Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However, real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS), leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity or response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP), we created a novel, green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics, ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems, including human stem cell-derived neurons and cardiomyocytes, primary neurons and astrocytes, and mouse neurons and astrocytes in ex vivo brain slices. These applications demonstrate oROS's capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress, G-protein coupled receptor (GPCR)-induced cell signaling, and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via Aß-putriscine-MAOB axis, highlighting the sensor's relevance in validating neurodegenerative disease models. oROS is a versatile tool, offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology, with significant implications for diseases associated with oxidative stress, such as cancer, neurodegenerative disorders, and cardiovascular diseases.

Differential effects of SORL1 deficiency on the endo-lysosomal network in human neurons and microglia.

The endosomal gene SORL1 is a strong Alzheimer's disease (AD) risk gene that harbours loss-of-function variants causative for developing AD. The SORL1 protein SORL1/SORLA is an endosomal receptor that interacts with the multi-protein sorting complex retromer to traffic various cargo through the endo-lysosomal network (ELN). Impairments in endo-lysosomal trafficking are an early cellular symptom in AD and a novel therapeutic target. However, the cell types of the central nervous system are diverse and use the ELN differently. If this pathway is to be effectively therapeutically targeted, understanding how key molecules in the ELN function in various cell types and how manipulating them affects cell-type specific responses relative to AD is essential. Here, we discuss an example where deficiency of SORL1 expression in a human model leads to stress on early endosomes and recycling endosomes in neurons, but preferentially leads to stress on lysosomes in microglia. The differences observed in these organelles could relate to the unique roles of these cells in the brain as neurons are professional secretory cells and microglia are professional phagocytic cells. Experiments to untangle these differences are fundamental to advancing the understanding of cell biology in AD and elucidating important pathways for therapeutic development. Human-induced pluripotent stem cell models are a valuable platform for such experiments. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

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.

Far-red and sensitive sensor for monitoring real time H2O2 dynamics with subcellular resolution and in multi-parametric imaging applications.

H2O2 is a key oxidant in mammalian biology and a pleiotropic signaling molecule at the physiological level, and its excessive accumulation in conjunction with decreased cellular reduction capacity is often found to be a common pathological marker. Here, we present a red fluorescent Genetically Encoded H2O2 Indicator (GEHI) allowing versatile optogenetic dissection of redox biology. Our new GEHI, oROS-HT, is a chemigenetic sensor utilizing a HaloTag and Janelia Fluor (JF) rhodamine dye as fluorescent reporters. We developed oROS-HT through a structure-guided approach aided by classic protein structures and recent protein structure prediction tools. Optimized with JF635, oROS-HT is a sensor with 635 nm excitation and 650 nm emission peaks, allowing it to retain its brightness while monitoring intracellular H2O2 dynamics. Furthermore, it enables multi-color imaging in combination with blue-green fluorescent sensors for orthogonal analytes and low auto-fluorescence interference in biological tissues. Other advantages of oROS-HT over alternative GEHIs are its fast kinetics, oxygen-independent maturation, low pH sensitivity, lack of photo-artifact, and lack of intracellular aggregation. Here, we demonstrated efficient subcellular targeting and how oROS-HT can map inter and intracellular H2O2 diffusion at subcellular resolution. Lastly, we used oROS-HT with the green fluorescent calcium indicator Fluo-4 to investigate the transient effect of the anti-inflammatory agent auranofin on cellular redox physiology and calcium levels via multi-parametric, dual-color imaging.

A familial missense variant in the Alzheimer's disease gene SORL1 impairs its maturation and endosomal sorting.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset > 75 years. All offspring were affected with AD with ages at onset ranging from 53 years to 74 years. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

Pharmacologic enhancement of retromer rescues endosomal pathology induced by defects in the Alzheimer's gene SORL1.

The SORL1 gene (SORLA) is strongly associated with risk of developing Alzheimer's disease (AD). SORLA is a regulator of endosomal trafficking in neurons and interacts with retromer, a complex that is a "master conductor" of endosomal trafficking. Small molecules can increase retromer expression in vitro, enhancing its function. We treated hiPSC-derived cortical neurons that are either fully deficient, haploinsufficient, or that harbor one copy of SORL1 variants linked to AD with TPT-260, a retromer-enhancing molecule. We show significant increases in retromer subunit VPS26B expression. We tested whether endosomal, amyloid, and TAU pathologies were corrected. We observed that the degree of rescue by TPT-260 treatment depended on the number of copies of functional SORL1 and which SORL1 variant was expressed. Using a disease-relevant preclinical model, our work illuminates how the SORL1-retromer pathway can be therapeutically harnessed.

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.

Cell-type-specific regulation of APOE and CLU levels in human neurons by the Alzheimer's disease risk gene SORL1.

