Utility of protein-protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display.

Over the past 3 decades, a diverse collection of small protein domains have been used as scaffolds to generate general purpose protein-binding reagents using a variety of protein display and enrichment technologies. To expand the repertoire of scaffolds and protein surfaces that might serve this purpose, we have explored the utility of (i) a pair of anti-parallel alpha-helices in a small highly disulfide-bonded 4-helix bundle, the CC4 domain from reversion-inducing Cysteine-rich Protein with Kazal Motifs and (ii) a concave beta-sheet surface and two adjacent loops in the human FN3 domain, the scaffold for the widely used monobody platform. Using M13 phage display and next generation sequencing, we observe that, in both systems, libraries of ∼30 million variants contain binding proteins with affinities in the low µM range for baits corresponding to the extracellular domains of multiple mammalian proteins. CC4- and FN3-based binding proteins were fused to the N- and/or C-termini of Fc domains and used for immunostaining of transfected cells. Additionally, FN3-based binding proteins were inserted into VP1 of AAV to direct AAV infection to cells expressing a defined surface receptor. Finally, FN3-based binding proteins were inserted into the Pvc13 tail fiber protein of an extracellular contractile injection system particle to direct protein cargo delivery to cells expressing a defined surface receptor. These experiments support the utility of CC4 helices B and C and of FN3 beta-strands C, D, and F together with adjacent loops CD and FG as surfaces for engineering general purpose protein-binding reagents.

Normal and Sjogren's syndrome models of the murine lacrimal gland studied at single-cell resolution.

The lacrimal gland is of central interest in ophthalmology both as the source of the aqueous component of tear fluid and as the site of autoimmune pathology in the context of Sjogren's syndrome (SjS). To provide a foundational description of mouse lacrimal gland cell types and their patterns of gene expression, we have analyzed single-cell transcriptomes from wild-type (Balb/c) mice and from two genetically based SjS models, MRL/lpr and NOD (nonobese diabetic).H2b, and defined the localization of multiple cell-type-specific protein and mRNA markers. This analysis has uncovered a previously undescribed cell type, Car6+ cells, which are located at the junction of the acini and the connecting ducts. More than a dozen secreted polypeptides that are likely to be components of tear fluid are expressed by acinar cells and show pronounced sex differences in expression. Additional examples of gene expression heterogeneity within a single cell type were identified, including a gradient of Claudin4 along the length of the ductal system and cell-to-cell heterogeneity in transcription factor expression within acinar and myoepithelial cells. The patterns of expression of channels, transporters, and pumps in acinar, Car6+, and ductal cells make strong predictions regarding the mechanisms of water and electrolyte secretion. In MRL/lpr and NOD.H2b lacrimal glands, distinctive changes in parenchymal gene expression and in immune cell subsets reveal widespread interferon responses, a T cell-dominated infiltrate in the MRL/lpr model, and a mixed B cell and T cell infiltrate in the NOD.H2b model.

Seeing is believing: The development of optical coherence tomography.

The 2023 Lasker-DeBakey Clinical Medical Research Award is being presented to James Fujimoto, David Huang, and Eric Swanson for their invention and development of optical coherence tomography (OCT), an imaging technology that uses light to visualize microscopic structures within tissues such as the retina. OCT has dramatically changed the practice of ophthalmology and improved the lives of millions of people. It also has great potential in a wide range of other medical fields.

Bacterial meningitis in the early postnatal mouse studied at single-cell resolution.

Bacterial meningitis is a major cause of morbidity and mortality, especially among infants and the elderly. Here, we study mice to assess the response of each of the major meningeal cell types to early postnatal E. coli infection using single nucleus RNA sequencing (snRNAseq), immunostaining, and genetic and pharamacologic perturbations of immune cells and immune signaling. Flatmounts of the dissected leptomeninges and dura were used to facilitiate high-quality confocal imaging and quantification of cell abundances and morphologies. Upon infection, the major meningeal cell types - including endothelial cells (ECs), macrophages, and fibroblasts - exhibit distinctive changes in their transcriptomes. Additionally, ECs in the leptomeninges redistribute CLDN5 and PECAM1, and leptomeningeal capillaries exhibit foci with reduced blood-brain barrier integrity. The vascular response to infection appears to be largely driven by TLR4 signaling, as determined by the nearly identical responses induced by infection and LPS administration and by the blunted response to infection in Tlr4-/- mice. Interestingly, knocking out Ccr2, encoding a major chemoattractant for monocytes, or acute depletion of leptomeningeal macrophages, following intracebroventricular injection of liposomal clodronate, had little or no effect on the response of leptomeningeal ECs to E. coli infection. Taken together, these data imply that EC responses to infection are largely driven by the intrinsic EC response to LPS.

