The spatiotemporal dynamics of microglia across the human lifespan.

Microglia, the brain's resident macrophages, shape neural development and are key neuroimmune hubs in the pathological signatures of neurodevelopmental disorders. Despite the importance of microglia, their development has not been carefully examined in the human brain, and most of our knowledge derives from rodents. We aimed to address this gap in knowledge by establishing an extensive collection of 97 post-mortem tissues in order to enable quantitative, sex-matched, detailed analysis of microglia across the human lifespan. We identify the dynamics of these cells in the human telencephalon, describing waves in microglial density across gestation, infancy, and childhood, controlled by a balance of proliferation and apoptosis, which track key neurodevelopmental milestones. These profound changes in microglia are also observed in bulk RNA-seq and single-cell RNA-seq datasets. This study provides a detailed insight into the spatiotemporal dynamics of microglia across the human lifespan and serves as a foundation for elucidating how microglia contribute to shaping neurodevelopment in humans.

Replicative senescence dictates the emergence of disease-associated microglia and contributes to Aß pathology.

The sustained proliferation of microglia is a key hallmark of Alzheimer's disease (AD), accelerating its progression. Here, we aim to understand the long-term impact of the early and prolonged microglial proliferation observed in AD, hypothesizing that extensive and repeated cycling would engender a distinct transcriptional and phenotypic trajectory. We show that the early and sustained microglial proliferation seen in an AD-like model promotes replicative senescence, characterized by increased ßgal activity, a senescence-associated transcriptional signature, and telomere shortening, correlating with the appearance of disease-associated microglia (DAM) and senescent microglial profiles in human post-mortem AD cases. The prevention of early microglial proliferation hinders the development of senescence and DAM, impairing the accumulation of Aß, as well as associated neuritic and synaptic damage. Overall, our results indicate that excessive microglial proliferation leads to the generation of senescent DAM, which contributes to early Aß pathology in AD.

Inhibition of IL-34 Unveils Tissue-Selectivity and Is Sufficient to Reduce Microglial Proliferation in a Model of Chronic Neurodegeneration.

The proliferation and activation of microglia, the resident macrophages in the brain, is a hallmark of many neurodegenerative diseases such as Alzheimer's disease (AD) and prion disease. Colony stimulating factor 1 receptor (CSF1R) is critically involved in regulating microglial proliferation, and CSF1R blocking strategies have been recently used to modulate microglia in neurodegenerative diseases. However, CSF1R is broadly expressed by many cell types and the impact of its inhibition on the innate immune system is still unclear. CSF1R can be activated by two independent ligands, CSF-1 and interleukin 34 (IL-34). Recently, it has been reported that microglia development and maintenance depend on IL-34 signaling. In this study, we evaluate the inhibition of IL-34 as a novel strategy to reduce microglial proliferation in the ME7 model of prion disease. Selective inhibition of IL-34 showed no effects on peripheral macrophage populations in healthy mice, avoiding the side effects observed after CSF1R inhibition on the systemic compartment. However, we observed a reduction in microglial proliferation after IL-34 inhibition in prion-diseased mice, indicating that microglia could be more specifically targeted by reducing IL-34. Overall, our results highlight the challenges of targeting the CSF1R/IL34 axis in the systemic and central compartments, important for framing any therapeutic effort to tackle microglia/macrophage numbers during brain disease.

Targeting Microglial Population Dynamics in Alzheimer's Disease: Are We Ready for a Potential Impact on Immune Function?

