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* Buck Institute for Age Research, Novato, California, USA;
University of California, San Francisco, California, USA; and
Amersham Biosciences, Piscataway, New Jersey, USA
1 Correspondence: Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945, USA. E-mail: dgreenberg{at}buckinstitute.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: doublecortin • migration • ischemia • proteomics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, neurogenesis (2
, 3)
, and ischemia (4
, 5)
. Two-dimensional differential in-gel electrophoresis (DIGE) is a method for comparing protein expression in paired samples, which are differentially labeled with fluorescent dyes and electrophoresed together on the same gel (6)
. This facilitates comparison of proteomic profiles and can be combined with mass spectroscopy (MS) to identify differentially expressed proteins. DIGE has been used to detect changes in protein expression associated with signal transduction (7)
, oncogenesis (8
9
10)
, and drug toxicity (11
, 12)
.
We have used DIGE/MS to investigate proteins involved in neurogenesis in adult mammalian brain, which may serve to replace and restore the function of neurons lost as a result of injury (13)
. Injury-induced neurogenesis may be triggered by chemical signals that stimulate proliferation, migration, and differentiation of neuronal precursors. Factors that stimulate neuronal proliferation have been identified (14)
, but little is known about early changes they produce in their cellular targets or the mechanisms that underlie migration and differentiation. Nonetheless, these events likely involve altered expression of genes and proteins involved in cell replication, migration, and maturation. Several genes—including doublecortin (Dcx), lissencephaly-1, and reelin—have been implicated in neuronal migration because mutations in these genes cause disorders of neuronal migration or lissencephaly (15
, 16)
.
In this study, DIGE/MS revealed several differences in protein expression between immature neuronal cultures from embryonic rat cerebral cortex and older cultures enriched for mature neuronal cells. The protein with the most striking differential expression was stathmin (
µo, relay) (17)
, a cytosolic phosphoprotein identified in leukemia cells (18)
. Stathmin is expressed at high levels in brain neurons and glia (19)
. Stathmin expression is stimulated by growth factors (20)
, is developmentally regulated (21)
, and is greatest in immature or proliferating tissues (22)
. Stathmin interacts with tubulin, leading to microtubule destabilization (23)
. In adults, stathmin is prominently expressed in neurogenesis pathways including the subgranular zone (SGZ) of dentate gyrus, subventricular zone (SVZ), rostral migratory stream (RMS), and olfactory bulb (OB) (24
, 25)
. Moreover, stathmin expression is induced by brain lesions (26
27
28)
, suggesting possible involvement in injury-induced neurogenesis.
We report that 1) stathmin expression coincides with expression of the microtubule-stabilizing protein Dcx in adult rodent brain and delineates ectopic neuromigratory pathways in cerebral ischemia and 2) antisense inhibition of either stathmin or Dcx expression inhibits this migration in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Approximately 4 x 105 cells/mL were plated on 100 mm Falcon culture dishes coated with 100 µg/mL of poly-D-lysine and cultures were maintained in neurobasal medium containing 2% B27 supplement, 2 mM glutamine, 1% penicillin, and 1% streptomycin (Life Technology, Rockville, MD, USA) at 37°C in humidified 95% air/5% CO2. Medium was changed every 3–4 days. Cells harvested after 5–6 days in vitro without passaging yielded "immature" cultures characterized by the expression of a range of immature neuronal markers, as reported previously (30
, 31)
. "Mature" cultures were maintained in the presence of fibroblast growth factor (FGF-2) (20 ng/mL; R&D Systems, Minneapolis, MN, USA), trypsinized when confluent, and replated at the original density for up to four passage cycles (32
, 33)
, when mature neuronal markers were predominantly expressed.
Protein preparation and labeling
Cells were washed twice in phosphate-buffered saline (PBS) and homogenized on ice in lysis buffer (8 M urea, 4% CHAPS, 0.2% Bio-Lyte 3/10 ampholytes (Bio-Rad, Hercules, CA, USA) and 40 mM Tris-HCl), and insoluble material was removed by centrifugation. Cell lysates were dialyzed using PlusOne Mini Dialysis kits (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s instructions and protein concentration was determined using a Bio-Rad protein assay. Protein (100 µg/sample) was labeled with 400 pmol of the N-hydroxy succinimidyl (NHS) ester DIGE dye Cy3 or Cy5 (Amersham Biosciences) following the manufacturer’s recommended protocols. The labeling mixture was incubated on ice in the dark for 30 min and the reaction was terminated by adding 10 nmol of lysine for 10 min on ice. An equal volume of sample buffer (8 M urea, 4% CHAPS, 0.2% biolate, 40 mM Tris-HCl, 1% (v/v) pharmalytes, and 2 mg/mL DTT) was added for 15 min at room temperature, and Cy3 and Cy5 labeling reactions were mixed and used to rehydrate immobilized nonlinear pH gradient (IPG) strips, pH 3–10 (Bio-Rad).