Structure of WNT inhibitor adenomatosis polyposis coli down-regulated 1 (APCDD1), a cell-surface lipid-binding protein.

Diverse extracellular proteins negatively regulate WNT signaling. One such regulator is adenomatosis polyposis coli down-regulated 1 (APCDD1), a conserved single-span transmembrane protein. In response to WNT signaling in a variety of tissues, APCDD1 transcripts are highly up-regulated. We have determined the three-dimensional structure of the extracellular domain of APCDD1, and this structure reveals an unusual architecture consisting of two closely apposed ß-barrel domains (ABD1 and ABD2). ABD2, but not ABD1, has a large hydrophobic pocket that accommodates a bound lipid. The APCDD1 ECD can also bind to WNT7A, presumably via its covalently bound palmitoleate, a modification that is common to all WNTs and is essential for signaling. This work suggests that APCDD1 functions as a negative feedback regulator by titrating WNT ligands at the surface of responding cells.

Structural insights into plasmalemma vesicle-associated protein (PLVAP): Implications for vascular endothelial diaphragms and fenestrae.

In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low-molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration-presumably a coiled coil-held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP-i.e., the long dimension of the pore-and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP.

Signaling Pathways in Neurovascular Development.

During development, the central nervous system (CNS) vasculature grows to precisely meet the metabolic demands of neurons and glia. In addition, the vast majority of the CNS vasculature acquires a unique set of molecular and cellular properties-collectively referred to as the blood-brain barrier-that minimize passive diffusion of molecules between the blood and the CNS parenchyma. Both of these processes are controlled by signals emanating from neurons and glia. In this review, we describe the nature and mechanisms-of-action of these signals, with an emphasis on vascular endothelial growth factor (VEGF) and beta-catenin (canonical Wnt) signaling, the two best-understood systems that regulate CNS vascular development. We highlight foundational discoveries, interactions between different signaling systems, the integration of genetic and cell biological studies, advances that are of clinical relevance, and questions for future research.

The WNT7A/WNT7B/GPR124/RECK signaling module plays an essential role in mammalian limb development.

In central nervous system vascular endothelial cells, signaling via the partially redundant ligands WNT7A and WNT7B requires two co-activator proteins, GPR124 and RECK. WNT7A and RECK have been shown previously to play a role in limb development, but the mechanism of RECK action in this context is unknown. The roles of WNT7B and GPR124 in limb development have not been investigated. Using combinations of conventional and/or conditional loss-of-function alleles for mouse Wnt7a, Wnt7b, Gpr124 and Reck, including a Reck allele that codes for a protein that is specifically defective in WNT7A/WNT7B signaling, we show that reductions in ligand and/or co-activator function synergize to cause reduced and dysmorphic limb bone growth. Two additional limb phenotypes - loss of distal Lmx1b expression and ectopic growth of nail-like structures - occur with reduced Wnt7a/Wnt7b gene copy number and, respectively, with Reck mutations and with combined Reck and Gpr124 mutations. A third limb phenotype - bleeding into a digit - occurs with the most severe combinations of Wnt7a/Wnt7b, Reck and Gpr124 mutations. These data imply that the WNT7A/WNT7B-FRIZZLED-LRP5/LRP6-GPR124-RECK signaling system functions as an integral unit in limb development.

A transcriptome atlas of the mouse iris at single-cell resolution defines cell types and the genomic response to pupil dilation.

The iris controls the level of retinal illumination by controlling pupil diameter. It is a site of diverse ophthalmologic diseases and it is a potential source of cells for ocular auto-transplantation. The present study provides foundational data on the mouse iris based on single nucleus RNA sequencing. More specifically, this work has (1) defined all of the major cell types in the mouse iris and ciliary body, (2) led to the discovery of two types of iris stromal cells and two types of iris sphincter cells, (3) revealed the differences in cell type-specific transcriptomes in the resting vs. dilated states, and (4) identified and validated antibody and in situ hybridization probes that can be used to visualize the major iris cell types. By immunostaining for specific iris cell types, we have observed and quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre-expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre-expressing progenitors contribute only to stromal cells. This work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.

