Discovering novel mechanisms of human cortical development & disease using in vivo mouse model and in vitro human-derived cerebral organoids

This thesis combines three research studies with the common interest of identifying novel mechanisms underlying human cortical development. This aim is pursued from different angles, always basing the investigations on human induced pluripotent stem cell-derived 2D and 3D in vitro model systems that are partly combined with in vivo studies in the developing mouse cortex. Namely, in the pieces of work combined here, we 1) bring to light a neurodevelopmental role of a gene already implicated in adult nervous system function, 2) discover a novel mechanism that fine-tunes human neurogenesis, and 3) identify a novel gene whose mutations lead to a malformation of cortical development. The entirety of this work thus adds several aspects to the existing knowledge. In the first study, we identified a neurodevelopmental function of a gene mutated in patients with the progressive gait disorder hereditary spastic paraplegia (HSP). In this group of inherited neurodegenerative diseases, mutations in lipid, mitochondrial, cytoskeletal or transport proteins lead to degeneration of primary motor neurons, which, due to the length of their axons, are particularly sensitive to disruption of these processes. Here, were generated cerebral organoids (COs) derived from HSP patients with mutations in SPG11 coding for spatacsin. Previous work had shown impaired proliferation of SPG11 patient-derived neural progenitor cells (NPCs). We found a proliferation defect also in CO NPCs, leading to a thinner progenitor zone and premature neurogenesis due to increased asymmetric progenitor divisions, along with smaller size of patient-derived COs. Molecularly, we found a decrease in deactivated GSK3β and increase in P-βcatenin at the basis of the observed proliferation/neurogenesis imbalance. We thus confirmed the neurodevelopmental role of SPG11 that had previously been suggested from 2D human in vitro findings. Both the observed reduction in proliferating progenitors and in organoid size were rescued through inhibition of GSK3β, with the Food and Drug Administration (FDA) approved compound tideglusib only affecting patient COs. These rescue experiments thus stressed the opportunity that COs represent for drug testing and translation of findings to precision medicine. In the second study, we investigated the role of a novel posttranslational modification (PTM) termed AMPylation in neurogenesis. Using a novel probe for the detection of AMPylated proteins and a combination of mass spectrometry-based proteomics, immunohistochemistry, and acute interference with the expression of the AMPylating enzyme, we made several interesting findings: AMPylation takes place on a cell type-specific set of proteins, is responsive to the predominant environmental condition, and both AMPylator and targets localize to cell type-specific intracellular localizations. During the process of neuronal differentiation, the set of AMPylated proteins is completely remodeled, with a very high number of unique targets in neurons. These include metabolic enzymes as in all analyzed cell types and, additionally and specifically, cytoskeletal and motor proteins. Cytoskeletal and motor proteins in neural progenitors and neurons are known to be differentially modified by several PTMs whose correct establishment is highly important during neurodevelopment; AMPylation may thus be an additional one. To assess the role of AMPylation in neurodevelopment, we manipulated the expression of the AMPylating enzyme FICD in COs. Downregulation kept cells in a proliferating progenitor state, whereas overexpression increased neurogenesis. We thus suggest AMPylation as a novel PTM fine-tuning neurogenesis. The third study focused on the identification of new mechanisms underlying cortical malformations, aiming at a better understanding of how the human brain develops. In patients with periventricular heterotopia (PH), a neuronal migration disorder in which a subset of neurons fail to migrate to the developing cortical plate and instead form nodules of grey matter lining the lateral ventricles as their site of production, biallelic mutations in endothelin converting enzyme 2 (ECE2) were identified as candidate causative. Combining in vitro and in vivo models, we found a role for ECE2 in neuronal migration and cortical development. In the absence of ECE2, several processes of general importance to proper neuronal migration were disrupted. Namely, changes in progenitor cell polarity and morphology and in apical adherens junctions led to their delamination, restricting their use as a scaffold for neuronal migration. This resulted in ectopic neurons reminiscent of nodules in PH. Besides a deregulation of cytoskeletal, polarity, and apical adhesion proteins, extracellular matrix (ECM) proteins were reduced in absence of ECE2, suggesting its role in ECM production and underlining the necessity of ECM components for proper neuronal migration during cortical development. Moreover, we detected differential phosphorylation of several cytoskeletal, motor and adhesion proteins in the absence of ECE2, which is functionally in line with the former findings and suggests an additional involvement of ECE2 in the regulation of PTMs. Altogether, the studies presented here underline the heterogeneity and complexity of pathways and mechanisms that contribute to human cortical development and its disorders, converging on the regulation of cytoskeleton and transport within the involved cells and of the ECM on their outside

Discovering novel mechanisms of human cortical development disease using in vivo mouse model and in vitro human-derived cerebral organoids.

This thesis combines three research studies with the common interest of identifying novel mechanisms underlying human cortical development. This aim is pursued from different angles, always basing the investigations on human induced pluripotent stem cell-derived 2D and 3D in vitro model systems that are partly combined with in vivo studies in the developing mouse cortex. Namely, in the pieces of work combined here, we 1) bring to light a neurodevelopmental role of a gene already implicated in adult nervous system function, 2) discover a novel mechanism that fine-tunes human neurogenesis, and 3) identify a novel gene whose mutations lead to a malformation of cortical development. The entirety of this work thus adds several aspects to the existing knowledge. In the first study, we identified a neurodevelopmental function of a gene mutated in patients with the progressive gait disorder hereditary spastic paraplegia (HSP). In this group of inherited neurodegenerative diseases, mutations in lipid, mitochondrial, cytoskeletal or transport proteins lead to degeneration of primary motor neurons, which, due to the length of their axons, are particularly sensitive to disruption of these processes. Here, were generated cerebral organoids (COs) derived from HSP patients with mutations in SPG11 coding for spatacsin. Previous work had shown impaired proliferation of SPG11 patient-derived neural progenitor cells (NPCs). We found a proliferation defect also in CO NPCs, leading to a thinner progenitor zone and premature neurogenesis due to increased asymmetric progenitor divisions, along with smaller size of patient-derived COs. Molecularly, we found a decrease in deactivated GSK3β and increase in P-βcatenin at the basis of the observed proliferation/neurogenesis imbalance. We thus confirmed the neurodevelopmental role of SPG11 that had previously been suggested from 2D human in vitro findings. Both the observed reduction in proliferating progenitors and in organoid size were rescued through inhibition of GSK3β, with the Food and Drug Administration (FDA) approved compound tideglusib only affecting patient COs. These rescue experiments thus stressed the opportunity that COs represent for drug testing and translation of findings to precision medicine. In the second study, we investigated the role of a novel posttranslational modification (PTM) termed AMPylation in neurogenesis. Using a novel probe for the detection of AMPylated proteins and a combination of mass spectrometry-based proteomics, immunohistochemistry, and acute interference with the expression of the AMPylating enzyme, we made several interesting findings: AMPylation takes place on a cell type-specific set of proteins, is responsive to the predominant environmental condition, and both AMPylator and targets localize to cell type-specific intracellular localizations. During the process of neuronal differentiation, the set of AMPylated proteins is completely remodeled, with a very high number of unique targets in neurons. These include metabolic enzymes as in all analyzed cell types and, additionally and specifically, cytoskeletal and motor proteins. Cytoskeletal and motor proteins in neural progenitors and neurons are known to be differentially modified by several PTMs whose correct establishment is highly important during neurodevelopment; AMPylation may thus be an additional one. To assess the role of AMPylation in neurodevelopment, we manipulated the expression of the AMPylating enzyme FICD in COs. Downregulation kept cells in a proliferating progenitor state, whereas overexpression increased neurogenesis. We thus suggest AMPylation as a novel PTM fine-tuning neurogenesis. The third study focused on the identification of new mechanisms underlying cortical malformations, aiming at a better understanding of how the human brain develops. In patients with periventricular heterotopia (PH), a neuronal migration disorder in which a subset of neurons fail to migrate to the developing cortical plate and instead form nodules of grey matter lining the lateral ventricles as their site of production, biallelic mutations in endothelin converting enzyme 2 (ECE2) were identified as candidate causative. Combining in vitro and in vivo models, we found a role for ECE2 in neuronal migration and cortical development. In the absence of ECE2, several processes of general importance to proper neuronal migration were disrupted. Namely, changes in progenitor cell polarity and morphology and in apical adherens junctions led to their delamination, restricting their use as a scaffold for neuronal migration. This resulted in ectopic neurons reminiscent of nodules in PH. Besides a deregulation of cytoskeletal, polarity, and apical adhesion proteins, extracellular matrix (ECM) proteins were reduced in absence of ECE2, suggesting its role in ECM production and underlining the necessity of ECM components for proper neuronal migration during cortical development. Moreover, we detected differential phosphorylation of several cytoskeletal, motor and adhesion proteins in the absence of ECE2, which is functionally in line with the former findings and suggests an additional involvement of ECE2 in the regulation of PTMs. Altogether, the studies presented here underline the heterogeneity and complexity of pathways and mechanisms that contribute to human cortical development and its disorders, converging on the regulation of cytoskeleton and transport within the involved cells and of the ECM on their outside. <br

Discovering novel mechanisms of human cortical development & disease using in vivo mouse model and in vitro human-derived cerebral organoids

This thesis combines three research studies with the common interest of identifying novel mechanisms underlying human cortical development. This aim is pursued from different angles, always basing the investigations on human induced pluripotent stem cell-derived 2D and 3D in vitro model systems that are partly combined with in vivo studies in the developing mouse cortex. Namely, in the pieces of work combined here, we 1) bring to light a neurodevelopmental role of a gene already implicated in adult nervous system function, 2) discover a novel mechanism that fine-tunes human neurogenesis, and 3) identify a novel gene whose mutations lead to a malformation of cortical development. The entirety of this work thus adds several aspects to the existing knowledge. In the first study, we identified a neurodevelopmental function of a gene mutated in patients with the progressive gait disorder hereditary spastic paraplegia (HSP). In this group of inherited neurodegenerative diseases, mutations in lipid, mitochondrial, cytoskeletal or transport proteins lead to degeneration of primary motor neurons, which, due to the length of their axons, are particularly sensitive to disruption of these processes. Here, were generated cerebral organoids (COs) derived from HSP patients with mutations in SPG11 coding for spatacsin. Previous work had shown impaired proliferation of SPG11 patient-derived neural progenitor cells (NPCs). We found a proliferation defect also in CO NPCs, leading to a thinner progenitor zone and premature neurogenesis due to increased asymmetric progenitor divisions, along with smaller size of patient-derived COs. Molecularly, we found a decrease in deactivated GSK3β and increase in P-βcatenin at the basis of the observed proliferation/neurogenesis imbalance. We thus confirmed the neurodevelopmental role of SPG11 that had previously been suggested from 2D human in vitro findings. Both the observed reduction in proliferating progenitors and in organoid size were rescued through inhibition of GSK3β, with the Food and Drug Administration (FDA) approved compound tideglusib only affecting patient COs. These rescue experiments thus stressed the opportunity that COs represent for drug testing and translation of findings to precision medicine. In the second study, we investigated the role of a novel posttranslational modification (PTM) termed AMPylation in neurogenesis. Using a novel probe for the detection of AMPylated proteins and a combination of mass spectrometry-based proteomics, immunohistochemistry, and acute interference with the expression of the AMPylating enzyme, we made several interesting findings: AMPylation takes place on a cell type-specific set of proteins, is responsive to the predominant environmental condition, and both AMPylator and targets localize to cell type-specific intracellular localizations. During the process of neuronal differentiation, the set of AMPylated proteins is completely remodeled, with a very high number of unique targets in neurons. These include metabolic enzymes as in all analyzed cell types and, additionally and specifically, cytoskeletal and motor proteins. Cytoskeletal and motor proteins in neural progenitors and neurons are known to be differentially modified by several PTMs whose correct establishment is highly important during neurodevelopment; AMPylation may thus be an additional one. To assess the role of AMPylation in neurodevelopment, we manipulated the expression of the AMPylating enzyme FICD in COs. Downregulation kept cells in a proliferating progenitor state, whereas overexpression increased neurogenesis. We thus suggest AMPylation as a novel PTM fine-tuning neurogenesis. The third study focused on the identification of new mechanisms underlying cortical malformations, aiming at a better understanding of how the human brain develops. In patients with periventricular heterotopia (PH), a neuronal migration disorder in which a subset of neurons fail to migrate to the developing cortical plate and instead form nodules of grey matter lining the lateral ventricles as their site of production, biallelic mutations in endothelin converting enzyme 2 (ECE2) were identified as candidate causative. Combining in vitro and in vivo models, we found a role for ECE2 in neuronal migration and cortical development. In the absence of ECE2, several processes of general importance to proper neuronal migration were disrupted. Namely, changes in progenitor cell polarity and morphology and in apical adherens junctions led to their delamination, restricting their use as a scaffold for neuronal migration. This resulted in ectopic neurons reminiscent of nodules in PH. Besides a deregulation of cytoskeletal, polarity, and apical adhesion proteins, extracellular matrix (ECM) proteins were reduced in absence of ECE2, suggesting its role in ECM production and underlining the necessity of ECM components for proper neuronal migration during cortical development. Moreover, we detected differential phosphorylation of several cytoskeletal, motor and adhesion proteins in the absence of ECE2, which is functionally in line with the former findings and suggests an additional involvement of ECE2 in the regulation of PTMs. Altogether, the studies presented here underline the heterogeneity and complexity of pathways and mechanisms that contribute to human cortical development and its disorders, converging on the regulation of cytoskeleton and transport within the involved cells and of the ECM on their outside

Remote Sensing of Plant Biodiversity

This Open Access volume aims to methodologically improve our understanding of biodiversity by linking disciplines that incorporate remote sensing, and uniting data and perspectives in the fields of biology, landscape ecology, and geography. The book provides a framework for how biodiversity can be detected and evaluated—focusing particularly on plants—using proximal and remotely sensed hyperspectral data and other tools such as LiDAR. The volume, whose chapters bring together a large cross-section of the biodiversity community engaged in these methods, attempts to establish a common language across disciplines for understanding and implementing remote sensing of biodiversity across scales. The first part of the book offers a potential basis for remote detection of biodiversity. An overview of the nature of biodiversity is described, along with ways for determining traits of plant biodiversity through spectral analyses across spatial scales and linking spectral data to the tree of life. The second part details what can be detected spectrally and remotely. Specific instrumentation and technologies are described, as well as the technical challenges of detection and data synthesis, collection and processing. The third part discusses spatial resolution and integration across scales and ends with a vision for developing a global biodiversity monitoring system. Topics include spectral and functional variation across habitats and biomes, biodiversity variables for global scale assessment, and the prospects and pitfalls in remote sensing of biodiversity at the global scale

Remote Sensing of Plant Biodiversity

At last, here it is. For some time now, the world has needed a text providing both a new theoretical foundation and practical guidance on how to approach the challenge of biodiversity decline in the Anthropocene. This is a global challenge demanding global approaches to understand its scope and implications. Until recently, we have simply lacked the tools to do so. We are now entering an era in which we can realistically begin to understand and monitor the multidimensional phenomenon of biodiversity at a planetary scale. This era builds upon three centuries of scientific research on biodiversity at site to landscape levels, augmented over the past two decades by airborne research platforms carrying spectrometers, lidars, and radars for larger-scale observations. Emerging international networks of fine-grain in-situ biodiversity observations complemented by space-based sensors offering coarser-grain imagery—but global coverage—of ecosystem composition, function, and structure together provide the information necessary to monitor and track change in biodiversity globally. This book is a road map on how to observe and interpret terrestrial biodiversity across scales through plants—primary producers and the foundation of the trophic pyramid. It honors the fact that biodiversity exists across different dimensions, including both phylogenetic and functional. Then, it relates these aspects of biodiversity to another dimension, the spectral diversity captured by remote sensing instruments operating at scales from leaf to canopy to biome. The biodiversity community has needed a Rosetta Stone to translate between the language of satellite remote sensing and its resulting spectral diversity and the languages of those exploring the phylogenetic diversity and functional trait diversity of life on Earth. By assembling the vital translation, this volume has globalized our ability to track biodiversity state and change. Thus, a global problem meets a key component of the global solution. The editors have cleverly built the book in three parts. Part 1 addresses the theory behind the remote sensing of terrestrial plant biodiversity: why spectral diversity relates to plant functional traits and phylogenetic diversity. Starting with first principles, it connects plant biochemistry, physiology, and macroecology to remotely sensed spectra and explores the processes behind the patterns we observe. Examples from the field demonstrate the rising synthesis of multiple disciplines to create a new cross-spatial and spectral science of biodiversity. Part 2 discusses how to implement this evolving science. It focuses on the plethora of novel in-situ, airborne, and spaceborne Earth observation tools currently and soon to be available while also incorporating the ways of actually making biodiversity measurements with these tools. It includes instructions for organizing and conducting a field campaign. Throughout, there is a focus on the burgeoning field of imaging spectroscopy, which is revolutionizing our ability to characterize life remotely. Part 3 takes on an overarching issue for any effort to globalize biodiversity observations, the issue of scale. It addresses scale from two perspectives. The first is that of combining observations across varying spatial, temporal, and spectral resolutions for better understanding—that is, what scales and how. This is an area of ongoing research driven by a confluence of innovations in observation systems and rising computational capacity. The second is the organizational side of the scaling challenge. It explores existing frameworks for integrating multi-scale observations within global networks. The focus here is on what practical steps can be taken to organize multi-scale data and what is already happening in this regard. These frameworks include essential biodiversity variables and the Group on Earth Observations Biodiversity Observation Network (GEO BON). This book constitutes an end-to-end guide uniting the latest in research and techniques to cover the theory and practice of the remote sensing of plant biodiversity. In putting it together, the editors and their coauthors, all preeminent in their fields, have done a great service for those seeking to understand and conserve life on Earth—just when we need it most. For if the world is ever to construct a coordinated response to the planetwide crisis of biodiversity loss, it must first assemble adequate—and global—measures of what we are losing

Marine invertebrates and sound

Peer ReviewedPostprint (published version

2021 Student Symposium Research and Creative Activity Book of Abstracts

The UMaine Student Symposium (UMSS) is an annual event that celebrates undergraduate and graduate student research and creative work. Students from a variety of disciplines present their achievements with video presentations. It’s the ideal occasion for the community to see how UMaine students’ work impacts locally – and beyond. The 2021 Student Symposium Research and Creative Activity Book of Abstracts includes a complete list of student presenters as well as abstracts related to their works