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Recent single-cell RNA sequencing studies have revealed distinct microglial states in development and disease. These include proliferative-region-associated microglia (PAMs) in developing white matter and disease-associated microglia (DAMs) prevalent in various neurodegenerative conditions. PAMs and DAMs share a similar core gene signature. However, the extent of the dynamism and plasticity of these microglial states, as well as their functional significance, remains elusive, partly due to the lack of specific tools. Here, we generated an inducible Cre driver line, Clec7a-CreERT2, that targets PAMs and DAMs in the brain parenchyma. Utilizing this tool, we profiled labeled cells during development and in several disease models, uncovering convergence and context-dependent differences in PAM and DAM gene expression. Through long-term tracking, we demonstrated microglial state plasticity. Lastly, we specifically depleted DAMs in demyelination, revealing their roles in disease recovery. Together, we provide a versatile genetic tool to characterize microglial states in CNS development and disease.
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Plasticidad de la Célula , Microglía , Remielinización , Microglía/fisiología , Animales , Ratones , Plasticidad de la Célula/genética , Enfermedades Desmielinizantes/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales de Enfermedad , Encéfalo , Vaina de Mielina/metabolismo , Sustancia Blanca/patologíaRESUMEN
The arachnoid barrier delineates the border between the central nervous system and dura mater. Although the arachnoid barrier creates a partition, communication between the central nervous system and the dura mater is crucial for waste clearance and immune surveillance1,2. How the arachnoid barrier balances separation and communication is poorly understood. Here, using transcriptomic data, we developed transgenic mice to examine specific anatomical structures that function as routes across the arachnoid barrier. Bridging veins create discontinuities where they cross the arachnoid barrier, forming structures that we termed arachnoid cuff exit (ACE) points. The openings that ACE points create allow the exchange of fluids and molecules between the subarachnoid space and the dura, enabling the drainage of cerebrospinal fluid and limited entry of molecules from the dura to the subarachnoid space. In healthy human volunteers, magnetic resonance imaging tracers transit along bridging veins in a similar manner to access the subarachnoid space. Notably, in neuroinflammatory conditions such as experimental autoimmune encephalomyelitis, ACE points also enable cellular trafficking, representing a route for immune cells to directly enter the subarachnoid space from the dura mater. Collectively, our results indicate that ACE points are a critical part of the anatomy of neuroimmune communication in both mice and humans that link the central nervous system with the dura and its immunological diversity and waste clearance systems.
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Aracnoides , Encéfalo , Duramadre , Animales , Humanos , Ratones , Aracnoides/anatomía & histología , Aracnoides/irrigación sanguínea , Aracnoides/inmunología , Aracnoides/metabolismo , Transporte Biológico , Encéfalo/anatomía & histología , Encéfalo/irrigación sanguínea , Encéfalo/inmunología , Encéfalo/metabolismo , Duramadre/anatomía & histología , Duramadre/irrigación sanguínea , Duramadre/inmunología , Duramadre/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Perfilación de la Expresión Génica , Imagen por Resonancia Magnética , Ratones Transgénicos , Espacio Subaracnoideo/anatomía & histología , Espacio Subaracnoideo/irrigación sanguínea , Espacio Subaracnoideo/inmunología , Espacio Subaracnoideo/metabolismo , Líquido Cefalorraquídeo/metabolismo , Venas/metabolismoRESUMEN
The ability to assess cell proliferation and viability is essential for assessing new drug treatments, particularly in cancer drug discovery. This study describes a new method that uses a plate reader digital microscopy cell imaging and analysis system to assess cell proliferation and viability. This imaging system utilizes high throughput fluorescence microscopy with two fluorescent probes: cell membrane-impermeable SYTOX green and nuclear binding Hoechst-33342. Here we compare this technology to other known viability assays, namely: propidium iodide (PI)-based flow cytometry, and sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based plate reader assays. These methods were assessed based on their effectiveness in detecting the cell numbers of two adherent cell lines and one suspension cell line. Automated cell imaging was most accurate at measuring cell number in both adherent and suspension cell lines. The PI-based flow cytometry method was more difficult to use with adherent cells, while the SRB and MTT assays had difficulties when monitoring cells in suspension. Despite these challenges, it was possible to obtain similar results when quantifying the effect of cytotoxic compounds. This study demonstrates that the digital microscopy automated cell imaging system is an effective method for assessing cell proliferation and the cytotoxic effect of compounds on both adherent and suspension cell lines.
