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The Journal of Neuroscience, December 1, 1998, 18(23):9858-9869
Evidence for the Participation of the Neuron-Specific CDK5
Activator P35 during Laminin-Enhanced Axonal Growth
Gabriela
Paglini1,
Gustavo
Pigino1,
Patricia
Kunda1,
Gerardo
Morfini1,
Ricardo
Maccioni2,
Santiago
Quiroga3,
Adriana
Ferreira4, and
Alfredo
Cáceres1
1 Instituto Mercedes y Martín Ferreyra, Consejo
Nacional de Investigaciones Científicas y Técnicas
(CONICET), 5000 Cordoba, Argentina, 2 Laboratorio
Biología Celular y Molecular, Universidad de Chile, 5000 Santiago, Chile, 3 Departamento Quimica Biologica, CONICET,
Universidad Nacional de Cordoba, 5000 Cordoba, Argentina, and
4 Institute of Neuroscience and Department of Cell and
Molecular Biology, Northwestern University, Chicago, Illinois
60611
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ABSTRACT |
Cultures of cerebellar macroneurons were used to study the pattern
of expression, subcellular localization, and function of the neuronal
cdk5 activator p35 during laminin-enhanced axonal growth. The results
obtained indicate that laminin, an extracellular matrix molecule
capable of selectively stimulating axonal extension and promoting MAP1B
phosphorylation at a proline-directed protein kinase epitope,
selectively stimulates p35 expression, increases its association with
the subcortical cytoskeleton, and accelerates its redistribution to the
axonal growth cones. Besides, suppression of p35, but not of a highly
related isoform designated as p39, by antisense oligonucleotide
treatment selectively reduces cdk5 activity, laminin-enhanced axonal
elongation, and MAP1b phosphorylation. Taken collectively, the present
results suggest that cdk5/p35 may serve as an important regulatory
linker between environmental signals (e.g., laminin) and constituents
of the intracellular machinery (e.g., MAP1B) involved in axonal elongation.
Key words:
cdk5; p35; neurons; development; axon; growth cones; MAP1b; phosphorylation; antisense oligonucleotides; neuronal
cultures
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INTRODUCTION |
Developing neurons project axons
toward their targets often over relatively enormous distances. At the
distal end of these projections is the growth cone, a structure
containing dynamic protrusions that sample a complex extracellular
environment composed of adhesive substrates. Among these are a variety
of extracellular matrix components, such as laminin, collagen,
fibronectin, vitronectin, tenascin, and thrombospondin capable of
promoting process outgrowth, elongation, and/or branching (Reichardt
and Tomaselli, 1991 ).
Intracellular mechanisms provide structural elements, most notably
microtubules, to maintain elongating neuronal projections and the
transport of membranous organelles or cytoskeletal elements to the
active growing tip of neuritic processes (Mitchison and Kirschner,
1988; Tanaka and Sabry, 1995). As the neuronal specific organization of
microtubules depends on their high degree of spatial and temporal
differentiation, a great deal of attention has been devoted to identify
factors controlling microtubule organization in nerve cells. Current
evidence favors the view that the regulation of microtubule assembly
and stability during process formation is dependent on the expression
of a particular set of proteins, known as microtubule-associated
proteins or MAPs (Matus, 1988; Hirokawa, 1994; Maccioni and Cambiazo,
1995). Interestingly, all of the MAPs that have been directly
implicated in axonal formation, such as MAP1b and tau
(Cáceres and Kosik, 1990; Brugg et al., 1993; Esmaeli-Azad
et al., 1994; Harada et al., 1994; DiTella et al., 1996; Edelman et
al., 1996; Takei et al., 1997) are present at the distal end of growing
axons, including the central and peripheral regions of growth cones
(Ulloa et al., 1994; Brandt et al., 1995; Rocha and Avila, 1995;
DiTella et al., 1996). Besides, there is an emerging body of evidence
suggesting that the in vivo usage of MAPs is stimulated by
environmental factors (Drubin et al., 1985; Ferreira and Cáceres,
1991; DiTella et al., 1996). Despite that, it is not yet clear how
environmental clues, and particularly extracellular matrix molecules,
may modulate the functional activity of MAP1b or tau. One obvious
possibility is the local and temporal regulation of MAPs activity by
phosphorylation (Avila et al., 1994; Tanaka and Sabry, 1995).
To begin testing this hypothesis, in previous studies we have
shown that laminin, a molecule capable of selectively enhancing axonal
outgrowth and promoting MAP1b phosphorylation, accelerates the
redistribution of the cyclin-dependent kinase 5 (cdk5) to the axonal
growth cone and dramatically stimulates its activity (DiTella et al.,
1996, Pigino et al., 1997). In addition, our results showed that cdk5
suppression by antisense oligonucleotide treatment reduced axonal
elongation and decreased the phosphorylation status of MAP1b, as well
as its binding to microtubules (Pigino et al., 1997). In the present
study we present evidence about the mechanisms by which laminin may
regulate cdk5 activity and MAP1b phosphorylation. The results obtained
suggest that by regulating the expression and subcellular distribution
of p35, a brain-specific activator of cdk5 (Lew et al., 1994; Tsai et
al., 1994; Nikolic et al., 1995; Lee et al., 1996a; Chae et al.,
1997) laminin stimulates cdk5 activity, MAP1b phosphorylation, and
axonal elongation.
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MATERIALS AND METHODS |
Cell cultures. Dissociated cultures of cerebellar
macroneurons were prepared as described previously (Ferreira et al.,
1989; Cáceres et al., 1992; DiTella et al., 1996). Cells were
plated onto polylysine-coated glass coverslips (12 or 25 mm in
diameter) at densities ranging from 5000 to 15,000 cells per
cm2 and maintained with DMEM plus 10% horse
serum for 1 hr. The coverslips with the attached cells were then
transferred to 60 mm Petri dishes containing serum-free medium plus the
N2 mixture of Bottenstein and Sato (1979). All cultures were maintained
in a humidified 37°C incubator with 5% CO2.
To bind laminin to the substrate, polylysine-coated coverslips were
soaked in DMEM containing mouse EHS laminin (Life Technologies, Gaithersburg, MD; Sigma, St. Louis, MO; or Boehringer Mannheim, Indianapolis, IN) at a concentration of 10 µg/ml (unless otherwise specified) overnight at 4°C. In some experiments, laminin was directly added to the culture medium from a 1 mg/ml stock solution to
make a final concentration of 10 or 20 µg/ml (DiTella et al., 1996).
To block laminin activity, an affinity-purified rabbit polyclonal
antibody against  1-integrin (Carri et al., 1992) was directly added
to the tissue culture medium at concentrations ranging from 100 to 200 µg/ml.
