Maturation dynamics of the axon initial segment (AIS) of newborn dentate granule cells in young adult C57BL/6J mice

This published version will be available under a Creative Commons Attribution 4.0 International License (CC-BY) after a 6 month periodNewborn dentate granule cells (DGCs) are generated in the hippocampal dentate gyrus (DG) of rodents through a process called adult hippocampal neurogenesis, which is subjected to tight intrinsic and extrinsic regulation. The use of retroviruses encoding fluorescent proteins has allowed the characterization of the maturation dynamics of newborn DGCs, including their morphological development and the establishment and maturation of their afferent and efferent synaptic connections. However, the study of a crucial cellular compartment of these cells, namely, the axon initial segment (AIS), has remained unexplored to date. The AIS is not only the site of action potential initiation, but it also has a unique molecular identity that makes it one of the master regulators of neural plasticity and excitability. Here we examined the dynamics of AIS formation in newborn DGCs of young female adult C57BL/6J mice in vivo. Our data reveal notable changes in AIS length and thickness throughout cell maturation under physiological conditions and show that the most remarkable structural changes coincide with periods of intense morphological and functional remodeling. Moreover, we demonstrate that AIS development can be modulated extrinsically by both neuroprotective (environmental enrichment) and detrimental (lipopolysaccharide from Escherichia coli) stimuliThis work was supported by the Spanish Ministry of Economy and Competitiveness SAF-2017-82185-R and RYC-2015-171899 to M.L.-M. and SAF-2014-53040-P to J.Á., Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas to J.Á., Alzheimer's Association 2015-NIRG-340709 and AARG-17-528125 to M.L.-M., Association for Frontotemporal Degeneration 2016 Basic Science Pilot Grant Award to M.L.-M., and Fundación Ramón Areces and Banco de Santander to the Centro de Biología Molecular Severo Ochoa Institutional Grant

Maturation Dynamics of the Axon Initial Segment (AIS) of Newborn Dentate Granule Cells in Young Adult C57BL/6J Mice

Newborn dentate granule cells (DGCs) are generated in the hippocampal dentate gyrus (DG) of rodents through a process called adult hippocampal neurogenesis, which is subjected to tight intrinsic and extrinsic regulation. The use of retroviruses encoding fluorescent proteins has allowed the characterization of the maturation dynamics of newborn DGCs, including their morphological development and the establishment and maturation of their afferent and efferent synaptic connections. However, the study of a crucial cellular compartment of these cells, namely, the axon initial segment (AIS), has remained unexplored to date. The AIS is not only the site of action potential initiation, but it also has a unique molecular identity that makes it one of the master regulators of neural plasticity and excitability. Here we examined the dynamics of AIS formation in newborn DGCs of young female adult C57BL/6J mice in vivo Our data reveal notable changes in AIS length and thickness throughout cell maturation under physiological conditions and show that the most remarkable structural changes coincide with periods of intense morphological and functional remodeling. Moreover, we demonstrate that AIS development can be modulated extrinsically by both neuroprotective (environmental enrichment) and detrimental (lipopolysaccharide from Escherichia coli) stimuli.SIGNIFICANCE STATEMENT The hippocampal dentate gyrus (DG) of rodents generates newborn dentate granule cells (DGCs) throughout life. This process, named adult hippocampal neurogenesis, confers a unique degree of plasticity to the hippocampal circuit, and it is crucial for learning and memory. Here we studied, for the first time, the formation of a key cellular compartment of newborn DGCs, namely, the axon initial segment (AIS) in vivo Our data reveal remarkable AIS structural remodeling throughout the maturation of these cells under physiological conditions. Moreover, AIS development can be modulated extrinsically by both neuroprotective (environmental enrichment) and detrimental (lipopolysaccharide from Escherichia coli) stimuli.Spanish Ministry of Economy and Competitiveness SAF-2017-82185-R and RYC-2015-171899 to M.L.-M. and SAF-2014-53040-P to J.A´., Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas to J.A´., Alzheimer’s Association 2015-NIRG-340709 and AARG-17-528125 to M.L.-M., Association for Frontotemporal Degeneration 2016 Basic Science Pilot Grant Award to M.L.-M., and Fundación Ramón Areces and Banco de Santander to the Centro de Biología Molecular SeveroOcho

