Metabolic Reprogramming in Astrocytes Distinguishes Region-Specific Neuronal Susceptibility in Huntington Mice.
Cell metabolism
The basis for region-specific neuronal toxicity in Huntington disease is unknown. Here, we show that region-specific neuronal vulnerability is a substrate-driven response in astrocytes. Glucose is low in HdhQ(150/150) animals, and astrocytes in each brain region adapt by metabolically reprogramming their mitochondria to use endogenous, non-glycolytic metabolites as an alternative fuel. Each region is characterized by distinct metabolic pools, and astrocytes adapt accordingly. The vulnerable striatum is enriched in fatty acids, and mitochondria reprogram by oxidizing them as an energy source but at the cost of escalating reactive oxygen species (ROS)-induced damage. The cerebellum is replete with amino acids, which are precursors for glucose regeneration through the pentose phosphate shunt or gluconeogenesis pathways. ROS is not elevated, and this region sustains little damage. While mhtt expression imposes disease stress throughout the brain, sensitivity or resistance arises from an adaptive stress response, which is inherently region specific. Metabolic reprogramming may have relevance to other diseases.
10.1016/j.cmet.2019.03.004
Beclin1-driven autophagy modulates the inflammatory response of microglia via NLRP3.
Houtman Judith,Freitag Kiara,Gimber Niclas,Schmoranzer Jan,Heppner Frank L,Jendrach Marina
The EMBO journal
Alzheimer's disease is characterized not only by extracellular amyloid plaques and neurofibrillary tangles, but also by microglia-mediated neuroinflammation. Recently, autophagy has been linked to the regulation of the inflammatory response. Thus, we investigated how an impairment of autophagy mediated by BECN1/Beclin1 reduction, as described in Alzheimer's disease patients, would influence cytokine production of microglia. Acutely stimulated microglia from mice exhibited increased expression of IL-1beta and IL-18 compared to wild-type microglia. mice also contained enhanced IL-1beta levels. The investigation of the IL-1beta/IL-18 processing pathway showed an elevated number of cells with inflammasomes and increased levels of NLRP3 and cleaved CASP1/Caspase1 in microglia. Super-resolation microscopy revealed a very close association of NLRP3 aggregates and LC3-positive vesicles. Interestingly, CALCOCO2 colocalized with NLRP3 and its downregulation increased IL-1beta release. These data support the notion that selective autophagy can impact microglia activation by modulating IL-1beta and IL-18 production via NLRP3 degradation and thus present a mechanism how impaired autophagy could contribute to neuroinflammation in Alzheimer's disease.
10.15252/embj.201899430
The microbiota protects from viral-induced neurologic damage through microglia-intrinsic TLR signaling.
eLife
Symbiotic microbes impact the function and development of the central nervous system (CNS); however, little is known about the contribution of the microbiota during viral-induced neurologic damage. We identify that commensals aid in host defense following infection with a neurotropic virus through enhancing microglia function. Germfree mice or animals that receive antibiotics are unable to control viral replication within the brain leading to increased paralysis. Microglia derived from germfree or antibiotic-treated animals cannot stimulate viral-specific immunity and microglia depletion leads to worsened demyelination. Oral administration of toll-like receptor (TLR) ligands to virally infected germfree mice limits neurologic damage. Homeostatic activation of microglia is dependent on intrinsic signaling through TLR4, as disruption of TLR4 within microglia, but not the entire CNS (excluding microglia), leads to increased viral-induced clinical disease. This work demonstrates that gut immune-stimulatory products can influence microglia function to prevent CNS damage following viral infection.
10.7554/eLife.47117
Absence of CX3CR1 impairs the internalization of Tau by microglia.