Antisense oligonucleotides. Three antisense phosphorothioate
oligonucleotides (S-modified) were used in the present study. One of
them, designated RP1, consists of the sequence 5' CCCTTCGGCCGGACCACG 3', and it is the inverse complement of the nucleotides +1870/+1887 of
the rat cDNA for p35; the other one designated RP2 consists of the
sequence 5' GACGACGCGACGGACCCG 3', and it is the inverse complement of
the nucleotides +914/+931 of the rat cDNA for p35 (Lew et al., 1994;
Tsai et al., 1994). A third antisense oligonucleotide consists of the
sequence AGCCGGCGGTCCCTGTCG, and it is the inverse complement of the
nucleotides +1094/+1111 of the rat cDNA for p39, an isoform of p35
(Tang et al., 1995). Analysis of a gene data base (GenBank) showed that
the sequences selected have no significant homology with any other
known sequence. The oligonucleotides were purchased from Quality
Controlled Biochemicals (Hopkinton, MA); they were purified by reverse
chromatography, and they were taken up in serum-free medium as
described previously (Cáceres and Kosik, 1990; Cáceres et
al., 1991, 1992; DiTella et al., 1996). For all the experiments the
antisense oligonucleotides were preincubated with 2 µl of
Lipofectin reagent (1 mg/ml; Life Technologies) diluted in 100 µl of serum-free medium. The resulting oligonucleotide suspension was
then added to the primary cultured neurons at concentrations ranging
from 0.5 to 5 µM. Control cultures were treated with the
same concentration of the corresponding sense-strand oligonucleotides.
For some experiments cultures were treated with a combination of tau
and MAP1b antisense oligonucleotides as described previously (DiTella
et al., 1996; Pigino et al., 1997).
Primary antibodies. The following primary antibodies were
used in this study: a monoclonal antibody (mAb) against
tyrosinated  -tubulin (clone TUB-1A2, mouse IgG; Sigma) diluted
1:2000; a mAb against microtubule-associated protein MAP2 (clone AP14;
Cáceres et al., 1992) diluted 1:500; a mAb against an integral
Golgi membrane protein designated as TGN38 (clone 2F7.1; Oncogene
Research Products, Calbiochem, MA; see also, Horn and Banting, 1994)
diluted 1:200; an affinity-purified rabbit polyclonal antibody raised
against a peptide corresponding to amino acids 2-21 mapping at
the amino terminus of p35 (antibody N-20; Santa Cruz Biotechnology,
Santa Cruz, CA) diluted 1:50 or 1:100; an affinity-purified rabbit
polyclonal antibody raised against a peptide corresponding to amino
acid residues 289-307 mapping at the C terminus of p35 (antibody C-19, Santa Cruz Biotechnology) diluted 1:50 or 1:100; an affinity-purified polyclonal antibody raised against a peptide corresponding to amino
acid residues 349-367 mapping at the C terminus of the 39 kDa isoform
of p35 (Research Genetics, Huntsville, AL; see also Tang et al., 1995)
diluted 1:50; an affinity-purified rabbit polyclonal antibody raised
against a peptide corresponding to amino acid residues 284-291 mapping
at the C terminus of cdk5 diluted 1:10, 1:50, or 1:100 (Santa Cruz
Biotechnology; see also, Lee et al., 1996a); and a mAb raised
against a peptide corresponding to amino acid residues 181-198 mapping
at the C terminus of the mitotic inhibitor p27 (clone F-8; Santa Cruz
Biotechnology) diluted 1:200. We also used several antibodies against
MAP1b: mAb AA6, which recognizes a conserved nonphosphorylated and
nonphosphorylatable epitope on MAP1b (mouse IgG; Sigma; Brugg et al.,
1993; DiTella et al., 1996) diluted 1:50; mAb 150, which recognizes a
proline-directed protein kinase- (PDPK) phosphorylated epitope (mouse
IgM; Ulloa et al., 1993a,b, 1994; DiTella et al., 1996; Pigino et al.,
1997) diluted 1:200; and rabbit antiserum 531 that recognizes the PDPK phosphorylatable epitope when it is dephosphorylated (Ulloa et al.,
1993a,b, 1994; DiTella et al., 1996; Pigino et al., 1997) diluted
1:200.
Immunofluorescence. Cells were fixed before or after
detergent extraction under microtubule-stabilizing conditions and
processed for immunofluorescence as previously described (DiTella et
al., 1996; Pigino et al., 1997; see also Black et al., 1994). For some experiments a mild extraction protocol that preserves
cytoskeletal-membrane interactions was used (Nakata and Hirokawa,
1987; Brandt et al., 1995; Pigino et al., 1997). Cells were washed in
extraction buffer (80 mM PIPES, pH 6.8, 1 mM
MgCl2, 1 mM EGTA, 30% v/v glycerol, and 1 mM
GTP), incubated for 30 sec with extraction buffer containing 0.02%
saponin and washed with extraction buffer. Cell were then fixed for 1 hr at room temperature with 2% (w/v) paraformaldehyde, 0.1% (v/v)
glutaraldehyde in extraction buffer, washed with PBS, permeabilized
with 0.1% (v/v) Triton X-100 in PBS for 30 min, and finally washed in
PBS. The antibody staining protocol entailed labeling with the first
primary antibody, washing with PBS, staining with labeled secondary
antibody (fluorescein- or rhodamine-conjugated) and washing similarly;
the same procedure was repeated for the second primary antibody.
Incubations with primary antibodies were for 1 or 3 hr at room
temperature, whereas incubations with secondary antibodies were
performed during 1 hr at 37°C. The cells were analyzed with a Zeiss
LSM 410 confocal scanning microscope or with an inverted
microscope (Carl Zeiss Axiovert 35 M) equipped with epifluorescence and
differential interference contrast (DIC) optics and photographed using
40×, 63×, or 100× objectives (Carl Zeiss) with Tri X-Pan or T-MAX
400 ASA film (Eastman Kodak, Rochester, NY). Exposure times ranged from
45 to 60 sec.
For some experiments the relative intensities of tubulin, cdk5, p35, or
MAP1b immunofluorescence were evaluated in fixed unextracted cells or
in detergent-extracted cytoskeletons using quantitative fluorescence
techniques as described previously (DiTella et al., 1994, 1996; Pigino
et al., 1997). To image labeled cells, the incoming epifluorescence
illumination was attenuated with glass neutral density filters. Images
were formed on the faceplate of a Silicon Intensified Target (SIT)
camera (Hamamatsu, Middlesex, NJ), set for manual sensitivity, gain,
and black level. They were digitized directly into a
Metamorph/Metafluor Image Processor (Universal Imaging Corporation,
West Chester, PA) controlled by a host IBM-AT computer. Fluorescence
intensity measurements were performed pixel by pixel along the
longitudinal axis of identified neurons. Using this data, we then
calculated the average fluorescence intensity within the cell body,
inner, middle, and distal third of identified neurites (either minor
processes or axons). In addition, in some cases the distribution of
cdk5 and that of microtubules labeled with antibodies against
tyrosinated -tubulin were analyzed using high resolution
fluorescence microscopy and ratio image analysis with the image
processing menu of the Metamorph/Metafluor system.
