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Lysophosphatidic acid (LPA) is a membrane-derived lysophospholipid that can induce pleomorphic effects in neural progenitor cells (NPCs) from the cerebral cortex, including alterations in ionic conductance. LPA-induced, calcium-mediated conductance changes have been reported, however the underlying molecular mechanisms have not been determined. We show here that activation of specific cognate receptors accounts for nearly all intracellular calcium responses evoked by LPA in acutely cultured nestin-positive NPCs from the developing mouse cerebral cortex. Fast onset changes in intracellular calcium levels required release from thapsigargin-sensitive stores by a pertussis toxin-insensitive mechanism. The influx of extracellular calcium through Cd2+/Ni2+-insensitive influx pathways, about half of which were Gd3+ sensitive, contributed to the temporal diversity of responses. Quantitative RT-PCR revealed the presence of all five known LPA receptors in primary NPCs, with prominent expression of LPA1, LPA2, and LPA4. Combined genetic and pharmacological studies indicated that NPC responses were mediated by LPA1 (~30% of the cells), LPA2 (~30%), a combination of receptors on single cells (~30%), and non-LPA1,2,3 pathways (~10%). LPA responsivity was significantly reduced in more differentiated TuJ1+ cells within cultures. Calcium transients in a large proportion of LPA responsive NPCs were also initiated by the closely related signaling lipid sphingosine 1-phosphate (S1P). These data demonstrate for the first time the involvement of LPA receptors in mediating surprisingly diverse NPC calcium responses involving multiple receptor subtypes that function within a single cell. Compared to other known factors, lysophospholipids represent the major activator of calcium signaling identified within NPCs at this early stage in corticogenesis.
The importance of the bioactive lysophospholipid LPA in the nervous system is underscored by its biologically relevant concentrations in the developing brain (Das and Hajra, 1989), and the localization of its five known cognate G protein-coupled receptors, LPA1–5, in developing and mature brain (Hecht et al., 1996; Contos and Chun, 2001; Contos, 2002; McGiffert et al., 2002; Noguchi et al., 2003; Choi et al., 2010). Genetic deletion of the first identified receptor (LPA1) has revealed central nervous system phenotypes (Contos et al., 2000; Harrison et al., 2003; Estivill-Torrus et al., 2008). Furthermore, these mice reveal behavioral phenotypes reminiscent of schizophrenia, a neurological disorder with developmental etiology (Harrison et al., 2003). Recent in vitro and ex vivo studies have addressed the relationship of LPA receptor-mediated effects to cell fate during early corticogenesis (Kingsbury et al., 2003; Fukushima et al., 2007). Acute administration of LPA induces process retraction and inhibits migration in young cortical neurons (Fukushima, 2002), and stimulates clustering and nuclear migration in nestin-positive NPCs (Fukushima et al., 2000). Ex vivo gain-of-function studies revealed that exogenous LPA induces thickening and abnormal folding of the cortex through LPA1- and LPA2-dependent increases in neurons and decreases in programmed cell death (PCD) (Kingsbury et al., 2003). LPA1 activation promotes neuronal differentiation in long term neurosphere cultures derived from murine E12 telencephalon in the presence of bFGF (Fukushima et al., 2007). In many cases, the targets of exogenous LPA are unknown but may include NPCs, young neurons, and differentiating glia.
Ca2+i signaling has been demonstrated to regulate progenitor and differentiated cell production (LoTurco et al., 1995; Haydar et al., 2000; Weissman et al., 2004), and dysregulated Ca2+i signaling may underlie certain neurological diseases with developmental etiologies (Caviness et al., 1995). Tightly controlled regulation of NPC proliferation, PCD, and differentiation is critical for proper cortical development (Corbin et al., 2008). Fluctuations in Ca2+i play diverse roles in nervous system development including differentiation of neural cells (Gu and Spitzer, 1995; Spitzer et al., 2004), chemotaxis (Kumada and Komuro, 2004; Komuro and Kumada, 2005), proliferation (Lory et al., 2006), and morphology (Aizawa et al., 2004). These important neurodevelopmental processes are initiated by cells utilizing intracellular and/or extracellular Ca2+ in distinct patterns (Berridge et al., 2000; Meyer zu Heringdorf, 2004).
As a group, LPA receptors activate G proteins to modulate differentiation, migration, morphology, proliferation, survival, and cellular physiologies of many cell types (Gardell et al., 2006; Choi et al., 2010). All known LPA receptors are capable of modulating Ca2+i (An et al., 1998; Bandoh et al., 1999; Lee et al., 2006; Lee et al., 2007; Yanagida et al., 2007). Since receptor-mediated intracellular Ca2+i fluctuations play diverse roles in nervous system development (LoTurco et al., 1995; Haydar et al., 2000; Okada et al., 2003; Weissman et al., 2004), we tested whether exogenously applied LPA could evoke intracellular Ca2+i responses in NPCs and employed pharmacological and genetic approaches to identify the responsible LPA receptor subtypes, contrasting with possible non-GPCR mechanisms. We further addressed downstream mechanisms involved in LPA-induced calcium signaling.
