expression can be detected in the principal neurons of the CNS, e.g. the mitral cells of the olfactory bulb, the cortical and hippocampal pyramidal cells, the hypothalamic magnocellular neurosecretory cells, the Purkinje cells, the cells of the deep cerebellar nuclei, the motor neurons of the ventral horn in the spinal cord, and in general in the large neurons of the cerebral cortex, the midbrain, the brainstem and the spinal cord. The expressions of the three CaM genes are less intensive in small interneurons and are undetectable in most glial cells. CaM mRNA levels are much lower in areas that are poor in neuronal cell bodies, such as the molecular layers of the hippocampus, or the cerebral and the cerebellar cortices. Moreover, hybridization signal intensities are minimal in white matter structures, such as the corpus callosum, the cerebellar white matter or the internal capsule. The above data suggest that the CaM expression in most glial cells is at least an order of magnitude lower than that detected in neurons. Nevertheless, Palfi et al. [39] described considerable mRNA levels for all CaM genes in the choroid plexus and ependyma. Moreover, a strong CaM immunoreactivity was detected in reactive microglial cells in the hippocampus of kainic acid-treated mice [46]. Recently, Kovacs and Gulya [52] reported the presence of CaM I mRNA-positive small and medium-sized glial cells in the white matter of the adult rat spinal cord (Fig. 2).

CaM genes exhibit unique expressional patterns under physiological conditions Early studies suggested a coordinated expression pattern for the three CaM genes in the brain [21,22]. However, more extensive research revealed that they are actually transcribed in a gene-specific manner in both the mouse [38] and the rat [39]. A markedly differential expression was described, e.g. in the olfactory bulb, the basal ganglia, the hippocampus-dentate

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2833

Fig. 2. Glial CaM I mRNA expression in the white matter of the rat spinal cord. The adult rat spinal cord was fixed by transcardial perfusion. Twenty mm-thick cryostat sections were hybridized at low alkaline pH with a DIGlabelled RNA probe specific for the rat CaM I mRNAs. Hybridized RNA probes were visualized by using the NBT/BCIP detection system according to the manufacturer’s instructions (Boehringer-Mannheim GmbH, Germany). CaM I mRNAs are heavily expressed in medium-sized, astrocyte-like cells (arrows) and much smaller oligodendrocyte-like cells (arrowheads) in the lateral column (lateral funiculus) of the rat lumbar spinal cord white matter. Some of the larger processes of these cells are also labelled. Scale bar: 100 mm.

gyrus complex, some of the hypothalamic nuclei and the cerebellar cortex. In order to determine the CaM mRNA levels precisely in the brain, we have recently paid much attention to developing reliable methods for the quantification of mRNAs by in situ hybridization (ISH; [53–55]). The quantitative assessment is highly accurate when the signal intensities and the corresponding mRNA copy numbers are calculated for the same probe. On the other hand, when different probes are used (i.e. the amounts of different mRNA species are measured), the absolute relations of the quantities of the hybridized targets are influenced by additional ambiguous factors, the unique kinetic characteristics of each probe. Consequently, although it is generally accepted that their gross amounts are similar in magnitude in the brain [21,38,39,51], the absolute ratios of the mRNAs corresponding to the three CaM genes in different brain areas are still to be determined. In the rat pheochromocytoma cell line (PC12), the relative abundances of the CaM mRNAs are 1.7 kb (CaM I) > 1.4 kb (CaM II) > 2.3 kb (CaM III) > 4.2 kb (CaM I) > 1.0 kb species (CaM I and CaM III; [56]). Although both CaM I and CaM III genes are transcribed into three alternatively polyadenylated mRNA species, the ISH studies described above utilized only gene-specific probes which cannot differentiate between the various transcripts. The results of Northern analyses [21,57] provide an insight into the transcript-specific distribution of these CaM mRNAs. Unique patterns of the different mRNA species in various tissues and even gross brain parts, such as the cerebrum, the cerebellum, the brainstem and the spinal cord seem to emerge. For example, the ratios of the 4.2 kb versus the 1.7 kb CaM I mRNAs are 1.5, 1.5,

2834

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2.4 and 0.9 in the cerebrum, the cerebellum, the spinal cord and the testis, respectively [21]. Sensitive methods, such as the reverse transcription polymerase chain reaction (RT-PCR) or in situ PCR [58] should provide more detailed information on the transcript-specific distribution of the heterogeneous CaM mRNAs in the CNS. Nevertheless, the foregoing results advocate that alternative polyadenylation may provide means for differential spatial (and perhaps temporal) localization of the CaM mRNAs. 50-flanking sequences of the CaM genes have been identified The promoter-regulatory sequences corresponding to the three rat [13] and the three human CaM genes have been isolated and characterized [16,17,19]. The sequence of the promoter region of the CaM III gene suggests that the CaM III gene belongs among the housekeeping genes ensuring a strong and continuous basal transcription, albeit its expression may also be specifically regulated. On the other hand, analysis of the 50-flanking sequences of the CaM I and II genes revealed several putative regulatory elements, suggesting that these genes might be the primary targets for the regulated CaM gene expression [17,19]. However, it is not clear which of these regulatory elements (and other, so far undefined sequences) determine the generally similar tissue-and cell type-specific expressions of the three CaM genes, and which of them contributes to the assignment of the differential, gene-specific expression profiles. Fusion genes of the CaM II promoter segment from 294 to +68 bases and the b-galactosidase reporter gene [7] or the CaM III promoter segment from 877 to +103 bases and the lacZ reporter gene have been produced [59]. These constructs exhibit neuron-specific and more or less CaM gene-specific expression in transgenic mice. Nevertheless, unambiguous anomalies between the expression of transgenes and their endogenous CaM counterparts (e.g. the expression of the CaM III transgene was not observed in the external germinal cells of the developing cerebellum) indicate that additional elements, situated more distantly from the near vicinity of the 50-flanking regions, must also participate in the complete regulatory process.

CaM protein and its mRNAs are broadly colocalized in the brain Immunocytochemical analyses in the adult rodent brain demonstrate that CaM immunoreactivity is widely distributed and predominantly localized in grey matter structures [37,38,47,60,61]. In general, a strong reaction has been found in neuronal cell somata (especially in the cell nucleus) and neuritic processes throughout the brain, e.g. the cerebral cortex, the striatum, the hippocampus, the septum, the thalamus, the cerebellum and the brainstem nuclei. However, not all neurons exhibit the same degree of immunoreactivity. For example, significant variations are observed between the various cortical neurons [38] or, in contrast with the prominent staining of granule cells of the hippocampus, a very light staining is detected in the granule cells of the cerebellum, and many granule cells appear completely unstained [37]. The molecular layers of the cerebellum and hippocampus exhibit light staining, except where immunopositive dendritic processes are visible. Occasionally, glial cells are also labelled in the

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2835

hippocampal molecular layer [37,45,46]. In white matter structures, such as the corpus callosum or the cerebellar white matter, CaM immunoreactivity is restricted to the fibres. Comparison of these data with the results of ISH studies [38,39,47] indicates that the distributions of the CaM protein and mRNAs are parallel in the CNS. However, the above studies used various anti-CaM antibodies (possibly with different recognition properties) and different immunochytochemical protocols. Accordingly, slightly different CaM-immunolabelling patterns might be due to technical circumstances.

CaM is differentially targeted to subcellular compartments in both neuronal and glial cells At the subcellular level, CaM immunostaining is localized in the cell nucleus, as well as in the cytoplasm and cellular processes [37,38,47,61,62]. In general, the immunostaining is characterized by a distinctive granular appearance. In the neurons, the immunoreactivity is particularly intense in the cell nucleus and in the dendrites, the cytoplasm of the cell body is more lightly stained than the nucleus, and light immunoreactivity can also be found in the axons. Inside the nucleus, much less immunoreactivity is present in the nucleolus. Electron microscopic analysis confirmed the association of CaM with the nuclear chromatin, while the nucleolus remained unstained [37]. The reaction product was also detected overlying the membranes of several organelles, in postsynaptic densities and decorating both dendritic and axonal microtubules. In terms of glial expression, high levels of CaM were observed in the nuclei of glial cells [63]. Moreover, in certain astrocytes and reactive microglial cells, where CaM is readily detectable by immunocytochemistry, CaM is localized not only in the cell bodies, but in the glial processes as well [37,38,45–47]. The translocation of CaM at the protein level from the surrounding cytoplasm to the cell nucleus, and the regulation and function of the nuclear CaM pool, is an area of current interest [31,64,65]. Outside the nucleus, the other possible mechanism of protein delivery is by targeting its mRNA(s), rather than the protein itself, to specific intracellular compartments and ultimately translating it at the distant sites. It has become clear in the past decade that cytoplasmic mRNA transport contributes significantly to the establishment of localized protein pools in polarized cells. There is accumulating evidence that CaM mRNA targeting also takes place in various cells of the CNS.

