CRISPR-Cas9 knockin mice for genome editing and cancer modeling.

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.

Removal of a partial genomic duplication restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

BACKGROUND: Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. RESULTS: Using the ASD and anxiety mouse model Flailer, which contains a partial genomic duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700 bp genomic region in vitro and in vivo. Importantly, DN-CRISPRs have not been used to remove genomic regions using sgRNA with an offset greater than 300 bp. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene editing. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescue some of the mutant behaviors, while intracerebroventricular delivery, completely recovers the Flailer animal phenotype associated to anxiety and ASD. CONCLUSIONS: Our results demonstrate the potential of DN-CRISPR to efficiently remove larger genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

CD44 regulates dendrite morphogenesis through Src tyrosine kinase-dependent positioning of the Golgi.

The acquisition of proper dendrite morphology is a crucial aspect of neuronal development towards the formation of a functional network. The role of the extracellular matrix and its cellular receptors in this process has remained enigmatic. We report that the CD44 adhesion molecule, the main hyaluronan receptor, is localized in dendrites and plays a crucial inhibitory role in dendritic tree arborization in vitro and in vivo. This novel function is exerted by the activation of Src tyrosine kinase, leading to the alteration of Golgi morphology. The mechanism operates during normal brain development, but its inhibition might have a protective influence on dendritic trees under toxic conditions, during which the silencing of CD44 expression prevents dendritic shortening induced by glutamate exposure. Overall, our results indicate a novel role for CD44 as an essential regulator of dendritic arbor complexity in both health and disease.

Cyr61, a matricellular protein, is needed for dendritic arborization of hippocampal neurons.

The shape of the dendritic arbor is one of the criteria of neuron classification and reflects functional specialization of particular classes of neurons. The development of a proper dendritic branching pattern strongly relies on interactions between the extracellular environment and intracellular processes responsible for dendrite growth and stability. We previously showed that mammalian target of rapamycin (mTOR) kinase is crucial for this process. In this work, we performed a screen for modifiers of dendritic growth in hippocampal neurons, the expression of which is potentially regulated by mTOR. As a result, we identified Cyr61, an angiogenic factor with unknown neuronal function, as a novel regulator of dendritic growth, which controls dendritic growth in a ß1-integrin-dependent manner.

Experimental Investigation of Load-Bearing Capacity in EN AW-2024-T3 Aluminum Alloy Sheets Strengthened by SPIF-Fabricated Stiffening Rib.

The aluminum strength-to-weight ratio has become a highly significant factor in industrial applications. Placing stiffening ribs along the surface can significantly improve the panel's resistance to bending and compression in aluminum alloys. This study used single-point incremental forming (SPIF) to fabricate stiffening ribs for 1 mm and 3 mm thick aluminum alloy EN AW-2024-T3 sheets. A universal compression machine was used to investigate sheet deformation. The resulting deformation was examined using non-contact digital image correlation (DIC) based on several high-resolution cameras. The results showed that deformation progressively escalated from the edges toward the center, and the highest buckling values were confined within the non-strengthened area. Specimens with a larger thickness (3 mm) showed better effectiveness against buckling and bending for each applied load: 8 kN or 10 kN. Additionally, the displacement from the sheet surface decreased by 60% for sheets 3 mm thick and by half for sheets 1 mm thick, which indicated that thicker sheets could resist deformation better.

Mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) control the dendritic arbor morphology of hippocampal neurons.

Dendrites are the main site of information input into neurons. Their development is a multistep process controlled by mammalian target of rapamycin (mTOR) among other proteins. mTOR is a serine/threonine protein kinase that forms two functionally distinct complexes in mammalian cells: mTORC1 and mTORC2. However, the one that contributes to mammalian neuron development remains unknown. This work used short hairpin RNA against Raptor and Rictor, unique components of mTORC1 and mTORC2, respectively, to dissect mTORC involvement in this process. We provide evidence that both mTOR complexes are crucial for the proper dendritic arbor morphology of hippocampal neurons. These two complexes are required for dendritic development both under basal conditions and upon the induction of mTOR-dependent dendritic growth. We also identified Akt as a downstream effector of mTORC2 needed for proper dendritic arbor morphology, the action of which required mTORC1 and p70S6K1.