All animal protocols were approved by The Scripps Research Institute Animal Subjects Committee and are in accordance with NIH guidelines and public law. Wild-type C57Bl/6J, Balb/cByJ, and mixed background C57/129 mice were used during the course of these studies. Lpar1−/− (Contos et al., 2000), Lpar2−/− (Contos, 2002), and Lpar3−/− (Ye, 2005) mice were generated as previously described. Lpar1−/− and Lpar2−/− embryos were obtained by crossing heterozygous females to heterozygous or homozygous knockout males in a Balb/cByJ background (N>10). Lpar1−/−/Lpar2−/−/Lpar3−/− triple-null embryos were obtained by crossing Lpar1−/−/Lpar2−/−/Lpar3+/− females to Lpar1−/−/Lpar2−/−/Lpar3−/− males in a 129/SvJ, C57BL/6N mixed background (Ye, 2008). In all experiments, heterozygous and wild-type littermates were used as controls for homozygous embryos.
Timed-pregnant mice (day of plug = E0.5) were euthanized by cervical dislocation, E12.5 dorsal telencephalic regions from individual embryos were dissected in ice cold serum-free OptiMEM-I (Gibco BRL/Life Technologies, Rockville, MD) supplemented with 20 mM D-glucose, and 1% penicillin-streptomycin, in the absence of bFGF or EGF, as described previously (Dubin et al., 1999). In studies using knockout animals, the tail of each embryo was removed for genotyping. The dissected cortical regions of the telencephalon (which excludes ganglionic eminence, developing olfactory bulbs, and meninges) from individual embryos were placed in 1000 μL (or larger dependent on the volume of the tissue) OptiMEM-I in plastic centrifuge tubes, gently triturated by glass pipette into small clusters (<50 cells), centrifuged at 150×g, resuspended in 200 μL OptiMEM-I, and aliquotted to the center of three or four 12 mm diameter coverslips previously coated with Cell-Tak (Collaborative Research, Bedford, MA) in 24 well cluster plates. After settling 15 min in at 37°C in a 5% CO2 incubator, 1 mL OptiMEM-I was gently added to each well and coverslips were removed for Fura-2/AM loading beginning as early as 45 min later.
Alterations in Ca2+i were determined using the ratiometric calcium indicator dye Fura-2. Cells were loaded with Fura-2 acetoxymethyl ester (Fura-2/AM; 8 μM) in the presence of 1.5 μM pluronic acid F-127, and incubated for 30–45 min at room temperature in the dark in 5 mM Ca2+ buffer containing (in mM): 126 NaCl, 5 KCl, 1 MgCl2, 5 CaCl2, 10 HEPES, buffered to pH 7.4. Loading was done on one coverslip at a time to avoid cytotoxicity during prolonged incubations in Fura-2/AM and pluronic acid (data not shown). Just prior to testing, coverslips were briefly dipped in 5 mM Ca2+ buffer, the edge blotted on a Kimwipe tissue and then secured to the bottom of a laminar flow perfusion chamber RC-25 (Warner Instrument Corporation) with vacuum grease. Buffer (5 mM Ca2+) was gently added and the chamber mounted on an Axiovert 200M microscope. Cells chosen for imaging included those in semi-flat clusters where individual cells are clearly distinguishable, as well as around the perimeter of clusters in the plane of focus such that the imaged area was within a single cell avoiding overlying or underlying cells that might contribute to the signal. Cells were constantly perfused at room temperature (~23–25°C) at a rate of 3.3 ml/min (one chamber volume per 5–6 sec) (Lee et al., 2007). For experiments aimed at determining the component of the response mediated by Ca2+ influx, cells were acutely exposed to a Ca2+ depleted solution (in mM: 140 NaCl, 5 KCl, 1 MgCl2, 1 EGTA, 10 HEPES, pH 7.4) or pretreated for 20–50 min in 1 μM Thapsigargin and tested for LPA responsiveness in the presence or absence (to ensure stores were depleted) of extracellular calcium. All compounds were applied by gravity feed bath perfusion (AutoMate Scientific, Berkeley, CA). Fresh LPA-containing solutions were made from 1000x stocks just prior (within 100 sec) to use (i.e., addition to perfusion apparatus reservoir). Fura-2 imaging was performed as previously described (Lee et al., 2006; Lee et al., 2007). Briefly, images of Fura-2-loaded cells with the excitation wavelength alternating between 340 nm and 380 nm were captured with a cooled CCD camera every ~4 sec. Following subtraction of background fluorescence, the ratio of fluorescence intensity emitted after excitation at 340 nm and 380 nm (F340/F380) was calculated (MetaFluor, Molecular Devices). The computer screen of the field of imaged cells (50–255 cells per field) (Supplemental Figure 1A) was saved for later comparison with immunohistochemical labeling (Supplemental Figure 1B). For pharmacological experiments with the specific LPA1/LPA3 inhibitor Ki16425 (Ohta et al., 2003), cells were exposed for 3–12 min to 10 μM Ki16425 followed by 100 sec in 300 nM LPA together with 10 μM Ki16425 followed by ~30 sec in Ki16425 without agonist prior to washout. LPA was applied at 5 min intervals. A second LPA1/LPA3 inhibitor VPC 32183 (S) was tested similarly. Criteria for classification as a “responder” included the initiation of the response (increase or decrease in intracellular calcium) after a stable baseline within 100 sec after agonist solution reached the recording chamber. Experiments to test the role of pertussis toxin (PTX)-sensitive G proteins (e.g., Gαi/Gαo) were done after 4–8 hr incubation in the presence of 100 ng/ml PTX at 37°C. GdCl3 10–100 μM incubation was done on line after exposure to LPA.
