Parkinson's disease (PD) is a chronic, progressive neurodegenerative condition; characterized... more Parkinson's disease (PD) is a chronic, progressive neurodegenerative condition; characterized with the degeneration of the nigrostriatal dopaminergic pathway and neuroinflammation. During PD progression, microglia, the resident immune cells in the central nervous system (CNS) display altered activity, but their role in maintaining PD development has remained unclear to date. The purinergic P2Y12 receptor (P2Y12R), which is expressed on the microglia in the CNS has been shown to regulate microglial activity and responses; however, the function of the P2Y12R in PD is unknown. Here we show that MPTP-induced PD symptoms in mice are associated with marked neuroinflammatory changes and P2Y12R contribute to the activation of microglia and progression of the disease. Surprisingly, while pharmacological or genetic targeting of the P2Y12R augments acute mortality in MPTP-treated mice, these interventions protect against the neurodegenerative cell loss and the development of neuroinflammation in vivo. Pharmacological inhibition of receptors during disease development reverses the symptoms of PD and halts disease progression. We found that P2Y12R regulates ROCK and p38 MAPK activity and control cytokine production. Our principal finding is that the receptor has a dualistic role in PD: functional P2Y12Rs are essential to initiate a protective inflammatory response, since the lack of the receptor leads to reduced survival; however, at later stages of neurodegeneration, P2Y12Rs are apparently responsible for maintaining the activated state of microglia and stimulating pro-inflammatory cytokine response. Understanding protective and detrimental P2Y12R-mediated actions in the CNS may reveal novel approaches to control neuroinflammation and modify disease progression in PD. DATA AVAILABILITY: Data will be made available upon reasonable request.
Parkinson’s disease (PD) is a chronic, progressive neurodegenerative condition; characterized wit... more Parkinson’s disease (PD) is a chronic, progressive neurodegenerative condition; characterized with the degeneration of the nigrostriatal dopaminergic pathway and neuroinflammation. During PD progression, microglia, the resident immune cells in the central nervous system (CNS) display altered activity, but their role in maintaining PD development has remained unclear to date. The purinergic P2Y12 receptor (P2Y12R), which is exclusively expressed on the microglia in the CNS has been shown to regulate microglial activity and responses; however, the function of the P2Y12R in PD is unknown. Here we show that while pharmacological or genetic targeting of P2Y12R previous to disease onset augments acute mortality, these interventions protect against neurodegenerative cell loss and the development of neuroinflammation in vivo. Pharmacological inhibition of receptors during disease development reverses the symptoms of PD and halts disease progression. We found that P2Y12R regulate ROCK and p3...
The centrally-projecting Edinger-Westphal nucleus (EWcp) hosts a large population of neurons expr... more The centrally-projecting Edinger-Westphal nucleus (EWcp) hosts a large population of neurons expressing urocortin 1 (Ucn1) and about half of these neurons also express the leptin receptor (LepRb). Previously, we have shown that the peripheral adiposity hormone leptin signaling energy surfeit modulates EWcp neurons' activity. Here, we hypothesized that Ucn1/LepRb neurons in the EWcp would act as a crucial neuronal node in the brain-white adipose tissue (WAT) axis modulating efferent sympathetic outflow to the WAT. We showed that leptin bound to neurons of the EWcp stimulated STAT3 phosphorylation, and increased Ucn1-production in a time-dependent manner. Besides, retrograde transneuronal tract-tracing using pseudorabies virus (PRV) identified EWcp Ucn1 neurons connected to WAT. Interestingly, reducing EWcp Ucn1 contents by ablating EWcp LepRb-positive neurons with leptin-saporin, did not affect food intake and body weight gain, but substantially (+26%) increased WAT weight accompanied by a higher plasma leptin level and changed plasma lipid profile. We also found that ablation of EWcp Ucn1/LepRb neurons resulted in lower respiratory quotient and oxygen consumption one week after surgery, but was comparable to sham values after 3 and 5 weeks of surgery. Taken together, we report that EWcp/LepRb/Ucn1 neurons not only respond to leptin signaling but also control WAT size and fat metabolism without altering food intake. These data suggest the existence of a EWcp-WAT circuitry allowing an organism to recruit fuels without being able to eat in situations such as the fight-or-flight response.
Microglia are the resident immune cells of the brain with multiple homeostatic and regulatory rol... more Microglia are the resident immune cells of the brain with multiple homeostatic and regulatory roles. Emerging evidence also highlights the fundamental transformative role of microglia in brain development. While tightly controlled, bi-directional communication between microglia and neuronal progenitors or immature neurons has been postulated, the main sites of interaction and the underlying mechanisms remain elusive. By using correlated light and electron microscopy together with super-resolution imaging, here we provide evidence that microglial processes form specialized nanoscale contacts with the cell bodies of developing and immature neurons throughout embryonic, early postnatal and adult neurogenesis. These early developmental contacts are highly reminiscent to somatic purinergic junctions that are instrumental for microglia-neuron communication in the adult brain. We propose that early developmental formation of somatic purinergic junctions represents an ideal interface for mi...
