Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Basal pro− and anti−inflammatory gene expression in mixed−sex microglia is APOE genotype−dependent: ( A ) basal pro−inflammatory and ( B ) anti−inflammatory gene expression was evaluated in non−stimulated mixed−sex microglia from APOE3 and APOE4 genotypes. Gene expression between genotypes was analyzed using an unpaired Student’s t −test (N = 3 isolations, n = 6 plates). Data are represented as the mean ± standard deviation. *, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.001, and 0.0001, respectively. ns = not statistically significant ( p > 0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Comparison of gene expression between APOE3 and APOE4 genotypes: ( A ) pro− and ( B ) anti−inflammatory gene expression was evaluated in mixed−sex microglia from APOE3 and APOE4 genotypes following treatment with LPS (10 ng/mL) and LPS (10 ng/mL) + IFNg (10 ng/mL) for 6 h. Gene expression between genotypes was assessed by two−way ANOVA followed by Tukey’s multiple comparisons test (N = 3 isolations, n = 6 plates). Each data point represents the average mRNA expression from one isolation. Data are represented as the mean ± standard deviation. # denotes a significant increase compared to the respective genotype control (data not included in the graphs, p < 0.05). *, **, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.01, 0.001, and 0.0001, respectively. ( C ) Heat map comparing the mean expression of pro− and anti−inflammatory genes in control, LPS, and LPS + IFNg treated APOE3 and APOE4 PMG. * and ^ denote a significant ( p < 0.05) increase and decrease, respectively, in APOE4 gene expression compared to APOE3.

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Expressing, Isolation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: APOE4 genotype exacerbates release of pro− and anti−inflammatory cytokines: cytokine levels were measured following treatment with LPS or LPS + IFNg for 24 h. ( A ) Average levels of nitrite (±standard deviation) accumulated in the media were measured by GRIESS assay. Using two−way ANOVA, significant treatment and genotype effects were observed. # denotes a significant increase compared to the respective genotype control ( p < 0.05). ( B ) Basal levels of secreted cytokines were measured in non−stimulated microglia after 24 h by an MSD multiplex assay. Comparisons between genotypes were made using an unpaired Student’s t −test. ( C ) Cytokine levels in the media were measured after treatment with LPS or a combination of LPS and IFNg for 24 h. Two−way ANOVA was used to assess cytokine release in mixed−sex microglia from APOE3 and APOE4 genotypes (N = 3−4 isolations, n = 6−8 plates). Data are represented as the mean ±standard deviation, and each data point denotes the average of each experimental plate. # denotes a significant increase compared to the respective genotype control (data not included in the graphs, p < 0.05). *, **, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.01, 0.001, and 0.0001, respectively. ns = not statistically significant ( p >0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Standard Deviation, Griess Assay, Multiplex Assay

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Basal pro− and anti−inflammatory gene expression in sex−specific microglia is dependent on both the APOE genotype and sex: ( A ) basal pro−inflammatory and ( B ) anti−inflammatory gene expression was evaluated in non−stimulated sex−specific microglia from APOE3 and APOE4 genotypes. Gene expression between genotypes and sexes was analyzed using two−way ANOVA (N = 3 isolations, n = 6 plates). Data are represented as the mean ± standard deviation. *, **, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.01, 0.001, and 0.0001, respectively. ns = not statistically significant ( p > 0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Gene expression in sex-specific microglia is dependent on both the APOE genotype and sex: ( A ) pro− and ( B ) anti−inflammatory gene expression was evaluated in sex−specific microglia from APOE3 and APOE4 genotypes following LPS or LPS + IFNg treatment for 6 h. Gene expression between genotypes and sexes was analyzed using a three−way ANOVA (N = 3 isolations, n = 6 plates) followed by Tukey’s multiple comparisons test. Each data point represents the average mRNA expression from one isolation. Data are represented as the mean ± standard deviation. # denotes a significant increase compared to the respective genotype control (data not included in the graphs, p < 0.05). *, **, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.01, 0.001, and 0.0001, respectively. ns = not statistically significant ( p >0.05). ( C ) Heat map comparing the mean expression of pro− and anti−inflammatory genes in sex−specific APOE3 and APOE4 PMG treated with LPS and LPS + IFNg. * and ^ denote a significant ( p < 0.05) increase and decrease, respectively, in fold changes of gene expression compared to a genotype−matched male PMG.

