1 Department of Neurosurgery, Soroka University Medical Center, Beer-Sheva, Israel, 2 Life Sciences Institute, The Hebrew University of Jerusalem, Jerusalem, Israel, 3 Department of Morphology, Zlotowski Center for Neuroscience, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel and 4 Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
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: soreq{at}cc.huji.ac.il.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: alternative splicing neurogenesis neuronal migration radial glia readthrough acetylcholinesterase
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Proliferation, differentiation and programmed gene expression in the developing nervous system may all be subject to modulation by stress. Embryonic stress (e.g. ischemiahypoxia) attenuates neuronal migration to the cerebral neocortex, resulting 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 malformation and reduced head circumference (Mulder et al., 2002
). Improved understanding of the molecular mechanisms underlying 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 transcripts, 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 variant mRNA transcripts of a neuronally-expressed gene that is subject to transcription and splicing changes under both development and stress, and observe the outcome with respect to subsequent developmental events. The acetylcholinesterase gene (ACHE) emerges as an appropriate example for such 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 proliferation and neurite outgrowth, is widely considered a sensitive early marker of histochemical differentiation (Layer and Willbold, 1995). Thus, in the adult brain, acute stress induces overproduction 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 cholinergic neurotransmission (Soreq and Seidman, 2001
), but subsequent 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 oligonucleotide 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 cleavage, 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 pathophysiological roles for alternative splicing of AChE in cortical development.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CD1 and FVB/N mice were used for antisense and transgenic experiments 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, respectively). Embryos were removed and dissected in cold phosphate buffered saline (PBS). Heads (E1115) and brains (E1617) were immersed in 4% paraformaldehyde in PBS (48 h, 4°C), embedded in paraffin and sectioned at 4 µm in the coronal plane. Animal care followed institutional guidelines according to NIH published guidelines.
BrdU and Oligonucleotides Injections
Pregnant dams were injected intraperitoneally (i.p.) with bromodeoxyuridine (BrdU; 50 mg/kg in 7 mM NaOHsaline 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 3'-terminal nucleotides 5'-CTGCAATATTTTCTTGC*A*C*C-3' (stars) were 2-O-methylated for nuclease protection. Inversely oriented oligodeoxynucleotides (INV101) with the same sequence as the antisense, but oriented from 3' to 5' 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 µg/kg per injection). Animals were sacrificed 48 h after BrdU injection.
Immunohistochemistry
Sections 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 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 Immunoresearch 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 proliferating 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 x 100 µm rectangle at the apical portion of the VZ, positioned 100 µm lateral to the dorsomedial to medial cortical border, similar to that done for detection of AChE.
BrdU Labeling
Sections were treated with 100 µg/ml deoxyribonuclease in PBST (30 min), incubated with mouse anti-BrdU (Becton-Dickinson, Mississauga, 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 Hybridization
Previously detailed probes and procedure (Meshorer et al., 2002) were modified as follows. Cy5-conjugated streptavidin and Cy3-conjugated anti-digoxygenin were 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-immunofluorescence 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 Microscopy
Images of 1-µm-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 505545 or 560615 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 were maintained to reach the narrow depth of maximal signal intensity.
Regions of Analyses
Sectors of analysis were 200 µm wide and distant 100 µm 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 Analyses
At 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.
Immunoblots
Cerebral 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 Activity
Acetylthiocholine hydrolysis was measured spectrophotometrically as described (Kaufer et al., 1998). Iso-OMPA (tetraisopropylpyrophosphoramide) was used to block butyrylcholinesterase activity (5 x 105 M).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 proliferation, 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, cytoplasmic 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). Densitometric measurements of AChE expression demonstrated a gradual reduction in labeling intensity throughout the VZ during neurogenesis. Expression areas of AChE splice variants exhibited 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 determined 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 Differentiated Neurons Display Splicing Shift at the Ventricular Zone
A 2 h pulse of BrdU was used to distinguish between S+G2+M versus 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 incorporation into nuclei in the S-phase, while the earliest of these nuclei advance through G2 and initiate mitosis (Takahashi et al., 1992). Combined with in situ hybridization, anti-BrdU immunofluorescence 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).
