Journal: The Journal of Prevention of Alzheimer's Disease

Article Title: LMTK2 and CRB1 are two novel risk genes for Alzheimer's disease in Han Chinese

doi: 10.1016/j.tjpad.2025.100087

Figure Lengend Snippet: Effects of the rare variants R234P or S974G of LMTK2 on Tau aggregation, Aβ generation, and apoptosis in vitro . (A) Western blot analysis of phosphorylation levels of Tau and total Tau after Flag-LMTK2 and HA-Tau plasmids were co-transfected into HEK293 cells for 48 h. (B) Quantification of relative protein levels in A ( n = 6). (C) Representative images of K18-induced Tau aggregation after HEK293-Tau RD cells were transfected with LMTK2. After LMTK2 plasmid transfection for 24 h, K18 (1µg/µL) was added, induced for 48 h, and photographed by a fluorescence microscope. Scale bar: 20 µm (D) Quantification of cell with inclusions in C ( n = 6). (E) Western blot analysis of the effect of LMTK2 WT and variants on PP1C-GSK3β signaling pathway. (F) Quantification of relative protein levels in E ( n = 3). (G) ELISA detection of secreted Aβ after LMTK2 WT and variants were transfected into SH-SHY5Y-APPSwe cells ( n = 3). (H) Western blot analysis of BAX and Bcl2 expression after LMTK2 WT and variants were transfected into SH-SHY5Y-APPSwe cells. (I) Quantification of relative BAX and Bcl2 expression ( n = 6). Western blot data are representative of three independent experiments. Data are mean ± SEM, *, * and *** represent p < 0.05, p < 0.01, and p < 0.001, respectively, and ns represents no statistical significance (Student's t -test).

Article Snippet: The supernatants were collected, and the levels of Aβ40 and Aβ42 were determined in accordance with the operating instructions of the human Aβ40 ELISA kit (Elabscience, #E-EL-H0542) and human Aβ42 ELISA kit (Elabscience, #E-EL-H0543), respectively.

Techniques: In Vitro, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Expressing

Journal: The Journal of Prevention of Alzheimer's Disease

Article Title: LMTK2 and CRB1 are two novel risk genes for Alzheimer's disease in Han Chinese

doi: 10.1016/j.tjpad.2025.100087

Figure Lengend Snippet: Effect of rare variants in CRB1 on Aβ generation. (A) WB detection of APP and related secretory enzymes expression after high expression of different CRB1 plasmids in SH-SY5Y-APPSwe cells. (B) Quantification of related APP expression. (C-E) ELISA detection of the concentration of Aβ42 (C), Aβ40 (D), and the Aβ42/Aβ40 ratio (E) in the culture medium after the overexpression of different CRB1 plasmids in SH-SY5Y-APPSwe cells. Western blot data are representative of three independent experiments. Data are mean ± SEM, * and ** represent p < 0.05 and p < 0.01, respectively, and ns represents no statistical significance (Student's t -test).

Article Snippet: The supernatants were collected, and the levels of Aβ40 and Aβ42 were determined in accordance with the operating instructions of the human Aβ40 ELISA kit (Elabscience, #E-EL-H0542) and human Aβ42 ELISA kit (Elabscience, #E-EL-H0543), respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Over Expression, Western Blot

Journal: Translational psychiatry

Article Title: Pitolisant alleviates brain network dysfunction and cognitive deficits in a mouse model of Alzheimer's disease.

