Landscape of brain myeloid cell transcriptome along the spatiotemporal progression of Alzheimer's disease reveals distinct sequential responses to Aß and tau.

Human microglia are critically involved in Alzheimer's disease (AD) progression, as shown by genetic and molecular studies. However, their role in tau pathology progression in human brain has not been well described. Here, we characterized 32 human donors along progression of AD pathology, both in time-from early to late pathology-and in space-from entorhinal cortex (EC), inferior temporal gyrus (ITG), prefrontal cortex (PFC) to visual cortex (V2 and V1)-with biochemistry, immunohistochemistry, and single nuclei-RNA-sequencing, profiling a total of 337,512 brain myeloid cells, including microglia. While the majority of microglia are similar across brain regions, we identified a specific subset unique to EC which may contribute to the early tau pathology present in this region. We calculated conversion of microglia subtypes to diseased states and compared conversion patterns to those from AD animal models. Targeting genes implicated in this conversion, or their upstream/downstream pathways, could halt gene programs initiated by early tau progression. We used expression patterns of early tau progression to identify genes whose expression is reversed along spreading of spatial tau pathology (EC > ITG > PFC > V2 > V1) and identified their potential involvement in microglia subtype conversion to a diseased state. This study provides a data resource that builds on our knowledge of myeloid cell contribution to AD by defining the heterogeneity of microglia and brain macrophages during both temporal and regional pathology aspects of AD progression at an unprecedented resolution.

Distinct transcriptomic responses to Aß plaques, neurofibrillary tangles, and APOE in Alzheimer's disease.

INTRODUCTION: Omics studies have revealed that various brain cell types undergo profound molecular changes in Alzheimer's disease (AD) but the spatial relationships with plaques and tangles and APOE-linked differences remain unclear. METHODS: We performed laser capture microdissection of amyloid beta (Aß) plaques, the 50 µm halo around them, tangles with the 50 µm halo around them, and areas distant (> 50 µm) from plaques and tangles in the temporal cortex of AD and control donors, followed by RNA-sequencing. RESULTS: Aß plaques exhibited upregulated microglial (neuroinflammation/phagocytosis) and downregulated neuronal (neurotransmission/energy metabolism) genes, whereas tangles had mostly downregulated neuronal genes. Aß plaques had more differentially expressed genes than tangles. We identified a gradient Aß plaque > peri-plaque > tangle > distant for these changes. AD APOE ε4 homozygotes had greater changes than APOE ε3 across locations, especially within Aß plaques. DISCUSSION: Transcriptomic changes in AD consist primarily of neuroinflammation and neuronal dysfunction, are spatially associated mainly with Aß plaques, and are exacerbated by the APOE ε4 allele.

Endothelial Cells Are Heterogeneous in Different Brain Regions and Are Dramatically Altered in Alzheimer's Disease.

Vascular endothelial cells play an important role in maintaining brain health, but their contribution to Alzheimer's disease (AD) is obscured by limited understanding of the cellular heterogeneity in normal aged brain and in disease. To address this, we performed single nucleus RNAseq on tissue from 32 human AD and non-AD donors (19 female, 13 male) each with five cortical regions: entorhinal cortex, inferior temporal gyrus, prefrontal cortex, visual association cortex, and primary visual cortex. Analysis of 51,586 endothelial cells revealed unique gene expression patterns across the five regions in non-AD donors. Alzheimer's brain endothelial cells were characterized by upregulated protein folding genes and distinct transcriptomic differences in response to amyloid ß plaques and cerebral amyloid angiopathy. This dataset demonstrates previously unrecognized regional heterogeneity in the endothelial cell transcriptome in both aged non-AD and AD brain.SIGNIFICANCE STATEMENT In this work, we show that vascular endothelial cells collected from five different brain regions display surprising variability in gene expression. In the presence of Alzheimer's disease pathology, endothelial cell gene expression is dramatically altered with clear differences in regional and temporal changes. These findings help explain why certain brain regions appear to differ in susceptibility to disease-related vascular remodeling events that may impact blood flow.

Distinct Transcriptomic Responses to Aß plaques, Neurofibrillary Tangles, and APOE in Alzheimer's Disease.

INTRODUCTION: Omics studies have revealed that various brain cell types undergo profound molecular changes in Alzheimer's disease (AD) but the spatial relationships with plaques and tangles and APOE -linked differences remain unclear. METHODS: We performed laser capture microdissection of Aß plaques, the 50µm halo around them, tangles with the 50µm halo around them, and areas distant (>50µm) from plaques and tangles in the temporal cortex of AD and control donors, followed by RNA-sequencing. RESULTS: Aß plaques exhibited upregulated microglial (neuroinflammation/phagocytosis) and downregulated neuronal (neurotransmission/energy metabolism) genes, whereas tangles had mostly downregulated neuronal genes. Aß plaques had more differentially expressed genes than tangles. We identified a gradient Aß plaque>peri-plaque>tangle>distant for these changes. AD APOE ε4 homozygotes had greater changes than APOE ε3 across locations, especially within Aß plaques. DISCUSSION: Transcriptomic changes in AD consist primarily of neuroinflammation and neuronal dysfunction, are spatially associated mainly with Aß plaques, and are exacerbated by the APOE ε4 allele.

