Signaling involved in neurite outgrowth of postnatally born subventricular zone neurons in vitro
© Khodosevich and Monyer; licensee BioMed Central Ltd. 2010
Received: 21 September 2009
Accepted: 10 February 2010
Published: 10 February 2010
Neurite outgrowth is a key process during neuronal migration and differentiation. Complex intracellular signaling is involved in the initiation of neurite protrusion and subsequent elongation. Although, in general many constituents of the machinery involved in this multi-stage process are common for neurons in distinct brain areas, there are notable differences between specific neuronal subtypes.
We analyzed key intracellular components of neurite outgrowth signaling in postnatally born subventricular zone (SVZ) neurons in vitro. We showed that inhibitors of PI3K, Akt1, PKCζ and small GTPases significantly reduced neurite outgrowth. Transfection of SVZ-derived neurons with inactive forms of Rac1 or Cdc42 also decreased neurite length whereas transfection with constitutively active forms of Rac1, Cdc42 or Akt1 as well as with full-length PI3K or PKCζ increased neurite length. PI3K, Akt1 and PKCζ acted upstream of the small GTPases Rac1 and Cdc42, which in turn modulate lamellipodia formation of SVZ-derived neurons.
We showed the involvement of PI3K/Akt1/PKCζ/Rac1/Cdc42 pathway in neurite outgrowth of postnatally born SVZ neurons.
Newly born migrating neuroblasts usually have one neurite, which they use for active migration from the site of origin to their destination site. A complex activity of receptors, cell adhesion molecules as well as attractants and repellents modulate intracellular machinery regulating outgrowth of neurites.
Many signaling molecules have been identified to be involved in neurite outgrowth, from membrane receptors to cytoskeleton constituents [1–3]. The tip of neurite, neural growth cone, is enriched in actin filaments as well as different filament remodeling and adapter proteins [2–4]. Intracellular kinases, such as MAPK, ERK and PI3K, regulate formation of actin filaments while small GTPases link kinase signaling to actin cytoskeleton machinery [5, 6]. Distribution of different cell membrane and cytoplasm components determines the polarization of neuroblasts, and thus the directionality of migration . Much of the analysis of neurite outgrowth machinery has been done in rat embryonic hippocampal culture (e.g., [8–11]). However, analysis in other systems does not always correspond to hippocampal culture and sometimes are even in contradiction with the results obtained in hippocampal culture. For example, activation of small GTPase Rac1 promotes neurite extension in rat hippocampal culture  while its activation decreases the longest neurite length of rat cortical culture  and its inhibition promotes neurite outgrowth in chick dorsal root ganglion neuronal culture . Also, while activation of PI3K-Akt pathway in hippocampal culture induces neurite outgrowth [9, 10], stimulation of this pathway can inhibit neurite outgrowth or have no effect in neuronal-like PC12 cell line [14, 15]. Thus, intracellular signaling regulating neurite outgrowth varies among different neuronal cell types and has to be analyzed separately for each cell type.
The majority of neurons in mammalian brain are born and migrate to their destination site during embryonic development. There are, however, two postnatal brain regions that continue to produce neurons - subventricular zone of lateral ventricles (SVZ) and subgranular zone of hippocampus [16–18]. Postnatally generated SVZ neuroblasts migrate via the rostral migratory stream (RMS) to the olfactory bulb where they mature into distinct interneuron subtypes, namely granule and periglomerular cells. We recently described the generation of transgenic mice, in which EGFP is expressed in the entire RMS , and optimized a procedure for RNA isolation from in vivo fluorescent RMS neuroblasts . Using transgenic mice with the clearly EGFP labeled RMS, we isolated neuroblasts from two distinct locations, one in the immediate vicinity of the SVZ (posterior RMS, pRMS), and the other more rostral, closer to the bulb (anterior RMS, aRMS) . We showed that the majority of upregulated genes and pathways in cells from the aRMS are involved in neuroblast migration. However, different genes/pathways can affect various cellular processes involved in neuroblast migration, e.g. we found that GluA1 (AMPA receptor subunit 1) probably modulates neuroblast polarization while Vav3 (guanine nucleotide exchange factor) is needed for growth cone formation .