In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in... more In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
The functional contribution of microglia to normal brain development, healthy brain function, and... more The functional contribution of microglia to normal brain development, healthy brain function, and neurological disorders is increasingly recognized. However, until recently, the nature of intercellular interactions mediating these effects remained largely unclear. Recent findings show microglia establishing direct contact with different compartments of neurons. Although communication between microglia and neurons involves intermediate cells and soluble factors, direct membrane contacts enable a more precisely regulated, dynamic, and highly effective form of interaction for fine-tuning neuronal responses and fate. Here, we summarize the known ultrastructural, molecular, and functional features of direct microglia-neuron interactions and their roles in brain disease.
The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, bu... more The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has not been studied to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology and inflammatory responses. As opposed to systemic NKCC1 blockade, which decreased intracortical lipopolysaccharide (LPS)-induced cytokine levels, neuroinflammation was potentiated by both intracortically administered bumetanide and in the absence of microglial NKCC1, suggesting a considerable role for microglia to influence central NKCC1 actions. Correspondingly, microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player to shape microglial responses and brain inflam...
Selective elimination of microglia from the brain was shown to dysregulate neuronal Ca2+ signalin... more Selective elimination of microglia from the brain was shown to dysregulate neuronal Ca2+ signaling and to reduce the incidence of spreading depolarization (SD) during cerebral ischemia. However, the mechanisms through which microglia interfere with SD remained unexplored. Here, we identify microglia as essential modulators of the induction and evolution of SD in the physiologically intact brain in vivo. Confocal- and super-resolution microscopy revealed that a series of SDs induced rapid morphological changes in microglia, facilitated microglial process recruitment to neurons and increased the density of P2Y12 receptors (P2Y12R) on recruited microglial processes. In line with this, depolarization and hyperpolarization during SD were microglia- and P2Y12R-dependent. An absence of microglia was associated with altered potassium uptake after SD and increased the number of c-fos-positive neurons, independently of P2Y12R. Thus, the presence of microglia is likely to be essential to maint...
Proceedings of the National Academy of Sciences, 2019
Neuroimmune interactions may contribute to severe pain and regional inflammatory and autonomic si... more Neuroimmune interactions may contribute to severe pain and regional inflammatory and autonomic signs in complex regional pain syndrome (CRPS), a posttraumatic pain disorder. Here, we investigated peripheral and central immune mechanisms in a translational passive transfer trauma mouse model of CRPS. Small plantar skin–muscle incision was performed in female C57BL/6 mice treated daily with purified serum immunoglobulin G (IgG) from patients with longstanding CRPS or healthy volunteers followed by assessment of paw edema, hyperalgesia, inflammation, and central glial activation. CRPS IgG significantly increased and prolonged swelling and induced stable hyperalgesia of the incised paw compared with IgG from healthy controls. After a short-lasting paw inflammatory response in all groups, CRPS IgG-injected mice displayed sustained, profound microglia and astrocyte activation in the dorsal horn of the spinal cord and pain-related brain regions, indicating central sensitization. Genetic de...
Glucose is a major fuel for the central nervous system and hypoglycemia is a significant homeosta... more Glucose is a major fuel for the central nervous system and hypoglycemia is a significant homeostatic stressor, which elicits counterregulatory reactions. Hypothalamic metabolic-and stress-related neurons initiate these actions, however recruitment of glia in control such adaptive circuit remain unknown. Groups of fed-and fasted-, vehicle-injected, and fasted + insulin-injected male mice were compared in this study. Bolus insulin administration to fasted mice resulted in hypoglycemia, which increased hypothalamo-pituitary-adrenal (HPA) axis-and sympathetic activity, increased transcription of neuropeptide Y (Npy) and agouti-related peptide (Agrp) in the hypothalamic arcuate nucleus and activated IBA1+ microglia in the hypothalamus. Activated microglia were found in close apposition to hypoglycemia-responsive NPY neurons. Inhibition of microglia by minocycline increased counterregulatory sympathetic response to hypoglycemia. Fractalkine-CX3CR1 signaling plays a role in control of microglia during hypoglycemia, because density and solidity of IBA1-ir profiles was attenuated in fasted, insulin-treated, CX3CR1 KO mice, which was parallel with exaggerated neuropeptide responses and higher blood glucose levels following insulin administration. Hypoglycemia increased Il-1b expression in the arcuate nucleus, while IL-1a/b knockout mice display improved glycemic control to insulin administration. In conclusion, activated microglia in the arcuate nucleus interferes with central counterregulatory responses to hypoglycemia. These results underscore involvement of microglia in hypothalamic regulation of glucose homeostasis. Blood glucose concentration is under strict physiological control. Glucose levels falling below 3,5 mM provoke centrally integrated bodily responses to mobilize glucose from peripheral stores and increase gluconeogenesis aiming at restoration of normoglycemia 1. Due to impaired autonomic counterregulatory responses (CRR), a serious condition, hypoglycemia associated autonomic failure (HAAF), may occur in diabetic patients 2. Glucose concentration is monitored by multiple peripheral and central glucosensors in the hypothalamus and brainstem 3. These neurons are found in close surrounding of circumventricular organs, i.e. the median eminence and the area postrema, through which additional metabolic-related hormonal-and nutritional signals are received 4. Information in glucoregulatory control circuits then relayed to autonomic pre-ganglionic and neuroendocrine motor neurons, which regulate effectors such as chromaffin cells in the adrenal medulla, α and β cells in pancreatic islets and corticotropes/somatotropes in the anterior pituitary 1. Hypoglycemia, as a potential life threatening condition, results activation of stress-related neurocircuit, which shares some common effectors involved in glucose counterregulatory responses (sympatho-adrenal activation and glucocorticoid secretion) 5. Metabolic-and stress-related circuits are intimately connected with immune regulation. Microglial cells are local representatives of the innate immune system in the CNS, which might be recruited in physiological responses to homeostatic challenges 6,7. Microglia continuously screen tissue microenvironment via highly motile fine branches and become rapidly activated in response to subtle changes in synaptic activity or danger (damage)and pathogen-associated molecular patterns (DAMPs and PAMPs) 8,9 .
