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Extracellular vesicles (EVs) effectively suppress neuroinflammation and induce neuroprotective effects in different disease models. However, the mechanisms by which EVs regulate the neuroinflammatory response of microglia remains largely unexplored. Here, we addressed this issue by testing the action of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on immortalized human microglial cells. We found that EVs induced a rapid increase in intracellular Ca2+ and promoted significant ATP release in microglial cells after 20 min of treatment. Boyden chamber assays revealed that EVs promoted microglial migration by 20%. Pharmacological inhibition of different subtypes of purinergic receptors demonstrated that EVs activated microglial migration preferentially through the P2X4 receptor (P2X4R) pathway. Proximity ligation and co-immunoprecipitation assays revealed that EVs promote association between milk fat globule-epidermal growth factor-factor VIII (MFG-E8) and P2X4R proteins. Furthermore, pharmacological inhibition of αVβ3/αVβ5 integrin suppressed EV-induced cell migration and formation of lipid rafts in microglia. These results demonstrate that EVs promote microglial motility through P2X4R/MFG-E8-dependent mechanisms. Our findings provide novel insights into the molecular mechanisms through which EVs target human microglia that may be exploited for the development of new therapeutic strategies targeting disease-associated neuroinflammation.

Aim: Mechanosensitive Piezo1 ion channels emerged recently as important contributors to various vital functions including modulation of the blood supply to skeletal muscles. The specific Piezo1 channel agonist Yoda1 was shown to regulate the tone of blood vessels similarly to physical exercise. However, the direct role of Piezo1 channels in muscle function has been little studied so far. We therefore investigated the action of Yoda1 on the functional state of skeletal muscle precursors (satellite cells and myotubes) as well as on adult muscle fibers. Methods: Immunostaining, electrophysiological intracellular recordings and Ca2+ imaging experiments were performed to localize and assess the effect of the chemical activation of Piezo1 channels with Yoda1, on myogenic precursors, adult myofibers and at the adult neuromuscular junction. Results: Piezo1 channels were detected by immunostaining in satellite cells and myotubes as well as in adult myofibers. In the skeletal muscle precursors, Yoda1 treatment stimulated the differentiation and cell fusion rather than the proliferation of satellite cells. Moreover, in myotubes, Yoda1 induced significant [Ca2+ ]i transients, without detectable [Ca2+ ]i response in adult myofibers. Furthermore, although expression of Piezo1 channels was detected around the muscle endplate region, Yoda1 application did not alter either the nerve-evoked or spontaneous synaptic activity or muscle contractions in adult myofibers. Conclusion: Our data indicate that the chemical activation of Piezo1 channels specifically enhances the differentiation of skeletal muscle precursors, suggesting a possible new strategy to promote muscle regeneration.

Alzheimer's disease (AD) is an incurable disease, with complex pathophysiology and a myriad of proteins involved in its development. In this study, we applied quantitative targeted absolute proteomic analysis for investigation of changes in potential AD drug targets, biomarkers, and transporters in cerebral cortices of lipopolysaccharide (LPS)-induced neuroinflammation mouse model, familial AD mice (APdE9) with and without LPS treatment as compared to age-matched wild type (WT) mice. The ABCB1, ABCG2 and GluN1 protein expression ratios between LPS treated APdE9 and WT control mice were 0.58 (95% CI 0.44-0.72), 0.65 (95% CI 0.53-0.77) and 0.61 (95% CI 0.52-0.69), respectively. The protein expression levels of other proteins such as MGLL, COX-2, CytC, ABCC1, ABCC4, SLC2A1 and SLC7A5 did not differ between the study groups. Overall, the study revealed that systemic inflammation can alter ABCB1 and ABCG2 protein expression in brain in AD, which can affect intra-brain drug distribution and play a role in AD development. Moreover, the inflammatory insult caused by peripheral infection in AD may be important factor triggering changes in GluN1 protein expression. However, more studies need to be performed in order to confirm these findings. The quantitative information about the expression of selected proteins provides important knowledge, which may help in the optimal use of the mouse models in AD drug development and better translation of preclinical data to humans. (c) 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

Human stem cell-derived brain organoids provide a physiologically relevant in vitro 3D brain model for studies of neurological development that are unique to the human nervous system. Prior studies have reported protocols that support the maturation of microglia from mesodermal progenitors leading to innately developing microglia within the organoids. However, although microglia are known to support neuronal development in rodents, none of the previous studies have reported what is the impact of microglia on neuronal growth and maturation in human brain organoids. Here we show that incorporating microglial progenitors into the developing organoid supports neuronal maturation, the emergence of neurons capable of firing repetitive action potentials and the appearance of synaptic and neuronal bursting activity. Immunocompetent organoids enable experimental strategies for interrogating fundamental questions on microglial and neuronal diversity and function during human brain development.

Microglia are resident immune cells of the central nervous system and play critical roles during the development, homeostasis, and pathologies of the brain. Originated from yolk sac erythromyeloid progenitors, microglia immigrate into the embryonic brain parenchyma to undergo final postnatal differentiation and maturation driven by distinct chemokines, cytokines, and growth factors. Among them, TGFβ1 is an important regulator of microglial functions, mediating homeostasis, anti-inflammation, and triggering the expression of microglial homeostatic signature genes. Since microglia studies are mainly based on rodent cells and the isolation of homeostatic microglia from human tissue is challenging, human-induced pluripotent stem cells have been successfully differentiated into microglia-like cells recently. However, employed differentiation protocols strongly vary regarding used cytokines and growth factors, culture conditions, time span, and cell yield. Moreover, the incomplete differentiation of human microglia can hamper the similarity to primary human microglia and dramatically influence the outcome of follow-up studies with these differentiated cells. This review summarizes the current knowledge of the molecular mechanisms driving rodent microglia differentiation in vivo, further compares published differentiation protocols, and highlights the potential of TGFβ as an essential maturation factor.

Human stem cell-derived brain organoids provide a physiologically relevant in vitro 3D brain model for studies of neurological development that are unique to the human nervous system. Prior studies have reported protocols that support the maturation of microglia from mesodermal progenitors leading to innately developing microglia within the organoids. However, although microglia are known to support neuronal development in rodents, none of the previous studies have reported what is the impact of microglia on neuronal growth and maturation in human brain organoids. Here we show that incorporating microglial progenitors into the developing organoid supports neuronal maturation, the emergence of neurons capable of firing repetitive action potentials and the appearance of synaptic and neuronal bursting activity. Immunocompetent organoids enable experimental strategies for interrogating fundamental questions on microglial and neuronal diversity and function during human brain development.

Olfactory dysfunction manifests early in several neurodegenerative disorders. Olfaction is orchestrated by olfactory mucosal cells located in the upper nasal cavity. However, it is unclear how this tissue reflects key neurodegenerative features in Alzheimer’s disease. Here we report that Alzheimer’s disease olfactory mucosal cells obtained from live individuals secrete toxic amyloid-beta. We detail cell-type-specific gene expression patterns, unveiling 147 differentially expressed disease-associated genes compared to the cognitively healthy controls, and 5 distinct populations in globose basal cell -, myofibroblast-, and fibroblast/ stromal – like cells in vitro. Overall, coordinated alteration of RNA and protein metabolism, inflammatory processes and signal transduction were observed in multiple cell populations, suggesting a key role in pathophysiology. Our results demonstrate the potential of olfactory cell cultures in modelling Alzheimer’s disease advocate their use for diagnostic purposes. Moreover, for the first time we provide single cell data on olfactory mucosa in Alzheimer’s disease for investigating molecular and cellular mechanisms associated with the disease.