2-Dimensional gel electrophoresis
Isoelectric focusing was performed on 11 cm, pH 3–10 strips using a Protean isoelectric focusing cell apparatus (Bio-Rad) for 35 kV-h at 20°C. Strips were equilibrated for 20 min in 0.375 M Tris-HCl (pH 8.8), 6 M urea, 20% (v/v) glycerol, 4% (w/v) SDS, and 2% DTT, then for 10 min in the same buffer containing 2.5% iodoacetamide. Equilibrated immobilized pH gradient strips were loaded onto 8–16% Criterion gels and overlaid with ReadyPrep agarose in 0.375 M Tris-HCl (pH 8.8), 6 M urea, 20% (v/v) glycerol, and 4% (w/v) SDS containing bromphenol blue. Gels were electrophoresed in Criterion cells (Bio-Rad) at 50 mA/gel at room temperature until free dye migrated off the bottom of the gels.
Image analysis
Two-dimensional gels were scanned using a Typhoon 8600 (Amersham) at excitation and emission wavelengths appropriate for the stain. Cy3 and Cy5 images were merged and analyzed using DeCyder software (Amersham Biosciences). Gels were fixed in 10% methanol/7% acetic acid (v/v) overnight, post-stained with Fast Stain Colloidal Coomassie (Zoion Biotech, Shrewsbury, MA, USA), and scanned using a GS-710 calibrated imaging densitometer (Bio-Rad).
In-gel tryptic digestion
Protein spots of interest were manually excised from the gel and processed with a ProGest automatic in-gel digester robot (Genomic Solutions, Ann Arbor, MI, USA). Spots were destained and dehydrated with acetonitrile and the proteins were reduced with 10 mM DTT at 60°C for 30 min, alkylated with 100 mM iodoacetamide (37°C, 45 min), then incubated with 125–250 ng of sequencing grade trypsin (Promega, Madison, WI, USA) at 37°C for 4 h. The resulting tryptic peptides were extracted from the gel with aqueous 10% formic acid and analyzed by MS.
Protein analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)
Mass spectra of digested gel spots were obtained by MALDI-TOF MS on a Voyager DESTR-Plus (Applied Biosystems, Framingham, MA, USA). All mass spectra were acquired in positive ionization mode with reflectron optics. The instrument was equipped with a 337 nm nitrogen laser and operated under delayed extraction conditions: delay time, 190 ns; grid voltage, 66–70% of full acceleration voltage (20–25 kV). All peptide samples were prepared using a matrix solution consisting of 33 mM -cyano-4-hydroxycinnamic acid in acetonitrile/methanol (1/1; v/v); 1 µL of analyte (0.1–1 pmol of material) was mixed with 1 µL of matrix solution, then air-dried at room temperature on a stainless steel target. Typically, 50–100 laser shots were used to record each spectrum. The mass spectra obtained were calibrated externally with an equimolar mixture of angiotensin I, ACTH 1-17, ACTH 18-39, and ACTH 7-38.
