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Stroke is a leading cause of permanent disability worldwide. Despite intensive research over the last decades, key anti-inflammatory strategies that have proven beneficial in pre-clinical animal models have often failed in translation. The importance of neutrophils as pro- and anti-inflammatory peripheral immune cells has often been overlooked in ischemic stroke. However, neutrophils rapidly infiltrate into the brain parenchyma after stroke and secrete an array of pro-inflammatory factors including reactive oxygen species, proteases, cytokines, and chemokines exacerbating damage. In this study, we demonstrate the neuroprotective and anti-inflammatory effect of benserazide, a clinically used DOPA decarboxylase inhibitor, using both in vitro models of inflammation and in vivo mouse models of focal cerebral ischemia. Benserazide significantly attenuated PMA-induced NETosis in isolated human neutrophils. Furthermore, benserazide was able to protect both SH-SY5Y and iPSC-derived human cortical neurons when challenged with activated neutrophils demonstrating the clinical relevance of this study. Additional in vitro data suggest the ability of benserazide to polarize macrophages towards M2-phenotypes following LPS stimulation. Neuroprotective effects of benserazide are further demonstrated by in vivo studies where peripheral administration of benserazide significantly attenuated neutrophil infiltration into the brain, altered microglia/macrophage phenotypes, and improved the behavioral outcome post-stroke. Overall, our data suggest that benserazide could serve as a drug candidate for the treatment of ischemic stroke. The importance of our results for future clinical trials is further underlined as benserazide has been approved by the European Medicines Agency as a safe and effective treatment in Parkinson's disease when combined with levodopa.
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Benserazida , Accidente Cerebrovascular Isquémico , Fármacos Neuroprotectores , Neutrófilos , Benserazida/farmacología , Animales , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Humanos , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/inmunología , Accidente Cerebrovascular Isquémico/metabolismo , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Modelos Animales de Enfermedad , Recuperación de la Función/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
Discovery of meningeal lymphatic vessels (LVs) in the dura mater, also known as dural LVs (dLVs) that depend on vascular endothelial growth factor C expression, has raised interest in their possible involvement in Alzheimer's disease (AD). Here we find that in the APdE9 and 5xFAD mouse models of AD, dural amyloid-ß (Aß) is confined to blood vessels and dLV morphology or function is not altered. The induction of sustained dLV atrophy or hyperplasia in the AD mice by blocking or overexpressing vascular endothelial growth factor C, impaired or improved, respectively, macromolecular cerebrospinal fluid (CSF) drainage to cervical lymph nodes. Yet, sustained manipulation of dLVs did not significantly alter the overall brain Aß plaque load. Moreover, dLV atrophy did not alter the behavioral phenotypes of the AD mice, but it improved CSF-to-blood drainage. Our results indicate that sustained dLV manipulation does not affect Aß deposition in the brain and that compensatory mechanisms promote CSF clearance.
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Mutations in ubiquitously expressed presenilin genes (PSENs) lead to early-onset familial Alzheimer's disease (FAD), but patients carrying the mutation also suffer from heart diseases. To elucidate the cardiac myocyte specific effects of PSEN ΔE9, we studied cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) from patients carrying AD-causing PSEN1 exon 9 deletion (PSEN1 ΔE9). When compared with their isogenic controls, PSEN1 ΔE9 cardiomyocytes showed increased sarcoplasmic reticulum (SR) Ca2+ leak that was resistant to blockage of ryanodine receptors (RyRs) by tetracaine or inositol-3-reseceptors (IP3Rs) by 2-ABP. The SR Ca2+ leak did not affect electrophysiological properties of the hiPSC-CMs, but according to experiments and in silico simulations the leak induces a diastolic buildup of [Ca2+] near the perinuclear SR and reduces the releasable Ca2+ during systole. This demonstrates that PSEN1 ΔE9 induced SR Ca2+ leak has specific effects in iPSC-CMs, reflecting their unique structural and calcium signaling features. The results shed light on the physiological and pathological mechanisms of PSEN1 in cardiac myocytes and explain the intricacies of comorbidity associated with AD-causing mutations in PSEN1.