Removal of a genomic duplication by double-nicking CRISPR restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies. Using the ASD and anxiety mouse model Flailer, that contains a duplication working as a dominant negative for MyoVa, we demonstrate the use of DN-CRISPRs to remove a 700bp genomic duplication in vitro and in vivo . Importantly, DN-CRISPRs have not been used to remove more gene regions <100bp successfully and with high efficiency. We found that editing the flailer gene in primary cortical neurons reverts synaptic transport and transmission defects. Moreover, long-term depression (LTD), disrupted in Flailer animals, is recovered after gene edition. Delivery of DN-CRISPRs in vivo shows that local delivery to the ventral hippocampus can rescues some of the mutant behaviors, while intracerebroventricular delivery, completely recovers Flailer animal phenotype associated to anxiety and ASD. Our results demonstrate the potential of DN-CRISPR to efficiently (>60% editing in vivo) remove large genomic duplications, working as a new gene therapy approach for treating neurodegenerative diseases.

CLIP-170 and IQGAP1 cooperatively regulate dendrite morphology.

Dendritic arbors are compartments of neurons dedicated to receiving synaptic inputs. Their shape is an outcome of both the intrinsic genetic program and environmental signals. The microtubules and actin cytoskeleton are both crucial for proper dendritic morphology, but how they interact is unclear. The present study demonstrates that microtubule plus-end tracking protein CLIP-170 and actin-binding protein IQGAP1 regulate dendrite morphology of rat neurons by coordinating the interaction between microtubules and the actin cytoskeleton. Moreover, we show that mTOR kinase interacts with CLIP-170 and is needed for efficient formation of a protein complex containing CLIP-170 and IQGAP1. Dynamic microtubules, CLIP-170, and IQGAP1 are required for proper dendritic arbor morphology and PI3K-mTOR-induced increase in dendritic arbor complexity. Moreover, CLIP-170 and IQGAP1 knockdown modulates dendritic arbor growth via regulation of the actin cytoskeleton. We postulate that mTOR controls dendritic arbor morphology by enhancing cross talk between dynamic microtubules and actin through CLIP-170 and IQGAP1.

Developmental plasticity of the dendritic compartment: focus on the cytoskeleton.

Plasticity, the ability to undergo lasting changes in response to a stimulus, is an important attribute of neurons. It allows proper development and underlies learning, memory, and the recovery of the nervous system after severe injuries. Often, an outcome of neuronal plasticity is a structural plasticity manifested as a change of neuronal morphology. In this chapter, we focus on the structural plasticity of dendritic arbors and spines during development. Dendrites receive and compute synaptic inputs from other neurons. The number of dendrites and their branching pattern are strictly correlated with the function of a particular neuron and the geometry of the connections it receives. The development of proper dendritic tree morphology depends on the interplay between genetic programming and extracellular signals. Spines are tiny actin-rich dendritic protrusions that harbor excitatory synapses. No consensus has been reached regarding how dendritic spines form, and several models of spine morphogenesis exist. Nevertheless, most researchers agree that spinogenesis is an important target for structural plasticity. In this chapter, we discuss examples of such plasticity and describe the principles and molecular mechanisms underlying this process, focusing mostly on the regulation of the cytoskeleton during dendrito- and spinogenesis.

Expression of Ttyh1, a member of the Tweety family in neurons in vitro and in vivo and its potential role in brain pathology.

We have previously shown that Ttyh1 mRNA is expressed in neurons and its expression is up-regulated in the brain during epileptogenesis and epilepsy. In this study, we aimed to elucidate the role of Ttyh1 in neurons. We found widespread expression of Ttyh1 protein in neurons in vivo and in vitro. Ttyh1 immunoreactivity in vitro was frequently found in invaginations of dendritic spines; however, Ttyh1, seldom co-localized with synaptic markers in vivo. Silencing Ttyh1 expression with siRNA in hippocampal cultures resulted in alterations of MAP2 distribution along neurites causing it to appear in the form of chains of beads. Over-expression of Ttyh1 caused intense neuritogenesis and the formation of numerous filopodia-like protrusions. Similar protrusions were also produced in SH-SY5Y neuroblastoma cells over-expressing Ttyh1. Using a biotin-streptavidin pull-down assay and mass spectrometry, we identified proteins that can form complexes with Ttyh1 in the brain. Ttyh1 binding proteins are often expressed in the endoplasmic reticulum or the Golgi apparatus or are localized at synapses. Finally, we found increased expression of Ttyh1 in the inner molecular layer of the dentate gyrus in an animal model of epilepsy. On the basis of our findings, we propose Ttyh1 involvement in brain pathology.