Bolós Marta,Llorens-Martín María,Perea Juan Ramón,Jurado-Arjona Jerónimo,Rábano Alberto,Hernández Félix,Avila Jesús
Molecular neurodegeneration
BACKGROUND:Extracellular Tau is toxic for neighboring cells, and it contributes to the progression of AD. The CX3CL1/CX3CR1 axis is an important neuron/microglia communication mechanism. METHODS:We studied Tau clearance by microglia both in vitro (microglia primary cultures treated with Cy5-Tau, affinity chromatography to study the binding of Tau to CX3CR1, and Tau-CX3CL1 competition assays) and in vivo (stereotaxic injection of Cy5-Tau into WT and CX3CR1 mice). The expression of CX3CR1, CX3CL1 and the microglial phagocytic phenotype were studied in brain tissue samples from AD patients. RESULTS:Tau binding to CX3CR1 triggers the internalization of the former by microglia, whereas S396 Tau phosphorylation decreases the binding affinity of this protein to CX3CR1. Of note, the progressive increase in the levels of phosho-Tau occurred in parallel with an increase in CX3CR1. In addition, our studies suggest that the phagocytic capacity of microglia in brain tissue samples from AD patients is decreased. Furthermore, the CX3CR1/CX3CL1 axis may be impaired in late stages of the disease. CONCLUSIONS:Our data suggest that the CX3CR1/CX3CL1 axis plays a key role in the phagocytosis of Tau by microglia in vitro and in vivo and that it is affected as AD progresses. Taken together, our results reveal CX3CR1 as a novel target for the clearance of extracellular Tau.
10.1186/s13024-017-0200-1
Microglia in Brain Development, Homeostasis, and Neurodegeneration.
Bohlen Christopher J,Friedman Brad A,Dejanovic Borislav,Sheng Morgan
Annual review of genetics
Advances in human genetics have implicated a growing number of genes in neurodegenerative diseases, providing insight into pathological processes. For Alzheimer disease in particular, genome-wide association studies and gene expression studies have emphasized the pathogenic contributions from microglial cells and motivated studies of microglial function/dysfunction. Here, we summarize recent genetic evidence for microglial involvement in neurodegenerative disease with a focus on Alzheimer disease, for which the evidence is most compelling. To provide context for these genetic discoveries, we discuss how microglia influence brain development and homeostasis, how microglial characteristics change in disease, and which microglial activities likely influence the course of neurodegeneration. In all, we aim to synthesize varied aspects of microglial biology and highlight microglia as possible targets for therapeutic interventions in neurodegenerative disease.
10.1146/annurev-genet-112618-043515
Interleukin-1 receptor associated kinase (IRAK)-M -mediated type 2 microglia polarization ameliorates the severity of experimental autoimmune encephalomyelitis (EAE).
Liu Baozhu,Gu Yong,Pei Shanshan,Peng Yu,Chen Jinyu,Pham Lan V,Shen Hai-Ying,Zhang Jun,Wang Honghao
Journal of autoimmunity
Toll-like receptor 4 (TLR4) play a key role in activating the innate immune system during pathogen recognition. In the pathogenesis of multiple sclerosis (MS), activated TLR4 together with myeloid differentiation primary response gene 88 (MyD88) produce an inflammatory microenvironment that promotes the differentiation of microglia into the M1 phenotype, who plays a key role in the pathogenesis of MS. Interleukin-1 receptor-associated kinase (IRAK)-M is specifically expressed in microglia in central nervous system (CNS) and act as a negative regulator of TLR4-MyD88 signaling pathway. Moreover, previous studies have shown that IRAK-M promotes the differentiation of type 2 microglia; however, its role in MS has not been explored. In the present study, we demonstrated that IRAK-M expression is elevated during EAE, and IRAK-M mice significantly accelerated course and increased severity of disease, accompanied by a visible increase of the M1 microglia infiltrated. In conclusion, these data indicates that IRAK-M significantly improves EAE onset through down-regulation of the TLR4-MyD88 signaling pathway, which finally leads to differentiation of M2 phenotype in the microglia. Our study suggests that IRAK-M may be a potential therapeutic target for the treatment of MS.
10.1016/j.jaut.2019.04.020
PU.1 regulates Alzheimer's disease-associated genes in primary human microglia.