Alzheimer's disease (AD) is the most common form of dementia, affecting two-thirds of people with dementia in the world. To date, no disease-modifying treatments are available to stop or delay the progression of AD. This chronic neurodegenerative disease is dominated by a strong innate immune response, whereby microglia plays a central role as the main resident macrophage of the brain. Recent genome-wide association studies (GWASs) have identified single-nucleotide polymorphisms (SNPs) located in microglial genes and associated with a delayed onset of AD, highlighting the important role of these cells on the onset and/or progression of the disease. These findings have increased the interest in targeting microglia-associated neuroinflammation as a potentially disease-modifying therapeutic approach for AD. In this review we provide an overview on the contribution of microglia to the pathophysiology of AD, focusing on the main regulatory pathways controlling microglial population dynamics during the neuroinflammatory response, such as the colony-stimulating factor 1 receptor (CSF1R), its ligands (the colony-stimulating factor 1 and interleukin 34) and the transcription factor PU.1. We also discuss the current therapeutic strategies targeting proliferation to modulate microglia-associated neuroinflammation and their potential impact on peripheral immune cell populations in the short and long-term. Understanding the effects of immunomodulatory approaches on microglia and other immune cell types might be critical for developing specific, effective, and safe therapies for neurodegenerative diseases.

Poly(ADP-ribose)polymerases inhibitors prevent early mitochondrial fragmentation and hepatocyte cell death induced by H2O2.

Poly(ADP-ribose)polymerases (PARPs) are a family of NAD+ consuming enzymes that play a crucial role in many cellular processes, most clearly in maintaining genome integrity. Here, we present an extensive analysis of the alteration of mitochondrial morphology and the relationship to PARPs activity after oxidative stress using an in vitro model of human hepatic cells. The following outcomes were observed: reactive oxygen species (ROS) induced by oxidative treatment quickly stimulated PARPs activation, promoted changes in mitochondrial morphology associated with early mitochondrial fragmentation and energy dysfunction and finally triggered apoptotic cell death. Pharmacological treatment with specific PARP-1 (the major NAD+ consuming poly(ADP-ribose)polymerases) and PARP-1/PARP-2 inhibitors after the oxidant insult recovered normal mitochondrial morphology and, hence, increased the viability of human hepatic cells. As the PARP-1 and PARP-1/PARP-2 inhibitors achieved similar outcomes, we conclude that most of the PARPs effects were due to PARP-1 activation. NAD+ supplementation had similar effects to those of the PARPs inhibitors. Therefore, PARPs activation and the subsequent NAD+ depletion are crucial events in decreased cell survival (and increased apoptosis) in hepatic cells subjected to oxidative stress. These results suggest that the alterations in mitochondrial morphology and function seem to be related to NAD+ depletion, and show for the first time that PARPs inhibition abrogates mitochondrial fragmentation. In conclusion, the inhibition of PARPs may be a valuable therapeutic approach for treating liver diseases, by reducing the cell death associated with oxidative stress.

Onset of microglial entry into developing quail retina coincides with increased expression of active caspase-3 and is mediated by extracellular ATP and UDP.

Microglial cell precursors located in the area of the base of the pecten and the optic nerve head (BP/ONH) start to enter the retina of quail embryos at the 7th day of incubation (E7), subsequently colonizing the entire retina by central-to-peripheral tangential migration, as previously shown by our group. The present study demonstrates a precise chronological coincidence of the onset of microglial cell entry into the retina with a striking increase in death of retinal cells, as revealed by their active caspase-3 expression and TUNEL staining, in regions dorsal to the BP/ONH area, suggesting that dying retinal cells would contribute to the microglial cell inflow into the retina. However, the molecular mechanisms involved in this inflow are currently unclear. Extracellular nucleotides, such as ATP and UDP, have previously been shown to favor migration of microglia towards brain injuries because they are released by apoptotic cells and stimulate both chemotaxis and chemokinesis in microglial cells via signaling through purinergic receptors. Hence, we tested here the hypothesis that ATP and UDP play a role in the entry and migration of microglial precursors into the developing retina. For this purpose, we used an experimental model system based on organotypic cultures of E6.5 quail embryo retina explants, which mimics the entry and migration of microglial precursors in the in situ developing retina. Inhibition of purinergic signaling by treating retina explants with either apyrase, a nucleotide-hydrolyzing enzyme, or suramin, a broad spectrum antagonist of purinergic receptors, significantly prevents the entry of microglial cells into the retina. In addition, treatment of retina explants with either exogenous ATP or UDP results in significantly increased numbers of microglial cells entering the retina. In light of these findings, we conclude that purinergic signaling by extracellular ATP and UDP is necessary for the entry and migration of microglial cells into the embryonic retina by inducing chemokinesis in these cells.

