Retroviruses were produced as previously described. In summary, the Lipofectamine 2000 kit (Invitrogen) was used to transfect human embryonic kidney cells (HEK 293T) with retroviral constructs. The virus was collected 2 days after transfection by filtering the supernatant through 0.22 µm filter top and subsequent centrifugation at 19’4000 rpm/4°C for 2 h. Viral pellet was resuspended with 4 ml PBS and spun down with 20% sucrose cushion at 20,500 rpm/4°C for 2 h. The final viral pellet was resuspended in 40 μl PBS.
Wild-type (wt) mouse hippocampal NSPCs were isolated from adult mice (6–8 weeks old) as previously described. Proliferating NSPCs were cultured as a monolayer with 37°C/5% CO2 in DMEM/F-12 (Glutamax) media supplemented with N2 (Invitrogen), antibiotics (penicillin-streptomycin-fungizone; Invitrogen), Heparin (5 μg/ml; Sigma), human EGF (20 ng/ml; PeproTech) and human basic FGF-2 (20 ng/ml; PeproTech). Media was changed every 2 days. For neuronal differentiation, media without growth factors was used (DMEM/F-12, N2, PSF, and heparin). For the viral infection, approximately 50,000 wt adult hippocampal NSPCs were plated on coated Poly-L-ornithine (10 μg/ml; Sigma) and Laminin (5 μg/ml; Invitrogen) 12 well plates in proliferation medium. 24 h after plating, cells were infected with 1–2 μl NeuroD1-ERT2-IRES-GFP retrovirus.
NeuroD1-ERT2-IRES-GFP virus-infected NSPCs were spun down at 300 g for 5 min and the cell pellet was trypsinized using 0.05% trypsin in Versene for 5 min at 37°C. Afterwards a double volume of ovumucoid trypsin inhibitor mix was added for 5 min at room temperature. Cells were resuspended with 5 ml of media and spun down at 120 g for 5 min. Final cell pellet was resuspended in 1 ml 1 mM EDTA in PBS or DPBS and stored on ice prior to sorting. FACS for NeuroD1-ERT2-IRES-GFP+ (NeuroD1-GFP+) cells was performed using the BD FACS Aria III Cell Sorter. For negative control/background autofluorescence, wild-type hippocampal NSPCs were used.
NeuroD1-induced neuronal differentiation
NeuroD1-induced neuronal differentiation was optimized from a previously established protocol to enable in vitro differentiation of pure neuronal cultures. Proliferating NeuroD1ERT2-expressing GFP+ cells were trypsinized as described before and in parallel plated on coated (Poly-L-Lysine, 10 μg/ml and LAM) 6 well plates for metabolome analysis (330,000 cells/well in triplicates) and on coated 12 well plates (130,000 cells/well in triplicates) for the corresponding immunocytochemistry analyses. 2 days after cell plating, proliferating media was replaced with differentiating media (no growth factors) and 0.5 μM OH-TAM (Sigma; dissolved in 100% EtOH) was added. Addition of OH-TAM was defined as differentiation start (D0). Differentiation media was exchanged after 3 days. Cells were collected for metabolite extraction or in parallel for immunocytochemistry after the following time points: 6 h, 12 h, 1 day (D1), 2 days (D2), 3 days (D3), 4 days (D4), 5 days (D5), 6 days (D6) and 7 days (D7). To measure the proliferation rate during neuronal differentiation, cells on 12 well plates were pulsed with 10 mM EdU (Clik-it EdU Imaging kit; Life Technologies) for 1 h at 37°C prior to fixation.
At every given time point, cells were fixed with pre-warmed (37°C) 4% paraformaldehyde (PFA) for 15 min at room temperature. Fixed plates were stored in PBS at 4°C until immunocytochemistry was performed. Cells were treated with blocking and permeabilization buffer containing 3% donkey serum and 0.25% Triton X-100 in TBS for 30 min at room temperature. Cells were incubated with primary antibodies in blocking/permeabilization buffer overnight at 4°C. The following primary antibodies were used: goat α-SOX2 (1:200; Santa Cruz Biotechnology), rabbit α-GFAP (1:500; DAKO), mouse α-MAP2ab (1:500; Sigma). All secondary antibodies were applied for 1.5 h at room temperature in blocking/permeabilization buffer with the dilution 1:250 (Jackson Laboratories). Cell nuclei were detected with 4-6-diamidino-2-phenylindole (DAPI, 1:5000; Sigma). To analyze the cell proliferation rate, Click-it EdU Imaging kit was used according to the manufacturer's instructions.