CaM mRNAs are differentially localized in neuronal compartments A number of mRNA species (their number is currently estimated to be a few hundred) have been demonstrated to be targeted to the dendritic compartment in mammalian neurons (for reviews, see [66–73]). The activity-regulated cytoskeleton-associated protein (Arc) mRNA [74] has recently exemplified even activity-dependent mRNA trafficking. Abundant evidence indicates that the mRNAs found in dendrites are translated there: 1) the mRNA species translocate as part of a large macromolecular complex, the RNA granule, in which many

2836

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

components of the translational unit have been detected [75]. 2) Ribosomes and even polysomes are readily detected at the base of dendritic spines [76–78]. 3) Other components required for translation (tRNAs, initiation/elongation factors, etc.) are also present [79–82]. 4) There are reticular structures that may function in glycoprotein and membrane protein synthesis [83–85]. 5) Local protein synthesis was evaluated in a cell culture system which permits the isolation of living dendrites [86]. 6) Through the use of single dendrite transfection, local protein synthesis was also directly shown to occur in dendrites and growth cones [87,88]. It is noteworthy that transcriptional factors are also synthesized within dendrites, providing a direct signalling pathway between the distal dendrite and the nucleus [89–91]. CaM mRNAs are heavily distributed in the neuronal cell somata, while the cell nuclei remain unlabelled [62]. Zhang et al. [92] reported the initial evidence that the mRNAs of the CaM gene family are dendritically targeted in PC12 cells. Transcripts of the CaM I and CaM II genes were found within neurite extensions, whereas CaM III mRNAs predominated in the cell body. Berry and Brown [62] detected a transient distribution of the 4.2 kb CaM I mRNA in the apical dendrites of cortical, hippocampal and Purkinje neurons during early development (postnatal days 5 – 20) in the rat. They recommended functional significance for their observation as the brief dendritic localization coincided with the synaptic formation of these cells [93–95]. Palfi et al. [39] demonstrated in their quantitative experiments that CaM mRNAs are significantly more abundant in the molecular layers of the hippocampus and the cerebral and cerebellar cortices and the external plexiform layer of the olfactory bulb (consisting mainly of dendrites) than in the white matter areas (containing mostly axonal tracts) in the adult rat brain. The mRNA concentrations were consistently different; in all these areas, the highest level was revealed for that of the CaM I gene, and the lowest for that of the CaM II gene. These results suggest that the CaM mRNAs are localized dendritically even in the adult brain, and their targeting is gene-specific. Recently, we also demonstrated the presence of CaM mRNAs in dendrites of adult neurons by electron microscopic ISH (Fig. 3; previously unpublished data; manuscript in preparation). Furthermore, strong CaM I and CaM II gene expressions were determined in the striatal GABAergic cell line M26-1F [96]. Both CaM I and CaM II mRNAs were detected not only in the cell bodies, but also in the neurites of these cells. Most recently, Kortvely et al. [97] described a specific developmental pattern of dendritic CaM mRNA distribution in the rat brain. The molecular layers of the hippocampus and the cerebral cortex contained marked levels of all three CaM mRNAs on postnatal days 1–5. For example, the mRNA levels in cortical layer 1 as compared to layers 2–6 were 25%, 51% and 32% for CaM I, CaM II and CaM III mRNAs, respectively. Later in the development (postnatal days 5–20), the mRNA levels decreased more steeply for the CaM II and CaM III genes than for the CaM I gene, and by postnatal day 20 the expression patterns were similar to those observed in the adult rat brain (CaM I > CaM III > CaM II; [39]). Similar changes, but with different timing, were observed in the cerebellar molecular layer [97]. Interestingly, therefore, during early development the dendritic mRNA pool is richest in the CaM II mRNA, which becomes the least targeted species by adulthood. A prominent dendritic localization of CaM I mRNAs is obvious in primary cultures of hippocampal pyramidal neurons (Fig. 4; previously unpublished results).

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2837

Fig. 3. Electron microscopic detection of CaM I mRNAs in the adult rat hippocampus. An adult rat brain was fixed by transcardial perfusion. Fifty mm-thick free-floating vibratome sections at the level of the dorsal hippocampus were hybridized with a DIG-labelled RNA probe specific for the rat CaM I mRNAs. Hybridized probes were detected by incubating the sections with anti-DIG-immunogold conjugate (10 nm gold particle size, TAAB, UK). Nanogold particles are seen over the dendrite (d) and the perisynaptic region, mostly in astrocytic (a) processes. Original magnification: 8400.

On the other hand, various mRNAs have also been recognized to be directed towards axons and axon terminals (for reviews, see [72,73]). However, because of the lack of proteinsynthesizing ability of mammalian axons, the functional role of these mRNAs remains obscure. The presence of CaM mRNAs has not been reported in axons. In general, CaM mRNA levels are very low in the white matter areas. Putative regulatory elements have been identified in the 30-untranslated regions (UTRs) of the CaM mRNAs Although not coding for the actual protein sequence, the UTRs control diverse functions of eukaryotic mRNAs, such as their stability, translation efficiency, cytoplasmic localization and coding capacity [66,98]. To date, most control elements have been identified in the 30-UTR, although the regulatory role of the 50-UTR and even the coding region has also been implicated. For example, a trinucleotide repeat in the 50-UTR of the human CaM I gene is required for full expression [99]. In silico analysis of the rat CaM mRNA sequences reveals several putative cis-acting elements in the 30-, but not the 50-UTRs, identical to or resembling

2838

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

Fig. 4. Dendritic targeting of CaM I mRNAs in primary hippocampal cell culture. Rat hippocampal cells were cultured according to Brewer et al. [159] and Banker and Cowan [185]. On the tenth day, the cells were fixed and processed for ISH with a DIG-labelled RNA probe specific for the rat CaM I mRNAs. Hybridized RNA probes were visualized by use of the NBT/BCIP detection system according to the manufacturer’s instructions (Boehringer-Mannheim GmbH, Germany). Cell nuclei were counterstained with haematoxylin. A) The cytoplasms of the pyramidal cells are heavily labeled, whereas the nuclei are not labeled by ISH. CaM I mRNAs are also present in the neuronal processes. Note that some neurons are only faintly labeled and the haematoxylin-stained nuclei of several unlabeled (probably glial) cells are also present. B) Higher magnification of the neuron labeled with an asterisk in A. Arrows indicate neuronal processes with high CaM I mRNA contents. Scale bars indicate 50 mm.

those described in other mRNA species (Fig. 5; data previously unpublished or from [100,101]). Almost all eukaryotic mRNAs receive a polyadenylate (poly(A)) tail at their 30 end after their synthesis in the cell nucleus [102]. The most common signal defining the 30 cleavage and poly(A) tail processing site of the mRNA precursor, the hexameric AAUAAA sequence, is about 15 nucleotides upstream of the actual cleavage site. A single gene may have several distinct polyadenylation sites, resulting in 30 end heterogeneity among its transcripts [103,104]. The choice of the polyadenylation sites influences the properties of the mRNA by either including or excluding regulatory elements in the 30-UTR. Moreover, the length of the poly(A) tail itself is able to influence the translational efficiency or the half-life of the mRNA, for instance. Differential polyadenylation plays an essential role in the tissue-, developmental stage- or disease-specific expression patterns [105–109]. As mentioned before, the CaM I and CaM III genes (but not the CaM II gene) also make use of alternative polyadenylation, as both are transcribed into three mRNA species (Fig. 5). Both the CaM I and CaM III genes contain tandem arrays of polyadenylation sites, e.g. the CaM I gene is characterized by two AAUAAA and two AUUAAA sites (the latter is a less frequent processing site variant; [49]). Interestingly, the shortest transcript of the CaM III gene is polyadenylated after the non-canonical (rare) GAUAAA signal, which occurs in only 1.3% of

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2839

Fig. 5. Schematic representation and Northern blotting of the multiple rat CaM transcripts. Solid disc: 50-cap structure, solid box: coding region, solid square: DICE, open triangle: AUUUA destabilization element, solid triangle: UUAUUA U/A U/A destabilization element, ZIP: zip signal, CPE: UUUUUAU, general cytoplasmic polyadenylation element, cpe: UUUUAU, minimal cytoplasmic polyadenylation element. Polyadenylation signals are marked with their corresponding signal sequences. The most distal polyadenylation site of the 2.3 kb CaM III transcript has not yet been identified in the rat, although it is known in the human [18]. The accession numbers of sequences used to generate these maps are X13931, X13933, AF178845, AF176375, AF176375, X13833, X14265, X13817 and AF231407. The compilation is based on our previously unpublished data and the results of Pesole et al. [100] and Dalphin et al. [101]. Insert: Northern blot analysis of the CaM mRNAs in total RNA samples prepared from the adult rat cerebral cortex. I: CaM I, II: CaM II, III: CaM III, M: RNA markers.

the human genes [12,104]. An additional polyadenylation signal (AACAAA) is also found just two nucleotides downstream from this element; it is reported to function in only a few genes [110,111]. The two signals possibly act in a combined fashion. Both the rat and human CaM genes utilize the same polyadenylation signals at the same positions, suggesting their conserved function. Differential polyadenylation of CaM mRNAs under various (patho)physiological conditions has been reported. For example, the 2.3 kb CaM III transcript clearly

