Journal: Molecular Neurodegeneration

Article Title: Astrocytic APOE4 removal confers cerebrovascular protection despite increased cerebral amyloid angiopathy

doi: 10.1186/s13024-023-00610-x

Figure Lengend Snippet: Depletion of APOE in astrocytes diminishes certain disease-associated glial signatures. a – e , GFAP + astrocytic staining ( a ) and quantification of area coverage ( b ) in the cortex of 10-month-old 5X+AL- or 5X+AL+ mice. Scalebar: 300 µm. Representative images ( c ) and quantification of the number of astrocytes surrounding CAA ( d ) or plaques ( e ). Scalebar: 20 µm. Each point represents the number of astrocytes surrounding a single CAA or plaque normalized to that CAA or plaque area from n = 8–10 mice per group. f–j , IBA1 + microglial coverage ( f ) and quantification of area coverage ( g ) in the cortex overlying the hippocampus. Scalebar: 300 µm. Per plaque analysis of number of microglia clustering CAA ( h , i ) or plaques ( j ). Scalebar: 20 µm. Each point represents the number of microglia surrounding a single CAA or plaque normalized to that CAA or plaque area from n = 8–10 mice per group. k, l , Relative mRNA expression of homeostatic and disease-associated astrocytic ( k ) and microglial genes ( l ). Data imputed for Clec7a mRNA (column 10) but not used in statistical analysis. Each column represents an individual mouse. Data expressed as mean ± SD, student’s t -test (two-sided) performed for all statistical analyses except ( d ), ( e ), ( k – S100β ), and ( l – Cst7 , Cst3 , Itgax , Spp1 ) where Welch’s t -test was performed. ∆ = males, ○ = females. * P < 0.05, ** P < 0.01. No other statistical comparisons are significant unless indicated

Article Snippet: Primary antibodies include: biotinylated antibody HJ3.4 for human Aβ 1–13 (produced in-house, 2 µg/mL), rabbit Aβ 40 (Thermo Fisher, 44,136, 1:500), rabbit Aβ 42 (Thermo Fisher, 700,254, 1:500), rabbit APOE antibody (Cell signaling, #13,366, 1:500), biotinylated GFAP for astrocytes (Sigma-Aldrich, MAB3402B, 1:1000), rabbit Iba1 (Wako, 019–19,741, 1:5000), rabbit fibrinogen (Abcam, ab34269, 1:1000), and rat LAMP1 (Developmental Studies Hybridoma Bank, #1D4B, 1:400).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Microglial 25-hydroxycholesterol mediates neuroinflammation and neurodegeneration in a tauopathy mouse model

doi: 10.1101/2023.09.08.556884

Figure Lengend Snippet: A) Representative images from a double stain of the DAM markers ApoE (green) and Clec7a (red) in 9.5-month old wild type (WT; n=5), Ch25h KO (CKO; n=5), PS19 (T; n=20) and PS19/Ch25h KO (TCKO; n=20) mouse brain sections. Percentage of area covered by ApoE immunoreactivity (B) and ApoE immunoreactivity in Clec7a positive cells (C) was quantified in the hippocampus. D) Representative images from a double stain of Trem2 (red) and Clec7a (green). Percentage of area covered by Trem2 immunoreactivity (E) and ApoE immunoreactivity in Clec7a positive cells (F) was quantified in the hippocampus. G) Representative images of homeostatic microglia immunostained with P2ry12 in the hippocampus (Scale bar 50 µm). Total P2ry12 immunoreactivity area was analyzed using Imaris (H). Scale bar 30 µm. Data expressed as mean ± SD. One-way ANOVA with Tukey’s post hoc test (two-sided) was used for all statistical analysis *p<0.05, **p<0.01, p<0.001, ****p<0.0001.

Article Snippet: Tissue was then incubated overnight at 4°C with primary antibodies CD68 (rat monoclonal, 1:400; Biorad, MCA1957), Iba1 (rabbit polyclonal, 1:1000; FUJIFILM Wako’s, 019-19741), GFAP (chicken polyclonal, 1:1000; Abcam, ab4674), Clec7a or Dectin-1 (rat monoclonal, 1:100; Invivogen, mabg-mdect), Trem2 (sheep polyclonal, 1:400; R&D systems, AF1729), ApoE (mouse monoclonal HJ6.3, 1:1000; from D. Holtzman); Tmem119 (rabbit polyclonal, 1:250; Cell Signaling, 90840S), P2ry12 (rat monoclonal, 1;100; Biolegend, 848002), Cd11b (rat monoclonal, 1:400; BioLegend, 101202), p-STAT3 (rabbit polyclonal, 1:500; Cell Signaling, 9145S), p-p65 NF-kB (rabbit polyclonal, 1:3000; Cell Signaling, 3033S), MC1 antibody (mouse monoclonal, 1:500: gift from Peter Davis), CD3 (rat monoclonal, 1:500; Invitrogen, 14-0032-82).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Targeting apolipoprotein E and N-terminal amyloid β-protein precursor interaction improves cognition and reduces amyloid pathology in Alzheimer’s mice

