Functional Manipulations of Acetylcholinesterase Splice Variants Highlight Alternative Splicing Contributions to Murine Neocortical Development

Functional Manipulations ofAcetylcholinesterase Splice VariantsHighlight Alternative Splicing Contributionsto Murine Neocortical Development

Amir Dori1,2, Jonathan Cohen1, William F. Silverman3, Yaakov

Pollack4 and Hermona Soreq2

1Department of Neurosurgery, Soroka University Medical

Center, Beer-Sheva, Israel, 2Life Sciences Institute, The

Hebrew University of Jerusalem, Jerusalem, Israel,3Department of Morphology, Zlotowski Center for

Neuroscience, Faculty of Health Sciences, Ben-Gurion

University of the Negev, Beer-Sheva, Israel and 4Department of

Microbiology and Immunology, Faculty of Health Sciences,

Ben-Gurion University of the Negev, Beer-Sheva, Israel

Proliferation and differentiation of mammalian central nervoussystem progenitor cells involve concertedly controlled transcrip-tional and alternative splicing modulations. Searching for thedevelopmental implications of this programming, we manipulatedspecific acetylcholinesterase (AChE) splice variants in the embry-onic mouse brain. In wild type mice, ‘synaptic’ AChE-S appearedin migrating neurons, whereas the C-terminus cleaved off thestress-induced AChE-R variant associated with migratory radialglial fibers. Antisense suppression of AChE-R reduced neuronalmigration, allowing increased proliferation of progenitor cells. Incontrast, transgenic overexpression of AChE-R was ineffective,whereas transgenic excess of enzymatically active AChE-S orinactive AChE-Sin suppressed progenitors proliferation alone orboth proliferation and neuronal migration, respectively. Our findingsattribute to alternative splicing events an interactive major role inneocortical development.

Keywords: alternative splicing, neurogenesis, neuronal migration, radialglia, readthrough acetylcholinesterase

Introduction

Neocortex development involves generation of projection

neurons in the ventricular zone (VZ) in response to as yet

incompletely understood cues, from a progenitor neuroepithe-

lium that is mostly composed of radial glial cells (Tamamaki

et al., 2001; Malatesta et al., 2003). These progenitor cells

undergo interkinetic nuclear migration, whereby DNA replica-

tion occurs when the nucleus is in the basal (outer) portion of

the VZ, and division adjacent to the ventricular surface give rise

to two daughter cells (Fig. 1A) (Boulder Committee, 1970).

Neurogenesis in mice commences on embryonic day (E) 11,

with production of the first post-mitotic neurons and their exit

from the VZ, while their sister cells re-enter the cell cycle and

continue to proliferate (Takahashi et al., 1995). Clonally related

proliferating cells coalesce in small, gap junction-coupled

clusters that cycle synchronously (Bittman et al., 1997), giving

rise to a clonally related output of neurons (Cai et al., 1997). As

neurogenesis proceeds, proliferation slows under the influence

of cell cycle-related proteins (Delalle et al., 1999), and cell

divisions primarily yield post-mitotic neurons, while the pro-

portion of daughter cells that continue to proliferate is greatly

decreased (Takahashi et al., 1996).

Following mitosis, the neural progeny reach the developing

cortex, i.e. the cortical plate (CP), either by somatic trans-

location, whereby a neuronal cell body migrates within a

pial-contacting radial process (Nadarajah et al., 2001) or by

locomotion, whereby newborn neurons develop elongated

leading and trailing processes that are oriented in a radial

direction, and migrate along radial glial fibers (Rakic, 1972).

Their adherence to nearby radial glial fibers activates multiple

intercellular events, such as cell--cell recognition and trans-

membrane signal transduction, that facilitate their movement

along the fibers (Rakic et al., 1994). Migrating neurons regulate

andmaintain the function of radial glia as migratory guides while

radial glia regulate migration along their processes (Anton et al.,

1997). Migrating neurons first pass through a subventricular

zone (SVZ) occupied by a secondary proliferative population of

cells adjacent to the VZ, and then through an intermediate zone

(IZ). They enter the CP and reach its most superficial portion,

adjacent to a marginal zone (MZ), where radial glial fibers

arborize before terminating at the pial surface (Fig. 1A,B)

(Gadisseux et al., 1989). Neurons are therefore guided by radially

directed processes of their founder cells, reaching their appro-

priate destination in clonally related clusters (Noctor et al.,

2001). They are positioned in the CP in an ‘inside-out’ sequence,

with newly arriving cells settling superficial to those that arrived

earlier (Rakic, 1974). Laminar fate, representing an aspect of

neuronal phenotype, was shown to be specified by gene ex-

pression in the progenitor cells, accompanied by progressive

restriction of multipotency (Frantz and McConnell, 1996).

Proliferation, differentiation and programmed gene expres-

sion in the developing nervous system may all be subject to

modulation by stress. Embryonic stress (e.g. ischemia--hypoxia)

attenuates neuronal migration to the cerebral neocortex, re-

sulting in morphological changes that are often accompanied by

postnatal behavioral deficits (Tashima et al., 2001). Similarly,

prenatal maternal stress impairs development of the offspring,

reducing, for example, learning and behavioral performance

(Kofman, 2002), and increasing the incidence of brain malfor-

mation and reduced head circumference (Mulder et al., 2002).

Improved understanding of the molecular mechanisms under-

lying the effects of stress on brain development is therefore of

considerable importance.

A notable cascade common to both brain development and

stress responses involves alternative splicing of pre-mRNA tran-

scripts, e.g. glutamic acid decarboxylase (Kuppers et al., 2000), G

protein isoforms (Morishita et al., 1999), the transcriptional

repressor ATF3 (Hashimoto et al., 2002) or potassium channels

(Xie and Black, 2001). However, the relationships between these

splicing modifications and the physiological changes occurring

during development and under stress remained obscure.

One way to explore this question is to directly manipulate the

embryonic expression levels and/or properties of specific vari-

ant mRNA transcripts of a neuronally-expressed gene that is

subject to transcription and splicing changes under both de-

velopment and stress, and observe the outcome with respect to

subsequent developmental events. The acetylcholinesterase

gene (ACHE) emerges as an appropriate example for such

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a study. It is known, for example, that ACHE gene expression

undergoes major changes during development and that its AChE

protein product, reported previously to regulate cell prolifera-

tion and neurite outgrowth, iswidely considered a sensitive early

marker of histochemical differentiation (Layer and Willbold,

1995). Thus, in the adult brain, acute stress induces overproduc-

tion of the relatively rare soluble ‘readthrough’ AChE variant

AChE-R by alternative splicing of the AChE pre-mRNA (Fig. 1C,D)

(Kaufer et al., 1998). AChE overproduction acts in the short term

to reduce available acetylcholine (ACh) and attenuate choliner-

gic neurotransmission (Soreq and Seidman, 2001), but subse-

quent accumulation may last weeks after exposure (Meshorer

et al., 2002) and may induce vulnerability to head injury

(Shohami et al., 2000). It is plausible, therefore, that changes in

ACHE gene expression are involved in both development- and

stress-related responses of the mammalian brain.