2840

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

predominates in the rat cerebrum as 76% of the total CaM III mRNA pool is comprised of this species, while in the testis the corresponding ratio is only 35% [21]. Moreover, cAMP selectively allocates the polyadenylation site preferences of certain CaM transcripts in PC12 cells [112]. Developmental stage-specific alternative polyadenylation of the CaM transcripts has also been described during spermatogenesis [113]. The stability of the mRNAs in the cytoplasm varies from several minutes to several days, and thereby plays a particularly important role in the post-transcriptional regulation [114]. Among other mechanisms, the degradation of mRNAs can be initiated by deadenylation [115]. Specific cis-acting sequences and their cognate trans-acting factors often affect the size of the poly(A) tract. For example, A- and U-rich elements (AREs) are found in the 30-UTRs of many highly unstable mRNAs often clustered within 100 nucleotides upstream of the polyadenylation sites [103]. Both rat and human 4.2 kb CaM I transcripts contain 13 AREs, two of which are also present in the 1.7 kb species. Most of the AREs are found at the same position of the corresponding rat and human CaM I mRNAs [18,116], further emphasizing that they are functional components of some regulatory pathway(s) selectively controlling the half-lives of these mRNAs. Moreover, certain AREs appear to be critical parts of some cytoplasmic mRNA localization signals (see below and [117]). Although 30 processing is generally a nuclear reaction, the cytoplasmic extension of the poly(A) tail of different mRNAs has also been described [118–120]. This readenylation requires another U-rich signal sequence, lying upstream of the polyadenylation signal and termed the cytoplasmic polyadenylation element (CPE). The CPE-binding protein (the transacting factor of CPE) is associated with the postsynaptic density in neurons; CPEs within the 30-UTR of the Ca2+/CaM-dependent protein kinase II (CaM-KII) mRNA have been found to influence the efficiency of dendritic translation and thereby even synaptic plasticity [118,121]. The 30-UTRs of several rat CaM transcripts also contain CPEs, as do their human counterparts (Fig. 5). Translational control can be operated in a poly(A) tail-dependent manner, involving interaction with the poly(A)-binding protein [122], or in a poly(A) tail-independent manner. An example of the latter is reticulocyte 15-lipoxygenase (LOX) mRNA, where 30-UTR differentiation control elements (DICEs) are recognized by trans-acting factors and the resulting complex then inhibits the initiation pathway of the translation [123,124]. The CaM I and CaM III transcripts also contain DICEs. Each rat CaM I mRNA has a single DICE in the proximal 30-UTR, while the corresponding human sequence contains three. The 2.3 kb CaM III transcripts possess four DICEs in both the rat and the human. Since functional DICEs should consist of at least two, almost overlapping repeats of DICEs [123,124], the physiological significance of these sequences in the CaM transcripts is not clear. Additionally, most rat and human CaM DICEs are found at different positions along their mRNAs. As described earlier, CaM mRNAs belong in the subset of mRNAs that are targeted to specific intracellular domains. The majority of the responsible localization signals (zip codes) described so far lie in the 30-UTR (e.g., see [66,67,72,125,126]). The zip sequences are bound by trans-acting factors to form a transport complex, which is then moved along the cytoskeleton [75]. To date, few studies have been carried out to identify dendritic targeting elements in neurons. The 4.2 kb and the 1.7 kb rat CaM I transcripts contain a 70 nucleotide-long stretch

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2841

highly similar to the zip code occurring in the b-actin and angiotensin II receptor mRNAs [127,128]. Additionally, a 91% homologous element is present in the corresponding human sequences. Although its role has not yet been proved, this zip code might well be responsible for the prominent dendritic trafficking of the CaM I mRNAs [39,62]. On the other hand, as described earlier, the mRNAs of the other two CaM genes are also targeted to dendrites, especially during early development [97]. Since the only region highly conserved among these transcripts is the coding sequence, its role in targeting might be envisaged. Accordingly, the 70 nucleotide-long zip signal may act only in the adult, while other elements are responsible for the CaM mRNA localization in the developing brain. Our computational analysis has revealed several putative regulatory elements along the CaM transcripts. Interestingly, AREs are present only on the CaM I transcripts, while DICEs are characteristic of the CaM III transcripts. On the other hand, CPEs are found in the sequences of each CaM gene. Thus, there is a striking distribution and clustering of these signals in the 30-UTRs, while the 50-UTRs appear to be silent. Although the mere presence of signal sequences within mRNAs is often regarded as prima facia evidence, their actual physiological roles must be established in further in vitro and in vivo experiments. With the growing number of newly identified signal sequences in diverse mRNAs, the map of CaM transcripts will predictably be further decorated.

Dendritic CaM targets play an essential role in synaptic plasticity As detailed above, CaM is particularly abundant in dendrites. Considerable evidence indicates that, at least in some dendrites, the targeting of CaM involves mRNA delivery and its local translation, suggesting a high demand of CaM in this compartment. As a regulatory protein, CaM acts through its effector molecules. What sorts of CaM targets are present in the postsynaptic compartment and what are their functions? Probably the most potent dendritic CaM target is CaM-KII, the foremost component of the postsynaptic densities [26]. As part of the NMDA receptor signalling complex [129], CaM-KII is the main target for the postsynaptic Ca2+ current produced by activation of the NMDA receptors [130]. CaM-KII has a wide substrate range, including Ca2+ channels, Ca2+-ATPases, glutamate receptors [131,132], microtubules [133] and transcriptional factors [28]. Thus, CaM-KII is a key factor in the postsynaptic signalling, and it is also necessary for the generation of long-lasting forms of synaptic plasticity, such as LTP [43,130,134]. Novel CaM-binding proteins such as striatin [135], SG2NA [136], NAP-22 [137], calponin and caldesmon [138] have recently been shown to be particularly enriched in dendrites and postsynaptic densities. The function of these proteins is currently being investigated, but, they are likely to interact with the members of the surrounding cluster of signalling or cytoskeletal (microfilaments and microtubules) molecules, thereby contributing to the plasticity of the postsynaptic specialization. Thus, the effectors of CaM in the dendrites appear to be the components of the signalprocessing machinery. To enable the neuron to govern the activities of these distant and often fast-acting molecules, appropriate CaM levels are essential. It is straightforward to theorize

2842

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

that locally controlled translation of CaM from targeted mRNAs allows the highest fidelity of this process.

CaM I and II mRNAs are enriched in the end feet in certain astrocyte cells Messenger RNA trafficking directed towards glial cell processes has been described. For example, the mRNA for the myelin basic protein (MBP) is highly concentrated in the myelin compartment in oligodendrocytes [139], while the mRNA encoding the glial fibrillary acidic protein (GFAP) is strongly targeted to the processes in astrocytes ([140,141]; for more data, see [141–143]). Similarly to neurons, the targeted glial mRNA population is transported in RNA granules and most probably translated locally [142,144,145]. No evidence has so far been revealed on the glial targeting of the CaM mRNAs. However, in an analysis of the subcellular distribution of the CaM mRNAs at an electron microscopic level in the adult rat brain, we found that the CaM I and CaM II mRNAs are heavily accumulated is certain (but not all) astroglial end feet (Fig. 4; previously unpublished data; manuscript in preparation). The GFAP mRNA distribution is also particularly concentrated in the tips of the astrocytic processes in cultures [140]. The high levels of CaM mRNAs in the end feet suggest that CaM may be translated there. Potential local CaM targets enriched in the glial processes have already been detected in different glial cell types. For example, SG2NA [136] is present in astrocytes, while calponin [138] is more widely expressed, including the radial glia, the glia limitans, the Bergmann glia and mature astrocytes. The colocalization of calponin with GFAP and vimentin filaments [138,146] may suggest that these proteins regulate the motility and plasticity of glial extensions.

CaM genes are differentially controlled under experimental conditions The expressions of the three CaM genes were determined under a range of experimental conditions; the following studies are selected examples and do not completely cover the corresponding literature. Gannon and McEwen [22] found that adrenalectomy selectively decreased the level of CaM III mRNAs by 30% in the cerebral cortex and the hippocampus in the rat, but not those of CaM I and II. Corticosterone administration fully prevented the down-regulation of the CaM III gene. Water deprivation caused a slight decrease (by up to 15%) of the CaM mRNA contents in several brain areas in the rat [147], while a marked and significantly differential upregulation was observed in the supraoptic hypothalamic nucleus (by 38%, 26% and 69% for CaM I, II and III, respectively). Palfi et al. [148] reported that a transient ischaemic insult in the rat forebrain resulted in slight shifts (by 10–15%) of the CaM mRNA levels in the hippocampus-dentate gyrus complex; although small in magnitude, these modulations were statistically significant in the hippocampal molecular layer. The effects of several drugs and agents with known action on the CNS have been investigated. For example, chronic ethanol administration and its withdrawal altered the CaM mRNA levels with a gene-specific pattern in the rat brain [51]. Modified mRNA contents were mainly

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2843

found in the forebrain, the limbic, the hypothalamic and the cortical structures for CaM I, in the limbic and the hypothalamic structures for CaM II, and in the forebrain structures for CaM III. We observed a systematically differential regulation for the three genes: the CaM I and CaM III mRNA levels most often increased, while the CaM II levels decreased in the affected brain regions. The extents of the changes in most areas were not more than 10–20%; the most prominent alteration was one of +58%. Michelhaugh et al. [149] reported that intermittent amphetamine treatment significantly decreased the CaM I mRNA content in the dorsal striatum, the nucleus accumbens and the prefrontal cortex, and depressed the CaM II mRNA level in the dorsal striatum by up to 30%. In contrast, slight increases were determined for both CaM I and II mRNAs in the ventral mesencephalon. Meanwhile, the CaM III mRNA content remained remarkably constant in all areas. In the same brain regions, the alterations in CaM protein levels determined by radioimmunoassay were opposite to the changes in the mRNAs; moreover, the protein concentrations varied more dramatically, by up to 100%. A single subcutaneous injection of reserpine increased the CaM I 4.2 kb mRNA content in the rat total brain RNA by 30% [150]. ISH analysis confirmed increased expressions in the brainstem and the neocortex, while a slight decrease characterized the expression in the midbrain. In parallel, the CaM protein content rose by 60% in tissue samples, including the brainstem. Barron et al. [151] studied the effects of a single dose of gamma-hexachlorocyclohexane (a convulsant agent) and deltahexachlorocyclohexane (a CNS depressant) on the expressions of CaM I and II genes in the rat brain. The CaM mRNA levels were altered in a markedly gene-specific fashion, by up to 80%. In the case of CaM I, the changes were even transcript-specific, as the bulk of the discrepancies corresponded to those for the 4.2 kb species. Sola et al. [45] determined the effects of a systemic convulsant dose of kainate on the expressions of the three CaM genes in the mouse brain. All examined brain areas (the hippocampus, the parietal cortex and the caudate putamen) exhibited similar conversions for each CaM gene: the CaM I mRNAs increased by up to 90%, the CaM II mRNA decreased by up to 50%, while the CaM III mRNAs were mostly unaffected. Although radioimmunoassay did not detect significant adjustments of the CaM contents in any of the above brain areas, an increased immunoreactivity was determined in the hippocampal pyramidal cell layer, while numerous immunoreactive glial cells became evident. In PC12 cells, nerve growth factor (NGF) induces neuronal differentiation, and in parallel a differential upregulation of the CaM genes can be observed, as the level of the 1.4 kb CaM II mRNA increases earlier and to a greater extent (3-fold) than those of the other CaM mRNAs [56]. In another study in PC12 cells, cAMP treatment selectively upregulated the CaM I and the CaM II genes, while the expression of the CaM III gene remained stable [112]. Transcriptional control of the CaM I gene was transcript-specific, as the 1.7 kb mRNA species increased more extensively than the 4.2 kb species. As all these experiments seem to reflect unique examples with their own characteristics, the above data are obviously not easy to interpret. However, some general conclusions may be drawn: 1) The expression of the CaM III gene is often unaffected, strengthening the notion of its house-keeping nature. 2) Nonetheless, other examples clearly indicate that the basal CaM III gene expression can be altered in certain conditions. 3) The expressional profiles for the CaM I and CaM II genes can readily be readjusted in response to a wide range of stimuli, and the alterations in their mRNA abundances are often opposing in direction. 4) Even when

