Journal: Journal of Alzheimer's Disease Reports

Article Title: Therapeutic Potential of Microbiota Modulation in Alzheimer’s Disease: A Review of Preclinical Studies

doi: 10.3233/ADR-220097

Figure Lengend Snippet: Table summarizing treatments and measures of the primary studies reviewed

Article Snippet: Clostridium butyricum n =10; n = 10 WT: n unknown sex unknown 1×10 9 cfu mL - 1 /200μL/day , Fecal sample DNA extraction and amplification with 16S rRNA using Illumina MiSeq sequencing, PCA and LDA analysis , Levels of IL-1β and TNF-α (proinflammatory cytokines) were detected using ELISA. Microglia: immunofluorescence and western blot analysis with antibodies (CD11b, COX-2, p-NF-κB p65) , ELISA for Aβ 42 Histology with Staining using Fluoro-Jade C for degenerating neurons and Congo red for amyloidosis. , MWM NOR.

Techniques: DNA Extraction, Amplification, Sequencing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Western Blot, Staining, Immunohistochemistry, Transplantation Assay, Immunostaining, Expressing, Immunohistochemical staining, Software, Real-time Polymerase Chain Reaction, Incubation, Microscopy, Imaging, Confocal Microscopy

Journal: Nature Communications

Article Title: Interferon regulatory factor 1 (IRF-1) promotes intestinal group 3 innate lymphoid responses during Citrobacter rodentium infection

doi: 10.1038/s41467-022-33326-5

Figure Lengend Snippet: a Colonic cross sections of uninfected or C. rodentium (9 dpi) infected C57BL/6 mice stained with anti-IRF-1 (red), anti-Ep-CAM (green), and DAPI (blue) and analyzed by confocal microscopy (epithelium: yellow arrow heads; lamina propria cells: white arrow heads). Scale bars represent 100 µm. b – g Irf1 +/+ and Irf1 –/– mice were infected orally with 4 × 10 9 CFU of C. rodentium . b Weights of infected mice were recorded daily and plotted as percentage of baseline ( Irf1 +/+ : n = 19, Irf1 –/– n = 18). Day 4 pvalue: 0,0172; pvalues day5-day9: <0.0001. c , d Bacterial loads were measured via in vivo imaging ( n = 15/group). Day 8 p value: 0.0008; p values day5–day7: <0.0001. e Fecal bacterial loads (CFU/g stool) on 3, 5 and 8 dpi. ( Irf1 +/+ : n = 9, Irf1 –/– n = 7). f Dissemination of C. rodentium to livers ( Irf1 +/+ : n = 13, Irf1 –/– n = 11), spleens ( Irf1 +/+ : n = 13, Irf1 –/– n = 11), mLN ( Irf1 +/+ : n = 8, Irf1 –/– n = 6) and blood ( Irf1 +/+ : n = 8, Irf1 –/– n = 6) (9 dpi). g C. rodentium colonization of the colonic epithelial surface and penetration of the crypt bottom (white arrow heads) was visualized by staining cross sections by immunohistochemistry. Scale bar: 100 µm. h Irf1 +/+ and Irf1 –/– mice ( n = 3/group) were infected with a C. rodentium reporter-strain expressing M-Ruby-II. Tissue samples from distal colon (7 dpi) were analysed by label-free multiphoton microscopy (MPM; excitation at 810 nm) to define densities of bacterial clusters per MPM image (1/mm 2 ). Scale bar: 50 µm. Data is expressed as mean ± SEM. Two-tailed Mann–Whitney U test was used for statistical comparison. In ( e ) and ( f ) exact p values are provided in the plots. * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001. Source data are provided as a Source data file.

Article Snippet: LPMCs were isolated with the lamina propria dissociation kit mouse from Miltenyi Biotec according to the manufacturer’s instructions under use of a gentleMACS Octo Dissociator (Miltenyi Biotec) running the program m_intestine_01.