To examine the involvement of alternative splicing in cortical

development, we subjected mouse embryos to antisense oligo-

nucleotide suppression or to transgenic overexpression of

specific AChE splice variants, and quantified the effects on

cortical development. Here, we report that both ‘synaptic’ AChE

(AChE-S) and AChE-R mRNA are expressed by progenitor cells

in the VZ and undifferentiated cells in the CP. However, while

the membrane-associated AChE-S was detected in migrating

neurons, embryonic brain AChE-R undergoes C-terminal cleav-

age, similar to the modification characterizing the AChE-R

isoform found in blood (Grisaru et al., 2001), and appeared in

radial glial fibers. Transgenic manipulations of AChE variants,

moreover, induced changes in progenitor cell proliferation as

well as neuronal migration, suggesting physiological and path-

ophysiological roles for alternative splicing of AChE in cortical

development.

Materials and Methods

AnimalsCD1 and FVB/N mice were used for antisense and transgenic experi-

ments respectively. Vaginal plugs on post-mating morning designated

E0. Pregnant dams were anesthetized by intra-muscular injection of

a ketamin and xylazine mixture (50 and 10 mg/kg body wt, respect-

ively). Embryos were removed and dissected in cold phosphate buffered

saline (PBS). Heads (E11--15) and brains (E16--17) were immersed in 4%

paraformaldehyde in PBS (48 h, 4�C), embedded in paraffin and

sectioned at 4 lm in the coronal plane. Animal care followed in-

stitutional guidelines according to NIH published guidelines.

BrdU and Oligonucleotides InjectionsPregnant dams were injected intraperitoneally (i.p.) with bromo-

deoxyuridine (BrdU; 50 mg/kg in 7 mM NaOH--saline solution; Sigma,

St Louis, MO). Post-injection time points at 1, 2, 4, 12 and 48 h served to

detect labeled nuclei at S-phase, S+G2 (with a few mitotic cells in the

VZ), S+G2+M, G1 and post-mitotic cells in the VZ, or a cohort of neurons

‘born’ on E14, that migrated and reached the CP, respectively. EN101,

a 20-mer antisense-oligodeoxynucleotide was previously shown to

primarily suppress AChE-R mRNA (Cohen et al., 2002; Meshorer et al.,

2002) is targeted to exon-2 of mouse AChE mRNA. Its three 39-terminal

nucleotides 59-CTGCAATATTTTCTTGC*A*C*C-39 (stars) were 2-O-

methylated for nuclease protection. Inversely oriented oligodeoxy-

nucleotides (INV101) with the same sequence as the antisense, but

oriented from 39 to 59 served as control. Oligonucleotides were

dissolved in saline and injected i.p. three successive times at 12 h

intervals, initiated 12 h following BrdU injection on E14 (40 or 100 lg/kgper injection). Animals were sacrificed 48 h after BrdU injection.

ImmunohistochemistrySections were deparaffinized, microwave-treated (750 W, 15 min) in

0.01 M citric buffer, pH 6.0, and blocked (30 min) in 5% normal goat,

rabbit or horse serum in PBS with 0.5% Tween-20 (PBST) for AChE

Readthrough Peptide (ARP), AChE Synaptic Peptide (ASP) or AChE

N-terminus (N-trm), and nestin, respectively (Fig. 1D). Immunoreactions

(90 min, room temperature) were with rabbit anti-ARP (Sternfeld et al.,

2000), goat anti-ASP [Santa Cruz Biotechnology, Santa Cruz, CA; AChE

(C-16)] or goat anti-AChE [Santa Cruz N-terminal AChE (N-19)], 1:100 in

Figure 1. Cellular and histological architecture of the developing cortex. (A) Schematicdrawing of the cerebral cortex at the initiation (left) and during neurogenesis (right).Progenitor cells in the ventricular zone (VZ) are bipolar, and nuclear migration withinthem indicates the cell cycle phase according to the distance from the ventricularborder. Following mitosis, neurogenesis initiates by the production of a post-mitoticneuron (elongated gray cell with black nucleus), or a cell that will re-enter the cell cyclethrough G1. The progenitor cells extend their basal process in a radial direction to thepia matter, obtaining the form of radial glial cells. Post-mitotic neurons, produced fromradial glial cells migrate to the cortical plate (CP) along their processes througha secondary proliferative population at the subventricular zone (SVZ), tangentiallyoriented fibers in the intermediate zone (IZ) and through the CP until they reach theborder of the marginal zone (MZ) were the radial processes arborize. (B) Murine braindevelopment during the neurogenic interval. Boxes mark analyzed areas of interest (seeFig. 2). (C) Alternative-splicing of AChE pre-mRNA. The murine ACHE gene is shownwith numbers marking the exons and pseudointron 4 (49). Splicing of exons 2, 3 and 4yields the common N-terminus and catalytic domain, whereas exon 6 and pseuointron4 encode the variant-specific -S and -R C-termini, respectively. (D) Schematic drawingsrepresenting AChE variants and antibody specificities to the common N-terminus (N-trm) or distinct C-terminal domain. Arrow indicates the approximate catalytic sitewithin the common domain, and a presumed cleavage site of the distinct C-terminaldomain of AChE-R, producing the ARP AChE Readthrough Peptide.

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PBST containing 2.5% serum. Immunoreactivity for ARP was eliminated

by incubation of the antiserum with synthetic ARP (Sternfeld et al.,

2000) at a molar ratio of 1:5, attesting to specificity of the antiserum (not

shown). TUJ1 antibody (Lee et al., 1990) (generously provided by

Dr A. Frankfurter) and mouse anti-nestin (Developmental Studies

Hybridoma Bank, University of Iowa, Iowa City, IA) were 1:500 in PBST.

Secondary IgG were biotin-conjugated goat anti-rabbit for ARP, donkey

anti-goat for ASP or N-trm, and horse anti-mouse for nestin detection

(Vector), 1:200 in PBST containing 2.5% serum (1 h). TUJ1 detection

involved goat anti-mouse Cy3-conjugated IgG (Jackson Immuno-

research Laboratories, West Grove, PA), 1:200 in PBST. Biotinylated

antibodies were incubated with avidin-bound peroxidase complex

(ABC Elite, Vector Laboratories) for 1 h, rinsed with 0.05 M Tris, pH

7.6, and reacted for 90 s with 0.05% diaminobenzidine (Sigma) and

0.006% H2O2 in 0.05 M Tris, pH 7.6, with 0.05% nickel ammonium

sulfate. Selected sections were counterstained with Gill-2 hematoxylin

(Sigma).

