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The risk of early recurrent events after stroke remains high despite currently established secondary prevention strategies1. Risk is particularly high in patients with atherosclerosis, with more than 10% of patients experiencing early recurrent events1,2. However, despite the enormous medical burden of this clinical phenomenon, the underlying mechanisms leading to increased vascular risk and recurrent stroke are largely unknown. Here, using a novel mouse model of stroke-induced recurrent ischaemia, we show that stroke leads to activation of the AIM2 inflammasome in vulnerable atherosclerotic plaques via an increase of circulating cell-free DNA. Enhanced plaque inflammation post-stroke results in plaque destabilization and atherothrombosis, finally leading to arterioarterial embolism and recurrent stroke within days after the index stroke. We confirm key steps of plaque destabilization also after experimental myocardial infarction and in carotid artery plaque samples from patients with acute stroke. Rapid neutrophil NETosis was identified as the main source of cell-free DNA after stroke and NET-DNA as the causative agent leading to AIM2 inflammasome activation. Neutralization of cell-free DNA by DNase treatment or inhibition of inflammasome activation reduced the rate of stroke recurrence after experimental stroke. Our findings present an explanation for the high recurrence rate after incident ischaemic events in patients with atherosclerosis. The detailed mechanisms uncovered here provide clinically uncharted therapeutic targets for which we show high efficacy to prevent recurrent events. Targeting DNA-mediated inflammasome activation after remote tissue injury represents a promising avenue for further clinical development in the prevention of early recurrent events.
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The medical burden of stroke extends beyond the brain injury itself and is largely determined by chronic comorbidities that develop secondarily. We hypothesized that these comorbidities might share a common immunological cause, yet chronic effects post-stroke on systemic immunity are underexplored. Here, we identify myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Single-cell sequencing revealed persistent pro-inflammatory changes in monocytes/macrophages in multiple organs up to 3 months after brain injury, notably in the heart, leading to cardiac fibrosis and dysfunction in both mice and stroke patients. IL-1ß was identified as a key driver of epigenetic changes in innate immune memory. These changes could be transplanted to naive mice, inducing cardiac dysfunction. By neutralizing post-stroke IL-1ß or blocking pro-inflammatory monocyte trafficking with a CCR2/5 inhibitor, we prevented post-stroke cardiac dysfunction. Such immune-targeted therapies could potentially prevent various IL-1ß-mediated comorbidities, offering a framework for secondary prevention immunotherapy.
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Lesões Encefálicas , Imunidade Inata , Memória Imunológica , Inflamação , Interleucina-1beta , Camundongos Endogâmicos C57BL , Monócitos , Animais , Camundongos , Interleucina-1beta/metabolismo , Lesões Encefálicas/imunologia , Humanos , Masculino , Monócitos/metabolismo , Monócitos/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/imunologia , Cardiopatias/imunologia , Feminino , Receptores CCR2/metabolismo , Fibrose , Epigênese Genética , Imunidade TreinadaRESUMO
Ischemic stroke is a major global health issue and characterized by acute vascular dysfunction and subsequent neuroinflammation. However, the relationship between these processes remains elusive. In the current study, we investigated whether alleviating vascular dysfunction by restoring vascular nitric oxide (NO) reduces post-stroke inflammation. Mice were subjected to experimental stroke and received inhaled NO (iNO; 50 ppm) after reperfusion. iNO normalized vascular cyclic guanosine monophosphate (cGMP) levels, reduced the elevated expression of intercellular adhesion molecule-1 (ICAM-1), and returned leukocyte adhesion to baseline levels. Reduction of vascular pathology significantly reduced the inflammatory cytokines interleukin-1ß (Il-1ß), interleukin-6 (Il-6), and tumor necrosis factor-α (TNF-α), within the brain parenchyma. These findings suggest that vascular dysfunction is responsible for leukocyte adhesion and that these processes drive parenchymal inflammation. Reversing vascular dysfunction may therefore emerge as a novel approach to diminish neuroinflammation after ischemic stroke and possibly other ischemic disorders.
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AVC Isquêmico , Acidente Vascular Cerebral , Camundongos , Animais , Óxido Nítrico , Doenças Neuroinflamatórias , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/tratamento farmacológico , Fator de Necrose Tumoral alfa/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Molécula 1 de Adesão Intercelular/metabolismoRESUMO
Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy ("rNLS8" model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature (IL-1ß, TNF-α, NfκB) in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression.