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Expressing, Isolation, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: APOE genotype and sex-dependent inflammation are mediated through p65 activation: ( A ) representative images for immunocytochemistry of p65 in control and LPS + IFNg treated APOE3 and APOE4, male and female PMG (scale bar = 100 μm). ( B ) Quantification of total p65 and ( C ) quantification of nuclear p65. N = 3 isolations. Each data point denotes the average p65 integrated density/cell density for an individual isolation. A denotes a significant increase compared to the E3 male control ( p < 0.05). # denotes a significant increase compared to the respective genotype and sex control ( p < 0.05). Data are represented as the mean ± standard deviation. * and **** indicate statistical significance for the comparison and denote p < 0.05 and 0.0001, respectively. ns = not statistically significant ( p > 0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Activation Assay, Immunocytochemistry, Isolation, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: Apolipoprotein E Inhibits Cerebrovascular Pericyte Mobility through a RhoA Protein-mediated Pathway

doi: 10.1074/jbc.m114.625251

Figure Lengend Snippet: FIGURE 1. Human brain vascular pericytes produce lipidated apoE. A, conditioned media and cell lysates were collected from human brain vascular pericytes with the APOE 3/3 genotype or immortalized astrocytes from apoE3-TR mice. ApoE concentrations in the conditioned media were normalized againstthetotalproteinconcentrationsincelllysatesandcomparedbetweenpericytesandastrocytes.B,conditionedmediafrompericytesorastrocyteswere concentrated and subjected to fractionation by a Superose-6 size exclusion column run on FPLC. Fractions were subjected to analysis for apoE concentration by ELISA. The amount of apoE in each fraction was plotted as percent of total apoE, calculated by combining those in all fractions. C, the cholesterol levels associating with apoE produced by pericytes or astrocytes were analyzed by Amplex Red cholesterol assay after immunoprecipitation. The amount of cholesterol in each fraction was plotted as percent of total cholesterol, calculated by combining those in all fractions. D, the fractions used in B were further subjected to analysis for cholesterol level by Amplex Red cholesterol assay.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 protein (Fitzgerald).

Techniques: Fractionation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Produced, Amplex Red Cholesterol Assay, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Apolipoprotein E Inhibits Cerebrovascular Pericyte Mobility through a RhoA Protein-mediated Pathway

doi: 10.1074/jbc.m114.625251

Figure Lengend Snippet: FIGURE 4. ApoE suppresses two-dimensional pericyte migration in an isoform-dependent manner. A, a wound healing assay was performed in control and apoE knockdown pericytes in the presence or absence of purified apoE3 or apoE4 particles (5 ng/ml). Cells from half of each well from the mid- line (dashed line) were removed and left to heal for 24 h before reimaging. B, migrated cell numbers were calculated as the number of cells beyond the scratch line. Data are plotted as mean S.D. (n 7). *, p 0.05; ***, p 0.001; n.s., not significant.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 protein (Fitzgerald).

