www.elsevier.com/locate/ymcne

Mol. Cell. Neurosci. 28 (2005) 683–693

Characterization of the NF2 protein merlin and the ERM protein

ezrin in human, rat, and mouse central nervous system

Mikaela Grfnholm,a,* Tambet Teesalu,b Jaana Tyynel7,c Katja Piltti,d Tom Bfhling,e

Kirmo Wartiovaara,d Antti Vaheri,f and Olli Carpena,g,h

aNeuroscience Program, Biomedicum Helsinki, University of Helsinki and Helsinki University Central Hospital,

PB 63, Haartmaninkatu 8, 00014 Helsinki, FinlandbDepartment of Neurology and Neurosurgery, University of Tartu, 50406 Tartu, EstoniacDepartment of Biochemistry, University of Helsinki and Helsinki University Central Hospital, 00014 Helsinki, FinlanddInstitute of Biomedicine, Developmental Biology, University of Helsinki and Helsinki University Central Hospital, 00014 Helsinki, FinlandeDepartment of Pathology, University of Helsinki and Helsinki University Central Hospital, 00014 Helsinki, FinlandfDepartment of Virology, University of Helsinki and Helsinki University Central Hospital, 00014 Helsinki, FinlandgDepartment of Anatomy, University of Helsinki, 00014 Helsinki, FinlandhDepartment of Pathology, University of Turku and Turku University Hospital, 20520 Turku, Finland

Received 7 September 2004; revised 12 November 2004; accepted 19 November 2004

Available online 21 January 2005

The neurofibromatosis 2 (NF2) protein, merlin, is structurally related

to the ERM (ezrin-radixin-moesin) protein family of membrane-

cytoskeleton linkers and is mutated in nervous system tumors. Apart

from tumor suppressor activity, merlin’s functions are poorly under-

stood. We compared the localization and expression of merlin and ezrin

in developing and adult brain and in brain-derived progenitor cells.

Both proteins were widely but differentially expressed in human, rat,

and mouse brain. In brain tissue and neuronal progenitor cell cultures

merlin was predominantly found in neurons while ezrin was expressed

in astrocytes. Merlin expression was seen from E11 in mouse embryos,

whereas ezrin was present earlier. Both proteins were expressed in

embryonic mouse neurospheres, where ezrin was specifically localized

in filopodia of adherent neuronal progenitor cells. Subcellular analysis

demonstrated ezrin in fine filopodial structures in astrocytes, while

merlin was detected in neuronal synaptic junctions. The widespread

expression of merlin in brain and its association with protein kinase A

suggest a role for merlin in brain biology.

D 2004 Elsevier Inc. All rights reserved.

Introduction

The neurofibromatosis 2 (NF2) tumor suppressor protein merlin

(schwannomin) is structurally related to the ezrin-radixin-moesin

(ERM) family of proteins (Rouleau et al., 1993; Trofatter et al.,

1993). Merlin and ERM proteins serve as linkers between the actin

1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.mcn.2004.11.014

* Corresponding author. Fax: +358 9 47171964.

E-mail address: mikaela.gronholm@helsinki.fi (M. Grfnholm).

Available online on ScienceDirect (www.sciencedirect.com).

cytoskeleton and cell membrane components and are involved in

the organization of membrane extensions and in cell adhesion,

membrane traffic, cell signaling, and regulation of cell growth

(Bretscher et al., 2002). Biallelic inactivation of the NF2 gene,

which encodes for merlin, leads to the development of benign

tumors of the nervous system; schwannomas, meningiomas, and

some ependymomas in the dominantly inherited NF2 disease

(Louis et al., 1995), whereas increased expression of ezrin is

associated with enhanced cell growth and poor prognosis of

malignant tumors, including gliomas (Geiger et al., 2000; Khanna

et al., 2004; Makitie et al., 2001; Tynninen et al., 2004). Animal

models suggest a role for merlin across many different cell types

and in early fetal development (Giovannini et al., 2000; Kalamar-

ides et al., 2002; McClatchey et al., 1997, 1998).

To gain insight in the role of merlin in the central nervous

system (CNS), we compared the localization and expression of

merlin and one of the ERM proteins, ezrin, which has partial

functional homology with merlin and which forms heterotypic

interactions with merlin (Grfnholm et al., 1999). Both merlin and

ezrin are known to be present in the CNS (Arakawa et al., 1994;

Claudio et al., 1994, 1995; den Bakker et al., 1995, 1999; Gutmann

et al., 1995; Haase et al., 1994; Hara et al., 1994; Huynh et al.,

1994, 1996; Stemmer-Rachamimov et al., 1997) but systematic

comparison between their expression pattern has not been

performed, and the subcellular distribution of merlin has been

poorly characterized. There is also no knowledge on expression

of ezrin and merlin in neuronal progenitor cells. Further knowl-

edge of the expression pattern is important also in light of new

potential functions of merlin and ezrin in CNS cells. We have

recently shown that merlin binds to the RIh subunit of protein

kinase A, PKA (Grfnholm et al., 2003), a subunit almost

M. Gronholm et al. / Mol. Cell. Neurosci. 28 (2005) 683–693684

exclusively expressed in neurons of the CNS. A targeted

disruption of the gene encoding for RIh in mouse resulted in

defective hippocampal synaptic plasticity (Brandon et al., 1995;