Background: Extracellular ATP is a powerful trigger of neuroinflammation by activating immune cells via P2X7 receptors. Acetylcholine and nicotinic agonists inhibit ATP-triggered proinflammatory cytokines via the so-called “cholinergic anti-inflammatory pathway” (CAP). However, it remains unclear as to what stage of ATP-induced signaling cholinergic agents provide this anti-inflammatory effect. Using the specific property of P2X7 receptor to open a pathway permeable to large molecules, associated with activation of inflammasome, we studied the action of cholinergic agents on this key event in CAP activation. Methods: Freshly isolated mouse peritoneal mast cells and primary human macrophages were used. To assess P2X7 channel opening, the permeability to the fluorescent dye YO-PRO1 or ethidium bromide (EtBr) was measured by flow cytometry. Expression of nicotinic receptors was probed in macrophages with the fluorescently labeled α-bungarotoxin or with patch-clamp recordings. Results: ATP opened P2X7 ion channels in mast cells and macrophages permeable to YO-PRO1 or EtBr, respectively. This stimulatory effect in mast cells was inhibited by the specific P2X7 antagonist A839977 confirming that YO-PRO1 uptake was mediated via ATP-gated P2X7 ion channels. Cholinergic agents also slightly induced dye uptake to mast cells but not in macrophages, which expressed functional α7 nicotinic receptors. However, both in mast cells and in macrophages, acetylcholine and nicotine failed to inhibit the stimulatory effect of ATP on dye uptake. Conclusion: These data suggest that in immune cells, cholinergic agents do not act on P2X7 receptor-coupled large pore formation but can mediate the anti-inflammatory effect underlying CAP downstream of ATP-driven signaling.

Two of the key functions of arteries in the brain are (1) the well‐recognized supply of blood via the vascular lumen and (2) the emerging role for the arterial walls as routes for the elimination of interstitial fluid (ISF) and soluble metabolites, such as amyloid beta (Aβ), from the brain and retina. As the brain and retina possess no conventional lymphatic vessels, fluid drainage toward peripheral lymph nodes is mediated via transport along basement membranes in the walls of capillaries and arteries that form the intramural peri‐arterial drainage (IPAD) system. IPAD tends to fail as arteries age but the mechanisms underlying the failure are unclear. In some people this is reflected in the accumulation of Aβ plaques in the brain in Alzheimer's disease (AD) and deposition of Aβ within artery walls as cerebral amyloid angiopathy (CAA). Knowledge of the dynamics of IPAD and why it fails with age is essential for establishing diagnostic tests for the early stages of the disease and for devising therapies that promote the clearance of Aβ in the prevention and treatment of AD and CAA. This editorial is intended to introduce the rationale that has led to the establishment of the Clearance of Interstitial Fluid (ISF) and CSF (CLIC) group, within the Vascular Professional Interest Area of the Alzheimer's Association International Society to Advance Alzheimer's Research and Treatment.

BackgroundA system of lymphatic vessels has been recently characterized in the meninges, with a postulated role in ‘cleaning’ the brain via cerebral fluid drainage. As meninges are the origin site of migraine pain, we hypothesized that malfunctioning of the lymphatic system should affect the local trigeminal nociception. To test this hypothesis, we studied nociceptive and inflammatory mechanisms in the meninges of K14-VEGFR3-Ig mice lacking the meningeal lymphatic system.MethodsWe recorded the spiking activity of meningeal afferents and estimated the local mast cells infiltration, calcitonin gene-related peptide (CGRP) and cytokine levels (basal and stimulated), as well as the dural trigeminal innervation in freshly-isolated hemiskull preparations from K14-VEGFR3-Ig (K14) or wild type C57BL/6 mice (WT).ResultsWe found that the meningeal level of CGRP and of the pro-inflammatory cytokines IL12-p70 and TNFα (implicated in migraine) were reduced in the meninges of K14 mice. On the contrary, in the meninges of K14 mice, we found an increased level of the mast cell activator MCP-1 and, consistently, a larger number of dural mast cells. The other migraine-related pro-inflammatory cytokines did not differ between the two genotypes. The patterns of trigeminal innervation in meninges remained unchanged and we did not observe alterations in basal or ATP-induced nociceptive firing in the meningeal afferents.ConclusionsIn summary, the lack of meningeal lymphatic system does not induce migraine-like nociceptive state per se, but leads to a new balance between pro- and antiinflammatory factors implicated in migraine mechanisms.

Neuroinflammation is strongly induced by cerebral ischemia. The early phase after the onset of ischemic stroke is characterized by acute neuronal injury, microglial activation, and subsequent infiltration of blood-derived inflammatory cells, including macrophages. Therefore, modulation of the microglial/macrophage responses has increasingly gained interest as a potential therapeutic approach for the ischemic stroke. In our study, we investigated the effects of peripherally administered interleukin 13 (IL-13) in a mouse model of permanent middle cerebral artery occlusion (pMCAo). Systemic administration of IL-13 immediately after the ischemic insult significantly reduced the lesion volume, alleviated the infiltration of CD45+ leukocytes, and promoted the microglia/macrophage alternative activation within the ischemic region, as determined by arginase 1 (Arg1) immunoreactivity at 3 days post-ischemia (dpi). Moreover, IL-13 enhanced the expression of M2a alternative activation markers Arg1 and Ym1 in the peri-ischemic (PI) area, as well as increased plasma IL-6 and IL-10 levels at 3 dpi. Furthermore, IL-13 treatment ameliorated gait disturbances at day 7 and 14 and sensorimotor deficits at day 14 post-ischemia, as analyzed by the CatWalk gait analysis system and adhesive removal test, respectively. Finally, IL-13 treatment decreased neuronal cell death in a coculture model of neuroinflammation with RAW 264.7 macrophages. Taken together, delivery of IL-13 enhances microglial/macrophage anti-inflammatory responses in vivo and in vitro, decreases ischemia-induced brain cell death, and improves sensory and motor functions in the pMCAo mouse model of cerebral ischemia. Electronic supplementary material The online version of this article (10.1007/s13311-019-00761-0) contains supplementary material, which is available to authorized users.

MicroRNAs (miRNAs) are small non-coding RNAs that have well-characterized roles in cytoplasmic gene regulation, where they act by binding to mRNA transcripts and inhibiting their translation (i.e. post-transcriptional gene silencing, PTGS). However, miRNAs have also been implicated in transcriptional gene regulation and alternative splicing, events that are restricted to the cell nucleus. Here we performed nuclear-cytoplasmic fractionation in a mouse endothelial cell line and characterized the localization of miRNAs in response to hypoxia using small RNA sequencing. A highly diverse population of abundant miRNA species was detected in the nucleus, of which the majority (56%) was found to be preferentially localized in one compartment or the other. Induction of hypoxia resulted in changes in miRNA levels in both nuclear and cytoplasmic compartments, with the majority of changes being restricted to one location and not the other. Notably, the classical hypoxamiR (miR-210-3p) was highly up-regulated in the nuclear compartment after hypoxic stimulus. These findings reveal a previously unappreciated level of molecular complexity in the physiological response occurring in ischemic tissue. Furthermore, widespread differential miRNA expression in the nucleus strongly suggests that these small RNAs are likely to perform extensive nuclear regulatory functions in the general case.

Background Blocking the pro-nociceptive action of CGRP is one of the most promising approaches for migraine prophylaxis. The aim of this study was to explore a role for CGRP as a neuroprotective agent for central and peripheral neurons. Methods The viability of isolated rat trigeminal, cortical and cerebellar neurons was tested by fluorescence vital assay. Engagement of Nrf2 target genes was analyzed by qPCR. The neuroprotective efficacy of CGRP in vivo was tested in mice using a permanent cerebral ischemia model. Results CGRP prevented apoptosis induced by the amino acid homocysteine in all three distinct neuronal populations. Using a set of specific kinase inhibitors, we show the role of multi-kinase signaling pathways involving PKA and CaMKII in neuronal survival. Forskolin triggered a very similar signaling cascade, suggesting that cAMP is the main upstream trigger for multi-kinase neuroprotection. The specific CGRP antagonist BIBN4096 reduced cellular viability, lending further support to the proposed neuroprotective function of CGRP. Importantly, CGRP was neuroprotective against permanent ischemia in mice. Conclusion Our data show an unexpected ‘positive’ role for the endogenous pro-nociceptive migraine mediator CGRP, suggesting more careful examination of migraine prophylaxis strategy based on CGRP antagonism although it should be noted that homocysteine induced apoptosis in primary neuronal cell culture might not necessarily reproduce all the features of cell loss in the living organism.