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Amyotrophic lateral sclerosis (ALS) is a fatal movement disorder involving degeneration of motor neurons through dysfunction of the RNA-binding protein TDP-43. Pericytes, the perivascular cells of the blood-brain, blood-spinal cord, and blood-CSF barriers also degenerate in ALS. Indeed, pericytes are among the earliest cell types to show gene expression changes in pre-symptomatic animal models of ALS. This suggests that pericyte degeneration precedes neurodegeneration and may involve pericyte cell-autonomous TDP-43 dysfunction. Here we determined the effect of TDP-43 dysfunction in human brain pericytes on interleukin 6 (IL-6), a critical secreted inflammatory mediator reported to be regulated by TDP 43. Primary human brain pericytes were cultured from biopsy tissue from epilepsy surgeries and TDP-43 was silenced using siRNA. TDP-43 silencing of pericytes stimulated with pro-inflammatory cytokines, interleukin-1ß or tumour necrosis factor alpha, robustly suppressed the induction of IL-6 transcript and protein. IL-6 regulation by TDP-43 did not involve the assembly of TDP-43 nuclear splicing bodies, and did not occur via altered splicing of IL6. Instead, transcriptome-wide analysis by RNA-Sequencing identified a poison exon in the IL6 destabilising factor HNRNPD (AUF1) as a splicing target of TDP-43. Our data support a model whereby TDP-43 silencing favours destabilisation of IL6 mRNA, via enhanced AU-rich element-mediated decay by HNRNP/AUF1. This suggests that cell-autonomous deficits in TDP-43 function in human brain pericytes would suppress their production of IL-6. Given the importance of the blood-brain and blood-spinal cord barriers in maintaining motor neuron health, TDP-43 in human brain pericytes may represent a cellular target for ALS therapeutics.
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Esclerosis Amiotrófica Lateral , Proteínas de Unión al ADN , Interleucina-6 , Pericitos , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Encéfalo/metabolismo , Citocinas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Interleucina-6/metabolismo , Pericitos/metabolismo , Pericitos/patología , Médula Espinal/metabolismoRESUMEN
BACKGROUND: Pericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed. METHODS: To study the effects of neuroinflammation at the BBB, primary brain endothelial cells and pericytes were isolated from human biopsy tissue. Culture purity was examined using qPCR and immunocytochemistry. Electrical cell-substrate impedance sensing (ECIS) was used to determine the barrier properties of endothelial and pericyte cultures. Using immunocytochemistry, cytometric bead array, and ECIS, we compared the responses of endothelia and pericytes to a panel of inflammatory stimuli (IL-1ß, TNFα, LPS, IFN-γ, TGF-ß1, IL-6, and IL-4). Secretome analysis was performed to identify unique secretions of endothelia and pericytes in response to IL-1ß. RESULTS: Endothelial cells were pure, moderately proliferative, retained the expression of BBB-related junctional proteins and transporters, and generated robust TEER. Both endothelia and pericytes have the same pattern of transcription factor activation in response to inflammatory stimuli but respond differently at the secretion level. Secretome analysis confirmed that endothelia and pericytes have overlapping but distinct secretome profiles in response to IL-1ß. We identified several cell-type specific responses, including G-CSF and GM-CSF (endothelial-specific), and IGFBP2 and IGFBP3 (pericyte-specific). Finally, we demonstrated that direct addition of IL-1ß, TNFα, LPS, and IL-4 contributed to the loss of endothelial barrier integrity in vitro. CONCLUSIONS: Here, we identify important cell-type differences in the inflammatory response of brain pericytes and endothelia and provide, for the first time, a comprehensive profile of the secretions of primary human brain endothelia and pericytes which has implications for understanding how inflammation affects the cerebrovasculature.