Techniques: Infection, Staining, Confocal Microscopy, In Vivo Imaging, Immunohistochemistry, Expressing, Microscopy, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Science Advances

Article Title: Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection

doi: 10.1126/sciadv.adk9884

Figure Lengend Snippet: ( A ) Levels of HSPB1 were detected by immunoblot in primary mouse astrocytes (GFAP) but not in neurons (β-III-tubulin) or microglia (IBA1). ( B ) Primary mouse astrocytes were treated with 50 nM scramble (control) or HSPB1 siRNA, and HSPB1 was detected in astrocyte lysates or in concentrated conditioned medium. Fresh culture medium (non–cell exposed) was used as a control to discard the presence of HSPB1 or unspecific binding in serum. ( C ) Mouse astrocytes were transfected with either an empty vector or a construct to express V5-hHSPB1, which was detected by Western blotting with a V5 antibody in either astrocyte lysates or concentrated conditioned medium. ( D and E ) HSPB1 was detected in iPSC-derived astrocyte total cell lysates and conditioned medium in two control lines: C53 (D) and C70 (E). ( F to I ) Primary mouse astrocytes were treated with TNFα (30 ng/ml) and IL-1α (3 ng/ml) for 24 hours. (F) HSPB1 in lysates or in concentrated medium was detected by Western blotting, and levels of HSPB1 in medium were quantified relative to intracellular HSPB1 (G) or to intracellular GAPDH (H); intracellular HSPB1 levels were quantified relative to GAPDH (I); and LDH release to the medium was determined as a readout of cellular toxicity ( J ). Data are shown as means ± SD and were analyzed by unpaired Student’s t test in a minimum of three biological replicates. *** P < 0.001; ns, not significant.

Article Snippet: Astrocytes were treated with TNFα (30 ng/ml, R&D Systems, 410-MT-050) and/or IL-1α (3 ng/ml, Sigma-Aldrich, I3901), Baf-A1 (20 nM in DMSO, Alfa Aesar, J61835.M), rhHSPB1 [50 ng/ml in 0.1% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS), R&D Systems, 1580-HS], or heat-inactivated rhHSPB1 prepared by incubating at 99°C for 30 min. For live-cell experiments, rhHSPB1–Oregon Green (120 ng/ml) was used.

Techniques: Western Blot, Control, Binding Assay, Transfection, Plasmid Preparation, Construct, Derivative Assay

Journal: Science Advances

Article Title: Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection

doi: 10.1126/sciadv.adk9884

Figure Lengend Snippet: ( A ) SEC was used to fractionate the medium and to separate large particles such as EVs from soluble free proteins. ( B ) Astrocyte-conditioned medium was fractionated by SEC, and fractions were immunoblotted for CD81 (EV marker) and LCN2 (secreted as free protein). ( C ) The concentration and size distribution of particles contained in fractions 7 to 9 were analyzed by NTA. ( D ) Total protein concentration was determined by NanoDrop spectrophotometer in fractions 1 to 36. ( E ) Conditioned medium from astrocytes treated with control or TNFα + IL-1α for 24 hours was fractionated by SEC and immunoblotted for HSPB1. ( F ) Astrocyte medium was treated with 1% Triton X-100 for 1 hour before SEC, followed by detection of CD81 and HSPB1. ( G ) Medium of astrocytes expressing V5-hHSPB1 was treated with increasing concentrations of proteinase K, and levels of HSPB1 and LCN2 and bands resulting from its degradation were detected by Western blotting. ( H ) HSPB1, LAMP1, and LC3 were detected by immunofluorescence in astrocytes treated with control or TNFα + IL-1α for 24 hours, and colocalization was determined as Pearson’s correlation coefficient for HSPB1-LC3 ( I ) and HSPB1-LAMP1 ( J ). ( K to O ) Astrocytes were treated with dimethyl sulfoxide (DMSO) (control) or 20 nM Baf-A1 for 24 hours: Levels of NAG were detected in the medium (K); HSPB1 was detected in lysates or concentrated medium after concomitant treatment with control or TNFα + IL-1α and Baf-A1 (L), and the levels of HSPB1 in medium (M) or the levels of intracellular HSPB1 (N) were quantified relative to GADPH in lysates; and LDH levels in medium relative to intracellular levels (O). Data are shown as means ± SD and analyzed by two-way ANOVA with Tukey’s multiple comparisons test [(M) to (O)] or unpaired Student’s t test [(I) to (K)] in a minimum of three biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Astrocytes were treated with TNFα (30 ng/ml, R&D Systems, 410-MT-050) and/or IL-1α (3 ng/ml, Sigma-Aldrich, I3901), Baf-A1 (20 nM in DMSO, Alfa Aesar, J61835.M), rhHSPB1 [50 ng/ml in 0.1% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS), R&D Systems, 1580-HS], or heat-inactivated rhHSPB1 prepared by incubating at 99°C for 30 min. For live-cell experiments, rhHSPB1–Oregon Green (120 ng/ml) was used.

