Journal: Frontiers in Genetics

Article Title: Aberrant expression of the MID1 protein in neurons of Huntington’s disease brain

doi: 10.3389/fgene.2026.1753495

Figure Lengend Snippet: MID1 mRNA expression is aberrantly increased in HD-neurons. The MID1 expression was quantified in distinct cell populations isolated by MACS from the cortex of 15-week-old HdhQ150 knock-in mice (tg) and age-matched wild-type littermates (wt). The success of separation was quantified by qPCR analysis detecting GFAP as marker for astrocytes (A) , CD11b as marker for microglia (B) , and NeuN as neuronal marker (C) . (D) The marker proteins GFAP, NeuN, and CD11b were also detected on a Western blot of pooled samples from the tg animals (n = 7). MID1 was detected on the same blots. MID1 qPCR analysis revealed no statistically significant differences in (E) astrocytes ( p = 0.595) and (F) microglia ( p = 0.1966). (G) In neurons a significant upregulation of MID1 expression was detected (* p = 0.0104). MID1 levels were normalized to ACTB . Columns represent mean values ± SEM, p -values were calculated using an unpaired t-test with Welch’s correction (n wt = 7, n tg = 7).

Article Snippet: The Anti-ACSA-2 MicroBead Kit (130-097-678), the CD11b MicroBeads Kit (130-093-634), and the Neuron Isolation Kit (130-097-678) from Miltenyi Biotech were used for MACS.

Techniques: Expressing, Isolation, Knock-In, Marker, Western Blot

Journal: eNeuro

Article Title: Minimizing the Ex Vivo Confounds of Cell-Isolation Techniques on Transcriptomic and Translatomic Profiles of Purified Microglia

doi: 10.1523/ENEURO.0348-21.2022

Figure Lengend Snippet: Comparison of purity and yield among microglial cell isolation techniques. A , Schematic of the experimental design. Cx3cr1-NuTRAP brains were enzymatically and mechanically dissociated to create a single-cell suspension. Different microglial sorting techniques were compared with Cell-Input for purity, yield, and transcriptomic signatures. B , Representative flow cytometry plots of immunostained single-cell suspensions from input and after each of the sorting strategies shows a distinct population of eGFP + cells (identified as Cx3cr1 + microglia) and CD11b + CD45 + cells (identified as microglia per traditional cell surface markers, as shown by IHC in Extended Data ). Assessment of the loss of eGFP in circulating CD11b + CD45 + cells 60 d after Tam induction (Extended Data ) and the relative levels of CD45 mid and CD45 high cell populations from the CD11b + CD45 + each sort method and cell input from brain (Extended Data ) were also performed to further characterize the sort fractions. Representative plots of the gating strategies used for the Tyto sort (Extended Data ), FACSAria sort (Extended Data ), and flow cytometry (Extended Data ) are given as supplements. C , D , All sort positive fractions were enriched for ( C ) eGFP + singlets and ( D ) CD11b + CD45 + singlets (as compared with input; two-way ANOVA, main effect of TRAP fraction, * p < 0.05, *** p < 0.001; Extended Data ). When comparing positive fractions, the autoMACS positive fraction had lower % eGFP + singlets as compared with all other sort methods. FACSAria had higher percentage of eGFP + singlets than all other sort methods. FACSAria had higher percentage of CD11b + CD45 + singlets as compared with all other sort methods positive fractions (two-way ANOVA, Tukey’s post hoc test, * p < 0.05). E , Microglial yield was significantly higher in the MACSQuant Tyto positive fraction as compared with the autoMACS to MACSQuant Tyto and FACSAria positive fractions (one-way ANOVA, Tukey’s post hoc test, # p < 0.01). Created with BioRender.com.

Article Snippet: Cells from a single hemisphere of a Cx3cr1-NuTRAP mouse brain were pelleted at 300 × g for 10 min at 4°C and resuspended in 90 μl of 0.5% BSA in D-PBS with 10 μl of CD11b (Microglia) MicroBeads (#130-093-636, Miltenyi Biotec) per 10 7 total cells.