Significance Statement Neuronal networks are highly dynamic structures: cellular morphology and s... more Significance Statement Neuronal networks are highly dynamic structures: cellular morphology and synaptic strength is changing as information is processed and stored. This means that the demand for mitochondrial supply is changing in space and time in neurons. Since the architecture ultimately determines the output performance of these organelles, we hypothesized that the ultrastructure of axonal mitochondria could well be a major parameter of adjusting their performance to the actual energetic needs of the synapse. Our results, describing a cell type-independent and synapse-specific correlation between mitochondrial ultrastructure, mitochondrial molecular fingerprints, and synaptic performance, highlight the importance of activity-dependent ultrastructural plasticity of these organelles in neurons and suggest an even more prominent role for mitochondria in neuroplasticity than it was thought previously.
Microglia are instrumental for recognition and elimination of amyloid β1-42 oligomers (AβO), but ... more Microglia are instrumental for recognition and elimination of amyloid β1-42 oligomers (AβO), but the long-term consequences of AβO-induced inflammatory changes in the brain are unclear. Here, we explored microglial responses and transciptome-level inflammatory signatures in the rat hippocampus after chronic AβO challenge. Middle-aged Long Evans rats received intracerebroventricular infusion of AβO or vehicle for 4 weeks, followed by treatment with artificial CSF or MCC950 for the subsequent 4 weeks. AβO infusion evoked a sustained inflammatory response including activation of NF-κB, triggered microglia activation and increased the expression of pattern recognition and phagocytic receptors. Aβ1-42 plaques were not detectable likely due to microglial elimination of infused oligomers. In addition, we found upregulation of neuronal inhibitory ligands and their cognate microglial receptors, whilst downregulation of Esr1 and Scn1a, encoding estrogen receptor alpha and voltage-gated sodium-channel Na(v)1.1, respectively, was observed. These changes were associated with impaired hippocampus-dependent spatial memory and resembled early neurological changes seen in Alzheimer's disease. To investigate the role of inflammatory actions in memory deterioration, we performed MCC950 infusion, which specifically blocks the NLRP3 inflammasome. MCC950 attenuated AβO-evoked microglia reactivity, restored expression of neuronal inhibitory ligands, reversed downregulation of ERα and Na(v)1.1, and abolished memory impairments. Furthermore, MCC950 abrogated AβO-invoked reduction of serum IL-10. These findings provide evidence that in response to AβO infusion microglia change their phenotype, but the resulting inflammatory changes are sustained for at least one month after the end of AβO challenge. Lasting NLRP3-driven inflammatory alterations and altered hippocampal gene expression contribute to spatial memory decline.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2018
Objective— Circulating complement factors are activated by tissue damage and contribute to acute ... more Objective— Circulating complement factors are activated by tissue damage and contribute to acute brain injury. The deposition of MBL (mannose-binding lectin), one of the initiators of the lectin complement pathway, on the cerebral endothelium activated by ischemia is a major pathogenic event leading to brain injury. The molecular mechanisms through which MBL influences outcome after ischemia are not understood yet. Approach and Results— Here we show that MBL-deficient (MBL −/− ) mice subjected to cerebral ischemia display better flow recovery and less plasma extravasation in the brain than wild-type mice, as assessed by in vivo 2-photon microscopy. This results in reduced vascular dysfunction as shown by the shift from a pro- to an anti-inflammatory vascular phenotype associated with MBL deficiency. We also show that platelets directly bind MBL and that platelets from MBL −/− mice have reduced inflammatory phenotype as indicated by reduced IL-1α (interleukin-1α) content, as early as...
BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DA... more BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes. METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE. RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02). CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.
Neurotropic herpesviruses can establish lifelong infection in humans and contribute to severe dis... more Neurotropic herpesviruses can establish lifelong infection in humans and contribute to severe diseases including encephalitis and neurodegeneration. However, the mechanisms through which the brain's immune system recognizes and controls viral infections propagating across synaptically linked neuronal circuits have remained unclear. Using a well-established model of alphaherpesvirus infection that reaches the brain exclusively via retrograde transsynaptic spread from the periphery, and in vivo two-photon imaging combined with high resolution microscopy, we show that microglia are recruited to and isolate infected neurons within hours. Selective elimination of microglia results in a marked increase in the spread of infection and egress of viral particles into the brain parenchyma, which are associated with diverse neurological symptoms. Microglia recruitment and clearance of infected cells require cell-autonomous P2Y12 signalling in microglia, triggered by nucleotides released fro...
The P2X7R protein, a P2 type purinergic receptor functioning as a non-selective cation channel, i... more The P2X7R protein, a P2 type purinergic receptor functioning as a non-selective cation channel, is expressed in different cell types of the central nervous system in several regions of the brain. The activation of the P2X7R protein by ATP modulates excitatory neurotransmission and contributes to microglial activation, apoptosis and neuron-glia communication. Zinc is an essential micronutrient that is highly concentrated in the synaptic vesicles of glutamatergic hippocampal neurons where free zinc ions released into the synaptic cleft alter glutamatergic signal transmission. Changes in both P2X7R-mediated signaling and brain zinc homeostasis have been implicated in the pathogenesis of mood disorders. Here, we tested the hypothesis that extracellular zinc regulates P2X7R activity in the hippocampus. We observed that P2X7R is expressed in both neurons and glial cells in primary mouse hippocampal neuron-glia culture. Propidium iodide (PI) uptake through large pores formed by pannexins a...
Microglia are the primary immune cells of the central nervous system. However, recent data indica... more Microglia are the primary immune cells of the central nervous system. However, recent data indicate that microglia also contribute to diverse physiological and pathophysiological processes that extend beyond immune-related functions and there is a growing interest to understand the mechanisms through which microglia interact with other cells in the brain. In particular, the molecular processes that contribute to microglia-neuron communication in the healthy brain and their role in common brain diseases have been intensively studied during the last decade. In line with this, fate-mapping studies, genetic models and novel pharmacological approaches have revealed the origin of microglial progenitors, demonstrated the role of self-maintaining microglial populations during brain development or in adulthood, and identified the unexpectedly long lifespan of microglia that may profoundly change our view about senescence and age-related human diseases. Despite the exponentially increasing kn...
Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging, Jan 7, 2018
The purpose of this study was to evaluate a set of widely used nuclear medicine imaging agents as... more The purpose of this study was to evaluate a set of widely used nuclear medicine imaging agents as possible methods to study the early effects of systemic inflammation on the living brain in a mouse model of sepsis-associated encephalopathy (SAE). The lipopolysaccharide (LPS)-induced murine systemic inflammation model was selected as a model of SAE. C57BL/6 mice were used. A multimodal imaging protocol was carried out on each animal 4 h following the intravenous administration of LPS using the following tracers: [Tc][2,2-dimethyl-3-[(3E)-3-oxidoiminobutan-2-yl]azanidylpropyl]-[(3E)-3-hydroxyiminobutan-2-yl]azanide ([Tc]HMPAO) and ethyl-7-[I]iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([I]iomazenil) to measure brain perfusion and neuronal damage, respectively; 2-deoxy-2-[F]fluoro-D-glucose ([F]FDG) to measure cerebral glucose uptake. We assessed microglia activity on another group of mice using 2-[6-chloro-2-(4-[I]iodophenyl)-imidazo[1,2-a]pyridin-3-yl]-N-et...
SummaryThe basal forebrain cholinergic system is widely assumed to control cortical functions via... more SummaryThe basal forebrain cholinergic system is widely assumed to control cortical functions via non-synaptic transmission of a single neurotransmitter, acetylcholine. Yet, using immune-electron tomographic, molecular anatomical, optogenetic and physiological techniques, we find that mouse hippocampal cholinergic terminals invariably establish synapses and their vesicles dock at synapses only. We demonstrate that these synapses do not co-release but co-transmit GABA and acetylcholine via different vesicles, whose release is triggered by distinct calcium channels. This co-transmission evokes fast composite postsynaptic potentials, which are mutually cross-regulated by presynaptic auto-receptors and display different short-term plasticity. The GABAergic component alone effectively suppresses hippocampal sharp wave-ripples and epileptiform activity. The synaptic nature of the forebrain cholinergic system with differentially regulated, fast, GABAergic and cholinergic co-transmission su...