Developmental, cellular, and behavioral phenotypes in a mouse model of congenital hypoplasia of the dentate gyrus.

In the hippocampus, a widely accepted model posits that the dentate gyrus improves learning and memory by enhancing discrimination between inputs. To test this model, we studied conditional knockout mice in which the vast majority of dentate granule cells (DGCs) fail to develop - including nearly all DGCs in the dorsal hippocampus - secondary to eliminating Wntless (Wls) in a subset of cortical progenitors with Gfap-Cre. Other cells in the Wlsfl/-;Gfap-Cre hippocampus were minimally affected, as determined by single nucleus RNA sequencing. CA3 pyramidal cells, the targets of DGC-derived mossy fibers, exhibited normal morphologies with a small reduction in the numbers of synaptic spines. Wlsfl/-;Gfap-Cre mice have a modest performance decrement in several complex spatial tasks, including active place avoidance. They were also modestly impaired in one simpler spatial task, finding a visible platform in the Morris water maze. These experiments support a role for DGCs in enhancing spatial learning and memory.

Structure of the RECK CC domain, an evolutionary anomaly.

Five small protein domains, the CC-domains, at the N terminus of the RECK protein, play essential roles in signaling by WNT7A and WNT7B in the context of central nervous system angiogenesis and blood-brain barrier formation and maintenance. We have determined the structure of CC domain 4 (CC4) at 1.65-Å resolution and find that it folds into a compact four-helix bundle with three disulfide bonds. The CC4 structure, together with homology modeling of CC1, reveals the surface locations of critical residues that were shown in previous mutagenesis studies to mediate GPR124 binding and WNT7A/WNT7B recognition and signaling. Surprisingly, sequence and structural homology searches reveal no other cell-surface or secreted domains in vertebrates that resemble the CC domain, a pattern that is in striking contrast to other ancient and similarly sized domains, such as Epidermal Growth Factor, Fibronectin Type 3, Immunoglobulin, and Thrombospondin type 1 domains, which are collectively present in hundreds of proteins.

A mouse model for kinesin family member 11 (Kif11)-associated familial exudative vitreoretinopathy.

During mitosis, Kif11, a kinesin motor protein, promotes bipolar spindle formation and chromosome movement, and during interphase, Kif11 mediates diverse trafficking processes in the cytoplasm. In humans, inactivating mutations in KIF11 are associated with (1) retinal hypovascularization with or without microcephaly and (2) multi-organ syndromes characterized by variable combinations of lymphedema, chorioretinal dysplasia, microcephaly and/or mental retardation. To explore the pathogenic basis of KIF11-associated retinal vascular disease, we generated a Kif11 conditional knockout (CKO) mouse and investigated the consequences of early postnatal inactivation of Kif11 in vascular endothelial cells (ECs). The principal finding is that postnatal EC-specific loss of Kif11 leads to severely stunted growth of the retinal vasculature, mildly stunted growth of the cerebellar vasculature and little or no effect on the vasculature elsewhere in the central nervous system (CNS). Thus, in mice, Kif11 function in early postnatal CNS ECs is most significant in the two CNS regions-the retina and cerebellum-that exhibit the most rapid rate of postnatal growth, which may sensitize ECs to impaired mitotic spindle function. Several lines of evidence indicate that these phenotypes are not caused by reduced beta-catenin signaling in ECs, despite the close resemblance of the Kif11 CKO phenotype to that caused by EC-specific reductions in beta-catenin signaling. Based on prior work, defective beta-catenin signaling had been the only known mechanism responsible for monogenic human disorders of retinal hypovascularization. The present study implies that retinal hypovascularization can arise from a second and mechanistically distinct cause.

A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture.

Vascular endothelial cells (ECs) derived from the central nervous system (CNS) variably lose their unique barrier properties during in vitro culture, hindering the development of robust assays for blood-brain barrier (BBB) function, including drug permeability and extrusion assays. In previous work (Sabbagh et al., 2018) we characterized transcriptional and accessible chromatin landscapes of acutely isolated mouse CNS ECs. In this report, we compare transcriptional and accessible chromatin landscapes of acutely isolated mouse CNS ECs versus mouse CNS ECs in short-term in vitro culture. We observe that standard culture conditions are associated with a rapid and selective loss of BBB transcripts and chromatin features, as well as a greatly reduced level of beta-catenin signaling. Interestingly, forced expression of a stabilized derivative of beta-catenin, which in vivo leads to a partial conversion of non-BBB CNS ECs to a BBB-like state, has little or no effect on gene expression or chromatin accessibility in vitro.

