APOE Christchurch-mimetic therapeutic antibody reduces APOE-mediated toxicity and tau phosphorylation.

INTRODUCTION: We discovered that the APOE3 Christchurch (APOE3Ch) variant may provide resistance to Alzheimer's disease (AD). This resistance may be due to reduced pathological interactions between ApoE3Ch and heparan sulfate proteoglycans (HSPGs). METHODS: We developed and characterized the binding, structure, and preclinical efficacy of novel antibodies targeting human ApoE-HSPG interactions. RESULTS: We found that one of these antibodies, called 7C11, preferentially bound ApoE4, a major risk factor for sporadic AD, and disrupts heparin-ApoE4 interactions. We also determined the crystal structure of a Fab fragment of 7C11 and used computer modeling to predict how it would bind to ApoE. When we tested 7C11 in mouse models, we found that it reduced recombinant ApoE-induced tau pathology in the retina of MAPT*P301S mice and curbed pTau S396 phosphorylation in brains of systemically treated APOE4 knock-in mice. Targeting ApoE-HSPG interactions using 7C11 antibody may be a promising approach to developing new therapies for AD.

Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.

Lattice light sheet microscopy reveals 4D force propagation dynamics and leading-edge behaviors in an embryonic epithelium in Drosophila.

How pulsed contractile dynamics drive the remodeling of cell and tissue topologies in epithelial sheets has been a key question in development and disease. Due to constraints in imaging and analysis technologies, studies that have described the in vivo mechanisms underlying changes in cell and neighbor relationships have largely been confined to analyses of planar apical regions. Thus, how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface in three dimensions, including especially the apical-basal axis, is unclear. Here, we perform lattice light sheet microscopy (LLSM)-based analysis to determine how far and fast forces propagate across different apical-basal layers, as well as where topological changes initiate from in a columnar epithelium. These datasets are highly time- and depth-resolved and reveal that topology-changing forces are spatially entangled, with contractile force generation occurring across the observed apical-basal axis in a pulsed fashion, while the conservation of cell volumes constrains instantaneous cell deformations. Leading layer behaviors occur opportunistically in response to favorable phasic conditions, with lagging layers "zippering" to catch up as new contractile pulses propel further changes in cell topologies. These results argue against specific zones of topological initiation and demonstrate the importance of systematic 4D-based analysis in understanding how forces and deformations in cell dimensions propagate in a three-dimensional environment.

APOE3 Christchurch modulates ß-catenin/Wnt signaling in iPS cell-derived cerebral organoids from Alzheimer's cases.

A patient with the PSEN1 E280A mutation and homozygous for APOE3 Christchurch (APOE3Ch) displayed extreme resistance to Alzheimer's disease (AD) cognitive decline and tauopathy, despite having a high amyloid burden. To further investigate the differences in biological processes attributed to APOE3Ch, we generated induced pluripotent stem (iPS) cell-derived cerebral organoids from this resistant case and a non-protected control, using CRISPR/Cas9 gene editing to modulate APOE3Ch expression. In the APOE3Ch cerebral organoids, we observed a protective pattern from early tau phosphorylation. ScRNA sequencing revealed regulation of Cadherin and Wnt signaling pathways by APOE3Ch, with immunostaining indicating elevated ß-catenin protein levels. Further in vitro reporter assays unexpectedly demonstrated that ApoE3Ch functions as a Wnt3a signaling enhancer. This work uncovered a neomorphic molecular mechanism of protection of ApoE3 Christchurch, which may serve as the foundation for the future development of protected case-inspired therapeutics targeting AD and tauopathies.

Interface extension is a continuum property suggesting a linkage between AP contractile and DV lengthening processes.

In the early Drosophila embryo, the elongation of the anterior-posterior (AP) body axis is driven by cell intercalation in the germband epithelium. Neighboring cells intercalate through the contraction of AP interfaces (between AP neighbors) into higher-order vertices, which then resolve through the extension of new dorsal-ventral (DV) interfaces (between DV neighbors). Although interface contraction has been extensively studied, less is known about how new interfaces are established. Here we show that DV interface elongation behaviors initiate at the same time as AP contractions, and that DV interfaces which are newly created from resolution of higher-order vertices do not appear to possess a unique 'identity;' instead, all horizontal interfaces undergo lengthening, elongating through ratchetlike sliding behaviors analogous to those found in AP interfaces. Cortical F-actin networks are essential for high area oscillation amplitudes required for effective ratcheting. Our results suggest that, contrary to canonical models, the elongation of new DV interfaces is not produced by a mechanistically separate process. Instead, medial myosin populations drive oscillating radial forces in the cells to generate transient force asymmetries at all tricellular vertices, which-combined with planar polarized stabilization-produce directional ratcheted sliding to generate both AP interface contraction and DV interface elongation.