Immunochemistry for the nuclear antigen Ki67 was used to monitor

cell proliferation. Ki67, previously used to label dividing cells in the

human embryonic VZ (Weissman et al., 2003), is expressed by pro-

liferating cells during late G1, S, M and G2 phases of the cell cycle

(Gerdes et al., 1984; Scholzen and Gerdes, 2000), and is often used to

evaluate the proliferative fraction of solid tumors (Scholzen and Gerdes,

2000). The utility of Ki67 as a proliferative marker that is comparable to

BrdU labeling was previously tested for neurogenesis in the adult

dentate gyrus of the hippocampus, where its expression mimicked that

of BrdU when examined soon after exogenous BrdU administration.

Experimental increases in the number of mitotic cells by ischemia, or

their reductions by radiation produced parallel changes in BrdU and

Ki-67 labeling (Kee et al., 2002). Ki67 staining increases during S-phase,

reaches a peak during metaphase (du Manoir et al., 1991) and decreases

during ana- and telophase (Starborg et al., 1996). Quantification of Ki67

expression was compiled by measuring the mean sum of pixel values in

a 50 3 100 lm rectangle at the apical portion of the VZ, positioned

100 lm lateral to the dorsomedial to medial cortical border, similar to

that done for detection of AChE.

BrdU LabelingSections were treated with 100 lg/ml deoxyribonuclease in PBST

(30 min), incubated with mouse anti-BrdU (Becton-Dickinson, Missis-

sauga, Ontario, Canada; 1:100 in PBST, 2 h), followed by anti-mouse Cy2-

conjugated IgG (Jackson; 1:50 in PBST) or biotinylated goat anti-

mouse IgG. Processing was as described above.

In Situ HybridizationPreviously detailed probes and procedure (Meshorer et al., 2002) were

modified as follows. Cy5-conjugated streptavidin and Cy3-conjugated

anti-digoxygeninwere employed for detection of biotin- and digoxygenin-

labeled probes, respectively [1:200 in Tris-buffered saline with 0.1%

Tween-20 (TBST); Jackson]. In situ hybridization was combined with

TUJ1-immunofluorescence as detailed above, or with BrdU-immuno-

fluorescence applying fast-red reaction with alkaline-phosphatase

(AP)-conjugated streptavidin (Zymed Laboratories, San Francisco, CA;

1:25 in TBST, 1 h), followed by BrdU-immunofluorescence with Cy2-

conjugated anti-mouse IgG.

Confocal MicroscopyImages of 1-lm-thick sections were captured by excitation at 488, 543,

633 and 488 nm of Cy2, Cy3, Cy5 and Fast-Red, respectively. Emission

was measured with band-passes of 505--545 or 560--615 nm or long-

passes of 650 and 560 nm, respectively. The microscope’s detector and

amplifier were calibrated by referring to sections expected to have the

highest signal as 100% (E11 for ontogeny experiments, INV101 for

antisense experiments). The focus was adjusted to the point of maximal

intensity, and the detector and amplifier were adjusted to obtain the

optimal image. For subsequent sections, the focus was adjusted but the

same amplifier and detector values weremaintained to reach the narrow

depth of maximal signal intensity.

Regions of AnalysesSectors of analysis were 200 lm wide and distant 100 lm from the

medial edge of the lateral ventricle, within the posterior-medial portion

of the future somatosensory area. Digitized images were analyzed in

a ‘blind’ manner. BrdU-immunostaining was considered positive if nuclei

were darkly stained or at least three puncta were discerned.

Image AnalysesAt least three embryonic brains from at least three different litters were

analyzed for each group. Averaged cell counts were obtained by averaging

values from three or four non-consecutive sections from each brain, for

all analyzed brains in each group. Confocal signal was converted to

grayscale for intensity measurements of pixel values (Scion Image, Scion

Corporation, Frederick, MD). Analysis of variance (ANOVA; Statistica

software, StatSoft, Tulsa, OK) was used to compare multiple groups and

a one-tailed t-test (Microsoft Excel) was used to compare two groups.

ImmunoblotsCerebral homogenates yielding soluble AChE from E17 control and

transgenic embryos were processed as described (Birikh et al., 2003).

Immunodetection was with rabbit anti-ARP (1:250), goat anti-ASP

(1:500) and goat anti-N-trm (1:500).

Catalytic ActivityAcetylthiocholine hydrolysis was measured spectrophotometrically as

described (Kaufer et al., 1998). Iso-OMPA (tetraisopropylpyrophosphor-

amide) was used to block butyrylcholinesterase activity (5 3 10–5 M).

Results

Pre-AChE mRNA Splicing Shift at the Ventricular Zone

ACHE gene expression was first studied by in situ hybridization

in the VZ during the neurogenic interval (Fig. 1B). At the onset

of neurogenesis (E11), a time of intense progenitor cell pro-

liferation, cytoplasmic AChE-R and AChE-S mRNA (Fig. 1C) were

co-localized in most VZ cells (Fig. 2A). Expression was most

intense in the apical portion of the VZ, close to the ventricular

lumen. With the advance of neurogenesis, e.g. at E13, cyto-

plasmic expression of the AChE isoforms became pronounced

in clusters of adjoining cells in the basal portion of the VZ

(Fig. 2A,C). These AChE-expressing clusters, which included

from two to >20 cells, coalesced at various points along their

common borders. By E15, AChE-expressing cell clusters were

smaller, and by E17 they were limited to very small clusters or

single cells (Fig. 2A). The subcellular distribution of AChE

mRNA had also changed, so that intense signals were observed

primarily in the basal pole of labeled cells (Fig. 2B). Densito-

metric measurements of AChE expression demonstrated a grad-

ual reduction in labeling intensity throughout the VZ during

neurogenesis. Expression areas of AChE splice variants exhibi-

ted a parallel reduction (Fig. 2C), implying decreasing numbers

of expressing cells. Both AChE-R and AChE-S mRNA declined in

the VZ. However, some of the cell clusters at E13 maintained

the intense level of AChE-R expression (Fig. 2A, open arrow). A

statistically significant reduction in AChE-S expression, as de-

termined for both intensity and signal area, was observed from

E11 to E13, while reduction of AChE-R was delayed until E15,

apparently reflecting a transient dominance of AChE-R over

AChE-S at E13 (Fig. 2C).

Proliferating Cells but not Terminally DifferentiatedNeurons Display Splicing Shift at the Ventricular Zone

A 2 h pulse of BrdU was used to distinguish between S+G2+Mversus G1 or post-mitotic nuclei in order to determine the

proliferative profile of AChE expressing cells in the VZ. During

such a short pulse, BrdU is continuously available for incorpor-

ation into nuclei in the S-phase, while the earliest of these nuclei

advance through G2 and initiate mitosis (Takahashi et al., 1992).