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Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células Endoteliales/metabolismo , Mediadores de Inflamación/metabolismo , Pericitos/metabolismo , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/citología , Encéfalo/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/efectos de los fármacos , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/farmacología , Pericitos/efectos de los fármacosRESUMEN
BACKGROUND: Neuroinflammation and blood-brain barrier (BBB) disruption are common features of many brain disorders, including Alzheimer's disease, epilepsy, and motor neuron disease. Inflammation is thought to be a driver of BBB breakdown, but the underlying mechanisms for this are unclear. Brain pericytes are critical cells for maintaining the BBB and are immunologically active. We sought to test the hypothesis that inflammation regulates the BBB by altering pericyte biology. METHODS: We exposed primary adult human brain pericytes to chronic interferon-gamma (IFNγ) for 4 days and measured associated functional aspects of pericyte biology. Specifically, we examined the influence of inflammation on platelet-derived growth factor receptor-beta (PDGFRß) expression and signalling, as well as pericyte proliferation and migration by qRT-PCR, immunocytochemistry, flow cytometry, and western blotting. RESULTS: Chronic IFNγ treatment had marked effects on pericyte biology most notably through the PDGFRß, by enhancing agonist (PDGF-BB)-induced receptor phosphorylation, internalization, and subsequent degradation. Functionally, chronic IFNγ prevented PDGF-BB-mediated pericyte proliferation and migration. CONCLUSIONS: Because PDGFRß is critical for pericyte function and its removal leads to BBB leakage, our results pinpoint a mechanism linking chronic brain inflammation to BBB dysfunction.
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Cerebrospinal fluid (CSF) bathes and cushions the brain; however, it also serves a major role in the clearance of metabolic wastes and in the distribution of glucose, lipids, and amino acids. Unlike every other organ in the body, the brain parenchyma lacks a traditional lymphatic system to drain fluids and central nervous system (CNS) antigens. It was historically assumed that all brain wastes were removed by endogenous processing, such as phagocytosis and autophagy, while excess fluids drained directly into the blood. However, the twin discoveries of the glial-lymphatic (glymphatic) system and meningeal lymphatics have transformed our understanding of brain waste clearance. The glymphatic system describes the movement of fluids through the subarachnoid space (SAS), the influx along periarterial spaces into the brain parenchyma, and the ultimate efflux back into the SAS along perivenous spaces where it comes into direct contact with the meningeal lymphatics. The dura mater of the meninges contains a bona fide lymphatic network that can drain CSF that has entered the dura. Together, these pathways provide insights into the clearance of molecules and fluids from the brain, and show that the CNS is physically connected to the adaptive immune system. Here, we outline the glymphatic and lymphatic systems, and describe the cellular components that are important to their function.
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Decades of research have characterized diverse immune cells surveilling the CNS. More recently, the discovery of osseous channels (so-called 'skull channels') connecting the meninges with the skull and vertebral bone marrow has revealed a new layer of complexity in our understanding of neuroimmune interactions. Here we discuss our current understanding of skull and vertebral bone marrow anatomy, its contribution of leukocytes to the meninges, and its surveillance of the CNS. We explore the role of this hematopoietic output on CNS health, focusing on the supply of immune cells during health and disease.