doi: 10.1038/s41398-025-03358-8

Figure Lengend Snippet: Fig. 4 Pitolisant alleviates Aβ burden in 5xFAD mice. a Left: Levels of soluble Aβ40 in brain homogenate of 5xFAD mice measured by ELISA (n = 8 mice for WT + S; n = 8 mice for 5xFAD + S; n = 8 mice for 5xFAD + P; Mann Whitney test, P = 0.0002). Middle: Levels of insoluble Aβ40 in brain homogenate of 5xFAD mice measured by ELISA (n = 8 mice for WT + S; n = 8 mice for 5xFAD + S; n = 8 mice for 5xFAD + P; two- tailed unpaired t test, P = 0.0007). Right: Levels of soluble Aβ40 in plasma of 5xFAD mice measured by ELISA (n = 8 mice for WT + S; n = 8 mice for 5xFAD + S; n = 8 mice for 5xFAD + P; two-tailed unpaired t test, P = 0.6006). ND = not detected. b Left: Levels of soluble Aβ42 in brain homogenate of 5xFAD mice measured by ELISA (n = 8 mice for WT + S; n = 8 mice for 5xFAD + S; n = 8 mice for 5xFAD + P; Mann Whitney test, P = 0.0003). Middle: Levels of insoluble Aβ40 in brain homogenate of 5xFAD mice measured by ELISA (n = 8 mice for WT + S; n = 8 mice for 5xFAD + S; n = 8 mice for 5xFAD + P; two-tailed unpaired t test; P = 0.0009). Right: Levels of soluble Aβ40 in plasma of 5xFAD mice measured by ELISA (n = 8 mice for WT + S; n = 8 mice for 5xFAD + S; n = 8 mice for 5xFAD + P; Mann Whitney test, P = 0.7984). ND = not detected. c Representative images of Aβ (Thioflavin S) staining in the cortex (Ctx) and hippocampus (Hp). d Representative images of Aβ (6E10 antibody) staining in the cortex (Ctx) and hippocampus (Hp). e Left: quantification of thioflavin S positive area in the cortex (n = 8 mice for WT + S; n = 8 mice for 5xFAD + S; n = 8 mice for 5xFAD + P; two-tailed unpaired t test, P = 0.014). Right: quantification of thioflavin S positive area in the hippocampus (n = 8 mice for WT + S; n = 8 mice for 5xFAD + S; n = 8 mice for 5xFAD + P; two-tailed unpaired t test, P = 0.0097). ND = not detected. f Left: quantification of 6E10 positive area in the cortex (n = 8 mice for WT + S; n = 8 mice for 5xFAD + S; n = 8 mice for 5xFAD + P; Mann Whitney test, P = 0.0002). Right: quantification of 6E10 positive area in the hippocampus (n = 8 mice for WT + S; n = 8 mice for 5xFAD+S; n = 8 mice for 5xFAD + P; two-tailed unpaired t test, P = 0.0039). ND = not detected. Each dot represents an individual animal. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance.

Article Snippet: Commercial ELISA kits (Elabscience, E-EL-M3009 for mouse Aβ40, E-EL-M3010 for mouse Aβ42) were employed to quantify Aβ40 and Aβ42 levels in the brain tissue of 5xFAD mice.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Two Tailed Test, Clinical Proteomics, Staining

Journal: Ecotoxicology and environmental safety

Article Title: Dysregulated APP expression and α-secretase processing of APP is involved in manganese-induced cognitive impairment.

doi: 10.1016/j.ecoenv.2021.112365

Figure Lengend Snippet: Fig. 3. Effect of Mn exposure on the expression of ADAM10 and sAPPα. (A-B) ADAM10 mRNA levels in control and Mn-treated mouse hippocampi (A) and N2a cells (B) were determined by qRT-PCR. (C-D, G-H) Representative western blotting of ADAM10 in control and Mn-treated mouse hippocampi (C), N2a cells (D), APP OE N2a cells (G), and APP shRNA N2a cells (H). β-actin is used as a loading control. (E-F) sAPPα protein levels in control and Mn-treated mouse plasma (E) and N2a cells (F) were determined by ELISA. Values are given as mean ± SME pooled from five mice and three repeated cell experiments. *P < 0.05, **P < compared to control group. #P < 0.05, ##P < 0.01 compared to low-dose group.