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Señalización del Calcio , Calcio , Células Madre Pluripotentes Inducidas , Mutación , Miocitos Cardíacos , Presenilina-1 , Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Retículo Sarcoplasmático/metabolismo , Calcio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
Central nervous system infections have been suggested as a possible cause for neurodegenerative diseases, particularly sporadic cases. They trigger neuroinflammation which is considered integrally involved in neurodegenerative processes. In this review, we will look at data linking a variety of viral, bacterial, fungal, and protozoan infections to Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis and unspecified dementia. This narrative review aims to bring together a broad range of data currently supporting the involvement of central nervous system infections in the development of neurodegenerative diseases. The idea that no single pathogen or pathogen group is responsible for neurodegenerative diseases will be discussed. Instead, we suggest that a wide range of susceptibility factors may make individuals differentially vulnerable to different infectious pathogens and subsequent pathologies.
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Infecciones del Sistema Nervioso Central , Enfermedades Neurodegenerativas , Humanos , Enfermedades Neurodegenerativas/patología , AnimalesRESUMEN
Timely relief of edema and clearance of waste products, as well as promotion of anti-inflammatory immune responses, reduce ischemic stroke pathology, and attenuate harmful long-term effects post-stroke. The discovery of an extensive and functional lymphatic vessel system in the outermost meningeal layer, dura mater, has opened up new possibilities to facilitate post-stroke recovery by inducing dural lymphatic vessel (dLV) growth via a single injection of a vector encoding vascular endothelial growth factor C (VEGF-C). In the present study, we aimed to improve post-stroke outcomes by inducing dLV growth in mice. We injected mice with a single intracerebroventricular dose of adeno-associated viral particles encoding VEGF-C before subjecting them to transient middle cerebral artery occlusion (tMCAo). Behavioral testing, Gadolinium (Gd) contrast agent-enhanced magnetic resonance imaging (MRI), and immunohistochemical analysis were performed to define the impact of VEGF-C on the post-stroke outcome. VEGF-C improved stroke-induced behavioral deficits, such as gait disturbances and neurological deficits, ameliorated post-stroke inflammation, and enhanced an alternative glial immune response. Importantly, VEGF-C treatment increased the drainage of brain interstitial fluid (ISF) and cerebrospinal fluid (CSF), as shown by Gd-enhanced MRI. These outcomes were closely associated with an increase in the growth of dLVs around the region where we observed increased vefgc mRNA expression within the brain, including the olfactory bulb, cortex, and cerebellum. Strikingly, VEGF-C-treated ischemic mice exhibited a faster and stronger Gd-signal accumulation in ischemic core area and an enhanced fluid outflow via the cribriform plate. In conclusion, the VEGF-C-induced dLV growth improved the overall outcome post-stroke, indicating that VEGF-C has potential to be included in the treatment strategies of post-ischemic stroke. However, to maximize the therapeutic potential of VEGF-C treatment, further studies on the impact of an enhanced dural lymphatic system at clinically relevant time points are essential.
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Background: Development of synaptic activity is a key neuronal characteristic that relies largely on interactions between neurons and astrocytes. Although astrocytes have known roles in regulating synaptic function and malfunction, the use of human- or donor-specific astrocytes in disease models is still rare. Rodent astrocytes are routinely used to enhance neuronal activity in cell cultures, but less is known about how human astrocytes influence neuronal activity. Methods: We established human induced pluripotent stem cell-derived neuron-astrocyte cocultures and studied their functional development on microelectrode array. We used cell lines from 5 neurotypical control individuals and 3 pairs of monozygotic twins discordant for schizophrenia. A method combining NGN2 overexpression and dual SMAD inhibition was used for neuronal differentiation. The neurons were cocultured with human induced pluripotent stem cell-derived astrocytes differentiated from 6-month-old astrospheres or rat astrocytes. Results: We found that the human induced pluripotent stem cell-derived cocultures developed complex network bursting activity similar to neuronal cocultures with rat astrocytes. However, the effect of NMDA receptors on neuronal network burst frequency (NBF) differed between cocultures containing human or rat astrocytes. By using cocultures derived from patients with schizophrenia and unaffected individuals, we found lowered NBF in the affected cells. We continued by demonstrating how astrocytes from an unaffected individual rescued the lowered NBF in the affected neurons by increasing NMDA receptor activity. Conclusions: Our results indicate that astrocytes participate in the regulation of neuronal NBF through a mechanism that involves NMDA receptors. These findings shed light on the importance of using human and donor-specific astrocytes in disease modeling.