|
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 they were migrating from the VZ to the CP. Immunofluorescence 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 were TUJ1-negative (Fig. 4B,C). With further development, AChE expression in the CP appeared in clusters of intensely labeled cells, surrounded by moderately 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 undifferentiated cells, and declined as the number of differentiated cells increased. Although TUJ1 expression was essentially detected in the deeper portion of the CP, a few of these cells were seen at the superficial portion adjacent to the MZ (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 from AChE to TUJ1-expressing cells. In addition to this, some horizontally oriented TUJ1 cells were detected in the MZ, possibly Cajal-Retzius cells.
|
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).
|
|
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 extracted 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 immunoreactivity was observed to antibodies directed against ASP (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-transferasehuman 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 conservation 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).
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-1720).
Antisense Suppression of AChE-R mRNA Attenuates Neuronal 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 100 x 50 µm square samples in the apical portion of the VZ (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 µg/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).
|
AChE-R mRNA Destruction Increases Proliferation in the Ventricular Zone
The gradual reduction of AChE gene expression during neurogenesis, 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 the VZ of the mouse developing neocortex at E16. Few cells 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 treatment significantly increased Ki67 expression in the VZ (Fig. 8C,D), suggesting increased re-entry into the cell cycle vs exiting the cell cycle following reduction of AChE-R expression by EN101.
|
VZ neuronal progenitors express both AChE-S and AChE-R. The observed effect of EN101 described above, therefore, implied one of two possibilities: (i) AChE-R alone reduces proliferation; or (ii) ACh hydrolysis, common to AChE-R and AChE-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 transcriptasepolymerase 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 and TgS 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 development. Arrival of cells in the CP from both TgR or TgS overexpressing 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.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
While many neuronal mRNAs are subject to alternative splicing modulations during brain development (e.g. the clathrin assembly protein 3 (AP-3) (Ishihara-Sugano and Nakae, 1997), the protein tyrosine phosphatases PTP-SL and PTPBR7 (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 processing 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 and Proliferation
During murine brain development, alternative splicing modulation 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 production attenuated neuronal migration to the CP, suggesting causal involvement of the splice shift in this process. In addition, the antisense treatment increased neuronal progenitor proliferation. 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, progenitor 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 suggested 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 regulator 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 neurogenesis, 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 proliferation 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 elevation (Sugawara et al., 2002
). Taken together with the present work, this suggests a causal interrelationship between AChE alternative splicing and glial cell de-differentiation.
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 previously 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 hydrolysis. Alternatively, or in addition, AChE-Sin incorporation into progenitors' membranes might 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 demonstrated that the C-terminus of AChE-R, including ARP, is detached 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 cellcell 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 oriented 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 neuroepithelium (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, especially 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 Iß, and provided a possible mechanism 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 ß-neurexins, neuronal surface proteins (Ullrich et al., 1995
) involved with neuronal differentiation, axogenesis and neural network formation (Dean et al., 2003
). That overexpressed AChE-S suppresses neurexin Iß production 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 microstructures, 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 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 alternative splicing shifts which may be perturbed under environmental exposure to anticholinesterases (e.g. common agricultural insecticides) or traumatic experiences.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anton ES, Marchionni MA, Lee KF, Rakic P (1997) Role of GGF/neuregulin signaling in interactions between migrating neurons and radial glia in the developing cerebral cortex. Development 124:35013510.
Bataille S, Portalier P, Coulon P, Ternaux JP (1998) Influence of acetylcholinesterase on embryonic spinal rat motoneurones growth in culture: a quantitative morphometric study. Eur J Neurosci 10:560572.[CrossRef][ISI][Medline]
Beeri R, Andres C, Lev Lehman E, Timberg R, Huberman T, Shani M, Soreq H (1995) Transgenic expression of human acetylcholinesterase induces progressive cognitive deterioration in mice. Curr Biol 5:10631071.[ISI][Medline]
Birikh KR, Sklan EH, Shoham S, Soreq H (2003) Interaction of readthrough acetylcholinesterase with RACK1 and PKCbeta II correlates with intensified fear-induced conflict behavior. Proc Natl Acad Sci USA 100:283288.