Huang et al., 1995). Another report biochemically showed that

merlin forms a complex together with Caspr/paranodin and

integrin h1 in the CNS (Denisenko-Nehrbass et al., 2003). Ezrin

may have a role in neuronal morphogenesis and adhesion through

its interaction with the axonal cell adhesion molecule L1

(Dickson et al., 2002) and in the formation and function of

peripheral astrocytic processes (PAPs) (Derouiche and Frotscher,

2001).

In this study, we have thoroughly compared the expression and

localization of merlin and ezrin in the CNS. Human, rat, and mouse

brain tissues were analyzed by Western blotting and immunohis-

tochemistry of a human brain tissue array was performed.

Subcellular localization of both proteins was studied by immuno-

cytochemistry of primary rat hippocampal and neuronal progenitor

cell cultures and by rat brain fractionations.

Fig. 1. Merlin and ezrin expression in adult and developing human, mouse

and rat brain. (A) Western blot analysis of merlin and ezrin in human,

mouse and rat brain. U251 malignant glioma cell lysate (U251) and lysates

of human cortex (CO), cerebrum (CB), cerebellum (CL) and hippocampus

(HC) were analyzed. Merlin and ezrin are expressed in all samples studied.

(B) Analysis of fetal mouse lysates from E5 to E18. Merlin can be detected

from E11 while ezrin is expressed at variable levels from E5. (C) Analysis

of mouse brain lysates from E18 to 1 year old. Day (D), week (W), month

(M), year (Y). Merlin and ezrin are expressed at various levels. Merlin was

detected with rabbit antiserum A19 and ezrin with mAb 3C12.

Results

Western blot analysis of merlin and ezrin in developing and adult

brain

Western blot analysis of merlin and ezrin was performed on

lysates from various regions of adult human, mouse, and rat CNS

and on various stages of mouse development. Widespread

expression of merlin and ezrin was detected in adult samples

studied; human cortex, mouse and rat cerebrum and cerebellum,

and adult hippocampus (Fig. 1A). Analysis of fetal lysates of

mouse embryonic stages from E5 to E18 demonstrated strong

merlin expression from E11 onwards, whereas no expression was

seen before this stage. Various amounts of ezrin were present from

E5 with highest expression at E8, E11, E12, and E18 (Fig. 1B). In

a Western blot of brain from mouse E18 fetus to 1-year old, merlin

and ezrin were expressed at various levels. The strongest merlin

and ezrin expressions were seen at E18 and day 1, with a clear

reduction after day 1. Stronger merlin expression could also be

seen at the age of 2 months and a small ezrin increase around 2

weeks to 1 month (Fig. 1C).

Lysates of different human brain regions were immunoblotted

for merlin and ezrin (Fig. 2). Merlin reactivity was present in

most regions; in the brain stem, cerebellum, diencephalon, basal

ganglia, corpus callosum, hypophysis, and the optic nerve, while

weaker reactivity was detected in cortex of the temporal lobe,

globus pallidus, and hippocampus. A double band of merlin,

possibly representing differently phoshorylated forms (Alfthan et

al., 2004; Kissil et al., 2002; Xiao et al., 2002) was seen in brain

stem and diencephalon. Ezrin reactivity was detected in cerebral

cortex, basal ganglia, hippocampus, hypophysis, and the optic

nerve, while the reactivity was weak in the brain stem and

diencephalon. Comparison of grey and white matter of frontal

lobe, demonstrated stronger reactivity in the grey matter for both

merlin and ezrin.

Immunohistochemistry of human brain tissue array

We further studied the presence of merlin and ezrin by

immunohistochemistry in a human brain tissue array representing

40 different brain areas. Merlin was detected predominantly in

neurons and ezrin in astrocytes in most tissues studied (Fig. 3).

Merlin staining of neuronal cells was strongest in the frontal

cortex, medulla, thalamus (Figs. 3 and 4), and capsula interna

(not shown). Strong ezrin staining of astrocytes was seen in the

hippocampus, frontal cortex, thalamus (Fig. 3), parahippocampal

cortex, amygdala, insula, and corpus callosum (not shown). No

ezrin was detected in neurons in most tissues studied. However,

in the brain stem, medulla, mesencephalon, pons (Fig. 4), olivary

nuclei, substantia nigra, and nucleus dentatus (not shown) strong

neuronal ezrin immunoreactivity was seen. Astrocytes were

present in these brain regions, as shown by GFAP staining, but

they were ezrin-negative (Fig. 4). Merlin and ezrin staining was

consistent in tissue arrays from five different patients (ages 1.5

month, 44, 47, 53 and 78 year). As an exception, ezrin staining

of cerebellum from a 1.5-month-old child was different from

adult samples (Fig. 5). While ezrin staining was weak in all adult

brains, very strong ezrin staining was detected in the Purkinje cell

layer and in part of the molecular layer of the infant brain.