The identification of imaged cells as NPCs or differentiating neurons was made by immunohistochemical labeling for nestin and β-tubulin III (TuJ1), respectively. At the end of each imaging experiment, the coverslip was carefully removed from the chamber and submerged in 4% paraformaldehyde and kept at 4°C prior to staining. Coverslips were washed with PBS and incubated with blocking solution (2.5% BSA, 0.3% Triton X-100 in PBS) for 1 hr before antibody labeling. Primary antibodies were diluted 1:500 in blocking solution and applied to coverslips overnight at 4°C with agitation. After rinsing, coverslips were incubated in secondary antibodies (e.g., Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen Corp., Carlsbad, CA) and Cy3-conjugated anti-rabbit IgG (Millipore, Temecula, CA) at 1:500 for 2 hr. Following PBS rinses, all coverslips were counterstained with DAPI and mounted on glass microscope slides in Vectashield (Vector Laboratories, Burlingame, CA). Coverslips were examined and photographed using a Zeiss Axioskop fluorescence microscope with rhodamine, fluorescein, or DAPI filters.
Total RNA was isolated from embryos at E12.5 using RNeasy Protect Mini Kit (Qiagen). Approximately 5 μg of each sample was DNase-treated and primed with oligo(dT) prior to cDNA synthesis with Superscript II reverse transcriptase (Invitrogen). Targets were amplified with iQ sybr green supermix (Bio-Rad) on a Bio-Rad iCycler using gene-specific primer pairs that flank an intron. To obtain absolute quantification of the target genes to compare between receptor subtypes and across ages, standard curves were established using plasmid samples containing reference sequences at known concentrations.
A change in intracellular calcium level was scored as a response if the change over baseline was at least 20% unless the prior baseline revealed significant fluctuations and the threshold change in ratio was increased to 50%. After thapsigargin exposure, the latency of responses was often >100sec and the cutoff time for a response was increased from 100 to 180 sec since the onset of the slowly increasing responses was difficult to determine accurately. Occasionally adjacent fields with identical response profiles were observed and these were counted only once. Population data were compared using one-way ANOVA with Tukey’s post-hoc multiple comparison test (GraphPad Prism4) (Figures 2, ,6,6, ,77).
The specific LPA1/LPA3 inhibitor Ki16425 (3-(4-[4-([1-(2-chlorophenyl) ethoxy] carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfonyl) propanoic acid) was a gift from Kirin Brewery Co. (Takasaki, Japan). LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) and VPC 32183(S) ((S)-Phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy))-phenyl]-propyl) ester) were purchased from Avanti Polar-Lipids, Inc. (Alabaster, AL). LPA was thoroughly dissolved in Milli-Q water and used fresh or stored frozen as a 5 mM stock solution in glass tubes. LPA was diluted to the indicated concentrations in extracellular saline and applied at concentrations ranging from 10 nM to 1 μM. Stock solutions of 1 mM S1P (Biomol, Plymouth Meeting, PA) were made by dissolving in 100% methanol, aliquotting to glass tubes, and storing at −20°C prior to use. Thapsigargin (Sigma) was prepared from a 2 mM stock in DMSO. PTX (List Biological Laboratories, Inc., Campbell, CA) was dissolved in water at 100μg/ml. Glutamate, CdCl2 and NiCl2 were purchased from Sigma and dissolved in water as 5000x, 3000x, and 10,000x stock solutions. Pituitary adenylate cyclase activating polypeptide-38 (PACAP, Sigma) was reconstituted as 1 μM in water and diluted to 3 nM in buffer prior to testing.
Submicromolar LPA concentrations modulated intracellular Ca2+ levels (Ca2+i) in ~75% of cells maintained in Ca2+-containing solution (5 mM Ca2+ buffer). Since spontaneous fluctuations of Ca2+i were occasionally observed (consistent with previous reports (Owens and Kriegstein, 1998; Owens et al., 2000)), the criteria for an “LPA-dependent” response were based on the timing after LPA arrival to the recording chamber (latency ≤100 sec) and reversibility of the calcium response (for further criteria see Methods). Responses to 100–300 nM LPA were reversible and could often be elicited multiple times (Figure 1). Since clusters of NPCs can be electrically coupled in the developing cortex (LoTurco and Kriegstein, 1991), we addressed whether intercellular transmission of the Ca2+ signal overestimated the fraction of cells responding directly to LPA. Several lines of evidence indicated that nearly all cells are activated by LPA through cell-intrinsic mechanisms. First, responsive cells were observed to be randomly distributed throughout clusters adjacent to non-responsive cells (Supplemental Figure 1C). Second, large LPA-induced Ca2+i responses were observed in single cells of multi-cellular clusters. Third, the onset of Ca2+i responses observed in a cluster often revealed no significant timing differences (i.e., there was no evidence of waves). Fourth, other agonists evoked calcium responses in only a small proportion of cells in a cluster (e.g., ATP and PACAP), and these cells were non-adjacent. Finally, pharmacological studies (see below) indicated that activation of different cells in a cluster appeared to be receptor subtype-dependent. Occasionally two adjacent fields revealed nearly identical response profiles, but this was rare and could be due to fields overlapping a single cell. These results are consistent with the findings that VZ cells in mouse E12-E13 telencephalon slices had mean input resistances and cell capacitances consistent with a single-cell electrical compartment (i.e., not coupled) (Albrieux et al., 2004).