Bittman K, Owens DF, Kriegstein AR, LoTurco JJ (1997) Cell coupling and uncoupling in the ventricular zone of developing neocortex. J Neurosci 17:70377044.
Boulder Committee (1970) Embryonic vertebrate central nervous system: revised terminology. The Boulder Committee. Anat Rec 166:257261.[ISI][Medline]
Cai L, Hayes NL, Nowakowski RS (1997) Synchrony of clonal cell proliferation and contiguity of clonally related cells: production of mosaicism in the ventricular zone of developing mouse neocortex. J Neurosci 17:20882100.
Chenn A, McConnell SK (1995) Cleavage orientation and the asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis. Cell 82:631641.[ISI][Medline]
Cohen O, Erb C, Ginzberg D, Pollak Y, Seidman S, Shoham S, Yirmiya R, Soreq H (2002) Neuronal overexpression of readthrough acetylcholinesterase is associated with antisense-suppressible behavioral impairments. Mol Psychiatry 7:874885.[CrossRef][ISI][Medline]
Cohen O, Reichenberg A, Perry C, Ginzberg D, Pollmacher T, Soreq H, Yirmiya R (2003) Endotoxin-induced changes in human working and declarative memory associate with cleavage of plasma readthrough acetylcholinesterase. J Mol Neurosci 21:199212.[CrossRef][ISI][Medline]
Dean C, Scholl FG, Choih J, DeMaria S, Berger J, Isacoff E, Scheiffele P (2003) Neurexin mediates the assembly of presynaptic terminals. Nat Neurosci 6:708716.[CrossRef][ISI][Medline]
Delalle I, Takahashi T, Nowakowski RS, Tsai LH, Caviness VS (1999) Cyclin E-p27 opposition and regulation of the G1 phase of the cell cycle in the murine neocortical PVE: a quantitative analysis of mRNA in situ hybridization. Cereb Cortex 9:824832.
du Manoir S, Guillaud P, Camus E, Seigneurin D, Brugal G (1991) Ki-67 labeling in postmitotic cells defines different Ki-67 pathways within the 2c compartment. Cytometry 12:455463.[ISI][Medline]
Frantz GD, McConnell SK (1996) Restriction of late cerebral cortical progenitors to an upper-layer fate. Neuron 17:5561.[ISI][Medline]
Gadisseux JF, Evrard P, Misson JP, Caviness VS (1989) Dynamic structure of the radial glial fiber system of the developing murine cerebral wall. An immunocytochemical analysis. Brain Res Dev Brain Res 50:5567.[ISI][Medline]
Geisert EE Jr, Frankfurter A (1989) The neuronal response to injury as visualized by immunostaining of class III beta-tubulin in the rat. Neurosci Lett 102:137141.[CrossRef][ISI][Medline]
Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H (1984) Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133:17101715.
Grifman M, Galyam N, Seidman S, Soreq H (1998) Functional redundancy of acetylcholinesterase and neuroligin in mammalian neuritogenesis. Proc Natl Acad Sci USA 95:1393513940.
Grisaru D, Lev Lehman E, Shapira M, Chaikin E, Lessing JB, Eldor A, Eckstein F, Soreq H (1999) Human osteogenesis involves differentiation-dependent increases in the morphogenically active 3' alternative splicing variant of acetylcholinesterase. Mol Cell Biol 19:788795.
