Structural and Functional Brain Connectivity in Middle-Aged Carriers of Risk Alleles for Alzheimer\u27s Disease

Single nucleotide polymorphisms (SNPs) in APOE, COMT, BDNF, and KIBRA have been associated with age-related memory performance and executive functioning as well as risk for Alzheimer’s disease (AD). The purpose of the present investigation was to characterize differences in brain functional and structural integrity associated with these SNPs as potential endophenotypes of age-related cognitive decline. I focused my investigation on healthy, cognitively normal middle-aged adults, as disentangling the early effects of healthy versus pathological aging in this group may aid early detection and prevention of AD. The aims of the study were 1) to characterize SNP-related differences in functional connectivity within two resting state networks (RSNs; default mode network [DMN] and executive control network [ECN]) associated with memory and executive functioning, respectively; 2) to identify differences in the white matter (WM) microstructural integrity of tracts underlying these RSNs; and 3) to characterize genotype differences in the graph properties of an integrated functional-structural network. Participants (age 40-60, N = 150) underwent resting state functional magnetic resonance imaging (rs-fMRI), diffusion tensor imaging (DTI), and genotyping. Independent components analysis (ICA) was used to derive RSNs, while probabilistic tractography was performed to characterize tracts connecting RSN subregions. A technique known as functional-by-structural hierarchical (FSH) mapping was used to create the integrated, whole brain functional-structural network, or resting state structural connectome (rsSC). I found that BDNF risk allele carriers had lower functional connectivity within the DMN, while KIBRA risk allele carriers had poorer WM microstructural integrity in tracts underlying the DMN and ECN. In addition to these differences in the connectivity of specific RSNs, I found significant impairments in the global and local topology of the rsSC across all evaluated SNPs. Collectively, these findings suggest that integrating multiple neuroimaging modalities and using graph theoretical analysis may reveal network-level vulnerabilities that may serve as biomarkers of age-related cognitive decline in middle age, decades before the onset of overt cognitive impairment

Figure 7

Acrobat files containing the original scanned images of the Western blot used for Figure 7 and additional confirmatory Western blots as well as descriptions of the samples in each lane. The file containing the blot used in the figure is Sheahan_2023_05.22.12_Western.pdf

Figure 4

Acrobat files containing the original scanned images of the Western blots used for Figures 4A and 4B and additional confirmatory Western blots as well as descriptions of the samples in each lane. The files containing the blots used in the figures are Sheahan_2023_7.23.10a_Western.pdf (figure 4A) and Sheahan_2023_6.12.12_Western.pdf (figure 4B)

Table 2

Sheahan_2023_table2_stats.pzfx - Graphpad Prism file with data for the graphs and statistics presented in Table 2 Sheahan_2023_table2_data.xlsx - Excel spreadsheet of all the raw data used to build Table

Figures 1 and S3

Confocal Z-series .nd2 files (Nikon NIS) showing GFP fluorescence from dissected egg chambers. These files were used to create the maximum intensity projections displayed in figure 1 (UAS-GFPnls drd-gal4 stage 10B and 14 10.27.16.nd2) and figure S3 (UAS-GFPnls drd-gal4 stage 14 7.16.21.nd2 and UAS-GFPnls control stage 14 7.16.21.nd2). Files with “UAS-GFPnls drd-gal4” were experimental samples containing both the drd-GAL4 and UAS-GFP.nls transgenes, while files with “UAS-GFPnls control” were controls lacking the drd-GAL4 transgene. The “10.27.16” and “7.16.21” file sets were imaged on different days with different settings, so image intensities should not be compared between the two file sets

Figure 3

Sheahan_2023_7.23.10_Western.pdf - Acrobat file containing the original scanned image of the Western blot used for Figure 3 as well as a description of the samples in each lane

Figure 6

Sheahan_2023_figure6_stats.pzfx -Graphpad Prism file with data for the graphs and statistics presented in Figure 6 Sheahan_2023_figure6_data.xlsx - Excel spreadsheet of all the raw data used to build Figure

The Drosophila drop-dead gene is required for eggshell integrity.

The eggshell of the fruit fly Drosophila melanogaster is a useful model for understanding the synthesis of a complex extracellular matrix. The eggshell is synthesized during mid-to-late oogenesis by the somatic follicle cells that surround the developing oocyte. We previously reported that female flies mutant for the gene drop-dead (drd) are sterile, but the underlying cause of the sterility remained unknown. In this study, we examined the role of drd in eggshell synthesis. We show that eggs laid by drd mutant females are fertilized but arrest early in embryogenesis, and that the innermost layer of the eggshell, the vitelline membrane, is abnormally permeable to dye in these eggs. In addition, the major vitelline membrane proteins fail to become crosslinked by nonreducible bonds, a process that normally occurs during egg activation following ovulation, as evidenced by their solubility and detection by Western blot in laid eggs. In contrast, the Cp36 protein, which is found in the outer chorion layers of the eggshell, becomes crosslinked normally. To link the drd expression pattern with these phenotypes, we show that drd is expressed in the ovarian follicle cells beginning in mid-oogenesis, and, importantly, that all drd mutant eggshell phenotypes could be recapitulated by selective knockdown of drd expression in the follicle cells. To determine whether drd expression was required for the crosslinking itself, we performed in vitro activation and crosslinking experiments. The vitelline membranes of control egg chambers could become crosslinked either by incubation in hyperosmotic medium, which activates the egg chambers, or by exogenous peroxidase and hydrogen peroxide. In contrast, neither treatment resulted in the crosslinking of the vitelline membrane in drd mutant egg chambers. These results indicate that drd expression in the follicle cells is necessary for vitelline membrane proteins to serve as substrates for peroxidase-mediated cross-linking at the end of oogenesis

Figure 5

Sheahan_2023_figure5_stats.pzfx - Graphpad Prism file with data for the graphs and statistics presented in Figure 5 Sheahan_2023_figure5_data.xlsx -Excel spreadsheet of all the raw data used to build Figure 5 The subfolders contain confocal image .nd2 files (Nikon NIS) showing GFP fluorescence from ovaries immunostained against Vm26Ab. Images in “homozygote” subfolders are from drd1/drd1 females and images in “heterozygote” subfolders are from drd1/FM7c females. The images in the “size only” folders were taken at different camera settings and so were only used for measurements of oocyte dimensions and not VM brightness or width