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Combined with in situ hybridization, anti-BrdU immuno-

fluorescence demonstrated that the AChE mRNA-labeled

clusters included both BrdU positive and negative nuclei,

suggesting that the clusters comprise both S+G2+M and G1 or

post-mitotic cells (Fig. 3A). The majority of intensely AChE-

expressing cells were located at the basal portion of the VZ, i.e.

in the S-phase zone. Adjacent to the ventricular lumen, i.e. in the

G2+M zone, AChE expression was relatively sparse, with cells in

mitosis expressing the transcripts at their basal pole (Fig. 3A,

arrows and insets).

BrdU-negative cells in the VZ represent either proliferative

cells re-entering the cell cycle through G1 or post-mitotic cells

in the process of migration out of the VZ. To differentiate

between these two possibilities, in situ hybridization for

AChE-R mRNA was combined with immunofluorescence for

beta-III tubulin (TUJ1), an early marker of neurons (Geisert and

Frankfurter, 1989) (Fig. 3B). The AChE-R mRNA positive

clusters were TUJ1 negative, suggesting that AChE-expressing

cells that were refractory to the 2 h BrdU pulse were pro-

liferative cells, i.e. cells at G1 phase. Nevertheless, not all of the

non-AChE expressing cells were labeled by TUJ1, indicating the

existence of another or intermediate cell population.

AChE Gene Expression in Migrating Neurons

AChE expression patterns in the IZ were examined to assess the

potential involvement of the protein and its splice variants in

neuronal migration from the VZ to the cortical plate. Both AChE-

R and AChE-S mRNA were observed either as individual IZ cells

or as clusters, with reduced labeling compared with the CP or

VZ (Fig. 4A,B). These cells were radially oriented, suggesting that

theyweremigrating fromtheVZ to theCP. Immunofluorescence

Figure 3. AChE-S and AChE-R expressing cell clusters in ventricular zone include cellsin all phases of the cell cycle but not post-mitotic neurons. (A) AChE-R and AChE-Sgene expression (red) in cell clusters, one of each is marked by a white border line.BrdU immunofluorescence (green) in the ventricular zone (VZ) at E14 following a 2 hBrdU pulse injection. Gaps of non-expressing cells are marked by asterisks. AChE-expressing clusters (arrowheads) include BrdU-positive and negative nuclei. Basalpolar AChE expression is observed in cells near mitosis (arrows, inset). (B) AChE-Rlabeled clusters in the VZ are TUJ1-negative. TUJ1-positive cells in the VZ at E15(arrowheads) are located between the AChE-R clusters. Note the horizontal orientationof TUJ1 positive cells in the subventricular zone (SVZ), which are apparentlytangentially migrating neurons.

Figure 2. Transient splicing shift from AChE-S to AChE-R in ventricular zone cellclusters (A) AChE-R (blue) and AChE-S (red) mRNA signals during cortical development.Confocal images of double-labeling in situ hybridization. AChE-R mRNA signal intensitywas color coded, with yellow to blue color-gradient representing decreasing signal-intensity (right column, color bar). Intensely expressing cells or clusters (arrows), non-expressing cells (arrowheads) and AChE-R dominance over AChE-S expression (openarrow) are marked. (B) An enlargement of arrow-marked AChE expressing cells at E17in (A). (C) AChE-R and AChE-S signal intensities and distribution areas (mean±SD) andratios in the ventricular zone (VZ). Note transient peak of R/S ratio.

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with the TUJ1 antibody intensely labeled horizontally oriented

cells in the IZ that were apparently involved in tangential

migration (Fig. 4B,C). Indeed, combined AChE-R mRNA/TUJ1

labeling demonstrated that the AChE-R expressing cells in the IZ

wereTUJ1-negative (Fig. 4B,C).With further development, AChE

expression in the CP appeared in clusters of intensely labeled

cells, surrounded bymoderately expressing cells (Fig. 4A,B). The

intensely labeled cells were more prominent in the superficial

portion compared with the deep CP. This suggested that the

younger, newly arriving cells in the CP expressed more AChE

transcripts than earlier arriving cells that had already undergone

some differentiation. Subsequently, at E17, the intense signals in

the superficial cell layer of the CP became significantly higher

than those of the deep CP portion. Combined AChE-R mRNA/

TUJ1 immunofluorescence demonstrated a complementary

pattern, similar to that observed in the VZ (Fig. 4B,C). The

distribution of TUJ1 exhibited increasing density of labeled cells

from the superficial to deeper portions of the CP, implying again

that AChE was intensely expressed by the relatively undifferen-

tiated cells, and declined as the number of differentiated cells

increased. Although TUJ1 expression was essentially detected in

the deeper portion of theCP, a fewof these cellswere seen at the

superficial portion adjacent to theMZ (Fig. 4C).We assume these

cells to be young neurons that were about to be displaced by

incoming newly arriving migrating neurons or possibly cells in

transition fromAChE toTUJ1-expressing cells. In addition to this,

some horizontally oriented TUJ1 cells were detected in the MZ,

possibly Cajal-Retzius cells.

Distinct Localization Patterns of AChE Splice Variantsduring Cortical Development

AChE splice variants are identical in most of their sequence,

differing, primarily in their C-termini (30 residues of AChE-R

peptide, ARP and 39 residues of AChE-S peptide, ASP; Fig. 1D).

ARP, ASP and the common N- terminus all demonstrated

cytoplasmic immunostaining patterns in VZ cells, similar to

that of AChE mRNA, with a gradual decrease in intensity and

in the number of expressing cells, as well as reduced clustering

of intensely labeled cells (Fig. 5). Moreover, at E15, ARP-

immunoreactive cell processes were observed ascending from

the VZ (Fig. 5, arrows) and extending radially through the total

thickness of the cortical wall to terminate at the pial surface.

This pattern, which is characteristic of radial glia cells

(Gadisseux et al., 1989), was most readily observed in the

medial neocortex. There, fibers could be clearly traced into the

marginal zone (MZ), where they arborized before terminating

at the pial surface (Fig. 6A).

Developing Brain AChE-R is C-terminally Cleaved

The immunoreactivity of radial glia to antibodies targeted at the

C-terminus of AChE-R, but not to its N-terminus (Fig. 5),

suggested cleavage of AChE-R to separate the C-terminal

domain that includes ARP, from the core AChE-R protein (Fig.