Grisaru D, Deutsch V, Shapira M, Pick M, Sternfeld M, Melamed-Book N, Kaufer D, Galyam N, Gait MJ, Owen D, Lessing JB, Eldor A, Soreq H (2001) ARP, a peptide derived from the stress-associated acetylcholinesterase variant, has hematopoietic growth promoting activities. Mol Med 7:93105.[ISI][Medline]
Hartfuss E, Galli R, Heins N, Gotz M (2001) Characterization of CNS precursor subtypes and radial glia. Dev Biol 229:1530.[CrossRef][ISI][Medline]
Hashimoto Y, Zhang C, Kawauchi J, Imoto I, Adachi MT, Inazawa J, Amagasa T, Hai T, Kitajima S (2002) An alternatively spliced isoform of transcriptional repressor ATF3 and its induction by stress stimuli. Nucleic Acids Res 30:23982406.
Ichtchenko K, Nguyen T, Sudhof TC (1996) Structures, alternative splicing, and neurexin binding of multiple neuroligins. J Biol Chem 271:26762682.
Ishihara-Sugano M, Nakae H (1997) Developmentally regulated mRNA splicing of clathrin assembly protein 3 (AP-3). Brain Res Mol Brain Res 52:290298.[ISI][Medline]
Jamain S, Quach H, Betancur C, Rastam M, Colineaux C, Gillberg IC, Soderstrom H, Giros B, Leboyer M, Gillberg C, Bourgeron T (2003) Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism. Nat Genet 34:2729.[CrossRef][ISI][Medline]
Karpel R, Sternfeld M, Ginzberg D, Guhl E, Graessmann A, Soreq H (1996) Overexpression of alternative human acetylcholinesterase forms modulates process extensions in cultured glioma cells. J Neurochem 66:114123.[ISI][Medline]
Kaufer D, Friedman A, Seidman S, Soreq H (1998) Acute stress facilitates long-lasting changes in cholinergic gene expression. Nature 393:373377.[CrossRef][ISI][Medline]
Kee N, Sivalingam S, Boonstra R, Wojtowicz JM (2002) The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. J Neurosci Methods 115:97105.[CrossRef][ISI][Medline]
Kofman O (2002) The role of prenatal stress in the etiology of developmental behavioural disorders. Neurosci Biobehav Rev 26:457470.[CrossRef][ISI][Medline]
Kuppers E, Sabolek M, Anders U, Pilgrim C, Beyer C (2000) Developmental regulation of glutamic acid decarboxylase mRNA expression and splicing in the rat striatum by dopamine. Brain Res Mol Brain Res 81:1928.[ISI][Medline]
Lauder JM, Schambra UB (1999) Morphogenetic roles of acetylcholine. Environ Health Perspect 107:6569.[ISI][Medline]
Layer PG (1991) Cholinesterases during development of the avian nervous system. Cell Mol Neurobiol 11:733.[ISI][Medline]
Layer PG, Rommel S, Bulthoff H, Hengstenberg R (1988) Independent spatial waves of biochemical differentiation along the surface of chicken brain as revealed by the sequential expression of acetylcholinesterase. Cell Tissue Res 251:587595.[CrossRef][ISI][Medline]
Layer PG, Willbold E (1995) Novel functions of cholinesterases in development, physiology and disease. Prog Histochem Cytochem 29:199.[ISI][Medline]
Lee MK, Tuttle JB, Rebhun LI, Cleveland DW, Frankfurter A (1990) The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis. Cell Motil Cytoskeleton 17:118132.[ISI][Medline]
Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60:585595.[ISI][Medline]
Lev Lehman E, Deutsch V, Eldor A, Soreq H (1997) Immature human megakaryocytes produce nuclear-associated acetylcholinesterase. Blood 89:36443653.
Ma W, Maric D, Li BS, Hu Q, Andreadis JD, Grant GM, Liu QY, Shaffer KM, Chang YH, Zhang L, Pancrazio JJ, Pant HC, Stenger DA, Barker JL (2000) Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation. Eur J Neurosci 12:12271240.[CrossRef][ISI][Medline]
Malatesta P, Hack MA, Hartfuss E, Kettenmann H, Klinkert W, Kirchhoff F, Gotz M (2003) Neuronal or glial progeny: regional differences in radial glia fate. Neuron 37:751764.[ISI][Medline]
McConnell SK, Kaznowski CE (1991) Cell cycle dependence of laminar determination in developing neocortex. Science 254:282285.[ISI][Medline]
Meshorer E, Erb C, Gazit R, Pavlovsky L, Kaufer D, Friedman A, Glick D, Ben-Arie N, Soreq H (2002) Alternative splicing and neuritic mRNA translocation under long-term neuronal hypersensitivity. Science 295:508512.
