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.

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.

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.

An integration-free iPSC line, ICCSICi007-A, derived from a female Alzheimer's disease patient with the APOE-ε4/ε4 alleles.

The epsilon4 (ε4) allele of the APOE gene, which encodes the apolipoprotein E4 (ApoE4), is the strongest genetic risk factor known for late-onset Alzheimer´s disease (LOAD). Here, we present the characterization of an iPSC line generated from dermal fibroblasts of a female AD patient using Sendai viral vectors encoding the transcription factors OCT4, SOX2, KLF4 and c-MYC. The iPSCs maintained the original genotype, a normal karyotype, were free from Sendai viral vectors and reprogramming factors, presented a normal morphology, expressed endogenous pluripotency markers, and could be differentiated into ectodermal, mesodermal and endodermal cells, confirming its pluripotency.

Generation of an integration-free iPSC line, ICCSICi006-A, derived from a male Alzheimer's disease patient carrying the PSEN1-G206D mutation.

The familial form of Alzheimer's disease (FAD), which is caused by mutations in PRESENILIN 1 (PSEN1) and amyloid precursor protein (APP) genes, represents less than 5% of all AD cases and has an early-onset. We report the generation and characterization of an iPSC line derived from a FAD patient carrying the PSEN1-G206D mutation. The iPSC line maintained the original genotype, a normal karyotype, was free from Sendai viral vectors and reprogramming factors (OCT4, SOX2, KLF4 and c-MYC), presented a typical morphology, expressed endogenous pluripotency markers, and could be differentiated into ectodermal, mesodermal and endodermal cells, confirming its pluripotency.

A collection of four integration-free iPSC lines derived from diagnosed sporadic Alzheimer's disease patients with different APOE alleles.

Genetic polymorphism of apolipoprotein E (APOE) confers differential susceptibility to late-onset Alzheimer's disease (LOAD). The ε3 allele of APOE, the most common isoform, does not represent a risk factor for LOAD. In contrast, the ε4 allele is the strongest genetic risk factor for this disease. Here, we present the characterization of four iPSC lines generated from dermal fibroblasts of diagnosed sporadic AD patients using Sendai viral vectors encoding OCT4, SOX2, KLF4 and c-MYC. The iPSCs expressed endogenous pluripotency markers, could be differentiated into the three germ layers, maintained the original genotypes, and were free from Sendai vectors and reprogramming factors.

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.

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.

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.