Experimental Analysis of Perimeter Shear Strength of Composite Sandwich Structures.

The work concerns the experimental analysis of the process of destruction of sandwich structures as a result of circumferential shearing. The aim of the research was to determine the differences that occur in the destruction mechanism of such structures depending on the thickness and material of the core used. Specimens with a Rohacell foam core and a honeycomb core were made for the purposes of the research. The specimen destruction process was carried out in a static loading test with the use of a system introducing circumferential shear stress. The analysis of the tests results was made based on the load-displacement curves, the maximum load, and the energy absorbed by individual specimens. The tests indicated significant differences in the destruction mechanism of specimens with varied core material. The specimen with the honeycomb core was characterized by greater stiffness, which caused the damage to occur locally in the area subjected to the pressure of the punch. In specimens with the foam core, due to the lower stiffness of that core, the skins of the structure were bent, which additionally transfers compressive and tensile loads. This led to a higher maximum force that the specimens obtained at the time of destruction and greater energy absorption.

The Impact of 3D Printing Parameters on the Post-Buckling Behavior of Thin-Walled Structures.

This study presents the results of experimental research and numerical calculations regarding models of a typical torsion box fragment, which is a common thin-walled load-bearing structure used in aviation technology. A fragment of this structure corresponding to the spar wall was made using 3D printing. The examined system was subjected to twisting and underwent post-critical deformation. The research was aimed at determining the influence of the printing direction of the structure's individual layers on the system stiffness. The experimental phase was supplemented by nonlinear numerical analyses of the models of the studied systems, taking into account the details of the structure mapping using the laminate concept. The purpose of the calculations was to determine the usefulness of the adopted method for modeling the examined structures by assessing the compliance of numerical solutions with the results of the experiment.

Role of mTOR in physiology and pathology of the nervous system.

Mammalian target of rapamycin (mTOR) is a serine-threonine protein kinase that regulates several intracellular processes in response to extracellular signals, nutrient availability, energy status of the cell and stress. mTOR regulates survival, differentiation and development of neurons. Axon growth and navigation, dendritic arborization, as well as synaptogenesis, depend on proper mTOR activity. In adult brain mTOR is crucial for synaptic plasticity, learning and memory formation, and brain control of food uptake. Recent studies reveal that mTOR activity is modified in various pathologic states of the nervous system, including brain tumors, tuberous sclerosis, cortical displasia and neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases. This review presents current knowledge about the role of mTOR in the physiology and pathology of the nervous system, with special focus on molecular targets acting downstream of mTOR that potentially contribute to neuronal development, plasticity and neuropathology.

Experimental and Numerical Studies of Low-Profile, Triangular Grid-Stiffened Plates Subjected to Shear Load in the Post-Critical States of Deformation.

Constant developments in manufacturing technology have made it possible to introduce integrally stiffened elements into load-bearing, thin-walled structures. The application of thin-walled elements with integral stiffeners potentially increases buckling and critical loads to maintain the mass of the structure and lower production costs. This paper presents the results of experimental investigations and numerical Finite Element Modelling (FEM) analyses of low-profile, isosceles grid stiffened, aluminium alloy plates subjected to pure shear load. Conducted research included analysing buckling and post-buckling states of deformation, taking into account both geometrical and physical nonlinear effects. Use of the Digital Image Correlation (DIC) system during the experimental tests created representative equilibrium pathways and recorded displacement field distributions over the plate surface. The model was initially validated against the experimental results. The results for the stiffened plate were compared to the reference structure in the form of a smooth plate with equivalent mass. Comparative analyses included examining the displacement fields and stress efforts over the plates. The stiffening configuration under examination increased the critical buckling load by 300% in comparison to the unstiffened structure with the same mass. Obtained results also indicate potential problems with areas of concentrated stress in the case of an incorrect geometry design near to the boundary conditions.

Experimental and Numerical Investigations of Thin-Walled Stringer-Stiffened Panels Welded with RFSSW Technology under Uniaxial Compression.