Nerve cell connections called synapses are formed in interaction with astrocytes, the main non-neuronal cell type of the brain. In vitro work commonly uses rodent astrocytes to enhance activity in human-derived neuronal cell cultures, but differences in using rodent versus human astrocytes are not well understood. We found that the electrical activity of nerve cell networks in cultures consisting of human cortical nerve cells and human astrocytes is altered when the astrocytes are from patients with schizophrenia, relative to neurotypical individuals. The effect of human astrocytes on these networks differed from rodent astrocytes, indicating the potential importance of a fully human culture system.
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Schizophrenia (SCZ) is a neuropsychiatric disorder, caused by a combination of genetic and environmental factors. The etiology behind the disorder remains elusive although it is hypothesized to be associated with the aberrant response to neurotransmitters, such as dopamine and glutamate. Therefore, investigating the link between dysregulated metabolites and distorted neurodevelopment holds promise to offer valuable insights into the underlying mechanism of this complex disorder. In this study, we aimed to explore a presumed correlation between the transcriptome and the metabolome in a SCZ model based on patient-derived induced pluripotent stem cells (iPSCs). For this, iPSCs were differentiated towards cortical neurons and samples were collected longitudinally at various developmental stages, reflecting neuroepithelial-like cells, radial glia, young and mature neurons. The samples were analyzed by both RNA-sequencing and targeted metabolomics and the two modalities were used to construct integrative networks in silico. This multi-omics analysis revealed significant perturbations in the polyamine and gamma-aminobutyric acid (GABA) biosynthetic pathways during rosette maturation in SCZ lines. We particularly observed the downregulation of the glutamate decarboxylase encoding genes GAD1 and GAD2, as well as their protein product GAD65/67 and their biochemical product GABA in SCZ samples. Inhibition of ornithine decarboxylase resulted in further decrease of GABA levels suggesting a compensatory activation of the ornithine/putrescine pathway as an alternative route for GABA production. These findings indicate an imbalance of cortical excitatory/inhibitory dynamics occurring during early neurodevelopmental stages in SCZ. Our study supports the hypothesis of disruption of inhibitory circuits to be causative for SCZ and establishes a novel in silico approach that enables for integrative correlation of metabolic and transcriptomic data of psychiatric disease models.
