Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition.

The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.

The γ-secretase substrate proteome and its role in cell signaling regulation.

γ-Secretases mediate the regulated intramembrane proteolysis (RIP) of more than 150 integral membrane proteins. We developed an unbiased γ-secretase substrate identification (G-SECSI) method to study to what extent these proteins are processed in parallel. We demonstrate here parallel processing of at least 85 membrane proteins in human microglia in steady-state cell culture conditions. Pharmacological inhibition of γ-secretase caused substantial changes of human microglial transcriptomes, including the expression of genes related to the disease-associated microglia (DAM) response described in Alzheimer disease (AD). While the overall effects of γ-secretase deficiency on transcriptomic cell states remained limited in control conditions, exposure of mouse microglia to AD-inducing amyloid plaques strongly blocked their capacity to mount this putatively protective DAM cell state. We conclude that γ-secretase serves as a critical signaling hub integrating the effects of multiple extracellular stimuli into the overall transcriptome of the cell.

Proteome-wide analysis identifies plasma immune regulators of amyloid-beta progression.

While immune function is known to play a mechanistic role in Alzheimer's disease (AD), whether immune proteins in peripheral circulation influence the rate of amyloid-ß (Aß) progression - a central feature of AD - remains unknown. In the Baltimore Longitudinal Study of Aging, we quantified 942 immunological proteins in plasma and identified 32 (including CAT [catalase], CD36 [CD36 antigen], and KRT19 [keratin 19]) associated with rates of cortical Aß accumulation measured with positron emission tomography (PET). Longitudinal changes in a subset of candidate proteins also predicted Aß progression, and the mid- to late-life (20-year) trajectory of one protein, CAT, was associated with late-life Aß-positive status in the Atherosclerosis Risk in Communities (ARIC) study. Genetic variation that influenced plasma levels of CAT, CD36 and KRT19 predicted rates of Aß accumulation, including causal relationships with Aß PET levels identified with two-sample Mendelian randomization. In addition to associations with tau PET and plasma AD biomarker changes, as well as expression patterns in human microglia subtypes and neurovascular cells in AD brain tissue, we showed that 31 % of candidate proteins were related to mid-life (20-year) or late-life (8-year) dementia risk in ARIC. Our findings reveal plasma proteins associated with longitudinal Aß accumulation, and identify specific peripheral immune mediators that may contribute to the progression of AD pathophysiology.

IL-4 drives microglia and macrophages toward a phenotype conducive for tissue repair and functional recovery after spinal cord injury.

Macrophages and microglia play a key role in the maintenance of nervous system homeostasis. However, upon different challenges, they can adopt several phenotypes, which may lead to divergent effects on tissue repair. After spinal cord injury (SCI), microglia and macrophages show predominantly pro-inflammatory activation and contribute to tissue damage. However, the factors that hamper their conversion to an anti-inflammatory state after SCI, or to other protective phenotypes, are poorly understood. Here, we show that IL-4 protein levels are undetectable in the spinal cord after contusion injury, which likely favors microglia and macrophages to remain in a pro-inflammatory state. We also demonstrate that a single delayed intraspinal injection of IL-4, 48 hours after SCI, induces increased expression of M2 marker in microglia and macrophages. We also show that delayed injection of IL-4 leads to the appearance of resolution-phase macrophages, and that IL-4 enhances resolution of inflammation after SCI. Interestingly, we provide clear evidence that delayed administration of IL-4 markedly improves functional outcomes and reduces tissue damage after contusion injury. It is possible that these improvements are mediated by the presence of macrophages with M2 markers and resolution-phase macrophages. These data suggest that therapies aimed at increasing IL-4 levels could be valuable for the treatment of acute SCI, for which there are currently no effective treatments. GLIA 2016;64:2079-2092.

Neuregulin-1 promotes functional improvement by enhancing collateral sprouting in SOD1(G93A) ALS mice and after partial muscle denervation.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motoneurons, which is preceded by loss of neuromuscular connections in a "dying back" process. Neuregulin-1 (Nrg1) is a neurotrophic factor essential for the development and maintenance of neuromuscular junctions, and Nrg1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS. Our main goal was to investigate the role of Nrg1 type I (Nrg1-I) in SOD1(G93A) mice muscles. We overexpressed Nrg1-I by means of an adeno-associated viral (AAV) vector, and investigated its effect by means of neurophysiological techniques assessing neuromuscular function, as well as molecular approaches (RT-PCR, western blot, immunohistochemistry, ELISA) to determine the mechanisms underlying Nrg1-I action. AAV-Nrg1-I intramuscular administration promoted motor axon collateral sprouting by acting on terminal Schwann cells, preventing denervation of the injected muscles through Akt and ERK1/2 pathways. We further used a model of muscle partial denervation by transecting the L4 spinal nerve. AAV-Nrg1-I intramuscular injection enhanced muscle reinnervation by collateral sprouting, whereas administration of lapatinib (ErbB receptor inhibitor) completely blocked it. We demonstrated that Nrg1-I plays a crucial role in the collateral reinnervation process, opening a new window for developing novel ALS therapies for functional recovery rather than preservation.

Deep proteomic analysis of microglia reveals fundamental biological differences between model systems.

Using high-resolution quantitative mass spectrometry, we present comprehensive human and mouse microglia proteomic datasets consisting of over 11,000 proteins across six microglia groups. Microglia share a core protein signature of over 5,600 proteins, yet fundamental differences are observed between species and culture conditions. Mouse microglia demonstrate proteome differences in inflammation- and Alzheimer's disease-associated proteins. We identify differences in the protein content of ex vivo and in vitro cells and significant proteome differences associated with protein synthesis, metabolism, microglia marker expression, and environmental sensors. Culturing microglia induces rapidly increased growth, protein content, and inflammatory protein expression. These changes are restored by engrafting in vitro cells into the brain, with xenografted human embryonic stem cell (hESC)-derived microglia closely resembling microglia from the human brain. These data provide an important resource for the field and highlight important considerations needed when using model systems to study human physiology and pathology of microglia.

Xenografted human microglia display diverse transcriptomic states in response to Alzheimer's disease-related amyloid-ß pathology.

Microglia are central players in Alzheimer's disease pathology but analyzing microglial states in human brain samples is challenging due to genetic diversity, postmortem delay and admixture of pathologies. To circumvent these issues, here we generated 138,577 single-cell expression profiles of human stem cell-derived microglia xenotransplanted in the brain of the AppNL-G-F model of amyloid pathology and wild-type controls. Xenografted human microglia adopt a disease-associated profile similar to that seen in mouse microglia, but display a more pronounced human leukocyte antigen or HLA state, likely related to antigen presentation in response to amyloid plaques. The human microglial response also involves a pro-inflammatory cytokine/chemokine cytokine response microglia or CRM response to oligomeric Aß oligomers. Genetic deletion of TREM2 or APOE as well as APOE polymorphisms and TREM2R47H expression in the transplanted microglia modulate these responses differentially. The expression of other Alzheimer's disease risk genes is differentially regulated across the distinct cell states elicited in response to amyloid pathology. Thus, we have identified multiple transcriptomic cell states adopted by human microglia in a multipronged response to Alzheimer's disease-related pathology, which should be taken into account in translational studies.

Stem-cell-derived human microglia transplanted into mouse brain to study human disease.

Microglia are critically involved in complex neurological disorders with a strong genetic component, such as Alzheimer's disease, Parkinson's disease and frontotemporal dementia. Although mouse microglia can recapitulate aspects of human microglia physiology, they do not fully capture the human genetic aspects of disease and do not reproduce all human cell states. Primary cultures of human microglia or microglia derived from human induced pluripotent stem cells (PSCs) are difficult to maintain in brain-relevant cell states in vitro. Here we describe MIGRATE (microglia in vitro generation refined for advanced transplantation experiments, which provides a combined in vitro differentiation and in vivo xenotransplantation protocol to study human microglia in the context of the mouse brain. This article details an accurate, step-by-step workflow that includes in vitro microglia differentiation from human PSCs, transplantation into the mouse brain and quantitative analysis of engraftment. Compared to current differentiation and xenotransplantation protocols, we present an optimized, faster and more efficient approach that yields up to 80% chimerism. To quantitatively assess engraftment efficiency by flow cytometry, access to specialized flow cytometry is required. Alternatively, the percentage of chimerism can be estimated by standard immunohistochemical analysis. The MIGRATE protocol takes ~40 d to complete, from culturing PSCs to engraftment efficiency assessment.

Dual Role of Lysophosphatidic Acid Receptor 2 (LPA2) in Amyotrophic Lateral Sclerosis.

Lysophosphatidic acid (LPA) is a pleiotropic extracellular lipid mediator with many physiological functions that signal through six known G protein-coupled receptors (LPA1-6). In the central nervous system (CNS), LPA mediates a wide range of effects including neural progenitor cell physiology, neuronal cell death, axonal retraction, and inflammation. Since inflammation is a hallmark of most neurological conditions, we hypothesized that LPA could be involved in the physiopathology of amyotrophic lateral sclerosis (ALS). We found that LPA2 RNA was upregulated in post-mortem spinal cord samples of ALS patients and in the sciatic nerve and skeletal muscle of SOD1G93A mouse, the most widely used ALS mouse model. To assess the contribution of LPA2 to ALS, we generated a SOD1G93A mouse that was deficient in Lpar2. This animal revealed that LPA2 signaling accelerates disease onset and neurological decline but, unexpectedly, extended the lifespan. To gain insights into the early harmful actions of LPA2 in ALS, we studied the effects of this receptor in the spinal cord, peripheral nerve, and skeletal muscle of ALS mice. We found that LPA2 gene deletion increased microglial activation but did not contribute to motoneuron death, astrogliosis, degeneration, and demyelination of motor axons. However, we observed that Lpar2 deficiency protected against muscle atrophy. Moreover, we also found the deletion of Lpar2 reduced the invasion of macrophages into the skeletal muscle of SOD1G93A mice, linking LPA2 signaling with muscle inflammation and atrophy in ALS. Overall, these results suggest for the first time that LPA2 contributes to ALS, and its genetic deletion results in protective actions at the early stages of the disease but shortens survival thereafter.

Combined intramuscular and intraspinal transplant of bone marrow cells improves neuromuscular function in the SOD1G93A mice.

BACKGROUND: The simultaneous contribution of several etiopathogenic disturbances makes amyotrophic lateral sclerosis (ALS) a fatal and challenging disease. Here, we studied two different cell therapy protocols to protect both central and peripheral nervous system in a murine model of ALS. METHODS: Since ALS begins with a distal axonopathy, in a first assay, we performed injection of bone marrow cells into two hindlimb muscles of transgenic SOD1G93A mice. In a second study, we combined intramuscular and intraspinal injection of bone marrow cells. Fluorescence-activated cell sorting was used to assess the survival of the transplanted cells into the injected tissues. The mice were assessed from 8 to 16 weeks of age by means of locomotion and electrophysiological tests. After follow-up, the spinal cord was processed for analysis of motoneuron survival and glial cell reactivity. RESULTS: We found that, after intramuscular injection, bone marrow cells were able to engraft within the muscle. However, bone marrow cell intramuscular injection failed to promote a general therapeutic effect. In the second approach, we found that bone marrow cells had limited survival in the spinal cord, but this strategy significantly improved motor outcomes. Moreover, we also found that the dual cell therapy tended to preserve spinal motoneurons at late stages of the disease and to reduce microgliosis, although this did not prolong mice survival. CONCLUSION: Overall, our findings suggest that targeting more than one affected area of the motor system at once with bone marrow cell therapy results in a valuable therapeutic intervention for ALS.

Chemotherapeutic agent 5-fluorouracil increases survival of SOD1 mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a lethal motor neuron disease with no cure. Currently there are only two ALS drugs approved by the FDA, both with a limited therapeutic effect. In the search for drug candidates for ALS, we studied the effect of known stem cell mobilizing agents (treatment) and antimetabolite 5-fluorouracil (5-FU) (anti-treatment) in SOD1G93A model of ALS. Surprisingly, we found that anti-cancer drug 5-FU increases lifespan, delays the disease onset and improves motor performance in ALS mice. Although we were not able to demonstrate the mechanistic basis of the beneficial 5-FU action in ALS mice, our findings suggest that 5-FU or similar drugs are possible drug candidates for the treatment of motor neuron diseases through drug repurposing.

