Mice with cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks

AbstractN-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 s than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

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Mice with a cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks

10.21203/rs.3.rs-64344/v3 ◽

2021 ◽

Author(s):

Megumi Asada-Utsugi ◽

Kengo Uemura ◽

Masakazu Kubota ◽

Yasuha Noda ◽

Yasutaka Tashiro ◽

...

Keyword(s):

Memory Function ◽

Three Dimensional ◽

Excitatory Synapses ◽

Missense Mutations ◽

Glutamatergic Synapses ◽

Learning Tasks ◽

Transmission Electron ◽

Nmdar Activation ◽

And Function

Abstract N-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2 R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 sec than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

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Mice with a cleavage-resistant N-cadherin exhibit synapse anomaly in the hippocampus and outperformance in spatial learning tasks

10.21203/rs.3.rs-64344/v2 ◽

2020 ◽

Author(s):

Megumi Asada-Utsugi ◽

Kengo Uemura ◽

Masakazu Kubota ◽

Yasuha Noda ◽

Yasutaka Tashiro ◽

...

Keyword(s):

Memory Function ◽

Three Dimensional ◽

Excitatory Synapses ◽

Missense Mutations ◽

Glutamatergic Synapses ◽

Learning Tasks ◽

Transmission Electron ◽

Nmdar Activation ◽

And Function

Abstract N-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2 R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 sec than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

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Mice With A Cleavage-Resistant N-Cadherin Exhibit Synapse Anomaly in the Hippocampus and Outperformance in Spatial Memory Tasks

10.21203/rs.3.rs-64344/v1 ◽

2020 ◽

Author(s):

Megumi Asada-Utsugi ◽

Kengo Uemura ◽

Masakazu Kubota ◽

Yasuha Noda ◽

Yasutaka Tashiro ◽

...

Keyword(s):

Memory Function ◽

Three Dimensional ◽

Excitatory Synapses ◽

Missense Mutations ◽

Glutamatergic Synapses ◽

Synaptic Density ◽

Transmission Electron ◽

Nmdar Activation ◽

And Function

Abstract N-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2 R714G, I715D, or GD) that confers resistance to proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 sec than WT and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.

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Three-Dimensional Cultures of Normal and Malignant Human Breast Epithelial Cells to Achieve in vivo-like Architecture and Function

Cell Biology ◽

10.1016/b978-012164730-8/50018-6 ◽

2006 ◽

pp. 139-149

Author(s):

C MYERS ◽

H LIU ◽

E LEE ◽

M BISSELL

Keyword(s):

Epithelial Cells ◽

Three Dimensional ◽

Human Breast ◽

Breast Epithelial Cells ◽

Breast Epithelial ◽

And Function ◽

Human Breast Epithelial Cells

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Three-dimensional echocardiography. In vivo validation for right ventricular volume and function.

Circulation ◽

10.1161/01.cir.89.5.2342 ◽

1994 ◽

Vol 89 (5) ◽

pp. 2342-2350 ◽

Cited By ~ 108

Author(s):

L Jiang ◽

S C Siu ◽

M D Handschumacher ◽

J Luis Guererro ◽

J A Vazquez de Prada ◽

...

Keyword(s):

Right Ventricular ◽

Three Dimensional ◽

Ventricular Volume ◽

Right Ventricular Volume ◽

Dimensional Echocardiography ◽

Three Dimensional Echocardiography ◽

And Function ◽

In Vivo Validation

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Natural hybrid silica/protein superstructure at atomic resolution

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019140117 ◽

2020 ◽

Vol 117 (49) ◽

pp. 31088-31093

Author(s):

Stefan Görlich ◽

Abisheik John Samuel ◽

Richard Johannes Best ◽

Ronald Seidel ◽

Jean Vacelet ◽

...

Keyword(s):

Tertiary Structure ◽

Three Dimensional ◽

Inorganic Materials ◽

Atomic Resolution ◽

Axial Filament ◽

X Ray Crystallography ◽

Organic Hybrid ◽

Hybrid Silica ◽

Transmission Electron

Formation of highly symmetric skeletal elements in demosponges, called spicules, follows a unique biomineralization mechanism in which polycondensation of an inherently disordered amorphous silica is guided by a highly ordered proteinaceous scaffold, the axial filament. The enzymatically active proteins, silicateins, are assembled into a slender hybrid silica/protein crystalline superstructure that directs the morphogenesis of the spicules. Furthermore, silicateins are known to catalyze the formation of a large variety of other technologically relevant organic and inorganic materials. However, despite the biological and biotechnological importance of this macromolecule, its tertiary structure was never determined. Here we report the atomic structure of silicatein and the entire mineral/organic hybrid assembly with a resolution of 2.4 Å. In this work, the serial X-ray crystallography method was successfully adopted to probe the 2-µm-thick filaments in situ, being embedded inside the skeletal elements. In combination with imaging and chemical analysis using high-resolution transmission electron microscopy, we provide detailed information on the enzymatic activity of silicatein, its crystallization, and the emergence of a functional three-dimensional silica/protein superstructure in vivo. Ultimately, we describe a naturally occurring mineral/protein crystalline assembly at atomic resolution.

