Endogenous and transplanted neural/stem progenitor cells (NSCs) have been shown to exert a multifunctional therapeutic action in models of central nervous system damage. Among the mechanisms ascribed to the observed functional recovery, the release of neurotrophic/neuroprotective factors has been shown to exert a significant role. Here we show that signaling through metabotropic glutamate receptors (mGluRs) regulates the release of neuroprotective factors by NSCs in vitro. The specific activation of group I mGluRs (mGluR1 and mGluR5) increases the expression of leukemia inhibitory factor (LIF) and brain-derived neurotrophic factor (BDNF), but not of vascular endothelial growth factor (VEGF), at both mRNA and protein levels.

Further experiments will be needed to understand the importance of this mechanism in vivo to foster the neuroprotective capacity of both endogenous and transplanted NSCs.

Neural stem/progenitor cells (NSCs) represent a heterogeneous population of multipotent cells able to self-renew and differentiate into astrocytes, neurons and oligodendrocytes^[1][2]. NSCs derived from different sources induce remarkable functional recovery in vivo after transplantation into laboratory animals with experimental neurological diseases. However, the extent to which transplanted animals recover from disease is unlikely to be attributable to the observed scarce differentiation of grafted cells^[3].

Thus, it is anticipated that grafted NSCs exert at least some of their therapeutic benefits through a series of bystander effects that imply the release of tissue trophic and immune modulatory paracrine factors at the level of the microenvironment^[4][5][6][3].

In some pathological conditions (e.g. spinal cord injury), the CNS environment is substantially altered, often characterized by an increase in the extracellular concentration of glutamate, the major responsible of excitotoxicity and neurodegeneration following injuries^[7]. The effects of glutamate are mediated both by ionotropic receptors/channels (iGluRs)^[8] and by metabotropic receptors (mGluRs)^[9]. The activation of group I mGluRs (composed of mGluR1 and mGluR5) via the administration of specific agonists induces the proliferation of rodent embryonic stem cells and NSCs, as well as human NSCs, both in vitro and in vivo^{[10][11][12][13]}.

To analyze the expression and function of mGluRs in spinal cord derived-NSCs;

To demonstrate the prominent role of mGluR1 and mGluR5 in regulating the increase of intracellular Ca2+ in spinal cord derived-NSCs;

To demonstrate that Group I metabotropic glutamate receptor signaling regulates the release of BDNF and LIF by spinal cord derived-NSCs.

mGluRs expression and functional characterization in SC-NSCs

We isolated stably expandable NSCs from the spinal cords of adult mice and compared their proliferation and differentiation properties with those of previously characterized subventricular zone (SVZ)-derived NSCs^[14] (Fig. 1A and Suppl. Fig. 1). Previous work demonstrated that SVZ-NSCs express the transcripts of all mGluRs subtypes with the exception of mGluR2 and mGluR6^[15]. By means of a semiquantitative RT-PCR, here we observed that SC-NSCs expressed all the transcripts, with the exception of mGluR8. We also detected two different isoforms of mGluR1 in SC-NSCs, with only one transcript for mGluR5 (Fig. 1B). We confirmed the expression of mGluR1 and mGluR5 by immunocytochemistry (Fig. 1C).

We then performed live Ca2+ imaging to assess the functionality of the identified glutamate receptors on SC-NSCs (Suppl. Fig. 2). Treatment with 50 μM glutamate alone activated significant intracellular Ca2+ transients (Fig. 1D). We then observed a partial reduction of [Ca2+]i transients when glutamate was administered in the presence of the specific NMDA receptor antagonist AP5 or the specific AMPA receptor antagonist CNQX (Fig. 1D). Quantification of active cells did not show any significant difference, vs. glutamate alone (Fig. 1E). Importantly, we observed significant decrease of both the [Ca2+]i transients (Fig. 1D) as well as the proportion of responding cells (Fig. 1E) when glutamate was administered in the presence of the selective non-competitive antagonist of mGluR5 MPEP, or the selective competitive inhibitor of mGluR1 LY367385. Treatment with the specific agonist of group I metabotropic receptors DHPG also led to a significant increase of [Ca2+]i, (Fig. 1D and E). Instead, treatment with DHPG in the presence of either MPEP or LY367385 resulted into a dampened average [Ca2+]i transients (not shown), and decreased number of responding cells (Fig. 1E).

Therefore, glutamate induced an increase in [Ca2+]i within SC-NSCs and its action was highly dependent on the activity of group I mGluRs.