Article Snippet: Levels of Aβ40, Aβ42, and sAPPα were determined in mouse plasma, and levels of sAPPα were determined in N2a cells using a mouse Aβ40 ELISA kit (Cusabio CSB-E08300m, Wuhan, China), a mouse Aβ42 ELISA kit (Elabscience Biotechnology Co., Ltd. E-EL-M0068c, Wuhan, China), and a mouse/rat sAPPα assay kit (IBL JP27419, Japan) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, shRNA, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Ecotoxicology and environmental safety

Article Title: Dysregulated APP expression and α-secretase processing of APP is involved in manganese-induced cognitive impairment.

doi: 10.1016/j.ecoenv.2021.112365

Figure Lengend Snippet: Fig. 4. Effect of Mn exposure on the protein levels of BACE1, Aβ40, and Aβ42. (A–B, E–F) Representative western blots of BACE1, Aβ40, and Aβ42 in control and Mn- treated mouse hippocampi (A), N2a cells (B), APP OE N2a cells (E), and APP shRNA N2a cells (F). β-actin is used as a loading control. (C-D) Aβ40 (C) and Aβ42 (D) protein levels in control and Mn-treated mouse plasma were determined by ELISA. Data are presented as the mean ± SEM (n = 5) of five mice.

Article Snippet: Levels of Aβ40, Aβ42, and sAPPα were determined in mouse plasma, and levels of sAPPα were determined in N2a cells using a mouse Aβ40 ELISA kit (Cusabio CSB-E08300m, Wuhan, China), a mouse Aβ42 ELISA kit (Elabscience Biotechnology Co., Ltd. E-EL-M0068c, Wuhan, China), and a mouse/rat sAPPα assay kit (IBL JP27419, Japan) according to the manufacturer’s instructions.

Techniques: Western Blot, Control, shRNA, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Translational Psychiatry

Article Title: Melatonin alleviates depression-like behaviors and cognitive dysfunction in mice by regulating the circadian rhythm of AQP4 polarization

doi: 10.1038/s41398-023-02614-z

Figure Lengend Snippet: A Schematic diagram of the behavioral experimental procedure. B , C The latency to the first immobility and the duration of total immobility in the tail suspension test (TST). D The total travel distance during the open field test (OFT). E The percentage of sucrose consumption during the sucrose preference test (SPT). Control, n = 10; CUMS, n = 11; Mel, n = 14. F Tracing of locomotion for representative animals during the arena test. G The latency to finding the hidden platform in the Morris Water Maze test during the 5-days training. H , I The latency to finding the platform area and the percentage time spent in the platform quadrant in the probe test. Each group, n = 15. J – V Measurement of glymphatic function and AQP4 polarization. J Diagram of CSF tracer injection via the cisterna magna. K Representative images of CSF tracers in the whole brain and coronal slices. Scale bar, 2 mm. L , M Analysis of fluorescent area in the brains for the anesthesia and awake states. Anesthesia, each group n = 8. Awake, Control, n = 6; CUMS, n = 7; Mel, n = 8. N Diagram of stereotactic injection of exogenous mixture of human Aβ40 and Aβ42. O , P The brain concentration of exogenous human Aβ40 and Aβ42 measured by ELISA. Each group n = 7. Q Representative images of AQP4, DAPI, and GFAP immunostaining. T Representative images of AQP4 and CD31 immunostaining. Scale bars, 50 μm and 25 μm. R Quantitation of GFAP fluorescent intensity. S Quantitation of AQP4 polarization to Q . AQP4 polarization = donut-shaped area AQP4 / global AQP4. U Quantitation of AQP4 polarization to T . AQP4 polarization = vessel AQP4 (peak of the vertical line) / global AQP4. Collect 3–5 vessels from each of 6 mice ( R , S and U ). V Model of AQP4 fluorescence intensity plot along the yellow dotted line in ( T ). Signal within the vessels is the baseline intensity of AQP4. * #<0.05, ** ##<0.01, *** ###<0.001.

Article Snippet: For the Aβ analysis, tissues were first homogenized in 1 ml/g PBS and then assayed using human ELISA kits for Aβ40 (CSB-E08299h, Cusabio, China) and Aβ42 (CSB-E10684h, Cusabio, China).