Parkinson's disease (PD) is a chronic, progressive neurodegenerative condition; characterized... more Parkinson's disease (PD) is a chronic, progressive neurodegenerative condition; characterized with the degeneration of the nigrostriatal dopaminergic pathway and neuroinflammation. During PD progression, microglia, the resident immune cells in the central nervous system (CNS) display altered activity, but their role in maintaining PD development has remained unclear to date. The purinergic P2Y12 receptor (P2Y12R), which is expressed on the microglia in the CNS has been shown to regulate microglial activity and responses; however, the function of the P2Y12R in PD is unknown. Here we show that MPTP-induced PD symptoms in mice are associated with marked neuroinflammatory changes and P2Y12R contribute to the activation of microglia and progression of the disease. Surprisingly, while pharmacological or genetic targeting of the P2Y12R augments acute mortality in MPTP-treated mice, these interventions protect against the neurodegenerative cell loss and the development of neuroinflammation in vivo. Pharmacological inhibition of receptors during disease development reverses the symptoms of PD and halts disease progression. We found that P2Y12R regulates ROCK and p38 MAPK activity and control cytokine production. Our principal finding is that the receptor has a dualistic role in PD: functional P2Y12Rs are essential to initiate a protective inflammatory response, since the lack of the receptor leads to reduced survival; however, at later stages of neurodegeneration, P2Y12Rs are apparently responsible for maintaining the activated state of microglia and stimulating pro-inflammatory cytokine response. Understanding protective and detrimental P2Y12R-mediated actions in the CNS may reveal novel approaches to control neuroinflammation and modify disease progression in PD. DATA AVAILABILITY: Data will be made available upon reasonable request.
Parkinson’s disease (PD) is a chronic, progressive neurodegenerative condition; characterized wit... more Parkinson’s disease (PD) is a chronic, progressive neurodegenerative condition; characterized with the degeneration of the nigrostriatal dopaminergic pathway and neuroinflammation. During PD progression, microglia, the resident immune cells in the central nervous system (CNS) display altered activity, but their role in maintaining PD development has remained unclear to date. The purinergic P2Y12 receptor (P2Y12R), which is exclusively expressed on the microglia in the CNS has been shown to regulate microglial activity and responses; however, the function of the P2Y12R in PD is unknown. Here we show that while pharmacological or genetic targeting of P2Y12R previous to disease onset augments acute mortality, these interventions protect against neurodegenerative cell loss and the development of neuroinflammation in vivo. Pharmacological inhibition of receptors during disease development reverses the symptoms of PD and halts disease progression. We found that P2Y12R regulate ROCK and p3...
The centrally-projecting Edinger-Westphal nucleus (EWcp) hosts a large population of neurons expr... more The centrally-projecting Edinger-Westphal nucleus (EWcp) hosts a large population of neurons expressing urocortin 1 (Ucn1) and about half of these neurons also express the leptin receptor (LepRb). Previously, we have shown that the peripheral adiposity hormone leptin signaling energy surfeit modulates EWcp neurons' activity. Here, we hypothesized that Ucn1/LepRb neurons in the EWcp would act as a crucial neuronal node in the brain-white adipose tissue (WAT) axis modulating efferent sympathetic outflow to the WAT. We showed that leptin bound to neurons of the EWcp stimulated STAT3 phosphorylation, and increased Ucn1-production in a time-dependent manner. Besides, retrograde transneuronal tract-tracing using pseudorabies virus (PRV) identified EWcp Ucn1 neurons connected to WAT. Interestingly, reducing EWcp Ucn1 contents by ablating EWcp LepRb-positive neurons with leptin-saporin, did not affect food intake and body weight gain, but substantially (+26%) increased WAT weight accompanied by a higher plasma leptin level and changed plasma lipid profile. We also found that ablation of EWcp Ucn1/LepRb neurons resulted in lower respiratory quotient and oxygen consumption one week after surgery, but was comparable to sham values after 3 and 5 weeks of surgery. Taken together, we report that EWcp/LepRb/Ucn1 neurons not only respond to leptin signaling but also control WAT size and fat metabolism without altering food intake. These data suggest the existence of a EWcp-WAT circuitry allowing an organism to recruit fuels without being able to eat in situations such as the fight-or-flight response.
Microglia are the resident immune cells of the brain with multiple homeostatic and regulatory rol... more Microglia are the resident immune cells of the brain with multiple homeostatic and regulatory roles. Emerging evidence also highlights the fundamental transformative role of microglia in brain development. While tightly controlled, bi-directional communication between microglia and neuronal progenitors or immature neurons has been postulated, the main sites of interaction and the underlying mechanisms remain elusive. By using correlated light and electron microscopy together with super-resolution imaging, here we provide evidence that microglial processes form specialized nanoscale contacts with the cell bodies of developing and immature neurons throughout embryonic, early postnatal and adult neurogenesis. These early developmental contacts are highly reminiscent to somatic purinergic junctions that are instrumental for microglia-neuron communication in the adult brain. We propose that early developmental formation of somatic purinergic junctions represents an ideal interface for mi...