Isolation of growth cone particles by subcellular
fractionation. Fetal rat brain (18 d of gestation) was
fractionated according to Pfenninger et al. (1983) (see also Quiroga et
al., 1995) to obtain growth cone particles (GCPs). Briefly, the low
speed supernatant (L) of fetal brain homogenate (H) was loaded on a
discontinuous sucrose gradient in which the 0.75 M and 1 M sucrose layers were replaced with a single 0.83 M sucrose step. This facilitated collection of the
interface and increased GCP yield without decreasing purity (Lohse et
al., 1996). The 0.32 M/0.83 M interface or A
fraction, was collected, diluted with 0.32 M sucrose, and
pelleted to give the GCP fraction. This was resuspended in 0.32 M sucrose for experimentation. It is worth noting that this
preparation (GCPs) has been extensively characterized by electron
microscopic (Pfenniger et al., 1983; Li et al., 1992) and biochemical
methods. These studies have revealed that GCPs contain significant
amounts of c-src (Helmke and Pfenniger, 1996), tau, GAP-43 (Lohse et
al., 1996), and gc (Quiroga et al., 1995), but lack detectable
amounts of high molecular weight MAP2, glial fibrillar acidic protein,
and vimentin (Lohse et al., 1996).
Western blot analysis. Changes in the levels of p35, p39,
p27, cdk5, and microtubular proteins during the in vitro
development of cerebellar macroneurons were also analyzed by Western
blotting as previously described (DiTella et al., 1996; Pigino et al., 1997). Briefly, equal amounts of crude brain homogenates or whole-cell extracts from cultured cells were fractionated on 10% SDS-PAGE and
transferred to polivinylidene difluoride membranes in a
Tris-glycine buffer, 20% methanol. The filters were dried, washed
several times with TBS (10 mM Tris, pH 7.5, and 150 mM NaCl), and blocked for 1 hr in TBS containing 5% BSA.
The filters were incubated for 1 hr at 37°C with the primary
antibodies in TBS containing 5% BSA. The filters were then washed
three times (10 min each) in TBS containing 0.05% Tween 20 and
incubated with a secondary alkaline phosphatase-conjugated antibody
(ProtoBlot western blot alkaline phosphatase system; Promega, Madison,
WI) for 1 hr at 37°C. After five washes with TBS and 0.05% Tween 20, the blots were developed with bromochloroiodolylphosphate (15 µl of a
50 mg/ml stock solution) and nitroblue-tetrazolium (2.5 µl of a 75 mg/ml stock solution) in 10 ml of alkaline phosphatase-detection buffer
(100 mM Tris, 100 mM NaCl, and 5 mM MgCl2, pH 9.5). In addition, p35, p27, and -tubulin
protein levels were measured by quantitative immunoblotting as
described by Drubin et al. (1985) (see also DiTella et al., 1996). For
such a purpose, immunoblots were probed with the corresponding primary
antibody, followed by incubation with 125I protein A. Autoradiography was performed on Kodak X-omat AR film using
intensifying screens. Autoradiographs were aligned with immunoblots,
and p35 protein levels were quantitated by scintillation counting of
nitrocellulose blot slices.
Immunoprecipitation. For immunoprecipitation cells were
lysed in RIPA buffer containing 50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 5 mM EDTA, pH 8.0, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM
DTT, 1 mM PMSF, 1 µg/ml aprotinin, and 1 µM
leupeptin. Two hundred micrograms of total cellular protein were then
used for immunoprecipitation with the anti-p35 or anti-cdk5 rabbit antibodies. In all cases immunoprecipitation was performed as described
by Tsai et al. (1993) (see also Pigino et al., 1997) using Protein G
Plus agarose (Santa Cruz Biotechnology).
Northern blot analysis of cdk5 and p35 expression. Total RNA
from cultured cerebellar macroneurons was isolated sing the QuickPrep mRNA purification kit from Pharmacia (Pharmacia, Piscataway, NJ). Two
micrograms of Poly(A)'RNA were resolved by gel electrophoresis (1.2%
agarose) and blotted to Hybond N' membranes (Amersham International) by
capillary action. Blots were baked for 2 hr at 80°C and hybridized with a radiolabeled cDNA corresponding to either cdk5 or p35 (a generous gift of Dr. Jerry Wang, Hong Kong University; see Lew et al.,
1994) in 5% SSPE (750 mM NaCl, 50 mM
NaH2PO4, and 5 mM EDTA, pH
7.4) after prehybridization under the same conditions. Blots were
washed at 68°C in 2× SSPE for 10 min, twice in 1× SSPE for 10 min,
and then autoradiographed at  70°C. Exposures times were for 24 or
72 hr.
Morphometric analysis of neuronal shape parameters. Images
were digitized on a video monitor using Metamorph/Metafluor software. To measure neurite length, fixed unstained or antibody-labeled cells
were randomly selected and traced from a video screen using the
morphometric menu of the Metamorph as described previously (Cáceres et al., 1992; DiTella et al., 1994, 1996). The following neuritic shape parameters were evaluated: total length of minor processes per cell, total axonal length per cell, and total dendritic length per cell. All measurements were performed using DIC optics at a
final magnification of 768×. Differences among groups were analyzed by
the use of ANOVA and Student Newman-Keuls test.
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RESULTS |
Laminin stimulates p35 expression in cultured
cerebellar macroneurons
The monospecificity of the affinity-purified rabbit polyclonal
antibody C19 raised against a peptide corresponding to amino acid
residues 289-307 mapping at the C terminus of p35 is shown in Figure
1A. The antibody
recognizes a single band of ~35 kDa when whole-cell extracts of
cultured cerebellar macroneurons growing on polylysine are resolved in
SDS-PAGE, blotted, and immunostained with the anti-p35 antibody (Fig.
1A, lanes 1-3). The
staining generated by this antibody is greatly inhibited by
neutralization with purified peptide (Fig. 1A,
lane 4). Quantitative Western blot analysis
revealed that embryonic day 15 (E15) cerebellar cells express low
levels of p35 at the time of plating and that a significant increase
occurs after the cells have developed in culture for >2 d. This
increase in p35 protein levels closely parallels the increase in cdk5
activity and in axonal length observed after the initial establishment
of neuronal polarity in cultured cerebellar macroneurons (Table
1; see also Pigino et al., 1997).
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Figure 1.
