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Fig. 3. Cortical neuron differentiation of PS1-E120K- and control-derived iPSCs. (A) Schematic diagram showing our stepwise cortical neuronal differentiation protocol. Control and PS1-E120K patient-derived iPSC lines were differentiated into neural precursor cells (NPC) using the Dual SMAD inhibition method. Afterwards, NPCs were treated with neurotrophic factors including BDNF, GDNF and NT3 to induce cortical neurons. (B) Immunofluorescence analysis of control and PS1-E120K iPSC-derived NPCs, showing the expression of NPC markers, such as Nestin (green), SOX2 (red) and Musashi (green) with DAPI (blue). (C) Immunofluorescence analysis of control and AD-iPSC-derived cortical neurons (TUJ1 [red] and Map2 [green]). Cholinergic neurons (ChAT [red]) and cortical neurons (TBR1 [red] and CTIP2 [green]) at 10 weeks after differentiation were shown. (D) Ratios of each mature neuronal cell type relative to DAPI staining. Scale bar: 50 µm.. From: iPSC Modeling of Presenilin1 Mutation in Alzheimer's Disease with Cerebellar Ataxia.
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Fig. 1. Pedigree of the patient's family and brain MR images of the cousin of the patient and MR and Pittsburgh compound-B (PiB) positron emission tomography (PET) images of the patient. (A) Pedigree of the patient. I-2: grandmother of the patient, dementia; III-2: cousin of the patient, late onset cerebellar ataxia; III-10: the patient, dementia, spastic paraparesis, cerebellar ataxia. (B) Fluid attenuated inversion recovery (FLAIR) MR images of the cousin show severe ponto-medullo-cerebellar atrophy without atrophy in the medial temporal lobes. (C) T2-weighted axial MR images of the patient (III-10 in the pedigree) showing atrophy bilaterally of the medial temporal lobes and prominent volume loss in the cerebellum with enlargement of the fourth ventricle. (D) Increased amyloid deposition in the bilateral striatum, frontal and posterior cingulate cortices, and precuneus, but not in the cerebellum.. From: iPSC Modeling of Presenilin1 Mutation in Alzheimer's Disease with Cerebellar Ataxia.
Fig. 5. Elevated p-tau levels in the PS1-E120K iPSC-derived neurons. (A~C) Western blot analysis showing a significant increase of phosphorylated tau (p-tau) including AT8, AT180 and p-Tau (Ser400, Thr403/Ser404) and total tau (t-tau, Tau5) proteins in the PS1-E120K iPSC-derived neurons compared with the control group. (D~F) Quantification of the western blots on the expression of AT8 (D), AT180 (E) and p-Tau (F) showing a significant increase of the expression of each in the PS1-E120K iPSC-derived neurons. (G) Immunocytochemical analysis showing the expression of AT8 (red) and MAP2 (green), counter-stained with DAPI (blue), in the PS1-E120K iPSC-derived neurons at 10 weeks of neuronal differentiation. Note that PS1-E120K iPSC-derived neurons exhibit a dramatic increase of phosphorylated-tau (AT8) both in the soma (arrow) and neurites (arrowhead) compared with the control group. Scale bar: 10 µm. (H~J) Quantification of the immunocytochemical analysis on the expression and localization of AT8 proteins with respect to MAP2-positive neurons. MAP2 expression was normalized against DAPI (G), whereas AT8 expression was normalized against MAP2 expression (I, J). Note that significant increase of AT8 expression both in the soma (I) and neurite (J) regions in the PS1-E120K iPSC-derived neurons. Representative western blot and ICC images were obtained from three independent experiments.. From: iPSC Modeling of Presenilin1 Mutation in Alzheimer's Disease with Cerebellar Ataxia.