Effects of signaling via Group I metabotropic glutamate receptors and expression of neurotrophins by NSCs

We then sought to investigate whether cellular signaling via group I mGluRs had any effect on the expression of mRNAs encoding for some of the most popular NSC-associated neurotrophic factors that include leukemia inhibitory factor (LIF), brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF)^[16][17][5].

To this aim, we optimized our experiments with SC-NSCs in glutamate-free tissue culture media containing small amounts (≥0.5 mM) of glutamine (control), or in control media added with 50 μM glutamate or 100 μM DHPG in combination with the different mGluRs antagonists.

Glutamate and DHPG both led to a slight down-regulation of Vegf (Fig. 1F), whereas the addition of LY367385 resulted in a further reduction. DHPG instead induced a significant up-regulation of the expression levels of Lif and Bdnf, which returned to control levels upon treatment with MPEP or LY367385 (Fig. 1F).

To determine the specificity of group I mGluR signaling in regulating Lif and Bdnf expression, we also investigated the effects of AMPA and NMDA receptor activation on SC-NSCs. Neither AMPA nor NMDA were able to induce a significant variation in the expression of Vegf, Lif or Bdnf, compared to controls (Fig. 1F). Furthermore, AP5 and CNQX did not affect the expression of Vegf, Lif or Bdnf, neither in the presence of glutamate or DHPG, which altogether confirmed the specificity of the signaling through mGluR1 and mGluR5. We did observe an unexpected effect of LY367385 in the regulation of Vegf and Bdnf upon treatment with AMPA or NMDA, which might be attributable to some unknown interactions between mGluRs and iGluRs.

We then verified whether an augmented protein secretion into tissue culture supernatants by ELISA paralleled the observed increase in gene expression. The secretion of LIF and BDNF increased similarly to correspondent mRNAs when SC-NSCs were treated with DHPG (Fig. 1G), controls. LIF and BDNF levels slightly decreased also upon treatment with AP5 or CNQX (Fig. 1G), as compared to mRNA expression (Fig. 1G). The decrease in protein secretion was more pronounced when cells were treated with MPEP or LY367385. In particular, we observed that the presence of MPEP led to a greater decrease in BDNF expression, while LY367385 led to a more significant reduction of LIF.

Here, we show that SC-NSCs express several components of the mGluRs family^{[15][18][19][20][21][22]}. We also demonstrate that glutamate signaling increases the intracellular concentration of Ca2+ in SC-NSCs and show that most of the glutamate-induced increase in intracellular Ca2+ is attributable to the action of group I mGluRs (mGlur1 and mGluR5). The activation of group I mGluRs, but not of NMDA or AMPA-associated receptors, led to an increase in the expression of LIF and BDNF, both in terms of mRNA and protein.

Altogether, these results suggest a novel pathway through which endogenous and transplanted SC-NSCs might exert neuroprotective effects observed in different pre-clinical studies. The increased extracellular concentration of glutamate as a consequence of cellular damage might contribute to promoting protective effects in recruited or transplanted NSCs.

Our work suggests a novel role for group I mGluRs in the regulation of Ca2+ dynamics as well as LIF and BDNF expression in SC-derived NSCs. The release of neuroprotective factors has been shown to play a major role in the therapeutic potential of stem cells. These observations may, therefore, suggest the manipulation of group I mGluRs activity as a novel therapeutic approach.

Once validated in the appropriate context, stem cell licensing/priming via mGluRs might represent a new strategy to foster the intrinsic neuroprotective capacities of both endogenous and transplanted NSCs.

We reveal here a possible relation between the activation of group I mGluRs and the release of neurotrophic factors. This is an in vitro proof-of-concept study that deserves further confirmation both in vitro, e.g., by analyzing the intracellular signaling pathways regulating the described cellular responses, as well as in vivo in different contexts that include transplantation research and studies of neurogenic responses in health and disease.

The addition of 50 µM glutamate alone did not lead to an increase in LIF and BDNF expression. This observation could be explained by the presence of glutamate transporters that rapidly remove extracellular glutamate, as shown in SVZ-NSCs^[23]. Quantification of extracellular glutamate showed in fact that 50 μM glutamate was rapidly taken up by SC-NSCs. Alternatively, we may speculate an interaction occurring between mGluR1 and the ionotropic receptors that partially revert the effects of AMPA and NMDA. The activity of group I mGluRs, in fact, may alter the surface density of specific receptor subunits, as previously reported^[24]. Alternatively, the inhibition of mGluR1 might enhance the action of mGluR5 and so, indirectly, potentiate the effects of NMDA^[25].

The growth of SC-NSCs with media enriched of group I mGluR agonists (such as DHPG) or the genetic overexpression of mGluR1 and/or mGlur5 in NSCs may represent a valid approach to increase stem cells therapeutic efficacy in transplantation studies.