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Anticorpos Monoclonais , Filamentos Intermediários , Animais , Camundongos , Epitopos , Imunização , NF-kappa BRESUMO
Stroke is the second leading cause of death worldwide and a leading cause of disability. Clinical and experimental studies highlighted the complex role of the immune system in the pathophysiology of stroke. Ischemic brain injury leads to the release of cell-free DNA, a damage-associated molecular pattern, which binds to pattern recognition receptors on immune cells such as toll-like receptors and cytosolic inflammasome sensors. The downstream signaling cascade then induces a rapid inflammatory response. In this review, we are highlighting the characteristics of cell-free DNA and how these can affect a local as well as a systemic response after stroke. For this purpose, we screened literature on clinical studies investigating cell-free DNA concentration and properties after brain ischemia. We report the current understanding for mechanisms of DNA uptake and sensing in the context of post-stroke inflammation. Moreover, we compare possible treatment options targeting cell-free DNA, DNA-sensing pathways, and the downstream mediators. Finally, we describe clinical implications of this inflammatory pathway for stroke patients, open questions, and potential future research directions.
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Isquemia Encefálica , Acidente Vascular Cerebral , Humanos , Transdução de Sinais , Receptores Toll-Like/metabolismo , Mediadores da InflamaçãoRESUMO
Inflammation driven by DNA sensors is now understood to be important to disease pathogenesis. Here, we describe new inhibitors of DNA sensing, primarily of the inflammasome forming sensor AIM2. Biochemistry and molecular modeling has revealed 4-sulfonic calixarenes as potent inhibitors of AIM2 that likely work by binding competitively to the DNA-binding HIN domain. Although less potent, these AIM2 inhibitors also inhibit DNA sensors cGAS and TLR9 demonstrating a broad utility against DNA-driven inflammatory responses. The 4-sulfonic calixarenes inhibited AIM2-dependent post-stroke T cell death, highlighting a proof of concept that the 4-sulfonic calixarenes could be effective at combating post-stroke immunosuppression. By extension, we propose a broad utility against DNA-driven inflammation in disease. Finally, we reveal that the drug suramin, by virtue of its structural similarities, is an inhibitor of DNA-dependent inflammation and propose that suramin could be rapidly repurposed to meet an increasing clinical need.
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We present a simple and effective approach for non-blind image deblurring, combining classical techniques and deep learning. In contrast to existing methods that deblur the image directly in the standard image space, we propose to perform an explicit deconvolution process in a feature space by integrating a classical Wiener deconvolution framework with learned deep features. A multi-scale cascaded feature refinement module then predicts the deblurred image from the deconvolved deep features, progressively recovering detail and small-scale structures. The proposed model is trained in an end-to-end manner and evaluated on scenarios with simulated Gaussian noise, saturated pixels, or JPEG compression artifacts as well as real-world images. Moreover, we present detailed analyses of the benefit of the feature-based Wiener deconvolution and of the multi-scale cascaded feature refinement as well as the robustness of the proposed approach. Our extensive experimental results show that the proposed deep Wiener deconvolution network facilitates deblurred results with visibly fewer artifacts and quantitatively outperforms state-of-the-art non-blind image deblurring methods by a wide margin.
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A key observation of tissue injury, such as stroke and burn, is a state of systemic immunosuppression characterized by loss of T cells and rise of infections. Here, we present an in vitro model for cell-cell interactions between innate (macrophages) and adaptive (T cells) immune cells. This protocol facilitates bone marrow-derived macrophages (BMDMs) and splenic T cells in a coculture model. The procedure mimics injury-induced T cell death, which is driven by inflammasome activation in macrophages. For complete details on the use and execution of this protocol, please refer to Roth et al. (2021).
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Morte Celular/fisiologia , Técnicas de Cocultura/métodos , Macrófagos/citologia , Modelos Imunológicos , Linfócitos T/citologia , Animais , Feminino , Inflamassomos/imunologia , Inflamassomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Ferimentos e Lesões/imunologia , Ferimentos e Lesões/metabolismoRESUMO
The fifth generation (5G) of cellular networks improves the precision of user localization and provides the means to disclose location information to over-the-top (OTT) service providers. The network data analytics function (NWDAF) can further elaborate this information at an aggregated level using artificial intelligence techniques. These powerful features may lead to the improper use of user location information by mobile network operators (MNOs) and OTT service providers. Moreover, vulnerabilities at various layers may also leak user location information to eavesdroppers. Hence, the privacy of users is likely at risk, as location is part of their sensitive data. In this paper, we first go through the evolution of localization in cellular networks and investigate their effects on location privacy. Then, we propose a location-privacy-preserving integrated solution comprising virtual private mobile networks, an independent authentication and billing authority, and functions to protect wireless signals against location information leakage. Moreover, we advocate the continuous and detailed control of localization services by the user.