Techniques: Comparison, Cell Isolation, Suspension, Flow Cytometry

Journal: Nature Communications

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

doi: 10.1038/s41467-021-25344-6

Figure Lengend Snippet: a sAβ42s were added to empty wells (scale bar = 20 μm) or b 12-week-old iPSC neurons at the indicated concentrations. All wells were stained with X04 (blue), Aβ (green), NFL-H (green), and p-Tau S235 (red). Empty wells have Aβ precipitates but no XO4 positive structure. In iPSC neuron wells, there is a dose-dependent increase of X04 staining. A Subset of XO4 is also surrounded by dystrophic neurites (NFL-H and S235 positive axonal swellings). Scale bar = 50 μm. c Microglia treated with sAβ42s ranging from 0 to 5 μM and also treated in combination with IFNγ. The panel below shows a zoomed-in section. Aβ plaques are stained with X04 (blue), microglia are labeled with Actin (green), and IBA1 (red). Scale bar = 50 μm. IFNγ increases plaque formation and plaque interaction. e Quantification of X04 intensity . f Quantification of IBA1 number. d Neuron and astrocytes coculture and triculture of neurons, astrocytes, and microglia were treated with sAβ42s with or without pro-inflammatory cytokine combination (IFNy + IL1b + LPS). Representative images from indicated conditions are shown. The lower panel of zoomed images is shown. Aβ plaque was stained with X04 (blue), dystrophic neurites swellings were stained with NFL-H (green), and microglia were labeled with IBA1 (red). In triple culture, sAβ42s addition led to Aβ plaque formations surrounded by dystrophic neurites and encircled by microglia similar to plaque presentation in vivo. Scale bar = 20 μm. g The area of IBA1 overlap with X04 is quantified. Pro-inflammatory cytokines increased microglia association with plaque. h Microglia increased the X04 plaque area, as measured by the total area of X04 staining. Pro-inflammatory cytokine addition increased the plaque area furthermore. MAP2 areas were also quantified in ( i ). sAβ42s addition caused a severe reduction to neuron culture. Microglia culture provided 25% MAP2 protection from sAβ42s. This protection is lost when pro-inflammatory cytokine is added. Data are presented as mean values +/− SEM and n = 4 wells. Two-way ANOVA with ( e , f , h , i ) Tukey’s or ( g ) Dunnett’s multiple comparisons test.

Article Snippet: iPSC Microglia , BrainXell , E3/E3.

Techniques: Staining, Labeling, In Vivo

Journal: Nature Communications

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

doi: 10.1038/s41467-021-25344-6

Figure Lengend Snippet: a – d Neurons and astrocytes ( a , c ) or neurons, astrocytes, and microglia ( b , d ) were treated with 5 μM sAβ42s and small molecules from a focused screen of known neuroprotective agents at multiple concentrations (50, 25, 12.5, and 6.25 μM (double culture), 50, 12.5, 3.1, and 0.78 μM (triple culture). Results were graphed as Synapse % rescue versus MAP2 % rescue ( a , b ) and Beta III tubulin % rescue versus MAP2 % rescue ( c , d ). Small molecules that prevented toxicity in dendrites (MAP2), synapses (Synapsin 1/2), cell count (CUX2), or axons (NFL-H) at or above 30% were considered hits (red dotted line); anti-Aβ antibody used as a positive control that prevented all types of toxicity. e – g Further validation of top hits DLKi ( e ), Indirubin-3′-monoxime ( f ), and AZD0530 ( g ) from the focused screen by IC50 curves against MAP2 (blue), Synapsin 1/2 (green), CUX2 (red), and NFL-H (purple). h sAβ42s treatment induced expression of p-cJun (green) in the nucleus (HuCD, red). i Quantification of MAP2 (blue), HuC/D (red), p-cJun (green) staining indicated an increase in cJun phosphorylation with prolonged sAβ42s treatment. j 22-week-old iPSC neuron culture treated with sAβ42s showed dose-dependent, sustained phosphorylation of cJun as shown by western blot. GAPDH served as the loading control. k Western blot quantification of p-cJun induction normalized to GAPDH. l – o Inhibition of known components of DLK-JNK-cJun pathway using small molecules VX-680 ( l ), GNE-495 ( m ), PF06260933 ( n ), JNK-IN-8 ( o ) prevents sAβ42s-induced neural toxicity in all measured markers in a dose-dependent manner. Data are presented as mean values +/− SEM and n = 4 wells ( e – g , I , k – o ). IC50 curves fitted by Prism software ( e – g , m – o ).