1D). This is consistent with AChE found in blood (Grisaru et al.,

2001). Consistent with this observation, soluble proteins ex-

tracted from E17 cerebral cortex demonstrated an intense ARP

immunoreactive band of 18 kDa in addition to a 65 kDa band

that appears to be intact AChE (Fig. 6B). In contrast to this,

antibodies directed against the N-terminal domain (N-trm)

common to all AChE variants (Fig. 1D) revealed several slowly

migrating bands (Fig. 6B). These included a lightly labeled band

that paralleled the 65kD band shown with anti-ARP and

representing the non-cleaved AChE-R, while the most intense

band was of ~55 kDa, reflecting the core AChE-R domain

following removal of the C-terminus. Negligible immuno-

reactivity was observed to antibodies directed against ASP

Figure 4. AChE-R mRNA expression in the embryonic cortical plate precedes neuronal differentiation. (A) AChE-R (blue) and AChE-S (red) mRNAs at E15 and E17. Note non-homogeneous distribution of intense signals in cell clusters (arrowheads) in the superficial portion of the cortical plate (CP), and deep situated lightly stained cells (asterisk).Intermediate zone (IZ) clusters are seen at E15 (arrows). (B) The corresponding brain regions. (C) AChE-R mRNA (red) and immunolabeled TUJ1 (green) in the cortical wall at E15and E17. Note TUJ1-negative AChE-R-positive cell clusters (arrows) and TUJ1-positive AChE-R-negative cells (arrowheads) in the superficial and deep cortical plate, respectively.TUJ1-positive AChE-R-negative horizontally orientated cells, likely migrating tangentially, are seen in the IZ.

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(Fig. 6B), suggesting that ASP remained attached to AChE-S,

rendering it insoluble and therefore not extractable by this

procedure. These results indicate that AChE-R, but not AChE-S,

is subject to cleavage of its C-terminal domain in the brain and

that the vast majority of AChE-R in the developing cortex

undergoes C-terminal cleavage.

Anti-ARP, which was originally raised against a glutathione S-

transferase--human ARP fusion protein, was not immunoreactive

against synthetic ASP (not shown), attesting to its specificity, yet

displayed clear immunoreactivity to synthetic murine ARP (Fig.

6B). The immunoreactivity of these two distinct amino acid

sequences to the same antiserum suggested evolutionary con-

servation of ARP structural epitopes, despite the disparity in

sequence.

Anti-ARP Labels Migration-associated Glial Processes

With the thickening of the cortical wall, ARP-labeled fibers in

the IZ became arched (from medial to lateral), and resumed

a radial alignment, orthogonal to the pial surface as they entered

the CP (Fig. 6D-2,6). Immunohistochemistry for nestin, a marker

of radial glia (Lendahl et al., 1990), exhibited a similar pattern of

fibers in adjacent sections (Fig. 6D-1,5). This alignment is typical

of the morphology of radial glia (Gadisseux et al., 1989; Misson

et al., 1988). In contrast to the pronounced staining by anti-ASP

and anti-N-trm antisera, ARP labeling was faint in the cytoplasm

of migrating cells in the IZ (Fig. 6D-2,6).

Figure 6. Cleaved AChE-R C-terminus in migratory glial fibers and AChE-S in migratingcells perikarya. (A) E15, AChE Readthrough Peptide (ARP)-immunostained fibersascend from the ventricular zone (VZ, arrowheads), reaching the medial cortex marginalzone (MZ) where they branch and terminate at the pia matter. (B) Immunoblot analysis.Anti-ARP labels syntheticmurine ARP (mARP), 18 and 65 kDa bands from E17 cerebrum.Antibodies to N-terminus (N-trm) label 65 and 55 kDa proteins, whereas anti-AChESynaptic Peptide (ASP) shows negligible immunoreactivity. (C) Coronal section schemeof migratory pathways within the analyzed regions in the cortex (CTX), striatum (STR)and lateral cortical stream (LCS) at E17. (D) Immunohistochemical staining. (1--4)Cortical wall. (5--9) Intermediate zone, enlarged from the boxed regions in 1--4. (1, 5, 9,13, 17) Nestin. Note labeled radial glial processes (arrowheads), and dense bundle ofglial fibers in the LCS. (2, 6, 10, 14, 18) ARP. Note cells at the VZ/subventricular zone(SVZ), radial glial processes (arrowheads), LCS and STR fibers and lightly labeledsuperficial cortical plate (CP) cells. (3, 4, 7, 8, 11, 12, 15, 16, 19, 20) ASP and N-trm.Note cells at the VZ/SVZ, clusters of radially oriented cells in the intermediate zone (IZ,arrows), prominent staining in the CP superficial cell layer, and in perikarya of cells withinthe LCS area, oriented parallel to migratory direction (arrowheads).

Figure 5. Developmental decreases in AChE immunolabeling. Photomicrographsshow AChE Readthrough Peptide (ARP), AChE Synaptic Peptide (ASP) and N-terminus(N-trm) labeling in the ventricular zone (VZ). E13 cell clusters and E17 single cells aremarked by arrowheads. E15 ARP-immunostained radial fibers ascending from the VZinto the overlying subventricular zone (SVZ) are shown (arrows).

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Within the CP, intense immunoreactivity of ASP and the N-

terminus was observed in the cells at the superficial cell layer,

i.e. newly arriving CP cells, similar to the pattern of AChE gene

expression (Fig. 6D-3,4). In contrast, ARP immunoreactivity in

this cell layer was sparse, compared with its intensity in the VZ

(Fig. 6D-2).

To examine whether ARP immunoreactivity is apparent in

other migration-associated glial processes, immunoreactivity

was examined in the lateral cortical stream (LCS) and in the

striatum (Fig. 6C). The dense glial bundles of the LCS were

strongly positive for both nestin and ARP (Fig. 6D-9,13 and

10,14, respectively), extending ventrolaterally from the lateral

edge of the VZ between the neocortex and the striatum. Both

nestin and ARP demonstrated ramification of this bundle into

fibers that assume an orthogonal orientation to the pial surface

as they penetrate the neocortex. In contrast, ASP and the

common N-terminus peptide were not detected in the LCS fiber

bundle or in its ramifications, but were labeled in the cytoplasm

of migrating cells in the region of the LCS (arrowheads in

Fig. 6D-15,16 and insets in 19,20, respectively). In the striatum,

both nestin and ARP labeled glial fiber processes extended from

the VZ area to the differentiating part of the striatum, which

were not immunolabeled by either the ASP or the common N-

trm antibodies (Fig. 6D-17--20).

Antisense Suppression of AChE-R mRNA AttenuatesNeuronal Migration

The distinct patterns of ARP and ASP immunostaining we

observed suggested that these two peptides and/or their

corresponding proteins may play distinct roles during neuronal

migration. To challenge this hypothesis, we labeled a cohort of

migrating cells with BrdU prior to their terminal mitosis in the

VZ (Fig. 7A). To reduce AChE-R during neuronal migration, we

employed mouse EN101, an antisense oligonucleotide capable

of inducing selective destruction of mouse neuronal AChE-R

mRNA (Cohen et al., 2002). Fluorescent double-labeling in situ

hybridization was performed to quantify AChE mRNA variants in

the cortical wall following EN101 injection during neuronal

migration. Cell density within the VZ as well as its thickness

were similar in control and EN101-treated brains. A reduction in

labeling was observed, however, which could not be attributed

to reduction in the number of AChE expressing cells. Signal

intensity for AChE-R mRNA, measured and compared in uniform

100350lmsquare samples in the apical portionof theVZ (121±12 cells; Fig. 7B) exhibited a 34% reduction following EN101

compared with control treatment with the inversely oriented

oligonucleotide sequence, INV101, both at 100 lg/kg (n = 10,

P < 0.05). In contrast, AChE-S mRNA labeling was reduced by

only 7% (n = 10), which was not statistically significant (Fig. 7B).