However, Purkinje cells were ezrin-negative. Merlin, on the other

hand, was expressed in Purkinje cells and we could see no

difference in the intensity of merlin immunoreactivity between

infant and adult brain.

Fig. 2. Western blot analysis of merlin and ezrin in human brain tissue

lysates. Spinal cord (SC), cauda equina (CE), medulla oblongata (MO),

olivary nuclei (ON), pons dorsal (PD), pons ventral (PV), cerebellum cortex

(CR), nucleus dentatus (ND), substantia nigra (SN), thalamus (TH),

mammillary bodies (MB), hypophysis (HY), frontal lobe grey matter

(FG), frontal lobe white matter (FW), temporal lobe cortex (TC), parietal

lobe cortex (PC), occipital lobe cortex (OC), insula (IN), enthorhinal cortex

(EC), nucleus caudatus head (NH), nucleus caudatus body (NB), putamen

(PU), globus pallidus (GP), hippocampus (HC), corpus callosum (CC),

internal capsule (IC), optic nerve (ON), choroid plexus (CP), kidney cortex

(KC). Merlin was detected with the rabbit antiserum 1398 and ezrin with

the mAb 3C12.

Fig. 3. Differential expression pattern of merlin and ezrin in human brain.

Immunohistochemical staining of human brain sections. Frontal cortex

(CO), hippocampus (HC), thalamus (TH) and cerebellum (CB). Merlin was

detected with the 1398NF2 rabbit antiserum and ezrin with the 3C12 mAb.

Preimmune serum and X63 were used as controls. Merlin staining is seen in

neurons, while ezrin expression is predominant in astrocytes (arrows).

Purkinje cells of the cerebellum (arrow heads) express merlin but not ezrin.

Scale bar = 10 Am.

M. Gronholm et al. / Mol. Cell. Neurosci. 28 (2005) 683–693 685

Merlin and ezrin in brain progenitor cells

Neuronal progenitor cells from mouse were cultured as floating,

multicellular neurospheres, which can self-renew and generate both

neuronal and glial cells. Neurospheres were cytocentrifuged onto

objective slides stained with merlin and ezrin antibodies. Partial

colocalization was seen between the two proteins (Figs. 6A–C).

When neurospheres were grown in the presence of serum, they

started to adhere to the substratum, migrate, and differentiate. In

attaching neurospheres, ezrin was predominantly seen in the outer

cell layer in short filopodia, while merlin was present in the cell

body (Figs. 6D–F). In migrating and differentiating cells, fine

ezrin-positive extensions could be seen (Figs. 6H–I) in cells

expressing the astrocyte marker GFAP (not shown). Ezrin-negative

and merlin-positive cells were also present (Fig. 6I), and these cells

expressed the neuronal marker TUJ-1 (not shown). Costaining of

the differentiating neuronal progenitor cells with glial and neuronal

markers confirmed the presence of merlin predominantly in

neurons and ezrin in astrocytes. Merlin staining was strongest in

TUJ-1 expressing neurons (Figs. 7A–C), which were ezrin-

negative (Figs. 7D–F). Ezrin however, was present in all GFAP-

positive cells (Figs. 7J–L), in which only weak merlin staining was

seen (Figs. 7G–I).

Subcellular distribution of merlin and ezrin in brain cells

In primary cultures of rat hippocampal cells, ezrin was

detected in astrocytes (Figs. 8A, C). The immunoreactivity

concentrated to cell extensions and in delicate peripheral

processes (Fig. 8D). Merlin staining in astrocytes was weak

and mainly detected in the cell body (Figs. 8B–C). In neurons, an

opposite staining pattern was detected. No ezrin was seen in

neurons while merlin staining was strong in the cell soma and

along extensions in a punctuate manner (Figs. 8A, C–D). To

further study merlin’s subcellular localization, double staining

with merlin and the presynaptic marker synapsin I or the

postsynaptic density marker PSD-95 was performed. Synapsin I

containing structures were not stained with merlin antibody (Figs.

8E–H), whereas merlin and PSD-95 colocalized in many

Fig. 5. Ezrin is strongly expressed in infant human cerebellum.

Immunohistochemical staining of three human cerebellar specimens, aged

1.5 month, 53 and 78 year. Ezrin was detected with 3C12 mAb and merlin

with 1398NF2 rabbit antiserum. Merlin staining is similar in all specimens.