Most nestin-immunoreactive cells responded to low nanomolar concentrations of LPA (Figure 1). LPA as low as 3 nM produced responses in ~25% of the cells (Figure 1A, 1B traces a-c). At 100 nM, ~75% of the cells responded, similar to proportions observed with higher (300–1000 nM) concentrations of LPA. The percentage of responsive cells increased as the field of cells was exposed to higher concentrations (e.g., 10–100 nM LPA) (Figure 1B, traces d,e (10 nM), f,g (30 nM), and h (100 nM)). Thus, the proportion of responding cells (Figure 1A), as well as the magnitude of the response to LPA (not shown), increased in a concentration dependent manner. F340/F380 ratios achieved in these experiments were far from saturating since the average maximal ratio change elicited by 1 μM ionomycin was ~20-fold the baseline (130 cells from 2 separate experiments), an increase not observed after LPA exposure (data not shown). In most cells, the extent of tachyphylaxis to a single concentration (300 nM) of LPA was minimal (Figure 1C, traces e,f), but there was extensive variability from cell to cell (Figure 1C). After 5 min extensive washout, the response to a second, 300 nM LPA challenge ranged from near zero (Figure 1C, traces a,b) to ~120% (not shown) of the initial response. LPA-induced responses showed no apparent sensitization in these experiments, a result that is essential for drawing conclusions from pharmacological experiments using Ki16425 (see below).
About 75% of wild-type cells responded to LPA at 100–1000 nM (72.4 ± 2.9%, n=36; Figure 2). The high proportion of cells responding to LPA with increases in Ca2+i is in contrast to the percentage of cells responding to S1P, ATP, and glutamate (~20–50%) (Figure 2; Supplementary Table 1a). PACAP-dependent signaling is an early event in corticogenesis (Suh et al., 2001) and receptors are capable of modulating intracellular calcium levels (Nicot and DiCicco-Bloom, 2001). Preliminary experiments indicated that PACAP elicited responses in ~25% of cells, and these cells were also sensitive to LPA (data not shown).
Temporal heterogeneity of Ca2+i responses was observed in individual cells bathed in Ca2+ containing solution (Figure 3). Intracellular calcium was increased in nearly every responsive cell (typical examples are shown in Figure 3a-f), however a small population of cells (1–2%) revealed a reversible decrease in Ca2+ (Figure 3g). In calcium containing buffer, a minority of cells revealed an increase in Ca2+i after a short lag which decreased to basal levels within ~100 sec of response onset (Figure 3a), similar to the response to LPA in the absence of extracellular calcium (see below). Most cells responded to LPA with temporally complex fluctuations in Ca2+ (Figure 3b-f). Transient increases could be followed by an “undershoot” of the basal Ca2+i that slowly returned to baseline (Figure 3f). Longer lasting responses could support oscillations (Figure 3b,c) or be followed by a secondary increase that was smaller than the initial response (Figure 3d). The mechanism underlying the decrease in calcium levels is not known but may involve stimulation of transport molecules that decrease cytosolic calcium. The heterogeneity of responses may reflect the components of the calcium “toolkit” (Berridge et al., 2000) expressed by individual cells or receptor-specific Ca2+ signaling pathways (see Discussion).
The striking heterogeneity of observed LPA-induced responses suggested that the duration of responses might be dependent on both release from intracellular stores and influx of external Ca2+. We therefore next determined the dependence of the LPA response profile on release of Ca2+ from intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to LPA (300 nM) were observed in a similar proportion of cells as in the presence of Ca2+ (61± 6% in 3 separate experiments, 50–200 cells/experiment) but were far less variable in time course, usually characterized by a fast transient rise in Ca2+i after a 15–30 sec delay. The time between half-maximal response rise and fall (t50) was 34 ± 7 sec (3 separate experiments) and the time to decay to basal levels in the continued presence of LPA was 50–100 sec. In the presence of extracellular Ca2+, the response time course was significantly more prolonged (t50 was 101 ± 9 sec, 14 separate experiments; p<0.0005 time for response to decay in 5 mM Ca2+ buffer vs. 0 mM Ca2+ buffer). The dependence of the initial fast transient rise in intracellular Ca2+ and influx of external calciumon intracellular store release was investigated by depleting stores using the Ca2+-ATPase inhibitor thapsigargin. Thapsigargin (1 μM) completely abolished LPA-induced effects when cells were tested in the absence of calcium, confirming the depletion of stores (data not shown). In the presence of extracellular Ca2+, the fast transient response was abolished. However, under these conditions, reversible, slowly rising increases were observed (Supplemental Figure 2). Further evidence that Ca2+ influx through plasma membrane ion channels contributed to the duration of the Ca2+i response was the reversible inhibition of the prolonged response by short exposure of external Gd3+ (10–100 μM) in about half the cells (Supplemental Figure 3). Since Gd3+ is known to block Cav channels (Biagi and Enyeart, 1990), we tested whether LPA-induced responses could be inhibited by the Cav channel blockers Cd2+ and Ni2+ (Hille, 2001). There was no obvious effect of 300 μM Cd2+ and 100 μM Ni2+ (applied before and during 300 nM LPA exposure) on LPA responsiveness, indicating that Ca2+ influx is not mediated by Cd2+- or Ni2+-sensitive channels (Supplemental Figure 4).
