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Human Neural Stem Cells Alleviate Alzheimer-like Pathology in a Mouse Model

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Human Neural Stem Cells Alleviate Alzheimer-like Pathology in a Mouse Model

Il-Shin Lee et al. Mol Neurodegener.

Abstract

Background: Alzheimer's disease (AD) is an inexorable neurodegenerative disease that commonly occurs in the elderly. The cognitive impairment caused by AD is associated with abnormal accumulation of amyloid-β (Aβ) and hyperphosphorylated tau, which are accompanied by inflammation. Neural stem cells (NSCs) are self-renewing, multipotential cells that differentiate into distinct neural cells. When transplanted into a diseased brain, NSCs repair and replace injured tissues after migration toward and engraftment within lesions. We investigated the therapeutic effects in an AD mouse model of human NSCs (hNSCs) that derived from an aborted human fetal telencephalon at 13 weeks of gestation. Cells were transplanted into the cerebral lateral ventricles of neuron-specific enolase promoter-controlled APPsw-expressing (NSE/APPsw) transgenic mice at 13 months of age.

Results: Implanted cells extensively migrated and engrafted, and some differentiated into neuronal and glial cells, although most hNSCs remained immature. The hNSC transplantation improved spatial memory in these mice, which also showed decreased tau phosphorylation and Aβ42 levels and attenuated microgliosis and astrogliosis. The hNSC transplantation reduced tau phosphorylation via Trk-dependent Akt/GSK3β signaling, down-regulated Aβ production through an Akt/GSK3β signaling-mediated decrease in BACE1, and decreased expression of inflammatory mediators through deactivation of microglia that was mediated by cell-to-cell contact, secretion of anti-inflammatory factors generated from hNSCs, or both. The hNSC transplantation also facilitated synaptic plasticity and anti-apoptotic function via trophic supplies. Furthermore, the safety and feasibility of hNSC transplantation are supported.

Conclusions: These findings demonstrate the hNSC transplantation modulates diverse AD pathologies and rescue impaired memory via multiple mechanisms in an AD model. Thus, our data provide tangible preclinical evidence that human NSC transplantation could be a safe and versatile approach for treating AD patients.