Instead, ezrin expression is very high in the Purkinje cell layer and parts of

the molecular layer in the cerebellum from 1.5 month old as compared to

other specimens. Merlin is detected in Purkinje cells (arrow) which are

ezrin-negative.

Fig. 4. Ezrin is expressed in neurons in human medulla oblongata, pons,

and mesencephalon. Immunohistochemical staining of medulla oblongata

(MO), pons (PO), and mesencephalon (ME) as in Fig. 3. Merlin was

detected with 1398NF2 rabbit antiserum, ezrin with the 3C12 mAb and

GFAP with GFAP mAb. Preimmune serum and X63 were used as

controls. Neurons in these tissues express both merlin and ezrin. GFAP-

positive astrocytic cells are not stained with ezrin antibody. Scale bar =

10 Am.

M. Gronholm et al. / Mol. Cell. Neurosci. 28 (2005) 683–693686

punctuate structures (Figs. 8I–L). Since merlin is known to bind

to PKA-RIh in brain, we costained cells with RIh rabbit

antiserum and PSD-95. Also RIh colocalized with PSD-95 (Figs.

8M–P). Punctuate merlin-positive structures which were negative

for PSD-95 (Fig. 8L) and synapsin I were also seen (Fig. 8H). The

presence of merlin and RIh in synaptic junctions was confirmed by

synaptic fractionations of rat brain lysates (Fig. 9). Merlin and RIhwere detected in synaptosomes and in the synaptic junction/PSD-

fraction. Weak ezrin reactivity was detected in synaptosomes and

in the synaptic vesicle fraction. Synaptophysin, PSD-95 and

SNAP-25 were used as fractionation controls.

Discussion

The present study demonstrates that both merlin and ezrin

are widely expressed in the CNS of mouse, rat and human.

However, the homologous proteins have a markedly different

expression pattern. In neuronal progenitor cell neurospheres both

proteins are present and partly colocalize, but as cells start to

differentiate and mature they become restricted to different cell

types. Merlin is more prominent in neurons and ezrin almost

exclusively found in astrocytes in most brain parts studied by

immunohistochemistry and in rat hippocampus and mouse

progenitor cell cultures.

Fig. 6. Localization of merlin and ezrin in mouse neurospheres. Neurospheres from E11 mouse brain were allowed to attach for 1 h (A–C), 5 h (D–F) or 10 h

(G–I) and stained with merlin A19 rabbit antiserum and ezrin 3C12 mAb. In spheres, an overlapping staining pattern of merlin and ezrin is seen. As spheres

attach, ezrin is present in the outer layer of cells and localized to short filopodia (F, insert). When cells start to migrate and differentiate, ezrin is concentrated to

filopodia, while merlin is present in the cell body of ezrin-expressing cells and in a neuronlike ezrin-negative cell (I, arrow). Western blot analysis of merlin and

ezrin in neurosphere lysates. Merlin was detected with A19 and ezrin with Ez9 rabbit antiserum. Scale bar = 10 Am.

M. Gronholm et al. / Mol. Cell. Neurosci. 28 (2005) 683–693 687

Expression and distribution of merlin and ezrin in adult tissues

In previous immunohistochemical and cell culture studies,

merlin mRNA or protein was detected in neurons (Claudio et al.,

1995; Gutmann et al., 1995; Huynh et al., 1996), but not in glial

cells (den Bakker et al., 1999; Derouiche and Frotscher, 2001) in

accordance to our study. However, two previous studies (Huynh et

al., 1996; Stemmer-Rachamimov et al., 1997) detected merlin in

glial cells of mouse E13 spinal cord and adult human brain,

respectively. In the study by Stemmer-Rachamimov et al. (1997),

astrocytes but not neurons of the cerebral cortex were merlin-

positive, in contrast to the present results. There are also conflicting

results of the cerebellar staining of merlin. The Purkinje cells are

positive in human and rat according to den Bakker et al. (1999) and

Gutmann et al. (1995), but Stemmer-Rachamimov et al. (1997)

could not detect merlin in human Purkinje cells, which were

merlin-positive in our study. Granular cells were negative in human

(den Bakker et al., 1999; Stemmer-Rachamimov et al., 1997) but

positive in rat (Gutmann et al., 1995) while molecular layers were

negative (den Bakker et al., 1999; Gutmann et al., 1995). The

reason for discrepant results is not known, but it may be related to

the different antibodies and methods used.

In accordance with our study, ezrin expression has previously

been detected in rat brain (Derouiche and Frotscher, 2001; Scherer

et al., 2001), where it was present specifically in fine filopodia of

astrocytes (Derouiche and Frotscher, 2001) and within radial glia in

prenatal human cerebrum (Johnson et al., 2002). We did not detect

ezrin in neurons in most regions of adult rat or human brain. An

exception was adult human brain stem, where neurons were ezrin-

and merlin-positive.