Comprehensive analysis of a mouse model of spontaneous uveoretinitis using single-cell RNA sequencing.

Autoimmune uveoretinitis is a significant cause of visual loss, and mouse models offer unique opportunities to study its disease mechanisms. Aire-/- mice fail to express self-antigens in the thymus, exhibit reduced central tolerance, and develop a spontaneous, chronic, and progressive uveoretinitis. Using single-cell RNA sequencing (scRNA-seq), we characterized wild-type and Aire-/- retinas to define, in a comprehensive and unbiased manner, the cell populations and gene expression patterns associated with disease. Based on scRNA-seq, immunostaining, and in situ hybridization, we infer that 1) the dominant effector response in Aire-/- retinas is Th1-driven, 2) a subset of monocytes convert to either a macrophage/microglia state or a dendritic cell state, 3) the development of tertiary lymphoid structures constitutes part of the Aire-/- retinal phenotype, 4) all major resident retinal cell types respond to interferon gamma (IFNG) by changing their patterns of gene expression, and 5) Muller glia up-regulate specific genes in response to IFN gamma and may act as antigen-presenting cells.

Roles of HIFs and VEGF in angiogenesis in the retina and brain.

Vascular development in the mammalian retina is a paradigm for CNS vascular development in general, and its study is revealing fundamental mechanisms that explain the efficacy of antiangiogenic therapies in retinal vascular disease. During development of the mammalian retina, hypoxic astrocytes are hypothesized to secrete VEGF, which attracts growing endothelial cells as they migrate radially from the optic disc. However, published tests of this model using astrocyte-specific deletion of Vegf in the developing mouse retina appear to contradict this theory. Here, we report that selectively eliminating Vegf in neonatal retinal astrocytes with a Gfap-Cre line that recombines with approximately 100% efficiency had no effect on proliferation or radial migration of astrocytes, but completely blocked radial migration of endothelial cells, strongly supporting the hypoxic astrocyte model. Using additional Cre driver lines, we found evidence for essential and partially redundant actions of retina-derived (paracrine) and astrocyte-derived (autocrine) VEGF in controlling astrocyte proliferation and migration. We also extended previous studies by showing that HIF-1α in retinal neurons and HIF-2α in Müller glia play distinct roles in retinal vascular development and disease, adding to a growing body of data that point to the specialization of these 2 hypoxia-sensing transcription factors.

Defining the binding interface of Amyloid Precursor Protein (APP) and Contactin3 (CNTN3) by site-directed mutagenesis.

The Amyloid Precursor Protein (APP) and Contactin (CNTN) families of cell-surface proteins have been intensively studied in the context of neural development and neuropsychiatric diseases. Earlier studies demonstrated both genetic and biochemical interactions between the extracellular domains of APP and CNTN3, but their precise binding interfaces were not defined. In the present study, we have used binding assays between APP-alkaline phosphatase (AP) fusion proteins and CNTN-Fc fusion proteins, together with alanine substitution mutagenesis, to show that: (i) the second Fibronectin domain (Fn(2)) in CNTN3 mediates APP binding; (ii) the copper binding domain (CuBD) in APP mediates CNTN3 binding; and (iii) the most important amino acids for APP-CNTN3 binding reside on one face of CNTN3-Fn(2) and on one face of APP-CuBD. These experiments define the regions of direct contact that mediate the binding interaction between APP and CNTN3.

Molecular determinants in Frizzled, Reck, and Wnt7a for ligand-specific signaling in neurovascular development.