A, Specificity of the
affinity-purified rabbit polyclonal antibody C19 diluted 1:200 (0.5 µg/ml) as revealed by Western blot analysis of cell extracts obtained
from cerebellar macroneurons cultured on polylysine for 12 (lane
1) or 48 (lane 2) hr. The antibody labels a
single immunoreactive protein species with an apparent molecular weight
of 35 kDa. The staining generated by this antibody is completely
abolished by neutralization with the corresponding purified peptide
(lane 3). Ten micrograms of total cellular protein were
loaded in each lane. B, Western blot analysis of
whole-cell extracts reacted with the C19 antibody (0.5 µg/ml)
revealed that laminin significantly increases p35 protein levels in
cultured cerebellar macroneurons. Lanes
1-3, Extracts obtained 4, 12, and 24 hr after
plating from cells growing on polylysine; lanes
4-6, idem but from cells growing on laminin. The
blot was also labeled with a mAb against tyrosinated -tubulin
diluted 1:500. Ten micrograms of total cellular protein were loaded in
each lane. C, Northern blot analysis of p35 mRNA
expression in cerebellar macroneurons cultured on polylysine for 12 (lane 1) or 24 (lane 2) hr, or on a
laminin-containing substrate for 4 (lane 3) or 24 (lane 4) hr. D, An equivalent
Northern blot to that shown in C but probed with a
radiolabeled cDNA probe for cdk5. Four micrograms of total RNA were
loaded in each lane. E, Lanes
1-4, Western blot analysis of whole tissue
extracts obtained from the cerebellar cortex of postnatal days 1 (lane 1), 7 (lane 2), 14 (lane
3), and adult (lane 4) rats, probed with
the affinity-purified peptide antibody against p39 diluted 1:100 (1 µg/ml); each lane was loaded with 10 µg of total protein.
Lane 5, Western blot showing the relative levels of p39
and p35 in a cerebellar extract obtained from an E18 rat embryo; the
blot was reacted with the anti-p39 antibody diluted 1:50 (2 µg/ml)
and with the N20 antibody diluted 1:200 (0.5 µg/ml); the lane was
loaded with 10 µg of total cellular protein. Lanes
6, 7, Western blot showing the relative levels
of p39 in cell extracts from cerebellar macroneurons cultured on
polylysine (lane 6) or laminin (lane
7) for 48 hr; the p39 antibody was used at a
concentration of 2 µg/ml, and 20 µg of total cellular protein were
loaded in each lane. F, Western blot showing the
relative levels of the mitotic inhibitor p27 in cell extracts from
cerebellar macroneurons cultured on polylysine (lane 1)
or laminin (lane 2) for 24 hr; 10 µg of total cellular
protein were loaded in each lane.
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More importantly, our results show that when cerebellar macroneurons
are cultured on a laminin-containing substrate, a dramatic increase in
p35 protein levels is detected (Fig. 1B, lanes
1-6; Table 1). This phenomenon is also
accompanied by an acceleration in the time course of p35 expression;
thus, Western blot analysis of whole-cell extracts obtained from
neurons cultured in the presence of laminin for 4, 10, or 20 hr and
probed with the anti-p35 antibodies revealed a considerable and
significant increase in the levels of p35 when compared with the ones
detected at equivalent time points in neurons maintained on polylysine
alone (Fig. 1B). The increase in p35 protein levels
detected in cerebellar macroneurons growing on laminin is significantly
inhibited when the cells are incubated with an affinity-purified rabbit
polyclonal antibody against -1 integrin (Table 1).
It is unlikely that the increase in p35 protein levels induced by
laminin is the result of an overall increase in protein synthesis,
because no changes in the levels of cdk5 (Pigino et al., 1997), casein
kinase II (Pigino et al., 1997), MAP1b (DiTella et al., 1996), MAP2
(Pigino et al., 1997), -tubulin (Fig. 1B, lanes 1-6), or kinesin heavy chain
(data not shown) are detected between neurons growing on laminin or
polylysine alone. Northern blot analysis provided additional support to
these findings and revealed that laminin rapidly stimulates p35 mRNA
expression in cultured cerebellar macroneurons (Fig. 1C); as
expected, this treatment has no effect on cdk5 mRNA levels (Fig.
1D).
It has recently been shown that neurons not only express p35, but also
a highly related isoform, designated as p39 (Tang et al., 1995).
Therefore, it became of interest to determine whether laminin enhances
the expression of p39. To test for this possibility we prepared a
polyclonal antibody against p39 using as immunogen a peptide
corresponding to amino acid residues 349-367 mapping at the C terminus
of the 39 kDa isoform; this region of the p39 molecule shows no
significant homology with the corresponding one of p35. Figure
1E shows that the p39 polyclonal antibody recognizes a single band of ~40 kDa in Western blots of
whole-brain homogenates. This analysis revealed that in
the cerebral cortex or cerebellum the expression of the
p39-immunoreactive protein species is lower at late embryonic and early
postnatal days but that it increases significantly afterward, reaching
a peak during the second postnatal week, in which the highest levels
are detected (Fig. 1C, lanes 1-5). In addition, Western blots of cell
extracts obtained from cultures of cerebellar macroneurons revealed
that these neurons express very low levels of p39 at the time of
plating but that a significant increase occurs after the cells have
developed in vitro for >4 d (data not shown). More
importantly, our results show that there are no significant differences
in the levels of p39 between neurons cultured on polylysine alone or
polylysine plus laminin (Fig. 1E, lanes
6-7).
Based on these observations it is likely that the increase in cdk5
activity observed in neurons growing on laminin (Pigino et al., 1997)
is associated with an enhanced and selective expression of p35.
However, other possibilities were also considered. For example, an
enhancement of cdk5 activity may also result from a reduced expression
of the mitotic inhibitor p27, whose expression increases in parallel
with terminal neuronal differentiation and that is capable of
suppressing cdk5 activity (Lee et al., 1996b). We think this is
unlikely because no differences in p27 protein levels were detected
between neurons growing on laminin or polylysine alone (Fig.
1F, lanes 1-2).
Alternatively, and because laminin may stimulate cdk5 activity by
enhancing the expression of other cdk5 activators, like p67 (Shetty et
al., 1994), we decided to examine the consequences of p35 suppression
by antisense oligonucleotide treatment on the activity of cdk5 in
cultured cerebellar macroneurons growing on laminin. Two different
nonoverlapping phosphorothioate antisense oligonucleotides were
fabricated. The sequences selected were specific for p35, but not for
p39. As controls, we also obtained sense oligonucleotide sequences
corresponding to both of the antisense sequences. The oligonucleotides
were added directly to the culture medium in the presence of
Lipofectin (see Materials and Methods) and renewed every 6 hr.
Figure 2A shows that
the addition of the p35 antisense oligonucleotide RP1 at a dose of 5 µM, but not of the corresponding sense-strand
oligonucleotide, to cells growing on laminin produces a significant
reduction in p35 protein levels as determined by Western blot analysis
of whole-cell homogenates reacted with either the rabbit antiserum C-19
or the N-20 antibody. Inhibition of p35 expression becomes evident
after 16 hr of antisense treatment, and by 36 hr only trace amounts of
protein are detected in the cell homogenates. In addition, to test for
specificity, quantitative fluorescence measurements were performed, and
the relative levels of p35, p39, cdk5, ERK1, and several cytoskeletal proteins were evaluated at different time points after the addition of
the antisense or sense oligonucleotides. The results obtained, shown in
Figure 2B, indicate that although both of the
antisense sequences are effective in reducing p35 protein levels in a
dose-dependent manner, neither of the sense sequences detectably
altered p35 protein levels. In addition, neither of the antisense
sequences nor the sense sequences resulted in diminutions of the levels of cdk5, p39, ERK1, tubulin, MAP1b, MAP2, or tau protein levels (Fig.