Methods to study adult hippocampal neurogenesis in humans and across the phylogeny

The hippocampus hosts the continuous addition of new neurons throughout life—a phenomenon named adult hippocampal neurogenesis (AHN). Here we revisit the occurrence of AHN in more than 110 mammalian species, including humans, and discuss the further validation of these data by single-cell RNAseq and other alternative techniques. In this regard, our recent studies have addressed the long-standing controversy in the field, namely whether cells positive for AHN markers are present in the adult human dentate gyrus (DG). Here we review how we developed a tightly controlled methodology, based on the use of high-quality brain samples (characterized by short postmortem delays and ≤24 h of fixation in freshly prepared 4% paraformaldehyde), to address human AHN. We review that the detection of AHN markers in samples fixed for 24 h required mild antigen retrieval and chemical elimination of autofluorescence. However, these steps were not necessary for samples subjected to shorter fixation periods. Moreover, the detection of labile epitopes (such as Nestin) in the human hippocampus required the use of mild detergents. The application of this strictly controlled methodology allowed reconstruction of the entire AHN process, thus revealing the presence of neural stem cells, proliferative progenitors, neuroblasts, and immature neurons at distinct stages of differentiation in the human DG. The data reviewed here demonstrate that methodology is of utmost importance when studying AHN by means of distinct techniques across the phylogenetic scale. In this regard, we summarize the major findings made by our group that emphasize that overlooking fundamental technical principles might have consequences for any given research fieldAssociation for Frontotemporal Degeneration; Banco de Santander; Center for Networked Biomedical Research on Neurodegenerative Diseases; Consejo Nacional de Ciencia y Tecnología (CONACYT), Grant/Award Number: 385084; European Research Council, Grant/Award Number: ERC-CoG2020-101001916; Fundacion Ram on Areces; Secretaria de Educacion, Ciencia Tecnología e Innovacion (SECTEI) of the Regional Government of Ciudad de México (CDMX), Grant/Award Number: SECTEI/159/2021; Spanish Ministry of Economy and Competitiveness, Grant/Award Numbers: PID2020-113007RB-I00, RYC-2015-171899, SAF-2017-82185-R; The Alzheimer's Association, Grant/Award Numbers: 2015-NIRG-340709, AARG-17-528125, AARG-17-528125-RAPI

GSK-3β orchestrates the inhibitory innervation of adult-born dentate granule cells in vivo

Adult hippocampal neurogenesis enhances brain plasticity and contributes to the cognitive reserve during aging. Adult hippocampal neurogenesis is impaired in neurological disorders, yet the molecular mechanisms regulating the maturation and synaptic integration of new neurons have not been fully elucidated. GABA is a master regulator of adult and developmental neurogenesis. Here we engineered a novel retrovirus encoding the fusion protein Gephyrin:GFP to longitudinally study the formation and maturation of inhibitory synapses during adult hippocampal neurogenesis in vivo. Our data reveal the early assembly of inhibitory postsynaptic densities at 1 week of cell age. Glycogen synthase kinase 3 Beta (GSK-3β) emerges as a key regulator of inhibitory synapse formation and maturation during adult hippocampal neurogenesis. GSK-3β-overexpressing newborn neurons show an increased number and altered size of Gephyrin+ postsynaptic clusters, enhanced miniature inhibitory postsynaptic currents, shorter and distanced axon initial segments, reduced synaptic output at the CA3 and CA2 hippocampal regions, and impaired pattern separation. Moreover, GSK-3β overexpression triggers a depletion of Parvalbumin+ interneuron perineuronal nets. These alterations might be relevant in the context of neurological diseases in which the activity of GSK-3β is dysregulatedPID2020-113007RB-I00, SAF-2017-82185-R, PID2020-112824GB-10

Activity-Dependent Reconnection of Adult-Born Dentate Granule Cells in a Mouse Model of Frontotemporal Dementia

