Primary neurospheres were passaged one time to expand the limited amount of starting material and to ensure a self-renewing progenitor population while minimizing effects of any differentiated cells that remain after dissection. Secondary neurospheres were differentiated in 96-well plates with one library compound per well for three days. Cultures were then processed for immunoreactivity against a commonly used neuron-specific marker, βIII tubulin (Tuj1), using chemiluminescent detection (). Approximately 7-9% of cells normally differentiate towards a neuronal lineage and express Tuj1; this assay reliably detects molecules which enhance expression of this marker, as shown by the addition of the neurotrophin BDNF (Supplementary Fig. 1a
online). Importantly, this enhancement in Tuj1 immunoreactivity need not be due to increased neurogenesis; compounds which influence neurite branching or axonal outgrowth would also register as positive hits. Any potential candidates need to be tested in secondary assays to validate functional effects on the phenotype of interest.
P-Ser enhances neuronal differentiation of neural stem cells
For the primary screen using Tuj1 immunoreactivity as a readout, we assayed several hundred bioactive molecules (including kinase/phosphatase inhibitors and orphan ligands, see Methods
) and identified 64 compounds which were classified into two groups: “enhancers” which increased Tuj1 signals, and “inhibitors” which decreased Tuj1 signals (Supplementary Table 1
online). Most of the “inhibitors” exhibited general cytotoxicity and were not studied further; others have been described as exerting negative effects on neuronal function, e.g., p38 MAP kinase inhibitors (Xia et al., 1995
; Dougherty et al., 2005
). Among the “enhancers”, several are known to improve neuronal survival, e.g., tobacco smoke constituents (Soto-Otero et al., 1998
). Fourteen compounds were chosen for secondary screening, of which five (four “enhancers” and one “inhibitor”, Supplementary Fig. 1b-d
online) were confirmed to modulate neuronal differentiation. A novel enhancer identified from the orphan ligand library is L-serine-O-phosphate (phosphoserine or P-Ser). P-Ser increased the number of Tuj1+ cells in differentiating E12.5 cortical cultures () in a dose-dependent manner (). Similar effects were observed in cultures derived from mouse postnatal day one (P1) cortex (). These results suggest that the effects of P-Ser are not limited to a single developmental stage. The increased neuronal identity of P-Ser treated cultures was further confirmed by elevated expression of the neurogenic bHLH transcription factor NeuroD in differentiated E12.5 neurospheres, as determined by RT-PCR ().
The neurogenic phenotype of P-Ser could be due to effects on progenitor proliferation, cell fate commitment, and/or neuronal survival. We first assayed the effects of P-Ser on progenitor proliferation using the neurosphere assay. P-Ser treatment of secondary neurospheres (see below) reduced proliferation, based on cell number measurements () and neurosphere size (). This finding was confirmed by a 33% decrease in BrdU labeling of P-Ser treated attached progenitor cultures ().
P-Ser slows progenitor proliferation and enhances neurogenic commitment
The findings above could represent an effect on neural stem cells, other progenitors within the culture, or both. Therefore, we also analyzed production of new stem cells during P-Ser treatment. Primary neurospheres can be dissociated and replated, as single cells, to form secondary neurospheres. Neurospheres form aggregates at cell densities >5,000 cells/mL, which can confound analysis of the true proliferative potential of the plated cells (Singec et al., 2006
). In contrast, at a cell density of 1,000 cells/mL nearly all newly formed neurospheres are clonal in origin, i.e. derived from a single plated cell ((Groszer et al., 2001
); T.K.K. and H.I.K. unpublished observations). Thus, at this cell density, the neurosphere assay can be used as a reliable estimate of stem cell proliferation (see Discussion
). Secondary neurospheres were grown under clonal conditions with or without P-Ser. These spheres were then dissociated to single cells and allowed to form tertiary neurospheres under optimal conditions. The number of tertiary neurospheres formed is a direct measure of stem cell proliferation. We found that there were fewer tertiary spheres formed from P-Ser treated secondary spheres than from control secondary spheres (). Nearly all spheres were tripotent, giving rise to neurons, astrocytes and oligodendrocytes (Supplementary Fig. 2a
online), suggesting that P-Ser inhibits proliferation of multipotent sphere-forming progenitors.