Many aircraft structures are thin walled and stringer-stiffened, and therefore, prone to a loss of stability. This paper deals with accurate and validated stability analysis of the model of aircraft skin under compressive loading. Both experimental and numerical analyzes are conducted. Two different methods of joining panel elements are considered. In the first case, the panel is fabricated using rivets. In the second variant, the refill friction stir spot welding technique is used. Both types of panels are loaded in axial compression in a uniaxial tensile testing machine. The geometrically and physically nonlinear finite element analyzes of the panels were carried out in ABAQUS/Standard. The Digital Image Correlation measurement system ARAMIS has been utilized to monitor the buckling behavior and failure mode in the skin-stringer interface of the stiffened panels. The results of experiments and the digital image correlation system are presented and compared to the numerical simulations.

Accumulation and dynamics of proteins of the MCM family during mouse oogenesis and the first embryonic cell cycle.

We describe the localization of three proteins of the minichromosome maintenance (MCM) family, Mcm2, -6 and -7 in mouse ovarian oocytes. We showed that Mcm proteins are stored in two forms: soluble and insoluble. Soluble Mcm2, -6 and -7 were uniformly distributed in the nuclei of ovarian oocytes. Insoluble Mcm2 and Mcm7 (but not Mcm6) were detected in the nuclei of resting, growing and fully-grown transcribing oocytes. In transcriptionally inactive fully-grown oocytes, Mcm2 underwent redistribution and Mcm7 disappeared. A similar effect was observed when transcription in growing oocytes was inhibited with alpha-amanitin. We postulate that in mouse oogenesis, the insoluble Mcm proteins are engaged in processes related to regulation of transcription and/or chromatin organization. In oocytes preparing for meiotic maturation, aggregates of the insoluble form of Mcm2 fragmented, dispersed and ultimately disappeared from the nuclei. Numerous Mcm2-positive deposits were observed in the cytoplasm of maturing oocytes. In the one-cell embryo, insoluble Mcm2 appeared in the G1 nucleus, persisted in the S phase and was undetectable in the G2 nucleus. Such behavior of Mcm2 supports its involvement in chromatin licensing in the first embryonic cell cycle.

Selective FcγR Co-engagement on APCs Modulates the Activity of Therapeutic Antibodies Targeting T Cell Antigens.

The co-engagement of fragment crystallizable (Fc) gamma receptors (FcγRs) with the Fc region of recombinant immunoglobulin monoclonal antibodies (mAbs) and its contribution to therapeutic activity has been extensively studied. For example, Fc-FcγR interactions have been shown to be important for mAb-directed effector cell activities, as well as mAb-dependent forward signaling into target cells via receptor clustering. Here we identify a function of mAbs targeting T cell-expressed antigens that involves FcγR co-engagement on antigen-presenting cells (APCs). In the case of mAbs targeting CTLA-4 and TIGIT, the interaction with FcγR on APCs enhanced antigen-specific T cell responses and tumoricidal activity. This mechanism extended to an anti-CD45RB mAb, which led to FcγR-dependent regulatory T cell expansion in mice.

Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons.

Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by 5-ethynyl-2'-deoxyuridine (EdU) to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. We also apply Div-Seq to identify and profile rare newborn neurons in the adult spinal cord, a noncanonical neurogenic region. sNuc-Seq and Div-Seq open the way for unbiased analysis of diverse complex tissues.

In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9.

Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain.

Tuberous sclerosis complex neuropathology requires glutamate-cysteine ligase.

INTRODUCTION: Tuberous sclerosis complex (TSC) is a genetic disease resulting from mutation in TSC1 or TSC2 and subsequent hyperactivation of mammalian Target of Rapamycin (mTOR). Common TSC features include brain lesions, such as cortical tubers and subependymal giant cell astrocytomas (SEGAs). However, the current treatment with mTOR inhibitors has critical limitations. We aimed to identify new targets for TSC pharmacotherapy. RESULTS: The results of our shRNA screen point to glutamate-cysteine ligase catalytic subunit (GCLC), a key enzyme in glutathione synthesis, as a contributor to TSC-related phenotype. GCLC inhibition increased cellular stress and reduced mTOR hyperactivity in TSC2-depleted neurons and SEGA-derived cells. Moreover, patients' brain tubers showed elevated GCLC and stress markers expression. Finally, GCLC inhibition led to growth arrest and death of SEGA-derived cells. CONCLUSIONS: We describe GCLC as a part of redox adaptation in TSC, needed for overgrowth and survival of mutant cells, and provide a potential novel target for SEGA treatment.