Techniques: Migration, Wound Healing Assay, Control, Knockdown, Purification

Journal: bioRxiv

Article Title: APOE traffics to astrocyte lipid droplets and modulates triglyceride saturation and droplet size

doi: 10.1101/2023.04.28.538740

Figure Lengend Snippet: ( A ) Cartoon schematic of the FPP assay to test the topology of fluorescently-tagged proteins in cells. Cells are treated with 30 µM digitonin for 1 minute, which selectively permeabilizes the plasma membrane but not the ER. After permeabilization, cells are treated with 50 µg/mL proteinase K, which enters the permeabilized plasma membrane and degrades all cytoplasmic-facing fluorophores (green). Because the ER membrane is not permeabilized, proteinase K does not enter into the ER lumen and ER lumen-facing fluorophores are retained (red). (B) Representative confocal slices of FPP performed on primary cortical rat astrocytes transiently transfected with APOE3-mEm, the ER marker TagBFP2-KDEL, and labelled for LDs with BODIPY 665/676. After digitonin permeabilization and proteinase K treatment, APOE signal on the surface of LDs is lost, but the luminal ER marker fluorescence is retained. A Gaussian filter with a radius of 1 pixel was applied to all images to improve visibility for print. Scale bars: 10 µm (left), 5 µm for zoom (right). (C) For APOE3 as well as two representative LD proteins, PLIN2 and LiveDrop, the fluorescence intensity ratio was calculated by dividing the mean fluorescence intensity after both permeabilization and proteinase K treatment by the mean fluorescence intensity after permeabilization. The ER ratio was calculated by measuring the whole cell intensity of the ER fluorescence at the two timepoints. The LD ratio was calculated by measuring the fluorescence on the surface of BODIPY-labelled LDs at the two timepoints. Ratios close to 1 indicate minimal loss of signal after proteinase K treatment, as observed with the ER marker TagBFP2-KDEL. Lower ratios indicate loss of fluorescence upon proteinase K treatment. N = 8-18 cells per condition, collected from three independent experiment. *p<0.05, **** p<0.0001. Dig., 30 µM digitonin. PK, +50 µg/mL Proteinase K. P-values were calculated via the Wilcoxon rank sum test and Bonferonni-corrected for multiple comparisons.

Article Snippet: A plasmid containing human APOE3-TurboGFP was purchased from Origene (Cat# RG200395).

Techniques: Membrane, Transfection, Marker, Fluorescence

Journal: bioRxiv

Article Title: APOE traffics to astrocyte lipid droplets and modulates triglyceride saturation and droplet size

doi: 10.1101/2023.04.28.538740

Figure Lengend Snippet: ( A ) Representative frames from fast Airyscan movies showing the localization of LD-associated APOE relative to the ER after 4 hours of treatment with 400 µM OA in TRAE3-H cells. Cells were transfected with APOE3-mEm and the ER marker TagBFP2-KDEL and labelled for LDs with BODIPY 665/676. In the merged images, the ER is in magenta and APOE is in green. The yellow lines across the merged images indicate the line of pixels used to create the linescan graphs to the right of the images. In the linescan graphs, the relative fluorescence intensity of BODIPY-labelled LDs is in cyan, APOE3-mEm is green, and the ER is magenta. Two different localization patterns were observed: “half rings”, in which APOE partially covers the LD surface and colocalizes with the ER and “full rings”, where APOE fully encloses the surface of the LD and only partially colocalizes with the ER. Scale bars, 500 nm. (B) Immunogold electron micrographs of endogenous APOE localization at membrane contact sites between the ER and LDs in TRAE3-H cells treated with 400 µM OA for 5 hours. The blue arrow points to a direct membrane contact between the ER and an LD. Yellow arrows mark APOE localized to the cytoplasmic face of the ER membrane. The red arrow marks APOE localized to the cytoplasmic surface of the LD. Scale bars, 200 nm. (C) Representative frames from confocal FRAP movies of APOE3-Em on the surface of BODIPY 665/676 labelled LDs in primary rat cortical astrocytes during an OA pulse (200 µM OA for 4 hours) or an OA pulse-chase (200 µM OA for 4 hours followed by 2 hours chase in complete media – OA). APOE fluorescence was bleached at the 0 s timepoint. Scale bar, 1 µm. (D) Normalized intensity of APOE signal within the bleach ROI over time, with t = 0 sec denoting the time at which APOE was bleached. The bold center line is the mean normalized intensity, and the upper and lower bounds of the ribbon represent ± standard deviation (SD). N = 28 cells per condition, collected from three independent experiments. (E) Comparison of the rate constant of recovery k between OA pulse and pulse-chase conditions. The rate constant was derived by fitting each recovery curve to the equation y = C (1 – e - k t ). N = 28 cells per condition, collected from three independent experiments. * p<0.05 (F) Comparison of the mobile fraction between OA pulse and pulse-chase conditions. The mobile fraction was derived by fitting each recovery curve to the equation y = C (1 – e - k t ), where C is equal to the asymptote of the curve i.e. the mobile fraction. N = 28 cells per condition, collected from three independent experiments. **** p<0.0001. P-values were calculated via the Wilcoxon rank-sum test. (G) Schematic illustrating interpretation of the results of the FRAP experiment. When APOE on the LD is bleached during the OA pulse, it recovers very rapidly with a high mobile fraction. This indicates that bleached APOE on the LD is rapidly exchanged for unbleached APOE. After a short washout, LD-associated APOE recovers slowly or not at all, indicating that unbleached APOE molecules are unable to replace bleached ones on the LD. We hypothesize LD-APOE exchanges with APOE on the cytoplasmic face of the ER via membrane bridges during OA loading. These bridges are reduced or lost after OA washout, preventing exchange of APOE between LDs and the ER.