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Alzheimer's disease (AD) is the most prevalent cause of dementia characterized by a progressive cognitive decline. Addressing neuroinflammation represents a promising therapeutic avenue to treat AD; however, the development of effective antineuroinflammatory compounds is often hindered by their limited blood-brain barrier (BBB) permeability. Consequently, there is an urgent need for accurate, preclinical AD patient-specific BBB models to facilitate the early identification of immunomodulatory drugs capable of efficiently crossing the human AD BBB. This study presents a unique approach to BBB drug permeability screening as it utilizes the familial AD patient-derived induced brain endothelial-like cell (iBEC)-based model, which exhibits increased disease relevance and serves as an improved BBB drug permeability assessment tool when compared to traditionally employed in vitro models. To demonstrate its utility as a small molecule drug candidate screening platform, we investigated the effects of diacetylbis(N(4)-methylthiosemicarbazonato)copper(II) (CuII(atsm)) and a library of metal bis(thiosemicarbazone) complexesâa class of compounds exhibiting antineuroinflammatory therapeutic potential in neurodegenerative disorders. By evaluating the toxicity, cellular accumulation, and permeability of those compounds in the AD patient-derived iBEC, we have identified 3,4-hexanedione bis(N(4)-methylthiosemicarbazonato)copper(II) (CuII(dtsm)) as a candidate with good transport across the AD BBB. Furthermore, we have developed a multiplex approach where AD patient-derived iBEC were combined with immune modulators TNFα and IFNγ to establish an in vitro model representing the characteristic neuroinflammatory phenotype at the patient's BBB. Here, we observed that treatment with CuII(dtsm) not only reduced the expression of proinflammatory cytokine genes but also reversed the detrimental effects of TNFα and IFNγ on the integrity and function of the AD iBEC monolayer. This suggests a novel pathway through which copper bis(thiosemicarbazone) complexes may exert neurotherapeutic effects on AD by mitigating BBB neuroinflammation and related BBB integrity impairment. Together, the presented model provides an effective and easily scalable in vitro BBB platform for screening AD drug candidates. Its improved translational potential makes it a valuable tool for advancing the development of metal-based compounds aimed at modulating neuroinflammation in AD.
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Enfermedad de Alzheimer , Tiosemicarbazonas , Humanos , Barrera Hematoencefálica/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Cobre/metabolismo , Enfermedades Neuroinflamatorias , Tiosemicarbazonas/farmacología , Tiosemicarbazonas/metabolismo , Tiosemicarbazonas/uso terapéutico , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Several lines of evidence indicate the involvement of neuroinflammatory processes in the pathophysiology of schizophrenia (SCZ). Microglia are brain resident immune cells responding toward invading pathogens and injury-related products, and additionally, have a critical role in improving neurogenesis and synaptic functions. Aberrant activation of microglia in SCZ is one of the leading hypotheses for disease pathogenesis, but due to the lack of proper human cell models, the role of microglia in SCZ is not well studied. We used monozygotic twins discordant for SCZ and healthy individuals to generate human induced pluripotent stem cell-derived microglia to assess the transcriptional and functional differences in microglia between healthy controls, affected twins and unaffected twins. The microglia from affected twins had increased expression of several common inflammation-related genes compared to healthy individuals. Microglia from affected twins had also reduced response to interleukin 1 beta (IL1ß) treatment, but no significant differences in migration or phagocytotic activity. Ingenuity Pathway Analysis (IPA) showed abnormalities related to extracellular matrix signaling. RNA sequencing predicted downregulation of extracellular matrix structure constituent Gene Ontology (GO) terms and hepatic fibrosis pathway activation that were shared by microglia of both affected and unaffected twins, but the upregulation of major histocompatibility complex (MHC) class II receptors was observed only in affected twin microglia. Also, the microglia of affected twins had heterogeneous response to clozapine, minocycline, and sulforaphane treatments. Overall, despite the increased expression of inflammatory genes, we observed no clear functional signs of hyperactivation in microglia from patients with SCZ. We conclude that microglia of the patients with SCZ have gene expression aberrations related to inflammation response and extracellular matrix without contributing to increased microglial activation.
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Brain functionality relies on finely tuned regulation of gene expression by networks of non-coding RNAs (ncRNAs) such as the one composed by the circular RNA ciRS-7 (also known as CDR1as), the microRNA miR-7, and the long ncRNA Cyrano. We describe ischemia-induced alterations in the ncRNA network both in vitro and in vivo and in transgenic mice lacking ciRS-7 or miR-7. Our data show that cortical neurons downregulate ciRS-7 and Cyrano and upregulate miR-7 expression during ischemia. Mice lacking ciRS-7 exhibit reduced lesion size and motor impairment, while the absence of miR-7 alone results in increased ischemia-induced neuronal death. Moreover, miR-7 levels in pyramidal excitatory neurons regulate neurite morphology and glutamatergic signaling, suggesting a potential molecular link to the in vivo phenotype. Our data reveal the role of ciRS-7 and miR-7 in modulating ischemic stroke outcome, shedding light on the pathophysiological function of intracellular ncRNA networks in the brain.
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MicroARNs , ARN Largo no Codificante , Ratones , Animales , MicroARNs/genética , MicroARNs/metabolismo , ARN no Traducido , ARN Circular , Transducción de Señal , ARN Largo no Codificante/metabolismo , IsquemiaRESUMEN
Central nervous system (CNS) accumulation of fibrillary deposits made of Amyloid ß (Aß), hyperphosphorylated Tau or α-synuclein (α-syn), present either alone or in the form of mixed pathology, characterizes the most common neurodegenerative diseases (NDDs) as well as the aging brain. Compelling evidence supports that acute neurological disorders, such as traumatic brain injury (TBI) and stroke, are also accompanied by increased deposition of toxic Aß, Tau and α-syn species. While the contribution of these pathological proteins to neurodegeneration has been experimentally ascertained, the cellular and molecular mechanisms driving Aß, Tau and α-syn-related brain damage remain to be fully clarified. In the last few years, studies have shown that Aß, Tau and α-syn may contribute to neurodegeneration also by inducing and/or promoting blood-brain barrier (BBB) disruption. These pathological proteins can affect BBB integrity either directly by affecting key BBB components such as pericytes and endothelial cells (ECs) or indirectly, by promoting brain macrophages activation and dysfunction. Here, we summarize and critically discuss key findings showing how Aß, Tau and α-syn can contribute to BBB damage in most common NDDs, TBI and stroke. We also highlight the need for a deeper characterization of the role of these pathological proteins in the activation and dysfunction of brain macrophages, pericytes and ECs to improve diagnosis and treatment of acute and chronic neurological disorders.
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Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Accidente Cerebrovascular , Humanos , alfa-Sinucleína/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/patología , Células Endoteliales/patología , Enfermedades Neurodegenerativas/patología , Accidente Cerebrovascular/patología , Proteínas tau/metabolismoRESUMEN
BACKGROUND: Air pollution is recognized as an emerging environmental risk factor for neurological diseases. Large-scale epidemiological studies associate traffic-related particulate matter (PM) with impaired cognitive functions and increased incidence of neurodegenerative diseases such as Alzheimer's disease. Inhaled components of PM may directly invade the brain via the olfactory route, or act through peripheral system responses resulting in inflammation and oxidative stress in the brain. Microglia are the immune cells of the brain implicated in the progression of neurodegenerative diseases. However, it remains unknown how PM affects live human microglia. RESULTS: Here we show that two different PMs derived from exhausts of cars running on EN590 diesel or compressed natural gas (CNG) alter the function of human microglia-like cells in vitro. We exposed human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMGLs) to traffic related PMs and explored their functional responses. Lower concentrations of PMs ranging between 10 and 100 µg ml-1 increased microglial survival whereas higher concentrations became toxic over time. Both tested pollutants impaired microglial phagocytosis and increased secretion of a few proinflammatory cytokines with distinct patterns, compared to lipopolysaccharide induced responses. iMGLs showed pollutant dependent responses to production of reactive oxygen species (ROS) with CNG inducing and EN590 reducing ROS production. CONCLUSIONS: Our study indicates that traffic-related air pollutants alter the function of human microglia and warrant further studies to determine whether these changes contribute to adverse effects in the brain and on cognition over time. This study demonstrates human iPSC-microglia as a valuable tool to study functional microglial responses to environmental agents.
