Journal: Cell research

Article Title: An App knock-in rat model for Alzheimer's disease exhibiting Aβ and tau pathologies, neuronal death and cognitive impairments.

doi: 10.1038/s41422-021-00582-x

Figure Lengend Snippet: Fig. 1 Aβ pathology in AppNL-G-F rat brains. a Gene-editing strategy to generate knock-in rat with three familial App mutations, using CRISPR- Cas9 technologies. Upper: App wild-type allele, targeting vector and targeted allele. The donor molecule comprises a 5′ homologous arm upstream of exon 16, and a 3′ homologous arm downstream of exon 17. Lower: Diagram showing all the important sites on the APP sequence. Green arrows: cleavage sites by 3 secretases. *: key mutation sites identified by human genetics and 3 humanized amino acids G676R, F681Y, and R684H. b, c Expression of full-length amyloid precursor protein (FL-APP). FL-APP containing humanized Aβ sequence was detected by Western blotting using antibody 6E10 (specific for human Aβ sequence) in 6-month-old wild type (WT), AppNL-G-F/NL-G-F (Homo) and AppNL-G-F/WT

Article Snippet: Sections were permeabilized and blocked in PBS containing 0.2% Triton X-100 and 10% normal goat serum at room temperature for 2 h. Sections were incubated overnight at 4 °C with the following primary antibodies: anti-Aβ antibody (1:1000, Cell Signaling Technology, 2454), 6E10 (1:1000, Covance, 39320) anti-Aβ oligomer antibody (OMAB) (1:1000, Agrisera, AS10932), anti-RIPK1 (1:200, BD Biosciences, 610459), anti-RIPK3 (1:500, Stata Crus, 374639), anti-MLKL (1:200, EnoGene, E11-11361C), antipMLKL (1:200, Abcam, ab196436), PSD95 (1:500, NeuroMab, 73-028), Synaptophysin1 (1:400, SYSY, 101002), MC1 (1:20; a gift from P. Davies, Albert Einstein College of Medicine, New York, New York, USA), anti-GFAP (1:1000, Millipore, MAB3402), anti-Iba1 (1:1000, Wako, 019-19741).

Techniques: Knock-In, CRISPR, Plasmid Preparation, Sequencing, Mutagenesis, Expressing, Western Blot

Journal: Cell research

Article Title: An App knock-in rat model for Alzheimer's disease exhibiting Aβ and tau pathologies, neuronal death and cognitive impairments.

doi: 10.1038/s41422-021-00582-x

Figure Lengend Snippet: Fig. 3 Enhanced gliosis in AppNL-G-F rat brains. a–d Microgliosis and astrocytosis in AppNL-G-F rats. Microglia marker Iba1 (a, b) or astrocyte marker GFAP (c, d) was detected in cortical lysates from 6-month-old WT and Homo rats using Western blotting. Representative immunoblots and quantification bar graphs are shown in the left (a, c) and right panels (b, d) respectively. n = 4 brains. e, f Representative microphotographs of microgliosis and astrocytosis in the cortex of AppNL-G-F rats. Gliosis were detected by triple staining of frozen sections from 6-month-old homozygous rat, using fluorostyryl benzene (FSB, for Aβ plaque), and GFAP (astrocytes) and Iba1 (microglia) antibodies. Scale, 300 μm. The boxed areas in the upper panels are shown in the panels below at a higher magnification (e). Scale bars, 40 μm. The numbers of astrocytes and microglia per sections are quantified and those of WT were normalized to 1 (f). n = 3 (Homo) or 4 (WT) rats. g Representative microphotographs of microgliosis and astrocytosis in the hippocampus of 6-month-old AppNL-G-F rats. The boxed areas in the upper panels are shown in the panels below at a higher magnification. h The numbers of astrocytes and microglia per sections are quantified and those of WT were normalized to 1. n = 6 rats.

Article Snippet: Sections were permeabilized and blocked in PBS containing 0.2% Triton X-100 and 10% normal goat serum at room temperature for 2 h. Sections were incubated overnight at 4 °C with the following primary antibodies: anti-Aβ antibody (1:1000, Cell Signaling Technology, 2454), 6E10 (1:1000, Covance, 39320) anti-Aβ oligomer antibody (OMAB) (1:1000, Agrisera, AS10932), anti-RIPK1 (1:200, BD Biosciences, 610459), anti-RIPK3 (1:500, Stata Crus, 374639), anti-MLKL (1:200, EnoGene, E11-11361C), antipMLKL (1:200, Abcam, ab196436), PSD95 (1:500, NeuroMab, 73-028), Synaptophysin1 (1:400, SYSY, 101002), MC1 (1:20; a gift from P. Davies, Albert Einstein College of Medicine, New York, New York, USA), anti-GFAP (1:1000, Millipore, MAB3402), anti-Iba1 (1:1000, Wako, 019-19741).