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Segurança Computacional , Privacidade , Inteligência Artificial , HumanosRESUMO
Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer's disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as microbial metabolites which promote Aß deposition. Germ-free (GF) AD mice exhibit a substantially reduced Aß plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice increased the Aß plaque load to levels of conventionally colonized (specific pathogen-free [SPF]) animals and SCFA supplementation to SPF mice even further exacerbated plaque load. This was accompanied by the pronounced alterations in microglial transcriptomic profile, including upregulation of ApoE. Despite increased microglial recruitment to Aß plaques upon SCFA supplementation, microglia contained less intracellular Aß. Taken together, our results demonstrate that microbiota-derived SCFA are critical mediators along the gut-brain axis which promote Aß deposition likely via modulation of the microglial phenotype.
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Ácidos Graxos Voláteis/metabolismo , Microbioma Gastrointestinal , Microglia/metabolismo , Placa Amiloide/metabolismo , Doença de Alzheimer/metabolismo , Animais , Feminino , Masculino , Camundongos , Organismos Livres de Patógenos EspecíficosRESUMO
Loss of lymphocytes, particularly T cell apoptosis, is a central pathological event after severe tissue injury that is associated with increased susceptibility for life-threatening infections. The precise immunological mechanisms leading to T cell death after acute injury are largely unknown. Here, we identified a monocyte-T cell interaction driving bystander cell death of T cells in ischemic stroke and burn injury. Specifically, we found that stroke induced a FasL-expressing monocyte population, which led to extrinsic T cell apoptosis. This phenomenon was driven by AIM2 inflammasome-dependent interleukin-1ß (IL-1ß) secretion after sensing cell-free DNA. Pharmacological inhibition of this pathway improved T cell survival and reduced post-stroke bacterial infections. As such, this study describes inflammasome-dependent monocyte activation as a previously unstudied cause of T cell death after injury and challenges the current paradigms of post-injury lymphopenia.
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Coinfecção/imunologia , Proteínas de Ligação a DNA/imunologia , Tolerância Imunológica/imunologia , Inflamassomos/imunologia , Transdução de Sinais/imunologia , Animais , Apoptose/imunologia , Infecções Bacterianas/imunologia , Queimaduras/imunologia , Queimaduras/microbiologia , Coinfecção/microbiologia , Humanos , Interleucina-1beta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/microbiologia , Linfócitos T/imunologiaRESUMO
Stroke causes severe and long-lasting symptoms in patients. Besides focal deficits such as speech impairment and limb weakness, stroke also results in neuropsychiatric symptoms, including fatigue, anxiety, and depression, which are debilitating and often impair post-stroke rehabilitation. However, in experimental stroke research, the study of neuropsychiatric symptoms and their therapeutic targeting has so far been largely neglected, which can be mainly attributed to the lack of appropriate tools to investigate such deficits in mice. Here, we report that neuropsychiatric symptoms can be differentiated from focal deficits and specifically modulated independent of treating the primary lesion. In order to achieve this, we developed a novel behavior analysis tool by assessing test performance of various tests, combining outcome parameters to cover functional domains of focal and neuropsychiatric symptoms, and finally weighted results into a time point-specific score. This weighted score enabled us to clearly differentiate focal deficits and neuropsychiatric symptoms and detect these until the chronic phase after stroke. Using this analysis tool, we detected that neutralizing systemic cytokines (TNF-α, IL-1ß and IL-6) specifically ameliorated neuropsychiatric symptoms but did not affect focal deficits or lesion volume. Hence, most conventional studies analyzing only focal deficits and lesion volume as primary outcome measures would have missed these significant and translationally relevant therapeutic effects. We anticipate that these findings will encourage more detailed analyses of neuropsychiatric symptoms particularly for anti-inflammatory therapies in stroke and that the presented weighted composite score will facilitate this development.