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Médula Ósea , Sistema Nervioso Central , Meninges , Cráneo , CabezaRESUMEN
Inflammation is a common feature of neurological diseases. During neuroinflammation, neutrophils are recruited to the brain vasculature, where myeloperoxidase can produce hypochlorous acid and the less well-studied oxidant hypothiocyanous acid (HOSCN). In this study, we exposed primary brain endothelial cells (BECs) to HOSCN and observed a rapid loss of transendothelial electrical resistance (TEER) at sublethal concentrations. Decreased barrier function was associated with a loss of tight junctions at cellular contacts and a concomitant loss of dynamic microtubules. Both tight junction and cytoskeletal disruptions were visible within 30 min of exposure, whereas significant loss of TEER took more than 1 h. The removal of the HOSCN after 30 min prevented subsequent barrier dysfunction. These results indicate that BECs are sensitive to HOSCN, resulting in the eventual loss of barrier function. We hypothesise that this mechanism may be relevant in neutrophil transmigration, with HOSCN facilitating blood-brain barrier opening at the sites of egress. Furthermore, this mechanism may be a way through which neutrophils, residing in the vasculature, can influence neuroinflammation in diseases.
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When modeling disease in the laboratory, it is important to use clinically relevant models. Patient-derived human brain cells grown in vitro to study and test potential treatments provide such a model. Here, we present simple, highly reproducible coordinated procedures that can be used to routinely culture most cell types found in the human brain from single neurosurgically excised brain specimens. The cell types that can be cultured include dissociated cultures of neurons, astrocytes, microglia, pericytes and brain endothelial and neural precursor cells, as well as explant cultures of the leptomeninges, cortical slice cultures and brain tumor cells. The initial setup of cultures takes ~2 h, and the cells are ready for further experiments within days to weeks. The resulting cells can be studied as purified or mixed population cultures, slice cultures and explant-derived cultures. This protocol therefore enables the investigation of human brain cells to facilitate translation of neuroscience research to the clinic.
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Células-Madre NeuralesRESUMEN
It remains unclear how immune cells from skull bone marrow niches are recruited to the meninges. Here we report that cerebrospinal fluid (CSF) accesses skull bone marrow via dura-skull channels, and CSF proteins signal onto diverse cell types within the niches. After spinal cord injury, CSF-borne cues promote myelopoiesis and egress of myeloid cells into meninges. This reveals a mechanism of CNS-to-bone-marrow communication via CSF that regulates CNS immune responses.
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Médula Ósea , Cráneo , Médula Ósea/fisiología , Líquido Cefalorraquídeo , Cabeza , Meninges , Células Mieloides/metabolismoRESUMEN
Parkinson's disease (PD) is characterised by the progressive loss of midbrain dopaminergic neurons and the presence of aggregated α-synuclein (α-syn). Pericytes and microglia, two non-neuronal cells contain α-syn in the human brain, however, their role in disease processes is poorly understood. Pericytes, found surrounding the capillaries in the brain are important for maintaining the blood-brain barrier, controlling blood flow and mediating inflammation. In this study, primary human brain pericytes and microglia were exposed to two different α-synuclein aggregates. Inflammatory responses were assessed using immunocytochemistry, cytometric bead arrays and proteome profiler cytokine array kits. Fixed flow cytometry was used to investigate the uptake and subsequent degradation of α-syn in pericytes. We found that the two α-syn aggregates are devoid of inflammatory and cytotoxic actions on human brain derived pericytes and microglia. Although α-syn did not induce an inflammatory response, pericytes efficiently take up and degrade α-syn through the lysosomal pathway but not the ubiquitin-proteasome system. Furthermore, when pericytes were exposed the ubiquitin proteasome inhibitor-MG132 and α-syn aggregates, there was profound cytotoxicity through the production of reactive oxygen species resulting in apoptosis. These results suggest that the observed accumulation of α-syn in pericytes in human PD brains likely plays a role in PD pathogenesis, perhaps by causing cerebrovascular instability, under conditions of cellular stress.