We next measured the effects of P-Ser on neurogenesis in proliferating neurospheres. We found that while P-Ser decreased cell number and neurosphere size (), there was an increase in the percentage of Tuj1+ cells within the proliferating spheres (, and Supplementary Fig. 2b
online). Upon differentiation, secondary spheres grown in P-Ser generated more Tuj1+ cells than control cultures (). As these P-Ser treated spheres were passaged continually, the relative percentages of Tuj1+ cells generated upon differentiation increased compared to controls (). Furthermore, spheres grown in the presence of P-Ser contained elevated levels of the neuronal fate-determining bHLH transcription factor Neurogenin2 (Ngn2, ), as determined by RT-PCR. Together, these results strongly suggest that P-Ser directly enhances commitment of NSCs towards a neurogenic fate.
This possibility was confirmed via clonal analysis. If P-Ser instructs commitment of NSCs towards a particular lineage, then tertiary neurospheres derived from treated secondary spheres should show a predisposition towards generating that fate. Therefore, secondary neurospheres were grown at clonal density in the absence or presence of P-Ser. These neurospheres were then passaged to form tertiary neurospheres at clonal density, in the absence of P-Ser, and subsequently differentiated to determine their neurogenic capacity. We found that P-Ser treated spheres gave rise to tertiary spheres that were twice as neurogenic as control-derived tertiary spheres ().
We also tested whether P-Ser affects neuronal survival. To this end, we derived neuronal cultures from E14.5 mouse cortex, treated with P-Ser or water, and quantified the neurons at multiple time points. Compared to control conditions in which the number of Tuj1+ cells with neuronal morphology decreased over time (), P-Ser treatment caused an increase in the number of these cells remaining in the cultures at both time points assayed (). This rescue was especially pronounced by three days post-plating, suggesting that P-Ser has neuroprotective effects under these conditions (). Consistent with this possibility, P-Ser treatment resulted in a substantial decrease in the total number of apoptotic nuclei as indicated by TUNEL assay (), in agreement with the percentage of Tuj1+ cells undergoing apoptosis after three days ().
P-Ser improves survival in differentiating neuron cultures
We then investigated potential molecular mechanisms through which P-Ser could act to modulate these phenotypes. One possibility is that P-Ser influences the serine biosynthesis pathway, which has been postulated to be important for neurogenesis (Furuya et al., 2000
; Yoshida et al., 2004
). Another possibility is that the neurogenic effects of P-Ser may be mediated by group III metabotropic glutamate receptors, for which it is an agonist (Conn and Pin, 1997
). The latter hypothesis is supported by our findings that another group III receptor agonist (L-AP4, (Conn and Pin, 1997
)) produced similar effects as P-Ser on neurosphere size, whereas a group III receptor antagonist (UBP1112, (Conway et al., 2001
)) reversed the effects of P-Ser ().
The effects of P-Ser are mediated by metabotropic glutamate receptor 4
The effects of these compounds were also observed on differentiating secondary neurospheres derived from P1 cortex (). Here, treatment with either P-Ser or L-AP4 caused a >2-fold increase in the number of Tuj1+ cells generated (). UBP1112 again abolished this increase (). No enhancement of neuronal differentiation was observed in E12 neurospheres following treatment with either 10μM of D-serine-O-phosphate or D-AP4 (Supplementary Fig. 3a
online), which are >300-fold less potent at this class of receptors than their L enantiomers (Davies and Watkins, 1982
; Thomsen and Suzdak, 1993
). Thus, group III metabotropic receptors appear to mediate the effects of P-Ser in both proliferating and differentiating neurosphere cultures.