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RGD-modified injectable hydrogel maintains islet beta-cell survival and function

Journal of Applied Biomaterials & Functional Materials ◽

10.1177/2280800020963473 ◽

2020 ◽

Vol 18 ◽

pp. 228080002096347

Author(s):

Tianshu Lan ◽

Jingyi Guo ◽

Xiaoming Bai ◽

Zengjiong Huang ◽

Zhimin Wei ◽

...

Keyword(s):

Extracellular Matrix ◽

Islet Transplantation ◽

Pancreatic Beta Cells ◽

Three Dimensional ◽

High Concentration ◽

Β Cells ◽

Glucose Levels ◽

And Function

Objective: A potential solution for islet transplantation and drug discovery vis-à-vis treating diabetes is the production of functional islets in a three-dimensional extracellular matrix. Although several scaffold materials have been reported as viable candidates, a clinically applicable one that is injectable and can maintain long-term functionality and survival of islet pancreatic beta-cells (β-cells) is far from being established. Results: In the current study, we evaluated a ready-to-use and injectable hydrogel’s impact on β-cells’ function and viability, both in vitro and in vivo. We found that β-cells in high concentration with hydrogels functionalized via Arg-Gly-Asp (RGD) demonstrated better viability and insulin secretory capacity in vitro. Moreover, it is a biocompatible hydrogel that can maintain β-cell proliferation and vascularization without stimulating inflammation after subcutaneous injection. Meanwhile, modifying the hydrogel with RGD can maintain β-cells’ secretion of insulin, regulating the blood glucose levels of mice with streptozotocin-induced diabetes. Conclusions: Thus, these preliminary results indicate that this RGD-modified hydrogel is a potential extracellular matrix for islet transplantation at extrahepatic sites, and they also provide a reference for future tissue engineering study.

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Adding exogenous biglycan or decorin improves tendon formation for equine peritenon and tendon proper cells in vitro

BMC Musculoskeletal Disorders ◽

10.1186/s12891-020-03650-2 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Monica Y. Pechanec ◽

Tannah N. Boyd ◽

Keith Baar ◽

Michael J. Mienaltowski

Keyword(s):

Material Properties ◽

Quantitative Polymerase Chain Reaction ◽

Tendon Repair ◽

Three Dimensional ◽

Collagen Content ◽

Matrix Assembly ◽

Post Injury ◽

Transmission Electron

Abstract Background Tendon injuries amount to one of the leading causes of career-ending injuries in horses due to the inability for tendon to completely repair and the high reinjury potential. As a result, novel therapeutics are necessary to improve repair with the goal of decreasing leg lameness and potential reinjury. Small leucine-rich repeat proteoglycans (SLRPs), a class of regulatory molecules responsible for collagen organization and maturation, may be one such therapeutic to improve tendon repair. Before SLRP supplementation can occur in vivo, proper evaluation of the effect of these molecules in vitro needs to be assessed. The objective of this study was to evaluate the effectiveness of purified bovine biglycan or decorin on tendon proper and peritenon cell populations in three-dimensional tendon constructs. Methods Equine tendon proper or peritenon cell seeded fibrin three-dimensional constructs were supplemented with biglycan or decorin at two concentrations (5 nM or 25 nM). The functionality and ultrastructural morphology of the constructs were assessed using biomechanics, collagen content analysis, transmission electron microscopy (TEM), and gene expression by real time – quantitative polymerase chain reaction (RT-qPCR). Results SLRP supplementation affected both tendon proper and peritenon cells-seeded constructs. With additional SLRPs, material and tensile properties of constructs strengthened, though ultrastructural analyses indicated production of similar-sized or smaller fibrils. Overall expression of tendon markers was bolstered more in peritenon cells supplemented with either SLRP, while supplementation of SLRPs to TP cell-derived constructs demonstrated fewer changes in tendon and extracellular matrix markers. Moreover, relative to non-supplemented tendon proper cell-seeded constructs, SLRP supplementation of the peritenon cells showed increases in mechanical strength, material properties, and collagen content. Conclusions The SLRP-supplemented peritenon cells produced constructs with greater mechanical and material properties than tendon proper seeded constructs, as well as increased expression of matrix assembly molecules. These findings provide evidence that SLRPs should be further investigated for their potential to improve tendon formation in engineered grafts or post-injury.

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