5×fad mouse brain tissue with trizol  (Thermo Fisher)

Journal: Neural Regeneration Research

Article Title: Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

doi: 10.4103/1673-5374.382254

Figure Lengend Snippet: The differentially expressed genes list obtained by RNA-seq in the dentate gyrus of 5×FAD-Rbm8a and 5×FAD-Ctrl group

Article Snippet: The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen, Carlsbad, CA, USA, Cat# 15596018) according to the manufacturer’s protocol, and synthesized using a cDNA Synthesis Kit (Transgene Biotech, Beijing, China, Cat# AT311-03).

Techniques:

Journal: Neural Regeneration Research

Article Title: Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

doi: 10.4103/1673-5374.382254

Figure Lengend Snippet: Neurons but not glial cells were infected by the AAV2-GFP virus in the DG region of the hippocampus. (A, B) Schematic representation of stereotactic injection of virus into the DG of 5×FAD mice brain. Mice were injected with the virus at P60 and killed at P120 (A). AAV2-GFP or AAV2- Rbm8a -GFP were injected into the DG of the dorsal hippocampus (B). (C, D) Representative images to show the association between GFP signal (green) and astrocytes labeled by GFAP (C) or microglia labeled by IBA1 (D). The box in the image indicates the enlarged image on the right. Left three images 10×, scale bar: 100 µm; and right images 63×, scale bar: 10 µm. (E) Representative confocal images of neurons stained with NeuN (red) indicates the neurons are infected by virus. The box in the image indicates the enlarged image on the right. Solid arrows indicate the overlap between NeuN and GFP signal. Left three images 10×, scale bar: 100 µm; and right images 63×, scale bar: 10 µm. (F, G) The virus mainly infected neurons and not glial cells (5×FAD-Ctrl: 47.8% neurons, 52.2% other cells, 0% astrocyte and microglia; 5×FAD- Rbm8a : 51.7% neurons, 48.3% other cells, 0% astrocyte and microglia) (F). The total number of NeuN-positive cells infected by virus in the DG region showed a significant increase in 5×FAD- Rbm8a group mice (G). n = 3/group, and each data point represents the average score from three sections per animal, ** P < 0.01 (unpaired Student’s t -test). Data are expressed as the mean ± SD. FAD: Familial Alzheimer’s disease; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; IBA1: ionized calcium-binding adapter molecule 1; NeuN: a mature neuron marker; SD: standard deviation; DG: dentate gyrus.

Article Snippet: The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen, Carlsbad, CA, USA, Cat# 15596018) according to the manufacturer’s protocol, and synthesized using a cDNA Synthesis Kit (Transgene Biotech, Beijing, China, Cat# AT311-03).

Techniques: Infection, Virus, Injection, Labeling, Staining, Binding Assay, Marker, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

doi: 10.4103/1673-5374.382254

Figure Lengend Snippet: Reduced soluble Aβ and plaque formation in the DG of 5×FAD mice with Rbm8a overexpression. (A) Confocal images of virus expression (green) and distribution of plaques (white) in the DG of 5×FAD- Rbm8a and 5×FAD-Ctrl group mice. 10×, scale bar: 100 µm. (B–D) Quantification of plaque-covered area (B), plaque number (C), and average plaque area (D). n = 6 female or male 5×FAD mice per group, and each data point represents the average score from three sections per animal, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey correction). (E, F) Enzyme-linked immunosorbent assay was performed to analyze the levels of Aβ 40 (E) and Aβ 42 (F), suggesting a decrease in the soluble fraction of plaques in the DG of 5×FAD- Rbm8a mice compared with the control group. n = 4 female 5×FAD mice per group, ** P < 0.01 (unpaired Student’s t -test). Data are expressed as the mean ± SD. Aβ: Amyloid beta; FAD: familial Alzheimer’s disease; SD: standard deviation; DG: dentate gyrus.

Article Snippet: The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen, Carlsbad, CA, USA, Cat# 15596018) according to the manufacturer’s protocol, and synthesized using a cDNA Synthesis Kit (Transgene Biotech, Beijing, China, Cat# AT311-03).

