Fig 2: Recovery of deficient astrocyte signaling mitigates network hypersynchrony and behavior hyperactivity in early AD(A and B) BOLD FC matrices (A) and graphs quantifying FC (Z scores ± SEMs) (B) between the anterior and posterior cingulate cortex (indicated in matrices by asterisk) upon modulation of astrocyte calcium activity in AppNL control and AppNL-F mice expressing mCherry or DREADDs (GFAP promoter) (N = 8/group). ***p < 0.001, ****p < 0.0001, 1-way ANOVA with Sidak correction.(C) Seizure susceptibility (seizure score ± SEM) to a subconvulsive dose of PTZ (30 mg/kg) for AppNL control (N = 15/group) and AppNL-F mice (N = 16/group) expressing mCherry or DREADDs. *p < 0.05, **p < 0.01, ****p < 0.0001, Kruskal-Wallis test with Dunn’s correction.(D and E) Behavior analyses of day/night activity of AppNL control and AppNL-F mice expressing mCherry (AppNL control mCherry N = 15, AppNL-F mCherry N = 16) or DREADDs (AppNL control DREADDs N = 18, AppNL-F DREADDs N = 19) over 24 h (counts of photobeam crosses ± SEMs). ****p < 0.0001, 1-way ANOVA with Sidak correction.

Fig 3: Astrocytes show decreased calcium signaling at early stages of amyloid pathology(A) Representative ?F/F0 calcium traces of astrocytes in the cingulate cortex of 3-month-old AppNL control and AppNL-F mice expressing the calcium reporter GCaMP6f in addition to either control AAV (mCherry) or DREADDs in astrocytes (GFAP promoter). Scale bar, 20 µm.(B) Heatmaps show astrocyte calcium activity (?F/F0 exceeding 2*SD baseline, y axis) over time (800 s, x axis) in AppNL control mice expressing mCherry (N = 5 mice, 223 cells) or DREADDs (N = 4 mice, 263 cells), and AppNL-F mice expressing mCherry (N = 4 mice, 291 cells) or DREADDs (N = 4 mice, 236 cells).(C–E) Graphs show percentage of active cells (X2(3, N = 1014) = 119, p < 0.0001), frequency (number of peaks per minute ± SEM), and signal amplitude (?F/F0±SEM). **p < 0.01, ***p < 0.001, ****p < 0.0001, Kruskal-Wallis test with Dunn’s correction.(F and G) Representative images and quantification of astrocytic IP3R2 (IP3R2+GFAP density/100 µm2 ± SEM) and GFAP (percentage area GFAP ± SEM) of immunofluorescent staining in mouse (N = 6 AppNL controls, N = 6 AppNL-F mice, 5 slices per mouse) and human AD cortical tissue (N = 3 controls, N = 4 AD patients, 4 slices per sample). Scale bar, 20 µm. *p < 0.05, **p < 0.01, 2-sample t test.

Fig 4: Transplanted astrocytes survive and develop processes in neonate and adult NSG mice but spread out only in neonate NSG mice, without evidence for disruption of blood brain barrier or xenotransplanted astrocyte migration outside of the brain.200,000 NHAs were injected into neonate brain at postnatal day 1 (PND1) or 50,000 NHAs into each striatum of adult (5–6 weeks) NSG mice. At 4 weeks (A), 8 weeks (B) and 12 weeks (C) post xenotransplantation in neonates, NHA expression is shown by human GFAP (huGFAP, red) with DAPI (blue) staining. Data is representative of 2 mice per time point. Representative images of neonatal prefrontal cortex (PFC, D), thalamus (TH, E), and hippocampus (HPC, F) immuno-stained for human GFAP (huGFAP, red) or human Nuclei (huNuclei, red) and DAPI (blue) at 12 weeks. Representative images of adult striatum immune-stained for human GFAP (G, red) or huNuclei (H, red) and DAPI (blue) at 4 weeks 5 days post xenotransplantation. Inset: higher magnification of square. (I) Representative image of negative staining control for rabbit or mouse IgG primary and corresponding secondary of adult non HIV-infected mouse striatum with DAPI (blue). Data is representative of 2 mice per time point. (J-K) Evaluation of cerebrovascular permeability in the striatum following intracranial implantation of astrocytes by microinjection in adult animals. Animals were transcardially perfused with a fluorescence tracer (FITC) conjugated to 10kDa dextrans in the following groups of animals: naïve, 4hrs after exposure to bacterial LPS (i.p 6mg/kg), 8hrs or 5 days following microinjections of transplanted exogenous astrocytes. (J) Representative confocal images of striatal areas at the injection coordinates (relative to Bregma +0.5 A/P, +1.8 M/L, -3.0 D/V). (K) Image analysis measurements of vascular permeability using pixel intensity (as a function of area) in the parenchymal areas of the injection site. Results are shown as the average ±SEM; multiple group comparisons were determined by one-way ANOVA with post-hoc Dunnett's test * = p<0.001, n = 3–4 per condition, NS, denotes no significance. (L) Adult mice were injected with NHAs without huPBMC reconstitution and real-time PCR hu cytoB mtDNA products extracted from the blood within 5 hours of injection were analyzed by electrophoresis. MS: blood from non-NHA injected animal, MS + NHA: blood from animal microinjected with NHAs, MS + huPBMCs: animals reconstituted with huPBMCs, NTC: no template control. (M) Adult mice were injected with NHAs without huPBMC reconstitution and real-time PCR DNA products (human GFAP, human GAPDH, mouse GFAP and mouse GAPDH) were analyzed by electrophoresis at day 5 post xenotransplantation. n = 3. MS: spleen from 1 non-NHA injected animal, HEK: human HEK293T cells. Scale bars: A-J, 50µm; G insert, 10µm.

Fig 5: Generation of hiPSC-astrocytes. (a) Schematic of hiPSC differentiation and RFP viral transduction. (b) Expression of RFP (left), ALDH1L1 (middle), GFAP and S100B (right) in astrocytes before engraftment (here shown for D20–D22). Scale = 200 µm [Color figure can be viewed at wileyonlinelibrary.com]