In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in... more In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
The functional contribution of microglia to normal brain development, healthy brain function, and... more The functional contribution of microglia to normal brain development, healthy brain function, and neurological disorders is increasingly recognized. However, until recently, the nature of intercellular interactions mediating these effects remained largely unclear. Recent findings show microglia establishing direct contact with different compartments of neurons. Although communication between microglia and neurons involves intermediate cells and soluble factors, direct membrane contacts enable a more precisely regulated, dynamic, and highly effective form of interaction for fine-tuning neuronal responses and fate. Here, we summarize the known ultrastructural, molecular, and functional features of direct microglia-neuron interactions and their roles in brain disease.
The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, bu... more The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has not been studied to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology and inflammatory responses. As opposed to systemic NKCC1 blockade, which decreased intracortical lipopolysaccharide (LPS)-induced cytokine levels, neuroinflammation was potentiated by both intracortically administered bumetanide and in the absence of microglial NKCC1, suggesting a considerable role for microglia to influence central NKCC1 actions. Correspondingly, microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player to shape microglial responses and brain inflam...
Selective elimination of microglia from the brain was shown to dysregulate neuronal Ca2+ signalin... more Selective elimination of microglia from the brain was shown to dysregulate neuronal Ca2+ signaling and to reduce the incidence of spreading depolarization (SD) during cerebral ischemia. However, the mechanisms through which microglia interfere with SD remained unexplored. Here, we identify microglia as essential modulators of the induction and evolution of SD in the physiologically intact brain in vivo. Confocal- and super-resolution microscopy revealed that a series of SDs induced rapid morphological changes in microglia, facilitated microglial process recruitment to neurons and increased the density of P2Y12 receptors (P2Y12R) on recruited microglial processes. In line with this, depolarization and hyperpolarization during SD were microglia- and P2Y12R-dependent. An absence of microglia was associated with altered potassium uptake after SD and increased the number of c-fos-positive neurons, independently of P2Y12R. Thus, the presence of microglia is likely to be essential to maint...
Proceedings of the National Academy of Sciences, 2019
Neuroimmune interactions may contribute to severe pain and regional inflammatory and autonomic si... more Neuroimmune interactions may contribute to severe pain and regional inflammatory and autonomic signs in complex regional pain syndrome (CRPS), a posttraumatic pain disorder. Here, we investigated peripheral and central immune mechanisms in a translational passive transfer trauma mouse model of CRPS. Small plantar skin–muscle incision was performed in female C57BL/6 mice treated daily with purified serum immunoglobulin G (IgG) from patients with longstanding CRPS or healthy volunteers followed by assessment of paw edema, hyperalgesia, inflammation, and central glial activation. CRPS IgG significantly increased and prolonged swelling and induced stable hyperalgesia of the incised paw compared with IgG from healthy controls. After a short-lasting paw inflammatory response in all groups, CRPS IgG-injected mice displayed sustained, profound microglia and astrocyte activation in the dorsal horn of the spinal cord and pain-related brain regions, indicating central sensitization. Genetic de...
Glucose is a major fuel for the central nervous system and hypoglycemia is a significant homeosta... more Glucose is a major fuel for the central nervous system and hypoglycemia is a significant homeostatic stressor, which elicits counterregulatory reactions. Hypothalamic metabolic-and stress-related neurons initiate these actions, however recruitment of glia in control such adaptive circuit remain unknown. Groups of fed-and fasted-, vehicle-injected, and fasted + insulin-injected male mice were compared in this study. Bolus insulin administration to fasted mice resulted in hypoglycemia, which increased hypothalamo-pituitary-adrenal (HPA) axis-and sympathetic activity, increased transcription of neuropeptide Y (Npy) and agouti-related peptide (Agrp) in the hypothalamic arcuate nucleus and activated IBA1+ microglia in the hypothalamus. Activated microglia were found in close apposition to hypoglycemia-responsive NPY neurons. Inhibition of microglia by minocycline increased counterregulatory sympathetic response to hypoglycemia. Fractalkine-CX3CR1 signaling plays a role in control of microglia during hypoglycemia, because density and solidity of IBA1-ir profiles was attenuated in fasted, insulin-treated, CX3CR1 KO mice, which was parallel with exaggerated neuropeptide responses and higher blood glucose levels following insulin administration. Hypoglycemia increased Il-1b expression in the arcuate nucleus, while IL-1a/b knockout mice display improved glycemic control to insulin administration. In conclusion, activated microglia in the arcuate nucleus interferes with central counterregulatory responses to hypoglycemia. These results underscore involvement of microglia in hypothalamic regulation of glucose homeostasis. Blood glucose concentration is under strict physiological control. Glucose levels falling below 3,5 mM provoke centrally integrated bodily responses to mobilize glucose from peripheral stores and increase gluconeogenesis aiming at restoration of normoglycemia 1. Due to impaired autonomic counterregulatory responses (CRR), a serious condition, hypoglycemia associated autonomic failure (HAAF), may occur in diabetic patients 2. Glucose concentration is monitored by multiple peripheral and central glucosensors in the hypothalamus and brainstem 3. These neurons are found in close surrounding of circumventricular organs, i.e. the median eminence and the area postrema, through which additional metabolic-related hormonal-and nutritional signals are received 4. Information in glucoregulatory control circuits then relayed to autonomic pre-ganglionic and neuroendocrine motor neurons, which regulate effectors such as chromaffin cells in the adrenal medulla, α and β cells in pancreatic islets and corticotropes/somatotropes in the anterior pituitary 1. Hypoglycemia, as a potential life threatening condition, results activation of stress-related neurocircuit, which shares some common effectors involved in glucose counterregulatory responses (sympatho-adrenal activation and glucocorticoid secretion) 5. Metabolic-and stress-related circuits are intimately connected with immune regulation. Microglial cells are local representatives of the innate immune system in the CNS, which might be recruited in physiological responses to homeostatic challenges 6,7. Microglia continuously screen tissue microenvironment via highly motile fine branches and become rapidly activated in response to subtle changes in synaptic activity or danger (damage)and pathogen-associated molecular patterns (DAMPs and PAMPs) 8,9 .