Article Snippet: A plasmid containing human APOE3-TurboGFP was purchased from Origene (Cat# RG200395).

Techniques: Transfection, Marker, Fluorescence, Membrane, Pulse Chase, Standard Deviation, Comparison, Derivative Assay

Journal: bioRxiv

Article Title: APOE traffics to astrocyte lipid droplets and modulates triglyceride saturation and droplet size

doi: 10.1101/2023.04.28.538740

Figure Lengend Snippet: ( A ) Cartoon illustrating the conditions used in the APOE rescue experiment. Endogenous APOE3 protein is present in cells transfected with a non-targeting siRNA. APOE protein is depleted upon APOE knockdown. The siRNA used to knock down APOE targets the mRNA sequence encoding the N-terminal signal peptide. The RNAi-resistant full-length APOE (RR FL) rescue construct consists of APOE3 with synonymous mutations in the signal peptide that impart resistance to the APOE siRNA. The LD-only APOE construct has the signal sequence removed, making it insensitive to the APOE siRNA , and replaced with the hairpin domain of the LD protein GPAT4. This version of APOE only targets LDs and never enters the ER lumen. (B) Western blot of lysates of TRAE3-H cells transfected with the indicated siRNA and transduced with the indicated lentivirus. The same samples were run on two separate SDS-PAGE gels, with 20 µg of total protein loaded into each well. Gels were transferred onto nitrocellulose membranes at the same time and then blotted with anti-HA or anti-APOE antibody together with an anti-tubulin antibody. Both the RR FL and LD-only APOE constructs are expressed in an endogenous APOE knockdown background. Moreover, the HA tag obstructs the epitope of the APOE antibody, allowing endogenous APOE and exogenous, HA-tagged APOE to be distinguished. (C) Representative confocal slices of cells transfected with non-targeting siRNA or APOE siRNA and transduced with an empty vector control, RR FL APOE, or LD-only APOE. Cells were subjected to an OA pulse-chase as described in 5A, fixed, and stained for LDs with BODIPY 493/507. Scale bar, 10 µm. (D-F) Quantification of LD parameters the conditions described in (A) after an OA pulse-chase assay. (D) Total LD area was measured as the area of the entire LD mask per cell in µm 2 . (E) Average LD size was calculated as the mean LD area per cell in µm 2 . (F) Number of LDs per cell. Each data point represents one cell, and each color represents data collected from a separate, independent experiment. N = 60 cells per condition and independent experiment. ns p>0.05, **** p<0.0001. P-values were calculated using a clustered Wilcox rank sum test via the Rosner-Glynn-Lee method and Bonferonni-corrected for multiple comparisons.