SORL1 is implicated in the pathogenesis of Alzheimer's disease (AD) through genetic studies. To interrogate the roles of SORL1 in human brain cells, SORL1-null induced pluripotent stem cells (iPSCs) were differentiated to neuron, astrocyte, microglial, and endothelial cell fates. Loss of SORL1 leads to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. SORL1 loss induces a neuron-specific reduction in apolipoprotein E (APOE) and clusterin (CLU) and altered lipid profiles. Analyses of iPSCs derived from a large cohort reveal a neuron-specific association between SORL1, APOE, and CLU levels, a finding validated in postmortem brain. Enhancement of retromer-mediated trafficking rescues tau phenotypes observed in SORL1-null neurons but does not rescue APOE levels. Pathway analyses implicate transforming growth factor ß (TGF-ß)/SMAD signaling in SORL1 function, and modulating SMAD signaling in neurons alters APOE RNA levels in a SORL1-dependent manner. Taken together, these data provide a mechanistic link between strong genetic risk factors for AD.

A familial missense variant in the Alzheimer's Disease gene SORL1 impairs its maturation and endosomal sorting.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset >75 years. All offspring were affected with AD with ages at onset ranging from 53yrs-74yrs. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

Human microglia show unique transcriptional changes in Alzheimer's disease.

Microglia, the innate immune cells of the brain, influence Alzheimer's disease (AD) progression and are potential therapeutic targets. However, microglia exhibit diverse functions, the regulation of which is not fully understood, complicating therapeutics development. To better define the transcriptomic phenotypes and gene regulatory networks associated with AD, we enriched for microglia nuclei from 12 AD and 10 control human dorsolateral prefrontal cortices (7 males and 15 females, all aged >60 years) before single-nucleus RNA sequencing. Here we describe both established and previously unrecognized microglial molecular phenotypes, the inferred gene networks driving observed transcriptomic change, and apply trajectory analysis to reveal the putative relationships between microglial phenotypes. We identify microglial phenotypes more prevalent in AD cases compared with controls. Further, we describe the heterogeneity in microglia subclusters expressing homeostatic markers. Our study demonstrates that deep profiling of microglia in human AD brain can provide insight into microglial transcriptional changes associated with AD.

Amyloid beta peptides (Aß) from Alzheimer's disease neuronal secretome induce endothelial activation in a human cerebral microvessel model.

In Alzheimer's disease (AD), secretion and deposition of amyloid beta peptides (Aß) have been associated with blood-brain barrier dysfunction. However, the role of Aß in endothelial cell (EC) dysfunction remains elusive. Here we investigated AD mediated EC activation by studying the effect of Aß secreted from human induced pluripotent stem cell-derived cortical neurons (hiPSC-CN) harboring a familial AD mutation (Swe+/+) on human brain microvascular endothelial cells (HBMECs) in 2D and 3D perfusable microvessels. We demonstrated that increased Aß levels in Swe+/+ conditioned media (CM) led to stress fiber formation and upregulation of genes associated with endothelial inflammation and immune-adhesion. Perfusion of Aß-rich Swe+/+ CM induced acute formation of von Willebrand factor (VWF) fibers in the vessel lumen, which was attenuated by reducing Aß levels in CM. Our findings suggest that Aß peptides can trigger rapid inflammatory and thrombogenic responses within cerebral microvessels, which may exacerbate AD pathology.

Cell-type-specific regulation of APOE levels in human neurons by the Alzheimer's disease risk gene SORL1.

SORL1 is strongly implicated in the pathogenesis of Alzheimer's disease (AD) through human genetic studies that point to an association of reduced SORL1 levels with higher risk for AD. To interrogate the role(s) of SORL1 in human brain cells, SORL1 null iPSCs were generated, followed by differentiation to neuron, astrocyte, microglia, and endothelial cell fates. Loss of SORL1 led to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. Intriguingly, SORL1 loss led to a dramatic neuron-specific reduction in APOE levels. Further, analyses of iPSCs derived from a human aging cohort revealed a neuron-specific linear correlation between SORL1 and APOE RNA and protein levels, a finding validated in human post-mortem brain. Pathway analysis implicated intracellular transport pathways and TGF- ß/SMAD signaling in the function of SORL1 in neurons. In accord, enhancement of retromer-mediated trafficking and autophagy rescued elevated phospho-tau observed in SORL1 null neurons but did not rescue APOE levels, suggesting that these phenotypes are separable. Stimulation and inhibition of SMAD signaling modulated APOE RNA levels in a SORL1-dependent manner. These studies provide a mechanistic link between two of the strongest genetic risk factors for AD.