2844

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

the expressions of all three CaM genes are altered in a similar way (e.g. some are upregulated, while the others are at least not down-regulated), the CaM protein concentration does not necessarily follow this trend (i.e. it rises in this example). 5) Expressional changes are not only gene-specific, but may be transcript-specific for the CaM I gene. The regulatory function of CaM is now recognized to operate through the action of subcellular microdomains, several of which exist in a single cell. When a particular stimulus delivered by a single population of the CaM microdomains in the cell initiates a feedback modification of the expressions of the CaM genes, it inevitably interferes with other regulatory stimuli. With regard to the possible number of CaM microdomains in the cell, there must be an extensive regulatory convergence for CaM gene transcription. Thus, the resultant expressional level might already be considerably different from that originally evoked by the experimental stimulus. The actual CaM expressional level in other (non-affected) cells in the surrounding environment could further mask the experimental effects when the regional mRNA level is measured. All the hybridization experiments referred to above were carried out by film autoradiography or equivalent methods, and most CaM protein measurements were made by radioimmunoassay, i.e. methods that are capable only of resolving the regional mRNA or protein levels. Consequently, the detected brain area-specific changes in both the CaM gene expression and the protein level might be quite different from those of the directly implicated neurons. It is now clear that other strategies, capable of providing more specific information, should contribute to a better understanding of the regulation of the CaM genes. Even careful selection of the experimental systems might appreciably facilitate the interpretation of the results. For example, the hippocampal formation is a well-characterized structure with a not too complex cellular composition; here, even conventional analysis methods might provide cell type-specific results [45,148]. Synaptosome or synaptodendrosome preparations, although representing various cell types, may offer an insight into dendrite-specific alterations [71,152–155]. With their inherent limitations, homogeneous populations of in vitro systems, such as embryonic stem cells [156–158], primary neuronal cultures [159] or neuronal cell lines derived from different sources, such as the hippocampus [160–163], the striatum [164,165], the cerebellum [166] or the periphery [167,168], might be suitable systems with which to answer particular experimental issues. A few studies describing the characteristics of the CaM gene expression have already demonstrated the effectiveness of in vitro strategies [56,92,96,112]. One of the most promising approaches is the use of high-resolution fluorescence ISH analysis [169–173], which can provide quantitative data corresponding to single cells, even in their natural tissue environments. Another useful technique is the antisense RNA amplification method, which includes an approach to the analysis of mRNA levels in single cells that have been phenotypically characterized on the basis of electrophysiology, morphology or protein expression [174,175].

CaM genes are under unique developmental regulation Several authors have analyzed the developmental expressional pattern of the CaM gene family. Cimino et al. [176] described marked differences in the total CaM mRNA levels on

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2845

the first postnatal day in the various brain areas; the levels became more uniform by postnatal day 32. MacManus et al. [177] acquired the first indication for the differential developmental expression of the CaM genes in different rat tissues. Ni et al. [116] described the developmental expression of the CaM I gene in the rat brain; most notably, they found that the 1.7 kb mRNA species appeared to correlate with the proliferating and developing cerebellar granule neurons, while the 4.2 kb mRNA species was present in the mature granule neuron population. Berry and Brown [57] reported that maximum CaM protein levels were attained on postnatal days 10–15 in the cerebral hemispheres, the thalamus, the colliculi and the brainstem in the rat; the protein levels declined thereafter in all regions except the thalamus. Northern blot analysis of the total CaM mRNA in the same regions indicated an early increase (postnatal days 5–15) and a maintained CaM gene expression afterwards. The 4.2 kb CaM I mRNA species exhibited a marked increase during postnatal days 5–15, and remained at this elevated level in the cerebral hemispheres and thalamus, whereas it subsequently decreased in the colliculi and the brainstem. Furthermore, Berry and Brown [62] detected a temporal dendritic localization of the 4.2 kb CaM I mRNA in the pyramidal cells of the cerebral cortex (postnatal days 5–15) and the hippocampus (postnatal days 5–20), and in the Purkinje neurons (postnatal days 15–20) in the rat. Thus, different neurons targeted the CaM I message to dendrites at varying times, though these coincided with synaptogenesis [93–95] in these brain areas. In parallel, polyribosomes were shown to dramatically accumulate under growing spine synapses in the dentate gyrus of the rat (60% of the synapses had one or more polyribosomes between 1 and 7 days of age; [178]). Kortvely et al. [97] followed the CaM gene expression by quantitative ISH in the postnatal rat brain. A widespread and differential developmental pattern characterized the distribution of the CaM mRNAs. The expressional patterns of the different brain areas were classified into three developmental profiles. Prominent dendritic mRNA targeting corresponding to all three CaM genes in the molecular layers of the hippocampus, the cerebral and the cerebellar cortices was reported on postnatal days 1–20 [97]. By postnatal day 20, a characteristic rearrangement in the dendritic CaM mRNA pool (predominated by the CaM I transcripts) was obvious.

CaM protein appears at some degree of neuronal maturation CaM immunoreactivity appears early in the brainstem, but later in the cerebral and the cerebellar cortices and the hippocampus in the mouse [179]. The major proliferative layers present during early development, such as the matrix cells in the cerebral cortex and the cells in the external germinal layer in the cerebellum, do not show the CaM immunoreactivity. In the cerebral cortex, the migrating cells and the cells in the cortical plate are also negative, while the deep cortical cells, which have probably settled in their final position become positive. These results suggest that detectable CaM appears only at some degree of maturation in neurons. In contrast with the above data, CaM unquestionably plays essential regulatory roles in the cell cycle and cell proliferation [29,30]. The CaM gene expression increases during the cell cycle [180,181], and is high in foetal and neoplastic tissues [177].

2846

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

Moreover, clearly detectable levels of CaM mRNAs have been found in the proliferative layers of the developing brain [116,176]. The CaM protein distribution during Purkinje cell development has been described in great detail [37,62]. CaM is clearly present in the apical cones from postnatal day 6, and becomes detectable in the primary dendrites by postnatal days 10–15. First in Purkinje cell maturation, newly synthesized CaM protein is transported from the soma to the dendrites. However, by postnatal days 15–20, there is a transient switch in the CaM synthesis pattern: as the dendritic CaM I mRNA targeting becomes evident [62], its local translation probably contributes significantly to the dendritic CaM pool. The study by Palfi et al. [39] suggests that dendritic targeting may be more general in Purkinje cells, involving other CaM mRNAs, and persisting even in the mature cerebellum.

Conclusions and perspectives As CaM controls a wide array of target molecules in the cells, some of them obviously exerting opposing effects, fine-tuning of the active CaM pools is exceptionally complex as compared with that of most other proteins. Regulation is carried out at multiple levels from Ca2+ currents through transcriptional control to protein delivery and storage. Since the conservation of the CaM gene family through the vertebrate lineage was discovered, differential utilization of the three CaM genes has been indirectly proposed. Thus, a hunt for clear evidence in support of this hypothesis has begun. As the mammalian brain possesses an extremely high CaM content and a versatile range of cell types, it is a uniquely beneficial system with which to explore the potentials of the multigene nature of CaM. Consequently, in this review we have focused on studies on the brain or brain cells and attempted to integrate the accumulated research data at the level of CaM mRNA transcription and distribution. The results indicate that: 1) Unique, gene-specific expressions of the three CaM genes are apparent in various areas in the developing and adult rodent brain under physiological conditions. The expressional levels may be not only gene-specific, but even transcript-specific, although our understanding is restricted by the limits of the current detection techniques. 2) To establish the local CaM pools in distant intracellular compartments (dendrites and glial processes), besides the classical perinuclear synthesis and protein transport pathway, local protein synthesis from differentially targeted mRNAs is also employed in certain brain cells. The 4.2 kb and possibly the 1.7 kb CaM I mRNA species are potent targets for dendritic translocation in both developing and adult neurons, while the 1.4 kb CaM II mRNA seems to be the most abundant species in the very early development of dendrites, and it is also heavily localized in the end feet of some mature astroglial cells. 3) Even though the experimental approaches used so far have not permitted an analysis of expressional alterations in single cells, the detected changes probably being largely masked by the surrounding tissue environment, a few studies have suggested that the CaM genes are controlled in a unique, gene-specific fashion when responding to certain external stimuli. 4) Several regulatory elements have been identified on the CaM genes and mRNAs, but their functional analysis is far from complete.