Techniques: Suspension, Control, Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunostaining, Quantitation Assay, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Lysosomal Dysfunction in a Mouse Model of Sandhoff Disease Leads to Accumulation of Ganglioside-Bound Amyloid-β Peptide

doi: 10.1523/JNEUROSCI.4860-11.2012

Figure Lengend Snippet: Accumulation of APP-CTFs and lipid-associated Aβ in HEXB KO mice. A, Western blot analysis, using 369 antibody to detect full-length APP and APP-CTFs and 22C11 antibody to detect sAPPα, shows significant increases in the levels of α-CTF and β-CTF in HEXB KO mice relative to wild-type (WT). No significant changes were observed in the levels of full-length APP or sAPPα levels. (For APP, α- and β-CTF analysis, n = 6 for WT, n = 7 for HZ, n = 7 for KO. For sAPPα, n = 3 for WT, n = 4 for HZ, n = 4 for KO). B, ELISA measurement of Aβ40 and Aβ42 levels shows significant increase in the levels of lipid-associated Aβ40 and Aβ42 in the HEXB KO mice (n = 6 for WT; n = 7 for HZ; n = 7 for KO). Shown are mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001). C, Western blot analysis using Aβ40-specific antibody (FCA3340) shows increase in the levels of Aβ40 in the soluble and lipid-associated fractions of HEXB KO mice. The levels of Aβ42 were not detectable by Western blotting. D, Brain sections were costained with antibodies that recognize either the N terminus (22C11) or C terminus (369) of APP. Although the signals from APP-C terminus (369) antibody seem to accumulate in the cortex (d) and hippocampus (h) of HEXB KO mice, no change is observed in the signals from APP-N terminus (22C11) antibody (c, g). Scale bars: a–h, 200 μm.

Article Snippet: The levels of soluble and lipid-bound Aβ40 and 42 were measured using ELISA kits (Wako Chemicals; no. 294-64701 for Aβ40 and 292-64501 for Aβ42), according to the manufacturer's instructions.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Neuroscience

Article Title: Lysosomal Dysfunction in a Mouse Model of Sandhoff Disease Leads to Accumulation of Ganglioside-Bound Amyloid-β Peptide

doi: 10.1523/JNEUROSCI.4860-11.2012

Figure Lengend Snippet: Region-specific accumulation of Aβ-like immunoreactivity in HEXB KO mice. Coronal brain sections of wild-type and HEXB KO mice were stained with NeuN antibody (a–j), Aβ42-specific antibody (FCA3542) (a1–j1), or Aβ40-specific antibody (FCA3340) (a2–j2). NeuN-positive neurons appeared swollen with vacuolar changes in the cortex (b, b′), subiculum (d, d′), dentate gyrus (f), and CA3 region of the hippocampus (h) of HEXB KO mice. Similarly, iAβ-LIR accumulated in the cortex (b1, b1′, b2, b2′), subiculum (d1, d1′, d2, d2′), dentate gyrus (f1, f2), and CA3 region of the hippocampus (h1, h2). The morphology of NeuN-positive neurons appeared normal in the CA1 region of the hippocampus (j), and iAβ-LIR was not observed in this region (j1, j2). DG, Dentate gyrus. Scale bars: a–j, a1–d1, g1–j1, a2–d2, g2–j2, 500 μm; a′–d′, a1′–d1′, a2′–d2′, e1, f1, e2, f2, 200 μm.

Article Snippet: The levels of soluble and lipid-bound Aβ40 and 42 were measured using ELISA kits (Wako Chemicals; no. 294-64701 for Aβ40 and 292-64501 for Aβ42), according to the manufacturer's instructions.

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: Lysosomal Dysfunction in a Mouse Model of Sandhoff Disease Leads to Accumulation of Ganglioside-Bound Amyloid-β Peptide

doi: 10.1523/JNEUROSCI.4860-11.2012

Figure Lengend Snippet: Ganglioside-bound Aβ (GAβ) immunoreactivity colocalizes with iAβ-LIR. A, Coronal brain sections from wild-type (a–c, g–i) and HEXB KO mice (d–f, j–l) were costained with GAβ antibody (4396C) and Aβ42-specific antibody (FCA3542). GAβ immunoreactivity as detected by 4396C antibody colocalized with iAβ-LIR in the subiculum and cortex of HEXB KO mice (f, l). Scale bars: a–l, 200 μm. B, Coronal brain sections from wild-type and HEXB KO mice were costained with GFAP (red)- and Aβ40 and Aβ42 (green)-specific antibodies (FCA3340 and FCA3542, respectively). Increased GFAP immunoreactivity and reactive gliosis were observed in the cortex (b), subiculum (d), and CA3 region of the hippocampus (f, h) of HEXB KO mice. Scale bars: a–h, 500 μm.