Human-Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons and Glia for the Elucidation of Pathogenic Mechanisms in Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disorder and a mechanistically complex disease. For the last decade, human models of AD using induced pluripotent stem cells (iPSCs) have emerged as a powerful way to understand disease pathogenesis in relevant human cell types. In this review, we summarize the state of the field and how this technology can apply to studies of both familial and sporadic studies of AD. We discuss patient-derived iPSCs, genome editing, differentiation of neural cell types, and three-dimensional organoids, and speculate on the future of this type of work for increasing our understanding of, and improving therapeutic development for, this devastating disease.

Advancements in high-resolution 3D microscopy analysis of endosomal morphology in postmortem Alzheimer's disease brains.

Abnormal endo-lysosomal morphology is an early cytopathological feature of Alzheimer's disease (AD) and genome-wide association studies (GWAS) have implicated genes involved in the endo-lysosomal network (ELN) as conferring increased risk for developing sporadic, late-onset AD (LOAD). Characterization of ELN pathology and the underlying pathophysiology is a promising area of translational AD research and drug development. However, rigorous study of ELN vesicles in AD and aged control brains poses a unique constellation of methodological challenges due in part to the small size of these structures and subsequent requirements for high-resolution imaging. Here we provide a detailed protocol for high-resolution 3D morphological quantification of neuronal endosomes in postmortem AD brain tissue, using immunofluorescent staining, confocal imaging with image deconvolution, and Imaris software analysis pipelines. To demonstrate these methods, we present neuronal endosome morphology data from 23 sporadic LOAD donors and one aged non-AD control donor. The techniques described here were developed across a range of AD neuropathology to best optimize these methods for future studies with large cohorts. Application of these methods in research cohorts will help advance understanding of ELN dysfunction and cytopathology in sporadic AD.

Single Cell RNA Sequencing Reveals Human Tooth Type Identity and Guides In Vitro hiPSC Derived Odontoblast Differentiation (iOB).

Over 90% of the U.S. adult population suffers from tooth structure loss due to caries. Most of the mineralized tooth structure is composed of dentin, a material produced and mineralized by ectomesenchyme derived cells known as odontoblasts. Clinicians, scientists, and the general public share the desire to regenerate this missing tooth structure. To bioengineer missing dentin, increased understanding of human tooth development is required. Here we interrogate at the single cell level the signaling interactions that guide human odontoblast and ameloblast development and which determine incisor or molar tooth germ type identity. During human odontoblast development, computational analysis predicts that early FGF and BMP activation followed by later HH signaling is crucial. Application of this sci-RNA-seq analysis generates a differentiation protocol to produce mature hiPSC derived odontoblasts in vitro (iOB). Further, we elucidate the critical role of FGF signaling in odontoblast maturation and its biomineralization capacity using the de novo designed FGFR1/2c isoform specific minibinder scaffolded as a C6 oligomer that acts as a pathway agonist. We find that FGFR1c is upregulated in functional odontoblasts and specifically plays a crucial role in driving odontoblast maturity. Using computational tools, we show on a molecular level how human molar development is delayed compared to incisors. We reveal that enamel knot development is guided by FGF and WNT in incisors and BMP and ROBO in the molars, and that incisor and molar ameloblast development is guided by FGF, EGF and BMP signaling, with tooth type specific intensity of signaling interactions. Dental ectomesenchyme derived cells are the primary source of signaling ligands responsible for both enamel knot and ameloblast development.

Neighborhood matters: Altered lipid metabolism in APOE4 microglia causes problems for neurons.

In this issue of Cell Stem Cell, Victor et al. reveal that human microglia harboring the Alzheimer's disease risk allele APOE4 have altered lipid metabolism and cellular activation. This dampens neuronal network activity, underscoring the importance of these brain-resident immune cells and highlighting a novel pathway for therapeutic intervention.

Evaluation of a Selective Chemical Probe Validates That CK2 Mediates Neuroinflammation in a Human Induced Pluripotent Stem Cell-Derived Microglial Model.

Novel treatments for neurodegenerative disorders are in high demand. It is imperative that new protein targets be identified to address this need. Characterization and validation of nascent targets can be accomplished very effectively using highly specific and potent chemical probes. Human induced pluripotent stem cells (hiPSCs) provide a relevant platform for testing new compounds in disease relevant cell types. However, many recent studies utilizing this platform have focused on neuronal cells. In this study, we used hiPSC-derived microglia-like cells (MGLs) to perform side-by-side testing of a selective chemical probe, SGC-CK2-1, compared with an advanced clinical candidate, CX-4945, both targeting casein kinase 2 (CK2), one of the first kinases shown to be dysregulated in Alzheimer's disease (AD). CK2 can mediate neuroinflammation in AD, however, its role in microglia, the innate immune cells of the central nervous system (CNS), has not been defined. We analyzed available RNA-seq data to determine the microglial expression of kinases inhibited by SGC-CK2-1 and CX-4945 with a reported role in mediating inflammation in glial cells. As proof-of-concept for using hiPSC-MGLs as a potential screening platform, we used both wild-type (WT) MGLs and MGLs harboring a mutation in presenilin-1 (PSEN1), which is causative for early-onset, familial AD (FAD). We stimulated these MGLs with pro-inflammatory lipopolysaccharides (LPS) derived from E. coli and observed strong inhibition of the expression and secretion of proinflammatory cytokines by simultaneous treatment with SGC-CK2-1. A direct comparison shows that SGC-CK2-1 was more effective at suppression of proinflammatory cytokines than CX-4945. Together, these results validate a selective chemical probe, SGC-CK2-1, in human microglia as a tool to reduce neuroinflammation.