Given the importance of microglia to inflammatory, phagocytic and synaptic modulatory processes, their function is vital in physiological and pathological brain. The impairment of microglia in Alzheimer's disease has been demonstrated on genetic, epigenetic, transcriptional and functional levels using unbiased systems level approaches. Recent studies have highlighted the immense phenotypic diversity of microglia, including the ability to adopt distinct and dynamic phenotypes in ageing and disease. We review the origins and functions of healthy microglia and the established and emerging models and techniques available for their study. Furthermore, we highlight recent advances on the role, heterogeneity and dysfunction of microglia in Alzheimer's disease and discuss the potential for therapeutic interventions targeting microglia. Microglia-selective molecular fingerprints will guide detailed functional analysis of microglial subsets and may aid in the development of therapies specifically targeting microglia.

ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs type 4) is a metalloprotease capable to degrade chondroitin sulfate proteoglycans leading to cartilage destruction during arthritis or to neuroplasticity during spinal cord injury (SCI). Although ADAMTS-4 is an inflammatory-regulated enzyme, its role during inflammation has never been investigated. The aim of this study was to investigate the role of ADAMTS-4 in neuroinflammation. First, we evidenced an increase of ADAMTS-4 expression in the ischemic brain hemisphere of mouse and human patients suffering from ischemic stroke. Then, we described that ADAMTS-4 has predominantly an anti-inflammatory effect in the CNS. Treatment of primary microglia or astrocyte cultures with low doses of a human recombinant ADAMTS-4 prior to LPS exposure decreased NO production and the synthesis/release of pro-inflammatory cytokines including NOS2, CCL2, TNF-α, IL-1β and MMP-9. Accordingly, when cell cultures were transfected with silencing siRNA targeting ADAMTS-4 prior to LPS exposure, the production of NO and the synthesis/release of pro-inflammatory cytokines were increased. Finally, the feasibility of ADAMTS-4 to modulate neuroinflammation was investigated in vivo after permanent middle cerebral artery occlusion in mice. Although ADAMTS-4 treatment did not influence the lesion volume, it decreased astrogliosis and macrophage infiltration, and increased the number of microglia expressing arginase-1, a marker of alternatively activated cells with inflammation inhibiting functions. Additionally, ADAMTS-4 increased the production of IL-10 and IL-6 in the peri-ischemic area. By having anti-inflammatory and neuroregenerative roles, ADAMTS-4 may represent an interesting target to treat acute CNS injuries, such as ischemic stroke, SCI or traumatic brain injury. GLIA 2016;64:1492-1507.

This project was supported by the Finnish Academy (grant 277442). AR and RG were supported by the Program of Competitive Growth of Kazan Federal University and the subsidy (6.2313.2017/4.6) allocated to Kazan Federal University for the state assignment in the sphere of scientific activities. TM was supported by Academy of Finland (grant 298071). DN and VG were supported by the EDUFI program., Extracellular ATP activates inflammasome and triggers the release of multiple cytokines in various immune cells, a process primarily mediated by P2X7 receptors. However, the expression and functional properties of P2X7 receptors in native mast cells in tissues such as meninges where migraine pain originates from have not been explored. Here we report a novel model of murine cultured meningeal mast cells and using these, as well as easily accessible peritoneal mast cells, studied the mechanisms of ATP-mediated mast cell activation. We show that ATP induced a time and dose-dependent activation of peritoneal mast cells as analyzed by the uptake of organic dye YO-PRO1 as well as 4,6-diamidino-2-phenylindole (DAPI). Both YO-PRO1 and DAPI uptake in mast cells was mediated by the P2X7 subtype of ATP receptors as demonstrated by the inhibitory effect of P2X7 antagonist A839977. Consistent with this, significant YO-PRO1 uptake was promoted by the P2X7 agonist 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP). Extracellular ATP-induced degranulation of native and cultured meningeal mast cells was shown with Toluidine Blue staining. Taken together, these data demonstrate the important contribution of P2X7 receptors to ATP-driven activation of mast cells, suggesting these purinergic mechanisms as potential triggers of neuroinflammation and pain sensitization in migraine., Finnish Academy [277442]; Program of Competitive Growth of Kazan Federal University; Academy of Finland [298071]; EDUFI program; [6.2313.2017/4.6]

The Finnish-variant late infantile neuronal ceroid lipofuscinosis, also known as CLN5 disease, is caused by mutations in the CLN5 gene. Cln5 is strongly expressed in the developing brain and expression continues into adulthood. CLN5, a protein of unknown function, is implicated in neurodevelopment but detailed investigation is lacking. Using Cln5−/− embryos of various ages and cells harvested from Cln5−/− brains we investigated the hitherto unknown role of Cln5 in the developing brain. Loss of Cln5 results in neuronal differentiation deficits and delays in interneuron development during in utero period. Specifically, the radial thickness of dorsal telencephalon was significantly decreased in Cln5−/− mouse embryos at embryonic day 14.5 (E14.5), and expression of Tuj1, an important neuronal marker during development, was down-regulated. An interneuron marker calbindin and a mitosis marker p-H3 showed down-regulation in ganglionic eminences. Neurite outgrowth was compromised in primary cortical neuronal cultures derived from E16 Cln5−/− embryos compared with WT embryos. We show that the developmental deficits of interneurons may be linked to increased levels of the repressor element 1-silencing transcription factor, which we report to bind to glutamate decarboxylase (Gad1), which encodes GAD67, a rate-limiting enzyme in the production of gamma-aminobutyric acid (GABA). Indeed, adult Cln5−/− mice presented deficits in hippocampal parvalbumin-positive interneurons. Furthermore, adult Cln5−/− mice presented deficits in hippocampal parvalbumin-positive interneurons and showed age-independent cortical hyper excitability as measured by electroencephalogram and auditory-evoked potentials. This study highlights the importance of Cln5 in neurodevelopment and suggests that in contrast to earlier reports, CLN5 disease is likely to develop during embryonic stages.

Astrocytes are the gatekeepers of neuronal energy supply. In neurodegenerative diseases, bio-energetics demand increases and becomes reliant upon fatty acid oxidation as a source of energy. Defective fatty acid oxidation and mitochondrial dysfunctions correlate with hippocampal neurodegeneration and memory deficits in Alzheimer’s disease (AD), but it is unclear whether energy metabolism can be targeted to prevent or treat the disease. Here we show for the first time an impairment in fatty acid oxidation in human astrocytes derived from induced pluripotent stem cells of AD patients. The impairment was corrected by treatment with a synthetic peroxisome proliferator activated receptor delta (PPARβ/δ) agonist GW0742 which acts to regulate an array of genes governing cellular metabolism. GW0742 enhanced the expression of CPT1a, the gene encoding for a rate-limiting enzyme of fatty acid oxidation. Similarly, treatment of a mouse model of AD, the APP/PS1-mice, with GW0742 increased the expression of Cpt1a and concomitantly reversed memory deficits in a fear conditioning test. Although the GW0742-treated mice did not show altered astrocytic glial fibrillary acidic protein-immunoreactivity or reduction in amyloid beta (Aβ) load, GW0742 treatment increased hippo-campal neurogenesis and enhanced neuronal differentiation of neuronal progenitor cells. Furthermore, GW0742 prevented Aβ-induced impairment of long-term potentiation in hippocampal slices. Collectively, these data suggest that PPARβ/δ-agonism alleviates AD related deficits through increasing fatty acid oxidation in astrocytes and improves cognition in a transgenic mouse model of AD.