Although LPA-induced Ca2+ transients are likely to be mediated by activation of Gαq, calcium signaling can also be modulated by Gαi/Gαo activation in other systems (Lee et al., 2007). Therefore we tested whether LPA-induced responses were sensitive to PTX. There were no differences observed in the proportion of LPA-sensitive cells or the character of the averaged response compared to control cells following a 4–6 hr pre-incubation with PTX (100ng/ml) (Supplemental Figure 5A). PTX-mediated inhibition of Gαi in NPCs was confirmed by cAMP assay (Supplemental Figure 5B).
The percentage of TuJ1+ cells that did not immunolabel for nestin (cells differentiating into neurons) in E12.5 cluster cultures ranged from 1 to 15%, with an average of 9.8 +/− 2.1% (n=10 separate cultures). The proportion of TuJ1+ cells responsive to LPA (25 ± 5%) was significantly reduced compared to the proportion of all cells in these same cultures (78.6 ± 2.8% (n=10); p<5e-8). The responsiveness of TuJ1+ cells to S1P was also reduced (20 ± 6%; p<0.002) from that observed for all cells (53 ± 6%, n=9). TuJ1+ cells revealed a spectrum of heterogeneity similar to nestin+ cells (transient, prolonged, prolonged with oscillatory component or subsequent increases or decreases after return of response to baseline). These data suggest that at this stage in cortical development, calcium signaling is modulated by LPA (as well as S1P) in most neuroblasts, but that most differentiating neurons lose this ability. They further suggest that an in vivo critical period for bioactive lipid signaling in cortical development may exist.
Previous results have revealed prominent expression of LPA1 and LPA2 in early embryonic cortices (Contos et al., 2000; Contos and Chun, 2001; McGiffert et al., 2002), as well as LPA3 (McGiffert et al., 2002; Ohuchi et al., 2008). Since that time, two novel Ki16425-insensitive LPA receptors have been identified that mobilize Ca2+ in recombinant expression systems (Noguchi et al., 2003; Lee et al., 2006; Lee et al., 2007) and may be expressed in the embryonic brain (Ohuchi et al., 2008). We therefore used quantitative RT-PCR to quantify the mRNA expression in E12.5 mouse telencephalon. As previously reported, LPA1 and LPA2 were the predominant receptors expressed, however significant levels of LPA3, LPA4, and LPA5 were also observed. The relative abundance was 1~2~4 3, 5 (Figure 4). The profile observed from acutely isolated cells was similar to that observed up to 8 hr in culture, indicating that during the course of the calcium imaging studies there was little change in the LPA receptor mRNA expressed by cells in culture.
To determine whether LPA-induced calcium mobilization was mediated by a single receptor or multiple receptors, cortical NPCs were treated with the selective antagonist Ki16425. This compound effectively inhibits LPA1 and LPA3 and is inactive against the other known LPA receptors at low micromolar concentrations (Ohta et al., 2003; Lee et al., 2006; Lee et al., 2007). To avoid any potential complications of tachyphylaxis, cultures were first tested in the presence of antagonist. Cultures were incubated with the antagonist (10 μM) for at least 5 min before application of 300 nM LPA in the continued presence of antagonist. The percentage of responsive wild-type NPCs was reduced to 47 ± 4 % (n=7) from ~75% (observed in 0.1% DMSO vehicle controls) in the presence of this LPA1/3 antagonist. Importantly, extensive washout (>10 min) of Ki16425 resulted in a slowly developing responsiveness to LPA in these cells, consistent with the known reversible activity of Ki16425 (Ohta et al., 2003), which recovered to control proportions of sensitivity to LPA (74 ± 3% (n=4)). In the experiment shown in Figure 5, we incubated wild-type cultures for 15 min in Ki16425 (10 μM) and challenged cells with LPA (300 nM; dark gray bars) after 5–7 and 10–12 min in the presence of Ki16425 (10 μM; light gray bar) followed by extensive rinsing and subsequent challenges with 300 nM LPA. A variety of outcomes were observed. In the first class of outcomes (examples in Figure 5a,b,c), responses were not observed in the presence of Ki16425 but were observed only during washout of Ki16425. Traces a and b reveal responses from individual cells that responded to LPA only after 10 min of washout. Trace c shows similar responses at 5 and 10 min after washout. These data are consistent with the expression of LPA1/3, but not LPA2 or other Ki16425-insensitive receptors. In the second class (Figure 5d,e), small non-sensitizing responses were observed during Ki16425 exposure and larger calcium signals were apparent after washout, consistent with the expression of Ki16425-insensitive receptors and LPA1/3. It is not likely that