Rustenhoven Justin,Smith Amy M,Smyth Leon C,Jansson Deidre,Scotter Emma L,Swanson Molly E V,Aalderink Miranda,Coppieters Natacha,Narayan Pritika,Handley Renee,Overall Chris,Park Thomas I H,Schweder Patrick,Heppner Peter,Curtis Maurice A,Faull Richard L M,Dragunow Mike
Molecular neurodegeneration
BACKGROUND:Microglia play critical roles in the brain during homeostasis and pathological conditions. Understanding the molecular events underpinning microglial functions and activation states will further enable us to target these cells for the treatment of neurological disorders. The transcription factor PU.1 is critical in the development of myeloid cells and a major regulator of microglial gene expression. In the brain, PU.1 is specifically expressed in microglia and recent evidence from genome-wide association studies suggests that reductions in PU.1 contribute to a delayed onset of Alzheimer's disease (AD), possibly through limiting neuroinflammatory responses. METHODS:To investigate how PU.1 contributes to immune activation in human microglia, microarray analysis was performed on primary human mixed glial cultures subjected to siRNA-mediated knockdown of PU.1. Microarray hits were confirmed by qRT-PCR and immunocytochemistry in both mixed glial cultures and isolated microglia following PU.1 knockdown. To identify attenuators of PU.1 expression in microglia, high throughput drug screening was undertaken using a compound library containing FDA-approved drugs. NanoString and immunohistochemistry was utilised to investigate the expression of PU.1 itself and PU.1-regulated mediators in primary human brain tissue derived from neurologically normal and clinically and pathologically confirmed cases of AD. RESULTS:Bioinformatic analysis of gene expression upon PU.1 silencing in mixed glial cultures revealed a network of modified AD-associated microglial genes involved in the innate and adaptive immune systems, particularly those involved in antigen presentation and phagocytosis. These gene changes were confirmed using isolated microglial cultures. Utilising high throughput screening of FDA-approved compounds in mixed glial cultures we identified the histone deacetylase inhibitor vorinostat as an effective attenuator of PU.1 expression in human microglia. Further characterisation of vorinostat in isolated microglial cultures revealed gene and protein changes partially recapitulating those seen following siRNA-mediated PU.1 knockdown. Lastly, we demonstrate that several of these PU.1-regulated genes are expressed by microglia in the human AD brain in situ. CONCLUSIONS:Collectively, these results suggest that attenuating PU.1 may be a valid therapeutic approach to limit microglial-mediated inflammatory responses in AD and demonstrate utility of vorinostat for this purpose.
10.1186/s13024-018-0277-1
Increased expression and altered subcellular distribution of cathepsin B in microglia induce cognitive impairment through oxidative stress and inflammatory response in mice.
Ni Junjun,Wu Zhou,Stoka Veronika,Meng Jie,Hayashi Yoshinori,Peters Christoph,Qing Hong,Turk Vito,Nakanishi Hiroshi
Aging cell
During normal aging, innate immunity progresses to a chronic state. However, how oxidative stress and chronic neuroinflammation arise during aging remains unclear. In this study, we found that genetic ablation of cathepsin B (CatB) in mice significantly reduced the generation of reactive oxygen species (ROS) and neuroinflammation and improved cognitive impairment during aging. In cultured microglia, pharmacological inhibition of CatB significantly reduced the generation of mitochondria-derived ROS and proinflammatory mediators induced by L-leucyl-L-leucine methyl ester (LLOMe), a lysosome-destabilizing agent. In the CatB-overexpressing microglia after treatment with LLOMe, which mimicked the aged microglia, CatB leaked in the cytosol is responsible for the degradation of the mitochondrial transcription factor A (TFAM), resulting in the increased generation of mitochondria-derived ROS and proinflammatory mediators through impaired mtDNA biosynthesis. Furthermore, intralateral ventricle injection of LLOMe-treated CatB-overexpressing microglia induced cognitive impairment in middle-aged mice. These results suggest that the increase and leakage of CatB in microglia during aging are responsible for the increased generation of mitochondria-derived ROS and proinflammatory mediators, culminating in memory impairment.
10.1111/acel.12856
Genome-wide RNAseq study of the molecular mechanisms underlying microglia activation in response to pathological tau perturbation in the rTg4510 tau transgenic animal model.