Endothelial Cells are Heterogeneous in Different Brain Regions and are Dramatically Altered in Alzheimer's Disease.

Vascular endothelial cells play an important role in maintaining brain health, but their contribution to Alzheimer's disease (AD) is obscured by limited understanding of the cellular heterogeneity in normal aged brain and in disease. To address this, we performed single nucleus RNAseq on tissue from 32 AD and non-AD donors each with five cortical regions: entorhinal cortex, inferior temporal gyrus, prefrontal cortex, visual association cortex and primary visual cortex. Analysis of 51,586 endothelial cells revealed unique gene expression patterns across the five regions in non-AD donors. Alzheimer's brain endothelial cells were characterized by upregulated protein folding genes and distinct transcriptomic differences in response to amyloid beta plaques and cerebral amyloid angiopathy (CAA). This dataset demonstrates previously unrecognized regional heterogeneity in the endothelial cell transcriptome in both aged non-AD and AD brain. Significance Statement: In this work, we show that vascular endothelial cells collected from five different brain regions display surprising variability in gene expression. In the presence of Alzheimer's disease pathology, endothelial cell gene expression is dramatically altered with clear differences in regional and temporal changes. These findings help explain why certain brain regions appear to differ in susceptibility to disease-related vascular remodeling events that may impact blood flow.

MEK1/2 activity modulates TREM2 cell surface recruitment.

Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer's disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface. Inhibitors of the kinases MEK1/2 displayed the strongest and most consistent increases in cell surface TREM2 protein, identifying a previously unreported pathway for TREM2 regulation. Unexpectedly, inhibitors of the downstream effector ERK kinases did not have the same effect, suggesting that noncanonical MEK signaling regulates TREM2 trafficking. In addition, siRNA knockdown experiments confirmed that decreased MEK1 and MEK2 were required for this recruitment. In iPSC-derived microglia, MEK inhibition increased cell surface TREM2 only modestly, so various cytokines were used to alter iPSC microglia phenotype, making cells more sensitive to MEK inhibitor-induced TREM2 recruitment. Of those tested, only IFN-gamma priming prior to MEK inhibitor treatment resulted in greater TREM2 recruitment. These data identify the first known mechanisms for increasing surface TREM2 protein and TREM2-regulated function in human myeloid cells and are the first to show a role for MEK1/MEK2 signaling in TREM2 activity.

Arrayed CRISPR reveals genetic regulators of tau aggregation, autophagy and mitochondria in Alzheimer's disease model.

Alzheimer's disease (AD) is a common neurodegenerative disease with poor prognosis. New options for drug discovery targets are needed. We developed an imaging based arrayed CRISPR method to interrogate the human genome for modulation of in vitro correlates of AD features, and used this to assess 1525 human genes related to tau aggregation, autophagy and mitochondria. This work revealed (I) a network of tau aggregation modulators including the NF-κB pathway and inflammatory signaling, (II) a correlation between mitochondrial morphology, respiratory function and transcriptomics, (III) machine learning predicted novel roles of genes and pathways in autophagic processes and (IV) individual gene function inferences and interactions among biological processes via multi-feature clustering. These studies provide a platform to interrogate underexplored aspects of AD biology and offer several specific hypotheses for future drug discovery efforts.

Microglial Drug Targets in AD: Opportunities and Challenges in Drug Discovery and Development.

Alzheimer's disease (AD) is a large and increasing unmet medical need with no disease-modifying treatment currently available. Genetic evidence from genome-wide association studies (GWASs) and gene network analysis has clearly revealed a key role of the innate immune system in the brain, of which microglia are the most important element. Single-nucleotide polymorphisms (SNPs) in genes predominantly expressed in microglia have been associated with altered risk of developing AD. Furthermore, microglia-specific pathways are affected on the messenger RNA (mRNA) expression level in post-mortem AD tissue and in mouse models of AD. Together these findings have increased the interest in microglia biology, and numerous scientific reports have proposed microglial molecules and pathways as drug targets for AD. Target identification and validation are generally the first steps in drug discovery. Both target validation and drug lead identification for central nervous system (CNS) targets and diseases entail additional significant obstacles compared to peripheral targets and diseases. This makes CNS drug discovery, even with well-validated targets, challenging. In this article, we will illustrate the special challenges of AD drug discovery by discussing the viability/practicality of possible microglia drug targets including cluster of differentiation 33 (CD33), KCa3.1, kynurenines, ionotropic P2 receptor 7 (P2X7), programmed death-1 (PD-1), Toll-like receptors (TLRs), and triggering receptor expressed in myeloid cells 2 (TREM2).