Article Snippet: The levels of soluble and lipid-bound Aβ40 and 42 were measured using ELISA kits (Wako Chemicals; no. 294-64701 for Aβ40 and 292-64501 for Aβ42), according to the manufacturer's instructions.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Lysosomal Dysfunction in a Mouse Model of Sandhoff Disease Leads to Accumulation of Ganglioside-Bound Amyloid-β Peptide

doi: 10.1523/JNEUROSCI.4860-11.2012

Figure Lengend Snippet: Dysfunction in lysosomal proteolysis leads to the accumulation of iAβ-LIR in the endosomal–lysosomal system and undegraded autophagosomes. A, Coronal brain sections of wild-type and HEXB KO mice were costained with Aβ42-specific antibodies (FCA3542) and markers of early endosomes (EEA1), late endosomes (Rab7), and lysosomes (Lamp-1). iAβ-LIR colocalized partially with EEA1 (a–f), Rab7 (g–l), and Lamp-1 (m–r) in HEXB KO mice. Images were taken from the subiculum. Scale bars, 50 μm. B, Immunohistochemical analysis shows the colocalization of iAβ42 (Pan1G6) with LC3 in undegraded autophagosomes in HEXB KO mice (d–f). The autophagosomal marker (LC3) colocalizes with the early endosomal marker (EEA1) (j–l) and with the lysosomal marker (Lamp-1) (p–r) in HEXB KO mice. Scale bars, 50 μm. C, Western blot analysis shows an increase in the LC3II/LC3I ratio and slight increases in the levels of p62 and Rab7 in HEXB KO mice relative to wild type (n = 3 for WT; n = 3 for HZ; n = 3 for KO). Shown are mean ± SEM.

Article Snippet: The levels of soluble and lipid-bound Aβ40 and 42 were measured using ELISA kits (Wako Chemicals; no. 294-64701 for Aβ40 and 292-64501 for Aβ42), according to the manufacturer's instructions.

Techniques: Immunohistochemical staining, Marker, Western Blot

Journal: The Journal of Neuroscience

Article Title: Lysosomal Dysfunction in a Mouse Model of Sandhoff Disease Leads to Accumulation of Ganglioside-Bound Amyloid-β Peptide

doi: 10.1523/JNEUROSCI.4860-11.2012

Figure Lengend Snippet: Human gangliosidoses brains accumulate intraneuronal ganglioside-bound Aβ. A, Immunostaining of human brain cortical sections using the antibodies 4G8, FC3542 (Aβ42-specific antibody), and FC3340 (Aβ40-specific antibody) demonstrates that Aβ42 accumulates in SD (b) and TS brains (e). Extracellular Aβ-like immunoreactivity structures were observed in the 45-year-old TS brain (e). The arrows indicate accumulation of intraneuronal Aβ-LIR. Scale bars: a–f, 200 μm; g–i, 500 μm. B, Cortical sections obtained from control (1 year) (a, b), control (27 years) (i, j), GM1 (1 year) (c, d), SD (1 year) (e, f), TS (4 years) (g, h), TS (27 years) (k, l), TS (45 years) (m, n), and AD (75 years) (o, p) were stained with 4396C antibody to detect ganglioside-bound Aβ. Scale bars: a, b, o, p, 200 μm; c–n, 50 μm.

Article Snippet: The levels of soluble and lipid-bound Aβ40 and 42 were measured using ELISA kits (Wako Chemicals; no. 294-64701 for Aβ40 and 292-64501 for Aβ42), according to the manufacturer's instructions.

Techniques: Immunostaining, Staining