Azithromycin preserves adult hippocampal neurogenesis and behavior in a mouse model of sepsis.

The mammalian hippocampus can generate new neurons throughout life. Known as adult hippocampal neurogenesis (AHN), this process participates in learning, memory, mood regulation, and forgetting. The continuous incorporation of new neurons enhances the plasticity of the hippocampus and contributes to the cognitive reserve in aged individuals. However, the integrity of AHN is targeted by numerous pathological conditions, including neurodegenerative diseases and sustained inflammation. In this regard, the latter causes cognitive decline, mood alterations, and multiple AHN impairments. In fact, the systemic administration of Lipopolysaccharide (LPS) from E. coli to mice (a model of sepsis) triggers depression-like behavior, impairs pattern separation, and decreases the survival, maturation, and synaptic integration of adult-born hippocampal dentate granule cells. Here we tested the capacity of the macrolide antibiotic azithromycin to neutralize the deleterious consequences of LPS administration in female C57BL6J mice. This antibiotic exerted potent neuroprotective effects. It reversed the increased immobility time during the Porsolt test, hippocampal secretion of pro-inflammatory cytokines, and AHN impairments. Moreover, azithromycin promoted the synaptic integration of adult-born neurons and functionally remodeled the gut microbiome. Therefore, our data point to azithromycin as a clinically relevant drug with the putative capacity to ameliorate the negative consequences of chronic inflammation by modulating AHN and hippocampal-related behaviors.

GSK-3ß 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-3ß) emerges as a key regulator of inhibitory synapse formation and maturation during adult hippocampal neurogenesis. GSK-3ß-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-3ß 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-3ß is dysregulated.

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 field.

GSK-3ß S9A overexpression leads murine hippocampal neural precursors to acquire an astroglial phenotype in vivo.

The addition of new neurons to the existing hippocampal circuitry persists in the adult dentate gyrus (DG). During this process, named adult hippocampal neurogenesis (AHN), adult hippocampal progenitor cells (AHPs) give rise to newborn dentate granule cells (DGCs). The acquisition of a neuronal lineage by AHPs is tightly regulated by numerous signaling molecules and transcription factors. In this regard, glycogen synthase kinase 3ß (GSK-3ß) is a master regulator of the maturation of AHPs in vitro. Here we analyzed the cell-autonomous effects of overexpressing a constitutively active form of GSK-3ß (GSK-3ß S9A) in AHPs in vivo. To this end, we stereotaxically injected a GSK-3ß S9A-encoding retrovirus (GSK-3ß-V5) into the DG of young adult C57BL6/J Ola Hsd female mice and studied the cell lineage acquisition, migratory and marker expression patterns, and the morphological maturation of the infected cells over time. Strikingly, GSK-3ß S9A-transduced cells expressed glial fibrillary acidic protein (GFAP) and NG2, thereby acquiring an immature astroglial phenotype, which differed markedly from the neuronal phenotype observed in cells transduced with a control retrovirus that encoded GFP. Accordingly, the morphology and migration patterns of cells transduced by the two retroviruses are remarkably divergent. These observations support the role of GSK-3ß as a cornerstone that regulates the balance between new astocytes/neurons generated in the adult murine DG.

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.

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.

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, TauVLW 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 TauVLW mice. In addition, retroviruses that encode either PSD95:GFP or Syn:GFP revealed striking alterations in the afferent and efferent connectivity of newborn TauVLW 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 TauVLW 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 TauVLW 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 TauVLW mice and FTD patients.SIGNIFICANCE STATEMENT We show, for the first time to our knowledge, that the population of dentate granule cells is disconnected from other regions of the brain in the neurodegenerative disease frontotemporal dementia (FTD). These alterations were observed in FTD patients and in a mouse model of this disease. Moreover, we tested the therapeutic potential of two strategies, environmental enrichment and chemoactivation, to stimulate the activity of these neurons in mice. We found that some of the alterations were reversed by these therapeutic interventions.

The Social Component of Environmental Enrichment Is a Pro-neurogenic Stimulus in Adult c57BL6 Female Mice.

In rodents, the hippocampal dentate gyrus gives rise to newly generated dentate granule cells (DGCs) throughout life. This process, named adult hippocampal neurogenesis (AHN), converges in the functional integration of mature DGCs into the trisynaptic hippocampal circuit. Environmental enrichment (EE) is one of the most potent positive regulators of AHN. This paradigm includes the combination of three major stimulatory components, namely increased physical activity, constant cognitive stimulation, and higher social interaction. In this regard, the pro-neurogenic effects of physical activity and cognitive stimulation have been widely addressed in adult rodents. However, the pro-neurogenic potential of the social aspect of EE has been less explored to date. Here we tackled this question by specifically focusing on the effects of a prolonged period of social enrichment (SE) in adult female C57BL6 mice. To this end, 7-week-old mice were housed in groups of 12 per cage for 8 weeks. These mice were compared with others housed under control housing (2-3 mice per cage) or EE (12 mice per cage plus running wheels and toys) conditions during the same period. We analyzed the number and morphology of Doublecortin-expressing (DCX+) cells. Moreover, using RGB retroviruses that allowed the labeling of three populations of newborn DGCs of different ages in the same mouse, we performed morphometric, immunohistochemical, and behavioral determinations. Both SE and EE increased the number and maturation of DCX+ cells, and caused an increase in dendritic maturation in certain populations of newborn DGCs. Moreover, both manipulations increased exploratory behavior in the Social Interaction test. Unexpectedly, our data revealed the potent neurogenesis-stimulating potential of SE in the absence of any further cognitive stimulation or increase in physical activity. Given that an increase in physical activity is strongly discouraged under certain circumstances, our findings may be relevant in the context of enhancing AHN via physical activity-independent mechanisms.

Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer's disease.

The hippocampus is one of the most affected areas in Alzheimer's disease (AD)1. Moreover, this structure hosts one of the most unique phenomena of the adult mammalian brain, namely, the addition of new neurons throughout life2. This process, called adult hippocampal neurogenesis (AHN), confers an unparalleled degree of plasticity to the entire hippocampal circuitry3,4. Nonetheless, direct evidence of AHN in humans has remained elusive. Thus, determining whether new neurons are continuously incorporated into the human dentate gyrus (DG) during physiological and pathological aging is a crucial question with outstanding therapeutic potential. By combining human brain samples obtained under tightly controlled conditions and state-of-the-art tissue processing methods, we identified thousands of immature neurons in the DG of neurologically healthy human subjects up to the ninth decade of life. These neurons exhibited variable degrees of maturation along differentiation stages of AHN. In sharp contrast, the number and maturation of these neurons progressively declined as AD advanced. These results demonstrate the persistence of AHN during both physiological and pathological aging in humans and provide evidence for impaired neurogenesis as a potentially relevant mechanism underlying memory deficits in AD that might be amenable to novel therapeutic strategies.

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.

Exercising New Neurons to Vanquish Alzheimer Disease.

Alzheimer disease (AD) is the most common type of dementia in individuals over 65 years of age. The neuropathological hallmarks of the condition are Tau neurofibrillary tangles and Amyloid-ß senile plaques. Moreover, certain susceptible regions of the brain experience a generalized lack of neural plasticity and marked synaptic alterations during the progression of this as yet incurable disease. One of these regions, the hippocampus, is characterized by the continuous addition of new neurons throughout life. This phenomenon, named adult hippocampal neurogenesis (AHN), provides a potentially endless source of new synaptic elements that increase the complexity and plasticity of the hippocampal circuitry. Numerous lines of evidence show that physical activity and environmental enrichment (EE) are among the most potent positive regulators of AHN. Given that neural plasticity is markedly decreased in many neurodegenerative diseases, the therapeutic potential of making certain lifestyle changes, such as increasing physical activity, is being recognised in several non-pharmacologic strategies seeking to slow down or prevent the progression of these diseases. This review article summarizes current evidence supporting the putative therapeutic potential of EE and physical exercise to increase AHN and hippocampal plasticity both under physiological and pathological circumstances, with a special emphasis on neurodegenerative diseases and AD.

The Role of Microglia in the Spread of Tau: Relevance for Tauopathies.

Tauopathies are neurodegenerative diseases which course with the accumulation of Tau, mainly in neurons. In addition, Tau accumulates in a hyperphosphorylated and aggregated form. This protein is released into the extracellular space and spreads following a stereotypical pattern, inducing the development of the disease through connected regions of the brain. Microglia-the macrophages of the brain-are involved in maintaining brain homeostasis. They perform a variety of functions related to the surveillance and clearance of pathological proteins, among other dead cells and debris, from the extracellular space that could compromise brain equilibrium. This review focuses on the role played by microglia in tauopathies, specifically in Alzheimer's disease (AD), and how the uncoupling of activation/phagocytosis functions can have fatal consequences leading to the development of the pathology.

Anti-IL17 treatment ameliorates Down syndrome phenotypes in mice.

Down syndrome (DS) is characterized by structural and functional anomalies that are present prenatally and that lead to intellectual disabilities. Later in life, the cognitive abilities of DS individuals progressively deteriorate due to the development of Alzheimer's disease (AD)-associated neuropathology (i.e., ß-amyloid (Aß) plaques, neurofibrillary tangles (NFTs), neurodegeneration, synaptic pathology, neuroinflammation and increased oxidative stress). Increasing evidence has shown that among these pathological processes, neuroinflammation plays a predominant role in AD etiopathology. In AD mouse models, increased neuroinflammation appears earlier than Aß plaques and NFTs, and in DS and AD models, neuroinflammation exacerbates the levels of soluble and insoluble Aß species, favoring neurodegeneration. The Ts65Dn (TS) mouse, the most commonly used murine model of DS, recapitulates many alterations present in both DS and AD individuals, including enhanced neuroinflammation. In this study, we observed an altered neuroinflammatory milieu in the hippocampus of the TS mouse model. Pro-inflammatory mediators that were elevated in the hippocampus of this model included pro-inflammatory cytokine IL17A, which has a fundamental role in mediating brain damage in neuroinflammatory processes. Here, we analyzed the ability of an anti-IL17A antibody to reduce the neuropathological alterations that are present in TS mice during early neurodevelopmental stages (i.e., hippocampal neurogenesis and hypocellularity) or that are aggravated in later-life stages (i.e., cognitive abilities, cholinergic neuronal loss and increased cellular senescence, APP expression, Aß peptide expression and neuroinflammation). Administration of anti-IL17 for 5 months, starting at the age of 7 months, partially improved the cognitive abilities of the TS mice, reduced the expression of several pro-inflammatory cytokines and the density of activated microglia and normalized the APP and Aß1-42 levels in the hippocampi of the TS mice. These results suggest that IL17-mediated neuroinflammation is involved in several AD phenotypes in TS mice and provide a new therapeutic target to reduce these pathological characteristics.