the responses observed after Ki16435 washout are due to sensitization since significantly enhanced responses were not observed after DMSO in control experiments (data not shown), however, we cannot rule out this possibility in some cases. In the third class (Figure 5f,g), tachyphylaxis of the LPA response during the continued presence of Ki16425 is observed followed by large responses after antagonist washout consistent with the expression of both LPA2 (and/or Ki16425 insensitive receptor(s) revealing tachyphylaxis) and LPA1/3. Finally, in the fourth class (Figure 5h), LPA responses revealed little tachyphylaxis both in the presence and absence of Ki16425, however under the latter condition, large responses had reproducibly different profiles than those in Ki16425 (e.g., oscillatory calcium levels) consistent with the presence of both Ki16425-sensitive and insensitive receptor populations. Class 1 is consistent with LPA sensitive cell populations expressing LPA1/3 but not LPA2/4/5 (which would have been observed during antagonist exposure), and Classes 2–4 are consistent with cells expressing LPA1/3 as well as Ki16425 insensitive receptors (possibly LPA2 and/or LPA4/5 or non-receptor mediated mechanisms). The character of Ca2+i responses observed in the presence (“Ki16425-insensitive”) and absence of Ki16425 included transient, prolonged, oscillatory, and decreased responses in roughly similar proportions (data not shown). Similar results were obtained with a second, structurally distinct LPA1/3 antagonist VPC 32183 (S) (data not shown).
Since there were detectable levels of LPA3 in cortical tissue used for these studies, and in situ studies indicated a low level of expression throughout the embryonic brain (McGiffert et al., 2002), we used both genetic and pharmacological tools to investigate whether LPA3 modulated Ca2+i. Significantly fewer Ca2+i responses were observed in cultures derived from mice lacking LPA1 compared to those from littermate controls after 3 min DMSO exposure (Lpar1−/−: 44 ± 4% (n=8) vs. Lpar1+/−: 68 ±3% (n=10), p<0.01). Lpar1+/− embryos were phenotypically similar to wild-type, indicating a lack of haploinsufficiency. The ~30% reduction in LPA sensitivity in cells lacking LPA1 (Figure 6; Supplementary Table 1b) was similar to the percentage of LPA responses observed during application of Ki16425 (10 μM) (Figure 6, “Control + Ki” (blue bar with white stripes) compared to Lpar1−/− (solid blue bar)). In an attempt to address whether LPA3 plays a role in calcium signaling in these cultures, we tested whether LPA responsiveness could be acquired after at least 15 min washout of Ki16425 in Lpar1−/−-derived cultures. Although Ki16425 may slightly reduce the proportion of responsive cells from Lpar1−/− littermates (Figure 6, “Lpar1−/− + Ki”, 33 ± 7%, n=6) compared to DMSO-treated controls (Figure 6, “Lpar1−/−”), the difference was not significant (P>0.05). However, inspecting the phenotypes of individual cells, a rare Lpar1−/− population (that included nestin+/TuJ1−, nestin+/TuJ1+, and nestin−/TuJ1+ cells) was observed that exhibited no response to two exposures of LPA (300 nM) during 13 min incubation in Ki16425 (10 μM), but LPA sensitivity developed after extensive washout of the antagonist (data not shown), suggesting the presence of LPA3-dependent responses in a small proportion of cells. The responses remaining after LPA1 block or genetic deletion included the full complement of heterogeneous profiles as observed in control cells (data not shown). These results indicate that LPA1 is the predominant Ki16425-sensitive receptor activating Ca2+i responses in NPCs.
We next used genetic approaches to determine whether LPA-evoked Ki16425-insensitive calcium −/− responses were entirely mediated by LPA2. About 40% of the nestin+ cells derived from Lpar2 embryos revealed an LPA response (Figure 6, “Lpar2−/−”), a reduction of ~30% compared to littermate controls (Lpar2+/−: 64 ± 4% (n=11), P<0.01). In the presence of Ki16425 to block LPA1 (and any LPA3) receptors, only ~10% of the Lpar2−/− cells still responded to 300 nM LPA (Figure 6, “Lpar2−/− + Ki”, orange striped bar), consistent with the hypothesis that LPA1 and LPA2 play predominant roles in LPA induced calcium signaling. The remaining sensitivity to LPA is likely mediated by activation of another Ki16425-insensitive receptor (e.g., LPA4 or LPA5) since similar proportions of cells were activated by LPA 300 nM in NPCs derived from mice lacking all three receptors LPA1, LPA2, and LPA3 (Lpar1−/−; Lpar2−/−; Lpar3−/−) (Figure 6, orange bar). These results indicate that LPA2 is the predominant Ki16425-insensitive receptor activating Ca2+ responses in NPCs, but one or more additional receptors are functionally expressed.