A PtdIns(3,4,5)P3 dispersal switch engages cell ratcheting at specific cell surfaces.

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating contractile forces before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be ratcheting mechanisms that drive processive transformations in cell shape. Previous work has shown that force generation is coordinated with endocytic remodeling; however, how ratcheting becomes engaged at specific cell surfaces remains unclear. Here, we report that PtdIns(3,4,5)P3 is a critical lipid-based cue for ratcheting engagement. The Sbf RabGEF binds to PIP3, and disruption of PIP3 reveals a dramatic switching behavior in which medial ratcheting is activated and epithelial cells begin globally constricting apical surfaces. PIP3 enrichments are developmentally regulated, with mesodermal cells having high apical PIP3 while germband cells have higher interfacial PIP3. Finally, we show that JAK/STAT signaling constitutes a second pathway that combinatorially regulates Sbf/Rab35 recruitment. Our results elucidate a complex lipid-dependent regulatory machinery that directs ratcheting engagement in epithelial tissues.

Cell ratcheting through the Sbf RabGEF directs force balancing and stepped apical constriction.

During Drosophila melanogaster gastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a trafficking pathway initiated by Sbf is disrupted, contractile steps become reversible. Sbf localizes to tubular, apical surfaces and associates with Rab35, where it promotes Rab GTP exchange. Interestingly, when Sbf/Rab35 function is compromised, the apical plasma membrane becomes deeply convoluted, and nonuniform cell behaviors begin to emerge. Consistent with this, Sbf/Rab35 appears to prefigure and organize the apical surface for efficient Myosin function. Finally, we show that Sbf/Rab35/CME directs the plasma membrane to Rab11 endosomes through a dynamic interaction with Rab5 endosomes. These results suggest that periodic ratcheting events shift excess membrane from cell apices into endosomal pathways to permit reshaping of actomyosin networks and the apical surface.

Vertex sliding drives intercalation by radial coupling of adhesion and actomyosin networks during Drosophila germband extension.

Oriented cell intercalation is an essential developmental process that shapes tissue morphologies through the directional insertion of cells between their neighbors. Previous research has focused on properties of cell-cell interfaces, while the function of tricellular vertices has remained unaddressed. Here, we identify a highly novel mechanism in which vertices demonstrate independent sliding behaviors along cell peripheries to produce the topological deformations responsible for intercalation. Through systematic analysis, we find that the motion of vertices connected by contracting interfaces is not physically coupled, but instead possess strong radial coupling. E-cadherin and Myosin II exist in previously unstudied populations at cell vertices and undergo oscillatory cycles of accumulation and dispersion that are coordinated with changes in cell area. Additionally, peak enrichment of vertex E-cadherin/Myosin II coincides with interface length stabilization. Our results suggest a model in which asymmetric radial force balance directs the progressive, ratcheted motion of individual vertices to drive intercalation.

Planar polarized Rab35 functions as an oscillatory ratchet during cell intercalation in the Drosophila epithelium.

The coordination between membrane trafficking and actomyosin networks is essential to the regulation of cell and tissue shape. Here, we examine Rab protein distributions during Drosophila epithelial tissue remodeling and show that Rab35 is dynamically planar polarized. Rab35 compartments are enriched at contractile interfaces of intercalating cells and provide the first evidence of interfacial monopolarity. When Rab35 function is disrupted, apical area oscillations still occur and contractile steps are observed. However, contractions are followed by reversals and interfaces fail to shorten, demonstrating that Rab35 functions as a ratchet ensuring unidirectional movement. Although actomyosin forces have been thought to drive interface contraction, initiation of Rab35 compartments does not require Myosin II function. However, Rab35 compartments do not terminate and continue to grow into large elongated structures following actomyosin disruption. Finally, Rab35 represents a common contractile cell-shaping mechanism, as mesoderm invagination fails in Rab35 compromised embryos and Rab35 localizes to constricting surfaces.Various stages of tissue morphogenesis involve the contraction of epithelial surfaces. Here, the authors identify the Rab GTPase Rab35 as an essential component of this contractile process, which functions as a membrane ratchet to ensure unidirectional movement of intercalating cells.