NSCs derivation

Adult C57BL/6J mice were culled by cervical dislocation and the spinal cords and brains collected and dissected in ice cold sterile phosphate buffered saline (PBS 1X). After carefully removing the meninges, whole spinal cords were cut into small pieces with a blade and dissociated in digestion medium (EBSS 1X, 200 mg/L Cysteine, 200 mg/L EDTA (Sigma), 2 U/ml Papain) for 45 min at 37°C.

After centrifugation, the pellet was mechanically dissociated and re-suspended in 0.9 M sucrose in EBSS, centrifuged again and subsequently resuspended in 200 μl of Complete Growth Medium (CGM, mouse NeuroCult basal medium, Stem Cell Technologies Catalog #05700 with the addition of mouse NeuroCult proliferation supplements, Stem Cell Technologies Catalog #05701) in the presence of heparin 0.002% (Sigma #H3393), basic fibroblast growth factor (bFGF) (10 ng/ml) (Peprotech #100-18B-1000), epidermal growth factor (EGF) (20 ng/ml) (Peprotech #AF-100-15-1000) and antibiotics (Pen/Strep, Invitrogen #1514012). The cell suspension was slowly added on top of 4% BSA in EBSS, and centrifuged 7 min at 200 g. The myelin-free pellet was then resuspended in CGM, plated in a 25 cm2 tissue culture flask at the density of 8000 cells/cm2, and incubated at 37°C, 5% CO2. After the first 6 passages of mechanical dissociation, cells were enzymatically dissociated once every 4–5 days with Accumax (Ebioscience 00-4666-56) for 10 min at 37°C and plated at 8000 cells/cm2.

Starting from passage 10, NSCs were grown in a glutamate free, custom-made medium (NB medium) composed of Neurobasal (Invitrogen, #12349015), bovine serum albumin 200 g/L, N2 supplements (Thermo Fisher Scientific 17502048), heparin, EGF (20 ng/ml), FGF (10 ng/ml), antibiotics (Pen/Strep, Invitrogen #1514012) and 2 mM Glutamine (35050-038). DHPG (#0805), AP5 (#0105), CNQX (#1045), MPEP (#1212) and LY 367385 (#1237) were purchased from Tocris Bioscience. As for SVZ-derived NSCs, the region containing the lateral ventricles was cut into 1 mm3 pieces and resuspended with a digestion solution containing Earle's Balanced Salt Solution (EBSS), Ethylenediaminetetraacetic acid (EDTA), Cysteine and Papain. After centrifugation, the pellet was re-suspended with complete growth medium until the formation of neurospheres.

For the differentiation protocol (Suppl. Fig. 1E-H) NSCs were collected, centrifuged, counted, resuspended in differentiation medium (CGM, mouse NeuroCult differentiation medium, Stem Cell Technologies) and plated on Matrigel-coated glass coverslips (placed inside 24 multi-well plates) with a density of 80.000 cells/cm2 in 130 of medium. Cells were then placed in the incubator (37°C, 5% CO2) for 30 min to homogenously settle onto the coverslips. Then the wells were filled with differentiation medium and cells were incubated at 37°C, 5% CO2 for 6 days. Half of the medium was changed to fresh medium after 3 days.

Gene expression analysis

Total RNA was extracted following TRIzol® reagent (Life Technologies, #15596-026) manufacturer description. 1 μg of RNA was converted into cDNA using high capacity cDNA reverse transcription kit (Applied Biosystem, #4368813). For the semi-quantitative analysis, 100 ng cDNA was amplified according to manufacturer description (Life Technologies, #10342-053) by using the primers listed in [Castiglione, #86]. Reaction conditions included an initial denaturation step (94 C/ 3 min) followed by 30–33 cycles of (94 C/45 s; 55 C/30 s; 72 C/30 s), followed by a final extension step (72 C/10 min). Samples were run using a 2% agarose gel in TBE.

For quantitative qPCR, 10 ng of cDNA was amplified using TaqMan® Fast Universal PCR Master Mix (Life Technologies, #4352042) and the following primers: Vegf (Life Technologies, Mm01281449_m1), Lif (Life Technologies, Mm00434762_g1) and Bdnf (Life Technologies, Mm00434762_g1). 96 well plates were used and the samples read with a 7500 Fast Real-Time PCR system machine (Applied Biosystems). The Ct method was used for quantification of gene expression. Expression levels were normalized to β-actin mRNA. All the experiments have been performed at least three times (from n=3 independent biological replicates).