Misson JP, Edwards MA, Yamamoto M, Caviness VS, Jr. (1988) Mitotic cycling of radial glial cells of the fetal murine cerebral wall: a combined autoradiographic and immunohistochemical study. Brain-Res 466:183190.[Medline]
Morishita R, Shinohara H, Ueda H, Kato K, Asano T (1999) High expression of the gamma5 isoform of G protein in neuroepithelial cells and its replacement of the gamma2 isoform during neuronal differentiation in the rat brain. J Neurochem 73:23692374.[CrossRef][ISI][Medline]
Mulder EJ, Robles de Medina PG, Huizink AC, Van den Bergh BR, Buitelaar JK, Visser GH (2002) Prenatal maternal stress: effects on pregnancy and the (unborn) child. Early Hum Dev 70:314.[CrossRef][ISI][Medline]
Nadarajah B, Brunstrom JE, Grutzendler J, Wong RO, Pearlman AL (2001) Two modes of radial migration in early development of the cerebral cortex. Nat Neurosci 4:143150.[CrossRef][ISI][Medline]
Noctor SC, Flint AC, Weissman TA, Dammerman RS, Kriegstein AR (2001) Neurons derived from radial glial cells establish radial units in neocortex. Nature 409:714720.[CrossRef][ISI][Medline]
Paoletti F, Mocali A, Vannucchi AM (1992) Acetylcholinesterase in murine erythroleukemia (Friend) cells: evidence for megakaryocyte-like expression and potential growth-regulatory role of enzyme activity. Blood 79:28732879.[Abstract]
Perrier AL, Massoulie J, Krejci E (2002) PRiMA: the membrane anchor of acetylcholinesterase in the brain. Neuron 33:275285.[ISI][Medline]
Perry C, Sklan EH, Birikh K, Shapira M, Trejo L, Eldor A, Soreq H (2002) Complex regulation of acetylcholinesterase gene expression in human brain tumors. Oncogene 21:84288441.[CrossRef][ISI][Medline]
Pick M, Flores-Flores C, Grisaru D, Shochat S, Deutsch V, Soreq H (2004) Blood cells-specific acetylcholinesterase splice variations under changing stimuli. Int J Dev Neurosci (in press).
Rakic P (1972) Mode of cell migration to the superficial layers of fetal monkey neocortex. J-Comp-Neurol 145:6183.[ISI][Medline]
Rakic P (1974) Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition. Science 183:425427.[ISI][Medline]
Rakic P, Cameron RS, Komuro H (1994) Recognition, adhesion, transmembrane signaling and cell motility in guided neuronal migration. Curr Opin Neurobiol 4:6369.[Medline]
Schambra UB, Sulik KK, Petrusz P, Lauder JM (1989) Ontogeny of cholinergic neurons in the mouse forebrain. J-Comp-Neurol 288:101122.[CrossRef][ISI][Medline]
Schlaggar BL, De Carlos JA, O'Leary DD (1993) Acetylcholinesterase as an early marker of the differentiation of dorsal thalamus in embryonic rats. Brain Res Dev Brain Res 75:1930.[ISI][Medline]
Scholzen T, Gerdes J (2000) The Ki-67 protein: from the known and the unknown. J Cell Physiol 182:311322.[CrossRef][ISI][Medline]
Sharma KV, Koenigsberger C, Brimijoin S, Bigbee JW (2001) Direct evidence for an adhesive function in the noncholinergic role of acetylcholinesterase in neurite outgrowth. J Neurosci Res 63:165175.[CrossRef][ISI][Medline]
Shohami E, Kaufer D, Chen Y, Seidman S, Cohen O, Ginzberg D, Melamed-Book N, Yirmiya R, Soreq H (2000) Antisense prevention of neuronal damages following head injury in mice. J Mol Med 78:228236.[CrossRef][ISI][Medline]
Song JY, Ichtchenko K, Sudhof TC, Brose N (1999) Neuroligin 1 is a postsynaptic cell-adhesion molecule of excitatory synapses. Proc Natl Acad Sci USA 96:11001105.