Background: Neuroinflammation and biometal dyshomeostasis are key pathological features of several neurodegenerative diseases, including Alzheimer's disease (AD). Inflammation and biometals are linked at the molecular level through regulation of metal buffering proteins such as the metallothioneins. Even though the molecular connections between metals and inflammation have been demonstrated, little information exists on the effect of copper modulation on brain inflammation. Methods: We demonstrate the immunomodulatory potential of the copper bis(thiosemicarbazone) complex Cu-II(atsm) in an neuroinflammatory model in vivo and describe its anti-inflammatory effects on microglia and astrocytes in vitro. Results: By using a sophisticated in vivo magnetic resonance imaging (MRI) approach, we report the efficacy of Cu-II(atsm) in reducing acute cerebrovascular inflammation caused by peripheral administration of bacterial lipopolysaccharide (LPS). Cu-II(atsm) also induced anti-inflammatory outcomes in primary microglia [significant reductions in nitric oxide (NO), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor (TNF)] and astrocytes [significantly reduced NO, MCP-1, and interleukin 6 (IL-6)] in vitro. These anti-inflammatory actions were associated with increased cellular copper levels and increased the neuroprotective protein metallothionein-1 (MT1) in microglia and astrocytes. Conclusion: The beneficial effects of Cu-II(atsm) on the neuroimmune system suggest copper complexes are potential therapeutics for the treatment of neuroinflammatory conditions.

Ischemic stroke is amongst the leading causes of death and disabilities. The available treatments are suitable for only a fraction of patients and thus novel therapies are urgently needed. Blockage of one of the cerebral arteries leads to massive and persisting inflammatory reaction contributing to the nearby neuronal damage. Targeting the detrimental pathways of neuroinflammation has been suggested to be beneficial in conditions of ischemic stroke. Nuclear receptor 4A-family (NR4A) member Nurr1 has been shown to be a potent modulator of harmful inflammatory reactions, yet the role of Nurr1 in cerebral stroke remains unknown. Here we show for the first time that an agonist for the dimeric transcription factor Nurr1/retinoid X receptor (RXR), HX600, reduces microglia expressed proinflammatory mediators and prevents inflammation induced neuronal death in in vitro co-culture model of neurons and microglia. Importantly, HX600 was protective in a mouse model of permanent middle cerebral artery occlusion and alleviated the stroke induced motor deficits. Along with the anti-inflammatory capacity of HX600 in vitro, treatment of ischemic mice with HX600 reduced ischemia induced Iba-1, p38 and TREM2 immunoreactivities, protected endogenous microglia from ischemia induced death and prevented leukocyte infiltration. These anti-inflammatory functions were associated with reduced levels of brain lysophosphatidylcholines (lysoPCs) and acylcarnitines, metabolites related to proinflammatory events. These data demonstrate that HX600 driven Nurr1 activation is beneficial in ischemic stroke and propose that targeting Nurr1 is a novel candidate for conditions involving neuroinflammatory component.

Exosomes are small extracellular vesicles that modulate important functions in physiology and under pathological conditions of the central nervous system (CNS). Exosomal contents, proteins, lipids and various RNA species, are altered during disease. The fact that exosomes are released into the blood stream from blood cells and endothelial cells responding to CNS diseases as well as from the brain and spinal cord, and that they express markers which allow their tracking to the cell of origin, makes the use of exosomes for diagnostic purposes and biomarker discovery particularly appealing. While the utilization of exosomes for diagnostics in diseases affecting the CNS are still in the early stages of discovery and development, it is expected that through further research and fervent development of protocols relating to isolation and purification the true potential of exosomes derived from the CNS will be harnessed for more effective clinical disease diagnosis. In this review we begin with a short introduction to the origin, composition and function of exosomes in the CNS. Next we discuss the current status of methodologies related to isolation and detection of CNS exosomes. We end with an account of exosomes in diagnostics and biomarker discovery, which focuses on three diseases of the CNS: Alzheimer's disease, multiple sclerosis, and stroke. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

Cerebral stroke induces massive Th1-shifted inflammation both in the brain and the periphery, contributing to the outcome of stroke. A Th1-type response is neurotoxic whereas a Th2-type response is accompanied by secretion of anti-inflammatory cytokines, such as interleukin-4 (IL-4). Interleukin-33 (IL-33) is a cytokine known to induce a shift towards the Th2-type immune response, polarize macrophages/microglia towards the M2-type, and induce production of anti-inflammatory cytokines. We found that the plasma levels of the inhibitory IL-33 receptor, sST2, are increased in human stroke and correlate with a worsened stroke outcome, suggesting an insufficient IL-33-driven Th2-type response. In mouse, peripheral administration of IL-33 reduced stroke-induced cell death and improved the sensitivity of the contralateral front paw at 5days post injury. The IL-33-treated mice had increased levels of IL-4 in the spleen and in the peri-ischemic area of the cortex. Neutralization of IL-4 by administration of an IL-4 antibody partially prevented the IL-33-mediated protection. IL-33 treatment also reduced astrocytic activation in the peri-ischemic area and increased the number of Arginase-1 immunopositive microglia/macrophages at the lesion site. In human T-cells, IL-33 treatment induced IL-4 secretion, and the conditioned media from IL-33-exposed T-cells reduced astrocytic activation. This study demonstrates that IL-33 is protective against ischemic insult by induction of IL-4 secretion and may represent a novel therapeutic approach for the treatment of stroke.

Interleukin-33 (IL-33) is a member of the interleukin-1 cytokine family and highly expressed in the naïve mouse brain and spinal cord. Despite the fact that IL-33 is known to be inducible by various inflammatory stimuli, its cellular localization in the central nervous system and role in pathological conditions is controversial. Administration of recombinant IL-33 has been shown to attenuate experimental autoimmune encephalomyelitis progression in one study, yet contradictory reports also exist. Here we investigated for the first time the pattern of IL-33 expression in the contused mouse spinal cord and demonstrated that after spinal cord injury (SCI) IL-33 was up-regulated and exhibited a nuclear localization predominantly in astrocytes. Importantly, we found that treatment with recombinant IL-33 alleviated secondary damage by significantly decreasing tissue loss, demyelination and astrogliosis in the contused mouse spinal cord, resulting in dramatically improved functional recovery. We identified both central and peripheral mechanisms of IL-33 action. In spinal cord, IL-33 treatment reduced the expression of pro-inflammatory tumor necrosis factor-alpha and promoted the activation of anti-inflammatory arginase-1 positive M2 microglia/macrophages, which chronically persisted in the injured spinal cord for up to at least 42 days after the treatment. In addition, IL-33 treatment showed a tendency towards reduced T-cell infiltration into the spinal cord. In the periphery, IL-33 treatment induced a shift towards the Th2 type cytokine profile and reduced the percentage and absolute number of cytotoxic, tumor necrosis factor-alpha expressing CD4+ cells in the spleen. Additionally, IL-33 treatment increased expression of T-regulatory cell marker FoxP3 and reduced expression of M1 marker iNOS in the spleen. Taken together, these results provide the first evidence that IL-33 administration is beneficial after CNS trauma. Treatment with IL33 may offer a novel therapeutic strategy for patients with acute contusion SCI.

