Genetic and multi-omic risk assessment of Alzheimer's disease implicates core associated biological domains.

INTRODUCTION: Alzheimer's disease (AD) is the predominant dementia globally, with heterogeneous presentation and penetrance of clinical symptoms, variable presence of mixed pathologies, potential disease subtypes, and numerous associated endophenotypes. Beyond the difficulty of designing treatments that address the core pathological characteristics of the disease, therapeutic development is challenged by the uncertainty of which endophenotypic areas and specific targets implicated by those endophenotypes to prioritize for further translational research. However, publicly funded consortia driving large-scale open science efforts have produced multiple omic analyses that address both disease risk relevance and biological process involvement of genes across the genome. METHODS: Here we report the development of an informatic pipeline that draws from genetic association studies, predicted variant impact, and linkage with dementia associated phenotypes to create a genetic risk score. This is paired with a multi-omic risk score utilizing extensive sets of both transcriptomic and proteomic studies to identify system-level changes in expression associated with AD. These two elements combined constitute our target risk score that ranks AD risk genome-wide. The ranked genes are organized into endophenotypic space through the development of 19 biological domains associated with AD in the described genetics and genomics studies and accompanying literature. The biological domains are constructed from exhaustive Gene Ontology (GO) term compilations, allowing automated assignment of genes into objectively defined disease-associated biology. This rank-and-organize approach, performed genome-wide, allows the characterization of aggregations of AD risk across biological domains. RESULTS: The top AD-risk-associated biological domains are Synapse, Immune Response, Lipid Metabolism, Mitochondrial Metabolism, Structural Stabilization, and Proteostasis, with slightly lower levels of risk enrichment present within the other 13 biological domains. DISCUSSION: This provides an objective methodology to localize risk within specific biological endophenotypes and drill down into the most significantly associated sets of GO terms and annotated genes for potential therapeutic targets.

Characterization of covalent inhibitors that disrupt the interaction between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.

Characterization of covalent inhibitors that disrupt the interaction between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM.

Echinobase: leveraging an extant model organism database to build a knowledgebase supporting research on the genomics and biology of echinoderms.

Echinobase (www.echinobase.org) is a third generation web resource supporting genomic research on echinoderms. The new version was built by cloning the mature Xenopus model organism knowledgebase, Xenbase, refactoring data ingestion pipelines and modifying the user interface to adapt to multispecies echinoderm content. This approach leveraged over 15 years of previous database and web application development to generate a new fully featured informatics resource in a single year. In addition to the software stack, Echinobase uses the private cloud and physical hosts that support Xenbase. Echinobase currently supports six echinoderm species, focused on those used for genomics, developmental biology and gene regulatory network analyses. Over 38 000 gene pages, 18 000 publications, new improved genome assemblies, JBrowse genome browser and BLAST + services are available and supported by the development of a new echinoderm anatomical ontology, uniformly applied formal gene nomenclature, and consistent orthology predictions. A novel feature of Echinobase is integrating support for multiple, disparate species. New genomes from the diverse echinoderm phylum will be added and supported as data becomes available. The common code development design of the integrated knowledgebases ensures parallel improvements as each resource evolves. This approach is widely applicable for developing new model organism informatics resources.

A nomenclature for echinoderm genes.

Echinoderm embryos and larvae are prominent experimental model systems for studying developmental mechanisms. High-quality, assembled, annotated genome sequences are now available for several echinoderm species, including representatives from most classes. The increased availability of these data necessitates the development of a nomenclature that assigns universally interpretable gene symbols to echinoderm genes to facilitate cross-species comparisons of gene functions, both within echinoderms and across other phyla. This paper describes the implementation of an improved set of echinoderm gene nomenclature guidelines that both communicates meaningful orthology information in protein-coding gene symbols and names and establishes continuity with nomenclatures developed for major vertebrate model organisms, including humans. Differences between the echinoderm gene nomenclature guidelines and vertebrate guidelines are examined and explained. This nomenclature incorporates novel solutions to allow for several types of orthologous relationships, including the single echinoderm genes with multiple vertebrate co-orthologs that result from whole-genome-duplication events. The current version of the Echinoderm Gene Nomenclature Guidelines can be found at https://www.echinobase.org/gene/static/geneNomenclature.jsp Database URL https://www.echinobase.org/.