Reduced gene dosage is a common mechanism of neuropathologies caused by ATP6AP2 splicing mutations.

BACKGROUND: Mutations that alter splicing of X-linked ATP6AP2 cause a spectrum of neurodevelopmental and neurodegenerative pathologies including parkinsonism in affected males. All previously reported splicing mutations increase the level of a minor isoform with skipped exon 4 (Δe4) that encodes a functionally deficient protein. OBJECTIVES: We investigated the pathogenic mechanism of a novel c.168+6T>A variant reported in a family with X-linked intellectual disability, epilepsy, and parkinsonism. We also analyzed ATP6AP2 splicing defects in brains of carriers of a c.345C>T variant associated with X-linked spasticity and parkinsonism. METHODS: We generated induced pluripotent stem cells from patients with c.168+6T>A, reprogrammed them to neural progenitor cells and analyzed them by RNA-Seq and qRT-PCR. We also quantified ATP6AP2 isoforms in the brains of c.345C>T carriers by Nanostring nCounter. RESULTS: The c.168+6T>A increased skipping of ATP6AP2 exon 2 and usage of cryptic intronic donor splice sites. This results in out-of-frame splicing products and a reciprocal 50% reduction in functional full-length ATP6AP2 transcripts. Neural progenitors of patients with c.168+6T>A exhibited downregulated neural development gene networks. Analysis of blood transcriptomes of c.168+6T>A carriers identified potential biomarkers of ATP6AP2 deficiency in non-neural tissues. The c.345C>T variant increased exon 4 skipping with concomitant decrease of full length ATP6AP2 in brains of carriers. CONCLUSION: A common pathogenic consequence of splicing mutations affecting inclusion of different ATP6AP2 exons is reduction of the functional full-length transcript. The exacerbated ATP6AP2 splicing defect in brains of c.345C>T carriers is consistent with their CNS-restricted clinical presentations.

Use of AD Informer Set compounds to explore validity of novel targets in Alzheimer's disease pathology.

Introduction: A chemogenomic set of small molecules with annotated activities and implicated roles in Alzheimer's disease (AD) called the AD Informer Set was recently developed and made available to the AD research community: https://treatad.org/data-tools/ad-informer-set/. Methods: Small subsets of AD Informer Set compounds were selected for AD-relevant profiling. Nine compounds targeting proteins expressed by six AD-implicated genes prioritized for study by Target Enablement to Accelerate Therapy Development for Alzheimer's Disease (TREAT-AD) teams were selected for G-protein coupled receptor (GPCR), amyloid beta (Aß) and tau, and pharmacokinetic (PK) studies. Four non-overlapping compounds were analyzed in microglial cytotoxicity and phagocytosis assays. Results: The nine compounds targeting CAPN2, EPHX2, MDK, MerTK/FLT3, or SYK proteins were profiled in 46 to 47 primary GPCR binding assays. Human induced pluripotent stem cell (iPSC)-derived neurons were treated with the same nine compounds and secretion of Aß peptides (Aß40 and Aß42) as well as levels of phosphophorylated tau (p-tau, Thr231) and total tau (t-tau) peptides measured at two concentrations and two timepoints. Finally, CD1 mice were dosed intravenously to determine preliminary PK and/or brain-specific penetrance values for these compounds. As a final cell-based study, a non-overlapping subset of four compounds was selected based on single-concentration screening for analysis of both cytotoxicity and phagocytosis in murine and human microglia cells. Discussion: We have demonstrated the utility of the AD Informer Set in the validation of novel AD hypotheses using biochemical, cellular (primary and immortalized), and in vivo studies. The selectivity for their primary targets versus essential GPCRs in the brain was established for our compounds. Statistical changes in tau, p-tau, Aß40, and/or Aß42 and blood-brain barrier penetrance were observed, solidifying the utility of specific compounds for AD. Single-concentration phagocytosis results were validated as predictive of dose-response findings. These studies established workflows, validated assays, and illuminated next steps for protein targets and compounds.