Techniques: Over Expression, Virus, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

doi: 10.4103/1673-5374.382254

Figure Lengend Snippet: Reduced dystrophic neurites surrounding plaques following overexpression of Rbm8a in the DG of 5×FAD mice. (A) Representative image from 4-month-old brain sections stained with 6E10 (white) to label plaques and ATG9A (red) to label early-stage dystrophic neurites (DNs). The box in the image indicates the enlarged images on the right. Left images 10×, scale bar: 100 μm; and right images 63×, scale bar: 10 μm. (B, C) Quantification analysis of the number of ATG9A + GFP + DNs surrounding plaques (B) and the percentage of 6E10 + GFP + ATG9 + to GFP + 6E10 + (C). Both were decreased in the 5×FAD- Rbm8a group compared with the 5×FAD-Ctrl group. n = 3/group, and each data point represents the average score from three sections per animal. * P < 0.05, ** P < 0.01 (unpaired Student’s t -test). (D–F) Representative images from 4-month-old brain sections stained with 6E10 (white) to label plaques and LC3B (red) to label late-stage DNs (D). The box in the image indicates the enlarged images on the right. Left images 10×, scale bar: 100 μm; and right images 63×, scale bar: 10 μm. Quantification of the number of LC3B + GFP + dystrophic neurites surrounding plaques (E) and the percentage of 6E10 + GFP + LC3B + to GFP + 6E10 + in the DG (F) were both decreased in the 5×FAD- Rbm8a group compared with the control group. n = 3/group, and each data point represents the average score from three sections per animal, * P < 0.05 (unpaired Student’s t -test). Data are expressed as the mean ± SD. ATG9A: Autophagy related protein 9A; Aβ: amyloid-beta; DG: dentate gyrus; GFP: green inflorescence protein; LC3B: light chain 3B; SD: standard deviation.

Article Snippet: The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen, Carlsbad, CA, USA, Cat# 15596018) according to the manufacturer’s protocol, and synthesized using a cDNA Synthesis Kit (Transgene Biotech, Beijing, China, Cat# AT311-03).

Techniques: Over Expression, Staining, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

doi: 10.4103/1673-5374.382254

Figure Lengend Snippet: Reduced gliosis response of astrocytes and microglia in the DG of 5×FAD mice following Rbm8a overexpression. (A) Representative image from the DG of 5×FAD mice for Aβ (6E10, white) and microglia (IBA1, red). The box in the image indicates the enlarged image on the right. Left images 10×, scale bar: 100 µm; and right images 63×, scale bar: 10 µm. (B, C) The area occupied by IBA1-positive microglia was decreased (B), but there was no change in the number of IBA1-positive microglia associated with plaques (C) in the 5×FAD- Rbm8a group. n = 3/group, and each data point represents the average score from 3–5 sections per animal. (D) Representative images for astrocytes (GFAP) and Aβ (6E10). The box in the image indicates the enlarged image on the right. Left images 10×, scale bar: 100 µm; and right images 63×, scale bar: 10 µm. (E, F) The area occupied by GFAP-positive astrocytes was decreased (E), but there was no change in the number of GFAP-positive astrocytes associated with plaques (F) in the 5×FAD- Rbm8a group. n = 3/group, and each data point represents the average score from 3–5 sections per animal. * P < 0.05, ** P < 0.01 (unpaired Student’s t -test). Data are expressed as mean ± SD. Aβ: Amyloid-beta; DG: dentate gyrus; FAD: familial Alzheimer’s disease; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; IBA1: ionized calcium-binding adapter molecule 1; Rbm8a: RNA binding motif protein 8a; SD: standard deviation.

Article Snippet: The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen, Carlsbad, CA, USA, Cat# 15596018) according to the manufacturer’s protocol, and synthesized using a cDNA Synthesis Kit (Transgene Biotech, Beijing, China, Cat# AT311-03).