Alzheimer’s disease (AD) may disturb functions of the blood-brain barrier and change the disposition of drugs to the brain. This study assessed the disease-induced changes in drug transporters in the brain capillaries of transgenic AD mice. Eighteen drug transporters and four tight junction-associated proteins were analyzed by RT-qPCR in cortex, hippocampus and cerebellum tissue samples of 12–16-month-old APdE9, Tg2576 and APP/PS1 transgenic mice and their healthy age-matched controls. In addition, microvessel fractions enriched from 1-3-month-old APdE9 mice were analyzed using RT-qPCR and Western blotting. Brain transport of methotrexate in APdE9 mice was assessed by in vivo microdialysis. The expression profiles of studied genes were similar in brain tissues of AD and control mice. Instead, in the microvessel fraction in APdE9 mice, >2-fold alterations were detected in the expressions of 11 genes but none at the protein level. In control mice strains, >5-fold changes between different brain regions were identified for Slc15a2, Slc22a3 and occludin. Methotrexate distribution into hippocampus of APdE9 mice was faster than in controls. The expression profile of mice carrying presenilin and amyloid precursor protein mutations is comparable to controls, but clear regional differences exist in the expression of drug transporters in brain.

Inflammation is a major mechanism of acute brain injury and chronic neurodegeneration. This neuroinflammation is known to be substantially regulated by the transcription factor NF-κB, which is predominantly found in the form of heterodimer of p65 (RelA) and p50 subunit, with p50/p50 homodimers being also common. The p65 subunit has a transactivation domain, whereas p50 is chiefly involved in DNA binding. Binding of the p65/p50 heterodimers is thought to induce expression of numerous proinflammatory genes in microglia. Here we show that cultured microglia deficient for the gene (Nfkb1) encoding p50 subunit show reduced induction of proinflammatory mediators, increased expression of anti-inflammatory genes, and increased expression of CD45, an immunoregulatory molecule, in response to lipopolysaccharide (LPS) exposure, but increased capacity to take up β-amyloid (Aβ) which is associated with enhanced release of tumor necrosis factor alpha (TNFα). However, Nfkb1 deficiency strongly increases leukocyte infiltration and the expression of proinflammatory genes in response to intrahippocampal administration of LPS. Also, when crossing Nfkb1 deficient mice with APdE9 transgenic mice the expression of proinflammatory genes was strongly enhanced, whereas Aβ burden was slightly but significantly reduced. These alterations in expression of inflammatory mediators in Nfkb1 deficient mice were associated with reduced expression of CD45. Our data demonstrates a crucial and complex role p50 subunit of NF-κB in brain inflammation, especially in regulating the phenotype of microglia after acute and chronic inflammatory insults relevant to clinical conditions, contributing to both pro-inflammatory and anti-inflammatory responses of microglia, infiltration of leukocytes, and clearance of Aβ in Alzheimer's disease.

Developing new therapies for stroke is urgently needed, as this disease is the leading cause of death and disability worldwide, and the existing treatment is only available for a small subset of patients. The interruption of blood flow to the brain during ischemic stroke launches multiple immune responses, characterized by infiltration of peripheral immune cells, the activation of brain microglial cells, and the accumulation of immune mediators. Copper is an essential trace element that is required for many critical processes in the brain. Copper homeostasis is disturbed in chronic neurodegenerative diseases and altered in stroke patients, and targeted copper delivery has been shown to be protective against chronic neurodegeneration. This study was undertaken to assess whether the copper bis(thiosemicarbazone) complex, CuII(atsm), is beneficial in acute brain injury, in preclinical mouse models of ischemic stroke. We demonstrate that the copper complex CuII(atsm) protects neurons from excitotoxicity and N2a cells from OGD in vitro, and is protective in permanent and transient ischemia models in mice as measured by functional outcome and lesion size. Copper delivery in the ischemic brains modulates the inflammatory response, specifically affecting the myeloid cells. It reduces CD45 and Iba1 immunoreactivity, and alters the morphology of Iba1 positive cells in the ischemic brain. CuII(atsm) also protects endogenous microglia against ischemic insult and reduces the proportion of invading monocytes. These results demonstrate that the copper complex CuII(atsm) is an inflammation-modulating compound with high therapeutic potential in stroke and is a strong candidate for the development of therapies for acute brain injury.

Alzheimer’s disease (AD) is characterized by extensive neuron loss that accompanies profound impairments in memory and cognition. We examined the neuronally directed effects of the retinoid X receptor agonist bexarotene in an aggressive model of AD. We report that a two week treatment of 3.5 month old 5XFAD mice with bexarotene resulted in the clearance of intraneuronal amyloid deposits. Importantly, neuronal loss was attenuated by 44% in the subiculum in mice 4 months of age and 18% in layer V of the cortex in mice 8 months of age. Moreover, bexarotene treatment improved remote memory stabilization in fear conditioned mice and improved olfactory cross habituation. These improvements in neuron viability and function were correlated with significant increases in the levels of post-synaptic marker PSD95 and the pre-synaptic marker synaptophysin. Moreover, bexarotene pretreatment improved neuron survival in primary 5XFAD neurons in vitro in response to glutamate-induced excitotoxicity. The salutary effects of bexarotene were accompanied by reduced plaque burden, decreased astrogliosis, and suppression of inflammatory gene expression. Collectively, these data provide evidence that bexarotene treatment reduced neuron loss, elevated levels of markers of synaptic integrity that was linked to improved cognition and in an aggressive model of AD., published version, peerReviewed

The Finnish variant of late infantile neuronal ceroid lipofuscinosis (CLN5 disease) belongs to a family of neuronal ceroid lipofuscinosis (NCLs) diseases. Vision loss is among the first clinical signs in childhood forms of NCLs. Mutations in CLN5 underlie CLN5 disease. The aim of this study was to characterize how the lack of normal functionality of the CLN5 protein affects the mouse retina. Scotopic electroretinography (ERG) showed a diminished c-wave amplitude in the CLN5 deficient mice already at 1 month of age, indicative of pathological events in the retinal pigmented epithelium. A- and b-waves showed progressive impairment later from 2 and 3 months of age onwards, respectively. Structural and immunohistochemical (IHC) analyses showed preferential damage of photoreceptors, accumulation of autofluorescent storage material, apoptosis of photoreceptors, and strong inflammation in the CLN5 deficient mice retinas. Increased levels of autophagy-associated proteins Beclin-1 and P62, and increased LC3b-II/LC3b-I ratio, were detected by Western blotting from whole retinal extracts. Photopic ERG, visual evoked potentials, IHC and cell counting indicated relatively long surviving cone photoreceptors compared to rods. In conclusion, CLN5 deficient mice develop early vision loss that reflects the condition reported in clinical childhood forms of NCLs. The vision loss in CLN5 deficient mice is primarily caused by photoreceptor degeneration., published version, peerReviewed

Proper lysosomal function is essential for cellular metabolism and health. Lysosomal storage diseases are a group of inherited disorders characterized by abnormal accumulation of proteins and lipids into lysosomes. One striking similarity in the pathogenesis of these diseases is anomalies in biometal ion homeostasis. Biometals, such as copper, iron, and zinc are needed for proper lysosomal function. On the other hand, accumulation of biometals in lysosomes leads to pathological events. Thus, understanding the events leading to alterations in the availability and distribution of biometals and thereby lysosomal deficits may provide the basis for halting degeneration associated with lysosomal storage disorders. This review summarizes the current knowledge on the pathophysiology of three lysosomal storage disorders that all share a neurodegenerative component, with a focus on the homeostasis of biometals. Modulation of biometal homeostasis is a promising therapeutic strategy to combat lysosomal deficits and neurodegeneration in conditions of biometal imbalance.