Wang Hong,Li Yupeng,Ryder John W,Hole Justin T,Ebert Philip J,Airey David C,Qian Hui-Rong,Logsdon Benjamin,Fisher Alice,Ahmed Zeshan,Murray Tracey K,Cavallini Annalisa,Bose Suchira,Eastwood Brian J,Collier David A,Dage Jeffrey L,Miller Bradley B,Merchant Kalpana M,O'Neill Michael J,Demattos Ronald B
Molecular neurodegeneration
BACKGROUND:Activation of microglia, the resident immune cells of the central nervous system, is a prominent pathological hallmark of Alzheimer's disease (AD). However, the gene expression changes underlying microglia activation in response to tau pathology remain elusive. Furthermore, it is not clear how murine gene expression changes relate to human gene expression networks. METHODS:Microglia cells were isolated from rTg4510 tau transgenic mice and gene expression was profiled using RNA sequencing. Four age groups of mice (2-, 4-, 6-, and 8-months) were analyzed to capture longitudinal gene expression changes that correspond to varying levels of pathology, from minimal tau accumulation to massive neuronal loss. Statistical and system biology approaches were used to analyze the genes and pathways that underlie microglia activation. Differentially expressed genes were compared to human brain co-expression networks. RESULTS:Statistical analysis of RNAseq data indicated that more than 4000 genes were differentially expressed in rTg4510 microglia compared to wild type microglia, with the majority of gene expression changes occurring between 2- and 4-months of age. These genes belong to four major clusters based on their temporal expression pattern. Genes involved in innate immunity were continuously up-regulated, whereas genes involved in the glutamatergic synapse were down-regulated. Up-regulated innate inflammatory pathways included NF-κB signaling, cytokine-cytokine receptor interaction, lysosome, oxidative phosphorylation, and phagosome. NF-κB and cytokine signaling were among the earliest pathways activated, likely driven by the RELA, STAT1 and STAT6 transcription factors. The expression of many AD associated genes such as APOE and TREM2 was also altered in rTg4510 microglia cells. Differentially expressed genes in rTg4510 microglia were enriched in human neurodegenerative disease associated pathways, including Alzheimer's, Parkinson's, and Huntington's diseases, and highly overlapped with the microglia and endothelial modules of human brain transcriptional co-expression networks. CONCLUSION:This study revealed temporal transcriptome alterations in microglia cells in response to pathological tau perturbation and provides insight into the molecular changes underlying microglia activation during tau mediated neurodegeneration.
10.1186/s13024-018-0296-y
Dual microglia effects on blood brain barrier permeability induced by systemic inflammation.
Haruwaka Koichiro,Ikegami Ako,Tachibana Yoshihisa,Ohno Nobuhiko,Konishi Hiroyuki,Hashimoto Akari,Matsumoto Mami,Kato Daisuke,Ono Riho,Kiyama Hiroshi,Moorhouse Andrew J,Nabekura Junichi,Wake Hiroaki
Nature communications
Microglia survey brain parenchyma, responding to injury and infections. Microglia also respond to systemic disease, but the role of blood-brain barrier (BBB) integrity in this process remains unclear. Using simultaneous in vivo imaging, we demonstrated that systemic inflammation induces CCR5-dependent migration of brain resident microglia to the cerebral vasculature. Vessel-associated microglia initially maintain BBB integrity via expression of the tight-junction protein Claudin-5 and make physical contact with endothelial cells. During sustained inflammation, microglia phagocytose astrocytic end-feet and impair BBB function. Our results show microglia play a dual role in maintaining BBB integrity with implications for elucidating how systemic immune-activation impacts neural functions.
10.1038/s41467-019-13812-z
Microglia Biology: One Century of Evolving Concepts.
Prinz Marco,Jung Steffen,Priller Josef
Cell
Microglia were first recognized as a distinct cell population in the CNS one century ago. For a long time, they were primarily considered to be phagocytes responsible for removing debris during CNS development and disease. More recently, advances in imaging and genetics and the advent of single-cell technologies provided new insights into the much more complex and fascinating biology of microglia. The ontogeny of microglia was identified, and their functions in health and disease were better defined. Although many questions about microglia and their roles in human diseases remain unanswered, the prospect of targeting microglia for the treatment of neurological and psychiatric disorders is tantalizing.
10.1016/j.cell.2019.08.053
Histone Deacetylases 1 and 2 Regulate Microglia Function during Development, Homeostasis, and Neurodegeneration in a Context-Dependent Manner.
