Journal: Nature Communications

Article Title: DNA methylation signatures of Alzheimer’s disease neuropathology in the cortex are primarily driven by variation in non-neuronal cell-types

doi: 10.1038/s41467-022-33394-7

Figure Lengend Snippet: Characteristics of the BDR samples profiled in this study

Article Snippet: Briefly, following tissue homogenization and nuclei purification using sucrose gradient centrifugation we used a FACS Aria III cell sorter (BD Biosciences) to simultaneously collect populations of NeuN+ (neuronal-enriched) (R&D systems, Cat No: NL2864R, dilution: 1:10) and SOX10+ (oligodendrocyte-enriched) (Millipore, Cat No: MAB377X, dilution: 1:1000) immunolabeled populations from bulk DLPFC tissue prior to genomic profiling, with the double-negative fraction and an aliquot of the “total” nuclei fraction (analogous to “bulk” cortex) also being collected from each tissue sample (Supplementary Fig. ).

Techniques: Purification

Journal: Nature Communications

Article Title: DNA methylation signatures of Alzheimer’s disease neuropathology in the cortex are primarily driven by variation in non-neuronal cell-types

doi: 10.1038/s41467-022-33394-7

Figure Lengend Snippet: Using linear regression models controlling for major covariates (see Methods) we show that a levels of tau pathology (measured using Braak NFT stage) are significantly associated with the proportion of NeuN+ cells (effect size = −2.74, SE = 0.705, P = 1.15E–04), SOX10+ cells (effect size = 1.60, SE = 0.423, P = 1.72E–04) and NeuN–/SOX10– cells (effect size = −2.00, SE = 0.687, P = 0.004) in the DLPFC ( N = 597 donors) using cell proportion estimates derived from “bulk” DNA methylation data. b In contrast no associations ( P > 0.008) between levels of tau pathology and cell proportion estimates derived from “bulk” DNA methylation data were observed in the OCC ( N = 598 donors). Boxplots of the estimated proportion of each cell-type across Braak NFT stages are shown, where the middle box represents the interquartile range (IQR), the middle line represents the median, and the whisker lines represent the minimum (quartile 1 –1.5 × IQR) and the maximum (quartile 3 + 1.5 × IQR). Tau pathology (Braak NFT stage) is shown on the x- axis split by cell-type and estimated cell proportions are shown on the y -axis. A similar pattern of results was found for levels of amyloid pathology as shown in Supplementary Fig. .

Article Snippet: Briefly, following tissue homogenization and nuclei purification using sucrose gradient centrifugation we used a FACS Aria III cell sorter (BD Biosciences) to simultaneously collect populations of NeuN+ (neuronal-enriched) (R&D systems, Cat No: NL2864R, dilution: 1:10) and SOX10+ (oligodendrocyte-enriched) (Millipore, Cat No: MAB377X, dilution: 1:1000) immunolabeled populations from bulk DLPFC tissue prior to genomic profiling, with the double-negative fraction and an aliquot of the “total” nuclei fraction (analogous to “bulk” cortex) also being collected from each tissue sample (Supplementary Fig. ).

Techniques: Derivative Assay, DNA Methylation Assay, Whisker Assay

Journal: Nature Communications

Article Title: DNA methylation signatures of Alzheimer’s disease neuropathology in the cortex are primarily driven by variation in non-neuronal cell-types

doi: 10.1038/s41467-022-33394-7

Figure Lengend Snippet: We compared effect sizes for the 334 overlapping tau-associated DMPs identified in our “bulk” cortex meta-analysis with those at the same sites in an analysis of purified DLPFC nuclei populations from low (Braak NFT stage 0 to II) and high (Braak NFT stage >V) tau-pathology donors. Shown is a comparison of effect sizes between the meta-analysis (bulk, N = 2013 individuals]) and the a total nuclei (bulk) nuclei fraction ( N = 26) (direction of effect = 87% concordant, sign-test P = 7.24E–46); b NeuN+ (neuron-enriched) nuclei fraction ( N = 27) (direction of effect = 60% concordant, sign-test P = 7.59E–05), c SOX10+ (oligodendrocyte-enriched) nuclei fraction ( N = 28) (direction of effect = 67% concordant, sign-test P = 2.15E–10), and d double-negative (microglia- and astrocyte-enriched) nuclei population ( N = 21) (direction of effect = 96% concordant, sign-test P = 1.2E–75). The x- axis shows effect sizes from the bulk cortex meta-analysis and the y -axis shows effect sizes for those same DMPs in each purified nuclei population. Gray dashed line represents y = x . e Bar-plots of the mean absolute relative effect sizes in each purified nuclei population compared to the bulk cortex across the 334 tau-associated DMPs, with error bars denoting the 95% confidence intervals.

Article Snippet: Briefly, following tissue homogenization and nuclei purification using sucrose gradient centrifugation we used a FACS Aria III cell sorter (BD Biosciences) to simultaneously collect populations of NeuN+ (neuronal-enriched) (R&D systems, Cat No: NL2864R, dilution: 1:10) and SOX10+ (oligodendrocyte-enriched) (Millipore, Cat No: MAB377X, dilution: 1:1000) immunolabeled populations from bulk DLPFC tissue prior to genomic profiling, with the double-negative fraction and an aliquot of the “total” nuclei fraction (analogous to “bulk” cortex) also being collected from each tissue sample (Supplementary Fig. ).

Techniques: Purification, Comparison

Journal: Biology Open

Article Title: Neuronal cell culture from transgenic zebrafish models of neurodegenerative disease

doi: 10.1242/bio.036475

Figure Lengend Snippet: Workflow for culturing zebrafish neurons. Zebrafish embryos from 24 hpf or 48 hpf aged embryos were collected, dechorionated (with fine forceps) and placed into microtubes with E3 medium and 16 µM tricaine. Embryos were then washed three times with ice-cold E3 medium before being placed into 1× trypsin (in PBS) and pipetted intermittently for 30 min within a 37°C water bath. FBS was then added to stop dissociation and the tubes were then centrifuged for 3 min at 180 rcf (1000 rpm). The supernatant was removed and the cell pellet was resuspended in HBSS. Using a hemacytometer, approximately 500,000 cells were plated onto 12 mm coverslips pre-coated with poly-D-lysine and cultured in neurobasal media. Half this media was changed daily.

Article Snippet: Cells were placed in neuron-enriching Neurobasal™ media supplemented with 2% B27, L-alanyl-L-glutamine and antimycotic (Invitrogen).

Techniques: Cell Culture