Background: Accumulation of amyloid β (Aβ) is one of the main hallmarks of Alzheimer’s disease (AD). The enhancement of Aβ clearance may provide therapeutic means to restrict AD pathology. The cellular responses to different forms of Aβ in monocytic cells are poorly known. We aimed to study whether different forms of Aβ induce inflammatory responses in monocytic phagocytes and how Aβ may affect monocytic cell survival and function to retain phagocytosis in Aβ-laden environment. Methods: Monocytic cells were differentiated from bone marrow hematopoietic stem cells (HSC) in the presence of macrophage-colony stimulating factor. Monocytic cells were stimulated with synthetic Aβ42 and intracellular calcium responses were recorded with calcium imaging. The formation of reactive oxygen species (ROS), secretion of cytokines and cell viability were also assessed. Finally, monocytic cells were introduced to native Aβ deposits ex vivo and the cellular responses in terms of cell viability, pro-inflammatory activation and phagocytosis were determined. The ability of monocytic cells to phagocytose Aβ plaques was determined after intrahippocampal transplantation in vivo. Results: Freshly solubilized Aβ induced calcium oscillations, which persisted after removal of the stimulus. After few hours of aggregation, Aβ was not able to induce oscillations in monocytic cells. Instead, lipopolysaccharide (LPS) induced calcium responses divergent from Aβ-induced response. Furthermore, while LPS induced massive production of pro-inflammatory cytokines, neither synthetic Aβ species nor native Aβ deposits were able to induce pro-inflammatory activation of monocytic cells, contrary to primary microglia. Finally, monocytic cells retained their viability in the presence of Aβ and exhibited phagocytic activity towards native fibrillar Aβ deposits and congophilic Aβ plaques. Conclusion: Monocytic cells carry diverse cellular responses to Aβ and inflammatory stimulus LPS. Even though Aβ species cause specific responses in calcium signaling, they completely lack the ability to induce pro-inflammatory phenotype of monocytic cells. Monocytes retain their viability and function in Aβ-laden brain.

Chronic psychosocial stress is increasingly recognized as a risk factor for late-onset Alzheimer's disease (LOAD) and associated cognitive deficits. Chronic stress also primes microglia and induces inflammatory responses in the adult brain, thereby compromising synapse-supportive roles of microglia and deteriorating cognitive functions during aging. Substantial evidence demonstrates that failure of microglia to clear abnormally accumulating amyloid-beta (Aβ) peptide contributes to neuroinflammation and neurodegeneration in AD. Moreover, genome-wide association studies have linked variants in several immune genes, such as TREM2 and CD33, the expression of which in the brain is restricted to microglia, with cognitive dysfunctions in LOAD. Thus, inflammation-promoting chronic stress may create a vicious cycle of aggravated microglial dysfunction accompanied by increased Aβ accumulation, collectively exacerbating neurodegeneration. Surprisingly, however, little is known about whether and how chronic stress contributes to microglia-mediated neuroinflammation that may underlie cognitive impairments in AD. This review aims to summarize the currently available clinical and preclinical data and outline potential molecular mechanisms linking stress, microglia and neurodegeneration, to foster future research in this field.

Protein disulfide isomerase (PDI) is an oxidoreductase assisting oxidative protein folding in the endoplasmic reticulum of all types of cells, including neurons and glia. In neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), up-regulation of PDI is an important part of unfolded protein response (UPR) that is thought to represent an adaption reaction and thereby protect the neurons. Importantly, studies on animal models of familial ALS with mutant Cu/Zn superoxide dismutase 1 (SOD1) have shown that the mutant SOD1 in astrocytes or microglia strongly regulates the progression of the disease. Here, we found an early up-regulation of PDI in microglia of transgenic (tg) mutant SOD1 mice, indicating that in addition to neurons, UPR takes place in glial cells in ALS. The observation was supported by the finding that also the expression of a UPR marker GADD34 (growth arrest and DNA damage-inducible protein) was induced in the spinal cord glia of tg mutant SOD1 mice. Because mutant SOD1 can cause sustained activation of NADPH oxidase (NOX), we investigated the role of PDI in UPR-induced NOX activation in microglia. In BV-2 microglia, UPR resulted in NOX activation with increased production of superoxide and increased release of tumor necrosis factor-α. The phenomenon was recapitulated in primary rat microglia, murine macrophages and human monocytes. Importantly, pharmacological inhibition of PDI or its down-regulation by short interfering RNAs prevented NOX activation in microglia and subsequent production of superoxide. Thus, results strongly demonstrate that UPR, caused by protein misfolding, may lead to PDI-dependent NOX activation and contribute to neurotoxicity in neurodegenerative diseases including ALS.

Glial cells in Alzheimer’s disease (AD) have been shown to be capable of clearing or at least restricting the accumulation of toxic amyloid beta (Aβ) deposits. Recently, bone marrow (BM)–derived monocytic cells have been recognized in experimental studies to be superior in their phagocytic properties when compared to their brain endogenous counterparts. In human AD, BM-derived monocytic cells may have deficiencies in their capacity to restrict plaque growth. Therefore, enhancement of phagocytic properties of cells of monocyte origin, both brain endogenous microglia and BM-derived monocytic cells, offers an attractive therapeutic approach to fight off AD. Transgenic mouse models with aberrant Aβ deposition offer a valuable tool for discovery of novel pathways to facilitate cell-mediated Aβ uptake. This article reviews the most recent findings on the phagocytic capacity of cells with monocytic origin in various transgenic AD models and describes the methods to study phagocytic activity of these cells.

Proliferation and activation of microglial cells is a neuropathological characteristic of brain injury and neurodegeneration, including Alzheimer's disease. Microglia act as the first and main form of immune defense in the nervous system. While the primary function of microglia is to survey and maintain the cellular environment optimal for neurons in the brain parenchyma by actively scavenging the brain for damaged brain cells and foreign proteins or particles, sustained activation of microglia may result in high production of proinflammatory mediators that disturb normal brain functions and even cause neuronal injury. Glycogen synthase kinase-3βhas been recently identified as a major regulator of immune system and mediates inflammatory responses in microglia. Glycogen synthase kinase-3βhas been extensively investigated in connection to tau and amyloidβtoxicity, whereas reports on the role of this enzyme in neuroinflammation in Alzheimer's disease are negligible. Here we review and discuss the role of glycogen synthase-3βin immune cells in the context of Alzheimer's disease pathology.

Bone marrow (BM)-derived monocytes contribute to the development of microglial reaction around beta-amyloid (Abeta) plaques in Alzheimer's disease (AD) and possibly clear Abeta. Therefore, it is of great importance to separate the proinflammatory actions of monocytic cells from Abeta phagocytic effects. We used minocycline (mino) to systemically downregulate microglial activation and studied proliferation, expression of markers for activated microglia, and Abeta removal in vitro and in vivo. Mino did not affect proliferation or phagocytic activity of BM-derived cells toward Abeta in vitro. Intrahippocampal LPS injection used to induce inflammation and increase recruitment of BM cells from periphery, reduced Abeta burden in BM-transplanted AD transgenic mice. All engrafted cells expressed CD45, approximately 50% expressed Iba-1, and

We assessed baseline and KCl-stimulated glutamate release by using microdialysis in freely moving young adult (7 months) and middle-aged (17 months) transgenic mice carrying mutated human amyloid precursor protein and presenilin genes (APdE9 mice) and their wild-type littermates. In addition, we assessed the age-related development of amyloid pathology and spatial memory impaired in the water maze and changes in glutamate transporters. APdE9 mice showed gradual spatial memory impairment between 6 and 15 months of age. The stimulated glutamate release declined very robustly in 17-month-old APdE9 mice as compared to 7-month-old APdE9 mice. This age-dependent decrease in stimulated glutamate release was also evident in wild-type mice, although it was not as robust as in APdE9 mice. When compared to individual baselines, all aged wild-type mice showed 25% or greater increase in glutamate release upon KCl stimulation, but none of the aged APdE9 mice. There was an age-dependent decline in VGLUT1 levels, but not in the levels of VGLUT2, GLT-1 or synaptophysin. Astrocyte activation as measured by glial acidic fibrillary protein was increased in middle-aged APdE9 mice. Blunted pre-synaptic glutamate response may contribute to memory deficit in middle-aged APdE9 mice.