Figures

Fig. 1
Fig. 1
Transplantation of hNSCs into NSE/APPsw transgenic mice. a Schematic drawing of a representative coronal brain section, illustrating the wide distribution of hNuMA+ grafted cells (red dots). Asterisks indicate the transplantation sites. Lowercase letters represent the site captured in the respective photomicrographs shown in (b–j). be GFP-expressing hNSCs (b and e) or hNuMA+ cells (c and d) are largely engrafted from the LV or third ventricle (3 V) to the SVZ, and from dorsal 3 V (D3V) to the thalamus and hypothalamus. f Some hnestin+ cells are detected in the thalamus. gj The grafted cells migrate along the white matter tracts: BrdU+ cells in the corpus callosum (cc; g); hNuC+ cells in the cingulum (h); and hNuMA+ cells in the external capsule (i), and toward the cortex (j). kn Most hNuMA+ cells express hnestin in the cortex (k) and external capsules (l), whereas a few hNuMA+ cells are co-localized with either TUJ1+ or Olig2+ cells in the SVZ (m) and thalamus (n), respectively. Arrows in N indicate hNuMA/Olig2 double-labeled cells. oq A few hNuMA+ cells express DCX (o) or PDGFR-α (p) in the white matter tracts, and GFAP (q) in the cortex. Arrowheads in Q indicate co-localization of hNuMA and GFAP, representing z-stack confocal images (three small images in the right panel of q). r Differentiation patterns of grafted hNSCs in transgenic mice (n = 4, where n is the number of mice). In km, o, and p, z-stack images are built and compiled to maximal intensity projections. Scale bars, 50 μm
Fig. 2
Fig. 2
Behavioral phenotypes of hNSC- or vehicle-injected NSE/APPsw transgenic mice and vehicle-injected wild-type mice. a Ambulatory and stereotypic activity of the three groups in a 30 min open field test (APP-NSC, n = 24; APP-Veh, n = 20; and WT-Veh, n = 24). b Ambulatory and stereotypic locomotor activity over 22 h (APP-NSC, n = 12; APP-Veh, n = 8; and WT-Veh, n = 14). c Latency to fall in the accelerating rotarod task (APP-NSC, n = 26; APP-Veh, n = 20; and WT-Veh, n = 23). d and e Results from the water maze test 6 weeks after hNSC or vehicle injection. The latency to reach the hidden platform (escape latency) during the learning phase was recorded d. Target quadrant occupancy and platform crossing during the probe trial were measured in the three groups (APP-NSC, n = 21; APP-Veh, n = 19; and WT-Veh, n = 19; e). f and g Results from the water maze test 12 weeks post-transplantation. Escape latency during the learning phase in the three groups (f). Target quadrant occupancy and platform crossing during the probe trial (APP-NSC, n = 12; APP-Veh, n = 10; and WT-Veh, n = 10; g). The number of mice (n) in each group is indicated. All data represent mean ± SEM. Error bars indicate ± SEM. *p < 0.05, **p < 0.01
Fig. 3
Fig. 3
hNSC transplantation reduces tau phosphorylation via Trk-induced Akt/GSK3β signaling in the NSE/APPsw transgenic mouse brain. ac Photomicrographs of AT180 immunostaining in the brains of vehicle-injected wild-type (WT-Veh; a), vehicle-injected transgenic (APP-Veh; b), and hNSC-injected transgenic (APP-NSC; c) mice. dg AT180 immunoreactivity in the stratum radiatum of the hippocampal CA1 region (HIPP; inside the dotted rectangle in d and e) and the posterior parietal cortex (CTX; f and g) in hNSC- and vehicle-injected transgenic mice. Or, oriens layer; Pyr, pyramidal cell layer; Rad, stratum radiatum. h Immunohistochemical image analyses of AT180 immunoreactivity comparing hNSC graft (NSC, n = 4) and vehicle injection (Veh, n = 4) in the transgenic mice. i Levels of phosphorylated tau (AT180, AT8, pTau, and PHF13) on western blot image analyses in brains of vehicle-injected (n = 5) and hNSC-injected (n = 6) transgenic mice. j Levels of neurotrophins (BDNF, NTF3, NGF, and NTF4) on western blot image analyses in brains of vehicle-injected (n = 3) and hNSC-injected (n = 4) transgenic mice. k Levels of phosphorylated TrkA/B, Akt and GSK3β using western blot analysis on brains of vehicle-injected (n = 3) and hNSC-injected (n = 3) transgenic mice. l and mRelative levels of phosphorylated tau (l) and kinases related to Trk-dependent Akt/GSK3β signaling (m) using western blot image analyses from Aβ42-treated PC12 cells in the presence of hNSC-derived CM (NSC, n = 3) and fibroblast-derived CM (Fib, n = 3). Scale bars, 100 μm. The number of mice (n) in H-K is indicated. The number of experiments (n) in L and M is indicated. All data represent mean ± SEM. Error bars indicate ± SEM. *p < 0.05, **p < 0.01
Fig. 4
Fig. 4
hNSC transplantation decreases brain intracellular soluble Aβ42 levels by regulating BACE1 in NSE/APPsw transgenic mice. a Photomicrographs of Aβ plaque (6E10) staining in hNSC-injected (NSC) and vehicle-injected (Veh) transgenic mice. Scale bar, 1 mm. b The number of Aβ plaques per unit area (left) and the percent area of the Aβ plaque load (right) between hNSC graft (NSC) and vehicle injection (Veh; n = 3 per group). cl Representative images of Aβ42 immunostaining in the brains of vehicle-injected wild-type mice (WT-Veh; c), and of vehicle-injected (APP-Veh; d) and hNSC-injected (APP-NSC; e) transgenic mice. Aβ42 immunostaining of each group in the pyramidal cell layer of the hippocampal CA1 region (HIPP; fh) and the posterior parietal cortex (CTX; ik). The relative levels of Aβ42 immunostaining in vehicle-injected (Veh) and hNSC-injected (NSC) transgenic mice (n = 4 per group; l). Scale bar, 100 μm (e and h) and 50 μm (k). m Levels of detergent-soluble (Sol) Aβ40/42 and detergent-insoluble (Insol) Aβ40/42 in the brains of hNSC-injected (NSC) and vehicle-injected (Veh) transgenic mice using ELISA kits (n = 4 per group). n and o Relative levels of BACE1 (NSC, n = 6; Veh, n = 5; n) and APP CTF-β/CTF-α (NSC, n = 4; Veh, n = 3; o) using western blot analyses in brains of hNSC-injected (NSC) and vehicle-injected (Veh) transgenic mice. p APPsw-expressing SK-N-MC cells treated with hNSC-derived (NSC) and fibroblast-derived (Fib) CM (n = 3 per group, where n is the number of experiments). Western blot image analysis of Aβ in the culture media of these cells, and BACE1, APP CTF-β/CTF-α, and phosphorylated Akt and GSK3β in cells of both groups. The number of mice (n) in (b) and (l-o) is indicated. All data represent mean ± SEM. Error bars indicate ± SEM. *p < 0.05
Fig. 5
Fig. 5
hNSC transplantation suppresses astrogliosis and microgliosis in the NSE/APPsw transgenic mouse brain. ac Representative images of GFAP+ astrogliosis in the hippocampus of vehicle-injected wild-type mice (WT-Veh; a), and of vehicle-injected (APP-Veh; b) and hNSC-injected (APP-NSC; c) transgenic mice, at 7 weeks post-transplantation. Scale bar, 100 μm (c). df Representative images of Iba1+ microgliosis in the brains of vehicle-injected wild-type mice (d), and of vehicle-injected (e) and hNSC-injected (f) transgenic mice, at 7 weeks post-transplantation. Scale bar, 100 μm (f). GaIb The hNSC transplantation substantially decreases the numbers of Iba1+ cells in the cortex (Gb) and hippocampus (Hb and Ib) of transgenic mice compared with those in their vehicle-injected cohorts (Ga, Ha, and Ia). Scale bar, 100 μm (Ib). gcl, granular cell layer in the dentate gyrus of the hippocampus. JaKb The numbers of F4/80+ (Ja and Jb) and CD11b+ microglia (Ka and Kb) appear to decrease in the hippocampus of hNSC-injected transgenic mice compared with those in their vehicle-injected cohorts. Scale bar, 50 μm (Jb and Kb). l Relative levels of the optical density of GFAP immunoreactivity in the hippocampus of hNSC-injected (NSC, n = 3) and vehicle-injected (Veh, n = 3) transgenic mice. m Relative levels of Iba1 immunoreactivity, quantified as a percentage of the area occupied using ImageJ, in the cortex (CTX) and hippocampus (HIPP) of hNSC- and vehicle-injected transgenic mice (n = 3 per group). The number of mice (n) in L and M is indicated. All data represent mean ± SEM. Error bars indicate ± SEM. *p < 0.05
Fig. 6
Fig. 6
hNSCs reduce the expression of pro-inflammatory mediators. a Comparison of the expression of Il1b, Il6, Tnfa, and iNOS mRNA levels in the brains of hNSC-injected (NSC, n = 7) and vehicle-injected (Veh, n = 6) NSE/APPsw transgenic mice. b and d The expression of Il1b, Il6, and Tnfa in LPS-stimulated BV2 microglial cells co-cultured with hNSCs (BV2 + NSC) compared with that in single cultures of LPS-stimulated BV2 cells (BV2) on Transwell permeable supports (n = 3 per group; b). The hNSCs co-cultured with BV2 cells (NSC + BV2) express TGFB1, IL4, and IL13 (d). c and e In mixed cultures, the change of Il1b, Il6, Tnfa, and iNOS expression in LPS-stimulated BV2 cells (BV2 + NSC) co-cultured with hNSCs compared with that in single cultures of LPS-stimulated BV2 cells (BV2; n = 3 per group; c). The hNSCs co-cultured with BV2 cells (NSC + BV2) express TGFB1, IL4, IL13, CX3CL1, CD47, and CD200 (e). f The relative expression of mRNA for Il1b, Il6, Tnfa, and iNOS in Tgfbr2-, Sirpa-, and Cd200r1-knockdown BV2 cells co-cultured with hNSCs compared with that in non-functioning negative siRNA (siNega)-transfected BV2 cells in mixed co-culture in the presence of LPS (n = 3 per group). The number of mice (n) in A is indicated. The number of experiments (n) in (b, c), and (f) is indicated. All data represent mean ± SEM. Error bars indicate ± SEM. *p < 0.05, **p < 0.01
Fig. 7
Fig. 7
hNSC transplantation increases synaptic density and decreases apoptosis in the NSE/APPsw transgenic mouse brain. a-h Photomicrographs of SVP and PSD95 immunostaining (green and red puncta, respectively) in the posterior parietal cortex (a-d) and striatum radiatum of the hippocampal CA1 region (e-f) in vehicle-injected (Veh; a, c, e, and g) and hNSC-injected (NSC; b, d, f, and h) transgenic mice at 7 weeks post-transplantation. Scale bar, 25 μm (h). i and j Results of image analyses using confocal microscopy for synaptic density (SVP and PSD95) in the cortex (CTX) and hippocampus (HIPP) comparing hNSC- and vehicle-injected transgenic mice (n = 4 per group; I, n = 3 per group; j). k and l Western blot analysis of SVP (k) and PSD95 (l) in both groups (n = 3 per group). m and n Representative images of TUNEL+ cells in the posterior parietal cortex of vehicle-injected (m) and hNSC- injected (n) transgenic mice at 7 weeks post-transplantation. Scale bar, 100 μm (h). o Quantification of the number of TUNEL+ cells of the posterior parietal cortex in both groups (n = 3 per group). p The relative level of active caspase-3 in the whole brains of hNSC- and vehicle-injected mice on western blot analysis (n = 4 per group). The number of mice (n) in (i-l, o, and p) is indicated. All data represent mean ± SEM. Error bars indicate ± SEM. *p < 0.05

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