Expression of merlin and ezrin during development and

differentiation

Merlin and ezrin may be developmentally regulated and show

changes in expression levels during development versus mature

Fig. 7. Localization and expression of merlin and ezrin in mouse neuronal progenitor cells. Neurospheres cultured in the presence of serum for 5 days were

stained with A19 rabbit antiserum for merlin, Ez9 rabbit antiserum for ezrin, TUJ-1 to detect neurons and GFAP to detect astrocytes. Merlin (C) but not ezrin

(F) is seen in cells positive for TUJ-1. Merlin antibody stains GFAP-positive cells weakly (I) while all GFAP-positive cells are ezrin-positive (L). Scale bar =

10 Am (J).

M. Gronholm et al. / Mol. Cell. Neurosci. 28 (2005) 683–693688

cells (Gutmann et al., 1995; Huynh et al., 1996). From mouse E8

through E10, the CNS is composed of mostly undifferentiated

neuroepithelia but by E11–E13 the brain and spinal cord are

developed. We could detect an abrupt neoexpression of merlin at

E11 in lysates from whole fetus, which interestingly coincides with

the onset of neurogenesis. Previously, merlin mRNA was detected

from E8 in mouse (Huynh et al., 1994) and in rat most tissues

express merlin mRNA from E12, while expression becomes

increasingly restricted to neuronal tissues by E15 and E17

(Gutmann et al., 1995). Huynh et al. (1996) detected the merlin

protein in a wide range of embryonic mouse tissues after E10. This

expression appeared to be increased in fully differentiated tissues

suggesting that merlin might play an important role during

morphogenesis and organogenesis.

In primary cultures from rat hippocampus, both merlin and ezrin

were present in GFAP- and TUJ-1-positive cells in 1- to 3-day

cultures (not shown), but no neuronal ezrin was seen in cultures

grown for more than 3 days. This is in accordance with previous

studies which showed that ezrin expression was high in young

cultured neurons but dropped off dramatically after the first week of

growth (Dickson et al., 2002; Paglini et al., 1998; Zhang and

Hutchins, 1997). Neuronal localization of ERM proteins has been

detected in dorsal root ganglia of chick embryo (Takahashi et al.,

1999) but has not been detected in adult rat CNS or prenatal human

cerebrum (Derouiche and Frotscher, 2001; Johnson et al., 2002).

The expression pattern of merlin and ezrin in cultured mouse

neuronal progenitor cells mimicked the pattern seen in differ-

entiated CNS cells. Both merlin and ezrin were expressed in the

Fig. 8. Localization of merlin and ezrin in primary rat hippocampal cultures. Seven day primary rat hippocampal cultures were stained with A19 rabbit

antiserum (merlin), 3C12 mAb (ezrin), RIh rabbit antiserum, PSD-95 mAb, and synapsin I mAb. Merlin is seen predominantly in neurons, which are ezrin-

negative. Merlin staining is detected in the cell body and in extensions in a punctuate pattern (A, D). Also, ezrin-positive astrocytes show weak merlin staining

in the cell body (C). Ezrin is localized in the cell body and in fine filopodia in astrocytes (B, D). There is no colocalization between synapsin I and merlin (G,

H) while PSD-95 is present in most but not all merlin-positive (K, L) and RIh-positive (O, P) punctuate structures. Scale bar = 10 Am.

M. Gronholm et al. / Mol. Cell. Neurosci. 28 (2005) 683–693 689

undifferentiated neurosphere cells, but when differentiation was

initiated the expression became restricted to different cell types.

Merlin, but not ezrin, was expressed in neurons while ezrin

expression was strong in astrocytes, which were merlin-negative.

Fig. 9. Merlin and RIh is present in the synaptic junction/PSD fraction of

rat brain. Western blot analysis of rat brain fractions. Total brain (tot),

microsomal pellet (S2), postnuclear sup (P2), synaptosomes (syn), synaptic

vesicles (ves), and synaptic junction/PSD (jnt) were blotted for merlin with

A19 rabbit antiserum, ezrin with 3C12 mAb and RIh rabbit antiserum.

Synaptophysin (synaptoph), PSD-95, and SNAP-25 were detected as

fractionation controls. Merlin and RIh, but not ezrin, are seen in the

synaptic junction/PSD fraction. Weak ezrin reactivity is seen in the synaptic

vesicle fraction, which is merlin and RIh-negative.

Neither protein was detected in oligodendrocytes or microglia (not

shown). A potential role for merlin and ezrin in the differentiation

process remains to be elucidated.

Subcellular distribution of merlin and ezrin in CNS cells

We have previously shown that ezrin and merlin interact with

each other (Grfnholm et al., 1999), but the lack of coexpression of

the two proteins after the onset of mouse neuronal progenitor cell

differentiation and in adult brain makes ezrin an unlikely

interaction partner for merlin in the adult CNS. The only

exceptions in the differential expression pattern were neurons of

the brain stem. In cell cultures merlin and ezrin colocalize at the

cell membrane in regions of dynamic cytoskeleton remodeling

(Gonzalez-Agosti et al., 1996; Grfnholm et al., 1999; Sainio et al.,

1997). In this study, no colocalization of merlin and ezrin in actin-

rich structures could be seen. Ezrin, but no merlin was present in

the fine filopodial structures in migrating neuronal progenitor cells

and mature astrocytes. On the other hand, merlin, but not ezrin,

was detected in another actin-rich structure, the neuronal dendritic

spine.