Systematic comparison of sea urchin and sea star developmental gene regulatory networks explains how novelty is incorporated in early development.

The extensive array of morphological diversity among animal taxa represents the product of millions of years of evolution. Morphology is the output of development, therefore phenotypic evolution arises from changes to the topology of the gene regulatory networks (GRNs) that control the highly coordinated process of embryogenesis. A particular challenge in understanding the origins of animal diversity lies in determining how GRNs incorporate novelty while preserving the overall stability of the network, and hence, embryonic viability. Here we assemble a comprehensive GRN for endomesoderm specification in the sea star from zygote through gastrulation that corresponds to the GRN for sea urchin development of equivalent territories and stages. Comparison of the GRNs identifies how novelty is incorporated in early development. We show how the GRN is resilient to the introduction of a transcription factor, pmar1, the inclusion of which leads to a switch between two stable modes of Delta-Notch signaling. Signaling pathways can function in multiple modes and we propose that GRN changes that lead to switches between modes may be a common evolutionary mechanism for changes in embryogenesis. Our data additionally proposes a model in which evolutionarily conserved network motifs, or kernels, may function throughout development to stabilize these signaling transitions.

Developmental transcriptomes of the sea star, Patiria miniata, illuminate how gene expression changes with evolutionary distance.

Understanding how changes in developmental gene expression alter morphogenesis is a fundamental problem in development and evolution. A promising approach to address this problem is to compare the developmental transcriptomes between related species. The echinoderm phylum consists of several model species that have significantly contributed to the understanding of gene regulation and evolution. Particularly, the regulatory networks of the sea star, Patiria miniata (P. miniata), have been extensively studied, however developmental transcriptomes for this species were lacking. Here we generated developmental transcriptomes of P. miniata and compared these with those of two sea urchins species. We demonstrate that the conservation of gene expression depends on gene function, cell type and evolutionary distance. With increasing evolutionary distance the interspecies correlations in gene expression decreases. The reduction is more severe in the correlations between morphologically equivalent stages (diagonal elements) than in the correlation between morphologically distinct stages (off-diagonal elements). This could reflect a decrease in the morphological constraints compared to other constraints that shape gene expression at large evolutionary divergence. Within this trend, the interspecies correlations of developmental control genes maintain their diagonality at large evolutionary distance, and peak at the onset of gastrulation, supporting the hourglass model of phylotypic stage conservation.

Genomic resources for the study of echinoderm development and evolution.

Echinoderms are important research models for a wide range of biological questions. In particular, echinoderm embryos are exemplary models for dissecting the molecular and cellular processes that drive development and testing how these processes can be modified through evolution to produce the extensive morphological diversity observed in the phylum. Modern attempts to characterize these processes depend on some level of genomic analysis; from querying annotated gene sets to functional genomics experiments to identify candidate cis-regulatory sequences. Given how essential these data have become, it is important that researchers using available datasets or performing their own genome-scale experiments understand the nature and limitations of echinoderm genomic analyses. In this chapter we highlight the current state of echinoderm genomic data and provide methodological considerations for common approaches, including analysis of transcriptome and functional genomics datasets.

Analysis of sea star larval regeneration reveals conserved processes of whole-body regeneration across the metazoa.

BACKGROUND: Metazoan lineages exhibit a wide range of regenerative capabilities that vary among developmental stage and tissue type. The most robust regenerative abilities are apparent in the phyla Cnidaria, Platyhelminthes, and Echinodermata, whose members are capable of whole-body regeneration (WBR). This phenomenon has been well characterized in planarian and hydra models, but the molecular mechanisms of WBR are less established within echinoderms, or any other deuterostome system. Thus, it is not clear to what degree aspects of this regenerative ability are shared among metazoa. RESULTS: We characterize regeneration in the larval stage of the Bat Star (Patiria miniata). Following bisection along the anterior-posterior axis, larvae progress through phases of wound healing and re-proportioning of larval tissues. The overall number of proliferating cells is reduced following bisection, and we find evidence for a re-deployment of genes with known roles in embryonic axial patterning. Following axial respecification, we observe a significant localization of proliferating cells to the wound region. Analyses of transcriptome data highlight the molecular signatures of functions that are common to regeneration, including specific signaling pathways and cell cycle controls. Notably, we find evidence for temporal similarities among orthologous genes involved in regeneration from published Platyhelminth and Cnidarian regeneration datasets. CONCLUSIONS: These analyses show that sea star larval regeneration includes phases of wound response, axis respecification, and wound-proximal proliferation. Commonalities of the overall process of regeneration, as well as gene usage between this deuterostome and other species with divergent evolutionary origins reveal a deep similarity of whole-body regeneration among the metazoa.