Fig. 2. Generation of iPSCs from an AD patient harboring a PSEN1 (E120K) mutation, and an eldely normal subject. (A) Established iPSC lines from both control and PS1-E120K patient showing the expression of pluripotent stem cell markers, such as OCT4 (red), SOX2 (green), SSEA4 (red) and TRA-1-81 (red). (B) Reverse transcription PCR (RT-PCR) showing the expression of pluripotency markers (OCT4, SOX2, NANOG, SSEA4 AND TRA-1-81) in both iPSC lines. (C) Genomic DNA sequences showing the presence of the heterozygous E120K mutation (GAA to AAA) in the PSEN1 gene of the PS1-E120K-iPSC line. (D) Immunofluorescence analysis showing the potential of iPSC lines to form three germ layers, including ectoderm (type III β-tubulin [TUJ1], green), mesoderm (smooth muscle actin [SMA], green), and endoderm (α-fetoprotein [AFP], red). Scale bar: 100 µm. (E) Karyotype analysis of the control and PS1-E120K iPSC lines. (F) Reverse-transcription PCR analysis showing the absence of integration of the Sendai virus vectors. (G) PCR analysis showing no contamination by mycoplasma.. From: iPSC Modeling of Presenilin1 Mutation in Alzheimer's Disease with Cerebellar Ataxia.
Fig. 4. Increase of Aβ deposits in the PS1-E120K iPSC-derived neurons, examined at 6 and 10 weeks after neuronal differentiation. ELISA detection of Aβ42 (A) and Aβ40 (B) secreted from the PS1-E120K iPSC-derived neurons into the medium (extracellular), which was measured at 48 hours after the last medium change. Levels of Aβ42 (A) and the ratio of Aβ42/Aβ40 (C) showed a dramatic increase in the PS1-E120K iPSC-derived neurons at both 6 and 10 weeks of neuronal differentiation. Intracellular Aβ42 (D) and Aβ40 (E) levels were measured in a total of 1 µg proteins from 10 week-differentiated neurons. Levels of the intracellular Aβ42 (D) and the ratio of Aβ42/Aβ40 (F) showed a significant increase in the PS1-E120K iPSC-derived neurons. (G, H) Detection of Aβ deposits using an antibody against 6E10 (shown in green) co-stained with MAP2 (red) (G) or using an antibody against 4G8 (shown in red) co-stained with and TUJ1 (green) (H) and DAPI (blue) at 10 weeks of neuronal differentiation. The bottom panels show the z-stack images of the 6E10 and 4G8-positive Aβ deposits (arrows) in the PS1-E120K iPSC-derived neurons. Scale bar: 10 µm. (I, J) Western blot analysis showing a significant increase of total APP expression levels in the PS1-E120K iPSC-derived neurons compared to the control. Representative ELISA, western blot and ICC images were obtained from three independent experiments.. From: iPSC Modeling of Presenilin1 Mutation in Alzheimer's Disease with Cerebellar Ataxia.
Fig. 6. Impaired balance of mitochondria fission and fusion along with defective autophagy in the PS1-E120K iPSC-derived neurons. (A) Representative western blot image from three independent experiments. (B~I) Quantification of marker proteins associated with mitochondrial fission (Drp1, p-Drp1 and Fis1) and fusion (OPA1, Mfn1and Mfn2). Note that fission-related proteins were increased, whereas fusion-related proteins were decreased in the PS1-E120K cells compared with the control cells. Mitophagy-related protein PINK1 and PARKIN showed dramatic increases in the PS1-E120K cells compared with the control cells. (J~K) Q-PCR analysis revealing the mRNA expression of fission and fusion-related genes in 10 weeks differentiated neurons from iPSCs. (L) Representative western blot image from three independent experiments. (M~Q) Quantification of the expression of ubiquitination (Ub) and autophagy-related proteins (p62, Beclin1, LC3B, LAMP2). Note that levels of Ub and LC3B were significantly increased, whereas the LAMP2 signal was significantly decreased in the PS1-E120K iPSC-derived neurons. (R) Immunocytochemical staining showing the expression of LC3b (red) and DAPI (blue) in both iPSC-derived neurons at 10 weeks of neuronal differentiation. Note that PS1-E120K iPSC-derived neurons exhibit a dramatic increase of LC3b in the periphery of soma area (arrow), compared with the control group. (S) Autophagy flux assay showing a significant increase in LC3B-II expression after treatment with 10 uM chloroquine (CQ) in the PS1-E120K iPSC-derived NPC compared with the control. (T~P) CytoID (green) stained with DAPI (blue) after CQ treatment showing a dramatic increase in number of autophagic vesicles in the PS1-E120K iPSC-derived NPC compared with the control. Representative western blot and ICC images were obtained from three independent experiments. Scale bar: 10 µm.. From: iPSC Modeling of Presenilin1 Mutation in Alzheimer's Disease with Cerebellar Ataxia.
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