Enabling structure-based drug design of Tyk2 through co-crystallization with a stabilizing aminoindazole inhibitor.

BACKGROUND: Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds. RESULTS: A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5-2.6 Å resolution. This structure revealed initial "hot-spot" regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods. CONCLUSIONS: Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.

Minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR): quantitative measures for selectivity ratios obtained by screening assays.

Development of inhibitor compounds selective against undesirable targets is critical in drug discovery. Selectivity ratios for candidate compounds are evaluated by dividing potencies from two assays assessing the off-target and target. Because all potency measurements have underlying uncertainty, understanding error propagation is essential to interpreting selectivity data. Assay noise introduces ambiguity in the statistical significance of selectivity ratios, particularly at low replicate numbers when compounds are often prioritized for subsequent testing. The ability to differentiate potency results for any pair of compounds in one assay is evaluated using a metric called minimum significant ratio (MSR). Potency results of one compound tested in a pair of assays can be differentiated by the minimum significant selectivity ratio (MSSR). To differentiate selectivity ratios for any pair of compounds, we extend this concept by proposing two new parameters called the minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR). Importantly, these tools can be used after a single selectivity measurement. We describe these methods and illustrate their usefulness using structure-activity relationship data from a Janus kinase inhibitor project, in which these tools informed a cogent retesting strategy and enabled rapid and objective decision making.

Inhibition of interleukin-1beta converting enzyme (ICE or caspase 1) by aspartyl acyloxyalkyl ketones and aspartyl amidooxyalkyl ketones.

A series of acyloxyalkyl and amidooxyalkyl ketones appended to a carbobenzyloxy aspartic acid core have been prepared. The most potent of these new inhibitors was 4i with a K(i) of 0.5 microM. These two series provide an improved understanding of the binding requirements for the hydrophobic prime side of ICE.

Succinic acid amides as P2-P3 replacements for inhibitors of interleukin-1beta converting enzyme (ICE or caspase 1).

Succinic acid amides have been found to be effective P2-P3 scaffold replacements for peptidic ICE inhibitors. Heteroarylalkyl fragments occupying the P4 position provided access to compounds with nM affinities. Utilization of an acylal prodrug moiety was required to overcome biopharmaceutical issues which led to the identification of 17f, a potential clinical candidate.

2,4-Diaminopyrimidine MK2 inhibitors. Part II: Structure-based inhibitor optimization.

We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC(50) values as low as 19nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFalpha production in peripheral human monocytes.

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode.

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.

Rational mutagenesis to support structure-based drug design: MAPKAP kinase 2 as a case study.

BACKGROUND: Structure-based drug design (SBDD) can provide valuable guidance to drug discovery programs. Robust construct design and expression, protein purification and characterization, protein crystallization, and high-resolution diffraction are all needed for rapid, iterative inhibitor design. We describe here robust methods to support SBDD on an oral anti-cytokine drug target, human MAPKAP kinase 2 (MK2). Our goal was to obtain useful diffraction data with a large number of chemically diverse lead compounds. Although MK2 structures and structural methods have been reported previously, reproducibility was low and improved methods were needed. RESULTS: Our construct design strategy had four tactics: N- and C-terminal variations; entropy-reducing surface mutations; activation loop deletions; and pseudoactivation mutations. Generic, high-throughput methods for cloning and expression were coupled with automated liquid dispensing for the rapid testing of crystallization conditions with minimal sample requirements. Initial results led to development of a novel, customized robotic crystallization screen that yielded MK2/inhibitor complex crystals under many conditions in seven crystal forms. In all, 44 MK2 constructs were generated, ~500 crystals were tested for diffraction, and ~30 structures were determined, delivering high-impact structural data to support our MK2 drug design effort. CONCLUSION: Key lessons included setting reasonable criteria for construct performance and prioritization, a willingness to design and use customized crystallization screens, and, crucially, initiation of high-throughput construct exploration very early in the drug discovery process.

Current in vitro kinase assay technologies: the quest for a universal format.

