Differential calmodulin gene expression in the rodent brain

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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

0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0024 -3205 (02 )01544 -8

* Corresponding author. Tel./fax: +36-62-544049.

E-mail address: [email protected] (K. Gulya).

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) 2829–28552830

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

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].

A. Palfi et al. / Life Sciences 70 (2002) 2829–2855 2831

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 micro-

glial 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 ex-

pression was described, e.g. in the olfactory bulb, the basal ganglia, the hippocampus-dentate


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,

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 DIG-

labelled 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.


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-galactosidasereporter 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 immunore-

activity 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 (espe-

cially 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


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-immunolabel-

ling 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


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).


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 protein-

synthesizing 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

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�.


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 cleavageand 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

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.


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)phy-

siological conditions has been reported. For example, the 2.3 kb CaM III transcript clearly

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.


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-UTRsof 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 trans-

acting 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-UTRdifferentiation control elements (DICEs) are recognized by trans-acting factors and the re-

sulting 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 physiolo-

gical 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 cytoske-

leton [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


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], mi-

crotubules [133] and transcriptional factors [28]. Thus, CaM-KII is a key factor in the post-

synaptic 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 signal-

processing 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


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 inves-

tigated. For example, chronic ethanol administration and its withdrawal altered the CaMmRNA

levels with a gene-specific pattern in the rat brain [51]. Modified mRNA contents were mainly


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 ImRNAcontent 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 themidbrain. In parallel, the CaMprotein

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 delta-

hexachlorocyclohexane (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]. Tran-

scriptional 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


the expressions of all three CaM genes are altered in a similar way (e.g. some are up-

regulated, 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 auto-

radiography or equivalent methods, and most CaM protein measurements were made by radio-

immunoassay, 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-spe-

cific 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 dif-

ferent 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 phenotypi-

cally 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


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].


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 con-

servation 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 evi-

dence 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 accu-

mulated 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.


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 consid-

erably. 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.

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Differential calmodulin gene expression in the rodent brain

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