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Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Animais , Isquemia Encefálica/complicações , Citocinas , Humanos , Comportamento de Doença , Camundongos , Acidente Vascular Cerebral/complicaçõesRESUMO
RATIONALE: Arterial inflammation manifested as atherosclerosis is the leading cause of mortality worldwide. Genome-wide association studies have identified a prominent role of HDAC (histone deacetylase)-9 in atherosclerosis and its clinical complications including stroke and myocardial infarction. OBJECTIVE: To determine the mechanisms linking HDAC9 to these vascular pathologies and explore its therapeutic potential for atheroprotection. METHODS AND RESULTS: We studied the effects of Hdac9 on features of plaque vulnerability using bone marrow reconstitution experiments and pharmacological targeting with a small molecule inhibitor in hyperlipidemic mice. We further used 2-photon and intravital microscopy to study endothelial activation and leukocyte-endothelial interactions. We show that hematopoietic Hdac9 deficiency reduces lesional macrophage content while increasing fibrous cap thickness thus conferring plaque stability. We demonstrate that HDAC9 binds to IKK (inhibitory kappa B kinase)-α and ß, resulting in their deacetylation and subsequent activation, which drives inflammatory responses in both macrophages and endothelial cells. Pharmacological inhibition of HDAC9 with the class IIa HDAC inhibitor TMP195 attenuates lesion formation by reducing endothelial activation and leukocyte recruitment along with limiting proinflammatory responses in macrophages. Transcriptional profiling using RNA sequencing revealed that TMP195 downregulates key inflammatory pathways consistent with inhibitory effects on IKKß. TMP195 mitigates the progression of established lesions and inhibits the infiltration of inflammatory cells. Moreover, TMP195 diminishes features of plaque vulnerability and thereby enhances plaque stability in advanced lesions. Ex vivo treatment of monocytes from patients with established atherosclerosis reduced the production of inflammatory cytokines including IL (interleukin)-1ß and IL-6. CONCLUSIONS: Our findings identify HDAC9 as a regulator of atherosclerotic plaque stability and IKK activation thus providing a mechanistic explanation for the prominence of HDAC9 as a vascular risk locus in genome-wide association studies. Its therapeutic inhibition may provide a potent lever to alleviate vascular inflammation. Graphical Abstract: A graphical abstract is available for this article.
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Artérias/enzimologia , Aterosclerose/enzimologia , Histona Desacetilases/metabolismo , Quinase I-kappa B/metabolismo , Placa Aterosclerótica , Proteínas Repressoras/metabolismo , Acetilação , Idoso , Idoso de 80 Anos ou mais , Animais , Artérias/efeitos dos fármacos , Artérias/patologia , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/patologia , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Ativação Enzimática , Feminino , Fibrose , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Quinase I-kappa B/genética , Mediadores da Inflamação/metabolismo , Migração e Rolagem de Leucócitos , Macrófagos/enzimologia , Macrófagos/patologia , Masculino , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Monócitos/enzimologia , Monócitos/patologia , Ligação Proteica , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transdução de SinaisRESUMO
Microglial dysfunction is a key pathological feature of Alzheimer's disease (AD), but little is known about proteome-wide changes in microglia during the course of AD and their functional consequences. Here, we performed an in-depth and time-resolved proteomic characterization of microglia in two mouse models of amyloid ß (Aß) pathology, the overexpression APPPS1 and the knock-in APP-NL-G-F (APP-KI) model. We identified a large panel of Microglial Aß Response Proteins (MARPs) that reflect heterogeneity of microglial alterations during early, middle and advanced stages of Aß deposition and occur earlier in the APPPS1 mice. Strikingly, the kinetic differences in proteomic profiles correlated with the presence of fibrillar Aß, rather than dystrophic neurites, suggesting that fibrillar Aß may trigger the AD-associated microglial phenotype and the observed functional decline. The identified microglial proteomic fingerprints of AD provide a valuable resource for functional studies of novel molecular targets and potential biomarkers for monitoring AD progression or therapeutic efficacy.