Article Snippet: A plasmid containing human APOE3-TurboGFP was purchased from Origene (Cat# RG200395).

Techniques: Transfection, Knockdown, Sequencing, Construct, Western Blot, Transduction, SDS Page, Plasmid Preparation, Control, Pulse Chase, Staining

Journal: Journal of genetic syndrome & gene therapy

Article Title: Gene Therapy Targeting LDL Cholesterol but not HDL Cholesterol Induces Regression of Advanced Atherosclerosis in a Mouse Model of Familial Hypercholesterolemia

doi:

Figure Lengend Snippet: Structure of helper-dependent adenoviral vectors expressing APOE3. A. Schematic presentation of helper-dependent adenoviral vectors (HDAd) containing phosphoenolpyruvate carboxykinase (PEPCK) promoter on C4HSU backbone. L-ITR and R-ITR indicate left and right adenovirus (Ad) inverted terminal repeat sequence, respectively; Ψ, Ad packaging signal; PEPCKpr, PEPCK promoter; AI intron, human apolipoprotein AI intron, hGH polyA, human growth hormone polyadenylation signal; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element; LCR, human APOE gene liver control region. B. HDAd containing human APOE gene promoter and liver specific enhancer, LCR. In HDAd-E-E3, the fragment containing exon 2–4 was removed and replaced with APOE3 cDNA. HDAd-gE3 contains APOE3 5′ flanking region as well as all exons. C. HDAd containing human APOAI promoter and 3′ flanking region. In HDAd-AI-E3, the APOAI gene corresponding to exon 2 to exon 4 was removed and replaced with APOE3 cDNA. D. HDAd containing human APOE3 gene and LCR on pΔ21 backbone. HPRT, intron region of human genomic HPRT; C346, cosmid C346 human genomic stuffer sequence.

Article Snippet: Human apoE3 levels were quantified by an ELISA using polyclonal goat anti-apoE antibody (Chemicon, 1:1000) and apoE3 (Calbiochem) as standard [ 18 ].

Techniques: Expressing, Sequencing

Journal: Journal of genetic syndrome & gene therapy

Article Title: Gene Therapy Targeting LDL Cholesterol but not HDL Cholesterol Induces Regression of Advanced Atherosclerosis in a Mouse Model of Familial Hypercholesterolemia

doi:

Figure Lengend Snippet: Plasma cholesterol levels. Six to eight week old Apoe−/− mice received i.v. injection of 5 × 1012 v.p./kg of HDAd expressing apoE3 and plasma cholesterol was measured over the following 32 weeks. A. PEPCK expression cassette. *p<0.05 vs. PBS, **p<0.001 vs. PBS, †p<0.01 vs. HDAd-P-E3, ‡p<0.05 vs. HDAd-P-E3 and HDAd-PW-E3. All time points in the HDAd-PW-E3 and HDAd-PWL-E3 groups were significantly different from the PBS group after vector treatment (p<0.001), but not indicated. B. APOAI expression cassette. p<0.001 vs. PBS, group, except in the HDAd-AI-E3 group 1 week after treatment. There was no statistical significance among treatment groups. C. APOE expression cassette. Plasma cholesterol levels in all treatment groups were significantly lower than those in the PBS group (p<0.001) except 1 week after treatment in the HDAd-E-E3 and HDAd-ghE3 groups. *p<0.05 vs. HDAd-EW-E3. **p<0.01 vs. HDAd-EW-E3 and HDAd-gE3. D. Plasma cholesterol levels in mice treated with HDAd-gE3 on pΔ21 backbone. *p<0.05 vs. HDAd-gE3, **p<0.01 vs. HDAd-gE3. n=5/group.