EchinoBase: Tools for Echinoderm Genome Analyses.

The echinoderms are a phylum of invertebrate deuterostome animals that constitute important research models for a number of biological disciplines. EchinoBase ( www.echinobase.org ) is an echinoderm-specific genome database and web information system that provides a platform for the interrogation and exploration of echinoderm genomic data. This chapter outlines the datasets available on EchinoBase; from assembled genomes and genome annotations, to spatial and quantitative expression data, as well as functional genomics datasets. We also highlight the bioinformatic tools available on the website to facilitate rapid inquiries using these data (genome browsers, precompiled BLAST databases, etc.), and suggest optimized strategies for performing these inquiries. We conclude with a perspective on how one could integrate various genomic resources to predict putative noncoding regulatory regions. The available datasets and analyses they permit provide the basic components required for developing an understanding of how echinoderm genomes are regulated, especially during early development, and provides a platform for comparative genomic inquiries among species in this phylum.

Genome-wide use of high- and low-affinity Tbrain transcription factor binding sites during echinoderm development.

Sea stars and sea urchins are model systems for interrogating the types of deep evolutionary changes that have restructured developmental gene regulatory networks (GRNs). Although cis-regulatory DNA evolution is likely the predominant mechanism of change, it was recently shown that Tbrain, a Tbox transcription factor protein, has evolved a changed preference for a low-affinity, secondary binding motif. The primary, high-affinity motif is conserved. To date, however, no genome-wide comparisons have been performed to provide an unbiased assessment of the evolution of GRNs between these taxa, and no study has attempted to determine the interplay between transcription factor binding motif evolution and GRN topology. The study here measures genome-wide binding of Tbrain orthologs by using ChIP-sequencing and associates these orthologs with putative target genes to assess global function. Targets of both factors are enriched for other regulatory genes, although nonoverlapping sets of functional enrichments in the two datasets suggest a much diverged function. The number of low-affinity binding motifs is significantly depressed in sea urchins compared with sea star, but both motif types are associated with genes from a range of functional categories. Only a small fraction (∼10%) of genes are predicted to be orthologous targets. Collectively, these data indicate that Tbr has evolved significantly different developmental roles in these echinoderms and that the targets and the binding motifs in associated cis-regulatory sequences are dispersed throughout the hierarchy of the GRN, rather than being biased toward terminal process or discrete functional blocks, which suggests extensive evolutionary tinkering.

Echinoderm development and evolution in the post-genomic era.

The highly recognizable animals within the phylum Echinodermata encompass an enormous disparity of adult and larval body plans. The extensive knowledge of sea urchin development has culminated in the description of the exquisitely detailed gene regulatory network (GRN) that governs the specification of various embryonic territories. This information provides a unique opportunity for comparative studies in other echinoderm taxa to understand the evolution and developmental mechanisms underlying body plan change. This review focuses on recent work that has utilized new genomic resources and systems-level experiments to address questions of evolutionary developmental biology. In particular, we synthesize the results of several recent studies from various echinoderm classes that have explored the development and evolution of the larval skeleton, which is a major feature that distinguishes the two predominant larval subtypes within the Phylum. We specifically examine the ways in which GRNs can evolve, either through cis regulatory and/or protein-level changes in transcription factors. We also examine recent work comparing evolution across shorter time scales that occur within and between species of sea urchin, and highlight the kinds of questions that can be addressed by these comparisons. The advent of new genomic and transcriptomic datasets in additional species from all classes of echinoderm will continue to empower the use of these taxa for evolutionary developmental studies.

Proteomic Analysis of Dhh1 Complexes Reveals a Role for Hsp40 Chaperone Ydj1 in Yeast P-Body Assembly.