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2847

For an overall understanding of the function of the CaM gene family, much work still remains to be done. One of the main areas of interest is to clarify the role of the regulatory sequences on both the CaM genes and the mRNAs. It should be ascertained how the gene regulatory elements determine the actual transcriptional levels of the three CaM genes, and how the UTRs govern the targeting, stability and translational efficiency of the CaM mRNAs. Quantitative detection of the mRNA levels at the cellular level is another important issue where new sensitive strategies might improve our current level of comprehension considerably. For example, it would be interesting to examine how certain neurons alter their CaM transcription, while other, morphologically similar, but functionally different cells maintain their expression level, or how cells rearrange their subcellular CaM mRNA targeting pattern for a particular stimulus. The green fluorescent protein (GFP)-human CaM III fusion gene has recently been constructed [182] and expressed in Hela cells. The fusion protein was found to have similar biochemical properties to those of wild-type CaM. In transgenic animals, tagging the endogenous CaM genes by similar means would reveal the intracellular distribution of the CaM subpopulations corresponding to single CaM genes. These experiments would shed light on whether the differential expression detected in the mRNA levels is indeed reflected or not in the protein allocation pattern.

Note added in proof While this manuscript was in the process of submission, a review on the roles of the Ca2+/CaM system in neuronal hyperexcitability was published elsewhere (Sola´ et al., Int. J. Biochem. Cell Biol. 33 (2001) 439–455).

Acknowledgments This work was supported by grants from the National Scientific Research Fund, Hungary (OTKA T034621) and the Ministry of Health, Hungary (57/2000) to K.G.

References 1. Cheung WY. Calmodulin plays a pivotal role in cellular regulation. Science 1980;207:19 – 27. 2. Chien YH, Dawid IB. Isolation and characterization of calmodulin genes from Xenopus laevis. Mol Cell Biol 1984;4:507 – 13. 3. Putkey JA, Ts’ui KF, Tanaka T, Lagace L, Stein JP, Lai EC, Means AR. Chicken calmodulin genes. A species comparison of cDNA sequences and isolation of a genomic clone. J Biol Chem 1983;258:11864 – 70. 4. Bender PK, Dedman JR, Emerson Jr CP. The abundance of calmodulin mRNAs is regulated in phosphorylase kinase-deficient skeletal muscle. J Biol Chem 1988;263:9733 – 7. 5. Danchin A, Sezer O, Glaser P, Chalon P, Caput D. Cloning and expression of mouse-brain calmodulin as an activator of Bordetella pertussis adenylate cyclase in Escherichia coli. Gene 1989;80:145 – 9. 6. Kato K. Finding new genes in the nervous system by cDNA analysis. Trends Neurosci 1992;15:319 – 23.

2848

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

7. Matsuo K, Ikeshima H, Shimoda K, Umezawa A, Hata J, Maejima K, Nojima H, Takano T. Expression of the rat calmodulin gene II in the central nervous system: a 294-base promoter and 68-base leader segment mediates neuron-specific gene expression in transgenic mice. Mol Brain Res 1993;20:9 – 20. 8. Skinner TL, Kerns RT, Bender PK. Three different calmodulin-encoding cDNAs isolated by a modified 50-RACE using degenerate oligodeoxyribonucleotides. Gene 1994;151:247 – 51. 9. Nojima H, Sokabe H. Structure of rat calmodulin processed genes with implications for a mRNA-mediated process of insertion. J Mol Biol 1986;190:391 – 400. 10. Nojima H, Kishi K, Sokabe H. Multiple calmodulin mRNA species are derived from two distinct genes. Mol Cell Biol 1987;7:1873 – 80. 11. Nojima H, Sokabe H. Structure of a gene for rat calmodulin. J Mol Biol 1987;193:439 – 45. 12. Sherbany AA, Parent AS, Brosius J. Rat calmodulin cDNA. DNA 1987;6:267 – 72. 13. Nojima H. Structural organization of multiple rat calmodulin genes. J Mol Biol 1989;208:269 – 82. 14. Mori M, Andoh Y, Nojima H, Serikawa T. Chromosomal mapping of calmodulin genes in the rat. Mamm Genome 1994;5:824 – 6. 15. Berchtold MW, Egli R, Rhyner JA, Hameister H, Strehler EE. Localization of the human bona fide calmodulin genes CALM1, CALM2, and CALM3 to chromosomes 14q24 – q31, 2p21.1 – p21.3, and 19q13.2 – q13.3. Genomics 1993;16:461 – 5. 16. Koller M, Schnyder B, Strehler EE. Structural organization of the human CaMIII calmodulin gene. Biochim Biophys Acta 1990;1087:180 – 9. 17. Rhyner JA, Ottiger M, Wicki R, Greenwood TM, Strehler EE. Structure of the human CALM1 calmodulin gene and identification of two CALM1-related pseudogenes CALM1P1 and CALM1P2. Eur J Biochem 1994;225:71 – 82. 18. Senterre-Lesenfants S, Alag AS, Sobel ME. Multiple mRNA species are generated by alternate polyadenylation from the human calmodulin-I gene. J Cell Biochem 1995;58:445 – 54. 19. Toutenhoofd SL, Foletti D, Wicki R, Rhyner JA, Garcia F, Tolon R, Strehler EE. Characterization of the human CALM2 calmodulin gene and comparison of the transcriptional activity of CALM1, CALM2 and CALM3. Cell Calcium 1998;23:323 – 38. 20. Fischer R, Koller M, Flura M, Mathews S, Strehler-Page MA, Krebs J, Penniston JT, Carafoli E, Strehler EE. Multiple divergent mRNAs code for a single human calmodulin. J Biol Chem 1988;263:17055 – 62. 21. Ikeshima H, Yuasa S, Matsuo K, Kawamura K, Hata J, Takano T. Expression of three nonallelic genes coding calmodulin exhibits similar localization on the central nervous system of adult rats. J Neurosci Res 1993; 36:111 – 9. 22. Gannon MN, McEwen BS. Distribution and regulation of calmodulin mRNAs in rat brain. Mol Brain Res 1994;22:186 – 92. 23. Friedberg F, Rhoads AR. Evolutionary aspects of calmodulin. IUBMB Life 2001;51:215 – 21. 24. Toutenhoofd SL, Strehler EE. The calmodulin multigene family as a unique case of genetic redundancy: multiple levels of regulation to provide spatial and temporal control of calmodulin pools? Cell Calcium 2000;28:83 – 96. 25. Means AR, Tash JS, Chafouleas JG. Physiological implications of the presence, distribution, and regulation of calmodulin in eukaryotic cells. Physiol Rev 1982;62:1 – 39. 26. Kennedy MB. Regulation of neuronal function by calcium. Trends Neurosci 1989;12:417 – 20. 27. Klee CB, Crouch TH, Richman PG. Calmodulin. Annu Rev Biochem 1980;49:489 – 515. 28. Schulman H, Hyman SE. In: Zigmond MJ, Bloom FE, Landis SC, Roberts JL, Squire LR, editors. Intracellular signaling. 1st ed. San Diego: Academic Press; 1999. p. 269 – 316. 29. Means AR, VanBerkum MF, Bagchi I, Lu KP, Rasmussen CD. Regulatory functions of calmodulin. Pharmacol Ther 1991;50:255 – 70. 30. Rasmussen CD, Means AR. Calmodulin, cell growth and gene expression. Trends Neurosci 1989;12:433 – 8. 31. Agell N, Aligue R, Alemany V, Castro A, Jaime M, Pujol MJ, Rius E, Serratosa J, Taules M, Bachs O. New nuclear functions for calmodulin. Cell Calcium 1998;23:115 – 21. 32. Chin D, Means AR. Calmodulin: a prototypical calcium sensor. Trends Cell Biol 2000;10:322 – 8.