Microglial Activation Promotes Cell Survival in Organotypic Cultures of Postnatal Mouse Retinal Explants.

The role of microglia during neurodegeneration remains controversial. We investigated whether microglial cells have a neurotoxic or neuroprotective function in the retina. Retinal explants from 10-day-old mice were treated in vitro with minocycline to inhibit microglial activation, with LPS to increase microglial activation, or with liposomes loaded with clodronate (Lip-Clo) to deplete microglial cells. Flow cytometry was used to assess the viability of retinal cells in the explants and the TUNEL method to show the distribution of dead cells. The immunophenotypic and morphological features of microglia and their distribution were analyzed with flow cytometry and immunocytochemistry. Treatment of retinal explants with minocycline reduced microglial activation and simultaneously significantly decreased cell viability and increased the presence of TUNEL-labeled cell profiles. This treatment also prevented the migration of microglial cells towards the outer nuclear layer, where cell death was most abundant. The LPS treatment increased microglial activation but had no effect on cell viability or microglial distribution. Finally, partial microglial removal with Lip-Clo diminished the cell viability in the retinal explants, showing a similar effect to that of minocycline. Hence, cell viability is diminished in retinal explants cultured in vitro when microglial cells are removed or their activation is inhibited, indicating a neurotrophic role for microglia in this system.

DNA damage, poly(ADP-Ribose) polymerase activation, and phosphorylated histone H2AX expression during postnatal retina development in C57BL/6 mouse.

PURPOSE: The purpose of this study was to investigate the incidence of DNA damage during postnatal development of the retina and the relationship between DNA damage and cell death. METHODS: DNA damage in the developing postnatal retina of C57BL/6 mice was assessed by determining the amounts of 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is indicative of DNA oxidation and related to the formation of DNA single-strand breaks (SSBs), and phosphorylated histone H2AX (γ-H2AX), a marker of DNA double-strand breaks (DSBs). Poly(ADP-ribose) polymerase (PARP) activation was measured by ELISA and Western blotting. The location of γ-H2AX-positive and dying cells was determined by immunofluorescence and TUNEL assays. RESULTS: Oxidative DNA damage was maintained at low levels during high PARP activation between postnatal days 0 (P0) and P7. Phosphorylated histone H2AX gradually increased between P0 and P14 and decreased thereafter. Phosphorylated histone H2AX-positive cells with cell death morphology or TUNEL positivity were more abundant at P7 than at P14. CONCLUSIONS: Oxidative DNA damage in postnatal retina increases during development. It is low during the first postnatal week when PARP-1 activity is high but increases thereafter. The rise in DSBs when PARP activity is downregulated may be attributable to accumulated oxidative damage and SSBs. At P7 and P14, γ-H2AX-positive cells are repairing naturally occurring DNA damage, but some are dying (mostly at P7), probably due to an accumulation of irreparable DNA damage.

Expression of inducible nitric oxide synthase (iNOS) in microglia of the developing quail retina.