doi: 10.1016/j.jbc.2023.104846

Figure Lengend Snippet: 6KApoEp treatment dampens amyloidogenic APP processing without altering BACE1 expression . A , Western blot is shown using anti-N-terminal amyloid-β 1–17 ( Aβ ) monoclonal antibody ( mAb 82E1 ), which detects amyloidogenic APP cleavage fragments, including phospho-C99 ( pC99 ) and nonphospho-C99 ( C99 ) as well as Aβ monomer and oligomers. Western blot is also shown using anti-C-terminal BACE1 (β-secretase) polyclonal antibody ( pAb BACE1 ). Actin is included as a loading control, and densitometry values are indicated below each lane. Equal amounts of total protein were loaded per lane. B and C , densitometry data are shown for ratios of pC99, C99, or Aβ to actin. D , abundance of Aβ oligomers in the detergent-soluble brain homogenate fraction (measured by sandwich ELISA) is shown. E , densitometry data are shown for ratios of BACE1 to actin. Data were obtained from APP/PS1/E2 mice that received vehicle ( APP/PS1/E2-V ) or 6KApoEp ( APP/PS1/E2-6K A poEp ), APP/PS1/E3 mice that received vehicle ( APP/PS1/E3-V ) or 6KApoEp ( APP/PS1/E3-6K A poEp ), and APP/PS1/E4 mice that received vehicle ( APP/PS1/E4-V ) or 6KApoEp ( APP/PS1/E4-6K A poEp ) for 3 months starting at 12 months of age. Western blotting data for ( B , C , and E ) included each mouse ( n = 4 per group with two of each sex), and quantitative data were averaged. Sandwich ELISA data for ( D ) included each mouse ( n = 8 per group with four of each sex), and measured data were averaged. Statistical comparisons for ( B ) are between groups for each protein. Statistical comparisons for ( C – E ) are between groups. ∗∗∗ p ≤ 0.001 for APP/PS1/E2-V or APP/PS1/E3-V versus APP/PS1/E4-V mice; †† p < 0.01; ††† p ≤ 0.001 for each 6KApoEp- versus each vehicle-treated APP/PS1/E2/E3/E4 mice ( Tables S16–S19 ). V , vehicle.

Article Snippet: Electrophoresed proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) that were blocked at ambient temperature for 1 h. Membranes were then hybridized at ambient temperature for 1 h with primary antibodies as follows: anti-N-terminal APP pAb (1:2000 dilution, IBL); anti-sodium-potassium ATPase mAb (1:20,000 dilution, EP1845Y; abcam), anti-heat-shock protein 90 mAb (1:1000 dilution, C45G5; Cell Signaling Technology), anti-N-terminal Aβ 1–16 mAb (1:500 dilution, 82E1; IBL); anti-C-terminal-BACE1 pAb (1:400 dilution; IBL); anti-phospho-p44/42 MAPK mAb (1:2000 dilution, D13.14.4E; Cell Signaling Technology); anti-p44/42 MAPK mAb (1:3000 dilution, 137F5; Cell Signaling Technology); anti-phospho-p38 MAPK (Thr180/Tyr182) mAb (1:1000 dilution, D3F9; Cell Signaling Technology); anti-p38 MAPK pAb (1:1500 dilution, Cell Signaling Technology); anti-C-terminal apoE pAb, (1:1000 dilution, A299; IBL); anti-human apoE LDLR binding domain pAb (1:1000 dilution; IBL), anti-DLK/MAP3K12 pAb (1:1500 dilution, GeneTex, Inc), or anti-β-actin mAb (1:5000 dilution, 13E5; Cell Signaling Technology; as a loading control).

Techniques: Expressing, Western Blot, Sandwich ELISA, Mouse Assay

Journal: The Journal of Biological Chemistry

Article Title: Targeting apolipoprotein E and N-terminal amyloid β-protein precursor interaction improves cognition and reduces amyloid pathology in Alzheimer’s mice

doi: 10.1016/j.jbc.2023.104846

Figure Lengend Snippet: 6KApoEp inhibits apoE-N-terminal APP interaction . A and B , brain homogenates from the vehicle-treated and 6KApoEp-treated APP/PS1/E2/E3/E4 mouse groups were immunoprecipitated with anti-N-terminal APP polyclonal antibody ( pAb , A ) or anti-C-terminal apoE pAb ( B ), and apoE ( A ) and APP ( holo , B ) were determined by Western blotting with anti-C-terminal apoE pAb or anti-N-terminal APP pAb. The left six lanes denote precipitates in each blot. The right six lanes denote inputs. IgG H , immunoglobulin heavy chain; IgG L , immunoglobulin light chain.