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Enfermedad de Parkinson , alfa-Sinucleína , Apoptosis , Citocinas/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Pericitos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/metabolismo , Proteoma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
Platelet-derived growth factor-BB (PDGF-BB):PDGF receptor-ß (PDGFRß) signalling in brain pericytes is critical to the development, maintenance and function of a healthy blood-brain barrier (BBB). Furthermore, BBB impairment and pericyte loss in Alzheimer's disease (AD) is well documented. We found that PDGF-BB:PDGFRß signalling components were altered in human AD brains, with a marked reduction in vascular PDGFB. We hypothesised that reduced PDGF-BB:PDGFRß signalling in pericytes may impact on the BBB. We therefore tested the effects of PDGF-BB on primary human brain pericytes in vitro to define pathways related to BBB function. Using pharmacological inhibitors, we dissected distinct aspects of the PDGF-BB response that are controlled by extracellular signal-regulated kinase (ERK) and Akt pathways. PDGF-BB promotes the proliferation of pericytes and protection from apoptosis through ERK signalling. In contrast, PDGF-BB:PDGFRß signalling through Akt augments pericyte-derived inflammatory secretions. It may therefore be possible to supplement PDGF-BB signalling to stabilise the cerebrovasculature in AD.
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Enfermedad de Alzheimer , Pericitos , Enfermedad de Alzheimer/metabolismo , Becaplermina/metabolismo , Becaplermina/farmacología , Encéfalo/metabolismo , Humanos , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/farmacologíaRESUMEN
INTRODUCTION: Neutrophil accumulation is a well-established feature of Alzheimer's disease (AD) and has been linked to cognitive impairment by modulating disease-relevant neuroinflammatory and vascular pathways. Neutrophils express high levels of the oxidant-generating enzyme myeloperoxidase (MPO), however there has been controversy regarding the cellular source and localisation of MPO in the AD brain. MATERIALS AND METHODS: We used immunostaining and immunoassays to quantify the accumulation of neutrophils in human AD tissue microarrays and in the brains of APP/PS1 mice. We also used multiplexed immunolabelling to define the presence of NETs in AD. RESULTS: There was an increase in neutrophils in AD brains as well as in the murine APP/PS1 model of AD. Indeed, MPO expression was almost exclusively confined to S100A8-positive neutrophils in both human AD and murine APP/PS1 brains. The vascular localisation of neutrophils in both human AD and mouse models of AD was striking and driven by enhanced neutrophil adhesion to small vessels. We also observed rare infiltrating neutrophils and deposits of MPO around plaques. Citrullinated histone H3, a marker of neutrophil extracellular traps (NETs), was also detected in human AD cases at these sites, indicating the presence of extracellular MPO in the vasculature. Finally, there was a reduction in the endothelial glycocalyx in AD that may be responsible for non-productive neutrophil adhesion to the vasculature. CONCLUSION: Our report indicates that vascular changes may drive neutrophil adhesion and NETosis, and that neutrophil-derived MPO may lead to vascular oxidative stress and be a relevant therapeutic target in AD.
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Enfermedad de Alzheimer , Trampas Extracelulares , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/metabolismo , Trampas Extracelulares/metabolismo , Humanos , Ratones , Neutrófilos/metabolismo , Peroxidasa/metabolismoRESUMEN
BACKGROUND: Macrophage migration inhibitory factor (MIF) is an important immuno-regulatory cytokine and is elevated in inflammatory conditions. Neutrophils are the first immune cells to migrate to sites of infection and inflammation, where they generate, among other mediators, the potent oxidant hypochlorous acid (HOCl). Here, we investigated the impact of MIF on HOCl production in neutrophils in response to phagocytic stimuli. METHODS: Production of HOCl during phagocytosis of zymosan was determined using the specific fluorescent probe R19-S in combination with flow cytometry and live cell microscopy. The rate of phagocytosis was monitored using fluorescently-labeled zymosan. Alternatively, HOCl production was assessed during phagocytosis of Pseudomonas aeruginosa by measuring the oxidation of bacterial glutathione to the HOCl-specific product glutathione sulfonamide. Formation of neutrophil extracellular traps (NETs), an oxidant-dependent process, was quantified using a SYTOX Green plate assay. RESULTS: Exposure of human neutrophils to MIF doubled the proportion of neutrophils producing HOCl during early stages of zymosan phagocytosis, and the concentration of HOCl produced was greater. During phagocytosis of P. aeruginosa, a greater fraction of bacterial glutathione was oxidized to glutathione sulfonamide in MIF-treated compared to control neutrophils. The ability of MIF to increase neutrophil HOCl production was independent of the rate of phagocytosis and could be blocked by the MIF inhibitor 4-IPP. Neutrophils pre-treated with MIF produced more NETs than control cells in response to PMA. CONCLUSION: Our results suggest a role for MIF in potentiating HOCl production in neutrophils in response to phagocytic stimuli. We propose that this newly discovered activity of MIF contributes to its role in mediating the inflammatory response and enhances host defence.
