Synapsin

Western blotting was used to detect protein expression levels. Cell or tissue samples were lysed with RIPA containing the protease inhibitor PMSF and cocktail, and then centrifuged at 12,000g for 10 min. Supernatants were boiled in SDS loading buffer and protein separated using SDS–PAGE. Proteins were then transferred to nitrocellulose membranes. Five percent skim milk powder was used to block, and proteins of interest were labeled overnight with primary antibody at 4 °C. After three times wash with washing buffer, the second antibody was incubated at room temperature for 1 h followed by another three washes, protein expression levels were visualized by using the Odyssey system. The primary antibodies used for Western blotting include PSD95 (1:1000; Cell Signaling Technology, USA), NR1 (1:1000; Millipore, USA), NR2B (1:1000; Millipore, USA), NR2A (1:1000; abcam, UK), GluA1 (1:1000; Millipore, USA), GluA2 (1:1000; Millipore, USA), Synapsin 1(1:1000; Millipore, USA), Synaptophysin (1:1000, sigma), actin (1:1000; Abcam), LaminB1(1:1000; Abcam), NF-κB p65(1:500; Cell Signaling Technology, USA).
The nuclear and cytoplasmic proteins preparation kit (P1200, Pulilai) was used to separate the nuclear and cytoplasmic components according to the manufacturer’s procedures for subsequent experiments.

Cells were fixed with 4 % paraformaldehyde and stained in PBS containing 0.05 % Triton-X100 and 5 % FBS. Cells were incubated with primary antibodies PKM2 (Origene, 1:500, TA347018), p-PKM2 (Eubio, 1:500, #11456-2), H3T11-P (Abcam, 1:100, ab5168), cleaved caspase 3 (Cell Signaling, 1:1000, #9664), β-III-tubulin (BioLegend, 1:1000, #802001), Synapsin (Merck, 1:750, #574778), PSD95 (ThermoFisher, 1:300, MA1046), NeuN (CellSignaling, 1:100, #24307T) at 4°C overnight and incubated with secondary antibodies for two hours at room temperature, followed by 10 minutes of DNA staining with DAPI (ThermoFisher, 300nM, D21490). Images were taken with the Leica DMi8 microscope and analyzed in FIJI. Nuclear expression of p-PKM2 was measured as IntDen in region-of-interests (ROIs) set based on DAPI; total neuronal expression was measured as IntDen in ROIs based on MAP2. For assessment of synapse-like structures, neurons were transduced with Synapsin-RFP (Addgene #22909). Neuronal morphology was assessed based on β-tubulin using the Neuroanatomy SNT plugin in ImageJ, measuring neurite length from the cell body to the furthest connection, and the complexity of the branching on these neurites.

The antibodies used in this study were NANOG (R&D Systems, AF1997, 1:500), OCT4 (STEMCELL Technologies, 60093, 1:1000), SOX1 (STEMCELL Technologies, 60095, 1:1000), SOX17 (R&D Systems, AF1924, 1:500), DESMIN (Invitrogen, PA5–16705, 1:500), MAP2 (Proteintech, 17490–1-AP, 1:500), TUJ1 (Neuromics, CH23005, 1:500), GFAP (STEMCELL Technologies, 60128, 1:500), TBR1 (Abcam, ab31940, 1:500), FOXG1 (Abcam, ab18259, 1:500), SYNAPSIN (EMD Millipore, 5747777, 1:500), VGLUT1 (Synaptic Systems, 135311, 1:500), GAD 65/67 (Sigma-Aldrich, G5163, 1:500), PGRN (R&D Systems, AF2420, 1:1000) (for immunofluorescence); PGRN (Sigma-Aldrich, HPA008763, 1:1000), CTSD (R&D Systems, AF1014, 1:1000), ACTIN (Novus Biologicals, NB600–532, 1:10,000) (for conventional western blot); V5 (Abcam, ab27671, 1:500), ACTIN (Novus Biologicals, NB600–532, 1:10,000) (for automated WES western blot) (Table S1).

vGluT1 (Synaptic systems, #135 303), pan-axonal neurofilament marker (SMI312, Sigma-Aldrich, # NE1022), Synapsin 1 (EMD Millipore, #1543), phospho (Ser62, Ser67) Synapsin 1 (EMD Millipore, #AB9848), alpha-bungarotoxin (Sigma-Aldrich, #B137), alpha-bungarotoxin-594 (Thermo Fisher #B-13423), FM1-43 (Thermo Fisher #T35356), Fluo4 (Thermo Fisher #F14217), tetrodotoxin (Tocris #1078), bicuculline (Tocris #0130), RuBi-nicotine (Tocris, #3855), D-AP-5 (Tocris #0106), and CNQX (Tocris #1045).