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Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Humanos , Material Particulado/toxicidad , Material Particulado/análisis , Microglía/química , Células Madre Pluripotentes Inducidas/química , Automóviles , Especies Reactivas de Oxígeno , Emisiones de Vehículos/toxicidad , Emisiones de Vehículos/análisisRESUMEN
INTRODUCTION: Historically, astrocytes were seen primarily as a supportive cell population within the brain; with neurodegenerative disease research focusing exclusively on malfunctioning neurons. However, astrocytes perform numerous tasks that are essential for maintenance of the central nervous system`s complex processes. Disruption of these functions can have negative consequences; hence, it is unsurprising to observe a growing amount of evidence for the essential role of astrocytes in the development and progression of neurodegenerative diseases. Targeting astrocytic functions may serve as a potential disease-modifying drug therapy in the future. AREAS COVERED: The present review emphasizes the key astrocytic functions associated with neurodegenerative diseases and explores the possibility of pharmaceutical interventions to modify these processes. In addition, the authors provide an overview of current advancement in this field by including studies of possible drug candidates. EXPERT OPINION: Glial research has experienced a significant renaissance in the last quarter-century. Understanding how disease pathologies modify or are caused by astrocyte functions is crucial when developing treatments for brain diseases. Future research will focus on building advanced models that can more precisely correlate to the state in the human brain, with the goal of routinely testing therapies in these models.
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Astrocitos , Desarrollo de Medicamentos , Enfermedades Neurodegenerativas , Humanos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/fisiopatología , Animales , Desarrollo de Medicamentos/métodos , Terapia Molecular Dirigida , Progresión de la Enfermedad , Encéfalo/fisiopatología , Neuronas/efectos de los fármacosRESUMEN
The blood-brain barrier (BBB) has a key function in maintaining homeostasis in the brain, partly modulated by transporters, which are highly expressed in brain endothelial cells (BECs). Transporters mediate the uptake or efflux of compounds to and from the brain and they can also challenge the delivery of drugs for the treatment of Alzheimer's disease (AD). Currently there is a limited understanding of changes in BBB transporters in AD. To investigate this, we generated brain endothelial-like cells (iBECs) from induced pluripotent stem cells (iPSCs) with familial AD (FAD) Presenilin 1 (PSEN1) mutation and identified AD-specific differences in transporter expression compared to control (ctrl) iBECs. We first characterized the expression levels of 12 BBB transporters in AD-, Ctrl-, and isogenic (PSEN1 corrected) iBECs to identify any AD specific differences. We then exposed the cells to focused ultrasound (FUS) in the absence (FUSonly) or presence of microbubbles (MB) (FUS+MB), which is a novel therapeutic method that can be used to transiently open the BBB to increase drug delivery into the brain, however its effects on BBB transporter expression are largely unknown. Following FUSonly and FUS+MB, we investigated whether the expression or activity of key transporters could be modulated. Our findings demonstrate that PSEN1 mutant FAD (PSEN1AD) possess phenotypical differences compared to control iBECs in BBB transporter expression and function. Additionally, we show that FUSonly and FUS+MB can modulate BBB transporter expression and functional activity in iBECs, having potential implications on drug penetration and amyloid clearance. These findings highlight the differential responses of patient cells to FUS treatment, with patient-derived models likely providing an important tool for modelling therapeutic effects of FUS.
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Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/terapia , Enfermedad de Alzheimer/metabolismo , Células Endoteliales/metabolismo , Preparaciones Farmacéuticas/metabolismo , Encéfalo/metabolismo , Barrera Hematoencefálica , Proteínas de Transporte de Membrana/metabolismoRESUMEN
LRRK2-G2019S is one of the most common Parkinson's disease (PD)-associated mutations and has been shown to alter microglial functionality. However, the impact of LRRK2-G2019S on transcriptional profile of human induced pluripotent stem cell-derived microglia-like cells (iMGLs) and how it corresponds to microglia in idiopathic PD brain is not known. Here we demonstrate that LRRK2-G2019S carrying iMGL recapitulate aspects of the transcriptional signature of human idiopathic PD midbrain microglia. LRRK2-G2019S induced subtle and donor-dependent alterations in iMGL mitochondrial respiration, phagocytosis and cytokine secretion. Investigation of microglial transcriptional state in the midbrains of PD patients revealed a subset of microglia with a transcriptional overlap between the in vitro PD-iMGL and human midbrain PD microglia. We conclude that LRRK2-G2019S iMGL serve as a model to study PD-related effects in human microglia.
