Journal: Alzheimer's & Dementia : Translational Research & Clinical Interventions

Article Title: Monomeric C‐reactive protein induces the cellular pathology of Alzheimer's disease

doi: 10.1002/trc2.12319

Figure Lengend Snippet: The effects of monomeric C‐reactive proteins (mCRP) on the amyloid beta (Aβ)42 secretion in primary cortical neurons. Postnatal day 0 to 2 cortical neurons from mice expressing different apolipoprotein E ( APOE ) genotype were isolated and cultured for 14 days. The neurons were treated with medium only or adding different concentrations of mCRP or pentameric CRP (pCRP) for 16 hours. A, Primary neurons from different APOE mice were treated with control medium versus different concentrations of mCRP. Cell culture media were collected and the expression levels of Aβ42 were detected by enzyme‐linkedimmunosorbent assay (ELISA). Values were the mean ± standard error (SE) from all experiments by using ANOVA with with Tukey's post hoc testing. Within each genotype, compared to the untreated neurons, differences of Aβ42 level with each concentration of mCRP are shown with statistical significance ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, and ∗∗∗∗ P < .0001. Within each concentration of mCRP, the comparisons of different APOE genotypes were conducted by using analysis of variance (ANOVA) with Tukey's post hoc testing. Statistical significance is shown with # P < .05, ## P < .01, ### P < .001, and #### P < .0001. B, Primary neurons from different APOE mice were treated with control medium versus different concentrations of pCRP. Cell culture media were collected and the expression levels of Aβ42 were detected by ELISA. Values were the mean ± SE from all experiments by using ANOVA with with Tukey's post hoc testing. C, Real‐time polymerase chain reaction (PCR) assays were conducted to reveal the expressions of amyloid precursor protein (APP) in neurons. Values are expressed relative to untreated condition in each genotype, which were set as 1. Values were the mean ± SE from all experiments. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with ∗ P < .05 and ∗∗ P < .01. D, Real‐time PCR assays were conducted to reveal the expressions of β‐secretase (BACE1) in neurons. Values are expressed relative to untreated condition in each genotype, which were set as 1. Values were the mean ± SE from all experiments. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with ∗ P < .05. E, Western blot assays for the expressions of APP and BACE1 in different primary APOE neurons treated with mCRP were performed and quantified after normalization against β‐actin. The representative photographs are shown. Values were the mean ± SE. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with ∗∗∗ P < .001

Article Snippet: On day 14 of culture, E. coli recombinant mCRP or human blood purified pCRP (LeeBio, #140‐11A) was added in cell culture medium followed by cell incubation.

Techniques: Expressing, Isolation, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Alzheimer's & Dementia : Translational Research & Clinical Interventions

Article Title: Monomeric C‐reactive protein induces the cellular pathology of Alzheimer's disease

doi: 10.1002/trc2.12319

Figure Lengend Snippet: The effects of monomeric C‐reactive proteins (mCRP) and pentameric CRP (pCRP) on the phosphorylated tau (p‐tau) expression in primary cortical neurons. Postnatal day 0 to 2 cortical neurons from mice expressing different apolipoprotein E ( APOE ) genotype were isolated and cultured for 14 days. The neurons were treated with medium only or adding different concentrations of mCRP for different time. A, Neurons from wild‐type (WT) mice or mice expressing different APOE genotypes were treated with control medium versus different concentrations of mCRP. The neurons were fixed, incubated with the p‐tau antibody, PHF1, to detect cellular tauopathy. The representative images were shown (scale bar: 50 μm). B, The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± standard error (SE) from all experiments and compared using analysis of variance (ANOVA) with Tukey's post hoc testing. Within each genotype, differences with different concentrations of mCRP for each genotype were shown with statistical significance ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001. Within the same concentration of mCRP treated neurons, the differences among different APOE neurons were compared with ANOVA with Tukey's post hoc testing. Statistical significance is shown # P < .05. C, The neurons were treated with control medium or 5 μg/mL mCRP for different incubation time. The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± SE from all experiments by using ANOVA with Tukey's post hoc testing. Within each genotype, differences with statistical significance for each genotype were shown with ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001. D, The neurons were treated with control medium versus different concentrations of pCRP. The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± SE from all experiments. E, The neurons were treated with control medium versus 5 μg/mL pCRP for different incubation time. The level of tau phosphorylation was normalized by NeuN and quantitated. Values were the mean ± SE from all experiments. F, Real‐time polymerase chain reaction (PCR) assay was conducted to reveal the expressions of CDK5 in neurons. Values are expressed relative to untreated APOE ε2 mouse primary neurons, which were set as 1. Values were the mean ± SE from all experiments by using ANOVA with Tukey's post hoc testing. Within each genotype, differences with statistical significance were shown with ∗ P < .05. G, Real‐time PCR assay was conducted to reveal the expressions of GSK3β in neurons. Values are expressed relative to untreated APOE ε2 mouse primary neurons, which were set as 1. Values were the mean ± SE from all experiments by using ANOVA with Tukey's post hoc testing. Within each genotype, differences with statistical significance were shown with ∗ P < .05 and ∗∗ P < .01. H, Western blot assays for the expressions of GSK3β, pGSK3β, and CDK5 in neurons were performed and quantified after normalization against β‐actin. The representative photographs are shown. Values were the mean ± SE. Within each genotype, compared to the untreated neurons, differences with statistical significance were shown with ∗ P < .05 and ∗∗ P < .01

Article Snippet: On day 14 of culture, E. coli recombinant mCRP or human blood purified pCRP (LeeBio, #140‐11A) was added in cell culture medium followed by cell incubation.

Techniques: Expressing, Isolation, Cell Culture, Control, Incubation, Phospho-proteomics, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot