Peripheral expression of brain-penetrant progranulin rescues pathologies in mouse models of frontotemporal lobar degeneration.

Progranulin (PGRN) haploinsufficiency is a major risk factor for frontotemporal lobar degeneration with TAR DNA-binding protein 43 (TDP-43) pathology (FTLD-GRN). Multiple therapeutic strategies are in clinical development to restore PGRN in the CNS, including gene therapy. However, a limitation of current gene therapy approaches aimed to alleviate FTLD-associated pathologies may be their inefficient brain exposure and biodistribution. We therefore developed an adeno-associated virus (AAV) targeting the liver (L) to achieve sustained peripheral expression of a transferrin receptor (TfR) binding, brain-penetrant (b) PGRN variant [AAV(L):bPGRN] in two mouse models of FTLD-GRN, namely, Grn knockout and GrnxTmem106b double knockout mice. This therapeutic strategy avoids potential safety and biodistribution issues of CNS-administered AAVs and maintains sustained concentrations of PGRN in the brain after a single dose. AAV(L):bPGRN treatment reduced several FTLD-GRN-associated pathologies including severe motor function deficits, aberrant TDP-43 phosphorylation, dysfunctional protein degradation, lipid metabolism, gliosis, and neurodegeneration in the brain. The potential translatability of our findings was tested in an in vitro model using cocultured human induced pluripotent stem cell (hiPSC)-derived microglia lacking PGRN and TMEM106B and wild-type hiPSC-derived neurons. As in mice, aberrant TDP-43, lysosomal dysfunction, and neuronal loss were ameliorated after treatment with exogenous TfR-binding protein transport vehicle fused to PGRN (PTV:PGRN). Together, our studies suggest that peripherally administered brain-penetrant PGRN replacement strategies ameliorate FTLD-GRN relevant phenotypes including TDP-43 pathology, neurodegeneration, and behavioral deficits. Our data provide preclinical proof of concept for the use of this AAV platform for treatment of FTLD-GRN and potentially other CNS disorders.

Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome.

During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12, and the tyrosine kinases of the TAM family Mer tyrosine kinase (MerTK)/Axl. In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.SIGNIFICANCE STATEMENT Microglia are the brain professional phagocytes and, in the adult hippocampal neurogenic niche, they remove newborn cells naturally undergoing apoptosis. Here we show that phagocytosis of apoptotic cells triggers a coordinated transcriptional program that alters their secretome, limiting neurogenesis both in vivo and in vitro In addition, chronic phagocytosis disruption in mice deficient for receptors P2Y12 and MerTK/Axl reduces adult hippocampal neurogenesis. In contrast, inducible MerTK downregulation transiently increases neurogenesis, suggesting that microglial phagocytosis provides a negative feedback loop that is necessary for the long-term maintenance of adult hippocampal neurogenesis. Therefore, we speculate that the effects of promoting engulfment/degradation of cell debris may go beyond merely removing corpses to actively promoting regeneration in development, aging, and neurodegenerative diseases.

Alzheimer's Risk Gene TREM2 Determines Functional Properties of New Type of Human iPSC-Derived Microglia.

Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer's disease (AD). Due to the many limitations to study microglia in situ or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discovery.

ProMoIJ: A new tool for automatic three-dimensional analysis of microglial process motility.

Microglia, the immune cells of the central nervous system, continuously survey the brain to detect alterations and maintain tissue homeostasis. The motility of microglial processes is indicative of their surveying capacity in normal and pathological conditions. The gold standard technique to study motility involves the use of two-photon microscopy to obtain time-lapse images from brain slices or the cortex of living animals. This technique generates four dimensionally-coded images which are analyzed manually using time-consuming, non-standardized protocols. Microglial process motility analysis is frequently performed using Z-stack projections with the consequent loss of three-dimensional (3D) information. To overcome these limitations, we developed ProMoIJ, a pack of ImageJ macros that perform automatic motility analysis of cellular processes in 3D. The main core of ProMoIJ is formed by two macros that assist the selection of processes, automatically reconstruct their 3D skeleton, and analyze their motility (process and tip velocity). Our results show that ProMoIJ presents several key advantages compared with conventional manual analysis: (1) reduces the time required for analysis, (2) is less sensitive to experimenter bias, and (3) is more robust to varying numbers of processes analyzed. In addition, we used ProMoIJ to demonstrate that commonly performed 2D analysis underestimates microglial process motility, to reveal that only cells adjacent to a laser injured area extend their processes toward the lesion site, and to demonstrate that systemic inflammation reduces microglial process motility. ProMoIJ is a novel, open-source, freely-available tool which standardizes and accelerates the time-consuming labor of 3D analysis of microglial process motility.

Correction: Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

[This corrects the article DOI: 10.1371/journal.pbio.1002466.].

Correction: Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

[This corrects the article DOI: 10.1371/journal.pbio.1002466.].

Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.

Lifestyle Shapes the Dialogue between Environment, Microglia, and Adult Neurogenesis.

Lifestyle modulates brain function. Diet, stress levels, and physical exercise among other factors influence the "brain cognitive reserve", that is, the capacity of the brain to maintain a normal function when confronting neurodegenerative diseases, injury, and/or aging. This cognitive reserve relays on several cellular and molecular elements that contribute to brain plasticity allowing adaptive responses to cognitive demands, and one of its key components is the hippocampal neurogenic reserve. Hippocampal neural stem cells give rise to new neurons that integrate into the local circuitry and contribute to hippocampal functions such as memory and learning. Importantly, adult hippocampal neurogenesis is well-known to be modulated by the demands of the environment and lifestyle factors. Diet, stress, and physical exercise directly act on neural stem cells and/or their progeny, but, in addition, they may also indirectly affect neurogenesis by acting on microglia. Microglia, the guardians of the brain, rapidly sense changes in the brain milieu, and it has been recently shown that their function is affected by lifestyle factors. However, few studies have analyzed the modulatory effect of microglia on adult neurogenesis in these conditions. Here, we review the current knowledge about the dialogue maintained between microglia and the hippocampal neurogenic cascade. Understanding how the communication between microglia and hippocampal neurogenesis is affected by lifestyle choices is crucial to maintain the brain cognitive reserve and prevent the maladaptive responses that emerge during disease or injury through adulthood and aging.