The rapidly growing interest in kinases as drug targets has prompted the development of many kinase assay technologies. These technologies can be grouped into three categories: radiometric assays, phospho-antibody-dependent fluorescence/luminescence assays, and phospho-antibody-independent fluorescence/luminescence assays. This article will review some of the major kinase assay technologies on the market, with particular emphasis on the newest systems. We will describe the physical principles, the practical advantages and drawbacks, and the potential applications of these technologies in kinase drug discovery. Most of these technologies are suitable for HTS, but only a few can be utilized for kinetic and mechanistic studies. Significant progress towards development of generic assays, free of radioisotopes and custom reagents such as phospho-specific antibodies, has been made in recent years. However, due to various limitations of each format, none of these generic assay technologies can yet claim to be truly universal. Several factors, including the intended applications, cost, timeline, expertise, familiarity, and comfort level, should be considered prior to pursuing a particular kinase assay technology.

Designing in vitro assays for drug discovery: a study with the human MAP3 kinase COT.

In drug discovery research, developing a validated and robust in vitro assay is crucial for high-throughput screening and subsequent hit characterization. There are many kinase assay technologies, so an assay developer has several decisions to make prior to undertaking experiments: which assay technology should be used? What are the advantages and disadvantages of a cascade assay compared with a direct assay format? Should the substrate be a peptide or a protein? When using a protein substrate, should it be physiologically relevant? In this perspective article, these questions will be addressed using examples from a study with the human mitogen-activated protein kinase kinase kinase COT.

Homogeneous time-resolved fluorescence and its applications for kinase assays in drug discovery.

The rapidly growing interest in kinases as potential targets for therapeutic intervention has prompted the development of many kinase assay technologies. One exciting example is homogeneous time-resolved fluorescence (HTRF). An HTRF assay utilizes the signal generated by the fluorescence resonance energy transfer between donor and acceptor molecules in close proximity. Dual-wavelength detection helps to eliminate media interference, and the final signal is proportional to the extent of product formation. Thus far, the reported applications of this technology for in vitro kinase assays have mainly focused on high-throughput screening. In this report, we extend the applications of HTRF technology to the areas of enzyme and inhibitor characterization, some aspects of which were previously believed impossible. We describe the methods developed for determining the kinetic parameters of an enzyme, such as K(m) and k(cat), and the procedures for inhibitor mechanistic studies including ATP competitiveness and slow-binding and dissociation kinetics. These assays can be readily applied to any kinase and are valuable in advancing a program through the early stages of drug discovery.

Comparative analysis of various in vitro COT kinase assay formats and their applications in inhibitor identification and characterization.

Cancer osaka thyroid (COT) is a member of the mitogen-activated protein kinase kinase kinase family of enzymes and plays a pivotal role in tumor necrosis factor-alpha production in macrophages. Consequently, COT is considered to be a promising target for antiinflammatory drug discovery. We describe here the development of in vitro COT assays in several formats and the advantages and disadvantages of each. A cascade assay requires very small amounts of enzyme and can provide a useful tool for high-throughput screening, but it is not desirable for compound mechanistic studies due to complicated kinetics. Direct assays are superior to cascade assays and are suitable for both compound screening and mechanistic studies. Among the direct assays, the homogeneous time-resolved fluorescence (HTRF) format is preferred over the radiometric format due to the robustness, throughput, and ease of use of the HTRF format. When the physiological protein substrate MEK1 (MAP/Erk kinase 1) was used to determine inhibitor potencies, false positives were observed due to compound interference by binding to MEK1. Using a MEK1 peptide substrate, these false positives were eliminated. In addition, we describe a simple method to study the ATP competitiveness of compounds. The knowledge gained through our studies with COT, and the methods described for our assays and compound mechanistic studies, can be readily applied to other kinase targets.

Caspase-dependent regulation and subcellular redistribution of the transcriptional modulator YY1 during apoptosis.

The transcriptional regulator Yin Yang 1 (YY1) controls many aspects of cell behavior and is essential for development. We analyzed the fate of YY1 during apoptosis and studied the functional consequences. We observed that this factor is rapidly translocated into the cell nucleus in response to various apoptotic stimuli, including activation of Fas, stimulation by tumor necrosis factor, and staurosporine and etoposide treatment. Furthermore, YY1 is cleaved by caspases in vitro and in vivo at two distinct sites, IATD(12)G and DDSD(119)G, resulting in the deletion of the first 119 amino acids early in the apoptotic process. This activity generates an N-terminally truncated YY1 fragment (YY1Delta119) that has lost its transactivation domain but retains its DNA binding domain. Indeed, YY1Delta119 is no longer able to stimulate gene transcription but interacts with DNA. YY1Delta119 but not the wild-type protein or the caspase-resistant mutant YY1D12A/D119A enhances Fas-induced apoptosis, suggesting that YY1 is involved in a positive feedback loop during apoptosis. Our findings provide evidence for a new mode of regulation of YY1 and define a novel aspect of the involvement of YY1 in the apoptotic process.