Datta Moumita,Staszewski Ori,Raschi Elena,Frosch Maximilian,Hagemeyer Nora,Tay Tuan Leng,Blank Thomas,Kreutzfeldt Mario,Merkler Doron,Ziegler-Waldkirch Stephanie,Matthias Patrick,Meyer-Luehmann Melanie,Prinz Marco
Immunity
Microglia as tissue macrophages contribute to the defense and maintenance of central nervous system (CNS) homeostasis. Little is known about the epigenetic signals controlling microglia function in vivo. We employed constitutive and inducible mutagenesis in microglia to delete two class I histone deacetylases, Hdac1 and Hdac2. Prenatal ablation of Hdac1 and Hdac2 impaired microglial development. Mechanistically, the promoters of pro-apoptotic and cell cycle genes were hyperacetylated in absence of Hdac1 and Hdac2, leading to increased apoptosis and reduced survival. In contrast, Hdac1 and Hdac2 were not required for adult microglia survival during homeostasis. In a mouse model of Alzheimer's disease, deletion of Hdac1 and Hdac2 in microglia, but not in neuroectodermal cells, resulted in a decrease in amyloid load and improved cognitive impairment by enhancing microglial amyloid phagocytosis. Collectively, we report a role for epigenetic factors that differentially affect microglia development, homeostasis, and disease that could potentially be utilized therapeutically.
10.1016/j.immuni.2018.02.016
Microglia control the spread of neurotropic virus infection via P2Y12 signalling and recruit monocytes through P2Y12-independent mechanisms.
Fekete Rebeka,Cserép Csaba,Lénárt Nikolett,Tóth Krisztina,Orsolits Barbara,Martinecz Bernadett,Méhes Előd,Szabó Bálint,Németh Valéria,Gönci Balázs,Sperlágh Beáta,Boldogkői Zsolt,Kittel Ágnes,Baranyi Mária,Ferenczi Szilamér,Kovács Krisztina,Szalay Gergely,Rózsa Balázs,Webb Connor,Kovacs Gabor G,Hortobágyi Tibor,West Brian L,Környei Zsuzsanna,Dénes Ádám
Acta neuropathologica
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 from affected neurons. In turn, we identify microglia as key contributors to monocyte recruitment into the inflamed brain, which process is largely independent of P2Y12. P2Y12-positive microglia are also recruited to infected neurons in the human brain during viral encephalitis and both microglial responses and leukocyte numbers correlate with the severity of infection. Thus, our data identify a key role for microglial P2Y12 in defence against neurotropic viruses, whilst P2Y12-independent actions of microglia may contribute to neuroinflammation by facilitating monocyte recruitment to the sites of infection.
10.1007/s00401-018-1885-0
The identity and function of microglia in neurodegeneration.
Song Wilbur M,Colonna Marco
Nature immunology
The predominant type of immune cell in the brain is the microglia, a type of tissue-resident macrophage. In a variety of neurodegenerative settings, microglia alter their transcriptional profile, morphology and function in similar ways; thus, these activated cells have been called 'degeneration- or disease-associated microglia' (DAM). These activated microglia can perform different functions and exert both positive effects and negative effects in different mouse disease models. In humans, mutations in genes expressed in microglia are linked to various neurodegenerative diseases. Here we provide an overview of the common microglial response to neurodegeneration and key contributing pathways; delineate the multifaceted functions of activated microglia spanning various diseases; and discuss insights from the study of human disease-associated genes. We argue that strong evidence from both mouse models and human genetics causally links the function of activated microglia to neurodegeneration.
10.1038/s41590-018-0212-1
The pro-remyelination properties of microglia in the central nervous system.
Lloyd Amy F,Miron Veronique E
Nature reviews. Neurology
Microglia are resident macrophages of the CNS that are involved in its development, homeostasis and response to infection and damage. Microglial activation is a common feature of neurological disorders, and although in some instances this activation can be damaging, protective and regenerative functions of microglia have been revealed. The most prominent example of the regenerative functions is a role for microglia in supporting regeneration of myelin after injury, a process that is critical for axonal health and relevant to numerous disorders in which loss of myelin integrity is a prevalent feature, such as multiple sclerosis, Alzheimer disease and motor neuron disease. Although drugs that are intended to promote remyelination are entering clinical trials, the mechanisms by which remyelination is controlled and how microglia are involved are not completely understood. In this Review, we discuss work that has identified novel regulators of microglial activation - including molecular drivers, population heterogeneity and turnover - that might influence their pro-remyelination capacity. We also discuss therapeutic targeting of microglia as a potential approach to promoting remyelination.