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2849

33. Manalan AS, Klee CB. Calmodulin. Adv Cyclic Nucleotide Protein Phosphorylation Res 1984;18:227 – 78. 34. Hoskins B, Burton CK, Liu DD, Porter AB, Ho IK. Regional and subcellular calmodulin content of rat brain. J Neurochem 1986;46:303 – 4. 35. Zhou LW, Moyer JA, Muth EA, Clark B, Palkovits M, Weiss B. Regional distribution of calmodulin activity in rat brain. J Neurochem 1985;44:1657 – 62. 36. Kakiuchi S, Yasuda S, Yamazaki R, Teshima Y, Kanda K, Kakiuchi R, Sobue K. Quantitative determinations of calmodulin in the supernatant and particulate fractions of mammalian tissues. J Biochem (Tokyo) 1982;92:1041 – 8. 37. Caceres A, Bender P, Snavely L, Rebhun LI, Steward O. Distribution and subcellular localization of calmodulin in adult and developing brain tissue. Neuroscience 1983;10:449 – 61. 38. Sola C, Tusell JM, Serratosa J. Comparative study of the pattern of expression of calmodulin messenger RNAs in the mouse brain. Neuroscience 1996;75:245 – 56. 39. Palfi A, Vizi S, Gulya K. Differential distribution and intracellular targeting of mRNAs corresponding to the three calmodulin genes in rat brain. A quantitative in situ hybridization study. J Histochem Cytochem 1999;47:583 – 600. 40. DeLorenzo RJ. Role of calmodulin in neurotransmitter release and synaptic function. Ann NY Acad Sci 1980;356:92 – 109. 41. DeLorenzo RJ. Calmodulin in neurotransmitter release and synaptic function. Fed Proc 1982;41:2265 – 72. 42. Liu YC, Storm DR. Regulation of free calmodulin levels by neuromodulin: neuron growth and regeneration. Trends Pharmacol Sci 1990;11:107 – 11. 43. Malenka RC, Kauer JA, Perkel DJ, Nicoll RA. The impact of postsynaptic calcium on synaptic transmission — its role in long-term potentiation. Trends Neurosci 1989;12:444 – 50. 44. Polak KA, Edelman AM, Wasley JW, Cohan CS. A novel calmodulin antagonist, CGS 9343B, modulates calcium-dependent changes in neurite outgrowth and growth cone movements. J Neurosci 1991;11: 534 – 42. 45. Sola C, Tusell JM, Serratosa J. Differential response of calmodulin genes in the mouse brain after systemic kainate administration. Neuroscience 1997;78:155 – 64. 46. Sola C, Tusell JM, Serratosa J. Calmodulin is expressed by reactive microglia in the hippocampus of kainic acid-treated mice. Neuroscience 1997;81:699 – 705. 47. Sola C, Barron S, Tusell JM, Serratosa J. The Ca2+/calmodulin signaling system in the neural response to excitability. Involvement of neuronal and glial cells. Prog Neurobiol 1999;58:207 – 32. 48. Matsuoka I, Giuili G, Poyard M, Stengel D, Parma J, Guellaen G, Hanoune J. Localization of adenylyl and guanylyl cyclase in rat brain by in situ hybridization: comparison with calmodulin mRNA distribution. J Neurosci 1992;12:3350 – 60. 49. Ni B, Rush S, Gurd JW, Brown IR. Molecular cloning of calmodulin mRNA species which are preferentially expressed in neurons in the rat brain. Mol Brain Res 1992;13:7 – 17. 50. Roberts-Lewis JM, Cimino M, Krause RGd, Tyrrell Jr DF, Davis LG, Weiss B, Lewis ME. Anatomical localization of calmodulin mRNA in the rat brain with cloned cDNA and synthetic oligonucleotide probes. Synapse 1990;5:247 – 54. 51. Vizi S, Palfi A, Gulya K. Multiple calmodulin genes exhibit systematically differential responses to chronic ethanol treatment and withdrawal in several regions of the rat brain. Mol Brain Res 2000;83:63 – 71. 52. Kovacs B, Gulya K. A color in situ hybridization method with improved sensitivity for the detection of lowabundance mRNAs. Acta Biol Szeged 2001;45:75 – 7. 53. Palfi A, Hatvani L, Gulya K. A new quantitative film autoradiographic method of quantifying mRNA transcripts for in situ hybridization. J Histochem Cytochem 1998;46:1141 – 9. 54. Vizi S, Gulya K. Calculation of maximal hybridization capacity (Hmax) for quantitative in situ hybridization: a case study for multiple calmodulin mRNAs. J Histochem Cytochem 2000;48:893 – 904. 55. Vizi S, Palfi A, Hatvani L, Gulya K. Methods for quantification of in situ hybridization signals obtained by film autoradiography and phosphorimaging applied for estimation of regional levels of calmodulin mRNA classes in the rat brain. Brain Res Protoc 2001;8:32 – 44.

2850

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

56. Bai G, Weiss B. The increase of calmodulin in PC12 cells induced by NGF is caused by differential expression of multiple mRNAs for calmodulin. J Cell Physiol 1991;149:414 – 21. 57. Berry F, Brown IR. Developmental expression of calmodulin mRNA and protein in regions of the postnatal rat brain. J Neurosci Res 1995;42:613 – 22. 58. Strappe PM, Wang TH, McKenzie CA, Lowrie S, Simmonds P, Bell JE. In situ polymerase chain reaction amplification of HIV-1 DNA in brain tissue. J Virol Methods 1998;70:119 – 27. 59. Shimoda K, Ikeshima H, Matsuo K, Hata J, Maejima K, Takano T. Spatial and temporal regulation of the rat calmodulin gene III directed by a 877-base promoter and 103-base leader segment in the mature and embryonal central nervous system of transgenic mice. Mol Brain Res 1995;31:61 – 70. 60. Seto-Ohshima A, Kitajima S, Sano M, Kato K, Mizutani A. Immunohistochemical localization of calmodulin in mouse brain. Histochemistry 1983;79:251 – 7. 61. Lin CT, Dedman JR, Brinkley BR, Means AR. Localization of calmodulin in rat cerebellum by immunoelectron microscopy. J Cell Biol 1980;85:473 – 80. 62. Berry FB, Brown IR. CaM I mRNA is localized to apical dendrites during postnatal development of neurons in the rat brain. J Neurosci Res 1996;43:565 – 75. 63. Vendrell M, Aligue R, Bachs O, Seratosa J. Presence of calmodulin and calmodulin-binding proteins in the nuclei of brain cells. J Neurochem 1991;57:622 – 8. 64. Bachs O, Agell N, Carafoli E. Calmodulin and calmodulin-binding proteins in the nucleus. Cell Calcium 1994;16:289 – 96. 65. Bachs O, Agell N, Carafoli E. Calcium and calmodulin function in the cell nucleus. Biochim Biophys Acta 1992;1113:259 – 70. 66. Gao FB. Messenger RNAs in dendrites: localization, stability, and implications for neuronal function. Bioessays 1998;20:70 – 8. 67. Kuhl D, Skehel P. Dendritic localization of mRNAs. [published erratum appears in Curr Opin Neurobiol 1999 Feb;9(1):142] Curr Opin Neurobiol 1998;8:600 – 6. 68. St Johnston D. The intracellular localization of messenger RNAs. Cell 1995;81:161 – 70. 69. Steward O. Targeting of mRNAs to subsynaptic microdomains in dendrites. Curr Opin Neurobiol 1995;5: 55 – 61. 70. Steward O. mRNA localization in neurons: a multipurpose mechanism? Neuron 1997;18:9 – 12. 71. Tian QB, Nakayama K, Okano A, Suzuki T. Identification of mRNAs localizing in the postsynaptic region. Mol Brain Res 1999;72:147 – 57. 72. Mohr E. Subcellular RNA compartmentalization. Prog Neurobiol 1999;57:507 – 25. 73. Sheetz MP, Pfister KK, Bulinski JC, Cotman CW. Mechanisms of trafficking in axons and dendrites: implications for development and neurodegeneration. Prog Neurobiol 1998;55:577 – 94. 74. Steward O, Worley PF. A cellular mechanism for targeting newly synthesized mRNAs to synaptic sites on dendrites. Proc Natl Acad Sci U S A 2001;98:7062 – 8. 75. Knowles RB, Sabry JH, Martone ME, Deerinck TJ, Ellisman MH, Bassell GJ, Kosik KS. Translocation of RNA granules in living neurons. J Neurosci 1996;16:7812 – 20. 76. Steward O, Levy WB. Preferential localization of polyribosomes under the base of dendritic spines in granule cells of the dentate gyrus. J Neurosci 1982;2:284 – 91. 77. Chicurel ME, Harris KM. Three-dimensional analysis of the structure and composition of CA3 branched dendritic spines and their synaptic relationships with mossy fiber boutons in the rat hippocampus. J Comp Neurol 1992;325:169 – 82. 78. Steward O, Falk PM, Torre ER. Ultrastructural basis for gene expression at the synapse: synapse-associated polyribosome complexes. J Neurocytol 1996;25:717 – 34. 79. Tiedge H, Brosius J. Translational machinery in dendrites of hippocampal neurons in culture. J Neurosci 1996;16:7171 – 81. 80. Kiebler MA, Hemraj I, Verkade P, Kohrmann M, Fortes P, Marion RM, Ortin J, Dotti CG. The mammalian staufen protein localizes to the somatodendritic domain of cultured hippocampal neurons: implications for its involvement in mRNA transport. J Neurosci 1999;19:288 – 97.