Using a neurite outgrowth culture assay, we analyzed several upregulated aRMS genes of the PI3K/Akt1/PKCζ/Rac1/Cdc42 pathway to establish their role for neurite outgrowth of postnatally generated SVZ/RMS neuroblasts. We found that activation of several proteins in this pathway enhanced neurite outgrowth while their inhibition decreased outgrowth. PI3K, Akt1 and PKCζ acted upstream of the small GTPases Rac1 and Cdc42, which in turn modulate lamellipodia formation and neurite elongation.
Development of morphological neuronal characteristics for postnatal SVZ/RMS culture
Neurite outgrowth analysis of SVZ/RMS-derived neurons following pharmacological treatment
We analyzed the involvement of different intracellular signaling molecules in neurite outgrowth of SVZ/RMS neuroblasts using protein inhibitors. For the analysis we chose 5 intracellular signaling molecules that are involved in neurite outgrowth/polarization of rat embryonic hippocampal cultures PI3K, Akt1, PKCζ, Rac1, Cdc42 [8–11]. Most of these genes are expressed in the postnatal SVZ and RMS at high levels and continue to be expressed in olfactory bulb according to the Allen Brain Atlas  (Additional file 1: Table S1). Expression of these genes in RMS was also shown in our previous microarray study .
Treatment with Raf1 inhibitor or rapamycin (inhibitor of mTOR kinase) did not influence neurite outgrowth (data not shown).
Neurite outgrowth analysis of SVZ/RMS-derived neurons following genetic manipulations
Involvement of small GTPase activation for neurite outgrowth of SVZ/RMS-derived neurons
Phosphatidylinositols are involved in the polarization of SVZ/RMS-derived neurons
Small GTPases are involved in lamellipodia formation of SVZ/RMS-derived neurons
Inhibition of PKCζ caused a stronger reduction in Rac1 and Cdc42 activation than inhibition of PI3K or Akt1 (Figure 4). This may be indicative of PKCζ acting downstream of PI3K and/or Akt1. The interaction of PKCζ with Cdc42 and Rac1 has been shown before [30–32], but differently from our results, Cdc42 and Rac1 acted upstream of PKCζ. In SVZ/RMS cultured neurons PKCζ can be envisaged to stabilize activated (GTP-bound) forms of Rac1 and Cdc42. Activation of both Rac1 and Cdc42 depended on PI3K/Akt1 activity (Figure 4). PI3K inhibitor reduced Cdc42 activation more than the Akt1 inhibitor, while reduction in GTP-bound Rac1 was stronger after application of the Akt1 inhibitor than the PI3K inhibitor. A possible scenario accounting for this findings may be that Cdc42 is activated downstream of PIP3 via PKCζ signaling, whilst activation of Rac1 involves a stronger recruitment of Akt1 (Figure 7). However, further experiments are required to directly prove the proposed model shown in Figure 7. Thus, it remains to be established whether activated versions of Rac1 and Cdc42 can rescue the phenotype caused by PI3K, Akt1 or PKCζ inhibitor. Conversely, activated forms of PI3K/Akt1/PKCζ should not have an effect on neurite outgrowth upon Cdc42 and Rac1 inhibition according to this model.
PI3K and Akt1 have been shown to be involved in neurite outgrowth in primary cultured neurons [11, 33]. In hippocampal cultures, the accumulation of PIP3,4,5, the main enzymatic product of PI3K, specifies the future axon. PIP3,4,5 is required for axon elongation, and the specific distribution of PIPs in developing neurons is necessary for neuronal polarization [11, 34]. Also for postnatal SVZ-derived neurons PI3K and Akt1 are required for neurite elongation as demonstrated in this study. However, affecting PIP3,4,5 distribution on cell membrane did not modify significantly neurite outgrowth while disturbing neuronal polarization. We hypothesize that for postnatal SVZ/RMS neurons PI3K activity is more important for neurite outgrowth while overall PIP distribution (which depends not only on PI3K, but also on many other proteins) is more important for neuronal polarization.