2C). Having shown that the p35 antisense oligonucleotides effectively and selectively inhibit p35 expression, we decided to
examine cdk5 activity in p35-suppressed cultured cerebellar macroneurons maintained on a laminin-containing substrate. For such a
purpose, cdk5 was immunoprecipitated from sense- and antisense-treated cultures; the ability of the cdk5 immunoprecipitates to phosphorylate histone H1 was then evaluated. The results obtained showed high cdk5
histone H1 kinase activity in the immunoprecipitates from sense-treated
cultures and a significant decrease of such an activity in the ones
obtained from the antisense-treated cultures (Fig. 2D,E).
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Figure 2.
A, Effect of the antisense
oligonucleotide RP1 (5 µM) on p35 protein levels as
revealed by Western blot analysis of cell extracts obtained from
cultured cerebellar macroneurons growing on laminin and reacted with
the C19 antibody diluted 1:200. Lane 1, Control
nontreated, 24 hr after plating; lane 2, sense-treated,
24 hr after plating; lane 3, RP1-treated, 12 hr after
plating; lane 4, RP1-treated, 24 hr after plating;
lane 5, RP1-treated, 36 hr after plating. Five
micrograms of total cellular protein were added in each lane.
B, Quantitative fluorescence measurements showing the
effect of the antisense oligonucleotides RP1 (5 or 2.5 µM) or RP2 (2.5 µm) on p35 immunofluorescence.
Fluorescence intensity measurements were performed in the cell body 4, 12, and 24 hr after plating; a total of 100 cells was measured for each
time point and experimental condition. C, Quantitative
fluorescence measurements showing the relative levels of tubulin, cdk5,
p35, ERK1, MAP2, and tau in control and RP1-treated cells. Fluorescence intensity measurements were performed
in the cell body of cells maintained in culture for 24 hr; a total of
at least 50 cells was analyzed for each protein and experimental
condition. D, cdk5 histone H1 activity in control
(lane 1) and RP1-treated (lane 2, 5 µM) cerebellar macroneurons. cdk5 was immunoprecipitated
from cell homogenates after 1 d in vitro (200 µg
of total cellular protein per sample) and kinase reactions performed as
described in Materials and Methods. Films were exposed for 1 d.
E, Graph showing quantitation of cdk5 histone H1
activity in control, sense, and RP1- or RP2-treated cultures, as
revealed by Cerenkow counting of samples prepared as described in
D.
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An additional possibility to consider is that the increase in p35
protein levels and the associated cdk5-enhanced histone H1 kinase
activity (Pigino et al., 1997) observed in neurons growing on laminin
are the result of an enhanced rate of neurite extension and not a
direct consequence of laminin activity. To distinguish between these
possibilities, the following experiment was performed. In previous
studies we have shown that the acute addition of laminin to the tissue
culture medium of neurons growing on polylysine stimulates cdk5
activity and increases the rate of neurite extension (DiTella et al.,
1996; Pigino et al., 1997). This enhanced neurite outgrowth response
can be prevented if cells are treated with a combination of antisense
oligonucleotides against the tau and MAP1b mRNAs (DiTella et al.,
1996). We took advantage of this situation to test whether soluble
laminin was capable of increasing p35 expression in neurons with low
levels of tau and MAP1b, as well as a with a reduced rate of axonal
growth. The results obtained clearly indicate that laminin is capable
of enhancing p35 expression independent of the magnitude of the neurite
outgrowth response that the cells display (Table
2).
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Table 2.
Changes in p35 protein levels after the acute addition of
laminin to control and tau/MAP1b suppressed cerebellar macroneurons
growing on polylysine
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The subcellular localization of p35 in developing
cerebellar macroneurons
Previous studies have shown that in cultured cortical neurons
(Nikolic et al., 1996) or cerebellar macroneurons (Pigino et al.,
1997), as well as during in situ cerebellar development
(Matsushita et al., 1995), cdk5 redistributes from the cell body toward
axonal growth cones during process formation. Although a similar
phenomenon has been described for p35 in cultured cortical neurons
(Nikolic et al., 1996), the situation appears to be different in the
case of in situ developing neurons because
immunohistochemical studies have failed to detect significant levels of
p35 immunoreactivity within axons or growth cones; by contrast, high
p35 immunolabeling was observed in cell bodies and dendrites of adult
neurons (Tomizawa et al., 1996). This apparent discrepancy may be
related with the fact that no immunocytochemical study has examined the
subcellular distribution of p35 in embryonic brain (or cerebellar)
tissue. Therefore, to clarify this point and obtain further evidences about the participation of p35 during laminin-stimulated cdk5 activation, the subcellular localization of p35 in cultured cerebellar macroneurons was analyzed by confocal microscopy; in addition, subcellular fractionation techniques were used to isolate growth cones
from the embryonic cerebral and cerebellar cortex and to determine by
Western blotting the presence of the cdk5/p35 kinase.
In neurons growing on polylysine, light immunoreactivity for p35 is
detected during the first 48 hr after plating; in these cells, most of
the labeling is found within the cell body, including the nucleus, but
absent from either minor processes or axons (Fig. 3A-D). However,
after 48 hr when most of the neurons have already extended an axon of
>120 µm, a significant change in the intensity of the labeling and
in the distributional pattern of p35 is detected in the majority of the
cells. Thus, at this stage, an intense staining of the cell body and
axonal growth cones is observed with the antibodies against p35 used at
concentrations of 0.2-0.5 µg/ml (Fig. 3E-G);
at neuritic tips this immunolabeling is particularly prominent within
the peripheral regions of growth cones. At higher antibody
concentrations (1-2 µg/ml) p35 is also detected in microspikes located within the distal third of growing axons, as well as within the
growth cones of minor processes, the neurites that will lately develop
into dendrites (Cáceres et al., 1991, 1992).
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Figure 3.
Distribution and subcellular localization of p35
in cerebellar macroneurons growing on polylysine.
A-D, Confocal micrographs showing the
distribution of tyrosinated -tubulin (A,
C) and p35 (B, D) in
cerebellar macroneurons maintained in culture for 36 hr. Note than in
both stage II (A, B) and in stage III
(C, D) neurons, p35 is mainly localized
to the cell body; there is no staining of minor processes or the axon.
E, F, Confocal images
showing the distribution of tyrosinated -tubulin
(E) and p35 (F) at the
distal end of a growing axon of a cerebellar macroneuron maintained in
culture for 48 hr. At this stage of neuritic development p35
immunolabeling is present in the cell body and within axonal growth
cones. G, Red-green
overlay of the images shown in E and F.
H, I, High-power confocal micrographs
showing the distribution of tyrosinated -tubulin
(H) and p35
(I) at the distal end of a growing axon.
Note that p35 immunolabeling is highly enriched at the growth cone
periphery in regions almost devoid of microtubules. For this experiment
the cells were extracted with 0.02% saponin before fixation. Scale
bars, 10 µm.
