Journal: bioRxiv

Article Title: Regulation of neuroinflammation by astrocyte-derived cholesterol

doi: 10.1101/2022.12.12.520161

Figure Lengend Snippet: (A, B) SREBP2 floxed mice (SB2flox) were crossed to GFAP-Cre +/- mice (SB2 -/- ) to generate mice that do not produce cholesterol specifically in astrocytes. SB2flox (control) and SREBP2-/- mice were given 2mg/kg LPS by I.P. injection. Hippocampal microglia were assessed for CD68 content after 7 days. (C) 3xTg-AD mice (AD) were crossed to SREBP2 fl/fl GFAP-Cre +/- mice (AD x SB2 -/- ). Confocal imaging on astrocytes labelled with GFAP (red) and FDFT1 (green) demonstrates down-regulation of the cholesterol synthesis pathway in AD x SB2 -/- astrocytes. (D) Representative images of astrocytes showing a change in morphology and spatial distribution. Analysis of cell volume shows a decrease in astrocyte volume after cholesterol knockdown. Quantitation of astrocyte branches shows a decrease in branch points, branch length and sholl intersections in AD x SB2-/- brains compared to AD brains. (E) Confocal imaging on brain slices of AD x SB2-/- shows a robust decrease in % area double labeled with IBA1 and CD68, suggesting a decrease in activated microglia and macrophages after cholesterol knockdown. (F) qPCR of inflammatory cytokines TNFα. (G) TNFα ELISA on brain tissue shows an increased inflammatory level in AD brains compared to wildtype brains. SB2 knockout in astrocytes of AD (ADxSB2-/-) brains significantly downregulated neuroinflammation. (h) Florescent cholesterol oxidase assay from fixed brain slices. SB2-/- decreases cholesterol in both AD (left) and LPS-treated (right) animals. (i) Filipin cholesterol stain. Values are expressed as mean. Each point is a biological replicate (n=3-6 animals per experiment). Comparison made with a Student’s T test; or ANOVA (G) *p≤0.05, **p≤0.01, ****p≤0.0001.

Article Snippet: For determining the CD68 content of hippocampal microglia, 40 μm PFA fixed brain sections from LPS injected animals and 3xTg AD animals (described above) were immuno-stained by applying an IBA1 antibody (E4O4W, Cell Signaling) at a 1:500 dilution and a CD68 antibody (FA-11, Bio Rad) at a 1:500 dilution in blocking solution (described above) overnight at 4 degrees.

Techniques: Injection, Imaging, Quantitation Assay, Labeling, Enzyme-linked Immunosorbent Assay, Knock-Out, Staining

Journal: bioRxiv

Article Title: Regulation of neuroinflammation by astrocyte-derived cholesterol

doi: 10.1101/2022.12.12.520161

Figure Lengend Snippet: (A) Mice were I.P. injected 2 times with saline or 5mg/kg LPS dissolved in saline. Brains were harvested 27 hours post-injection and examined for IBA1 immunoreactivity to assess microglial activation. SB2-/- mice demonstrated a similar increase in IBA1 expression in response to LPS. (B-E) qPCR of inflammatory cytokines IL1β (B), IL1α (C), INFA (D) and INFB (E) shows a trend of decreased inflammation in SB2-/- similar to TNFα (see ). (F) Cholesterol assay on brain slices from SREBP2 (SB2) flox (WT flox) and SB2-/- mice (in the absence of LPS). Under resting non-inflamed state, SB2-/- has no effect, suggesting that something other than SREBP2 is regulating cholesterol in the non-inflamed state. See for comparison to LPS treated animals. (G) Cholesterol determination as in panel F using Filipin staining. Values are expressed as mean each point is a biological replicate. Comparison made with a Student’s T test; p values shown or n.s.= not significant.

Article Snippet: For determining the CD68 content of hippocampal microglia, 40 μm PFA fixed brain sections from LPS injected animals and 3xTg AD animals (described above) were immuno-stained by applying an IBA1 antibody (E4O4W, Cell Signaling) at a 1:500 dilution and a CD68 antibody (FA-11, Bio Rad) at a 1:500 dilution in blocking solution (described above) overnight at 4 degrees.

Techniques: Injection, Activation Assay, Expressing, Cholesterol Assay, Staining

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in the immunofluorescence analysis of mitochondrial dynamics, mitochondrial biogenesis, synaptic mitophagy and autophagy proteins in mitophagy enhancer-treated and -untreated hAbKI mice.

Article Snippet: Iba1/AIF-12 Rabbit monoclonal (#17178S) 1:1000 , Cell Signaling Technology, Inc., MA , Donkey Anti-rabbit HRP 1:10,000 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Immunofluorescence

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in the immunoblotting analysis of mitochondrial dynamics, mitochondrial biogenesis, synaptic mitophagy and autophagy proteins in mitophagy enhancer-treated and -untreated hAbKI mice.

Article Snippet: Iba1/AIF-12 Rabbit monoclonal (#17178S) 1:1000 , Cell Signaling Technology, Inc., MA , Donkey Anti-rabbit HRP 1:10,000 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Western Blot

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Immunofluorescence analysis of hippocampal microglial, astrocytic and neuronal proteins in seven-month-old hAbKI mice and treated hAbKI mice with Urolithin A and EGCG. ( A ) Representative immunofluorescence images and ( B ) quantitative immunofluorescence analysis of microglial Iba1 and astrocytic protein GFAP and neuronal protein NeuN in hAbKI mice relative to treated hAbKI mice with Urolithin A and EGCG.

Article Snippet: Iba1/AIF-12 Rabbit monoclonal (#17178S) 1:1000 , Cell Signaling Technology, Inc., MA , Donkey Anti-rabbit HRP 1:10,000 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: High-Mobility Group Box 1 Inhibitor BoxA Alleviates Neuroinflammation-Induced Retinal Ganglion Cell Damage in Traumatic Optic Neuropathy

doi: 10.3390/ijms23126715

Figure Lengend Snippet: BoxA reduces microglial activation following ONC. ( a ) Retinal slices stained for DAPI (blue) and Iba1 (green). Scale bars: 50 µm. ( b ) Area of Iba1-positive staining per image (250 µm × 250 µm) for each group, n = 4. ( c ) The protein level of Iba1 was evaluated. Each piece of data represents the mean ± SD, n = 3. * p < 0.05, ** p < 0.001, *** p < 0.0001 vs. control normal eye, # p < 0.05 vs. TON+ BoxA.

Article Snippet: Sections were dewaxed using environmentally friendly dewaxing solution, heated at high pressure for antigen repair, blocked with goat serum at room temperature for 1 h, and sequentially incubated with primary antibody AIF-1/Iba1 ((E4O4W) XP ® Rabbit mAb, 17198T, Cell signaling, Boston, Massachusetts, USA) at 4 °C overnight and secondary antibody (Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor ® 488 Conjugate, 4412S, SIGMA, Darmstadt, Germany) at room temperature for 40 min. Stained sections were sealed with anti-fluorescence quenching agent containing DAPI (ab104139, abcam, Cambridge, UK) for the counterstaining of the nucleus.

Techniques: Activation Assay, Staining