Techniques: Over Expression, Binding Assay, RNA Binding Assay, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

doi: 10.4103/1673-5374.382254

Figure Lengend Snippet: Cognition but not depression and anxiety-like behavior were improved by Rbm8a overexpression in the DG of 5×FAD mice. (A–C) The open field test involved a 2-minute acclimation period, followed by a 10-minute exploration period (A). No notable differences were detected among the study cohorts with respect to total traveled distance (B) or time spent in the central zone (C). n = 11/group, and each data point represents one animal. (D–F) The Y-maze test schematic (D) revealed that mice in the 5×FAD- Rbm8a group exhibited a higher percentage of spontaneous alterations (E), yet with no meaningful difference in the number of arm entries (F) than those in the 5×FAD-Ctrl group. This finding supported a role for Rbm8a overexpression in the enhancement of memory function without affecting motor function. n = 11/group, and each data point represents one animal. (G, H) Schematic of the NORT method (G). Mice in the 5×FAD- Rbm8a group demonstrated an increased DI relative to those in the 5×FAD-Ctrl group (H), showing memory improvement induced by Rbm8a overexpression. n = 11/group, and each data point represents one animal. (I, J) Schematic of the CFC test (I). 5×FAD- Rbm8a mice exhibited a greater frequency of freezing during the testing phase (J) than 5×FAD-Ctrl mice, indicating a significant improvement in memory. n = 11/group, and each data point represents one animal, * P < 0.05, **** P < 0.0001 (unpaired Student’s t -test). (K, L) When comparing immobility times, no statistically significant differences were found between 5×FAD- Rbm8a mice and 5×FAD-Ctrl mice, according to both the tail suspension test (K) and forced swimming test (L). n = 11/group. Data are expressed as the mean ± SD. CFC: Contextual fear conditioning test; DG: dentate gyrus; DI: discrimination index; FAD: familial Alzheimer’s disease; NORT: novel object recognition test; SD: standard deviation.

Article Snippet: The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen, Carlsbad, CA, USA, Cat# 15596018) according to the manufacturer’s protocol, and synthesized using a cDNA Synthesis Kit (Transgene Biotech, Beijing, China, Cat# AT311-03).

Techniques: Over Expression, Suspension, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

doi: 10.4103/1673-5374.382254

Figure Lengend Snippet: Rbm8a overexpression increases the number of immature neurons in the DG of 5×FAD mouse brain. (A) Representative confocal images of newborn neurons (DCX, red). The box in the image indicates the enlarged image on the right. Left images 10×, scale bar: 100 µm; and right images 63×, scale bar: 10 µm. Solid arrows show DCX-positive cells infected by AAV-GFP virus. (B–D) Quantification analysis of the number of DCX + cells (B), percentage of DCX + GFP + to GFP + cells (C), and relative mRNA expression of DCX (D). For immunofluorescence staining test, n = 4/group, and each data point represents the average score from three sections per animal. For quantitative polymerase chain reaction test, n = 3/group, and each data point represents one animal. ** P < 0.01 (unpaired Student’s t- test). Data are expressed as mean ± SD. DCX: Doublecortin; DG: dentate gyrus; FAD: familial Alzheimer’s disease; GFP: green fluorescence protein; IF: immunofluorescence; SD: standard deviation.

Article Snippet: The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen, Carlsbad, CA, USA, Cat# 15596018) according to the manufacturer’s protocol, and synthesized using a cDNA Synthesis Kit (Transgene Biotech, Beijing, China, Cat# AT311-03).

Techniques: Over Expression, Infection, Virus, Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Fluorescence, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

doi: 10.4103/1673-5374.382254

Figure Lengend Snippet: Increased proliferation of neural progenitor cells induced by Rbm8a overexpression in the DG. (A) Schematic diagram of pretreatment with BrdU for 5×FAD mice. (B) Representative confocal images of proliferating neural progenitor cells infected by virus: SOX2 + BrdU + GFP + cells in DG. (C–F) Analysis of the data including number of BrdU + cells in the DG (C), percentage of SOX2 + BrdU + cells to SOX2 + cells in the DG (D), number of GFP + SOX2 + BrdU + cells in the DG (E), and percentage of GFP + SOX2 + BrdU + cells to SOX2 + GFP + cells in the DG (F). Images 10×, scale bar: 100 µm. n = 3/group, and each data point represents the average score from three sections per animal. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (unpaired Student’s t -test). Data are expressed as the mean ± SD. BrdU: 5-Bromo-2’-deoxyuridine; DG: dentate gyrus; FAD: familial Alzheimer’s disease; GFP: green fluorescence protein; Rbm8a: RNA binding motif protein 8a; SD: standard deviation; SOX2: SRY-box transcription factor 2.