Protein analysis by electrospray ionization (ESI-MS, MS/MS)
In some cases, proteolytic peptide mixtures were analyzed by reverse-phase nano-HPLC-MS/MS. Peptides were separated on an Ultimate nanocapillary HPLC system equipped with a PepMapTM C18 nano-column (75 µm IDx15 cm; Dionex, Sunnyvale, CA, USA) and CapTrap Micro guard column (0.5 µL bed volume; Michrom, Auburn, CA, USA). Peptide mixtures were loaded onto the guard column, washed for 5 min with loading solvent (0.05% formic acid in H2O; flow rate, 20 µL/min), transferred onto the analytical C18 nanocapillary HPLC column, and eluted at a flow rate of 300 nL/min using the following gradient: 2% solvent B in solvent A (0–5 min) and 2–70% solvent B in solvent A (5–55 min). Solvent A consisted of 0.05% formic acid in 98% H2O/2% acetonitrile and solvent B consisted of 0.05% formic acid in 98% acetonitrile/2% H2O. The column eluant was directly coupled to a QSTAR Pulsar-i quadrupole orthogonal TOF mass spectrometer (MDS Sciex, Concord, Ontario, Canada) equipped with a Protana nanospray ion source (ProXeon Biosystems, Odense, Denmark). The nanospray needle voltage was typically 2300 V in the HPLC-MS mode. Mass spectra (ESI-MS) and tandem mass spectra (ESI-MS/MS) were recorded in positive ion mode with a resolution of 12,000–15,000 FWHM. For collision-induced dissociation tandem MS, the mass window for precursor ion selection of the quadrupole mass analyzer was set to ±1 m/z. The precursor ions were fragmented in a collision cell using N2 as the collision gas. Spectra were calibrated in static nanospray mode using MS/MS fragment ions of a renin peptide standard (histidine immonium ion with m/z at 110.0713, and b8 ion with m/z at 1028.5312), providing a mass accuracy of 
50 ppm.
Analysis of peptide sequence
MS data were analyzed with the bioinformatics database systems RADARS (Genomic Solutions) (34)
and Mascot (Matrix Sciences, London, UK) (35)
. Routinely, MALDI-MS data were analyzed with RADARS using the search engine ProFound for peptide mass fingerprint matching against peptides from known protein sequences entered in publicly available protein databases (e.g., NCBI), using the following parameters: internal calibration using trypsin autolysis masses (m/z 842.5100 and 2211.1046), 100 ppm mass accuracy, two missed proteolytic cleavages allowed. In several cases, tryptic digestion extracts of proteins were analyzed by HPLC-ESI-MS and MS/MS; these data were submitted to the search engine Mascot, which analyzes peptide sequence information from tandem mass spectra. Both search engines provide a statistical scoring parameter. ProFound uses a so-called "expectation value" for data quality control that reflects the probability of a nonrandom (real) protein hit. For example, 1 x 10-2 is a 1-in-100 chance of being a random hit (confidence >99.0%) and 1 x 10-3 is a 1-in-1000 chance of being a random hit (confidence >99.9%). Protein matches are therefore considered significant when the expectation values are <5 x 10-2 (confidence >95%) (34)
. Mascot uses a probability-based MOWSE Score to evaluate data obtained from tandem mass spectra, in which a score >56 indicates that protein matches are significant (35)
.
Intracerebroventricular administration of reagents
A phosphorothioate antisense oligodeoxynucleotide (ODN) directed against the initial coding regions of stathmin (5'-ATATCAGAAGATGCCAT-3', nucleotides 185-169) and DCX (5'-CCAAAATCAAGTTCCAT-3', nucleotides 17-1) were designed based on rat stathmin (GenBank accession no. J04979) and DCX (GenBank accession no. MN053379) sequences. These and sense sequences for stathmin (5'-ATGGCATCTTCTGATAT-3', nucleotides 169-185) and DCX (5'-ATGGAACTTGATTTTGG-3', nucleotide 1-17) were synthesized commercially (Qiagen, Valencia, CA, USA) and purified by HPLC. For intracerebroventricular delivery of ODNs, 280–310 g male Sprague-Dawley rats were anesthetized by mask with 4% isoflurane in 70% N2O and 30% O2 and placed in stereotaxic frames with a rat head holder. Burr holes were drilled with a dental drill, which was irrigated continuously with saline at room temperature to prevent overheating of the underlying cortex. Artificial cerebrospinal fluid (aCSF; 128 mM NaCl, 2.5 mM KCl, 0.95 mM CaCl2, 1.99 mM MgCl) or ODNs (5 µM in aCSF) were infused into the left lateral ventricle (–0.8 mm anterior to the bregma, 1.3 mm lateral to the midline, 3.5 mm deep beneath the dura) in a volume of 3 µL over 2 min, and the needle was left in place for an additional 5 min. After injections, bone wounds were closed with bone wax. Anesthesia was discontinued and animals were returned to their cages.