Neuroinflammation Quantification for Spinal Cord Injury.

Spinal cord injury (SCI) leads to irreversible devastating neurological disabilities. Accumulated evidence in the literature indicates that the inflammatory response that occurs in the spinal cord following injury contributes importantly to spread tissue damage to healthy regions adjacent to the lesion site, and consequently, to increase neurological deficits. Therefore, targeting inflammation could lead to the development of new therapies to prevent tissue damage and neurological impairments after SCI. Inflammation is regulated, in part, by the expression of pro-inflammatory and anti-inflammatory cytokines synthesized, mainly, by glial cells. Hence, methodologies that could ease the quantification of multiple cytokines and immune cells from spinal cord tissue samples are needed to assess the potential of new anti-inflammatory therapies. In the present unit, we describe how to induce contusion injuries in the mouse spinal cord, as well as, two useful methodologies to assess neuroinflammation in lesioned spinal cord tissue samples. © 2018 by John Wiley & Sons, Inc.

Nintedanib decreases muscle fibrosis and improves muscle function in a murine model of dystrophinopathy.

Duchenne muscle dystrophy (DMD) is a genetic disorder characterized by progressive skeletal muscle weakness. Dystrophin deficiency induces instability of the sarcolemma during muscle contraction that leads to muscle necrosis and replacement of muscle by fibro-adipose tissue. Several therapies have been developed to counteract the fibrotic process. We report the effects of nintedanib, a tyrosine kinase inhibitor, in the mdx murine model of DMD. Nintedanib reduced proliferation and migration of human fibroblasts in vitro and decreased the expression of fibrotic genes such as COL1A1, COL3A1, FN1, TGFB1, and PDGFA. We treated seven mdx mice with 60 mg/kg/day nintedanib for 1 month. Electrophysiological studies showed an increase in the amplitude of the motor action potentials and an improvement of the morphology of motor unit potentials in the animals treated. Histological studies demonstrated a significant reduction of the fibrotic areas present in the skeletal muscles. Analysis of mRNA expression from muscles of treated mice showed a reduction in Col1a1, Col3a1, Tgfb1, and Pdgfa. Western blot showed a reduction in the expression of collagen I in skeletal muscles. In conclusion, nintedanib reduced the fibrotic process in a murine model of dystrophinopathy after 1 month of treatment, suggesting its potential use as a therapeutic drug in DMD patients.

CSF1R blockade slows the progression of amyotrophic lateral sclerosis by reducing microgliosis and invasion of macrophages into peripheral nerves.

Inflammation is a common neuropathological feature in several neurological disorders, including amyotrophic lateral sclerosis (ALS). We have studied the contribution of CSF1R signalling to inflammation in ALS, as a pathway previously reported to control the expansion and activation of microglial cells. We found that microglial cell proliferation in the spinal cord of SOD1(G93A) transgenic mice correlates with the expression of CSF1R and its ligand CSF1. Administration of GW2580, a selective CSF1R inhibitor, reduced microglial cell proliferation in SOD1(G93A) mice, indicating the importance of CSF1-CSF1R signalling in microgliosis in ALS. Moreover, GW2580 treatment slowed disease progression, attenuated motoneuron cell death and extended survival of SOD1(G93A) mice. Electrophysiological assessment revealed that GW2580 treatment protected skeletal muscle from denervation prior to its effects on microglial cells. We found that macrophages invaded the peripheral nerve of ALS mice before CSF1R-induced microgliosis occurred. Interestingly, treatment with GW2580 attenuated the influx of macrophages into the nerve, which was partly caused by the monocytopenia induced by CSF1R inhibition. Overall, our findings provide evidence that CSF1R signalling regulates inflammation in the central and peripheral nervous system in ALS, supporting therapeutic targeting of CSF1R in this disease.