Article Snippet: The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen, Carlsbad, CA, USA, Cat# 15596018) according to the manufacturer’s protocol, and synthesized using a cDNA Synthesis Kit (Transgene Biotech, Beijing, China, Cat# AT311-03).

Techniques: Over Expression, Infection, Virus, Fluorescence, RNA Binding Assay, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

doi: 10.4103/1673-5374.382254

Figure Lengend Snippet: Decreased expression of Adora2a and increased neurogenesis pathway by Rbm8a overexpression in the DG. (A) A differential gene clustering heatmap was created to compare the Rbm8a overexpression group with the control group. (B) The Volcano plot revealed 38 genes with significant differential expression levels, which included nine upregulated genes and 29 downregulated genes. Each individual gene is represented by a corresponding point visible on the plot. (C) The top five upregulated and downregulated genes with the most significant differential expression levels are shown. (D) A heatmap was generated for gene expression profiles of selected factors, including neurogenesis, amyloid protein formation, and proliferation, using RNA-seq data from 5×FAD mice with Rbm8a overexpression in the DG. (E) Enrichment analysis of genes in various diseases revealed that the number of genes related to neurodegenerative diseases ranked second. (F) A Venn diagram was generated to identify the intersection between genes enriched in neurodegenerative disease, cell proliferation, and neural cells. Adora2a was the single common gene among these intersecting sets. (G) The cAMP signaling pathway, alcoholism, and ligand-receptor interaction are all pathways enriched with Adora2a . (H, I) The relative mRNA expression levels of Adora2a (I) and Rbm8a (H) to GAPDH in the DG of 5×FAD-Ctrl and 5×FAD- Rbm8a mouse brain. n = 3/group. ** P < 0.01, *** P < 0.001 (unpaired Student’s t -test). Data are expressed as the mean ± SD. cAMP: Adenosine 3’,5’-cyclic monophosphate; DEGs: different expression genes; DG: dentate gyrus; FAD: familial Alzheimer’s disease; FC: fold change; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Rbm8a: RNA binding motif protein 8a; SD: standard deviation.

Article Snippet: The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen, Carlsbad, CA, USA, Cat# 15596018) according to the manufacturer’s protocol, and synthesized using a cDNA Synthesis Kit (Transgene Biotech, Beijing, China, Cat# AT311-03).

Techniques: Expressing, Over Expression, Generated, RNA Sequencing Assay, RNA Binding Assay, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Rbm8a regulates neurogenesis and reduces Alzheimer’s disease-associated pathology in the dentate gyrus of 5×FAD mice

doi: 10.4103/1673-5374.382254

Figure Lengend Snippet: Working Model: Rbm8a overexpression promotes neurogenesis, alleviates pathological phenotypes, and improves cognitive function in 5×FAD mice. (A) AAV2- Rbm8a -GFP and AAV2-GFP were injected into the DG of the hippocampus in 5×FAD mouse brain. Rbm8a overexpression in the DG improved neurogenesis and inhibited plaque formation in AD. (B) Rbm8a overexpression inhibits Adora2a expression, promotes neurogenesis, improves cognitive function, and alleviates pathological phenotypes, which is likely via the cAMP signaling pathway. BACE2: Beta-secretase 2; cAMP: adenosine 3’,5’-cyclic monophosphate; DG: dentate gyrus; GFP: green fluorescence protein; NPC: neural progenitor cell; Rbm8a: RNA binding motif 8a; RGC: radial glia cell.

Article Snippet: The cDNA was extracted from the DG of 5×FAD mouse brain tissue with TRIzol (Invitrogen, Carlsbad, CA, USA, Cat# 15596018) according to the manufacturer’s protocol, and synthesized using a cDNA Synthesis Kit (Transgene Biotech, Beijing, China, Cat# AT311-03).

Techniques: Over Expression, Injection, Expressing, Fluorescence, RNA Binding Assay