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To test whether the localization of p35 at the periphery of axonal
growth cones involves a plasma membrane association, a mild extraction
protocol using saponin was used. This procedure selectively removes
cytosolic proteins but retains cytoskeletal membrane interactions
(Nakata and Hirokawa, 1987; Brandt et al., 1995; Pigino et al., 1997).
In saponized cells, both p35 antibodies stain the actin-rich peripheral
regions of growth cones, as well as microspikes or filopodial
extensions emerging from lamellipodial veils (Fig.
3H,I).
Western blot analysis of growth cone particles isolated by subcellular
fractionation according to the procedures described by Pfenninger et
al. (1983) (Lohse et al., 1996; see Materials and Methods) and probed
with antibodies specific for cdk5 or p35, revealed the presence of the
cdk5/p35 kinase in growth cones obtained from either the cerebral
cortex or cerebellum (Fig.
4A); a very low signal
for p39 was also detected in these preparations (Fig. 4A). To test for the purity of the GCP fraction we
analyzed the distribution of several well established growth cone
markers, namely c-src (Helmke and Pfenninger, 1996) or gc (Quiroga
et al., 1995; Mascotti et al., 1997); both proteins were highly
enriched in the growth cone extracts containing cdk5 or p35 (Fig.
4A); by contrast high molecular weight MAP2 was
undetectable in these preparations (Fig. 4A).
Moreover, to discard possible contaminations of GCPs with membranes of
relatively low buoyant densities, we assayed GCPs for the presence of a
well established Golgi marker, such as the protein designated as TGN-38
(Horn and Banting, 1994). As shown in Figure 4A, this
protein was completely absent from this preparation.
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Figure 4.
A, Western blots of total brain
homogenates (H) and growth cone particles
(GCP) reacted with antibodies against cdk5 (dilution
1:200, 0.5 µg/ml), p35 (dilution 1:200, 0.5 µg/ml), p39 (dilution
1:50, 2 µg/ml), c-src (dilution 1:100, 1 µg/ml), MAP2 (dilution
1:500), and TGN38 (dilution 1:200). The lanes reacted with antibodies
against cdk5, p35, c-src, MAP2, and TGN38 were loaded with 10 µg of
total cellular protein; the ones reacted with the p39 antibody were
loaded with 75 µg of total cellular protein. The total brain
homogenate and the GCP were obtained from E18 rat embryos as described
in Materials and Methods. B, Average intensity
measurements of p35 immunofluorescence within the cell body of
cerebellar macroneurons cultured on polylysine
(Ctrl) or on a laminin-containing substrate
(Lam); immunofluorescence was performed in cells fixed
before extraction with detergents. A total of 50 cells was measured per
time point and experimental condition. Each value represents the
mean ± SEM. C, Average intensity measurements of
p35 immunofluorescence within growth cones (GC) of axons
of cerebellar macroneurons cultured on polylysine (Ctrl)
or a laminin-containing substrate (Lam).
Immunofluorescence was performed in cells fixed before (Ctrl
GC/Lam GC) or after saponin (Ctrl GC
Sap/Lam GC Sap) or Triton X-100 (Lam GC
Triton) extraction performed under conditions that stabilize
the cytoskeleton (see Materials and Methods). Note that in cells
growing on laminin a significant amount of p35 immunofluorescence
remains associated with the cytoskeleton after saponin extraction
performed under conditions that preserve cytoskeletal-membrane
interactions. A total of 50 cells was measured per time point and
experimental condition. Each value represents the mean ± SEM.
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In a complementary series of experiments we used quantitative
fluorescence techniques to measure the relative amount of p35 at
neuritic tips of axons from neurons growing on either polylysine or
laminin. The results obtained, which are shown in Figure 4, B and C, indicate that laminin selectively and
significantly increases the relative amount of p35 associated with the
subcortical cytoskeleton of axonal growth cones. This analysis also
revealed that laminin not only increases the intensity of p35
immunolabeling, but that it selectively accelerates the time course of
appearance of p35 immunofluorescence at the distal end of growing
axons. Under this experimental condition, it was possible to detect
high p35 immunolabeling within axonal growth cones, as soon as 4-6 hr
after plating, when a significant proportion of cells have already
developed an axon-like neurite of >80 µm.
p35 suppression inhibits laminin-enhanced axonal elongation and
MAP1b phosphorylation
In a previous study (Pigino et al., 1997) we have shown
that in neurons growing on laminin cdk5 and mode I phosphorylated MAP1b
(e.g., phosphorylation at PDPK epitope; Ulloa et al., 1993a,b) increase significantly at the distal end of growing axons in parallel with rapid axonal elongation. Besides, cdk5 suppression significantly reduces laminin-enhanced axonal elongation and the mode I of MAP1b phosphorylation (Ulloa et al., 1993a,b; Pigino et al., 1997). Based on
these observations we have proposed that at least one mechanism by
which cdk5 may participate in axonal extension involves the regulation
of MAP1b phosphorylation at the distal end of the growing axon.
Therefore, to obtain further evidences about this proposal we decided
to examine the consequences of p35 suppression on process formation and
MAP1b phosphorylation in cerebellar macroneurons growing on a
laminin-containing substrate.
Under this condition p35 suppression significantly and selectively
reduces laminin-stimulated axonal extension (Fig.
5A). This phenomenon becomes
evident 20-24 hr after the addition of the antisense oligonucleotides,
being highly coincident with the reduction in p35 protein levels (Fig.
5B). By contrast, no inhibition of axonal elongation was
observed when equivalent cultures were treated with the corresponding
sense oligonucleotides or with a p39 antisense oligonucleotide (Fig.
5A,B). We also failed to detect an
inhibition of axonal elongation when the RP1 or RP2 oligonucleotides
were added from the time of plating, and for a period of 24 hr, to
cerebellar macroneurons growing on polylysine alone (data not
shown).
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Figure 5.
A, Graph showing that suppression
of p35, but not of p39, selectively inhibits axonal elongation in
cerebellar macroneurons growing on a laminin-containing substrate.
Open squares, Axonal length in sense-treated cultures;
solid squares, idem but in RP1-treated cultures (5 µM); solid diamonds, idem but in p39
antisense-treated cultures (5 µM); total length of minor
processes per cell in p35 sense-treated cultures; idem but in
RP1-treated cultures. A total of 100 cells was measured per time point
and experimental condition. Each value represents the mean ± SEM.
B, Average intensity measurements of p35 and p39
immunofluorescence in cerebellar macroneurons treated with p35 (RP1, 5 µM) or p39 (5 µM) antisense
oligonucleotides. Measurements were performed in the cell body, and a
total of 50 cells were analyzed per time point and experimental
condition. Each value represents the mean ± SEM.
C, Average intensity measurements of
PDPK-dephosphorylated MAP1b immunofluorescence in p35 sense-
(S) and antisense- (AS) treated
cultures; cells were stained with the polyclonal antibody 531 (Ab 531).