Article Snippet: Electrophoresed proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) that were blocked at ambient temperature for 1 h. Membranes were then hybridized at ambient temperature for 1 h with primary antibodies as follows: anti-N-terminal APP pAb (1:2000 dilution, IBL); anti-sodium-potassium ATPase mAb (1:20,000 dilution, EP1845Y; abcam), anti-heat-shock protein 90 mAb (1:1000 dilution, C45G5; Cell Signaling Technology), anti-N-terminal Aβ 1–16 mAb (1:500 dilution, 82E1; IBL); anti-C-terminal-BACE1 pAb (1:400 dilution; IBL); anti-phospho-p44/42 MAPK mAb (1:2000 dilution, D13.14.4E; Cell Signaling Technology); anti-p44/42 MAPK mAb (1:3000 dilution, 137F5; Cell Signaling Technology); anti-phospho-p38 MAPK (Thr180/Tyr182) mAb (1:1000 dilution, D3F9; Cell Signaling Technology); anti-p38 MAPK pAb (1:1500 dilution, Cell Signaling Technology); anti-C-terminal apoE pAb, (1:1000 dilution, A299; IBL); anti-human apoE LDLR binding domain pAb (1:1000 dilution; IBL), anti-DLK/MAP3K12 pAb (1:1500 dilution, GeneTex, Inc), or anti-β-actin mAb (1:5000 dilution, 13E5; Cell Signaling Technology; as a loading control).

Techniques: Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Targeting apolipoprotein E and N-terminal amyloid β-protein precursor interaction improves cognition and reduces amyloid pathology in Alzheimer’s mice

doi: 10.1016/j.jbc.2023.104846

Figure Lengend Snippet: 6KApoEp is detected in brains from 6KApoEp-treated APP/PS1/E2/E3/E4 mice, and is stable in mouse plasma for 24 h at 37 °C. A , Western blots are shown using anti-low-density lipoprotein receptor ( LDLR ) binding domain (residues 133–152 of human apoE) polyclonal antibody ( pAb ) that is the matching epitope as 6KApoEp except for 6K. The right five lanes denote a series of 6KApoEp calibration peptides ( i.e. , 20, 40, 80, 160, and 320 ng). Brain homogenates were obtained from APP/PS1/E2 mice that received vehicle ( APP/PS1/E2-V ) or 6KApoEp ( APP/PS1/E2-6KApoEp ), APP/PS1/E3 mice that received vehicle ( APP/PS1/E3-V ) or 6KApoEp ( APP/PS1/E3-6KApoEp ), and APP/PS1/E4 mice that received vehicle ( APP/PS1/E4-V ) or 6KApoEp ( APP/PS1/E4-6KApoEp ) for 3 months starting at 12 months of age. B , Western blotting data for the calibration graph were obtained from a series of 6KApoEp calibration peptides ( i.e. , 20, 40, 80, 160, and 320 ng). Western blotting data for 3 bars were obtained from APP/PS1/E2 mice that received 6KApoEp ( APP/PS1/E2-6KApoEp ), APP/PS1/E3 mice that received 6KApoEp ( APP/PS1/E3-6KApoEp ), and APP/PS1/E4 mice that received 6KApoEp ( APP/PS1/E4-6KApoEp ) for 3 months starting at 12 months of age. Western blotting data for ( B ) included each mouse ( n = 4 per group with two of each sex), and quantitative data were averaged. C , the stability of 6KApoEp (40 ng) in mouse plasma was examined with a time of incubation ( i.e. , 0, 3, 6, 12, and 24 h) at 37 °C. D , the experiment was performed four times, and quantitative data were averaged ( Table S23 ).

Article Snippet: Electrophoresed proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) that were blocked at ambient temperature for 1 h. Membranes were then hybridized at ambient temperature for 1 h with primary antibodies as follows: anti-N-terminal APP pAb (1:2000 dilution, IBL); anti-sodium-potassium ATPase mAb (1:20,000 dilution, EP1845Y; abcam), anti-heat-shock protein 90 mAb (1:1000 dilution, C45G5; Cell Signaling Technology), anti-N-terminal Aβ 1–16 mAb (1:500 dilution, 82E1; IBL); anti-C-terminal-BACE1 pAb (1:400 dilution; IBL); anti-phospho-p44/42 MAPK mAb (1:2000 dilution, D13.14.4E; Cell Signaling Technology); anti-p44/42 MAPK mAb (1:3000 dilution, 137F5; Cell Signaling Technology); anti-phospho-p38 MAPK (Thr180/Tyr182) mAb (1:1000 dilution, D3F9; Cell Signaling Technology); anti-p38 MAPK pAb (1:1500 dilution, Cell Signaling Technology); anti-C-terminal apoE pAb, (1:1000 dilution, A299; IBL); anti-human apoE LDLR binding domain pAb (1:1000 dilution; IBL), anti-DLK/MAP3K12 pAb (1:1500 dilution, GeneTex, Inc), or anti-β-actin mAb (1:5000 dilution, 13E5; Cell Signaling Technology; as a loading control).

Techniques: Western Blot, Binding Assay, Incubation