Exosomes, nano-sized extracellular vesicles secreted by most cell types, are found everywhere in the body. The role of exosomes in cellular functions has in the past years developed from being considered little more than cellular trashcans, to being proven important intercellular messengers and notable contributors to both health and in disease. A vast number of studies have revealed the multiple, and somewhat controversial role of exosomes in Alzheimer's disease, the most common neurodegenerative disease. Exosomes have been shown to spread toxic amyloid-beta and hyperphosphorylated tau between cells, and they have been suspected of inducing apoptosis and thereby contributing to neuronal loss. On the other hand, exosomes seem to possess the ability to reduce brain amyloid-beta through microglial uptake, and they are known to transfer neuroprotective substances between cells. These features, among many others, make exosomes extremely interesting from the point of view of developing novel therapeutic approaches. The fact that exosomes derived from the central nervous system can be found in bodily fluids also makes them an appealing target for biomarker development, which is not limited only to Alzheimer's disease.

Improved functional recovery after spinal cord injury by transplantation of induced pluripotent stem cell-derived neural stem/progenitor cells (iPSC-NPCs) has been reported. However, beneficial effects of iPSC-based therapy have so far been produced mostly using genetically immunodeficient rodents. Because of the long time required for generation and characterization of iPSCs, the use of autologous iPSCs for treating patients with acute spinal cord injury (SCI) is not feasible. Therefore, it is of utmost importance to investigate the effect of iPSC-based therapy on functional recovery after SCI using pharmacologically immunosuppressed, immunocompetent animal models. Here we studied the functional outcome following subacute transplantation of human iPSC-derived NPCs into contused mouse spinal cord when tacrolimus was used as an immunosuppressive agent. We show that human iPSC-derived NPCs transplanted into pharmacologically immunosuppressed C57BL/6J mice exhibited poor long-term survival and failed to improve functional recovery after SCI as measured by Basso Mouse Scale (BMS) for locomotion and CatWalk gait analysis when compared to vehicle-treated animals. The scarce effect of iPSC-based therapy observed in the current study may be attributable to insufficient immunosuppressive effect, provided by monotherapy with tacrolimus in combination with immunogenicity of transplanted cells and complex microenvironment of the injured spinal cord. Our results highlight the importance of extensive preclinical studies of transplanted cells before the clinical application of iPSC-based cell therapy is achieved.

Oxidative stress has been implicated to play a role in a number of acute and chronic diseases including acute injuries of the central nervous system, neurodegenerative and cardiovascular diseases, and cancer. The redox-activated transcription factor Nrf2 has been shown to protect many different cell types and organs from a variety of toxic insults, whereas in many cancers, unchecked Nrf2 activity increases the expression of cytoprotective genes and, consequently, provides growth advantage to cancerous cells. Herein, we discuss current preclinical gene therapy approaches to either increase or decrease Nrf2 activity with a special reference to neurological diseases and cancer. In addition, we discuss the role of Nrf2 in stem cell therapy for neurological disorders.

Alzheimer's disease (AD) is characterized by a robust inflammatory response elicited by the accumulation and subsequent deposition of amyloid (Aβ) within the brain. The brain's immune cells migrate to and invest their processes within Aβ plaques but are unable to efficiently phagocytose and clear plaques from the brain. Previous studies have shown that treatment of myeloid cells with nuclear receptor agonists increases expression of phagocytosis-related genes. In this study, we elucidate a novel mechanism by which nuclear receptors act to enhance phagocytosis in the AD brain. Treatment of murine models of AD with agonists of the nuclear receptors PPARγ, PPARδ, LXR, and RXR stimulated microglial phagocytosisin vitroand rapidly induced the expression of the phagocytic receptors Axl and MerTK. In murine models of AD, we found that plaque-associated macrophages expressed Axl and MerTK and treatment of the cells with an RXR agonist further induced their expression, coincident with the rapid reduction in plaque burden. Further characterization of MerTK+/Axl+macrophages revealed that they also expressed the phagocytic receptor TREM2 and high levels of CD45, consistent with a peripheral origin of these cells. Importantly, in anex vivoslice assay, nuclear receptor agonist treatment reversed the AD-related suppression of phagocytosis through a MerTK-dependent mechanism. Thus, nuclear receptor agonists increase MerTK and Axl expression on plaque-associated immune cells, consequently licensing their phagocytic activity and promoting plaque clearance.

Alzheimer's disease (AD) is typified by a robust microglial-mediated inflammatory response within the brain. Indeed, microglial accumulation around plaques in AD is one of the classical hallmarks of the disease pathology. Although microglia have the capacity to remove β-amyloid deposits and alleviate disease pathology, they fail to do so. Instead, they become chronically activated and promote inflammation-mediated impairment of cognition and cytotoxicity. However, if microglial function could be altered to engage their phagocytic response, promote their tissue maintenance functions, and prevent release of factors that promote tissue damage, this could provide therapeutic benefit. This review is focused on the current knowledge of microglial homeostatic mechanisms in AD, and mechanisms involved in the regulation of microglial phenotype in this context.

Background Human intravenous immunoglobulin (hIVIG) preparation is indicated for treating primary immunodeficiency disorders associated with impaired humoral immunity. hIVIG is known for its anti-inflammatory properties and a decent safety profile. Therefore, by virtue of its constituent natural anti-amyloid beta antibodies and anti-inflammatory effects, hIVIG is deemed to mediate beneficial effects to patients of Alzheimer’s disease (AD). Here, we set out to explore the effects of hIVIG in a mouse model of AD. Methods We treated APP/PS1dE9 transgenic and wild-type mice with weekly injections of a high hIVIG dose (1 g/kg) or saline for 3 or 8 months. Treatment effect on brain amyloid pathology and microglial reactivity was assessed by ELISA, immunohistochemistry, RT-PCR, and confocal microscopy. Results We found no evidence for reduction in Aβ pathology; instead 8 months of hIVIG treatment significantly increased soluble levels of Aβ40 and Aβ42. In addition, we noticed a significant reduction in CD45 and elevation of Iba-1 markers in specific sub-populations of microglial cells. Long-term hIVIG treatment also resulted in significant suppression of TNF-α and increase in doublecortin positive adult-born neurons in the dentate gyrus. Conclusions Our data indicate limited ability of hIVIG to impact amyloid burden but shows changes in microglia, pro-inflammatory gene expression, and neurogenic effects. Immunomodulation by hIVIG may account for its beneficial effect in AD patients.

Accumulation of amyloid β (Aβ) is a major hallmark in Alzheimer’s disease (AD). Bone marrow derived monocytic cells (BMM) have been shown to reduce Aβ burden in mouse models of AD, alleviating the AD pathology. BMM have been shown to be more efficient phagocytes in AD than the endogenous brain microglia. Because BMM have a natural tendency to infiltrate into the injured area, they could be regarded as optimal candidates for cell-based therapy in AD. In this study, we describe a method to obtain monocytic cells from BM-derived haematopoietic stem cells (HSC). Mouse or human HSC were isolated and differentiated in the presence of macrophage colony stimulating factor (MCSF). The cells were characterized by assessing the expression profile of monocyte markers and cytokine response to inflammatory stimulus. The phagocytic capacity was determined with Aβ uptake assay in vitro and Aβ degradation assay of natively formed Aβ deposits ex vivo and in a transgenic APdE9 mouse model of AD in vivo. HSC were lentivirally transduced with enhanced green fluorescent protein (eGFP) to determine the effect of gene modification on the potential of HSC-derived cells for therapeutic purposes. HSC-derived monocytic cells (HSCM) displayed inflammatory responses comparable to microglia and peripheral monocytes. We also show that HSCM contributed to Aβ reduction and could be genetically modified without compromising their function. These monocytic cells could be obtained from human BM or mobilized peripheral blood HSC, indicating a potential therapeutic relevance for AD.