Article Snippet: Human apoE3 levels were quantified by an ELISA using polyclonal goat anti-apoE antibody (Chemicon, 1:1000) and apoE3 (Calbiochem) as standard [ 18 ].

Techniques: Injection, Expressing, Plasmid Preparation

Journal: Journal of genetic syndrome & gene therapy

Article Title: Gene Therapy Targeting LDL Cholesterol but not HDL Cholesterol Induces Regression of Advanced Atherosclerosis in a Mouse Model of Familial Hypercholesterolemia

doi:

Figure Lengend Snippet: Human ApoE3 levels in Apoe−/− mice after treatment with HDAd. A. PEPCK expression cassette. *p<0.05 vs. HDAd-P-E3 (n=5/group). B. APOAI cassette. *p<0.05 vs. HDAd-AIW-E3 at 2 weeks, **p<0.01 vs. HDAd-AIW-E3 and HDAd-AIWL at 1 week. C. APOE expression cassette. *p<0.05 vs. HDAd-EW-E3 and HDAdgE3, **p<0.01 vs. HDAd-EW-E3 and HDAd-gE3, and †p<0.05 vs. HDAd-EW-E3. D. Plasma apoE3 levels in mice treated with HDAd-gE3-D21. *p<0.05 vs. HDAd-gE3, **p<0.01 vs. HDAd-gE3.

Article Snippet: Human apoE3 levels were quantified by an ELISA using polyclonal goat anti-apoE antibody (Chemicon, 1:1000) and apoE3 (Calbiochem) as standard [ 18 ].

Techniques: Expressing

Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

doi: 10.1016/j.dadm.2017.08.003

Figure Lengend Snippet: The levels of apoE and α-syn in the hippocampus of apoE4 and apoE3-TR mice. Hippocampi of 4-month-old male and female apoE3 and apoE4 mice with or without α-syn were excised, homogenized, and subjected to apoE and α-syn immunoblotting and qRT-PCR, as described in Section . Representative apoE, α-syn, and loading standard β-tubulin bands of α-syn–deficient or normal α-syn males (A) and females (B) apoE3 (white bars) and apoE4 (black bars) mice are presented. Quantitations of the apoE protein levels (mean ± SEM; n = 5 per group) are normalized relative to the α-syn−/− apoE3 mice of each sex. As can be seen, the levels of apoE were higher in the apoE3 than the apoE4 mice, regardless of α-syn status or sex. qRT-PCR measurement of α-syn and apoE in males (C) and females (D) show no difference in mRNA levels of α-syn and apoE between the different genotypes (mean ± SEM; n = 4 per group). Abbreviations: apoE, apolipoprotein E; α-syn, α-synuclein; mRNA, messenger RNA; SEM, standard error of the mean. ∗ P <.05 for the effect of genotype on apoE levels.

Article Snippet: To minimize possible genetic drifting between the apoE4 and apoE3 mice, which were offspring of the homozygous apoE4 and apoE3 mice generated by Taconic around 2001, they were further crossed by us with Harlan C57Bl/6JOlaHsd mice, which unlike the standard Jackson laboratory C57Bl/6J ApoE tm1.1(APOE∗4)Adpmc mice (Jackson Laboratories, Bar Harbor, ME) turned out to be α-syn−/−.

Techniques: Western Blot, Quantitative RT-PCR

Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

doi: 10.1016/j.dadm.2017.08.003

Figure Lengend Snippet: The effects of apoE genotype and α-syn on the lipidation of apoE. Hippocampal homogenates of α-syn–deficient or normal α-syn apoE3 and apoE4 male (A) and female (B) mice were subjected to a blue native gel and stained with anti-apoE Ab as described in Section . Representative immunoblots of three mice per group are presented. As can be seen, the apoE3 mice, both male and female, contain higher levels of high molecular weight apoE and this effect is maintained independently of the presence or absence of α-syn. Abbreviations: Ab, antibody; apoE, apolipoprotein E; α-syn, α-synuclein.