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Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Humanos , Microglía , Enfermedad de Parkinson/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación , Expresión GénicaRESUMEN
Metabolic reprogramming is a hallmark of the immune cells in response to inflammatory stimuli. This metabolic process involves a switch from oxidative phosphorylation (OXPHOS) to glycolysis or alterations in other metabolic pathways. However, most of the experimental findings have been acquired in murine immune cells, and little is known about the metabolic reprogramming of human microglia. In this study, we investigate the transcriptomic, proteomic, and metabolic profiles of mouse and iPSC-derived human microglia challenged with the TLR4 agonist LPS. We demonstrate that both species display a metabolic shift and an overall increased glycolytic gene signature in response to LPS treatment. The metabolic reprogramming is characterized by the upregulation of hexokinases in mouse microglia and phosphofructokinases in human microglia. This study provides a direct comparison of metabolism between mouse and human microglia, highlighting the species-specific pathways involved in immunometabolism and the importance of considering these differences in translational research.
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Lipopolisacáridos , Microglía , Animales , Ratones , Humanos , Microglía/metabolismo , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Proteómica , Fosforilación Oxidativa , GlucólisisRESUMEN
Systemic inflammation triggers protective as well as pro-inflammatory responses in the brain based on neuronal and/or cytokine signaling, and it associates with acutely and protractedly disrupted cognition. However, the multiple mechanisms underlying the peripheral-central inflammatory signaling are still not fully characterized. We used intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) in freely moving mice with chronically implanted electrodes for recording of local field potentials (LFP) and electrocorticography (ECoG) in the hippocampus and neocortex, respectively. We show here that a sudden switch in the mode of network activity occurred in both areas starting at 10-15 min after the LPS injection, simultaneously with a robust change from exploration to sickness behavior. This switch in cortical mode commenced before any elevations in pro-inflammatory cytokines IL-1ß, TNFα, CCL2 or IL-6 were detected in brain tissue. Thereafter, this mode dominated cortical activity for the recording period of 3 h, except for a partial and transient recovery around 40 min post-LPS. These effects were closely paralleled by changes in ECoG spectral entropy. Continuous recordings for up to 72 h showed a protracted attenuation in hippocampal activity, while neocortical activity recovered after 48 h. The acute sickness behavior recovered by 72 h post-LPS. Notably, urethane (1.3 mg/kg) administered prior to LPS blocked the early effect of LPS on cortical activity. However, experiments under urethane anesthesia which were started 24 h post-LPS (with neuroinflammation fully developed before application of urethane) showed that both theta-supratheta and fast gamma CA1 activity were reduced, DG delta activity was increased, and sharp-wave ripples were abolished. Finally, we observed that experimental compensation of inflammation-induced hypothermia 24-48 h post-LPS promoted seizures and status epilepticus; and that LPS decreased the threshold of kainate-provoked seizures beyond the duration of acute sickness behavior indicating post-acute inflammatory hyperexcitability. Taken together, the strikingly fast development and initial independence of brain cytokines of the LPS-induced cortical mode, its spectral characteristics and simultaneity in hippocampus and neocortex, as well as inhibition by pre-applied urethane, strongly suggest that the underlying mechanisms are based on activation of the afferent vagus nerve and its mainly cholinergic ascending projections to higher brain areas.