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2851

81. Gardiol A, Racca C, Triller A. Dendritic and postsynaptic protein synthetic machinery. J Neurosci 1999; 19:168 – 79. 82. Steward O, Schuman EM. Protein synthesis at synaptic sites on dendrites. Annu Rev Neurosci 2001;24: 299 – 325. 83. Racca C, Gardiol A, Triller A. Dendritic and postsynaptic localizations of glycine receptor alpha subunit mRNAs. J Neurosci 1997;17:1691 – 700. 84. Martone ME, Pollock JA, Jones YZ, Ellisman MH. Ultrastructural localization of dendritic messenger RNA in adult rat hippocampus. J Neurosci 1996;16:7437 – 46. 85. Torre ER, Steward O. Protein synthesis within dendrites: glycosylation of newly synthesized proteins in dendrites of hippocampal neurons in culture. J Neurosci 1996;16:5967 – 78. 86. Torre ER, Steward O. Demonstration of local protein synthesis within dendrites using a new cell culture system that permits the isolation of living axons and dendrites from their cell bodies. J Neurosci 1992; 12:762 – 72. 87. Crino PB, Eberwine J. Molecular characterization of the dendritic growth cone: regulated mRNA transport and local protein synthesis. Neuron 1996;17:1173 – 87. 88. Kacharmina JE, Job C, Crino P, Eberwine J. Stimulation of glutamate receptor protein synthesis and membrane insertion within isolated neuronal dendrites. Proc Natl Acad Sci U S A 2000;97:11545 – 50. 89. Crino P, Khodakhah K, Becker K, Ginsberg S, Hemby S, Eberwine J. Presence and phosphorylation of transcription factors in developing dendrites. Proc Natl Acad Sci U S A 1998;95:2313 – 8. 90. Eberwine J, Miyashiro K, Kacharmina JE, Job C. Local translation of classes of mRNAs that are targeted to neuronal dendrites. Proc Natl Acad Sci U S A 2001;98:7080 – 5. 91. Eberwine J, Job C, Kacharmina JE, Miyashiro K, Therianos S. Transcription factors in dendrites: dendritic imprinting of the cellular nucleus. Results Probl Cell Differ 2001;34:57 – 68. 92. Zhang SP, Natsukari N, Bai G, Nichols RA, Weiss B. Localization of the multiple calmodulin messenger RNAs in differentiated PC12 cells. Neuroscience 1993;55:571 – 82. 93. Aghajanian GK, Bloom FE. The formation of synaptic junctions in developing rat brain: a quantitative electron microscopic study. Brain Res 1967;6:716 – 27. 94. Armstrong-James MA, Johnson FR. Post-natal development of synapses in the cerebral cortex of the rat. J Anat 1969;104:590. 95. Robain O, Bideau I, Farkas E. Developmental changes of synapses in the cerebellar cortex of the rat. A quantitative analysis. Brain Res 1981;206:1 – 8. 96. Palfi A, Tarcsa M, Varszegi S, Gulya K. Calmodulin gene expression in an immortalized striatal gabaergic cell line. Acta Biol Hung 2000;51:65 – 71. 97. Kortvely E, Palfi A, Bakota L, Gulya K. Ontogeny of calmodulin gene expression in rat brain. 2002 (submitted). 98. Decker CJ, Parker R. Diversity of cytoplasmic functions for the 30 untranslated region of eukaryotic transcripts. Curr Opin Cell Biol 1995;7:386 – 92. 99. Toutenhoofd SL, Garcia F, Zacharias DA, Wilson RA, Strehler EE. Minimum CAG repeat in the human calmodulin-1 gene 50 untranslated region is required for full expression. Biochim Biophys Acta 1998;1398: 315 – 20. 100. Pesole G, Liuni S, Grillo G, Licciulli F, Larizza A, Makalowski W, Saccone C. UTRdb and UTRsite: specialized databases of sequences and functional elements of 50 and 30 untranslated regions of eukaryotic mRNAs. Nucleic Acids Res 2000;28:193 – 6. 101. Dalphin ME, Stockwell PA, Tate WP, Brown CM. TransTerm, the translational signal database, extended to include full coding sequences and untranslated regions. Nucleic Acids Res 1999;27:293 – 4. 102. Wahle E. 30-end cleavage and polyadenylation of mRNA precursors. Biochim Biophys Acta 1995;1261: 183 – 94. 103. Gautheret D, Poirot O, Lopez F, Audic S, Claverie JM. Alternate polyadenylation in human mRNAs: a largescale analysis by EST clustering. Genome Res 1998;8:524 – 30. 104. Beaudoing E, Freier S, Wyatt JR, Claverie JM, Gautheret D. Patterns of variant polyadenylation signal usage in human genes. Genome Res 2000;10:1001 – 10.

2852

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

105. Edwalds-Gilbert G, Veraldi KL, Milcarek C. Alternative poly(A) site selection in complex transcription units: means to an end? Nucleic Acids Res 1997;25:2547 – 61. 106. Schwartz E, Gelfand JM, Mauch JC, Kligman LH. Generation of a tropoelastin mRNA variant by alternative polyadenylation site selection in sun-damaged human skin and ultraviolet B-irradiated fibroblasts. Biochem Biophys Res Commun 1998;246:217 – 21. 107. Abdel Wahab N, Gibbs J, Mason RM. Regulation of gene expression by alternative polyadenylation and mRNA instability in hyperglycaemic mesangial cells. Biochem J 1998;336:405 – 11. 108. O’Hare K. mRNA 30 ends in focus. Trends Genet 1995;11:255 – 7. 109. Beaudoing E, Gautheret D. Identification of alternate polyadenylation sites and analysis of their tissue distribution using EST data. Genome Res 2001;11:1520 – 6. 110. Tsui SK, Lee SM, Fung KP, Waye MM, Lee CY. Primary structures and sequence analysis of human ribosomal proteins L39 and S27. Biochem Mol Biol Int 1996;40:611 – 6. 111. Tsai SC, Haun RS, Tsuchiya M, Moss J, Vaughan M. Isolation and characterization of the human gene for ADP-ribosylation factor 3, a 20-kDa guanine nucleotide-binding protein activator of cholera toxin. J Biol Chem 1991;266:23053 – 9. 112. Bai G, Nichols RA, Weiss B. Cyclic AMP selectively up-regulates calmodulin genes I and II in PC12 cells. Biochim Biophys Acta 1992;1130:189 – 96. 113. Slaughter GR, Means AR. Analysis of expression of multiple genes encoding calmodulin during spermatogenesis. Mol Endocrinol 1989;3:1569 – 78. 114. Ross J. mRNA stability in mammalian cells. Microbiol Rev 1995;59:423 – 50. 115. Jacobson A, Peltz SW. Interrelationships of the pathways of mRNA decay and translation in eukaryotic cells. Annu Rev Biochem 1996;65:693 – 739. 116. Ni B, Landry CF, Brown IR. Developmental expression of neuronal calmodulin mRNA species in the rat brain analyzed by in situ hybridization. J Neurosci Res 1992;33:559 – 67. 117. Veyrune JL, Campbell GP, Wiseman J, Blanchard JM, Hesketh JE. A localisation signal in the 30 untranslated region of c-myc mRNA targets c-myc mRNA and beta-globin reporter sequences to the perinuclear cytoplasm and cytoskeletal-bound polysomes. J Cell Sci 1996;109:1185 – 94. 118. Wells DG, Richter JD, Fallon JR. Molecular mechanisms for activity-regulated protein synthesis in the synapto-dendritic compartment. Curr Opin Neurobiol 2000;10:132 – 7. 119. Dickson KS, Thompson SR, Gray NK, Wickens M. Poly(A) polymerase and the regulation of cytoplasmic polyadenylation. J Biol Chem 2001;10:10. 120. Richter JD. Cytoplasmic polyadenylation in development and beyond. Microbiol Mol Biol Rev 1999;63: 446 – 56. 121. Wu L, Wells D, Tay J, Mendis D, Abbott MA, Barnitt A, Quinlan E, Heynen A, Fallon JR, Richter JD. CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses. Neuron 1998;21:1129 – 39. 122. Wells SE, Hillner PE, Vale RD, Sachs AB. Circularization of mRNA by eukaryotic translation initiation factors. Mol Cell 1998;2:135 – 40. 123. Ostareck DH, Ostareck-Lederer A, Wilm M, Thiele BJ, Mann M, Hentze MW. mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 30 end. Cell 1997; 89:597 – 606. 124. Ostareck DH, Ostareck-Lederer A, Shatsky IN, Hentze MW. Lipoxygenase mRNA silencing in erythroid differentiation: The 30UTR regulatory complex controls 60S ribosomal subunit joining. Cell 2001;104: 281 – 90. 125. Mori Y, Imaizumi K, Katayama T, Yoneda T, Tohyama M. Two cis-acting elements in the 30 untranslated region of alpha-CaMKII regulate its dendritic targeting. Nat Neurosci 2000;3:1079 – 84. 126. Mohr E, Fuhrmann C, Richter D. VP-RBP, a protein enriched in brain tissue, specifically interacts with the dendritic localizer sequence of rat vasopressin mRNA. Eur J Neurosci 2001;13:1107 – 12. 127. Kislauskis EH, Zhu X, Singer RH. Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype. J Cell Biol 1994;127:441 – 51.