For intracerebroventricular delivery of the fluorescent lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 0.5% DiI (Molecular Probes; Eugene OR, USA) in 5 µL of DMSO was injected into the left lateral ventricle over 10 min using a Hamilton syringe; this was done 48 h after the injection of aCSF or ODNs. To prevent cell-to-cell transfer of the fluorophore, 0.1% EDTA was added to solutions used for DiI experiments (36)
. Rats were perfused 24 h after DiI administration with 0.9% saline and 4% paraformaldehyde in PBS (pH 7.5). Sagittal brain sections were cut and kept in PBS. DiI-labeled cells were visualized with a Nikon E300 epifluorescence microscope. Colocalization of DiI with other markers was detected using a Nikon PCM-2000 laser scanning confocal microscope and Simple PCI imaging software (Compix, Cranberry Township, PA, USA).
Western blot
Protein (100 µg per lane) was boiled at 100°C in SDS sample buffer for 5 min, electrophoresed by 12% SDS-PAGE, and transferred to PVDF membranes. These were incubated overnight at 4°C with the following primary antibodies: 1) affinity-purified goat polyclonal anti-stathmin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; 1:250); 2) affinity-purified goat polyclonal anti-doublecortin (DCX) (Santa Cruz Biotechnology; 1:200); 3) mouse monoclonal anti-polysialylated nerve cell adhesion molecule (PSA-NCAM) (Chemicon, Temecula, CA, USA;1:500); 4) mouse monoclonal anti-Hu (Molecular Probes; 1:250), which recognizes the Hu-family proteins HuC, HuD, and Hel-N1; 5) rat monoclonal antibody anti-ßIII tubulin (Caltag Laboratories, Burlingame, CA, USA; 1:20,000); and 6) mouse monoclonal anti-actin (Oncogene, Boston, MA, USA; 1:20,000). Membranes were washed with PBS containing 0.1% Tween-20, incubated at room temperature for 60 min with horseradish peroxidase-conjugated anti-mouse or rat (for monoclonal primary) or anti-goat (for polyclonal primary) secondary antibody (Santa Cruz Biotechnology; 1:3,000), and washed three times for 15 min with PBS/Tween-20. Peroxidase activity was visualized with a chemiluminescence substrate system (NEN Life Science Products Inc., Boston, MA, USA). Primary and secondary antibodies were removed with stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) at 50°C for 30 min and washed again with PBS/Tween-20 before the membrane was reprobed.
BrdU administration
BrdU (Sigma, St. Louis, MO, USA) was dissolved in saline and given as two intraperitoneal doses of 50 mg/kg each spaced 8 h apart; rats were killed 24 h later.
Focal cerebral ischemia model
Transient focal cerebral ischemia was induced using the suture occlusion technique (37)
. Male Sprague-Dawley rats weighing 280–310 g were anesthetized by mask with 4% isoflurane in 70% N2O/30% O2. The neck was incised in the midline, the right external carotid artery (ECA) was carefully exposed and dissected, and a 19 mm-long, 3-0 monofilament nylon suture was inserted from the ECA into the right internal carotid artery to occlude the origin of right middle cerebral artery (MCA). After 90 min, the suture was removed to allow reperfusion, the ECA was ligated, and the wound was closed. Animals were killed at various time thereafter. Sham-operated rats underwent an identical procedure except the suture was not inserted. Rectal temperature was maintained at 37.0 ± 0.5°C with a heating pad and lamp. Blood pressure and blood glucose concentration were monitored.
Immunohistochemistry
Brains were removed after perfusion with saline and 4% paraformaldehyde in PBS. Sections were incubated in 1% H2O2 in PBS for 15 min in blocking solution (2% goat serum, 0.3% Triton X-100, and 0.1% bovine serum albumin in PBS) for 2 h at room temperature and with affinity-purified goat polyclonal anti-stathmin (Santa Cruz Biotechnology; 1:200) at 4°C overnight. The secondary antibody was biotinylated donkey anti-goat IgG (Vectastain Elite ABC, Vector), diluted 1:200. The horseradish peroxidase reaction was detected with 0.05% diaminobenzidine (DAB) and 0.03% H2O2. Processing was stopped with H2O and sections were dehydrated through graded alcohols, cleared in xylene, and coverslipped in permanent mounting medium (Vector). Sections were examined with a Nikon E800 microscope.