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Trampas Extracelulares , Factores Inhibidores de la Migración de Macrófagos , Humanos , Ácido Hipocloroso , Oxidorreductasas Intramoleculares , Neutrófilos , FagocitosisRESUMEN
Neuroinflammation is a key component of virtually all neurodegenerative diseases, preceding neuronal loss and associating directly with cognitive impairment. Neuroinflammatory signals can originate and be amplified at barrier tissues such as brain vasculature, surrounding meninges and the choroid plexus. We designed a high content screening system to target inflammation in human brain-derived cells of the blood-brain barrier (pericytes and endothelial cells) to identify inflammatory modifiers. Screening an FDA-approved drug library we identify digoxin and lanatoside C, members of the cardiac glycoside family, as inflammatory-modulating drugs that work in blood-brain barrier cells. An ex vivo assay of leptomeningeal and choroid plexus explants confirm that these drugs maintain their function in 3D cultures of brain border tissues. These results suggest that cardiac glycosides may be useful in targeting inflammation at border regions of the brain and offer new options for drug discovery approaches for neuroinflammatory driven degeneration.
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Antiinflamatorios/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Plexo Coroideo/efectos de los fármacos , Digoxina/farmacología , Células Endoteliales/efectos de los fármacos , Inflamación/tratamiento farmacológico , Lanatosidos/farmacología , Meninges/efectos de los fármacos , Pericitos/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Células Cultivadas , Plexo Coroideo/metabolismo , Plexo Coroideo/patología , Evaluación Preclínica de Medicamentos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Ensayos Analíticos de Alto Rendimiento , Humanos , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Meninges/metabolismo , Meninges/patología , Pericitos/metabolismo , Pericitos/patología , Técnicas de Cultivo de TejidosRESUMEN
Neurological disorders are associated with increased oxidative stress. Reactive oxidants damage tissue and promote cell death, but it is apparent that oxidants can have more subtle effects on cell function through the modulation of redox-sensitive signalling pathways. Cells of the blood-brain barrier regulate the brain microenvironment but become dysfunctional during neurological disease. The blood-brain barrier is maintained by many cell types, and is modulated by redox-sensitive pathways, ranging from the cytoskeletal elements responsible for establishing a barrier, to growth factor and cytokine signalling pathways that influence neurovascular cells. During neurological disease, blood-brain barrier cells are exposed to exogenously generated oxidants from immune cells, as well as increasing endogenously oxidant production. These oxidants impair the function of the blood-brain barrier, leading to increased leakage and reduced blood flow. Reducing the impact of oxidants on the function of blood-brain barrier cells may provide new strategies for delaying the progression of neurological disease.