Fine ezrin-positive filopodial extensions were seen in the mouse

neuronal progenitor cells. When neurospheres were allowed to

attach microspikes extending from the border cells were seen. As

the border cells migrated away from the neurosphere, longer

extensions could be seen with strong ezrin immunoreactivity.

These structures were seen in GFAP-positive cells but not TUJ-1-

positive cells indicating that they are cells differentiating into glial

cells. This could indicate a role for ezrin in migration of diffe-

rentiating glial cells.

Glial cells play an important role in regulation of synapse

structure and function. Particularly, astrocytes extend long pro-

M. Gronholm et al. / Mol. Cell. Neurosci. 28 (2005) 683–693690

cesses that engulf the synaptic terminal. With these integrated

processes astrocytes can modulate synaptic activity and effectively

take up transmitters released by neurons (Haydon, 2001). In this

study, PAPs of cultured hippocampal astrocytes were strongly

labeled for ezrin in analogy with Derouiche and Frotscher (2001),

who suggested a potential role for ezrin in the regulation of PAPs

morphogenic properties and its connections to signaling pathways.

Merlin was localized to the cell body and along dendrites in a

punctuate manner but only weak merlin staining was detected in

axons of cultured neurons. This is in accordance with Western blot

results demonstrating stronger merlin reactivity in grey than in

white matter. In neurons, the signal transduction machinery is

localized to thousands of small compartments at presynaptic and

postsynaptic sites. PSDs are present at the tips of dendritic spines,

which are contact sites for most excitatory synapses in the brain.

They undergo morphological changes in response to activities that

are associated with neuronal plasticity, learning, and memory

functions (Yuste and Bonhoeffer, 2001). The PSD is localized to

the postsynaptic membrane in register with the active zones of the

presynaptic terminal and provides a structural framework for

localizing functional molecules, regulating adhesion, controlling

receptor clustering, and regulating receptor function (Siekevitz,

1985). Drugs that inhibit actin dynamics suppress long-term

potentiation (LTP) and block shape changes in dendritic filopodia

and spines (Kim and Lisman, 1999; Krucker et al., 2000). We

purified synaptic vesicles and junctions/PSD from rat brain and

detected merlin in the synaptic junction/PSD fraction. An

important signaling route involved in synaptic plasticity in the

PSD is the cAMP-PKA pathway (Bauman et al., 2004; Brandon et

al., 1997). We have previously shown that merlin is an A kinase

anchoring protein (AKAP) for RIh containing PKA (Grfnholm et

al., 2003), which is most abundantly expressed in brain (Clegg et

al., 1988; Solberg et al., 1991). We now show that RIh, like merlin,

colocalizes with PSD-95 along dendrites of cultured hippocampal

neurons (Fig. 8O) and is present in the synaptosome and PSD/

junction fraction of rat brain lysates. Hippocampal synaptic

plasticity including LTP and long-term depression (LTD) are

defective in mice carrying a targeted disruption of the gene

encoding for RIh (Brandon et al., 1995; Huang et al., 1995). Since

merlin and RIh are expressed in the same cell types in the CNS

including the hippocampus, have a similar subcellular distribution

and form a complex, one can speculate that merlin participates in

neuronal PKA signaling and memory functions.

In addition to PKA-RIh, merlin binds to beta II-spectrin/fodrin

(Scoles et al., 1998), actin, and tubulin (Brault et al., 2001; James

et al., 2001; Xu and Gutmann, 1998), all integral components of

the PSD (Kennedy, 1993) and with paranodin (Denisenko-

Nehrbass et al., 2003), which is enriched in synapses in the

hippocampus (Murai et al., 2002). Merlin also binds to integrins

(Obremski et al., 1998) and forms a complex with cadherins

(Lallemand et al., 2003), which are both components of synaptic

junctions. Cadherins have been suggested to play a role in

synaptic junction formation and synaptic plasticity (Tanaka et al.,

2000; Tang et al., 1998) and integrins in synaptic maturation

(Chavis and Westbrook, 2001). The widespread expression of

merlin in CNS and its specific interactions with signaling

molecules in neuronal cells suggest that merlin may be involved

in yet unknown functions in brain. It will be interesting to further

study merlin’s potential role in the regulation of actin rearrange-

ment in the dendritic spine and as a scaffolding protein in the

synaptic junction.