The effect of AChE-R mRNA reduction on cell migration was

evaluated by counting the number of BrdU immunoreactive

nuclei present in the dorsomedial CP 48 h after BrdU injection

(Fig. 7C). EN101 treatment significantly reduced the number of

BrdU immunoreactive cells in the CP by 26% compared with the

INV101 treatment (n = 13; ANOVA, P < 0.01; Fig. 7D). Lower

concentrations of EN101 or INV101 (40 lg/kg) failed to elicit

Figure 7. Antisense oligonucleotide suppression of AChE-R production attenuates cellarrival to the cortical plate. (A) Photomicrographs (top) and schematic drawings(bottom) of BrdU incorporation by progenitor cells into nuclei during S-phase, theirsubsequent inter-kinetic movement in the ventricular zone (VZ) and migration towardthe cortical plate. At E14, 12 h after BrdU injection, labeled nuclei have all moved awayfrom the ventricular surface, indicating that they have become post-mitotic. By 48 hpost-injection, many BrdU-immunostained nuclei have reached the cortical plate (CP).Note that the majority of labeled nuclei in the CP are darkly labeled, whereas those in theVZ/subventricular zone (SVZ) become lightly labeled. Arrows point to mitotic BrdU-labeled cells, arrowheads point to proliferating cells in the SVZ. (B) AChE-R and AChE-SmRNA following EN101 or control INV101 treatment. Histograms show signal intensityin 100 3 50 lm square sectors at the inner portion of the VZ (box). (C) BrdU-immunoreactive nuclei in the cortical plate. Coronal 200 lm cortical sectors, followingINV101 or EN101 treatment. (D) BrdU-positive cell counts. Attenuated migrationdisplayed dose dependence (40 versus 100 lg/kg) and sequence specificity (EN101versus INV101). Note that the total number of immunoreactive cells in the CPþIZremained unchanged.

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discernible effects on cell migration to the CP (Fig. 7D),

indicating dose-dependence. Consistent with this, significantly

more BrdU-immunoreactive cells were detected in the IZ under

EN101 treatment (at 100 lg/kg) compared with INV101 or low

dose EN101 (n = 13) (ANOVA, P < 0.05) (Fig. 7C,D). The total

number of BrdU-immunoreactive cells in the CP and IZ was

similar between all treated groups, demonstrating that post-

mitotic cell survival was unchanged (Fig. 7D). Therefore, the

EN101-mediated reduction of neuronal migration reflected

attenuated progression of cells from the IZ to the CP.

AChE-R mRNA Destruction Increases Proliferation inthe Ventricular Zone

The gradual reduction of AChE gene expression during neuro-

genesis, in parallel with the restriction of proliferation in the

VZ, suggested an involvement of AChE and the AChE-associated

migration process with proliferation of progenitor cells (Fig. 8A).

The effect of AChE-R on proliferation in the VZ was examined by

EN101 treatment in animals treated with BrdU 48 h prior to

sacrifice. Immunocytochemical labeling of the Ki67 nuclear

antigen appeared in most of the cells at the M-phase region in

theVZof themouse developing neocortex at E16. Fewcells in the

basal portion of the VZ were intensely stained, though, the

majority of labeled cells in that region exhibited light, punctate

nuclear staining (Fig. 8B). Compared with INV101, EN101 treat-

ment significantly increasedKi67expression in theVZ (Fig. 8C,D),

suggesting increased re-entry into the cell cycle vs exiting the cell

cycle following reduction of AChE-R expression by EN101.

Both Catalytic and Non-catalytic AChE Activities AffectProliferation and Migration

VZ neuronal progenitors express both AChE-S and AChE-R. The

observedeffect of EN101described above, therefore, impliedone

of two possibilities: (i) AChE-R alone reduces proliferation; or (ii)

AChhydrolysis, common toAChE-R andAChE-S is responsible. To

distinguish between these possibilities, we injected BrdU into

E14 transgenic mice overexpressing (i) the membrane adhering

AChE-S (TgS, Beeri et al., 1995); (ii) soluble AChE-R (TgR,

Sternfeld et al., 2000); or (iii) an enzymatically inactive form of

AChE-S (TgSin, Sternfeld et al., 1998) (Fig. 9A). The animals were

sacrificed 48 h later. Reverse transcriptase--polymerase chain

reaction was employed to confirm the expression of each of the

AChE variants, and acetylthiocholine hydrolysis measurements

confirmed increased catalytic activity in brain homogenates from

the TgR andTgS but not TgSin strains (Fig. 9B). ARP expression in

embryonic brains from the three transgenic and control groups,

appeared similar by immunoblot analysis (not shown), suggesting

that high AChE-R levels are maintained during cortical develop-

ment. Arrival of cells in the CP from both TgR or TgS over-

expressing embryos was unchanged relative to parent strain

controls. However, in TgSin embryos, the average number of

BrdU immunoreactive cells in the CP was significantly reduced

compared with control mice as well as those of TgS or TgR mice

(Fig. 9B), suggesting a role for ACh in neuronal migration.

The effect of overexpressing AChE splice variants on pro-

genitor cells proliferation was examined in the transgenic

mouse strains by Ki67 labeling and density quantification in

the VZ. Ki67 expression was reduced in both TgS and TgSin, but

not in TgR embryos compared with controls (Fig. 9B). This

suggests that the non- hydrolytic activity of AChE-S acts to

reduce proliferation in the VZ, whereas its hydrolytic activity

has a role in promoting neuronal migration to the CP.

Discussion

Using the regulation of AChE pre-mRNA processing as a case

study, we explored the involvement of alternative splicing

modulations in shaping the developing brain. We found tran-

sient changes in AChE pre-mRNA processing during murine

cortical development. Using transgenic and antisense manipu-

lations of these variants, we have further demonstrated causal

involvement of such changes in progenitor proliferation and the

shift toward neuronal migration and differentiation which

together shape the mammalian cortical plate.