Double-label immunohistochemistry
For double-label studies, sections were fixed with 4% paraformaldehyde in PBS for 1 h at room temperature, washed twice with PBS, and incubated with blocking solution, then with primary antibodies at 4°C overnight, and with secondary antibodies in blocking solution at room temperature for 2 h. The primary antibodies used were 1) affinity-purified goat polyclonal anti-stathmin (Santa Cruz Biotechnology; 1:200) 2) affinity-purified goat polyclonal anti-DCX (Santa Cruz Biotechnology; 1:200), 3) mouse monoclonal anti-BrdU (Roche, Indianapolis, IN, USA; 2 µg/mL), 4) mouse monoclonal anti-neuronal nuclear antigen (NeuN) (Chemicom; 1:250), 5) affinity-purified goat polyclonal anti-NeuroD (Santa Cruz Biotechnology; 1:200), 6) mouse monoclonal anti-GFAP (Sigma; 1:200), 7) mouse monoclonal anti-Hu (Molecular Probes; 1:250), 8) mouse monoclonal anti-von Willebrand factor (vWF; Sigma; 1:500), 9) mouse monoclonal anti-nestin (BD Bioscience PharMingen, San Diego, CA; 1:400), and 10) mouse monoclonal anti-2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase; Chemicom; 1:400). The secondary antibodies were rhodamine-conjugated rat-absorbed donkey anti-mouse IgG (Jackson ImmunoResearch; 1:200) and fluorescein isothiocyanate (FITC) -conjugated pig anti-goat or goat anti-rabbit IgG (Jackson ImmunoResearch; 1:200). Sections were mounted with Vectashield (Vector) and fluorescence signals were detected with a Nikon E800 microscope at excitation/emission wavelengths of 535/565 nm (rhodamine, red) and 470/505 (FITC, green). Results were recorded with a Magnifire digital camera (ChipCoolers. Warwick, RI, USA). A Nikon PCM-2000 laser scanning confocal microscope was used to produce a Z-series stack of high-magnification digitized images of representative neocortical and SVZ neurons acquired through consecutive focal planes. For each double-labeled neuron, two Z-series stacks were generated with FITC and TRITC filters. Parallel pairs of stacks were processed with a deconvolution subroutine and images displaying the colocalization of each fluoroprobe were generated using Imaris software (Bitplane AG, Zurich, Switzerland).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). A total of 1033 protein spots were identified, of which 185 (17.9%) showed 
2-fold differences in expression between immature and mature cultures. Among proteins showing increased expression in immature cultures, the magnitude of the increase was 2-fold in 81, 3-fold in 24, 4-fold in 12, and 5-fold in 4 cases. Among proteins showing increased expression in mature cultures, the magnitude of the increase was 2-fold in 104, 3-fold in 32, 4-fold in 10, and 5-fold in 2 cases.
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Identification of stathmin as a differentially expressed protein
Those proteins with threefold higher expression in immature compared with mature cultures are listed in Table 1
. The protein with the greatest difference in expression between immature and mature cultures migrated with a relative molecular mass (Mr) of 20 kDa and isoelectric point (pI) of 6.2, and showed a 7.6-fold higher expression in immature cultures (Fig. 1B, C
). This spot was excised from gels, digested with trypsin, and analyzed by MALDI-MS (Fig. 2
A). MALDI-MS peptide mass fingerprint data assigned six peptides to the protein stathmin (theoretical Mr=17 kDa and pI=5.9), with 40% coverage (60/149 amino acids) of its sequence. This assignment was confirmed by ESI-MS/MS. For example, Fig. 2B
shows a collision-induced dissociation (CID) tandem mass spectrum of a doubly charged precursor ion with [M+2H]2+ at m/z 456.712+ (M=911.40), corresponding to the peptide sequence SHEAEVLK (residues Ser-63 to Lys-70 of stathmin). The ESI-MS/MS spectrum revealed characteristic fragmentation, such as an almost complete b ion series at m/z 225.1, 354.1, 425.1, 554.2, 653.2, and 766.3 (corresponding to fragment ions, b2-b7), and y ions at 147.1, 260.2, 359.3, and 559.3, 688.3, 413.22+ (corresponding to fragment ions, y1-y3, and y5-y7). Table 2
shows a complete list of observed tryptic peptides leading to the identification of stathmin protein. The overall stathmin sequence coverage was increased to 52% (78/149 amino acids) when MALDI-MS and ESI-MS/MS results were combined.
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Differential expression of developmentally regulated neuronal proteins in immature and mature cortical cultures
Western blot analysis of immature cultures showed prominent expression of stathmin, confirming findings obtained with DIGE, and of the immature neuronal markers DCX, PSA-NCAM, Hu, and ßIII tubulin (Fig. 3
). In contrast, the mature neuronal marker NeuN was expressed at only low levels, in keeping with prior results (30
, 31)
. This expression pattern was reversed in mature cultures.