Alzheimer's disease is a progressive, irreversible brain disorder. Patients with Alzheimer's have problems with memory and other mental skills, which lead to more severe cognitive decline and, eventually, premature death. This is due to increasing numbers of nerve cells in the brain dying over time. A distinctive feature of Alzheimer's is the abnormally high accumulation of a protein called amyloid-ß, which forms distinctive clumps in the brain termed 'plaques'. The brain has a type of cells called the microglia that identify infections, toxic material and damaged cells, and prevent these from building up by clearing them away. In Alzheimer's disease, however, the microglia do not work properly, which is thought to contribute to the accumulation of amyloid-ß plaques. This means that people with mutations in the genes important for the microglia activity are also at higher risk of developing the disease. Although problems with the microglia play an important role in Alzheimer's, researchers still do not fully understand why microglia stop working in the first place. It is also not known exactly when and how the microglia change as Alzheimer's disease progresses. To unravel this mystery, Sebastian Monasor, Müller et al. carried out a detailed study of the molecular 'fingerprints' of microglia at each key stage of Alzheimer's disease. The experiments used microglia cells from two different strains of genetically altered mice, both of which develop the hallmarks of Alzheimer's disease, including amyloid-ß plaques, at similar rates. Analysis of the proteins in microglia cells from both strains revealed distinctive, large-scale changes corresponding to successive stages of the disease reflecting the gradual accumulation of plaques. Obvious defects in microglia function also appeared soon after plaques started to build up. Microscopy imaging of the brain tissue showed that although amyloid-ß plaques appeared at the same time, they looked different in each mouse strain. In one, plaques were more compact, while in the other, plaques appeared 'fluffier', like cotton wool. In mice with more compacted plaques, microglia recognized the plaques earlier and stopped working sooner, suggesting that plaque structure and microglia defects could be linked. These results shed new light on the role of microglia and their changing protein 'signals' during the different stages of Alzheimer's disease. In the future, this information could help identify people at risk for the disease, so that they can be treated as soon as possible, and to design new therapies to make microglia work again.
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Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Microglia/metabolismo , Proteoma/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Transgênicos , Microglia/patologiaRESUMO
Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut-brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut-brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Using in vivo wide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells.SIGNIFICANCE STATEMENT Previous studies have shown a bidirectional communication along the gut-brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut-brain axis and as a potential therapeutic to improve recovery after stroke.
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Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Ácidos Graxos Voláteis/administração & dosagem , Acidente Vascular Cerebral/imunologia , Animais , Encéfalo/metabolismo , Feminino , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/imunologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Acidente Vascular Cerebral/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
The C9orf72 repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). Non-canonical translation of the expanded repeat results in abundant poly-GA inclusion pathology throughout the CNS. (GA)149 -CFP expression in mice triggers motor deficits and neuroinflammation. Since poly-GA is transmitted between cells, we investigated the therapeutic potential of anti-GA antibodies by vaccinating (GA)149 -CFP mice. To overcome poor immunogenicity, we compared the antibody response of multivalent ovalbumin-(GA)10 conjugates and pre-aggregated carrier-free (GA)15 . Only ovalbumin-(GA)10 immunization induced a strong anti-GA response. The resulting antisera detected poly-GA aggregates in cell culture and patient tissue. Ovalbumin-(GA)10 immunization largely rescued the motor function in (GA)149 -CFP transgenic mice and reduced poly-GA inclusions. Transcriptome analysis showed less neuroinflammation in ovalbumin-(GA)10 -immunized poly-GA mice, which was corroborated by semiquantitative and morphological analysis of microglia/macrophages. Moreover, cytoplasmic TDP-43 mislocalization and levels of the neurofilament light chain in the CSF were reduced, suggesting neuroaxonal damage is reduced. Our data suggest that immunotherapy may be a viable primary prevention strategy for ALS/FTD in C9orf72 mutation carriers.
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Esclerose Lateral Amiotrófica , Proteína C9orf72/genética , Demência Frontotemporal , Microglia , Esclerose Lateral Amiotrófica/terapia , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , VacinaçãoRESUMO
The ubiquitin-proteasome system (UPS) is an established therapeutic target for approved drugs to treat selected hematologic malignancies. While drug discovery targeting the UPS focuses on irreversibly binding epoxyketones and slowly-reversibly binding boronates, optimization of novel covalent-reversibly binding warheads remains largely unattended. We previously reported α-ketoamides to be a promising reversible lead motif, yet the cytotoxic activity required further optimization. This work focuses on the lead optimization of phenoxy-substituted α-ketoamides combining the structure-activity relationships from the primed and the non-primed site of the proteasome ß5 subunit. Our optimization strategy is accompanied by molecular modeling, suggesting occupation of P1' by a 3-phenoxy group to increase ß5 inhibition and cytotoxic activity in leukemia cell lines. Key compounds were further profiled for time-dependent inhibition of cellular substrate conversion. Furthermore, the α-ketoamide lead structure 27 does not affect escape response behavior in Danio rerio embryos, in contrast to bortezomib, which suggests increased target specificity.