Images were taken on a Zeiss Observer Z1 microscope. To analyze proliferation rate 12 h and 1 day (D1) after differentiation start, we quantified EdU and DAPI-stained nuclei. Both stained nuclei were automatically counted by self-written Fiji macro program. Number of EdU-positive DAPI nuclei was compared to EdU-negative DAPI nuclei to calculate the proliferation rate. Statistical analysis was performed using Prism6. Ordinary one way ANOVA test was performed, followed by Turkey’s multiple comparisons test, p <0.0001, n= 3, SEM shown.
Quantification of neuronal and astroglial progeny with and without NeuroD1 induction
We plated 60,000 NeuroD1-GFP+ hippocampal NSPCs cells/well for each condition on glass coverslips coated with Poly-L-ornithine (50 μg/ml) and LAM (5 μg/ml) in proliferating media (DMEM/F-12 supplemented with N2, heparin and growth factors as previously described). After 24 h, we changed the media to differentiation media (DMEM/F-12 supplemented with N2, heparin but no growth factors) and cells were treated either with EtOH or OH-TAM (0.5 μM; SIGMA) for 2 days. During the whole differentiation process, cells were kept in differentiation media. After 7 days of differentiation, cells were fixed and stained with DAPI, anti-MAP2ab and anti-GFAP antibodies as previously described. Confocal images were taken on Olympus microscope. 5 images per well were taken. To quantify the amount of cells expressing MAP2ab and GFAP markers, we used self-written FiJi macro: a Z-stack was created with MAX intensity for all images. First, we counted DAPI particles by using the following functions: find edges, make binary, fill holes, watershed and analyze particles size 30–300, circularity 0.40–1.00. Then we analyzed MAP2ab and GFAP expression by setting a threshold and converting it to a mask. Threshold was adjusted for each condition to yield a better separation of MAP2ab and GFAP-positive cells and to eliminate any overlap in the channels. This mask was selected and laid over the DAPI particles. Then all DAPI particles overlaying the GFAP or MAP2ab mask were counted using the analyze particle function with size 30–300, circularity 0.30–1.00. The counted particles for MAP2ab and GFAP were divided by the DAPI counted particles to get the ratio of MAP2ab and GFAP-positive cells per condition. In total, 3,197 DAPI+ cells were counted for ETOH condition and 2249 DAPI+ for OH-TAM condition.
At every given time point, cells were washed with 75 mM ammonium carbonate (Sigma) pH 7.4 and snap-frozen on the plates with liquid nitrogen. Metabolites were extracted by treating the snap-frozen plates with hot extraction solution (70% EtOH) on heating block (75°C). The plate was re-extracted with hot extraction solution 2 more times, allowing for a high recovery rate. Note that the extraction efficiency is irrelevant for the analyses performed here given that we did not perform quantitative analyses but a qualitative comparison between conditions. Hence, full recovery is not a precondition to detect difference in metabolites across groups. Supernatant containing metabolites from all 3 extractions was pooled, spun down, and stored at -80°C until mass spectrometry measurements.
Mass spectrometry measurements and data analyses
For non-targeted metabolite profiling, metabolite samples were analyzed by flow injection analysis on an FIA Agilent Q-TOF 6550 QTOF instrument (Agilent, Santa Clara, CA) in negative mode at 4 GHz, high resolution in a m/z range of 50–1000. A 60:40 mixture of isopropanol:water supplemented with NH4F at pH 9.0, as well as 10 nM hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazine and 80 nM taurochloric acid for online mass calibration. Ions were putatively annotated to metabolites based on exact mass using a tolerance of 0.001 Da and the KEGG hsa database following the procedure described in. Notably, this procedure does not allow distinguishing metabolites with same molecular formula or weight. For full disclosure, all putative matches are reported in Table S1. All data analyses were performed using Matlab (The Mathworks, Natick).