New Beginnings in Alzheimer's Disease: The Most Prevalent Tauopathy.

Alzheimer's disease (AD) is characterized by the presence of two aberrant structures: namely senile plaques, composed of amyloid-ß peptide (Aß), and neurofibrillary tangles, composed of tau protein. In this regard, Aß and tau protein have been widely studied in research efforts aiming to find a therapy for AD. Aß and tau pathologies do not always overlap. The precursor of Aß is expressed in peripheral tissues and in the central nervous system (CNS), whereas tau is mainly a neuronal protein. Since AD is a disease of the CNS, it has been proposed that Aß may initiate the disease process, with tau being the executor. In this review, we will focus on future studies of tau pathology, although we will comment on new beginnings for AD, as other molecules other than Aß and tau may be involved in the onset of dementia.

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-3ß 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.

Cognitive Decline in Neuronal Aging and Alzheimer's Disease: Role of NMDA Receptors and Associated Proteins.

Molecular changes associated with neuronal aging lead to a decrease in cognitive capacity. Here we discuss these alterations at the level of brain regions, brain cells, and brain membrane and cytoskeletal proteins with an special focus in NMDA molecular changes through aging and its effect in cognitive decline and Alzheimer disease. Here, we propose that some neurodegenerative disorders, like Alzheimer's disease (AD), are characterized by an increase and acceleration of some of these changes.

Absence of CX3CR1 impairs the internalization of Tau by microglia.

BACKGROUND: Extracellular Tau is toxic for neighboring cells, and it contributes to the progression of AD. The CX3CL1/CX3CR1 axis is an important neuron/microglia communication mechanism. METHODS: We studied Tau clearance by microglia both in vitro (microglia primary cultures treated with Cy5-Tau, affinity chromatography to study the binding of Tau to CX3CR1, and Tau-CX3CL1 competition assays) and in vivo (stereotaxic injection of Cy5-Tau into WT and CX3CR1-/- mice). The expression of CX3CR1, CX3CL1 and the microglial phagocytic phenotype were studied in brain tissue samples from AD patients. RESULTS: Tau binding to CX3CR1 triggers the internalization of the former by microglia, whereas S396 Tau phosphorylation decreases the binding affinity of this protein to CX3CR1. Of note, the progressive increase in the levels of phosho-Tau occurred in parallel with an increase in CX3CR1. In addition, our studies suggest that the phagocytic capacity of microglia in brain tissue samples from AD patients is decreased. Furthermore, the CX3CR1/CX3CL1 axis may be impaired in late stages of the disease. CONCLUSIONS: Our data suggest that the CX3CR1/CX3CL1 axis plays a key role in the phagocytosis of Tau by microglia in vitro and in vivo and that it is affected as AD progresses. Taken together, our results reveal CX3CR1 as a novel target for the clearance of extracellular Tau.

GSK-3ß Overexpression Alters the Dendritic Spines of Developmentally Generated Granule Neurons in the Mouse Hippocampal Dentate Gyrus.

The dentate gyrus (DG) plays a crucial role in hippocampal-related memory. The most abundant cellular type in the DG, namely granule neurons, are developmentally generated around postnatal day P6 in mice. Moreover, a unique feature of the DG is the occurrence of adult hippocampal neurogenesis, a process that gives rise to newborn granule neurons throughout life. Adult-born and developmentally generated granule neurons share some maturational aspects but differ in others, such as in their positioning within the granule cell layer. Adult hippocampal neurogenesis encompasses a series of plastic changes that modify the function of the hippocampal trisynaptic network. In this regard, it is known that glycogen synthase kinase 3ß (GSK-3ß) regulates both synaptic plasticity and memory. By using a transgenic mouse overexpressing GSK-3ß in hippocampal neurons, we previously demonstrated that the overexpression of this kinase has deleterious effects on the maturation of newborn granule neurons. In the present study, we addressed the effects of GSK-3ß overexpression on the morphology and number of dendritic spines of developmentally generated granule neurons. To this end, we performed intracellular injections of Lucifer Yellow in developmentally generated granule neurons of wild-type and GSK-3ß-overexpressing mice and analyzed the number and morphologies of dendritic spines (namely, stubby, thin and mushroom). GSK-3ß overexpression led to a general reduction in the number of dendritic spines. In addition, it caused a slight reduction in the percentage, head diameter and length of thin spines, whereas the head diameter of mushroom spines was increased.