In contrast to the phenotype of postnatally generated dentate gyrus granule cells that grow dendrites and axons upon maturation, the majority of SVZ-generated neurons develop into axonless cells [16, 22]. Indeed, we found that only few polarized neurons exhibited Tau expression (Figure 1). The results were confirmed using anti-Tau antibodies from different suppliers. We propose that the longest neurite of the polarized neurons in SVZ/RMS cultures corresponds to the major dendrite of granule cells, the main subtype of neurons produced in the SVZ [16, 22].
In our lamellipodia analysis experiments we found that C. difficile protein Toxin A treatment resulted in formation of large lamellipodia around cell bodies of the neurons. Toxin A inhibits the activity not only of lamellipodia-regulating small GTPases, but also of the small GTPase Rho that stabilizes focal adhesion . The observed phenotype could be the consequence of Rho inhibition that impaired the stabilization of focal adhesion and, as a result, promoted the formation of large lamellipodia.
It will be interesting to see whether other intracellular molecules that were shown to be involved in neurite outgrowth of postnatally born SVZ neurons can also modify PI3K/Akt1/PKCζ/Rac1/Cdc42 pathway signaling. One prominent candidate is PTEN, which negatively regulates PIP3 generation and PI3K signaling, thus, inhibiting neuronal polarization [11, 36]. Since we showed the importance of PIP distribution for polarization of the SVZ/RMS neurons in vitro, the balance between PTEN/PI3K signaling may be important for proper neurite development. Another potentially interesting candidate is GSK3beta that was shown to inhibit axonal formation [36, 37]. PI3K/Akt1/PKCζ/Rac1/Cdc42 pathway signaling may be also affected by some extracellular cues such as trophic factors and repellents/attractants. For instance, semaphorin  and netrin  signaling affect neurite outgrowth in vivo, and BDNF was shown to promote axonal differentiation in vitro via LKB1/Strad .
Neurite outgrowth is a fundamental neuronal feature and plays an important role in neuronal development during embryogenesis and in the adult brain. Although in general the machinery for neurite outgrowth has many common constituents when comparing various neuronal cell types, there are differences as shown here that need to be studied to better understand neuronal functions at the cellular level.
In this study we analyzed intracellular signaling constituents involved in neurite outgrowth of postnatally born SVZ neurons. We showed that inhibition of PI3K, Akt1, PKCζ and small GTPases Rac1 and Cdc42 decreased neurite outgrowth. Since inhibition of PI3K, Akt1 and PKCζ resulted in a reduction of activated forms of Rac1 and Cdc42, we propose a model according to which the PI3K/Akt1/PKCζ cascade leads to the activation of the small GTPases Rac1 and Cdc42, thereby modulating the cytoskeleton machinery during neurite outgrowth (Figure 7).
For our experiments we used wild-type C57Bl/6 mice. All procedures with animals were performed according to the guidance of Heidelberg University Animal Care Committee.
Materials and reagents
All chemicals and cell culture reagents were purchased from Sigma-Aldrich (Germany) and Invitrogen (Germany), respectively, unless otherwise specified. The following protein inhibitors and phosphatidylinositols were used in our experiments: Wortmannin (Alexis Biochemicals, USA), PKCζ pseudosubstrate (Biotrend, Switzerland), PKCζ pseudosubstrate inhibitor myristoylated (Calbiochem, Germany), LY294002 (Alexis Biochemicals, USA), Rac1 inhibitor (Calbiochem, Germany), Akt inhibitor X (Calbiochem, Germany), Clostridium difficile Toxin A (Calbiochem, Germany), Raf1 kinase inhibitor (Calbiochem, Germany), manumycin A (Calbiochem, Germany), rapamycin (Calbiochem, Germany), phosphatidylinositol-(3,4,5)-P3 (PIP3,4,5) (Cayman chemical, USA), phosphatidylinositol-(3,4)-P2 (PIP3,4) (Cayman chemical, USA), phosphatidylinositol-(4,5)-P2 (PIP4,5) (Calbiochem, Germany).