Background Purified intravenous immunoglobulin (IVIG) obtained from the plasma of healthy humans is indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. IVIG contains naturally occurring auto-antibodies, including antibodies (Abs) against β-amyloid (Aβ) peptides accumulating in the brains of Alzheimer's disease (AD) patients. IVIG has been shown to alleviate AD pathology when studied with mildly affected AD patients. Although its mechanisms-of-action have been broadly studied, it remains unresolved how IVIG affects the removal of natively formed brain Aβ deposits by primary astrocytes and microglia, two major cell types involved in the neuroinflammatory responses. Methods We first determined the effect of IVIG on Aβ toxicity in primary neuronal cell culture. The mechanisms-of-action of IVIG in reduction of Aβ burden was analyzed with ex vivo assay. We studied whether IVIG solubilizes natively formed Aβ deposits from brain sections of APP/PS1 mice or promotes Aβ removal by primary glial cells. We determined the role of lysosomal degradation pathway and Aβ Abs in the IVIG-promoted reduction of Aβ. Finally, we studied the penetration of IVIG into the brain parenchyma and interaction with brain deposits of human Aβ in a mouse model of AD in vivo. Results IVIG was protective against Aβ toxicity in a primary mouse hippocampal neuron culture. IVIG modestly inhibited the fibrillization of synthetic Aβ1-42 but did not solubilize natively formed brain Aβ deposits ex vivo. IVIG enhanced microglia-mediated Aβ clearance ex vivo, with a mechanism linked to Aβ Abs and lysosomal degradation. The IVIG-enhanced Aβ clearance appears specific for microglia since IVIG did not affect Aβ clearance by astrocytes. The cellular mechanisms of Aβ clearance we observed have potential relevance in vivo since after peripheral administration IVIG penetrated to mouse brain tissue reaching highest concentrations in the hippocampus and bound selectively to Aβ deposits in co-localization with microglia. Conclusions Our results demonstrate that IVIG promotes recognition and removal of natively formed brain Aβ deposits by primary microglia involving natural Aβ Abs in IVIG. These findings may have therapeutic relevance in vivo as IVIG penetrates through the blood-brain barrier and specifically binds to Aβ deposits in brain parenchyma.

Alzheimer's disease (AD) is a dementing neurodegenerative disorder without a cure. The abnormal parenchymal accumulation of beta-amyloid (Abeta) is associated with inflammatory reactions involving microglia and astrocytes. Increased levels of Abeta and Abeta deposition in the brain are thought to cause neuronal dysfunction and underlie dementia. Microglia, the brain resident cells of monocytic origin, have a potential ability to phagocytose Abeta but they also react to Abeta by increased production of proinflammatory toxic agents. Microglia originate from hemangioblastic mesoderm during early embryonic stages and from bone marrow (BM)-derived monocytic cells that home the brain throughout the neonatal stage of development. Recent studies indicate that BM or blood-derived monocytes are recruited to the diseased AD brain, associate with the Abeta depositions, and are more efficient phagocytes of Abeta compared with resident microglia. The clearance of Abeta deposition by these cells has been recently under intensive investigation and can occur through several different mechanisms. Importantly, peripheral monocytic cells of patients with AD appear to be deficient in clearing Abeta. This review will summarize the findings on the role of blood-derived cells in AD and discuss their therapeutic potential for treating patients suffering from this devastating disease.

The role of microglia recruited from bone marrow (BM) into the CNS during the progression of Alzheimer's disease (AD) is poorly understood. To investigate whether beta-amyloid (Abeta) associated microglia are derived from blood monocytes, we transplanted BM cells from enhanced green fluorescent protein expressing mice into young or old transgenic AD mice and determined the engraftment of BM-derived cells into the brain and their relative distribution near Abeta deposits. When young transgenic mice were transplanted before the onset of AD-like pathology and the brains analyzed 6.5 months later, the number of engrafted cells was significantly higher than in age-matched wild type mice. Moreover, the number of BM-derived cells associated with Abeta was significantly higher than in old transgenic mice transplanted after the establishment of AD-like pathology. Local inflammation caused by intrahippocampal lipopolysaccharide injection significantly increased the engraftment of BM-derived cells in old AD mice and decreased the hippocampal Abeta burden. These results suggest that infiltration of BM-derived monocytic cells into the brain contributes to the development of microglial reaction in AD.

Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel protein associated with language development, synaptic plasticity, tissue remodeling, and angiogenesis. We investigated the expression and spatial localization of SRPX2 in normal mouse, rat, monkey, and human brain using in situ hybridization and immunohistochemistry. Antibody specificity was determined using in vitro siRNA based silencing of SRPX2. Cell type-specific expression was verified by double-labeling with oxytocin or vasopressin. Western blot was used to detect SRPX2 protein in rat and human plasma and cerebrospinal fluid. Unexpectedly, SRPX2 mRNA expression levels were strikingly higher in the hypothalamus as compared to the cortex. All SRPX2 immunoreactive (ir) neurons were localized in the hypothalamic paraventricular, periventricular, and supraoptic nuclei in mouse, rat, monkey, and human brain. SRPX2 colocalized with vasopressin or oxytocin in paraventricular and supraoptic neurons. Hypothalamic SRPX2-ir positive neurons gave origin to dense projections traveling ventrally and caudally towards the hypophysis. Intense axonal varicosities and terminal arborizations were identified in the rat and human neurohypophysis. SRPX2-ir cells were also found in the adenohypophysis. Light SRPX2-ir projections were observed in the dorsal and ventral raphe, locus coeruleus, and the nucleus of the solitary tract in mouse, rat and monkey. SRPX2 protein was also detected in plasma and CSF. Our data revealed intense phylogenetically conserved expression of SRPX2 protein in distinct hypothalamic nuclei and the hypophysis, suggesting its active role in the hypothalamo-pituitary axis. The presence of SRPX2 protein in the plasma and CSF suggests that some of its functions depend on secretion into body fluids. This article is protected by copyright. All rights reserved.

Background Granulocyte colony stimulating factor (GCSF) is protective in animal models of various neurodegenerative diseases. We investigated whether pegfilgrastim, GCSF with sustained action, is protective in a mouse model of amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease with manifestations of upper and lower motoneuron death and muscle atrophy accompanied by inflammation in the CNS and periphery. Methods Human mutant G93A superoxide dismutase (SOD1) ALS mice were treated with pegfilgrastim starting at the presymptomatic stage and continued until the end stage. After long-term pegfilgrastim treatment, the inflammation status was defined in the spinal cord and peripheral tissues including hematopoietic organs and muscle. The effect of GCSF on spinal cord neuron survival and microglia, bone marrow and spleen monocyte activation was assessed in vitro. Results Long-term pegfilgrastim treatment prolonged mutant SOD1 mice survival and attenuated both astro- and microgliosis in the spinal cord. Pegfilgrastim in SOD1 mice modulated the inflammatory cell populations in the bone marrow and spleen and reduced the production of pro-inflammatory cytokine in monocytes and microglia. The mobilization of hematopoietic stem cells into the circulation was restored back to basal level after long-term pegfilgrastim treatment in SOD1 mice while the storage of Ly6C expressing monocytes in the bone marrow and spleen remained elevated. After pegfilgrastim treatment, an increased proportion of these cells in the degenerative muscle was detected at the end stage of ALS. Conclusions GCSF attenuated inflammation in the CNS and the periphery in a mouse model of ALS and thereby delayed the progression of the disease. This mechanism of action targeting inflammation provides a new perspective of the usage of GCSF in the treatment of ALS.