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Barrera Hematoencefálica/citología , Inflamación/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Estrés Oxidativo/fisiología , Animales , Astrocitos/metabolismo , Astrocitos/patología , Barrera Hematoencefálica/enzimología , Barrera Hematoencefálica/metabolismo , Muerte Celular/efectos de los fármacos , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Humanos , Inflamación/enzimología , Inflamación/inmunología , Microglía/enzimología , Microglía/metabolismo , Enfermedades del Sistema Nervioso/enzimología , Enfermedades del Sistema Nervioso/fisiopatología , Neutrófilos/enzimología , Neutrófilos/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Pericitos/enzimología , Pericitos/metabolismo , Pericitos/patología , Transducción de Señal/genéticaRESUMEN
In Alzheimer's disease (AD), microglia are affected by disease processes, but may also drive pathogenesis. AD pathology-associated microglial populations have been identified with single-cell RNA-Seq, but have not been validated in human brain tissue with anatomical context. Here, we quantified myeloid cell markers to identify changes in AD pathology-associated microglial populations. We performed fluorescent immunohistochemistry on normal (n = 8) and AD (n = 8) middle temporal gyri, co-labelling the pan-myeloid cell marker, Iba1, with one of 11 markers of interest (MOIs): CD45, HLA-DR, CD14, CD74, CD33, CD206, CD32, CD163, P2RY12, TMEM119, L-Ferritin. Novel image analyses quantified the single-cell abundance of Iba1 and each MOI. Each cell was gated into one Iba1-MOI population (Iba1low MOIhigh, Iba1high MOIhigh, or Iba1high MOIlow) and the abundance of each population was compared between AD and control. Triple-labelling of L-Ferritin and Iba1 with a subset of MOIs was performed to investigate L-Ferritin-MOI co-expression on Iba1low cells. Iba1low MOIhigh myeloid cell populations delineated by MOIs CD45, HLA-DR, CD14, CD74, CD33, CD32, and L-Ferritin were increased in AD. Further investigation of the Iba1low MOIhigh populations revealed that their abundances correlated with tau, but not amyloid beta, load in AD. The Iba1low microglial population highly expressed L-Ferritin, reflecting microglial dysfunction. The L-Ferritinhigh CD74high HLA-DRhigh phenotype of the Iba1low population mirrors that of a human AD pathology-associated microglial subpopulation previously identified using single-cell RNA-Seq. Our high-throughput immunohistochemical data with anatomical context support the microglial dysfunction hypothesis of AD.
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Enfermedad de Alzheimer/patología , Corteza Cerebral/patología , Microglía/patología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Corteza Cerebral/metabolismo , Femenino , Ferritinas/metabolismo , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunohistoquímica , Lectinas Tipo C/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Proteínas de la Membrana/metabolismo , Microglía/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Análisis de la Célula IndividualRESUMEN
Brain pericytes and vascular smooth muscle cells (vSMCs) are a critical component of the neurovascular unit and are important in regulating cerebral blood flow and blood-brain barrier integrity. Identification of subtypes of mural cells in tissue and in vitro is important to any study of their function, therefore we identified distinct mural cell morphologies in neurologically normal post-mortem human brain. Further, the distribution of mural cell markers platelet-derived growth factor receptor-ß (PDGFRß), α-smooth muscle actin (αSMA), CD13, neural/glial antigen-2 (NG2), CD146 and desmin was examined. We determined that PDGFRß, NG2, CD13, and CD146 were expressed in capillary-associated pericytes. NG2, and CD13 were also present on vSMCs in large vessels, however abundant CD146 and desmin staining was also detected in vSMCs on large vessels, co-labelling with αSMA. To determine whether cultures recapitulated observations from tissue, primary human brain pericytes derived from neurologically normal autopsies were analysed for the presence of pericyte markers by immunocytochemistry, western blotting and qPCR. The proteins observed in brain pericytes in tissue (PDGFRß, αSMA, desmin, CD146, CD13, and NG2) were present in vitro, validating a panel of proteins that can be used to label brain pericytes and vSMCs in tissue and in vitro. Finally, we showed that the proteins CD146 and desmin that are expressed on large vessels in situ, are also selective markers of a smooth muscle cell phenotype in vitro.