Article Snippet: To minimize possible genetic drifting between the apoE4 and apoE3 mice, which were offspring of the homozygous apoE4 and apoE3 mice generated by Taconic around 2001, they were further crossed by us with Harlan C57Bl/6JOlaHsd mice, which unlike the standard Jackson laboratory C57Bl/6J ApoE tm1.1(APOE∗4)Adpmc mice (Jackson Laboratories, Bar Harbor, ME) turned out to be α-syn−/−.

Techniques: Staining, Western Blot, Molecular Weight

Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

doi: 10.1016/j.dadm.2017.08.003

Figure Lengend Snippet: The effects of apoE genotype on the levels of the synaptic marker synaptophysin, VGluT1, VGaT, and ApoER2 in α-syn–deficient female mice. Brains of apoE3, apoE4 homozygous, and apoE3/E4 heterozygous female mice were subjected to histologic staining with anti-synaptophysin (A), anti-VGluT1 (B), anti-VGaT (C), and anti-ApoER2 (D) Abs. Representative images (20 ×magnification) of the CA3 hippocampal subfield are presented in the upper part of each panel and show reduced levels in both apoE4 and apoE3/E4 mice compared with apoE3 mice. The results (mean ± SEM; n = 7–10 per group) of apoE3 mice (white bars), apoE4 mice (black bars), and apoE3/E4 (checkered bars) were quantified by computerized image analysis. qRT-PCR of the synaptic parameters (E) show no difference between apoE genotypes in the mRNA expression levels. The results shown were all normalized relative to control apoE3 mice (mean ± SEM; n = 4 per group). Abbreviations: Abs, antibodies; apoE, apolipoprotein E; α-syn, α-synuclein; mRNA, messenger RNA; SEM, standard error of the mean. ∗ P <.05 for the effect of genotype on the levels of the markers.

Article Snippet: To minimize possible genetic drifting between the apoE4 and apoE3 mice, which were offspring of the homozygous apoE4 and apoE3 mice generated by Taconic around 2001, they were further crossed by us with Harlan C57Bl/6JOlaHsd mice, which unlike the standard Jackson laboratory C57Bl/6J ApoE tm1.1(APOE∗4)Adpmc mice (Jackson Laboratories, Bar Harbor, ME) turned out to be α-syn−/−.

Techniques: Marker, Staining, Quantitative RT-PCR, Expressing

Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

doi: 10.1016/j.dadm.2017.08.003

Figure Lengend Snippet: The effects of apoE on the levels of Aβ42, phosphorylated tau, glial, and vascular marker genotype in α-syn–deficient female mice. Brains of apoE3, apoE4 homozygous, and apoE3/E4 heterozygous female mice were subjected to histologic staining with anti-Aβ42 (A), anti-AT8 mAb (B), which specifically recognizes the phosphorylated Ser202/Thr205 tau epitope, anti-GFAP (C), and anti-collagen IV (D) Abs. Representative images (10 ×magnification) of the CA3 hippocampal subfield are presented for Aβ42, AT8, and GFAP, whereas the collagen IV staining and analysis were performed in the stratum lacunosum molecular area of the hippocampus. The results (mean ± SEM; n = 7–10 per group) of apoE3 mice (white bars), apoE4 mice (black bars), and apoE3/E4 (checkered bars) were quantified by computerized image analysis as described in Section . As seen, the AD-related phenotypes of the apoE4 mice, namely, high levels of Aβ42, tau phosphorylation, and the glial marker GFAP, are not present in the heterozygous mice. The results shown are normalized relative to control apoE3 mice. In contrast, the levels of the vascular marker collagen IV are reduced in both apoE4 and apoE3/E4 mice. Abbreviations: Abs, antibodies; apoE, apolipoprotein E; α-syn, α-synuclein; SEM, standard error of the mean. ∗ P ≤.05 for the effect of genotype on the levels of the markers.

