Fig 2: The differential and brain region-dependent effects of long-term peripheral myeloid cell engraftment on astrocytic and neuronal properties. a Representative whole brain images of GFP (green) and common astrocyte marker glial fibrillary acidic protein (GFAP, red) cell deposition in monocyte-engrafted mice. b–c Representative immunofluorescence images of different astrocyte subtypes stained for GFAP (red) and S100ß (blue) in the in the cortex (B) and hippocampus (C) of control (WT CON), irradiated (GFP-BM CON), and monocyte-engrafted (GFP-BM REPOP) mice. GFP (green) stains BM-derived cells. d–g Quantification of S100ß+ and GFAP+ cell number in the cortex (D, F) and cell number in the hippocampus (e, g). h Quantification of GFAP staining intensity in the hippocampus. (i) Quantification of NeuN+ cell number in the whole cortex. (j) Representative immunofluorescence images stained for neuronal marker NeuN (red) and GFP (green, BM-derived cells) in the cortex and hippocampus. (k) Representative immunofluorescence images of microtubule associated protein 2 (MAP2, red) and GFP (green, BM-derived cells) in the cortex and hippocampus. (l) Representative immunofluorescence brain images of synaptic marker PSD95 (postsynaptic density protein 95, red) and GFP (green, BM-derived cells) in the cortex and hippocampus. L’ provides representative images of spots modeling using Imaris spots module software overlaid on PSD95+ staining. (m) Representative immunofluorescence brain images of synaptic marker SV2A (synaptic vesicle glycoprotein 2A, red) and GFP (green, BM-derived cells) in the cortex. (n–s) Quantification of the integrated density of MAP2 staining in the cortex (n) and hippocampus (o), total number of PSD95+ spots per field of view in the cortex (p) and hippocampus (q), and total number of SV2A+ spots per field of view in the cortex (r) and hippocampus (s). Data are represented as mean ± SEM (n = 4–9). *p < 0.05, **p < 0.01. ***p < 0.001. CTX cortex, HC hippocampus, GFP green fluorescent protein, IBA1 ionized calcium adapter molecule 1. Scale bar ~ 150 µm (B–C, j–left panel); ~ 200 µm (j–right panel); ~40 µm (k); ~ 30 µm (l, m) ~ 15 µm (L’)

Fig 3: Sequence alignment of the fourth luminal domains from human SV2A, SV2B and SV2C. Red boxes mark conserved residues and white boxes weakly conserved residues. Residues of SV2C that interact with both HcA1 and HcA2 are marked with a black arrowhead or a blue star, while residues that only interact with either HcA1 or HcA2 are marked with green or cyan arrowheads, respectively. Conserved glycosylation sites are marked with stars, with the glycosylation involved in interaction in vivo [10,16] shown in dark blue. Secondary structure elements are from SV2C in 6ES1. Observe that in 6ES1, only residues 474–567 of SV2C are seen.

Fig 4: Immunolabelings of hiPSC-derived Neurons at day 15 after thawing: (A–E) TUJ1 (green) and (A) SNAP25 (red); (B) SV2A (red); (C) VAMP2 (red); (D) GM1 (red) and (E) GT1b (red). Nuclei are stained in blue (DAPI). Scale bar: 25 µm.

Fig 5: Laser confocal microphotographs (A–C) of coronal sections through the CA1 field of the hippocampus. (A–C) Z stacks of 9 slices (0.5 μm thick) illustrating the distribution of SV2A (A), VGLUT1 (B), and GAD 65–67 (C) immunoreactive synaptic vesicles in the control and tbl mice. Graphs showing the number of synaptic vesicles identified by SV2A immunoreactivity (A), the synaptic glutamatergic vesicles identified by VGLUT1 expression (B), and the GABAergic endings identified with the GAD 65–67 antibody (C). The tbl CA1 possesses significantly fewer vesicles expressing SV2A (A, ***p = 4.0179E–14) and VGLUT1 (B, ***p = 1.68366E–12) as presynaptic markers, while no difference was found in GAD 65-67 expression (C, p = 0.3280439). Bar = 100 μm (A–C).