Measurements were performed in proximal (PAX) and
distal (DAX) axonal segments. Note that in the
p35 antisense-treated neurons, 531 immunolabeling increases at the
distal axonal segment. A total of 50 cells was analyzed per time point
and experimental condition; each value represents the mean ± SEM.
Oligonucleotides were used at a concentration of 5 µM.
D, Average intensity measurements of PDPK-phosphorylated
MAP1b immunofluorescence in p35 sense- (S) and
antisense- (AS) treated cultures; cells were stained
with the mAb 150. Measurements were performed in proximal
(PAX) and distal (DAX)
axonal segments. Note that in the p35 antisense-treated neurons mAb 150 immunolabeling decreases at the distal axonal segment; also note the
absence of mAB 150 immunolabeling in proximal axonal segments of both
sense- and antisense-treated cultures. Oligonucleotides were used at a
concentration of 5 µM. A total of 50 cells was analyzed
per time point and experimental condition; each value represents the
mean ± SEM.
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Our observations (Fig. 5C,D) also show that a 48 hr treatment of cerebellar macroneurons growing on a laminin-containing
substrate with the RP1 antisense oligonucleotide (5 µM)
significantly reduces the labeling generated by the mAb 150 that
recognizes a phosphorylated mode I epitope on the MAP1b molecule (Ulloa
et al., 1993a,b; Pigino et al., 1997) while it increases the one
observed with the rabbit antiserum 531, which recognizes a PDPK
phosphorylatable MAP1b epitope when it is dephosphorylated (Ulloa et
al., 1993a,b; Pigino et al., 1997).
Because at axonal tips the cdk5/p35 complex is highly enriched in
the lamellipodial veil of growth cones, we decided to examine whether
dephosphorylated MAP1b was also present in this subcellular compartment. In previous studies we have shown that dephosphorylated MAP1b distributes throughout the cell, localizing in the cell body,
minor neurites, and axonal processes; it should be noted that most of
the dephosphorylated MAP1b present in developing cerebellar
macroneurons is lost when cells are extracted with Triton X-100 under
microtubule stabilizing conditions before fixation (DiTella et al.,
1996; Pigino et al., 1997). However, when the cells are extracted with
0.02% saponin under conditions that preserve cytoskeletal-membrane interactions (Nakata and Hirokawa, 1987; Pigino et al., 1997) 531 immunolabeling is detected throughout the
neurons including neuritic tips (Fig. 6).
High resolution fluorescence microscopy, digital image processing, and
ratio image analysis of cells double labeled with the 531 antiserum and
an mAb against tyrosinated -tubulin clearly revealed the presence of
dephosphorylated MAP1b within the lamellipodial veils of growth cones;
confocal microscopy confirmed these observations and also revealed the
presence of intense 531 immunolabeling in microspikes emerging from the
periphery of growth cones (Fig. 6D,
insert; H).
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Figure 6.
A, B, Double
immunofluorescence micrographs showing the distribution of tyrosinated
-tubulin (A) and PDPK-dephosphorylated MAP1b
in a stage II cerebellar macroneuron cultured on polylysine. The cell
was extracted with saponin before fixation under conditions that
preserve cytoskeletal-membrane interactions. MAP1b was labeled with
the rabbit antiserum 531 used at a dilution of 1:500. For this
experiment the images were formed on the faceplate of an SIT camera and
digitized directly into the Metamorph Image Processor. Using image
B as numerator and image A as denominator, we
applied the ratio image menu of the processor to generate the image
shown in C, which clearly reveals the presence of
dephosphorylated MAP1b within the lamellipodial veil of growth cones.
D, A confocal image of a stage II cerebellar macroneuron
also showing the presence of PDPK-dephosphorylated MAP1b within the
lamellipodial veils of growth cones; the insert shows
the presence of 531 immunolabeling (dilution 1:500) within microspikes
located at the periphery of lamellipodial veils.
E, F, Double
immunofluorescence micrographs showing the distribution of tyrosinated
-tubulin (E) and PDPK dephosphorylated MAP1b (F)
within an axonal tip of a cerebellar macroneuron growing on polylysine
and prepared for immunocytochemistry as described previously.
G, Ratio image analysis clearly reveals the presence of
dephosphorylated MAP1b within the peripheral lamellipodial veil of
axonal growth cones. H, Confocal image also showing the
presence of PDPK-dephosphorylated MAP1b at the periphery of axonal
growth cones. Scale bars, 10 µm.
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DISCUSSION |
p35 participation in axonal elongation
p35 is a neuron-specific regulatory subunit of cdk5 that
is expressed in postmitotic neurons but is absent in proliferating neuronal progenitors (Lew et al., 1994; Tsai et al., 1994; Tomizawa et
al., 1996; Delalle et al., 1997). Based on the analysis of the
spatiotemporal pattern of p35 mRNA distribution (Tsai et al., 1994;
Delalle et al., 1997) and of the phenotype of mice lacking p35, a key
role for the cdk5/p35 kinase in proper neuronal migration has been
established (Chae et al., 1997). Thus, examination of mice lacking p35,
and, thus, p35/cdk5 activity, has revealed severe cortical lamination
defects, including a reversed packing order of cortical neurons, such
that earlier born neurons reside in superficial layers and lately
generated ones occupy deep layers (Chae et al., 1997).
A second major proposed role for the cdk5/p35 kinase is the regulation
of neuritic development (Nikolic et al., 1996; Delalle et al., 1997).
In favor of this view, previous studies have established the existence
of a high degree of temporal correlation among p35 mRNA expression, the
induction of cdk5 activity, and the formation of major axonal tracts
(Tsai et al., 1993; Delalle et al., 1997; Tomizawa et al., 1997);
besides, expression of a p35 antisense construct in cultured cortical
neurons reduces neurite outgrowth (Nikolic et al., 1996). Although the
present results are consistent with these observations, they suggest
that the cdk5/p35 complex, rather than having an essential role in
process formation (Nikolic et al., 1996), acts as a modulator of axonal
elongation. A recent study showing that in immortalized hippocampal
cells, p35 is sufficient but not essential for inducing neurite
outgrowth also favors this view (Xiong et al., 1997). In the case of
cerebellar macroneurons, we found a high degree of spatial and temporal
correlation between the expression of the cdk5/p35 kinase and axonal
elongation when the neurons were maintained on a laminin-containing
substrate; by contrast, such a correlation was not observed in neurons
growing on polylysine alone in which the expression of the cdk5/p35
kinase occurs after the initial extension of neurites and
the establishment of neuronal polarity (this study; see also Pigino et
al., 1997). Besides, and perhaps more importantly, two nonoverlapping
antisense oligonucleotides that effectively diminished the levels of
p35 were capable of selectively suppressing laminin-enhanced axonal elongation, without altering the normal extension of minor neurites or
the ability of cerebellar macroneurons to differentiate one of the
minor processes into an axon-like neurite. As with any study involving
the use of antisense oligonucleotides, it was important to establish
that the observed effects were not related to a diminution in the
health of the cultures. Several observations suggest that the p35
antisense oligonucleotides specifically and selectively blocked the
expression of p35. First, sequence analysis of the regions of the rat
p35 mRNA selected for designing the antisense oligonucleotides revealed
no significant homology with any other reported sequence. Besides, none
of the S-modified antisense oligonucleotides used in this study
contains four contiguous guanosines residues, which are believed to
increase oligomer affinity to proteins and, hence, generate nonspecific
inhibitory effects (Wagner, 1995). Second, the antisense
oligonucleotide treatment dramatically reduced p35 protein levels
without altering the levels of several other proteins, including cdk5,
p39, tubulin, MAP1b, MAP2, tau, and ERK1. Third, the effects of the
antisense oligonucleotides were dose-dependent and not observed when
the cells were treated with equivalent doses of the corresponding
"sense" oligonucleotides. Finally, the antisense treatment was not
effective in reducing axonal length when it was applied to neurons that
express low levels of p35 and, hence, display low cdk5 activity, e.g.,
neurons cultured on polylysine for up to 48 hr.