Frontotemporal dementia (FTD) is characterized by neuronal loss in the frontal and temporal lobes of the brain. Here, we provide the first evidence of striking morphological alterations in dentate granule cells (DGCs) of FTD patients and in a mouse model of the disease, Tau VLW mice. Taking advantage of the fact that the hippocampal dentate gyrus (DG) gives rise to newborn DGCs throughout the lifetime in rodents, we used RGB retroviruses to study the temporary course of these alterations in newborn DGCs of female Tau VLW mice. In addition, retroviruses that encode either PSD95:GFP or Syn:GFP revealed striking alterations in the afferent and efferent connectivity of newborn Tau VLW DGCs, and monosynaptic retrograde rabies virus tracing showed that these cells are disconnected from distal brain regions and local sources of excitatory innervation. However, the same cells exhibited a predominance of local inhibitory innervation. Accordingly, the expression of presynaptic and postsynaptic markers of inhibitory synapses was markedly increased in the DG of Tau VLW mice and FTD patients. Moreover, an increased number of neuropeptide Y-positive interneurons in the DG correlated with a reduced number of activated egr-1 + DGCs in Tau VLW mice. Finally, we tested the therapeutic potential of environmental enrichment and chemoactivation to reverse these alterations in mice. Both strategies reversed the morphological alterations of newborn DGCs and partially restored their connectivity in a mouse model of the disease. Moreover, our data point to remarkable morphological similarities between the DGCs of Tau VLW mice and FTD patients.Fil: Terreros Roncal, Julia. Universidad Autónoma de Madrid. Facultad de Ciencias; EspañaFil: Flor García, Miguel. Universidad Autónoma de Madrid. Facultad de Ciencias; EspañaFil: Moreno Jiménez, Elena P.. Universidad Autónoma de Madrid. Facultad de Ciencias; EspañaFil: Pallas Bazarra, Noemí. Universidad Autónoma de Madrid. Facultad de Ciencias; EspañaFil: Rábano, Alberto. No especifíca;Fil: Sah, Nirnath. National Institutes of Health; Estados UnidosFil: Van Praag, Henriette. National Institutes of Health; Estados UnidosFil: Giacomini, Damiana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Schinder, Alejandro Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Ávila, Jesús. Universidad Autónoma de Madrid. Facultad de Ciencias; EspañaFil: Llorens Martín, Maria. Universidad Autónoma de Madrid. Facultad de Ciencias; Españ

Neurogénesis hipocampal adulta en sujetos neurológicamente sanos y pacientes con enfermedades neurodegenerativas

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 10-11-2022Esta tesis tiene embargado el acceso al texto completo hasta el 10-05-2024La generación de nuevas neuronas en el hipocampo adulto se conoce como neurogénesis hipocampal adulta. La existencia de este fenómeno se ha demostrado en más de 120 especies de mamíferos, incluyendo el ser humano. Durante el proceso de neurogénesis, las nuevas neuronas inmaduras se originan a partir de las células madre neurales, las cuales se dividen y dan lugar a neuroblastos proliferativos. Tras la determinación al linaje neuronal, las nuevas neuronas inmaduras atraviesan un proceso de maduración durante el que modifican su posición y adquieren morfologías cada vez más complejas. Nuestros datos indican que el proceso de neurogénesis hipocampal adulta posee una naturaleza dinámica y tiene lugar hasta, al menos, los 90 años de edad en nuestra especie. Todos los elementos que conforman el proceso de neurogénesis adulta reciben el aporte trófico de distintos componentes del nicho neurogénico, entre los que destacan los vasos sanguíneos, astrocitos y microglía. A lo largo del envejecimiento fisiológico, se observa una disminución del número de neuronas inmaduras positivas para el marcador doublecortin (DCX), en paralelo a una disminución en la capacidad fagocítica de la microglía. En la presente tesis doctoral se ha estudiado la modulación de la neurogénesis hipocampal adulta humana no sólo a lo largo del envejecimiento fisiológico sino también durante el avance de varias enfermedades neurodegenerativas. En los pacientes con enfermedad de Alzheimer, se detecta una disminución en el número de neuronas inmaduras en los primeros estadios de la enfermedad, que se vuelve más acusado con el avance de la patología. En pacientes con esclerosis lateral amiotrófica, enfermedad de Huntington, demencia con cuerpos de Lewy, enfermedad de Parkinson y demencia frontotemporal se han detectado alteraciones a lo largo de todo el proceso de neurogénesis, así como en los distintos componentes del nicho neurogénico. Principalmente, se han encontrado alteraciones en el número de células madre, células proliferativas y neuronas inmaduras, las cuales presentan una morfología aberrante en los estadios iniciales y finales de su proceso de maduración. Además, se observan cambios en los componentes del nicho neurogénico, destacando la astrogliosis, el aumento de muerte celular y vascularización, y alteraciones en la morfología y funcionalidad de la microglía. El conjunto de estas modificaciones produce una firma específica, propia de cada enfermedad neurodegenerativa, sobre el proceso de neurogénesis hipocampal adulta y del nicho neurogénico. Estas alteraciones podrían estar relacionadas con los déficits cognitivos, emocionales y de memoria que presentan estos pacientes. Por último, esta tesis doctoral incluye el ensayo, en modelos animales de dos enfermedades neurodegenerativas distintas, de varias aproximaciones terapéuticas capaces de revertir las alteraciones en neurogénesis hipocampal adulta encontradasLos experimentos incluidos en la presente tesis doctoral han sido financiados gracias a los siguientes proyectos: The European Research Council (ERC) (ERC-CoG-2020-101001916), Ministerio de Economía y Competitividad (PID2020-113007RBI00, SAF-2017-82185-R, The Alzheimer ́s Association (2015-NIRG-340709, AARG-17-528125, and AARG-17-528125-RAPID), The Association for Frontotemporal Degeneration (2016 Basic Science Pilot Grant Award); and Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED, ISCIII