Significance Statement Neuronal networks are highly dynamic structures: cellular morphology and s... more Significance Statement Neuronal networks are highly dynamic structures: cellular morphology and synaptic strength is changing as information is processed and stored. This means that the demand for mitochondrial supply is changing in space and time in neurons. Since the architecture ultimately determines the output performance of these organelles, we hypothesized that the ultrastructure of axonal mitochondria could well be a major parameter of adjusting their performance to the actual energetic needs of the synapse. Our results, describing a cell type-independent and synapse-specific correlation between mitochondrial ultrastructure, mitochondrial molecular fingerprints, and synaptic performance, highlight the importance of activity-dependent ultrastructural plasticity of these organelles in neurons and suggest an even more prominent role for mitochondria in neuroplasticity than it was thought previously.
Microglia are instrumental for recognition and elimination of amyloid β1-42 oligomers (AβO), but ... more Microglia are instrumental for recognition and elimination of amyloid β1-42 oligomers (AβO), but the long-term consequences of AβO-induced inflammatory changes in the brain are unclear. Here, we explored microglial responses and transciptome-level inflammatory signatures in the rat hippocampus after chronic AβO challenge. Middle-aged Long Evans rats received intracerebroventricular infusion of AβO or vehicle for 4 weeks, followed by treatment with artificial CSF or MCC950 for the subsequent 4 weeks. AβO infusion evoked a sustained inflammatory response including activation of NF-κB, triggered microglia activation and increased the expression of pattern recognition and phagocytic receptors. Aβ1-42 plaques were not detectable likely due to microglial elimination of infused oligomers. In addition, we found upregulation of neuronal inhibitory ligands and their cognate microglial receptors, whilst downregulation of Esr1 and Scn1a, encoding estrogen receptor alpha and voltage-gated sodium-channel Na(v)1.1, respectively, was observed. These changes were associated with impaired hippocampus-dependent spatial memory and resembled early neurological changes seen in Alzheimer's disease. To investigate the role of inflammatory actions in memory deterioration, we performed MCC950 infusion, which specifically blocks the NLRP3 inflammasome. MCC950 attenuated AβO-evoked microglia reactivity, restored expression of neuronal inhibitory ligands, reversed downregulation of ERα and Na(v)1.1, and abolished memory impairments. Furthermore, MCC950 abrogated AβO-invoked reduction of serum IL-10. These findings provide evidence that in response to AβO infusion microglia change their phenotype, but the resulting inflammatory changes are sustained for at least one month after the end of AβO challenge. Lasting NLRP3-driven inflammatory alterations and altered hippocampal gene expression contribute to spatial memory decline.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2018
Objective— Circulating complement factors are activated by tissue damage and contribute to acute ... more Objective— Circulating complement factors are activated by tissue damage and contribute to acute brain injury. The deposition of MBL (mannose-binding lectin), one of the initiators of the lectin complement pathway, on the cerebral endothelium activated by ischemia is a major pathogenic event leading to brain injury. The molecular mechanisms through which MBL influences outcome after ischemia are not understood yet. Approach and Results— Here we show that MBL-deficient (MBL −/− ) mice subjected to cerebral ischemia display better flow recovery and less plasma extravasation in the brain than wild-type mice, as assessed by in vivo 2-photon microscopy. This results in reduced vascular dysfunction as shown by the shift from a pro- to an anti-inflammatory vascular phenotype associated with MBL deficiency. We also show that platelets directly bind MBL and that platelets from MBL −/− mice have reduced inflammatory phenotype as indicated by reduced IL-1α (interleukin-1α) content, as early as...
BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DA... more BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes. METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE. RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02). CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.
Neurotropic herpesviruses can establish lifelong infection in humans and contribute to severe dis... more Neurotropic herpesviruses can establish lifelong infection in humans and contribute to severe diseases including encephalitis and neurodegeneration. However, the mechanisms through which the brain's immune system recognizes and controls viral infections propagating across synaptically linked neuronal circuits have remained unclear. Using a well-established model of alphaherpesvirus infection that reaches the brain exclusively via retrograde transsynaptic spread from the periphery, and in vivo two-photon imaging combined with high resolution microscopy, we show that microglia are recruited to and isolate infected neurons within hours. Selective elimination of microglia results in a marked increase in the spread of infection and egress of viral particles into the brain parenchyma, which are associated with diverse neurological symptoms. Microglia recruitment and clearance of infected cells require cell-autonomous P2Y12 signalling in microglia, triggered by nucleotides released fro...
The P2X7R protein, a P2 type purinergic receptor functioning as a non-selective cation channel, i... more The P2X7R protein, a P2 type purinergic receptor functioning as a non-selective cation channel, is expressed in different cell types of the central nervous system in several regions of the brain. The activation of the P2X7R protein by ATP modulates excitatory neurotransmission and contributes to microglial activation, apoptosis and neuron-glia communication. Zinc is an essential micronutrient that is highly concentrated in the synaptic vesicles of glutamatergic hippocampal neurons where free zinc ions released into the synaptic cleft alter glutamatergic signal transmission. Changes in both P2X7R-mediated signaling and brain zinc homeostasis have been implicated in the pathogenesis of mood disorders. Here, we tested the hypothesis that extracellular zinc regulates P2X7R activity in the hippocampus. We observed that P2X7R is expressed in both neurons and glial cells in primary mouse hippocampal neuron-glia culture. Propidium iodide (PI) uptake through large pores formed by pannexins a...
Microglia are the primary immune cells of the central nervous system. However, recent data indica... more Microglia are the primary immune cells of the central nervous system. However, recent data indicate that microglia also contribute to diverse physiological and pathophysiological processes that extend beyond immune-related functions and there is a growing interest to understand the mechanisms through which microglia interact with other cells in the brain. In particular, the molecular processes that contribute to microglia-neuron communication in the healthy brain and their role in common brain diseases have been intensively studied during the last decade. In line with this, fate-mapping studies, genetic models and novel pharmacological approaches have revealed the origin of microglial progenitors, demonstrated the role of self-maintaining microglial populations during brain development or in adulthood, and identified the unexpectedly long lifespan of microglia that may profoundly change our view about senescence and age-related human diseases. Despite the exponentially increasing kn...
Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging, Jan 7, 2018
The purpose of this study was to evaluate a set of widely used nuclear medicine imaging agents as... more The purpose of this study was to evaluate a set of widely used nuclear medicine imaging agents as possible methods to study the early effects of systemic inflammation on the living brain in a mouse model of sepsis-associated encephalopathy (SAE). The lipopolysaccharide (LPS)-induced murine systemic inflammation model was selected as a model of SAE. C57BL/6 mice were used. A multimodal imaging protocol was carried out on each animal 4 h following the intravenous administration of LPS using the following tracers: [Tc][2,2-dimethyl-3-[(3E)-3-oxidoiminobutan-2-yl]azanidylpropyl]-[(3E)-3-hydroxyiminobutan-2-yl]azanide ([Tc]HMPAO) and ethyl-7-[I]iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([I]iomazenil) to measure brain perfusion and neuronal damage, respectively; 2-deoxy-2-[F]fluoro-D-glucose ([F]FDG) to measure cerebral glucose uptake. We assessed microglia activity on another group of mice using 2-[6-chloro-2-(4-[I]iodophenyl)-imidazo[1,2-a]pyridin-3-yl]-N-et...
SummaryThe basal forebrain cholinergic system is widely assumed to control cortical functions via... more SummaryThe basal forebrain cholinergic system is widely assumed to control cortical functions via non-synaptic transmission of a single neurotransmitter, acetylcholine. Yet, using immune-electron tomographic, molecular anatomical, optogenetic and physiological techniques, we find that mouse hippocampal cholinergic terminals invariably establish synapses and their vesicles dock at synapses only. We demonstrate that these synapses do not co-release but co-transmit GABA and acetylcholine via different vesicles, whose release is triggered by distinct calcium channels. This co-transmission evokes fast composite postsynaptic potentials, which are mutually cross-regulated by presynaptic auto-receptors and display different short-term plasticity. The GABAergic component alone effectively suppresses hippocampal sharp wave-ripples and epileptiform activity. The synaptic nature of the forebrain cholinergic system with differentially regulated, fast, GABAergic and cholinergic co-transmission su...
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Papers by Ádám Dénes