Inducible nitric oxide synthase (iNOS), which produce large amounts of nitric oxide (NO), is induced in macrophages and microglia in response to inflammatory mediators such as LPS and cytokines. Although iNOS is mainly expressed by microglia that become activated in different pathological and experimental situations, it was recently reported that undifferentiated amoeboid microglia can also express iNOS during normal development. The aim of this study was to investigate the pattern of iNOS expression in microglial cells during normal development and after their activation with LPS by using the quail retina as model. iNOS expression was analyzed by iNOS immunolabeling, western-blot, and RT-PCR. NO production was determined by using DAR-4M AM, a reliable fluorescent indicator of subcellular NO production by iNOS. Embryonic, postnatal, and adult in situ quail retinas were used to analyze the pattern of iNOS expression in microglial cells during normal development. iNOS expression and NO production in LPS-treated microglial cells were investigated by an in vitro approach based on organotypic cultures of E8 retinas, in which microglial cell behavior is similar to that of the in situ retina, as previously demonstrated in our laboratory. We show here that amoeboid microglia in the quail retina express iNOS during normal development. This expression is stronger in microglial cells migrating tangentially in the vitreal part of the retina and is downregulated, albeit maintained, when microglia differentiate and become ramified. LPS treatment of retina explants also induces changes in the morphology of amoeboid microglia compatible with their activation, increasing their lysosomal compartment and upregulating iNOS expression with a concomitant production of NO. Taken together, our findings demonstrate that immature microglial cells express iNOS during normal development, suggesting a certain degree of activation. Furthermore, LPS treatment induces overactivation of amoeboid microglia, resulting in a significant iNOS upregulation.

Microglial cells in organotypic cultures of developing and adult mouse retina and their relationship with cell death.

Organotypic cultures of retinal explants allow the detailed analysis of microglial cells in a cellular microenvironment similar to that in the in situ retina, with the advantage of easy experimental manipulation. However, the in vitro culture causes changes in the retinal cytoarchitecture and induces a microglial response that may influence the results of these manipulations. The purpose of this study was to analyze the influence of the retinal age on changes in retinal cytoarchitecture, cell viability and death, and microglial phenotype and distribution throughout the in vitro culture of developing and adult retina explants. Explants from developing (3 and 10 postnatal days, P3 and P10) and adult (P60) mouse retinas were cultured for up to 10 days in vitro (div). Dead or dying cells were recognized by TUNEL staining, cell viability was determined by flow cytometry, and the numbers and distribution patterns of microglial cells were studied by flow cytometry and immunocytochemistry, respectively. The retinal cytoarchitecture was better preserved at prolonged culture times (10 div) in P10 retina explants than in P3 or adult explants. Particular patterns of cell viability and death were observed at each age: in general, explants from developing retinas showed higher cell viability and lower density of TUNEL-positive profiles versus adult retinas. The proportion of microglial cells relative to the whole population of retinal cells was higher in explants fixed immediately after their dissection (i.e., non-cultured) from adult retinas than in those from developing retinas. This proportion was always higher in non-cultured explants than in explants at 10 div, suggesting the death of some microglial cells during the culture. Activation of microglial cells, as revealed by their phenotypical appearance, was observed in both developing and adult retina explants from the beginning of the culture. Immunofluorescence with the anti-CD68 antibody showed that some activated microglial cells were CD68-positive but others were CD68-negative. Flow cytometry using CD68-labeling revealed that the percentage of CD68-positive microglial cells was much higher in developing than in adult retina explants, despite the activation of microglia in both types of explants, indicating that CD68-labeling was more closely related to the maturity degree of microglia than to their activation. Some swollen activated microglial cells entered the outer nuclear layer in developing and adult cultured retinal explants, whereas this layer was devoid of microglia in non-cultured explants. There was no apparent correlation between the distribution of microglia and that of TUNEL-labeled profiles. However, some swollen activated microglial cells in the outer and inner nuclear layers engulfed clusters of cell nuclei that were negative or weakly positive for TUNEL. This engulfment activity of microglia mimicked that observed in degenerative pathologies of the retina. We conclude that organotypic cultures from developing retinas show a higher rate of cell viability and better preservation of the normal cytoarchitecture in comparison to those obtained from adult retinas. In addition, the features of microglial response in cultured retinal explants show them to be a useful model for studying interactions between microglial cells and degenerating neurons in retinal diseases.