Evidences for adult hippocampal neurogenesis in humans

The rodent hippocampus generates new neurons throughout life. This process, named adult hippocampal neurogenesis (AHN), is a striking form of neural plasticity that occurs in the brains of numerous mammalian species. Direct evidence of adult neurogenesis in humans has remained elusive, although the occurrence of this phenomenon in the human dentate gyrus has been demonstrated in seminal studies and recent research that have applied distinct approaches to birthdate newly generated neurons and to validate markers of adult-born neurons. Our data point to the persistence of AHN until the 10th decade of human life, as well as to marked impairments in this process in patients with Alzheimer's disease. Moreover, our work demonstrates that the methods used to process and analyze postmortem human brain samples can limit the detection of various markers of AHN to the point of making them undetectable. In this Dual Perspectives article, we highlight the critical methodological aspects that should be strictly controlled in human studies and the robust evidence that supports the occurrence of AHN in humans. We also put forward reasons that may account for current discrepancies on this topic. Finally, the unresolved questions and future challenges awaiting the field are highlighted.Spanish Ministry of Economy and Competitiveness SAF-2017-82185-R and RYC-2015-171899 to M.L.-M., Alzheimer’s Association 2015-NIRG-340709 and AARG-17-528125 to M.L.-M., Association for Frontotemporal Degeneration 2016 Basic Science Pilot Grant Award to M.L.-M., and Comunidad de Madrid PEJD-2017-PRE/BMD-3439 to M.L.-M. Institutional grants from Fundación Ramón Areces and Banco de Santander to Centro de Biología Molecular “Severo Ocho

Untold New Beginnings: Adult Hippocampal Neurogenesis and Alzheimer's Disease

Neurogenesis occurs in a limited number of brain regions during adulthood. Of these, the hippocampus has attracted great interest due to its involvement in memory processing. Moreover, both the hippocampus and the main area that innervates this structure, namely the entorhinal cortex, show remarkable atrophy in patients with Alzheimer's disease (AD). Adult hippocampal neurogenesis is a process that continuously gives rise to newborn granule neurons in the dentate gyrus. These cells coexist with developmentally generated granule neurons in this structure, and both cooperative and competition phenomena regulate the communication between these two types of cells. Importantly, it has been revealed that GSK-3β and tau proteins, which are two of the main players driving AD pathology, are cornerstones of adult hippocampal neurogenesis regulation. We have shown that alterations either promoting or impeding the actions of these two proteins have detrimental effects on the structural plasticity of granule neurons. Of note, these impairments occur both under basal conditions and in response to detrimental and neuroprotective stimuli. Thus, in order to achieve the full effectiveness of future therapies for AD, we propose that attention be turned toward identifying the pathological and physiological actions of the proteins involved in the pathogenesis of this condition.Spanish Ministry of Economy and Competitiveness (SAF-2014-53040-P (Jesu´s A´ vila) and RYC-2015-17189 (María Llorens-Mart´ın)); the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Spain) (Jesu´s Avila); the Alzheimer’s Association (2015-NIRG-340709 (Mar´ıa Llorens-Mart´ın)); and the Association for Frontotemporal Degeneration (2016 Basic Science Pilot Grant Award (María Llorens-Martín))