Article Snippet: iPSC Microglia , BrainXell , E3/E3.

Techniques: Cell Counting, Positive Control, Biomarker Discovery, Expressing, Staining, Phospho-proteomics, Western Blot, Control, Inhibition, Software

Journal: Nature Communications

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

doi: 10.1038/s41467-021-25344-6

Figure Lengend Snippet: a Schematic showing sAβ42s labeled by HiLyte-555 and pHrodo Green continuously fluoresce red, but only fluoresce green under intracellular pH 5 conditions. b Quantitative analysis of red Aβ plaque area and green internalized Aβ. Internalized green Aβ outpace the red extracellular Aβ plaque formation, indicating active Aβuptake throughout the 7 days and proceed before the appearance of red Aβ plaques. c Example images of the plaque formation time-lapse movie. Four different plaque formations are retrospectively labeled. sAβ42s are first internalized by microglia (green) before plaque formation (red) in the center of the cultured microglia. Scale bar = 100 μm. d iPSC-derived microglia were treated with 5 μM sAβ42s, then fixed and stained after 30 min, 6 h, 1 day, and 4 days. Microglia (IBA1, red) internalize small Aβ puncta (green; white—the second row) indicated by white arrowheads (green) after 30 min, then externalize these puncta as large aggregates that are faintly X04 positive (blue; white—bottom row) indicated by white arrows, then form large, extracellular X04-positive plaque structures surrounded by microglia from 1 to 6 days. e Human iPSC-derived microglia were treated with 5 μM soluble Aβ species and various dynamin inhibitors (Dynasore, Dynole 4a, Dynole 34-2) at 0.6 μM for 24 h and plaque-like structures (Methoxy-X04-positive) were quantified as a percentage of control. Treating with dynamin inhibitors decreased plaque formation approximately fourfold in all conditions. f A summary of steps of microglia plaque formation observed. Data are presented as mean values +/− SEM and n = 4 wells. One-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: iPSC Microglia , BrainXell , E3/E3.

Techniques: Labeling, Cell Culture, Derivative Assay, Staining, Control

Journal: Nature Communications

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

doi: 10.1038/s41467-021-25344-6

Figure Lengend Snippet: a , b iPSC neurons and astrocytes treated with 5 μM sAβ42s followed by serial dilutions of anti-gD and anti-Aβ antibodies with IgG1 and LALAPG backbones with and without iPSC microglia. Results for dendrite protection (MAP2 area) ( a ) and synapse protection (synapsin count) ( b ) were analyzed via IC50 curve fitting using Prism software. Microglia provide baseline protection as shown by an upward shift in the anti-gD graph when microglia are added (gD IgG1 alone, blue; gD IgG1 + microglia, dark blue). Anti-Aβ antibody backbones protect dendrites and synapses similarly without microglia (Anti-Aβ IgG1, red; Anti-Aβ LALAPG, green) and with microglia (Anti-Aβ IgG1, dark red; Anti-Aβ LALAPG, dark green). c , d Neuron, astrocyte, microglia triculture treated with 5 μM sAβ42s (solid lines) and pro-inflammatory cytokines (dashed lines), then serial dilutions of gD antibody (black lines) and anti-Aβ antibody (red lines) were added. c Basal dendrite protection (MAP2 area) is lost in a neuroinflammatory environment, and anti-Aβ shows dose-dependent efficacy. d Plaque formation (Methoxy-X04 total intensity) increases in pro-inflammatory conditions, however, anti-Aβ shows similar plaque reduction. e iPSC microglia (red) were treated with 5 sAβ42s and serial dilutions of anti-Aβ antibody; no cells wells were used as control (blue). The supernatant was collected and anti-Aβ ELISA was performed to measure total Aβ concentration. Anti-Aβ antibody treatment increases soluble Aβ species present in the culture supernatant. f Summary of sequential events in the iPSC AD model. Data are presented as mean values +/− SEM and n = 4 wells. Two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: iPSC Microglia , BrainXell , E3/E3.

Techniques: Software, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Nature Communications

Article Title: Improved modeling of human AD with an automated culturing platform for iPSC neurons, astrocytes and microglia

doi: 10.1038/s41467-021-25344-6

Figure Lengend Snippet: ApoE genotype status.

Article Snippet: iPSC Microglia , BrainXell , E3/E3.

Techniques: