@phdthesis{13107, abstract = {Within the human body, the brain exhibits the highest rate of energy consumption amongst all organs, with the majority of generated ATP being utilized to sustain neuronal activity. Therefore, the metabolism of the mature cerebral cortex is geared towards preserving metabolic homeostasis whilst generating significant amounts of energy. This requires a precise interplay between diverse metabolic pathways, spanning from a tissue-wide scale to the level of individual neurons. Disturbances to this delicate metabolic equilibrium, such as those resulting from maternal malnutrition or mutations affecting metabolic enzymes, often result in neuropathological variants of neurodevelopment. For instance, mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), have been associated with autism and microcephaly. However, despite recent progress in the field, the extent of metabolic restructuring that occurs within the developing brain and the corresponding alterations in nutrient demands during various critical periods remain largely unknown. To investigate this, we performed metabolomic profiling of the murine cerebral cortex to characterize the metabolic state of the forebrain at different developmental stages. We found that the developing cortex undergoes substantial metabolic reprogramming, with specific sets of metabolites displaying stage-specific changes. According to our observations, we determined a distinct temporal period in postnatal development during which the cortex displays heightened reliance on LNAAs. Hence, using a conditional knock-out mouse model, we deleted Slc7a5 in neural cells, allowing us to monitor the impact of a perturbed neuronal metabolic state across multiple developmental stages of corticogenesis. We found that manipulating the levels of essential LNAAs in cortical neurons in vivo affects one particular perinatal developmental period critical for cortical network refinement. Abnormally low intracellular LNAA levels result in cell-autonomous alterations in neuronal lipid metabolism, excitability, and survival during this particular time window. Although most of the effects of Slc7a5 deletion on neuronal physiology are transient, derailment of these processes during this brief but crucial window leads to long-term circuit dysfunction in mice. In conclusion, out data indicate that the cerebral cortex undergoes significant metabolic reorganization during development. This process involves the intricate integration of multiple metabolic pathways to ensure optimal neuronal function throughout different developmental stages. Our findings offer a paradigm for understanding how neurons synchronize the expression of nutrient-related genes with their activity to allow proper brain maturation. Further, our results demonstrate that disruptions in these precisely calibrated metabolic processes during critical periods of brain development may result in neuropathological outcomes in mice and in humans.}, author = {Knaus, Lisa}, issn = {2663 - 337X}, pages = {147}, publisher = {Institute of Science and Technology Austria}, title = {{The metabolism of the developing brain : How large neutral amino acids modulate perinatal neuronal excitability and survival}}, doi = {10.15479/at:ista:13107}, year = {2023}, } @article{12802, abstract = {Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.}, author = {Knaus, Lisa and Basilico, Bernadette and Malzl, Daniel and Gerykova Bujalkova, Maria and Smogavec, Mateja and Schwarz, Lena A. and Gorkiewicz, Sarah and Amberg, Nicole and Pauler, Florian and Knittl-Frank, Christian and Tassinari, Marianna and Maulide, Nuno and Rülicke, Thomas and Menche, Jörg and Hippenmeyer, Simon and Novarino, Gaia}, issn = {0092-8674}, journal = {Cell}, keywords = {General Biochemistry, Genetics and Molecular Biology}, number = {9}, pages = {1950--1967.e25}, publisher = {Elsevier}, title = {{Large neutral amino acid levels tune perinatal neuronal excitability and survival}}, doi = {10.1016/j.cell.2023.02.037}, volume = {186}, year = {2023}, } @misc{13126, abstract = {Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here, we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease.}, author = {Danzl, Johann G}, publisher = {Institute of Science and Technology Austria}, title = {{Research data for the publication "Imaging brain tissue architecture across millimeter to nanometer scales"}}, doi = {10.15479/AT:ISTA:13126}, year = {2023}, } @article{14257, abstract = {Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease.}, author = {Michalska, Julia M and Lyudchik, Julia and Velicky, Philipp and Korinkova, Hana and Watson, Jake and Cenameri, Alban and Sommer, Christoph M and Amberg, Nicole and Venturino, Alessandro and Roessler, Karl and Czech, Thomas and Höftberger, Romana and Siegert, Sandra and Novarino, Gaia and Jonas, Peter M and Danzl, Johann G}, issn = {1546-1696}, journal = {Nature Biotechnology}, publisher = {Springer Nature}, title = {{Imaging brain tissue architecture across millimeter to nanometer scales}}, doi = {10.1038/s41587-023-01911-8}, year = {2023}, } @article{12140, abstract = {Microglia are dynamic cells, constantly surveying their surroundings and interacting with neurons and synapses. Indeed, a wealth of knowledge has revealed a critical role of microglia in modulating synaptic transmission and plasticity in the developing brain. In the past decade, novel pharmacological and genetic strategies have allowed the acute removal of microglia, opening the possibility to explore and understand the role of microglia also in the adult brain. In this review, we summarized and discussed the contribution of microglia depletion strategies to the current understanding of the role of microglia on synaptic function, learning and memory, and behavior both in physiological and pathological conditions. We first described the available microglia depletion methods highlighting their main strengths and weaknesses. We then reviewed the impact of microglia depletion on structural and functional synaptic plasticity. Next, we focused our analysis on the effects of microglia depletion on behavior, including general locomotor activity, sensory perception, motor function, sociability, learning and memory both in healthy animals and animal models of disease. Finally, we integrated the findings from the reviewed studies and discussed the emerging roles of microglia on the maintenance of synaptic function, learning, memory strength and forgetfulness, and the implications of microglia depletion in models of brain disease.}, author = {Basilico, Bernadette and Ferrucci, Laura and Khan, Azka and Di Angelantonio, Silvia and Ragozzino, Davide and Reverte, Ingrid}, issn = {1662-5102}, journal = {Frontiers in Cellular Neuroscience}, keywords = {Cellular and Molecular Neuroscience}, publisher = {Frontiers Media}, title = {{What microglia depletion approaches tell us about the role of microglia on synaptic function and behavior}}, doi = {10.3389/fncel.2022.1022431}, volume = {16}, year = {2022}, } @article{12174, abstract = {Vacuolar-type H+-ATPase (V-ATPase) is a multimeric complex present in a variety of cellular membranes that acts as an ATP-dependent proton pump and plays a key role in pH homeostasis and intracellular signalling pathways. In humans, 22 autosomal genes encode for a redundant set of subunits allowing the composition of diverse V-ATPase complexes with specific properties and expression. Sixteen subunits have been linked to human disease. Here we describe 26 patients harbouring 20 distinct pathogenic de novo missense ATP6V1A variants, mainly clustering within the ATP synthase α/β family-nucleotide-binding domain. At a mean age of 7 years (extremes: 6 weeks, youngest deceased patient to 22 years, oldest patient) clinical pictures included early lethal encephalopathies with rapidly progressive massive brain atrophy, severe developmental epileptic encephalopathies and static intellectual disability with epilepsy. The first clinical manifestation was early hypotonia, in 70%; 81% developed epilepsy, manifested as developmental epileptic encephalopathies in 58% of the cohort and with infantile spasms in 62%; 63% of developmental epileptic encephalopathies failed to achieve any developmental, communicative or motor skills. Less severe outcomes were observed in 23% of patients who, at a mean age of 10 years and 6 months, exhibited moderate intellectual disability, with independent walking and variable epilepsy. None of the patients developed communicative language. Microcephaly (38%) and amelogenesis imperfecta/enamel dysplasia (42%) were additional clinical features. Brain MRI demonstrated hypomyelination and generalized atrophy in 68%. Atrophy was progressive in all eight individuals undergoing repeated MRIs. Fibroblasts of two patients with developmental epileptic encephalopathies showed decreased LAMP1 expression, Lysotracker staining and increased organelle pH, consistent with lysosomal impairment and loss of V-ATPase function. Fibroblasts of two patients with milder disease, exhibited a different phenotype with increased Lysotracker staining, decreased organelle pH and no significant modification in LAMP1 expression. Quantification of substrates for lysosomal enzymes in cellular extracts from four patients revealed discrete accumulation. Transmission electron microscopy of fibroblasts of four patients with variable severity and of induced pluripotent stem cell-derived neurons from two patients with developmental epileptic encephalopathies showed electron-dense inclusions, lipid droplets, osmiophilic material and lamellated membrane structures resembling phospholipids. Quantitative assessment in induced pluripotent stem cell-derived neurons identified significantly smaller lysosomes. ATP6V1A-related encephalopathy represents a new paradigm among lysosomal disorders. It results from a dysfunctional endo-lysosomal membrane protein causing altered pH homeostasis. Its pathophysiology implies intracellular accumulation of substrates whose composition remains unclear, and a combination of developmental brain abnormalities and neurodegenerative changes established during prenatal and early postanal development, whose severity is variably determined by specific pathogenic variants.}, author = {Guerrini, Renzo and Mei, Davide and Szigeti, Margit Katalin and Pepe, Sara and Koenig, Mary Kay and Von Allmen, Gretchen and Cho, Megan T and McDonald, Kimberly and Baker, Janice and Bhambhani, Vikas and Powis, Zöe and Rodan, Lance and Nabbout, Rima and Barcia, Giulia and Rosenfeld, Jill A and Bacino, Carlos A and Mignot, Cyril and Power, Lillian H and Harris, Catharine J and Marjanovic, Dragan and Møller, Rikke S and Hammer, Trine B and Keski Filppula, Riikka and Vieira, Päivi and Hildebrandt, Clara and Sacharow, Stephanie and Maragliano, Luca and Benfenati, Fabio and Lachlan, Katherine and Benneche, Andreas and Petit, Florence and de Sainte Agathe, Jean Madeleine and Hallinan, Barbara and Si, Yue and Wentzensen, Ingrid M and Zou, Fanggeng and Narayanan, Vinodh and Matsumoto, Naomichi and Boncristiano, Alessandra and la Marca, Giancarlo and Kato, Mitsuhiro and Anderson, Kristin and Barba, Carmen and Sturiale, Luisa and Garozzo, Domenico and Bei, Roberto and Masuelli, Laura and Conti, Valerio and Novarino, Gaia and Fassio, Anna}, issn = {1460-2156}, journal = {Brain}, keywords = {Neurology (clinical)}, number = {8}, pages = {2687--2703}, publisher = {Oxford University Press}, title = {{Phenotypic and genetic spectrum of ATP6V1A encephalopathy: A disorder of lysosomal homeostasis}}, doi = {10.1093/brain/awac145}, volume = {145}, year = {2022}, } @article{12268, abstract = {The complexity of the microenvironment effects on cell response, show accumulating evidence that glioblastoma (GBM) migration and invasiveness are influenced by the mechanical rigidity of their surroundings. The epithelial–mesenchymal transition (EMT) is a well-recognized driving force of the invasive behavior of cancer. However, the primary mechanisms of EMT initiation and progression remain unclear. We have previously showed that certain substrate stiffness can selectively stimulate human GBM U251-MG and GL15 glioblastoma cell lines motility. The present study unifies several known EMT mediators to uncover the reason of the regulation and response to these stiffnesses. Our results revealed that changing the rigidity of the mechanical environment tuned the response of both cell lines through change in morphological features, epithelial-mesenchymal markers (E-, N-Cadherin), EGFR and ROS expressions in an interrelated manner. Specifically, a stiffer microenvironment induced a mesenchymal cell shape, a more fragmented morphology, higher intracellular cytosolic ROS expression and lower mitochondrial ROS. Finally, we observed that cells more motile showed a more depolarized mitochondrial membrane potential. Unravelling the process that regulates GBM cells’ infiltrative behavior could provide new opportunities for identification of new targets and less invasive approaches for treatment.}, author = {Basilico, Bernadette and Palamà, Ilaria Elena and D’Amone, Stefania and Lauro, Clotilde and Rosito, Maria and Grieco, Maddalena and Ratano, Patrizia and Cordella, Federica and Sanchini, Caterina and Di Angelantonio, Silvia and Ragozzino, Davide and Cascione, Mariafrancesca and Gigli, Giuseppe and Cortese, Barbara}, issn = {2234-943X}, journal = {Frontiers in Oncology}, keywords = {Cancer Research, Oncology}, publisher = {Frontiers Media}, title = {{Substrate stiffness effect on molecular crosstalk of epithelial-mesenchymal transition mediators of human glioblastoma cells}}, doi = {10.3389/fonc.2022.983507}, volume = {12}, year = {2022}, } @article{10818, abstract = {Microglia cells are active players in regulating synaptic development and plasticity in the brain. However, how they influence the normal functioning of synapses is largely unknown. In this study, we characterized the effects of pharmacological microglia depletion, achieved by administration of PLX5622, on hippocampal CA3-CA1 synapses of adult wild type mice. Following microglial depletion, we observed a reduction of spontaneous and evoked glutamatergic activity associated with a decrease of dendritic spine density. We also observed the appearance of immature synaptic features and higher levels of plasticity. Microglia depleted mice showed a deficit in the acquisition of the Novel Object Recognition task. These events were accompanied by hippocampal astrogliosis, although in the absence ofneuroinflammatory condition. PLX-induced synaptic changes were absent in Cx3cr1−/− mice, highlighting the role of CX3CL1/CX3CR1 axis in microglia control of synaptic functioning. Remarkably, microglia repopulation after PLX5622 withdrawal was associated with the recovery of hippocampal synapses and learning functions. Altogether, these data demonstrate that microglia contribute to normal synaptic functioning in the adult brain and that their removal induces reversible changes in organization and activity of glutamatergic synapses.}, author = {Basilico, Bernadette and Ferrucci, Laura and Ratano, Patrizia and Golia, Maria T. and Grimaldi, Alfonso and Rosito, Maria and Ferretti, Valentina and Reverte, Ingrid and Sanchini, Caterina and Marrone, Maria C. and Giubettini, Maria and De Turris, Valeria and Salerno, Debora and Garofalo, Stefano and St‐Pierre, Marie‐Kim and Carrier, Micael and Renzi, Massimiliano and Pagani, Francesca and Modi, Brijesh and Raspa, Marcello and Scavizzi, Ferdinando and Gross, Cornelius T. and Marinelli, Silvia and Tremblay, Marie‐Ève and Caprioli, Daniele and Maggi, Laura and Limatola, Cristina and Di Angelantonio, Silvia and Ragozzino, Davide}, issn = {1098-1136}, journal = {Glia}, keywords = {Cellular and Molecular Neuroscience, Neurology}, number = {1}, pages = {173--195}, publisher = {Wiley}, title = {{Microglia control glutamatergic synapses in the adult mouse hippocampus}}, doi = {10.1002/glia.24101}, volume = {70}, year = {2022}, } @unpublished{11943, abstract = {Complex wiring between neurons underlies the information-processing network enabling all brain functions, including cognition and memory. For understanding how the network is structured, processes information, and changes over time, comprehensive visualization of the architecture of living brain tissue with its cellular and molecular components would open up major opportunities. However, electron microscopy (EM) provides nanometre-scale resolution required for full in-silico reconstruction1–5, yet is limited to fixed specimens and static representations. Light microscopy allows live observation, with super-resolution approaches6–12 facilitating nanoscale visualization, but comprehensive 3D-reconstruction of living brain tissue has been hindered by tissue photo-burden, photobleaching, insufficient 3D-resolution, and inadequate signal-to-noise ratio (SNR). Here we demonstrate saturated reconstruction of living brain tissue. We developed an integrated imaging and analysis technology, adapting stimulated emission depletion (STED) microscopy6,13 in extracellularly labelled tissue14 for high SNR and near-isotropic resolution. Centrally, a two-stage deep-learning approach leveraged previously obtained information on sample structure to drastically reduce photo-burden and enable automated volumetric reconstruction down to single synapse level. Live reconstruction provides unbiased analysis of tissue architecture across time in relation to functional activity and targeted activation, and contextual understanding of molecular labelling. This adoptable technology will facilitate novel insights into the dynamic functional architecture of living brain tissue.}, author = {Velicky, Philipp and Miguel Villalba, Eder and Michalska, Julia M and Wei, Donglai and Lin, Zudi and Watson, Jake and Troidl, Jakob and Beyer, Johanna and Ben Simon, Yoav and Sommer, Christoph M and Jahr, Wiebke and Cenameri, Alban and Broichhagen, Johannes and Grant, Seth G. N. and Jonas, Peter M and Novarino, Gaia and Pfister, Hanspeter and Bickel, Bernd and Danzl, Johann G}, booktitle = {bioRxiv}, publisher = {Cold Spring Harbor Laboratory}, title = {{Saturated reconstruction of living brain tissue}}, doi = {10.1101/2022.03.16.484431}, year = {2022}, } @unpublished{11950, abstract = {Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanoscopic synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS leverages fixation-compatible extracellular labeling and advanced optical readout, in particular stimulated-emission depletion and expansion microscopy, to comprehensively delineate cellular structures. It enables 3D-reconstructing single synapses and mapping synaptic connectivity by identification and tailored analysis of putative synaptic cleft regions. Applying CATS to the hippocampal mossy fiber circuitry, we demonstrate its power to reveal the system’s molecularly informed ultrastructure across spatial scales and assess local connectivity by reconstructing and quantifying the synaptic input and output structure of identified neurons.}, author = {Michalska, Julia M and Lyudchik, Julia and Velicky, Philipp and Korinkova, Hana and Watson, Jake and Cenameri, Alban and Sommer, Christoph M and Venturino, Alessandro and Roessler, Karl and Czech, Thomas and Siegert, Sandra and Novarino, Gaia and Jonas, Peter M and Danzl, Johann G}, booktitle = {bioRxiv}, publisher = {Cold Spring Harbor Laboratory}, title = {{Uncovering brain tissue architecture across scales with super-resolution light microscopy}}, doi = {10.1101/2022.08.17.504272}, year = {2022}, }