Rac1 and Cdc42 constitutively active (pcDNA3.1(+)hCdc42G12V and pcDNA3.1(+)hRac1G12V) and inactive (pcDNA3.1(+)hCdc42T17N and pcDNA3.1(+)hRac1T17N) constructs were purchased from UMR (University of Missouri Rolla, USA). Constitutively active Akt1 mutant construct, pUSEamp(+)myr-Akt1, was purchased from Upstate (USA).
PCMV-SPORT6-Pik3r1 and -Prkcz were purchased from Biocat (Heidelberg, Germany).
The following antibodies were used in our analysis: rabbit anti-EGFP antibody, 1:10000 (Molecular Probes, USA), mouse anti-beta-tubulin III, Tuj1, 1:500 (Covance, USA), mouse anti-Tau, 1:1000 (Chemicon, UK), rabbit anti-Tau 1:1000 (Santa Cruz, Germany), mouse anti-MAP2 1:500 (Chemicon, UK), Alexa 488-conjugated anti-rabbit and anti-mouse IgG (Invitrogen GmbH, Germany), anti-mouse and anti-rabbit Cy3 coupled and anti-mouse Cy5 coupled secondary antibody (Jackson Immuno Research Laboratories, USA).
Preparation of SVZ/RMS culture
SVZ/RMS areas were dissected from coronal sections of P6-10 wild-type mice. All steps of tissue processing were done in 1×Dissection Media (10×DM: 100 mM MgCl2, 10 mM kynurenic acid, 100 mM HEPES in 1×Hank's Balanced Salt Solution). Brains were placed in 1×DM media and coronal sections containing the anterior part of the SVZ and posterior part of the RMS were dissected using a blade. Regions around the lateral ventricles were isolated and washed in 1×DM. Dissected SVZ/RMS areas were incubated for 5 min with 30 U of papain (Worthington, USA) and 0.0005% DNase solution, and washed by trypsin inhibitor (Sigma-Aldrich, Germany) with 0.0005% DNAse in Neurobasal Media Supplemented [500 ml of Neurobasal media + 10 ml 50×B27-Supplement + 1.25 ml 200 mM L-Glutamate + 5 ml penicillin/streptomycin (100 U/ml)]. Cells were triturated through a fine tip, counted and plated at appropriate densities in Neurobasal Media with serum [500 ml of Neurobasal media + 50 ml FBS + 10 ml 50×B27-Supplement + 1.25 ml 200 mM L-Glutamate + 5 ml penicillin/streptomycin (100 U/ml)]. After 2 hours Neurobasal Media with serum was changed to Neurobasal Media Supplemented. Half of the media was changed every 4 days.
Neurite outgrowth assay
Cultured SVZ/RMS cells were treated for one or four days with different protein inhibitors: LY294002 - 30 μM, Wortmannin - 500 nM, PKCζ pseudosubstrate inhibitor - 0.1-0.5 μM, Akt inhibitor X - 500 nM, Clostridium difficile Toxin A - 100 ng/ml, Rac1 inhibitor - 50 μM, Raf1 kinase inhibitor - 2 μM, rapamycin - 50 pM; PIP3,4,5 - 10 μM; PIP3,4 - 10 μM; PIP4,5 - 10 μM. For positive apoptosis control inhibitor we used manumycin A (5 μM), an inhibitor of the Ras cell survival pathway . Cells were then fixed and stained with DAPI and anti-Tuj1 antibodies. For treated and untreated samples, the length of the longest neurite of Tuj1-positive cells was determined in 5 circles across the cell growth area (3 samples, number of cells > 100).