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Citocinas , Conducta de Enfermedad , Ratones , Animales , Citocinas/metabolismo , Lipopolisacáridos/toxicidad , Encéfalo/metabolismo , Inflamación/inducido químicamente , Convulsiones , Uretano/farmacologíaRESUMEN
2019 coronavirus disease (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms lasting from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to an infection of brain cells. We found that a small fraction of human induced pluripotent stem cell (iPSC)-derived neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), despite very low levels of its expression in neurons. The presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout infected cells, and an increase in the level of viral RNA in the culture medium (viral release) within the first 48 h of infection suggested that the infection was productive. Productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of the viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface spike protein, which can occur at the plasma membrane or from endosomes or lysosomes. We found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases, including transmembrane serine protease 2 (TMPRSS2). Inhibition of cathepsin L also did not significantly block infection. In contrast, the neuronal infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K), which regulates early to late endosome maturation. IMPORTANCE COVID-19 is a disease caused by the coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures, and encephalopathy, as well as anatomical abnormalities, such as changes in brain morphology. SARS-CoV-2 infection of the human brain has been documented, but it is unclear whether the observed neurological symptoms are linked to direct brain infection. The mechanism of virus entry into neurons has also not been characterized. Here, we investigated SARS-CoV-2 infection by using a human iPSC-derived neural cell model and found that a small fraction of cortical-like neurons was naturally susceptible to infection. The productive infection was ACE2 dependent and TMPRSS2 independent. We also found that the virus used the late endosomal and lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of cellular PIK5K.
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COVID-19 , Células Madre Pluripotentes Inducidas , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2 , COVID-19/fisiopatología , Endosomas/metabolismo , Endosomas/virología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Neuronas/virología , Síndrome Post Agudo de COVID-19/fisiopatología , Síndrome Post Agudo de COVID-19/virología , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus/efectos de los fármacos , Fosfotransferasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Astrocitos/virología , Células CultivadasRESUMEN
The PSEN1 ΔE9 mutation causes a familial form of Alzheimer's disease (AD) by shifting the processing of amyloid precursor protein (APP) towards the generation of highly amyloidogenic Aß42 peptide. We have previously shown that the PSEN1 ΔE9 mutation in human-induced pluripotent stem cell (iPSC)-derived astrocytes increases Aß42 production and impairs cellular responses. Here, we injected PSEN1 ΔE9 mutant astrosphere-derived glial progenitors into newborn mice and investigated mouse behavior at the ages of 8, 12, and 16 months. While we did not find significant behavioral changes in younger mice, spatial learning and memory were paradoxically improved in 16-month-old PSEN1 ΔE9 glia-transplanted male mice as compared to age-matched isogenic control-transplanted animals. Memory improvement was associated with lower levels of soluble, but not insoluble, human Aß42 in the mouse brain. We also found a decreased engraftment of PSEN1 ΔE9 mutant cells in the cingulate cortex and significant transcriptional changes in both human and mouse genes in the hippocampus, including the extracellular matrix-related genes. Overall, the presence of PSEN1 ΔE9 mutant glia exerted a more beneficial effect on aged mouse brain than the isogenic control human cells likely as a combination of several factors.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Animales , Humanos , Masculino , Ratones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Astrocitos/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Aprendizaje Espacial , EnvejecimientoRESUMEN
Cytosolic phospholipase A2 (cPLA2) is an enzyme regulating membrane phospholipid homeostasis and the release of arachidonic acid utilized in inflammatory responses. It represents an attractive target for the treatment of Alzheimer's disease (AD). Previously, we showed that lipopolysaccharide (LPS)-induced systemic inflammation caused abnormal lipid metabolism in the brain of a transgenic AD mouse model (APdE9), which might be associated with potential changes in cPLA2 activity. Here, we investigated changes in cPLA2 expression and activity, as well as the molecular mechanisms underlying these alterations due to chronic LPS administration in the cerebral cortex of female APdE9 mice as compared to saline- and LPS-treated female wild-type mice and saline-treated APdE9 mice. The study revealed the significant effects of genotype LPS treatment on cortical cPLA2 protein expression and activity in APdE9 mice. LPS treatment resulted in nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activation in the cortex of APdE9 mice. The gene expressions of inflammation markers Il1b and Tnfa were significantly elevated in the cortex of both APdE9 groups compared to the wild-type groups. The study provides evidence of the elevated expression and activity of cPLA2 in the brain cortex of APdE9 mice after chronic LPS treatment, which could be associated with NFkB activation.