Article Snippet: To minimize possible genetic drifting between the apoE4 and apoE3 mice, which were offspring of the homozygous apoE4 and apoE3 mice generated by Taconic around 2001, they were further crossed by us with Harlan C57Bl/6JOlaHsd mice, which unlike the standard Jackson laboratory C57Bl/6J ApoE tm1.1(APOE∗4)Adpmc mice (Jackson Laboratories, Bar Harbor, ME) turned out to be α-syn−/−.

Techniques: Marker, Staining

Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

doi: 10.1016/j.dadm.2017.08.003

Figure Lengend Snippet: The effects of apoE genotype on the levels of Aβ42, phosphorylated tau, VGluT1, and ApoER2 in α-syn expressing and α-syn–deficient female mice. Brains of apoE3 and apoE4 homozygous female mice that express normal levels of α-syn (left panel) or are α-syn–deficient (right panel) were subjected to histologic staining with anti-Aβ42, anti-AT8 mAb, which specifically recognizes the phosphorylated Ser202/Thr205 tau epitope, anti-VGluT1, and anti-ApoER2 Abs, as described in Section . The results (mean ± SEM; n = 5–9 per group) of apoE3 mice (white bars) and apoE4 mice (black bars) were quantified by computerized image analysis and are presented relative to the apoE3 mice of the corresponding α-syn background. This revealed that in α-syn+/+ mice, unlike the α-syn−/− mice, no statistically significant difference between apoE3 and apoE4 mice was observed. Abbreviations: Abs, antibodies; apoE, apolipoprotein E; α-syn, α-synuclein; SEM, standard error of the mean. ∗ P <.05 for the effect of genotype on the levels of the markers.

Article Snippet: To minimize possible genetic drifting between the apoE4 and apoE3 mice, which were offspring of the homozygous apoE4 and apoE3 mice generated by Taconic around 2001, they were further crossed by us with Harlan C57Bl/6JOlaHsd mice, which unlike the standard Jackson laboratory C57Bl/6J ApoE tm1.1(APOE∗4)Adpmc mice (Jackson Laboratories, Bar Harbor, ME) turned out to be α-syn−/−.

Techniques: Expressing, Staining

Journal: Alzheimer's & Dementia : Diagnosis, Assessment & Disease Monitoring

Article Title: The effects of apolipoprotein E genotype, α-synuclein deficiency, and sex on brain synaptic and Alzheimer's disease–related pathology

doi: 10.1016/j.dadm.2017.08.003

Figure Lengend Snippet: The effects of apoE genotype on performance of α-syn expressing and α-syn–deficient female mice in the novel object recognition test. ApoE3 and apoE4 homozygous female mice that are α-syn–deficient or express normal levels of α-syn were first exposed to two identical objects, followed by a delay of 24 hours, after which the mice were exposed to an old and a new object. The preference of the mice to the different objects was monitored, as described in Section . The results obtained are depicted as the percent of visits made to the novel object out of the total number of visits to both familiar and novel objects. White bars correspond to apoE3 mice, whereas black bars correspond to apoE4 mice (mean ± SEM; n = 10 mice). As seen, the α-syn–deficient apoE4 mice are unable to discriminate between old and novel objects. Abbreviations: apoE, apolipoprotein E; α-syn, α-synuclein; SEM, standard error of the mean. ∗ P <.05 for the effect of genotype.

Article Snippet: To minimize possible genetic drifting between the apoE4 and apoE3 mice, which were offspring of the homozygous apoE4 and apoE3 mice generated by Taconic around 2001, they were further crossed by us with Harlan C57Bl/6JOlaHsd mice, which unlike the standard Jackson laboratory C57Bl/6J ApoE tm1.1(APOE∗4)Adpmc mice (Jackson Laboratories, Bar Harbor, ME) turned out to be α-syn−/−.

Techniques: Expressing