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Amidas/síntese química , Antineoplásicos/síntese química , Neoplasias Hematológicas/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/síntese química , Ubiquitina/química , Amidas/administração & dosagem , Amidas/efeitos adversos , Sequência de Aminoácidos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Sítios de Ligação , Compostos de Boro/química , Bortezomib/administração & dosagem , Bortezomib/efeitos adversos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/efeitos adversos , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Peixe-Zebra/embriologiaRESUMO
The organization of brain areas in functionally connected networks, their dynamic changes, and perturbations in disease states are subject of extensive investigations. Research on functional networks in humans predominantly uses functional magnetic resonance imaging (fMRI). However, adopting fMRI and other functional imaging methods to mice, the most widely used model to study brain physiology and disease, poses major technical challenges and faces important limitations. Hence, there is great demand for alternative imaging modalities for network characterization. Here, we present a refined protocol for in vivo widefield calcium imaging of both cerebral hemispheres in mice expressing a calcium sensor in excitatory neurons. We implemented a stringent protocol for minimizing anesthesia and excluding movement artifacts which both imposed problems in previous approaches. We further adopted a method for unbiased identification of functional cortical areas using independent component analysis (ICA) on resting-state imaging data. Biological relevance of identified components was confirmed using stimulus-dependent cortical activation. To explore this novel approach in a model of focal brain injury, we induced photothrombotic lesions of the motor cortex, determined changes in inter- and intrahemispheric connectivity at multiple time points up to 56 days post-stroke and correlated them with behavioral deficits. We observed a severe loss in interhemispheric connectivity after stroke, which was partially restored in the chronic phase and associated with corresponding behavioral motor deficits. Taken together, we present an improved widefield calcium imaging tool accounting for anesthesia and movement artifacts, adopting an advanced analysis pipeline based on human fMRI algorithms and with superior sensitivity to recovery mechanisms in mouse models compared to behavioral tests. This tool will enable new studies on interhemispheric connectivity in murine models with comparability to human imaging studies for a wide spectrum of neuroscience applications in health and disease.
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Cálcio , Córtex Cerebral/fisiologia , Conectoma/métodos , Rede Nervosa/fisiologia , Neuroimagem/métodos , Imagem Óptica/métodos , Prosencéfalo/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Animais , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Córtex Motor/lesões , Córtex Motor/fisiopatologia , Rede Nervosa/diagnóstico por imagem , Rede Nervosa/fisiopatologia , Prosencéfalo/diagnóstico por imagem , Prosencéfalo/fisiopatologia , Acidente Vascular Cerebral/diagnóstico por imagemRESUMO
Microbiome alterations have been shown to affect stroke outcome. However, to what extent the presence of a gut microbiome per se is affecting post-stroke neuroinflammation has not been tested. By comparing germfree mice with recolonized (Ex-GF) and conventional SPF mice, we were able to demonstrate that bacterial colonization reduces stroke volumes. Bacterial colonization increased cerebral expression of cytokines as well as microglia/macrophage cell counts in contrast to improved stroke outcome. Interestingly, the microbiome-mediated brain protection was absent in lymphocyte-deficient mice. These findings support the concept of lymphocyte-driven protective neuroinflammation after stroke under control of the microbiome.
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Microbioma Gastrointestinal , Inflamação/imunologia , Neuroproteção , Acidente Vascular Cerebral/imunologia , Linfócitos T/imunologia , Animais , Imunidade , Inflamação/complicações , Inflamação/patologia , Camundongos , Fatores de Proteção , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/patologiaRESUMO
Stroke induces a multiphasic systemic immune response, but the consequences of this response on atherosclerosis-a major source of recurrent vascular events-have not been thoroughly investigated. We show that stroke exacerbates atheroprogression via alarmin-mediated propagation of vascular inflammation. The prototypic brain-released alarmin high-mobility group box 1 protein induced monocyte and endothelial activation via the receptor for advanced glycation end products (RAGE)-signaling cascade and increased plaque load and vulnerability. Recruitment of activated monocytes via the CC-chemokine ligand 2-CC-chemokine receptor type 2 pathway was critical in stroke-induced vascular inflammation. Neutralization of circulating alarmins or knockdown of RAGE attenuated atheroprogression. Blockage of ß3-adrenoreceptors attenuated the egress of myeloid monocytes after stroke, whereas neutralization of circulating alarmins was required to reduce systemic monocyte activation and aortic invasion. Our findings identify a synergistic effect of the sympathetic stress response and alarmin-driven inflammation via RAGE as a critical mechanism of exacerbated atheroprogression after stroke.