Techniques: Marker, Western Blot, Staining

Journal: Cell research

Article Title: An App knock-in rat model for Alzheimer's disease exhibiting Aβ and tau pathologies, neuronal death and cognitive impairments.

doi: 10.1038/s41422-021-00582-x

Figure Lengend Snippet: Fig. 5 Neuronal loss and brain atrophy in AppNL-G-F rats. a, d Neuronal loss in the hippocampus and cortex of AppNL-G-F rats. Brain sections from 12-month-old WT or homozygous (Homo) rat were stained with an antibody for NeuN, a neuronal marker. Quantitations of NeuN- positive cells in the hippocampus and cortex of stained sections are shown in (b, d) respectively. n = 5 (Homo) or 7 (WT) rats for hippocampus. n = 6 rats for cortex. Scale, 30 μm. e–h Enlarged ventricles in AppNL-G-F rats. Representative MRI images showing the larger ventricles in a 12- month-old Homo rat compared with its WT counterpart (e). The bright regions indicated by arrows are the lateral ventricles. 3D reconstruction of ventricles in WT and Homo rats (f). Volumetric quantitation of the left and right lateral ventricles (g, h). n = 10 rats.

Article Snippet: Sections were permeabilized and blocked in PBS containing 0.2% Triton X-100 and 10% normal goat serum at room temperature for 2 h. Sections were incubated overnight at 4 °C with the following primary antibodies: anti-Aβ antibody (1:1000, Cell Signaling Technology, 2454), 6E10 (1:1000, Covance, 39320) anti-Aβ oligomer antibody (OMAB) (1:1000, Agrisera, AS10932), anti-RIPK1 (1:200, BD Biosciences, 610459), anti-RIPK3 (1:500, Stata Crus, 374639), anti-MLKL (1:200, EnoGene, E11-11361C), antipMLKL (1:200, Abcam, ab196436), PSD95 (1:500, NeuroMab, 73-028), Synaptophysin1 (1:400, SYSY, 101002), MC1 (1:20; a gift from P. Davies, Albert Einstein College of Medicine, New York, New York, USA), anti-GFAP (1:1000, Millipore, MAB3402), anti-Iba1 (1:1000, Wako, 019-19741).

Techniques: Staining, Marker, Quantitation Assay

Journal: Cell research

Article Title: An App knock-in rat model for Alzheimer's disease exhibiting Aβ and tau pathologies, neuronal death and cognitive impairments.

doi: 10.1038/s41422-021-00582-x

Figure Lengend Snippet: Fig. 6 Increased apoptosis and necroptosis in AppNL-G-F rats. a–c Expression of apoptotic proteins in AppNL-G-F rats. a Hippocampal lysates from 12-month-old WT, Homo, and Hetero rats were immunoblotted using anti-cleaved caspase3, anti-procaspase3, anti-Bax and anti Bcl-2 antibodies. b, c Apoptosis was quantified by the ratios of cleaved caspase3 to procaspase3, as well as by the ratios of Bax to Bcl-2, shown in the right two panels. n = 4. d–g Expression of necrotic proteins in AppNL-G-F rats. d Hippocampal lysates from 12-month-old WT, Homo, and Hetero rats were immunoblotted using anti-RIPK1, anti-phosphorylated MLKL, anti-total MLKL (Upper panel), and anti-RIPK3 (Lower panel) antibodies. e–g The necroptosis levels were quantified by the ratio of RIPK1 to actin, the ratio of pMLKL to total MLKL, and the ratio of RIPK3 to actin. n = 4, Statistics: one-way ANOVA. h, i Representative fluorescent images of brain sections from WT and Homo rats stained with anti- RIPK1 and RIPK3 antibodies (h). Scale bars, 20 μm. Quantitative analyses of the immunoreactivity are shown below (i). n = 5. j–o Necrosomes in AppNL-G-F rats. Necrosome-like structures were immunostained by RIPK1, RIPK3 and MLKL antibodies. Representative fluorescent images and quantitative analyses of co-localization of necroptotic protein markers on the brain sections from 12-month-old WT and Homo rats are shown in the left (j, l, n) and right (k, m, o) panel respectively. Scale bars, 2 μm.

Article Snippet: Sections were permeabilized and blocked in PBS containing 0.2% Triton X-100 and 10% normal goat serum at room temperature for 2 h. Sections were incubated overnight at 4 °C with the following primary antibodies: anti-Aβ antibody (1:1000, Cell Signaling Technology, 2454), 6E10 (1:1000, Covance, 39320) anti-Aβ oligomer antibody (OMAB) (1:1000, Agrisera, AS10932), anti-RIPK1 (1:200, BD Biosciences, 610459), anti-RIPK3 (1:500, Stata Crus, 374639), anti-MLKL (1:200, EnoGene, E11-11361C), antipMLKL (1:200, Abcam, ab196436), PSD95 (1:500, NeuroMab, 73-028), Synaptophysin1 (1:400, SYSY, 101002), MC1 (1:20; a gift from P. Davies, Albert Einstein College of Medicine, New York, New York, USA), anti-GFAP (1:1000, Millipore, MAB3402), anti-Iba1 (1:1000, Wako, 019-19741).

Techniques: Expressing, Staining