10.1038/s41582-019-0184-2
Microglia Function in the Central Nervous System During Health and Neurodegeneration.
Colonna Marco,Butovsky Oleg
Annual review of immunology
Microglia are resident cells of the brain that regulate brain development, maintenance of neuronal networks, and injury repair. Microglia serve as brain macrophages but are distinct from other tissue macrophages owing to their unique homeostatic phenotype and tight regulation by the central nervous system (CNS) microenvironment. They are responsible for the elimination of microbes, dead cells, redundant synapses, protein aggregates, and other particulate and soluble antigens that may endanger the CNS. Furthermore, as the primary source of proinflammatory cytokines, microglia are pivotal mediators of neuroinflammation and can induce or modulate a broad spectrum of cellular responses. Alterations in microglia functionality are implicated in brain development and aging, as well as in neurodegeneration. Recent observations about microglia ontogeny combined with extensive gene expression profiling and novel tools to study microglia biology have allowed us to characterize the spectrum of microglial phenotypes during development, homeostasis, and disease. In this article, we review recent advances in our understanding of the biology of microglia, their contribution to homeostasis, and their involvement in neurodegeneration. Moreover, we highlight the complexity of targeting microglia for therapeutic intervention in neurodegenerative diseases.
10.1146/annurev-immunol-051116-052358
Microglia, Lifestyle Stress, and Neurodegeneration.
Madore Charlotte,Yin Zhuoran,Leibowitz Jeffrey,Butovsky Oleg
Immunity
Recent years have witnessed a revolution in our understanding of microglia biology, including their major role in the etiology and pathogenesis of neurodegenerative diseases. Technological advances have enabled the identification of microglial signatures in health and disease, including the development of new models to investigate and manipulate human microglia in vivo in the context of disease. In parallel, genetic association studies have identified several gene risk factors associated with Alzheimer's disease that are specifically or highly expressed by microglia in the central nervous system (CNS). Here, we discuss evidence for the effect of stress, diet, sleep patterns, physical activity, and microbiota composition on microglia biology and consider how lifestyle might influence an individual's predisposition to neurodegenerative diseases. We discuss how different lifestyles and environmental factors might regulate microglia, potentially leading to increased susceptibility to neurodegenerative disease, and we highlight the need to investigate the contribution of modern environmental factors on microglia modulation in neurodegeneration.
10.1016/j.immuni.2019.12.003
A Breakdown in Metabolic Reprogramming Causes Microglia Dysfunction in Alzheimer's Disease.
Baik Sung Hoon,Kang Seokjo,Lee Woochan,Choi Hayoung,Chung Sunwoo,Kim Jong-Il,Mook-Jung Inhee
Cell metabolism
Reactive microglia are a major pathological feature of Alzheimer's disease (AD). However, the exact role of microglia in AD pathogenesis is still unclear. Here, using metabolic profiling, we found that exposure to amyloid-β triggers acute microglial inflammation accompanied by metabolic reprogramming from oxidative phosphorylation to glycolysis. It was dependent on the mTOR-HIF-1α pathway. However, once activated, microglia reached a chronic tolerant phase as a result of broad defects in energy metabolisms and subsequently diminished immune responses, including cytokine secretion and phagocytosis. Using genome-wide RNA sequencing and multiphoton microscopy techniques, we further identified metabolically defective microglia in 5XFAD mice, an AD mouse model. Finally, we showed that metabolic boosting with recombinant interferon-γ treatment reversed the defective glycolytic metabolism and inflammatory functions of microglia, thereby mitigating the AD pathology of 5XFAD mice. Collectively, metabolic reprogramming is crucial for microglial functions in AD, and modulating metabolism might be a new therapeutic strategy for AD.
10.1016/j.cmet.2019.06.005