To address whether the deletion of LPA receptors specifically reduced LPA responsiveness, we tested NPCs derived from wild-type, single (Lpar1−/− and Lpar2−/−), and triple knockout animals (Lpar1−/−; Lpar2−/−; Lpar3−/−) for their responsiveness to the closely related signaling lipid S1P (1 μM) at the end of each experiment. The percentage of S1P responsive cells was similar in all LPA receptor genetic backgrounds, including Lpar1−/−; Lpar2−/−; Lpar3−/− (Figure 7). This indicates that the genetic and pharmacologic manipulations performed in this study are specific to LPA receptor-mediated Ca2+ responses rather than general signaling mechanisms. Calcium responses evoked by S1P revealed heterogeneity in response types as observed for LPA: ~20% were transient (response decayed to baseline within 100 sec), 60% extended beyond 100 sec including a secondary increase, 16% revealed oscillations, and 4% an undershoot. Furthermore, this provides evidence for potential functional redundancy between LPA and S1P signaling systems.
The importance of LPA signaling in brain development has been underscored by receptor expression studies and mouse knockout strategies. Specifically, LPA1 deficient mice exhibit defects in cerebral development (Estivill-Torrus et al., 2008), show altered responses in ex vivo cortical cultures (Kingsbury et al., 2003; Rehen et al., 2006), and show abnormal behaviors consistent with pathologies such as schizophrenia (Harrison et al., 2003) (see review by (Herr and Chun, 2007). During embryonic development, electrophysiological responses had been observed in NPCs (Dubin et al., 1999), yet their activating mechanism(s) was unknown. An expectation extending from LPA receptor gene expression in the cortical VZ was the existence of LPA receptor-mediated actions on cortical NPCs. This study demonstrates for the first time that diverse calcium responses are regulated by specific LPA receptors in the developing mammalian neocortex. Notably, the extracellular stimulus is in fact a lipid, and has a more pervasive effect on NPC calcium signaling than glutamate, PACAP, or ATP at this stage in early corticogenesis.
Submicromolar concentrations of LPA were found to modulate intracellular calcium levels in the majority of nestin-immunoreactive putative neuroblasts. The responses are mediated by multiple cognate receptors, but predominantly by LPA1 and LPA2. At the early stage of development chosen for these studies (E12.5), the cortical anlage consists of a proliferative neuroepithelium – the ventricular zone (VZ) – bordering the lateral ventricle and composed predominantly of NPCs, as compared to a narrow preplate below the pial surface composed of postmitotic neurons (Bayer and Altman, 1991; Dubin et al., 1999; Maric et al., 2000a; Wonders and Anderson, 2005). The cell population most affected by LPA is undifferentiated nestin-immunoreactive NPCs. Interestingly, we show that calcium signaling in individual NPCs is often complex and reveals a previously unrecognized heterogeneity and sensitivity: cells express different combinations of functional receptors and exhibit calcium responses with variable cell specific dynamics. Furthermore, a large proportion of LPA responsive cells are also sensitive to the bioactive phospholipid S1P. It is likely that the overlap of these systems serves to protect essential cellular processes during brain development.
The characteristics of the observed LPA-induced responses revealed striking heterogeneity and were dependent on both release of calcium from intracellular stores and influx of external calcium. In developing neuronal systems, temporal differences in Ca2+i fluctuations have been shown to differentially affect cellular behavior in developmental processes including neuronal differentiation, proliferation, and migration (Gu and Spitzer, 1995; Behar et al., 1996; Komuro and Rakic, 1998; Behar et al., 1999; Flint et al., 1999; Maric et al., 2000a; Owens et al., 2000; Opitz et al., 2002; Ciccolini et al., 2003; Spitzer et al., 2004; Weissman et al., 2004; Calderon et al., 2005; Komuro and Kumada, 2005; Kumada et al., 2006). Receptor-mediated and spontaneous Ca2+i fluctuations have been demonstrated to play diverse roles in nervous system development including differentiation of neural cell phenotype (Gu and Spitzer, 1995; Spitzer et al., 2000; Spitzer et al., 2004), chemotaxis (Kumada and Komuro, 2004; Komuro and Kumada, 2005) and proliferation (Lory et al., 2006). With regard to the developing mammalian cortex, Ca2+i signaling pathways have been demonstrated to regulate progenitor and differentiated cell production (LoTurco et al., 1995; Haydar et al., 2000; Weissman et al., 2004) and dysregulated Ca2+i signaling may underlie certain neurological diseases with developmental etiologies (Caviness et al., 1995). Thus, controlled regulation of calcium signaling is important for proper cortical development (Konur and Ghosh, 2005). To achieve differential Ca2+i signaling, cells encode the signal in distinct spatio-temporal patterns utilizing mechanisms that release Ca2+ from intracellular stores and/or that modulate the flux of Ca2+ across the plasma membrane (Berridge et al., 2000; Meyer zu Heringdorf, 2004). Thapsigargin-sensitive store release mediates the initial fast transient increase but does not appear to be required for activation of at least a proportion of calcium influx pathways. Although Cav channels play a role in Ca2+ signaling at E16 (Corlew et al., 2004), Cd2+ and Ni2+ sensitive channels do not appear to contribute significantly to the LPA induced responses in E12.5 acute cultures. This result is consistent with a previous study on early neurogenesis in rat reporting a paucity of dihydopyridine sensitive L-type Cav in progenitor cells (Maric et al., 2000b). The insensitivity to Cav channel antagonists and sensitivity to Gd3+ in about half the cells suggest that TRPC (TRPC1-3 TRPC6-7) channels may mediate a large proportion of the LPA-induced Ca2+ influx responses (Ramsey et al., 2006; Tai et al., 2009). Receptor-mediated LPA signaling thus broadens the repertoire of extracellular signals modulating calcium responses.