Our results also suggest that p35, and not the p39 isoform, is
involved in regulating axonal extension in neurons growing on a
laminin-containing substrate. Thus, no decreases in axonal length or in
the time course of axonal formation were detected in cerebellar
macroneurons treated with a p39 antisense oligonucleotide. This may not
be surprising because p39 is predominantly expressed in polarized
neurons, and not during the initial stages of process formation, both
in situ and in vitro (Cai et al., 1997; this
study). Although this analysis suggests that p39 has no essential role in neurite outgrowth and axonal extension, it does not rule out the
possibility of p39 being involved in the maintenance of more mature
neurites or in process formation in response to a different type of
environmental cue. In this regard, it is worth noting that in
immortalized hippocampal pyramidal cells p39, rather than p35, is both
necessary and sufficient for promoting neurite outgrowth in response to
basic fibroblast growth factor (Xiong et al., 1997).
Suppression of either cdk5 (Pigino et al., 1997) or p35 (this study),
but not of p39, significantly decreases the levels of PDPK-phosphorylated MAP1b. Because this phenomenon selectively occurs
at the distal end of growing axons, it is likely that the cdk5/p35
kinase regulates MAP1b phosphorylation within axonal growth cones.
Thus, the time course of appearance of the cdk5/p35 kinase within
growth cones is highly coincident with a decrease of
PDPK-dephosphorylated MAP1b and with an increase in PDPK-phosphorylated MAP1b. It is unlikely that the presence of the cdk5/p35 kinase within
axonal growth cones of cultured neurons (this study; see also Nikolic
et al., 1996; Pigino et al., 1997) represent some peculiarity related
with their in vitro development, because Western blot
analysis of growth cone particles clearly revealed the presence of both
proteins in this compartment. Our results, and those of Nikolic et al.
(1996), show that within growth cones most of the cdk5/p35 kinase is
associated with the subcortical cytoskeleton but not with microtubules.
This may be important in terms of previous studies that have shown that
dephosphorylated MAP1b binds to microfilaments (Pedrotti and Islam,
1996) and not to microtubules (DiTella et al., 1996); besides,
dephosphorylation kinetics suggests that the PDPK site, but not casein
kinase II sites, negatively regulates the association of MAP1b with
F-actin (Pedrotti and Islam, 1996). In accordance with that, we
detected a significant association of PDPK-dephosphorylated MAP1b with
the growth cone subcortical cytoskeleton in neurons displaying low
levels of p35 and, hence, of cdk5 activity. Therefore, it is tempting
to speculate that the presence of an active cdk5/p35 complex within the
growth cone subcortical cytoskeleton, as in the case of neurons growing
on laminin, will phosphorylate MAP1b, detaching it from microfilaments and allowing its interaction with microtubules, an event that seems to
be crucial for the participation of MAP1b in axonal elongation (DiTella
et al., 1996: Pigino et al., 1997). However, it is worth noting that
our results do not rule out the possibility of cdk5 participating in
axonal elongation by additional mechanisms (see Pigino et al., 1997 for
a discussion of this issue).
Laminin regulation of p35 expression
The present observations provide novel insights about the
mechanisms by which laminin may regulate axonal elongation.
Specifically, we show that laminin increases p35 mRNA and protein
levels, a phenomenon that in turn enhances cdk5 activity, MAP1b
phosphorylation, and axonal extension. The upregulation of p35 protein
levels in neurons growing on a laminin-containing substrate seems not
to be related with an overall increase in protein synthesis, because no
effects were observed in the levels of total tubulin, MAP1b, MAP2, tau,
cdk5, p39, and p27; nor it is the consequence of an increased rate of
growth, because laminin-stimulated p35 expression was also detected in
neurons displaying a reduced rate of axonal elongation, such as in
MAP1b- and tau-suppressed neurons. It is also unlikely that laminin
increases cdk5 activity by stimulating the expression of other cdk5
activators, because it was completely prevented in neurons treated with
antisense oligonucleotides specific for the p35 mRNA. Besides, our
results also suggest that laminin-stimulated cdk5 activity is not the
result of a reduced expression of a cdk5 inhibitor because no
differences in the levels of p27 (Lee et al., 1996b) were
detected between neurons growing on polylysine or laminin.
Our results suggest that one mechanism by which laminin increases
p35 protein levels may involve the stimulation of p35 mRNA expression;
this may not be an unusual phenomenon. In fact, a significant body of
evidence exists showing that integrin activation after ECM binding can
regulate expression of many genes (Ginsberg et al., 1995; Giancotti,
1997). For example, in anchorage-dependent cell lines, replating cells
on fibronectin triggers expression of early immediate genes, such as
c-fos and c-jun (Dike and Farmer, 1988; Dike and Ingber, 1996), whereas
in rat kidney cells, cyclin A expression is specifically
regulated by adhesion (Guadagno et al., 1993; see also Giancotti,
1997). In the specific case of neurons, a recent study has shown that
laminin stimulates the expression of two mitochondrial proteins during
process outgrowth (Weeks et al., 1997). Studies are in progress to
determine the mechanisms by which laminin increases p35 mRNA levels.
| |
FOOTNOTES |
Received June 8, 1998; revised Sept. 9, 1998; accepted Sept. 11, 1998.
This work was supported by grants from Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET),
CONICOR, Fundación Perez-Companc, Fundación
Antorchas (ABC Research Grant), and the Howard Hughes Medical Institute
under an International Research Scholar Program to A.C. It was also
supported by start-up funds from the Northwestern Institute of
Neuroscience to A.F. P.K. is supported by a doctoral fellowship from
CONICET. We thank Dr. L. Binder, N. Carri, and J. Avila for providing
antibodies and J. Wong for cDNA probes.
Correspondence should be addressed to Alfredo Cáceres, Instituto
Mercedes y Martín Ferreyra, Casilla de Correo 389, 5000 Córdoba, Argentina.
| |
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Abstract
Introduction