P-bodies (PB) are ribonucleoprotein (RNP) complexes that aggregate into cytoplasmic foci when cells are exposed to stress. Although the conserved mRNA decay and translational repression machineries are known components of PB, how and why cells assemble RNP complexes into large foci remain unclear. Using mass spectrometry to analyze proteins immunoisolated with the core PB protein Dhh1, we show that a considerable number of proteins contain low-complexity sequences, similar to proteins highly represented in mammalian RNP granules. We also show that the Hsp40 chaperone Ydj1, which contains an low-complexity domain and controls prion protein aggregation, is required for the formation of Dhh1-GFP foci on glucose depletion. New classes of proteins that reproducibly coenrich with Dhh1-GFP during PB induction include proteins involved in nucleotide or amino acid metabolism, glycolysis, transfer RNA aminoacylation, and protein folding. Many of these proteins have been shown to form foci in response to other stresses. Finally, analysis of RNA associated with Dhh1-GFP shows enrichment of mRNA encoding the PB protein Pat1 and catalytic RNAs along with their associated mitochondrial RNA-binding proteins. Thus, global characterization of PB composition has uncovered proteins important for PB assembly and evidence suggesting an active role for RNA in PB function.

Unidirectional P-body transport during the yeast cell cycle.

P-bodies belong to a large family of RNA granules that are associated with post-transcriptional gene regulation, conserved from yeast to mammals, and influence biological processes ranging from germ cell development to neuronal plasticity. RNA granules can also transport RNAs to specific locations. Germ granules transport maternal RNAs to the embryo, and neuronal granules transport RNAs long distances to the synaptic dendrites. Here we combine microfluidic-based fluorescent microscopy of single cells and automated image analysis to follow p-body dynamics during cell division in yeast. Our results demonstrate that these highly dynamic granules undergo a unidirectional transport from the mother to the daughter cell during mitosis as well as a constrained "hovering" near the bud site half an hour before the bud is observable. Both behaviors are dependent on the Myo4p/She2p RNA transport machinery. Furthermore, single cell analysis of cell size suggests that PBs play an important role in daughter cell growth under nutrient limiting conditions.

Identification and characterization of a drug-sensitive strain enables puromycin-based translational assays in Saccharomyces cerevisiae.

Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C-terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6∆, pdr1∆ and pdr3∆ (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin-based assays in yeast, which will enable new avenues of inquiry into protein production and maturation.

Melatonin: neuritogenesis and neuroprotective effects in crustacean x-organ cells.

Melatonin has both neuritogenic and neuroprotective effects in mammalian cell lines such as neuroblastoma cells. The mechanisms of action include receptor-coupled processes, direct binding and modulation of calmodulin and protein kinase C, and direct scavenging of free radicals. While melatonin is produced in invertebrates and has influences on their physiology and behavior, little is known about its mechanisms of action. We studied the influence of melatonin on neuritogenesis in well-differentiated, extensively-arborized crustacean x-organ neurosecretory neurons. Melatonin significantly increased neurite area in the first 24h of culture. The more physiological concentrations, 1 nM and 1 pM, increased area at 48 h also, whereas the pharmacological 1 µM concentration appeared to have desensitizing effects by this time. Luzindole, a vertebrate melatonin receptor antagonist, had surprising and significant agonist-like effects in these invertebrate cells. Melatonin receptors have not yet been studied in invertebrates. However, the presence of membrane-bound receptors in this population of crustacean neurons is indicated by this study. Melatonin also has significant neuroprotective effects, reversing the inhibition of neuritogenesis by 200 and 500 µM hydrogen peroxide. Because this is at least in part a direct action not requiring a receptor, melatonin's protection from oxidative stress is not surprisingly phylogenetically-conserved.

Androgen receptor function in motor neuron survival and degeneration.

Polyglutamine repeat expansion in the androgen receptor is responsible for the motor neuron degeneration in X-linked spinal and bulbar muscular atrophy (SBMA; Kennedy's disease). This mutation, like the other polyglutamine repeat expansions, has proven to be toxic itself by a gain-of-function effect; however, a growing body of evidence indicates that loss of androgen receptor normal function simultaneously contributes to SBMA disease pathology, and, conversely, that normal androgen receptor signaling mediates important trophic effects upon motor neurons. This review considers the trophic requirements of motor neurons, focusing upon the role of known neurotrophic factors in motor neuron disease natural history, and the interactions of androgen receptor signaling pathways with motor neuron disease pathogenesis and progression. A thorough understanding of androgen receptor signaling in motor neurons should provide important inroads toward the development of effective treatments for a variety of devastating motor neuron diseases.