Soreq H, Seidman S (2001) Acetylcholinesterase new roles for an old actor. Nat Rev Neurosci 2:294302.[CrossRef][ISI][Medline]
Starborg M, Gell K, Brundell E, Hoog C (1996) The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and heterochromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progression. J Cell Sci 109:143153.
Sternfeld M, Ming G, Song H, Sela K, Timberg R, Poo M, Soreq H (1998) Acetylcholinesterase enhances neurite growth and synapse development through alternative contributions of its hydrolytic capacity, core protein, and variable C termini. J Neurosci 18:12401249.
Sternfeld M, Shoham S, Klein O, Flores-Flores C, Evron T, Idelson GH, Kitsberg D, Patrick JW, Soreq H (2000) Excess read-through acetylcholinesterase attenuates but the synaptic variant intensifies neurodeterioration correlates. Proc Natl Acad Sci USA 97:86478652.
Sugawara K-i, Kurihara H, Negishi M, Saito N, Nakazato Y, Sasaki T, Takeuchi T (2002) Nestin as a marker for proliferative endothelium in gliomas. Lab Invest 82:345351.[CrossRef][ISI][Medline]
Takahashi T, Nowakowski RS, Caviness VS Jr (1992) BUdR as an S-phase marker for quantitative studies of cytokinetic behaviour in the murine cerebral ventricular zone. J Neurocytol 21:185197.[ISI][Medline]
Takahashi T, Nowakowski RS, Caviness VS Jr (1995) The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall. J Neurosci 15:60466057.[Abstract]
Takahashi T, Nowakowski RS, Caviness VS Jr (1996) The leaving or Q fraction of the murine cerebral proliferative epithelium: a general model of neocortical neuronogenesis. J Neurosci 16:61836196.
Tamamaki N, Nakamura K, Okamoto K, Kaneko T (2001) Radial glia is a progenitor of neocortical neurons in the developing cerebral cortex. Neurosci Res 41:5160.[CrossRef][ISI][Medline]
Tashima L, Nakata M, Anno K, Sugino N, Kato H (2001) Prenatal influence of ischemiahypoxia-induced intrauterine growth retardation on brain development and behavioral activity in rats. Biol Neonate 80:8187.[CrossRef][ISI][Medline]
Ullrich B, Ushkaryov YA, Sudhof TC (1995) Cartography of neurexins: more than 1000 isoforms generated by alternative splicing and expressed in distinct subsets of neurons. Neuron 14:497507.[ISI][Medline]
Van Den Maagdenberg AM, Bachner D, Schepens JT, Peters W, Fransen JA, Wieringa B, Hendriks WJ (1999) The mouse Ptprr gene encodes two protein tyrosine phosphatases, PTP-SL and PTPBR7, that display distinct patterns of expression during neural development. Eur J Neurosci 11:38323844.[CrossRef][ISI][Medline]
Weikert T, Rathjen FG, Layer PG (1990) Developmental maps of acetylcholinesterase and G4-antigen of the early chicken brain: long-distance tracts originate from AChE-producing cell bodies. J Neurobiol 21:482498.[CrossRef][ISI][Medline]
Weissman T, Noctor SC, Clinton BK, Honig LS, Kriegstein AR (2003) Neurogenic radial glial cells in reptile, rodent and human: from mitosis to migration. Cereb Cortex 13:550559.
Xie J, Black DL (2001) A CaMK IV responsive RNA element mediates depolarization-induced alternative splicing of ion channels. Nature 410:936939.[CrossRef][ISI][Medline]
|