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Abstract Epigenetic mechanisms occurring in the brain as well as alterations in the gut microbiome composition might contribute to Alzheimer’s disease (AD). Human amyloid precursor protein knock-in (KI) mice contain the Swedish and Iberian mutations (App NL-F ) or those two and also the Arctic mutation (App NL-G-F ). In this study, we assessed whether behavioral and cognitive performance in 6-month-old App NL-F , App NL-G-F , and C57BL/6J wild-type (WT) mice was associated with the gut microbiome, and whether the genotype modulates this association. The genotype effects observed in behavioral tests were test-dependent. The biodiversity and composition of the gut microbiome linked to various aspects of mouse behavioral and cognitive performance but differences in genotype modulated these relationships. These genotype-dependent associations include members of the Lachnospiraceae and Ruminococcaceae families. In a subset of female mice, we assessed DNA methylation in the hippocampus and investigated whether alterations in hippocampal DNA methylation were associated with the gut microbiome. Among other differentially methylated regions, we identified a 1 Kb region that overlapped ing 3′UTR of the Tomm40 gene and the promoter region of the Apoe gene that and was significantly more methylated in the hippocampus of App NL-G-F than WT mice. The integrated gut microbiome hippocampal DNA methylation analysis revealed a positive relationship between amplicon sequence variants (ASVs) within the Lachnospiraceae family and methylation at the Apoe gene. Hence, these microbes may elicit an impact on AD-relevant behavioral and cognitive performance via epigenetic changes in AD-susceptibility genes in neural tissue or that such changes in the epigenome can elicit alterations in intestinal physiology that affect the growth of these taxa in the gut microbiome.

The gut microbiome and the gut brain axis are potential determinants of Alzheimer’s disease (AD) etiology or severity and gut microbiota might coordinate with the gut-brain axis to regulate behavioral phenotypes in AD mouse models. Using 6-month-old human amyloid precursor protein (hAPP) knock-in (KI) mice, which contain the Swedish and Iberian mutations [APP NL-F (AppNL–F)] or the Arctic mutation as third mutation [APP NL-G-F (AppNL–G–F)], behavioral and cognitive performance is associated with the gut microbiome and APP genotype modulates this association. In this study, we determined the feasibility of behavioral testing of mice in a biosafety cabinet and whether stool from 6-month-old AppNL–G–F mice or AppNL–G–F crossed with human apoE4 targeted replacement mice is sufficient to induce behavioral phenotypes in 4-5 month-old germ-free C57BL/6J mice 4 weeks following inoculation. We also compared the behavioral phenotypes of the recipient mice with that of the donor mice. Finally, we assessed cortical Aβ levels and analyzed the gut microbiome in the recipient mice. These results show that it is feasible to behaviorally test germ-free mice inside a biosafety cabinet. However, the host genotype was critical in modulating the pattern of induced behavioral phenotypes as compared to those seen in the genotype- and sex-match donor mice. Male mice that received stool from AppNL–G–F and AppNL–G–F/E4 donor genotypes tended to have lower body weight as compared to wild type, an effect not observed among donor mice. Additionally, AppNL–G–F/E4 recipient males, but not females, showed impaired object recognition. Insoluble Aβ40 levels were detected in AppNL–G–F and AppNL–G–F/E4 recipient mice. Recipients of AppNL–G–F, but not AppNL–G–F/E4, donor mice carried cortical insoluble Aβ40 levels that positively correlated with activity levels on the first and second day of open field testing. For recipient mice, the interaction between donor genotype and several behavioral scores predicted gut microbiome alpha-diversity. Similarly, two behavioral performance scores predicted microbiome composition in recipient mice, but this association was dependent on the donor genotype. These data suggest that genotypes of the donor and recipient might need to be considered for developing novel therapeutic strategies targeting the gut microbiome in AD and other neurodegenerative disorders.

Older adult populations are at risk for zinc deficiency, which may predispose them to immune dysfunction and age-related chronic inflammation that drives myriad diseases and disorders. Recent work also implicates the gut microbiome in the onset and severity of age-related inflammation, indicating that dietary zinc status and the gut microbiome may interact to impact age-related host immunity. We hypothesize that age-related alterations in the gut microbiome contribute to the demonstrated zinc deficits in host zinc levels and increased inflammation. We tested this hypothesis with a multifactor two-part study design in a C57BL/6 mouse model. The two studies included young (2 month old) and aged (24 month old) mice fed either (1) a zinc adequate or zinc supplemented diet, or (2) a zinc adequate or marginal zinc deficient diet, respectively. Overall microbiome composition did not significantly change with zinc status; beta diversity was driven almost exclusively by age effects. Microbiome differences due to age are evident at all taxonomic levels, with more than half of all taxonomic units significantly different. Furthermore, we found 150 out of 186 genera were significantly different between the two age groups, with Bacteriodes and Parabacteroides being the primary taxa of young and old mice, respectively. These data suggest that modulating individual micronutrient concentrations does not lead to comprehensive microbiome shifts, but rather affects specific components of the gut microbiome. However, a phylogenetic agglomeration technique (ClaaTU) revealed phylogenetic clades that respond to modulation of dietary zinc status and inflammation state in an age-dependent manner. Collectively, these results suggest that a complex interplay exists between host age, gut microbiome composition, and dietary zinc status.

High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3’ and 5’ adaptors to purified small RNAs; ligation products that lack a small RNA molecule (adaptor-adaptor products) are intentionally depleted. After cDNA synthesis, a linear PCR step amplifies the DNA fragments. The 3’ PCR primers used here include unique 6-nucleotide sequences to allow for multiplexing up to 12 samples.

Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.