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Stathmin expression in neuroproliferative zones and associated migratory pathways of the adult brain
Labeling of newborn neurons with [3H]thymidine, BrdU, or retroviral vectors has shown that neurogenesis occurs primarily in two areas of adult mammalian brain, the SGZ of dentate gyrus and the rostral SVZ, and that the RMS transports newborn neurons from SVZ to the OB (38
, 39)
. We have found that DCX expression is localized to these regions (37
, 40)
, where stathmin expression has also been reported (24
, 25)
. We found expression of stathmin in SGZ, SVZ, RMS, and OB (Fig. 4
), as well as in the cerebral cortex, corpus callosum and cerebellum (Fig. 5
).
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Stathmin expression colocalizes with neuronal and oligodendroglial markers
The location and appearance of stathmin immunoreactive cells suggested a neuronal phenotype, but stathmin can be expressed in a variety of cell types (22)
. We examined the properties of stathmin-expressing cells in adult rat brain by double-label immunohistochemistry. Stathmin in SVZ colocalized with BrdU, consistent with expression in newly divided cells, and with the immature neuronal marker DCX (Fig. 6
). In gray matter regions including DG, SVZ, OB, and cerebral cortex, stathmin was expressed in cells that also stained for the immature neuronal markers NeuroD and Hu, but not the neuroepithelial cell marker nestin, the mature neuronal marker NeuN, the astroglial marker GFAP or the endothelial cell marker vWF. In white matter of the striatum and corpus callosum, stathmin was coexpressed with CNPase, indicative of an oligodendroglial phenotype.
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Stathmin, DCX, and the migratory phase of adult neurogenesis
Neurogenesis ultimately requires cell proliferation, migration, and neuronal differentiation. Many factors have been identified that can stimulate the proliferative phase of neurogenesis in adult brain, but less is known about possible mediators of migration. Neuronal migration depends on the assembly and disassembly of microtubules, which are thought to be involved in nuclear translocation within the migrating neuron (15)
. Microtubule-associated proteins regulate microtubule structure and function by promoting polymerization or depolymerization of microtubules. Because stathmin is a microtubule-destabilizing protein (23)
, we thought it might help regulate the migration of newborn neurons in the adult brain. The similarity between the distribution of stathmin and that of the microtubule-stabilizing factor DCX suggested that stathmin and DCX might have complementary roles in this regard. To test whether stathmin and DCX are required for normal migration of newborn neurons in the adult rat brain, we used DiI, injected by the intracerebroventricular route, to label cells in periventricular regions, including the SVZ (Fig. 7
), then followed the migration pattern of DiI-labeled cells by fluorescence microscopy (40)
. In untreated rat brains (not shown) or the brains of rats given stathmin or DCX sense ODNs, the pattern of DiI labeling from SVZ, via RMS, to the OB was similar to that of stathmin (Fig. 4)
or DCX (40)
immunohistochemistry, or of the SVZ/RMS/OB system as originally described (38
, 39
, 41)
. In rats given stathmin or DCX antisense ODNs, however, migration along the RMS was inhibited. Thus, 24 h after DiI administration, DiI-labeled cells in stathmin or DCX antisense-treated rats had traveled only about two-thirds of the distance traveled in sense-treated rats. In antisense-treated brains examined 72 h after DiI administration, DiI labeled-cells did reach the OB, albeit in reduced numbers compared with sense-treated brains (not shown). These results suggest that both stathmin and DCX are required for normal neuronal migration from SVZ to OB.