Experimental methods

Antibodies

All antibodies have been characterized. 1398NF2 rabbit

antiserum (den Bakker et al., 1995) and A19 rabbit antiserum

(Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) were used

to detect merlin. Specificity of merlin antibodies has been tested

(Muranen et al., in press). 3C12 mAb (Bohling et al., 1996) and

Ez9 rabbit antiserum (Alfthan et al., 2004) were used to detect

ezrin. Specificity of ezrin antibodies were tested by immunoblot-

ting lysates of mouse embryonic fibroblasts (MEFs) from ezrin

knockout mice. A band corresponding to ezrin was seen in the

MEF lysate from heterozygote but not from homozygote mice (not

shown). Additional antibodies used were TUJ-1 mAb (BAbCO/

Covance, Princeton, NJ), GFAP mAb (Chemicon International

Inc., Temecula, CA), synapsin I (Chemicon), PSD-95 mAb

(Chemicon), SNAP-25 mAb (Transduction Laboratories, Lexing-

ton, KY), synaptophysin mAb (Bibmeda, Foster City, CA) and

PKA-RIh rabbit antiserum (Calbiochem-Novabiochem, San

Diego, CA).

Protein studies

Different regions of human, mouse and rat brain were

homogenized in ELB-buffer (50 mM HEPES, pH 7.4, 150 mM

NaCl, 5 mM EDTA), 1% Nonidet P-40 and protease inhibitors.

Samples were normalized for protein concentration and equal

amounts of lysate in Laemmli sample buffer were subjected to

SDS-PAGE, transferred to nitrocellulose filters and immuno-

blotted with A19 merlin rabbit antiserum or 3C12 ezrin mAb.

Bound proteins were detected by enhanced chemiluminescence.

Pre-made Never fail Western blots (RNWAY laboratories,

Seoul, Korea), with 30 Ag of whole protein tissue lysates

from mouse embryo or mouse brain, were immunoblotted as

above. Never fail blot m202 includes developmental mouse

fetus from E5 to E18. E5–7 samples include maternal uterus

tissue, E8–10 samples are conceptus and E11–18 contain fetus

only. Never fail blot m207 includes whole brain from E18

fetus to 1 year old.

Collection of tissue samples

Tissues were collected during autopsies at the Department of

Pathology and Neuropathology of the Tartu University Clinics

according to procedures approved by the Ethics Committee of the

Faculty of Medicine, University of Tartu. Samples were obtained

from 5 patients (ages 1.5 month, and 44, 47, 53 and 78 year)

without evidence of neurological disease. Tissues were dissected

within 4–18 h after clinical death and either snap-frozen in liquid

nitrogen or fixed with freshly prepared 4% PFA in PBS (pH 7.4)

over night at 48C. Fixed tissues were dehydrated, paraffin-

embedded and sectioned at 6 Am. Areas of interest were

identified by histology and tissue arrays (Kononen et al., 1998)

with a 0.6-mm core diameter were constructed using a tissue

array instrument (Beecher Instruments Inc., Sun Prairie, WI). For

each location, 2–6 array cores were transferred into the recipient

block. Tissue arrays were sectioned using a rotary microtome at 6

Am and the sections were mounted in RNase-free conditions on

heat-treated Super Frost (+) slides (Menzel-Glaser R, Braunsch-

weig, Germany).

M. Gronholm et al. / Mol. Cell. Neurosci. 28 (2005) 683–693 691

Immunohistochemistry

Immunostaining of paraffin-embedded tissue sections was

performed using the avidin–biotin–peroxidase complex method

(Ultra Vision, Lab Vision Inc., Fremont, CA). For antigen retrieval,

the slides were incubated in 10 mM sodium citrate buffer (pH 6.0)

for 10 min at 958C. Merlin was detected with the 1398NF2 rabbit

antiserum (1:2000), ezrin with 3C12 mAb (1:1000), and GFAP

with a GFAP mAb (1:1000). Preimmune serum (1:1000) and X63

(ascites 1:2000) were used as controls. The slides were stained

using a LabVision Autostainer automatic immunostaining device

(Lab Vision Inc., Fremont, CA).

Neuronal progenitor cell cultures

Cells were collected by cutting brain lobes of E11 mouse fetus.

Tissue was suspended and cells were cultured in serum-free

DMEM, with 25% Nutrient mixture F-12 Ham (Sigma-Aldrich, St.