Techniques: Marker, Concentration Assay, Protein Concentration, Spectrophotometry, Control, Expressing, Western Blot, Immunofluorescence

Journal: Science Advances

Article Title: Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection

doi: 10.1126/sciadv.adk9884

Figure Lengend Snippet: ( A ) Primary astrocytes were pretreated with rhHSPB1 for 24 hours before treatment with either 0.1% BSA (vehicle) or rhHSPB1 (50 ng/ml) and either TNFα + IL-1α or control for further 24 hours. Created with BioRender.com . ( B ) LCN2, SerpinA3N, and iNOS were detected by Western blotting in astrocyte lysates, and levels were quantified relative to GADPH as a loading control ( C to E ). ( F and G ) LCN2 was detected in concentrated conditioned medium and quantified relative to intracellular GAPDH levels (G). ( H ) The concentration of nitrites in the astrocyte medium was measured as an indirect measure of NO using a Griess assay. ( I ) The levels of IL-6 in the astrocyte medium were determined by ELISA. ( J and K ) A cytokine array was used to determine levels of 38 cytokines in the medium in a membrane-based sandwich immunoassay. The average z scores for each of the 38 cytokines was represented in the graph (J) or shown individually for each cytokine in the heatmap (K), where cytokines are ranked from low to high fold change of TNFα + IL-1α/rhHSPB1 versus TNFα + IL-1α/vehicle. Cytokines with an asterisk showed significant increase (red) or decrease (green) in TNFα + IL-1α/rhHSPB1 versus TNFα + IL-1α/vehicle condition. Data are shown as means ± SD ( N = 3). Two-way ANOVA with Tukey’s or Sidak’s multiple comparisons test [(C), (D), (E), (G), (H), and (J)] or unpaired Student’s t test (I). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Astrocytes were treated with TNFα (30 ng/ml, R&D Systems, 410-MT-050) and/or IL-1α (3 ng/ml, Sigma-Aldrich, I3901), Baf-A1 (20 nM in DMSO, Alfa Aesar, J61835.M), rhHSPB1 [50 ng/ml in 0.1% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS), R&D Systems, 1580-HS], or heat-inactivated rhHSPB1 prepared by incubating at 99°C for 30 min. For live-cell experiments, rhHSPB1–Oregon Green (120 ng/ml) was used.

Techniques: Control, Western Blot, Concentration Assay, Griess Assay, Enzyme-linked Immunosorbent Assay, Membrane

Journal: Science Advances

Article Title: Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection

doi: 10.1126/sciadv.adk9884

Figure Lengend Snippet: ( A ) Organotypic brain slices were prepared from postnatal mouse brain, transduced at 0 DIV with AAVs to express hHSPB1 in astrocytes under the GFAP promoter for 20 DIV followed by treatment with TNFα + IL-1α for 24 hours. Created with BioRender.com . ( B ) Transduction of slices with AAV-GFAP-BFP (AAV-GFAP) and AAV-GFAP-hHSPB1:BFP (AAV-GFAP-hHSPB1) shows astrocyte-specific (GFAP + ) expression of BFP and hHSPB1 (note that HSPB1 antibody has some unspecific nuclear staining). ( C ) Western blotting to confirm expression of hHSPB1 and its secretion in brain slices. ( D to F ) Changes in LCN2 were quantified in lysates (E) and in conditioned medium (F) relative to intracellular GAPDH. ( G and H ). A cytokine array measured levels of 38 cytokines in the medium in a membrane-based sandwich immunoassay. The average z scores for each cytokine were represented in the graph (G) or shown individually in the heatmap (H), where cytokines are ranked from high to low fold change of control/AAV-GFAP versus TNFα + IL-1α/AAV-GFAP. Cytokines marked with a green asterisk were significantly decreased in TNFα + IL-1α/AAV-GFAP-hHSPB1 versus TNFα + IL-1α/AAV-GFAP conditions. ( I ) The hippocampus was separated and cultured for 19 days and then pretreated with rhHSPB1 (50 ng/ml) for 24 hours and with either control or TNFα + IL-1α and either vehicle (0.1% BSA) or rhHSPB1 (50 ng/ml) for further 24 hours. ( J ) Levels of LCN2 were detected by immunoblot in slice lysates and quantified relative to GAPDH ( K ). Data are shown as means ± SD. N = 5 [(E) and (F)], N = 3 [(G) and (H)], or N = 6 [(J) and (K)]. Two-way ANOVA with Tukey’s or Sidak’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001.