While many neuronal mRNAs are subject to alternative

splicing modulations during brain development (e.g. the cla-

thrin assembly protein 3 (AP-3) (Ishihara-Sugano and Nakae,

1997), the protein tyrosine phosphatases PTP-SL and PTPBR7

Figure 8. Antisense treatment increases proliferation in the ventricular zone andreduces cell arrival to the cortical plate. (A) Proliferating cells in the ventricular zone(VZ) can continue proliferating or migrate to the cortical plate (CP), possibly sendingsignals (broken arrow) that modulate proliferation. (B) Ki67-immunostaining (E16coronal section counterstained with hematoxylin). Adjacent to the ventricular border,where mitosis occurs, note the intense nuclear labeling of cells in metaphase (largearrowhead), anaphase (small arrowhead) and G2 or beginning of G1 (long arrow).Some basal VZ cells were intensely stained (open arrows), but most were lightlylabeled with punctated nuclear staining (small arrow). (C) Ki67-immunostainingfollowing INV101 or EN101 treatments. 100 3 50 lm sectors at the inner VZ (box)served for density measurements. (D) Ki67-immunoreactivity density in the VZfollowing EN101 treatment. *P\ 0.05. INV101 served as control.

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(Van Den Maagdenberg et al., 1999) and G protein isoforms

(Morishita et al., 1999), the information accumulated on AChE’s

splice variants and their putative roles in neuronal development

and functioning provides added value to this particular example.

First, the alternative splicing shift in AChE pre-mRNA process-

ing occurred in proliferating progenitors prior to their neuronal

commitment, marking a checkpoint between proliferation and

migration. Secondly, our analyses pointed at four distinct

functions for AChE in cortical development: (i) ACh hydrolysis,

common to AChE-S and AChE-R; (ii) non-catalytic structural

features of the core domain, also common to both variants;

(iii) migration-supportive properties of ARP, the cleavable C-

terminus of AChE-R; and (iv) adherent capacities of ASP, the

corresponding uncleaved C-terminus of AChE-S, which joins

AChE-S tetramers to a proline-rich membrane anchor (PRiMA)

structural subunit (Perrier et al., 2002) but also drives AChE-S to

the cell nucleus (Perry et al., 2002). In the following, we discuss

the implications of each of these roles for cortical development.

Concerted Effects on Progenitor Migration andProliferation

During murine brain development, alternative splicing modula-

tion yields a relative dominance of AChE-R, which we found to

be a pre-protein to its cleavable C-terminus ARP, compatible

with its cleavage under stress in the mouse and human blood

(Grisaru et al., 2001; Cohen et al., 2003; Pick et al., 2004). In the

developing cortex, ARP interacts with migration-supportive

radial glia, unlike the core AChE domain and the uncleaved

variant AChE-S, which persist in migrating and differentiating

neurons. Moreover, antisense suppression of AChE-R produc-

tion attenuated neuronal migration to the CP, suggesting causal

involvement of the splice shift in this process. In addition, the

antisense treatment increased neuronal progenitor prolifera-

tion. This could have reflected a proliferation-inhibitory effect

of AChE-R itself or of EN101-resistant AChE-S in the attenuated

progenitors. To distinguish between these possibilities, pro-

genitor proliferation and neuronal migration were compared in

transgenic mice overexpressing distinct AChE variants. AChE-R

excess had no effect, whereas both AChE-S and its genetically

inactivated mutant AChE-Sin suppressed proliferation, and

AChE-Sin further suppressed migration. These findings sug-

gested a non-catalytic, proliferation-inhibiting effect for AChE-S,

possibly acting through AChE-R or in an AChE-R-dependent

manner. Thus, reduction of AChE-R following EN101 treatment

abolished the capacity of AChE-S to attenuate proliferation.

Additionally, suppression of neuronal migration by AChE-Sin is

compatible with the assumption that ACh hydrolysis is pivotal

for neuronal migration, supporting the view of ACh as a regu-

lator of neuronal migration (Lauder and Schambra, 1999).

Role in Glial Cell Differentiation

The dynamic changes that take place in the VZ during cortical

development include increased cell cycle length (Takahashi

et al., 1995), reduction of symmetric mitotic divisions (Chenn

and McConnell, 1995), restriction in layer specification

(McConnell and Kaznowski, 1991) and change in radial glia

phenotype (Hartfuss et al., 2001). During brain development,

AChE expression in the VZ decreased at the end of neuro-

genesis, when radial glia transform to astrocytes (Hartfuss et al.,

2001). AChE involvement in cell proliferation was previously

proposed in several brain and hematopoietic cell types (Karpel

et al., 1996; Sharma et al., 2001; Perry et al., 2002). Its growth-

regulatory role in hematopoietic progenitors (Paoletti et al.,

1992; Lev Lehman et al., 1997) was more recently attributed to

ARP, the cleavable C-terminus of AChE-R (Grisaru et al., 2001).

Antisense suppression of AChE-R production enhanced pro-

liferation of cultured osteoblastoma cells as well (Grisaru et al.,

1999), suggesting a wide cell type specificity to this effect.

In the adult brain, AChE levels are very low in all types of

normal adult glia, but increase in astrocytic tumors, with a shift

in alternative splicing favoring AChE-R production in more

aggressive tumors (Perry et al., 2002), resembling nestin eleva-

tion (Sugawara et al., 2002). Taken together with the present

work, this suggests a causal interrelationship between AChE

alternative splicing and glial cell de-differentiation.

Figure 9. AChE splice-variant manipulations show involvement with both proliferationand migration. (A) Schematic representation of cell and cell-membrane binding andinteraction with acetylcholine (ACh) for AChE variants. Transgenic (Tg) strains over-expressing variant AChE are: TgS, overexpressing the membrane associated AChhydrolysing AChE-S; TgR, overexpressing the soluble ACh hydrolysing AChE-R; andTgSin, overexpressing the membrane-associated AChE-S which has been mutated bysequence insertion encoding seven amino acids to the catalytic site to abolish its AChhydrolytic activity. (B) Ellman’s reaction, cell arrival to the cortical plate (CP) and Ki67-immunoreactivity in the ventricular zone (VZ) at E16 following BrdU injection at E14.*P\ 0.05 when compared with control, TgR or TgS; **P\ 0.01 when comparedwith control or TgR.

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Clustering of AChE-expressing Cells

Following the initiation of neurogenesis, AChE was detectable

in clusters of VZ proliferating cells which included all phases of

the cell cycle, though were sparsely detected during mitosis,

and did not include post-mitotic neurons. This resembles pre-

viously shown cell clusters, thought to dynamically couple by

gap junctions during all phases of the cell cycle except M, and

contain radial glial cells but not migrating or post-mitotic

neurons (Bittman et al., 1997). Cell clustering during cortical

development likely reflects assembled clonally related dividing

cells (Cai et al., 1997) and includes cell clusters expressing

choline acetyltransferase (ChAT), the rate-limiting enzyme in

ACh synthesis (Schambra et al., 1989). That ACh stimulates

cortical precursor cell proliferation in vitro through muscarinic

receptor activation (Ma et al., 2000) may suggest that AChE,

expressed in such clusters, functions by hydrolyzing ACh and

terminating its activity as a morphogenic cue. That TgSin

embryos display reduced progenitors proliferation may suggest

additional mechanisms that are not dependent on ACh hydro-

lysis. Alternatively, or in addition, AChE-Sin incorporation into

progenitors’ membranesmight have limited the incorporation of

enzymatically active AChE to these sites, creating a cholinergic

imbalance.