To overexpress different genes of the intracellular signaling pathway, cultured SVZ/RMS cells were co-transfected with the pEGFP and the particular gene expression construct. As control we used pCMV-SPORT6 plasmid. Two μl of Lipofectamine 2000 (Invitrogen GmbH, Germany) were mixed with 1 μg of plasmid DNA. The mixture was incubated at room temperature for 30 min and applied to SVZ/RMS cultures. After 1 hour at 37°C, cells were washed and incubated for another hour in Neurobasal media with serum (see recipe above). Subsequently, the cells were washed with Neurobasal Media Supplemented and conditioned media was added back. After 4 days in culture, cells were fixed and stained using anti-Tuj1 and anti-EGFP antibodies. For each gene expression construct, the length of all neurites for double-labeled EGFP and Tuj1-positive cells was calculated (3 samples, number of cells > 100).
The effect of PIPs was examined by intracellular PIP delivery into the cultured SVZ/RMS cells. Four days after PIP delivery, cells were fixed and stained with DAPI and anti-Tuj1 antibodies. The number of Tuj1-positive cells having one or more long neurites was determined (3 samples, n > 100).
Intracellular delivery of PIPs
Stock solutions of PIPs and neomycin at 1 mM concentration were prepared in HEPES-buffered saline. PIPs were mixed with a carrier (neomycin) to 10 μM each in Neurobasal Media, incubated at room temperature for 10 min, followed by 10 s of bath sonication (SONOREX, Bandelin GmbH & Co. KG, Germany). PIP-carrier containing medium was applied to SVZ/RMS cultures.
Rac1 and Cdc42 pull down assays
Three million cells from the SVZ/RMS area were plated on 10 cm plates coated with poly-L-lysine. After 3 days in culture, different protein inhibitors were added (concentrations were the same as in neurite outgrowth assay, except of for PKCζ inhibitor - 0.5 μM), and cells were cultured for one additional day. Rac1-GTP and Cdc42-GTP concentrations were analyzed by Rac1 and Cdc42 pull-down kits (Cytoskeleton, USA) according to the manufacturer's recommendations.
Cultures were fixed with 4% paraformaldehyde for 1 hour and then blocked in 0.5-1% Triton and 1% normal goat serum. Primary and secondary antibodies were described above. Sections were mounted onto slides with Moviol and subsequently analyzed on an upright fluorescent microscope (Zeiss Axioplan 2).
For Western-blot analysis protein samples were boiled in SDS gel sample buffer. Denatured proteins were separated by SDS-PAGE, transferred onto PVDF membranes and probed with antibodies. For statistical analysis antibody signals were quantified using ImageJ software and values were normalized to the corresponding β-actin signals. Sample sizes were n ≥ 3 and statistical analysis was performed with paired t-test.
Analysis of cell death
Cell death in dissociated SVZ/RMS cultures was estimated by adapting a protocol from . Briefly, cultures were incubated for 20 min in 5 μg/ml of propidium iodide, fixed for 4 h in 4% PFA, stained with appropriate antibodies and analyzed on an upright fluorescent microscope (Zeiss Axioplan 2).
The major neurite length and total neurite length were measured using Image J software. The normality of distribution was analyzed by d'Agostino and Shapiro-Wilk tests. We used ANOVA test for multiple comparisons and t-test for pair-wise comparisons. Differences were considered significant at p < 0.05. The graphs show mean ± standard deviation. For all pharmacological analysis at least 3 independent experiments for each condition were used and cell count was performed on 5 randomly picked areas on a coverslip. For all genetic analysis at least 3 independent experiments for each condition were used and at least 100 cells were analyzed.
We thank R. Hinz-Hernkommer and I. Preugschat-Gumbrecht for technical assistance. This work was supported in part by the Schilling Foundation and SFB488 grants.
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