In order to examine the physiologically relevant receptor species, we analyzed the expression of the various members of the group III mGluRs by RT-PCR. In primary neurospheres, mGluR 7 and 8 were present at very low levels, whereas mGluR4 was moderately abundant (). In secondary neurospheres, only mGluR4 was detected (). The primer sets used were equally capable of detecting equimolar quantities of appropriate template in control reactions (Supplementary Fig. 3b
), suggesting that observed differences in mRNA quantity were not due to inefficient amplification by different primer sets. mGluR4 mRNA was present in the positive and negative fractions of secondary neurospheres FAC-sorted for the NSC marker LeX (Capela and Temple, 2002
) (Supplementary Fig. 3c
online), indicating the receptor is present in progenitors as well as more differentiated or committed cells. As a control, the progenitor-specific marker nucleostemin (Tsai and McKay, 2002
) was correctly detected only in the LeX(+) fraction (Supplementary Fig. 3c
online). According to the Allen Brain Atlas (http://www.brain-map.org
) (Lein et al., 2007
), mGluR4 was expressed by a subset of cells within the subventricular zone proximal to the lateral ventricles (one of two neurogenic germinal zones within the adult brain (Doetsch, 2003
), Supplementary Fig. 3d
online), as shown by non-isotopic in situ
hybridization. A specific probe for mGluR8 did not detect any positive cells within this region (Supplementary Fig. 3d
online). Thus, mGluR4 expression patterns suggest an important role for this receptor in mediating neural progenitor proliferation.
To confirm that mGluR4 is the target of P-Ser for progenitor proliferation, we used a loss-of-function approach by RNA interference. Two siRNAs (Methods
online) were designed to target mGluR4; both effectively reduced mGluR4 mRNA levels (). Dissociated primary spheres were transfected with mGluR4 siRNA vs. control siRNA and replated to form secondary spheres in the presence of either P-Ser or vehicle. As shown in , although P-Ser inhibits proliferation of cells transfected with control siRNA (as expected), this inhibition by P-Ser is lost in mGluR4 knockdown cells, indicating that the effect of P-Ser is indeed mediated though mGluR4. Interestingly, we found that mGluR4 knockdown produced larger spheres than control cells (), suggesting that mGluR4 may serve to restrict neural progenitor growth. Consistent with this hypothesis, mGluR4 knockdown led to hyperactivation of mTOR, a major regulator of cell growth and proliferation (Wullschleger et al., 2006
), as indicated by the greatly elevated levels of phospho-S389 S6K1 ().
We then tested the effects of mGluR4 knockdown on neuronal differentiation. Following transfection with control or receptor-specific siRNA duplexes, progenitors were differentiated in the absence or presence of P-Ser. As expected, cells transfected with control siRNA generated more Tuj1+ cells when treated with P-Ser (). Consistent with previous results, knockdown of mGluR4 abrogated the effect of P-Ser on neuronal differentiation (). Interestingly, we consistently observed an increase in O4+ cells in the receptor knockdown conditions, regardless of P-Ser treatment (). These data show that mGluR4 mediates the effects of P-Ser on neuronal differentiation and also suggests an unexpected role for mGluR4 in oligodendrocyte differentiation.
mGluR4 mediates the effects of P-Ser on differentiation
The experiments described above suggest that treatment with P-ser provides a simple method to enhance neuronal production from mouse neural progenitors. To determine whether such is also the case for human neural progenitors, and to investigate potential therapeutic application of these studies, we determined the effects of P-Ser on differentiating neural progenitors derived from human embryonic stem (hES) cells. As shown in , treatment with P-Ser doubled the number of neurons produced from two hES cell lines, HSF-1 (NIH UC01; karyotype XX) and HSF-6 (NIH UC06; karyotype XY). These results demonstrate that the neurogenic effects of P-Ser are neither species- nor gender- specific, and thus should prove broadly useful in directing hES cells for neural regeneration.
P-Ser increases neuronal differentiation of human embryonic stem cells