Article Snippet: Astrocytes were treated with TNFα (30 ng/ml, R&D Systems, 410-MT-050) and/or IL-1α (3 ng/ml, Sigma-Aldrich, I3901), Baf-A1 (20 nM in DMSO, Alfa Aesar, J61835.M), rhHSPB1 [50 ng/ml in 0.1% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS), R&D Systems, 1580-HS], or heat-inactivated rhHSPB1 prepared by incubating at 99°C for 30 min. For live-cell experiments, rhHSPB1–Oregon Green (120 ng/ml) was used.

Techniques: Transduction, Expressing, Staining, Western Blot, Membrane, Control, Cell Culture

Journal: Science Advances

Article Title: Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection

doi: 10.1126/sciadv.adk9884

Figure Lengend Snippet: ( A ) Primary mouse neurons were treated with rhHSPB1 (10 ng/ml) or vehicle (0.1% BSA) every 3 days from 3 DIV, fixed on 14 DIV, and stained for MAP2. ( B to G ) High-content confocal microscopy was used to determine changes in the number of segments (B), end points (C), primary branching points (D), secondary branching points (E), root points (F) per neuron, and the total length of all neurites for each neuron (G) ( N = 6). ( H ) Neurons were treated with conditioned medium from control or TNFα + IL-1α–treated astrocytes at 10 and 12 DIV, together with rhHSPB1 (50 ng/ml) or vehicle (0.1% BSA), and the medium was collected at 13 DIV. In parallel, neurons were treated with equivalent concentrations of TNFα + IL-1α. Cellular toxicity was determined by measuring LDH levels in neuron-conditioned medium ( N = 5). ( I ) Neurons were transduced at 6 DIV with AAVs to express WT-htau-EGFP or mutant P301L/S320F-htau-EGFP and treated with either vehicle (0.1% BSA) or rhHSPB1 (50 ng/ml) every 2 days and fixed at 12 DIV. Created with BioRender.com . ( J ) Representative images of neurons transduced with mutant P301L/S320F-htau-EGFP and treated with vehicle (0.1% BSA) or rhHSPB1 (50 ng/ml). ( K ) Graph shows the percentage of EGFP-positive neurons that show mutant htau aggregates. N = 6 with >1500 cells counted per experiment. ( L and M ) Total levels of tau were detected using an antibody that recognizes total tau, and the band corresponding to htau tagged to EGFP was quantified relative to β-actin. ( N ) Cellular toxicity was determined by measuring LDH levels in neuron-conditioned medium ( N = 4). Data are shown as means ± SD. Paired Student’s t test [(B) to (G) and (K)]; two-way ANOVA with Tukey’s multiple comparisons test [(H), (M), and (N)], * P < 0.05, ** P < 0.01.

Article Snippet: Astrocytes were treated with TNFα (30 ng/ml, R&D Systems, 410-MT-050) and/or IL-1α (3 ng/ml, Sigma-Aldrich, I3901), Baf-A1 (20 nM in DMSO, Alfa Aesar, J61835.M), rhHSPB1 [50 ng/ml in 0.1% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS), R&D Systems, 1580-HS], or heat-inactivated rhHSPB1 prepared by incubating at 99°C for 30 min. For live-cell experiments, rhHSPB1–Oregon Green (120 ng/ml) was used.

Techniques: Staining, Confocal Microscopy, Control, Mutagenesis, Transduction