Unraveling human adult hippocampal neurogenesis

Adult neurogenesis occurs in a few selected regions of the mammalian brain. One such region is the hippocampus, the so-called gateway to memory, where adult hippocampal neurogenesis (AHN) occurs. Here, we provide a comprehensive description of the methods used in our laboratory to unambiguously detect a population of immature neurons in the human hippocampus until the 10th decade of life. The criteria used to refine and develop the current protocol include obtaining post-mortem human samples of remarkable quality and under tightly controlled conditions for immunohistochemistry (IHC) studies, optimizing tissue processing and histological procedures, establishing criteria to reliably validate antibody signal and performing unbiased stereological cell counts. Moreover, we provide a detailed description of the parameters that, in our view, should be reported in human AHN studies. The opposing results obtained by introducing slight variations in the methodological conditions should be considered by future studies that seek to increase our knowledge of this fascinating process. By applying simple and inexpensive tissue pre-treatments, this protocol, which can be completed in 7 days, might be applicable to a variety of IHC studies performed on other tissues of human (or animal) origin.the Spanish Ministry of Economy and Competitiveness (SAF-2017-82185-R and RYC-2015-171899 (M.L.-M.); SAF-2014-53040-P (J.A.)); The Alzheimer´s Association (2015-NIRG-340709 and AARG-17-528125 (M.L.-M.)); The Association for Frontotemporal Degeneration (2016 Basic Science Pilot Grant Award (M.L.-M.)); the Comunidad de Madrid (PEJD-2017-PRE/BMD-3439 (M.L.-M.)); and the Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED, Spain) (J.A.). Institutional grants from the Fundación Ramón Areces and Banco de Santande

Absence of microglial CX3CR1 impairs the synaptic integration of adult-born hippocampal granule neurons

Microglia are immune cells that play a crucial role in maintaining brain homeostasis. Among the mechanisms of communication between microglia and neurons, the CX3CL1/CX3CR1 axis exerts a central modulatory role. Animals lacking CX3CR1 microglial receptor (CX3CR1−/− mice) exhibit marked alterations not only in microglia but also in neurons located in various regions of the brain. Here we show that microglial depletion of CX3CR1 leads to the deficient synaptic integration of adult-born granule neurons in the dentate gyrus (DG), both at the afferent and efferent level. Regarding the alterations in the former level, these cells show a reduced number of dendritic spines, which also exhibit morphological changes, namely enlargement and shortening. With respect to changes at the efferent level, these cells show a reduced area of axonal terminals. Both at the afferent and efferent level, synapses show ultrastructural enlargement, but they are depleted of synaptic vesicles, which suggests impaired functionality. We also show that selective increased microglial activation and extracellular matrix deposition in the zones in which the afferent synaptic contacts of these cells occur, namely in the molecular and the granule layer of the DG. In order to evaluate the impact of these structural alterations from a functional point of view, we performed a battery of behavioral tests related to hippocampal-dependent emotional behavior. We observed that female CX3CR1−/− mice exhibit a hyperactive, anxiolytic-like and depressive-like phenotype. These data shed light on novel aspects of the regulation of adult hippocampal neurogenesis by microglia that could be highly relevant for research into mood disorders.This study was supported by the Spanish Ministry of Economy and Competitiveness (SAF-2014-53040-P (Jesús Ávila) and RYC-2015-17189 (María Llorens-Martín)); the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Spain) (Jesús Ávila); the Alzheimer’s Association (USA) (2015-NIRG-340709 and 2017-AARG-528125 (María Llorens-Martín)); and the Association for Frontotemporal Degeneration (USA) (2016 Basic Science Pilot Grant Award (María Llorens-Martín)). These funding sources had no involvement in the design of the experiments, analysis of the data or manuscript preparation