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Stathmin expression in injury-induced adult neurogenesis
We and others have shown that after focal cerebral ischemia, neuronal proliferation in SGZ and SVZ is increased and neuronal migration is altered, such that some newborn neurons depart from the RMS and migrate into ischemic brain regions (40
, 42
43
44)
. These divagating neurons express DCX throughout their course (40)
. To determine whether stathmin expression is also a feature of injury-induced neuronal migration, we produced focal cerebral ischemia by MCA occlusion and evaluated the resulting distribution of stathmin immunoreactivity. Prior to ischemia and for up to 4 h thereafter, stathmin expression in sagittal brain sections through the striatum was confined to the SVZ (Fig. 8
). However, stathmin expression extended into the ischemic striatum beginning at 8 h and increased for up to at least 24 h. This time course is reminiscent of that observed earlier for DCX (40)
, and striatal expression of stathmin colocalized with expression of DCX. Cells both within the RMS and diverging from the RMS toward the ischemic striatum were stathmin immunopositive, and exhibited leading and trailing processes consistent with a migratory neuronal phenotype (15)
. In the ischemic cerebral cortex, increased numbers of stathmin immunoreactive cells that coexpressed DCX and Hu were also observed 8 h and longer after ischemia (Fig. 9
). Chains of stathmin- and DCX-expressing cells resembling those in the RMS and its extensions into the ischemic striatum were seen at the border between the corpus callosum and cerebral cortex, corresponding to the lateral cortical stream along which neurons migrate during forebrain development (45
, 46)
. We showed before that these cells originate in the SVZ, because they are labeled by DiI injected into the lateral ventricle (40)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neuronal migration in development can be resolved into several steps: leading edge extension, nuclear translocation, retraction of the trailing edge, and architectonic ordering at the ultimate destination (15)
. Cytoskeletal rearrangements are crucial for many of these events. For example, leading edge extension depends on polymerization of actin microfilaments, and nuclear translocation and retraction of the trailing edge involve microtubule assembly and disassembly, respectively (47)
. The role of microtubules has focused attention on microtubule-associated proteins as regulators of neuronal migration, especially because genetic defects in such proteins have been implicated in clinical disorders of neuronal migration. Thus, mutations in the gene for DCX, which promotes tubulin polymerization and stabilizes microtubules (48)
and is expressed in migrating neurons (48
, 49)
, cause migrational arrest of cortical neurons leading to X-linked lissencephaly and X-linked subcortical laminar heterotopia (50
, 51)
. Another candidate for a role in the regulation of neurogenesis-associated cell migration is stathmin, which promotes microtubule disassembly, sequesters tubulin, or both (52
, 53)
, and is expressed in SVZ and RMS (24
, 25)
.
In this study, we used 2-dimensional DIGE and MS techniques to identify stathmin as a protein that showed dramatically increased expression in immature compared with mature cortical cultures. Stathmin is regulated at the level of both expression and phosphorylation (52)
, and the stathmin antibody we used does not distinguish different phosphorylation states of the protein, so we cannot be certain which form or forms of stathmin are up-regulated in our immature cultures. However, our in vivo studies demonstrated that expression of stathmin was associated for the most part with cells of neuronal lineage in the dentate GCL, SVZ, and RMS of normal rat brain. Moreover, knockdown of stathmin expression with an antisense ODN inhibited the migration of DiI-labeled cells from SVZ, via the RMS, to the OB. This finding indicates that stathmin is not only a marker of migrating neurons but is required for normal migration, and parallel studies pointed to a similar role for DCX. Stathmin, like DCX (40)
, was expressed in neurons migrating ectopically into the ischemic striatum and cerebral cortex. This also suggests that stathmin and DCX expression correlate with cell function (migration) and not simply cell location.
Based on their opposing effects on microtubule stability (52)
and the apparent roles of microtubule assembly and disassembly in neuronal migration (47)
, DCX and stathmin may promote nuclear translocation and retraction of the trailing process, respectively, in SVZ-derived neurons transiting the RMS. Although young (2-month-old) stathmin-knockout mice exhibit no overt phenotype (54)
, it is doubtful that the analysis performed would have detected a reduction in cell migration during adult neurogenesis. Moreover, developmental absence of stathmin, as in stathmin-knockout mice, could elicit compensatory mechanisms that mask its normal function, such as increased expression of other stathmin-family proteins (52)
. Finally, neuropathological findings consisting of central and peripheral axonopathy are present in aged (20-month-old) stathmin-knockout mice (55)
, so other neurological abnormalities, including defects in neurogenesis, might also emerge in an age-dependent manner.
In addition to stathmin, our DIGE/MS study identified several additional proteins that may be candidates for a role in adult neurogenesis (Table 1)
. These include other cytoskeletal proteins (
-actin, 1-tubulin, ß3-tubulin, and cofilin 1); heat-shock proteins, which have been implicated in embryonic retinal neurogenesis (56)
; brain lipid binding protein, which is found in migrating neurons of the adult songbird telencephalon (57)
; and macrophage migration inhibitory factor, which is expressed in neuroproliferative zones of the embryonic and early postnatal rat brain (58)
. Like stathmin, these represent possible mediators of adult neurogenesis that may be suitable candidates for further investigation.
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ACKNOWLEDGMENTS |
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Received for publication September 15, 2003. Accepted for publication October 9, 2003.
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