ARP May Exert an Independent Migratory Effect

By E13, the neuroepithelium exhibits the radial glial phenotype

(Malatesta et al., 2003; Tamamaki et al., 2001), stretching fibers

to the pia matter. ARP was detected throughout the full length

of these cells, whereas the perikaryons of migrating cells in the

IZ and arriving cells in the CP were positive for both ASP and

the N-terminal core of AChE. Cleaved ARP was detected in the

mouse serum following forced swim stress, where its presence

accompanies blood cell progenitor proliferation (Grisaru et al.,

2001) and in humans following lipopolysaccharide (LPS)

exposure, concomitant with the psychological impact of such

exposure (Cohen et al., 2003). Migrating cells in the IZ express

AChE-R mRNA but not ARP, suggesting secretion of this soluble

peptide. Conversely, radial glial fibers are decorated for ARP but

not the common N-terminus, and protein blot analysis demon-

strated that the C-terminus of AChE-R, including ARP, is de-

tached from the larger, N-terminal portion of AChE. Combined

with the antisense and transgenic manipulations, these findings

support the notion that ARP participates in the neural migration

role of radial glia within the developing cortex.

Radial Migration of Intermediate Zone Neurons

AChE’s involvement in cell migration was proposed previously

based on its expression in migrating sensory rat dorsal thalamic

neurons (Schlaggar et al., 1993). Furthermore, an AChE-coated

substrate induced migration and clustering of cultured spinal

motoneurons (Bataille et al., 1998), suggesting an extracellular

effect of AChE on cell migration and cell--cell interaction. In our

study, transient in vivo antisense reduction of AChE-R reduced

cell arrival to the CP, with cells remaining on their way, in the IZ.

In contrast, constitutive overexpression of AChE-R in transgenic

mice did not elicit an increase in cell arrival to the CP. Also, ARP

levels were similar in control, TgR, TgS and TgSin embryos,

suggesting robust control over ARP in brain development, with

increased but limited production of AChE-R, in turn suggesting

that its transgenic overexpression did not contribute to neuronal

migration at that phase. Nevertheless, AChE-Sin overexpression

exhibited a reduction in cell arrival to the CP, suggesting that the

hydrolytic activity of AChE-S promotes neuronal migration.

Nevertheless, ChAT is expressed primarily in tangentially ori-

ented cells in the IZ (Schambra et al., 1989), suggesting that

these cells do not migrate radially, and indicating that ACh

hydrolysis may indirectly influence radial migration.

Cortical Plate Differentiation

Transient AChE expression was previously described in young

post-mitotic neurons in a superficial layer of the chick neuro-

epithelium (Layer et al., 1988), which later comes to cover the

entire surface of the embryonic chicken brain. Furthermore,

shortly after chick neurons initiate AChE expression, they

extend long projecting neurites (Layer, 1991) and establish

distant connections (Weikert et al., 1990). Murine ChAT

immunoreactivity was reported in the early arriving cells of

the margin between the IZ and CP (Schambra et al., 1989),

suggesting that ACh may possibly induce AChE expression. In

our study, AChE-R and AChE-S mRNAs were both expressed in

clusters of newly arriving, i.e. undifferentiated, neurons. ASP and

the common N-terminus exhibited similar immunoreactivity to

that of both AChE-S and AChE-R transcripts in the CP, whereas

ARP was located along radial glia. AChE-R secreted from

differentiating cells at the CP may hence regulate cell migration.

Conversely, the effect of AChE-S on neurite extension was

attributed to the adhesion properties of its neuroligin-like core

domain (Andres et al., 1997; Grifman et al., 1998; Sternfeld et al.,

1998), independently of its catalytic activity (for review see

Soreq and Seidman, 2001). The neuroligin family of brain-

specific mammalian AChE-homologues (Ichtchenko et al.,

1996), is of particular importance to brain development, espe-

cially in excitatory synapses (Ichtchenko et al., 1996; Song et al.,

1999). In PC12 cells, antisense suppression of AChE-R restricted

differentiation and neurite extension in a manner restorable by

transfected neuroligin-1 (Grifman et al., 1998). This suggested

redundant properties for AChE and neuroligins, possibly

through binding to neurexin Ib, and provided a possible mech-

anism for AChE’s involvement in neuronal differentiation and

network formation in the cortical plate. Mutated neuroligin

increases the risk of autism (Jamain et al., 2003), likely through

impaired interaction with b-neurexins, neuronal surface pro-

teins (Ullrich et al., 1995) involved with neuronal differentia-

tion, axogenesis and neural network formation (Dean et al.,

2003). That overexpressed AChE-S suppresses neurexin Ibproduction in embryonic motoneurons of TgS mice (Andres

et al., 1997), thus highlights the putative importance of the

alternative splicing shift for brain development.

Prenatal Stress and AChE Malexpression

In the adult brain, stress and blockade of AChE enhance ACh

release, with balance retrieved by AChE-R overproduction

(Kaufer et al., 1998). Our findings suggest that both ACh release

and AChE-R excess may interfere with cortical development.

This provides a tentative explanation to the effects shown for

acute, transient or chronic embryonic stress as well as anti-

AChE intoxication in later forming years. Even defects that are

morphologically non-apparent may result with aberrant micro-

structures, as may be the case with TgS mice which are subject

to early neurodegeneration. No structural or cortical lamination

abnormalities were observed in these mice; nevertheless, they

display progressive accumulation of pathologic, curled neuronal

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processes in the somatosensory cortex, whereas transgenic

excess of AChE-R attenuates this appearance (Sternfeld et al.,

2000). The developmental construction of the mammalian

cortical plate thus reflects a well-concerted balance of alter-

native splicing shifts which may be perturbed under environ-

mental exposure to anticholinesterases (e.g. common agricultural

insecticides) or traumatic experiences.

Notes

The authors are grateful to Dr A. Frankfurter for anti-TUJ1 antibodies.

This study was supported by US Army Medical Research and Material

Command Grant DAMD 17-99-1-9547, Israel Science Fund Grant 618/

02 (to H.S.). A.D. was a Post-Doctoral Fellow of the National Institute for

Psychobiology in Israel (Fellowship in Memory of Mrs Leah M. Smith),

at the Hebrew University.

Address correspondence to Hermona Soreq, Department of Biological

Chemistry, Institute of Life Sciences, The Edmond J. Safra Campus, The

Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.

Email: [email protected].

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