Some of the conclusions drawn from this study rely on the interpretation that Ki16425 selectively and completely antagonizes LPA1 and LPA3 in this system. In addition to reported data (Ohta et al., 2003), three lines of evidence indicate Ki16425 is effective under these conditions. First, only 10% of the cells derived from Lpar2−/− animals in the presence of Ki16425 were responsive to LPA (Figure 6) similar to the percentage responsive to LPA in the triple knockout, Lpar1−/−/Lpar2−/−/Lpar3−/−. Second, cells that were initially unresponsive to LPA in the presence of Ki16425, revealed LPA-induced Ca2+i responses after extensive washout of Ki16425 (Figure 5). Third, there was no difference in the percentage of responsive wild-type cells in the presence of a higher concentration of Ki16425 (30 μM, data not shown). Thus, we have defined “Ki16425-sensitive” responses as those that are observed upon washout of Ki16425 for >5 min and are >150% the initial response(s) to LPA in the presence of Ki16425. The validity of this criterion is based on the findings that only 2% of cells revealed a second response to 300 nM LPA that was more than 150% the initial response magnitude (data not shown) and there was no apparent time-dependent increase in LPA responsiveness after initiation of the assay (i.e., the response magnitude was similar whether LPA was applied within 1 min or >5 min after initiating the assay; data not shown). About half the wild-type cells produced a response in the presence of the LPA1/3 blocker. Importantly, in cells revealing tachyphylaxis of the first Ki16425-insensitive response, a response could be observed after extensive (>10 min) washout of Ki16425. These data demonstrate that a significant proportion of cells express both Ki16425- sensitive and insensitive Ca2+i receptors. This result provides the first direct evidence that different LPA receptor subtypes can produce similar downstream effects in the same cells.
Both pharmacologic and genetic approaches indicated that LPA1 and LPA2 mediated the majority of responses and at least 30% of cells revealed responses that were mediated by multiple receptor subtypes. The expression of LPA1 is highly enriched in the VZ with low expression in the postmitotic cortical plate. This expression declines dramatically over the course of embryonic corticogenesis such that in situ analyses identified very few labeled cells by E18 (Hecht et al., 1996). Furthermore, LPA-induced 35S-GTPγS labeling of tissue sections from embryonic brain decreases between E12 and E18 indicated that, overall, there appears to be a decrease in LPA receptor activation of G proteins during this period (Harada et al., 2004). This decrease in LPA receptor expression and activity with differentiation is consistent with the responsivity of cortical cells reported in this study whereby NPCs were more responsive than differentiated young neurons. Thus, a much higher percentage of nestin+ NPCs from the VZ (~75%) responded to LPA relative to postmitotic, TuJ1+ neurons (~25%) that primarily reside in the CP. This is likely to reflect a spatial, basal-to-apical signaling gradient during cortical development that may regulate such processes as migration of differentiating neuroblasts. Importantly, compared to known activators of NPC calcium signaling, the lysophospholipids LPA and S1P represent the broadest extracellular activator yet identified.
The known LPA receptors signal through heterotrimeric G proteins, many of which are shared by single receptor subtypes (Ishii et al., 2004; Gardell et al., 2006). Important exceptions include the lack of LPA3 coupling to Gα12/13 and the coupling of LPA4 to Gαs. Although intracellular signaling pathways show considerable cross talk, the interactions of Gα12/13 and Gαs with the Ca2+ signaling pathway in developing cortex is poorly defined. It is possible that in the present studies, LPA2 (but not LPA1 (or LPA3 if present)) will influence Ca2+i homeostasis via an interaction with Na+/H+ exchanger regulatory factor2 (NHERF2) to potentiate PLCβ activation (Oh et al., 2004). We found no evidence for a significant role of Gαi/Gαo activation of calcium signaling by LPA. The expression of multiple LPA receptor subtypes within the same cell and the responsivity of many neuroblasts to both LPA and S1P indicate that there are multiple levels of modulation that regulate lysophospholipid-mediated calcium signaling, with further complexity produced by preferences for distinct chemical forms of LPA by some receptor subtypes (Bandoh et al., 2000). The potential for LPA to influence cortical development through specific Ca2+i signaling pathways that may ultimately modulate transcription, cell cycle, cell death, chemotaxis, and neuronal differentiation could lead to a better understanding of basic cellular mechanisms underlying neurodevelopment, as well as those contributing to neuropsychiatric disorders with developmental etiologies.
The authors gratefully acknowledge Danielle Letourneau and Hope Mirendil for critical review of the manuscript, and Dr. Kathy Spencer, Dr. Bialong Xiao, and Takashi Miyamoto for help with calcium imaging experiments. This work was supported by the NIH (MH051699 and DA019674 (JC)).