Louis, MO), 2% B27 supplement (Gibco/Invitrogen, Carlsbad,

CA), 1% Glutamax (Gibco), 1% Penicillin-Streptamycin (Gibco),

250 Ag/ml Fungizone (Gibco) supplied with 20 ng/ml EGF

(Sigma-Aldrich) and 40 ng/ml FGF2 (Sigma-Aldrich) (Toma et

al., 2001). Neurospheres were cytocentrifuged onto objective slides

and fixed in 4% PFA. Alternatively, cells were washed with EGF/

FGF2-free medium and allowed to attach as spheres or mechan-

ically disrupted to single cells by pipetting up and down and plated

on poly-l-lysine (50 Ag/ml) (Sigma) at a density of 20000–40000

cells/well onto 24-well plates. Cells were grown for 3–7 days in D-

MEM with 2% serum prior to fixation in 4% PFA. Cells were

stained with A19 rabbit antiserum (1:200) for merlin, 3C12 mAb

(1:200) for ezrin, TUJ-1 (1:200) as a neuronal marker, and GFAP

(1:400) as an astrocyte marker followed by Alexa 488 anti-mouse

and Alexa 594 anti-rabbit antibodies (Molecular Probes, Eugene,

OR). Coverslips were mounted in DABCO (Sigma-Aldrich) and

Mowiol (Calbiochem-Novabiochem) and examined by immuno-

fluorescence microscopy (Zeiss Axiophot equipped with AxioCam

cooled CCD-camera, Carl Zeiss, Esslingen, Germany).

Rat embryo hippocampal cell culture and stainings

Hippocampus was prepared from rat brains of 19-day-old rat

embryos as described (Banker and Cowan, 1977). The cells were

plated on poly-l-lysine-coated (Sigma-Aldrich) coverslips at a

density of 5000 cells/cm2 and cultured in Neurobasal medium

(Invitrogen, Carlsbad, CA) with B27 supplement (Invitrogen) for 7

days. Cells were fixed with 3.5% PFA and stained with A19 rabbit

antiserum (1:200) for merlin, 3C12 mAb (1:200) for ezrin, RIhrabbit antiserum (1:100), PSD-95 (1:50) or synapsin I (1:200)

followed by Alexa 488 anti-mouse and Alexa 594 anti-rabbit

antibodies (Molecular Probes).

Isolation of synaptic junctions and vesicles from rat brain

Synaptic junctions and vesicles were prepared as previously

described with modifications (Cho et al., 1992; Jo et al., 1999;

Suopanki et al., 2002). Four rat brains were homogenized by ten

strokes with a teflon-glass homogenizer, using 1 g brain for 3 ml of

homogenization buffer (320 mM sucrose, 4 mM HEPES, pH 7.3,

0.5 mM CaCl2, 1 mM MgCl2, protease inhibitors). The homoge-

nate was centrifuged first at 1.500 g for 10 min to pellet nuclei. The

postnuclear supernatant was centrifuged at 14.000 � g for 15 min,

the supernatant (S2) kept for analysis and the resulting pellet (P2),

containing synaptosomes, resuspended in 1 ml homogenization

buffer and layered on a discontinuous sucrose density gradient

consisting of 3 ml each of 0.8 M, 1.0 M and 1.2 M sucrose solution

in homogenization buffer. Gradients were run for 2 h at 80,000 � g

in 48C. Synaptosomes were collected from the border between 1.0

M and 1.2 M sucrose. To lyse the lysosomes and mitochondria, the

synaptosomes were diluted 1:10 in ice-cold 0.1 mM CaCl2,homogenized with a dounce homogenizer three times and kept

on ice for 20 min. The lysate was centrifuged at 25,000 � g for 20

min at 48C, and the 25,000 � g supernatant was further centrifuged

for 2 h at 260,000 � g in 48C. The resulting pellet represents the

synaptic vesicle fraction, and it was dissolved in 4 mM HEPES, 0.1

mM CaCl2 and 5% SDS. The 25,000 � g pellet was extracted with

Triton: it was resuspended in 320 mM sucrose, 4 mM HEPES pH

7.3, 0.5% Triton X-100 and 0.1 mM CaCl2, incubated 15 min on

ice and centrifuged at 32,000 � g for 20 min. The pellet was

resuspended in 320 mM sucrose, 4 mM HEPES pH 7.3, 0.5%

Triton X-100 and 0.1 mM CaCl2 and centrifuged for 1 h at

200,000 � g. The resulting pellet represents the synaptic junctions,

and it was dissolved in 320 mM sucrose, 4 mM HEPES pH 7.3,

0.5% Triton X-100 and 0.1 mM CaCl2. Samples were normalized

for protein content and equal amounts of lysate in Laemmli sample

buffer were subjected to SDS-PAGE, transferred to nitrocellulose

filters and immunoblotted with A19 merlin rabbit antiserum, 3C12

ezrin mAb, RIh rabbit antiserum, PSD-95 mAb, SNAP-25, or

synaptophysin. Purity of the preparations was checked by

immunoblotting with the above mentioned biochemical markers

and by electron microscopy. Isolations were repeated three times.

Acknowledgments

We thank Tuula Halmesvaara and Helena Ahola for skillful

technical assistance and Juha-Kuja Panula and Heikki Rauvala for

hippocampal cells. This work was supported by the United States

Army Neurofibromatosis Research Grant DAMD17-00-0550,

Finsk-Norska Medicinska Stiftelsen and Medicinska Under-

stfdsffreningen Liv och H7lsa.

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