The molecular basis of Wnt-Frizzled specificity is a central question in developmental biology. Reck, a multi-domain and multi-functional glycosylphosphatidylinositol-anchored protein, specifically enhances beta-catenin signaling by Wnt7a and Wnt7b in cooperation with the 7-transmembrane protein Gpr124. Among amino acids that distinguish Wnt7a and Wnt7b from other Wnts, two clusters are essential for signaling in a Reck- and Gpr124-dependent manner. Both clusters are far from the site of Frizzled binding: one resides at the amino terminus and the second resides in a protruding loop. Within Reck, the fourth of five tandem repeats of an unusual domain with six-cysteines (the CC domain) is essential for Wnt7a stimulation: substitutions P256A and W261A in CC4 eliminate this activity without changing protein abundance or surface localization. Mouse embryos carrying ReckP256A,W261A have severe defects in forebrain angiogenesis, providing the strongest evidence to date that Reck promotes CNS angiogenesis by specifically stimulating Wnt7a and Wnt7b signaling.

Dlg1 activates beta-catenin signaling to regulate retinal angiogenesis and the blood-retina and blood-brain barriers.

Beta-catenin (i.e., canonical Wnt) signaling controls CNS angiogenesis and the blood-brain and blood-retina barriers. To explore the role of the Discs large/membrane-associated guanylate kinase (Dlg/MAGUK) family of scaffolding proteins in beta-catenin signaling, we studied vascular endothelial cell (EC)-specific knockout of Dlg1/SAP97. EC-specific loss of Dlg1 produces a retinal vascular phenotype that closely matches the phenotype associated with reduced beta-catenin signaling, synergizes with genetically-directed reductions in beta-catenin signaling components, and can be rescued by stabilizing beta-catenin in ECs. In reporter cells with CRISPR/Cas9-mediated inactivation of Dlg1, transfection of Dlg1 enhances beta-catenin signaling ~4 fold. Surprisingly, Frizzled4, which contains a C-terminal PDZ-binding motif that can bind to Dlg1 PDZ domains, appears to function independently of Dlg1 in vivo. These data expand the repertoire of Dlg/MAGUK family functions to include a role in beta-catenin signaling, and they suggest that proteins other than Frizzled receptors interact with Dlg1 to enhance beta-catenin signaling.

Hypoxia tolerance in the Norrin-deficient retina and the chronically hypoxic brain studied at single-cell resolution.

The mammalian CNS is capable of tolerating chronic hypoxia, but cell type-specific responses to this stress have not been systematically characterized. In the Norrin KO (NdpKO ) mouse, a model of familial exudative vitreoretinopathy (FEVR), developmental hypovascularization of the retina produces chronic hypoxia of inner nuclear-layer (INL) neurons and Muller glia. We used single-cell RNA sequencing, untargeted metabolomics, and metabolite labeling from 13C-glucose to compare WT and NdpKO retinas. In NdpKO retinas, we observe gene expression responses consistent with hypoxia in Muller glia and retinal neurons, and we find a metabolic shift that combines reduced flux through the TCA cycle with increased synthesis of serine, glycine, and glutathione. We also used single-cell RNA sequencing to compare the responses of individual cell types in NdpKO retinas with those in the hypoxic cerebral cortex of mice that were housed for 1 week in a reduced oxygen environment (7.5% oxygen). In the hypoxic cerebral cortex, glial transcriptome responses most closely resemble the response of Muller glia in the NdpKO retina. In both retina and brain, vascular endothelial cells activate a previously dormant tip cell gene expression program, which likely underlies the adaptive neoangiogenic response to chronic hypoxia. These analyses of retina and brain transcriptomes at single-cell resolution reveal both shared and cell type-specific changes in gene expression in response to chronic hypoxia, implying both shared and distinct cell type-specific physiologic responses.

Beta-catenin signaling regulates barrier-specific gene expression in circumventricular organ and ocular vasculatures.

The brain, spinal cord, and retina are supplied by capillaries that do not permit free diffusion of molecules between serum and parenchyma, a property that defines the blood-brain and blood-retina barriers. Exceptions to this pattern are found in circumventricular organs (CVOs), small midline brain structures that are supplied by high permeability capillaries. In the eye and brain, high permeability capillaries are also present in the choriocapillaris, which supplies the retinal pigment epithelium and photoreceptors, and the ciliary body and choroid plexus, the sources of aqueous humor and cerebrospinal fluid, respectively. We show here that (1) endothelial cells in these high permeability vascular systems have very low beta-catenin signaling compared to barrier-competent endothelial cells, and (2) elevating beta-catenin signaling leads to a partial conversion of permeable endothelial cells to a barrier-type state. In one CVO, the area postrema, high permeability is maintained, in part, by local production of Wnt inhibitory factor-1.