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2853

128. Thekkumkara TJ, Thomas WG, Motel TJ, Baker KM. Functional role for the angiotensin II receptor (AT1A) 30-untranslated region in determining cellular responses to agonist: evidence for recognition by RNA binding proteins. Biochem J 1998;329:255 – 64. 129. Kennedy MB. Signal-processing machines at the postsynaptic density. Science 2000;290:750 – 4. 130. Nicoll RA, Kauer JA, Malenka RC. The current excitement in long-term potentiation. Neuron 1988;1:97 – 103. 131. Kitamura Y, Miyazaki A, Yamanaka Y, Nomura Y. Stimulatory effects of protein kinase C and calmodulin kinase II on N-methyl-D-aspartate receptor/channels in the postsynaptic density of rat brain. J Neurochem 1993;61:100 – 9. 132. McGlade-McCulloh E, Yamamoto H, Tan SE, Brickey DA, Soderling TR. Phosphorylation and regulation of glutamate receptors by calcium/calmodulin-dependent protein kinase II. Nature 1993;362:640 – 2. 133. Marcum JM, Dedman JR, Brinkley BR, Means AR. Control of microtubule assembly-disassembly by calcium-dependent regulator protein. Proc Natl Acad Sci U S A 1978;75:3771 – 5. 134. Lisman J. The CaM kinase II hypothesis for the storage of synaptic memory. Trends Neurosci 1994;17: 406 – 12. 135. Castets F, Bartoli M, Barnier JV, Baillat G, Salin P, Moqrich A, Bourgeois JP, Denizot F, Rougon G, Calothy G, Monneron A. A novel calmodulin-binding protein, belonging to the WD-repeat family, is localized in dendrites of a subset of CNS neurons. J Cell Biol 1996;134:1051 – 62. 136. Castets F, Rakitina T, Gaillard S, Moqrich A, Mattei MG, Zinedin MA. SG2NA, and striatin are calmodulinbinding, WD repeat proteins principally expressed in the brain. J Biol Chem 2000;275:19970 – 7. 137. Iino S, Kobayashi S, Maekawa S. Immunohistochemical localization of a novel acidic calmodulin-binding protein, NAP-22, in the rat brain. Neuroscience 1999;91:1435 – 44. 138. Agassandian C, Plantier M, Fattoum A, Represa A, der Terrossian E. Subcellular distribution of calponin and caldesmon in rat hippocampus. Brain Res 2000;887:444 – 9. 139. Brophy PJ, Boccaccio GL, Colman DR. The distribution of myelin basic protein mRNAs within myelinating oligodendrocytes. Trends Neurosci 1993;16:515 – 21. 140. Medrano S, Steward O. Differential mRNA localization in astroglial cells in culture. J Comp Neurol 2001; 430:56 – 71. 141. Landry CF, Watson JB, Kashima T, Campagnoni AT. Cellular influences on RNA sorting in neurons and glia: an in situ hybridization histochemical study. Mol Brain Res 1994;27:1 – 11. 142. Carson JH, Kwon S, Barbarese E. RNA trafficking in myelinating cells. Curr Opin Neurobiol 1998;8: 607 – 12. 143. Sarthy PV, Fu M, Huang J. Subcellular localization of an intermediate filament protein and its mRNA in glial cells. Mol Cell Biol 1989;9:4556 – 9. 144. Ainger K, Avossa D, Morgan F, Hill SJ, Barry C, Barbarese E, Carson JH. Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes. J Cell Biol 1993;123:431 – 41. 145. Barbarese E, Koppel DE, Deutscher MP, Smith CL, Ainger K, Morgan F, Carson JH. Protein translation components are colocalized in granules in oligodendrocytes. J Cell Sci 1995;108:2781 – 90. 146. Plantier M, Fattoum A, Menn B, Ben-Ari Y, Der Terrossian E, Represa A. Acidic calponin immunoreactivity in postnatal rat brain and cultures: subcellular localization in growth cones, under the plasma membrane and along actin and glial filaments. Eur J Neurosci 1999;11:2801 – 12. 147. Palfi A, Gulya K. Water deprivation upregulates the three calmodulin genes in exclusively the supraoptic nucleus of the rat brain. Mol Brain Res 1999;74:111 – 6. 148. Palfi A, Simonka JA, Pataricza M, Tekulics P, Lepran I, Papp G, Gulya K. Postischemic calmodulin gene expression in the rat hippocampus. Life Sci 2001;68:2373 – 81. 149. Michelhaugh SK, Pimputkar G, Gnegy ME. Alterations in calmodulin mRNA expression and calmodulin content in rat brain after repeated, intermittent amphetamine. Mol Brain Res 1998;62:35 – 42. 150. Ni B, Brown IR. Modulation of a neuronal calmodulin mRNA species in the rat brain stem by reserpine. Neurochem Res 1993;18:185 – 92. 151. Barron S, Tusell JM, Serratosa J. Effect of hexachlorocyclohexane isomers on calmodulin mRNA expression in the central nervous system. Mol Brain Res 1995;30:279 – 86.

2854

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

152. Rao A, Steward O. Evaluation of RNAs present in synaptodendrosomes: dendritic, glial, and neuronal cell body contribution. J Neurochem 1993;61:835 – 44. 153. Wu K, Carlin R, Siekevitz P. Binding of L-[3H]glutamate to fresh or frozen synaptic membrane and postsynaptic density fractions isolated from cerebral cortex and cerebellum of fresh or frozen canine brain. J Neurochem 1986;46:831 – 41. 154. Rao A, Steward O. Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes. J Neurosci 1991;11:2881 – 95. 155. Terrian DM, Johnston D, Claiborne BJ, Ansah-Yiadom R, Strittmatter WJ, Rea MA. Glutamate and dynorphin release from a subcellular fraction enriched in hippocampal mossy fiber synaptosomes. Brain Res Bull 1988;21:343 – 51. 156. Bain G, Ray WJ, Yao M, Gottlieb DI. Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture. Biochem Biophys Res Commun 1996;223:691 – 4. 157. Okabe S, Forsberg-Nilsson K, Spiro AC, Segal M, McKay RD. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech Dev 1996;59:89 – 102. 158. Strubing C, Ahnert-Hilger G, Shan J, Wiedenmann B, Hescheler J, Wobus AM. Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons. Mech Dev 1995;53:275 – 87. 159. Brewer GJ, Torricelli JR, Evege EK, Price PJ. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res 1993;35:567 – 76. 160. Beaman-Hall CM, Wainer BH, Eves E, Bohn MC. Expression of glucocorticoid and mineralocorticoid receptors in an immortalized hippocampal neuronal cell line. Brain Res 1996;726:141 – 52. 161. Berger F, Gage FH, Vijayaraghavan S. Nicotinic receptor-induced apoptotic cell death of hippocampal progenitor cells. J Neurosci 1998;18:6871 – 81. 162. Eves EM, Tucker MS, Roback JD, Downen M, Rosner MR, Wainer BH. Immortal rat hippocampal cell lines exhibit neuronal and glial lineages and neurotrophin gene expression. Proc Natl Acad Sci U S A 1992;89: 4373 – 7. 163. Eves EM, Kwon J, Downen M, Tucker MS, Wainer BH, Rosner MR. Conditional immortalization of neuronal cells from postmitotic cultures and adult CNS. Brain Res 1994;656:396 – 404. 164. Wainwright MS, Perry BD, Won LA, O’Malley KL, Wang WY, Ehrlich ME, Heller A. Immortalized murine striatal neuronal cell lines expressing dopamine receptors and cholinergic properties. J Neurosci 1995;15: 676 – 88. 165. Giordano M, Takashima H, Herranz A, Poltorak M, Geller HM, Marone M, Freed WJ. Immortalized GABAergic cell lines derived from rat striatum using a temperature-sensitive allele of the SV40 large T antigen. [published erratum appears in Exp Neurol 1994 Apr;126(2):313] Exp Neurol 1993;124:395 – 400. 166. Seigel GM, Rollins J, Mhyre TR, Poles T, Fideli U, Holliday J. Immortalized cerebellar cells can be induced to display mature neuronal characteristics. Neuroscience 1996;74:511 – 8. 167. Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A 1976;73:2424 – 8. 168. Wood JN, Bevan SJ, Coote PR, Dunn PM, Harmar A, Hogan P, Latchman DS, Morrison C, Rougon G, Theveniau M, et al. Novel cell lines display properties of nociceptive sensory neurons. Proc R Soc Lond B Biol Sci 1990;241:187 – 94. 169. Senatorov VV, Trudeau VL, Hu B. Immunofluorescence in situ hybridization (IFISH) in neurones retrogradely labelled with rhodamine latex microspheres. Brain Res Protoc 1997;1:49 – 56. 170. Durrant I, Brunning S, Eccleston L, Chadwick P, Cunningham M. Fluorescein as a label for non-radioactive in situ hybridization. Histochem J 1995;27:94 – 9. 171. Breininger JF, Baskin DG. Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification. J Histochem Cytochem 2000;48:1593 – 9. 172. Zaidi AU, Enomoto H, Milbrandt J, Roth KA. Dual fluorescent in situ hybridization and immunohistochemical detection with tyramide signal amplification. J Histochem Cytochem 2000;48:1369 – 75.

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855

2855

173. Yang H, Wanner IB, Roper SD, Chaudhari N. An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs. J Histochem Cytochem 1999;47:431 – 46. 174. Kacharmina JE, Crino PB, Eberwine J. Preparation of cDNA from single cells and subcellular regions. Methods Enzymol 1999;303:3 – 18. 175. Eberwine J, Yeh H, Miyashiro K, Cao Y, Nair S, Finnell R, Zettel M, Coleman P. Analysis of gene expression in single live neurons. Proc Natl Acad Sci U S A 1992;89:3010 – 4. 176. Cimino M, Chen JF, Weiss B. Ontogenetic development of calmodulin mRNA in rat brain using in situ hybridization histochemistry. Dev Brain Res 1990;54:43 – 9. 177. MacManus JP, Gillen MF, Korczak B, Nojima H. Differential calmodulin gene expression in fetal, adult, and neoplastic tissues of rodents. Biochem Biophys Res Commun 1989;159:278 – 82. 178. Steward O, Falk PM. Protein-synthetic machinery at postsynaptic sites during synaptogenesis: a quantitative study of the association between polyribosomes and developing synapses. J Neurosci 1986;6:412 – 23. 179. Seto-Ohshima A, Kitajima S, Sano M, Kato K, Mizutani A. Immunohistochemical appearance of calmodulin in the developing brain: a comparison with neuron specific enolase. Cell Struct Funct 1984;9:337 – 44. 180. MacManus JP, Braceland BM, Rixon RH, Whitfield JF, Morris HP. An increase in calmodulin during growth of normal and cancerous liver in vivo. FEBS Lett 1981;133:99 – 102. 181. Soriano M, Pujol MJ, Bachs O. Possible cyclic AMP-dependence of the prereplicative surge of cytosolic calmodulin in proliferatively activated rat liver cells. J Cell Physiol 1988;135:345 – 9. 182. Li CJ, Heim R, Lu P, Pu Y, Tsien RY, Chang DC. Dynamic redistribution of calmodulin in HeLa cells during cell division as revealed by a GFP-calmodulin fusion protein technique. J Cell Sci 1999;112:1567 – 77. 183. Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711. 184. Program Manual for the EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 1RQ, England. Additional code by Peter Rice, The Sanger Centre, Hinxton, England and other members of the EGCG team. 185. Banker GA, Cowan WM. Rat hippocampal neurons in dispersed cell culture. Brain Res 1977;126:342 – 97.