Immunocytochemistry

Unless differently specified, NSCs were fixed with pre-warmed 2% PFA + 2% sucrose in PBS for 5–10 min at RT and subsequently washed 3 times with PBS and conserved at 4°C with 0.005% PBS sodium azide. Fixed cells were then incubated with blocking solution [PBS + 10% Normal Goat Serum (NGS, PAA #B11-035)] with 0.3% Triton X-100, for 1.5 h at RT. The blocking solution was then removed and the cells were incubated either O/N at 4°C or 2 h at RT with the primary antibody (see Suppl. Info.) diluted in PBS-NGS 1% with 0.3% Triton X-100. Cells were then washed twice with PBS and incubated 1 h at RT with the appropriate Alexa Fluor 488, 546 or 647 secondary antibody diluted 1:1000 in PBS-NGS 1% with 0.3% Triton X-100. Cells were washed three times in 1X PBS and incubated for 3 min with 4',6-diamidino-2-phenylindole (DAPI) at RT in the dark. Finally, cells were washed twice with PBS, once with distilled water and mounted on glass microscope slides with mounting medium (DAKO, #S3023). Slides were stored at 4 or -20°C.

ELISA protein binding assay

ELISA protein binding assays were performed according to the manufacturer's protocol (R&D DBNT00 and R&D MLF00). Medium samples were previously collected and stored at -80°C and used undiluted. Absorbance was measured at 450 nm using a microplate reader (Infinite M200 Pro-TECAN). Concentrations were extrapolated by using GraphPad Prism 4.0 software.

Ca2+ imaging analysis

NSCs were seeded at a density of 100.000 cells/cm2 on laminin coated-glass bottom culture dishes (MatTek Corporation) and primed for 3–5 days with a medium containing Neurobasal (Gibco 21103-049), N2 supplement (Thermo Fisher Scientific 17502-048), 10 ng/ml bFGF (PeproTech #100-18B-1000), 1 μg/ml laminin (Roche 11243217001), 5 μg/ml heparin (Sigma #H3393), 1.5% glucose, glutamine (Thermo Fisher Scientific 35050-038), and antibiotics (Pen/Strep, Invitrogen #151401). NSCs were loaded with 5 μM Fluo-4AM (Life Technologies, F-14217) for 30 min at 37°C and washed twice for 15 min at 37°C with Tyrode’s solution, an isotonic solution resembling the composition of the cerebrospinal fluid and containing 129 mM NaCl, 5 mM KCl, 2 mM CaCl2, 3 mM MgCl2, 30 mM Glucose, 25 mM Hepes. The chamber was then mounted on the stage of a Leica DMI 6000B inverted live imaging microscope within a heated (37°C) and CO2 conditioned box. Images were acquired with a frequency of 2 frames per second (fps). The chamber was connected with a perfusion system to allow a continuous and regular flow of solutions when necessary. NSCs were recorded for a total of 160 s: 15 s of basal recording, 105 s of stimulation and 40 s of wash out. Cells were also imaged for the same time length during basal conditions to quantify their Ca2+ spontaneous activity at the beginning and at the end of each experimental session. Each acquired time-lapse was then analyzed with a custom-made macro using the software Fiji. Individual regions of interest (ROIs) corresponding to the soma of each imaged cell were automatically acquired. Additional ROIs were manually added into 5 cell-free areas of the imaged field to compensate for fluorescent background. The fluorescent changes over time within each ROI were then measured and the average background subtracted. The first 10 s of recording were used to calculate the average basal fluorescence (F0). Fluorescence values for each time frame were then calculated as F= ΔF/F0, where ΔF is Fi-F0, with Fi being the fluorescence value of an ROI at any given time point. Fluorescent values were then plotted over time. The number of active cells was calculated as the percentage of cells showing a ΔF/F0 > 0.4 in at least 5 consecutive frames (2.5 s).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 4.0 software. One-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test correction was used for multiple group comparison and repeated measure two-way ANOVA was used to analyze differences in behavioural tests, in not differently indicated.

This work was funded by the Italian Ministry of Health (GR08-7 to SP), the EU 7th Framework Programme [FP7/2007–2013] under Grant Agreement No. 280772, “Implantable Organic Nanoelectronics (I-ONE-FP7)” project, the European Research Council (ERC) under the ERC-2010-StG grant agreement n° 260511-SEM_SEM, the Evelyn Trust (RG 69865 to SP), the Bascule Charitable Trust (RG 75149 to SP) and a core support grant from the Wellcome Trust and Medical Research Council to the Wellcome Trust – MRC Cambridge Stem Cell Institute.

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The authors declare no conflicts of interest.

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