RESUMEN
Germinal matrix hemorrhage (GMH) is a pathology that occurs in infancy, with often devastating long-term consequences. Posthemorrhagic hydrocephalus (PHH) can develop acutely, while periventricular leukomalacia (PVL) is a chronic sequala. There are no pharmacological therapies to treat PHH and PVL. We investigated different aspects of the complement pathway in acute and chronic outcomes after murine neonatal GMH induced at postnatal day 4 (P4). Following GMH-induction, the cytolytic complement membrane attack complex (MAC) colocalized with infiltrating red blood cells (RBCs) acutely but not in animals treated with the complement inhibitor CR2-Crry. Acute MAC deposition on RBCs was associated with heme oxygenase-1 expression and heme and iron deposition, which was reduced with CR2-Crry treatment. Complement inhibition also reduced hydrocephalus and improved survival. Following GMH, there were structural alterations in specific brain regions linked to motor and cognitive functions, and these changes were ameliorated by CR2-Crry, as measured at various timepoints through P90. Astrocytosis was reduced in CR2-Crry-treated animals at chronic, but not acute, timepoints. At P90, myelin basic protein and LAMP-1 colocalized, indicating chronic ongoing phagocytosis of white matter, which was reduced by CR2-Crry treatment. Data indicate acute MAC-mediated iron-related toxicity and inflammation exacerbated the chronic effects of GMH.
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Hidrocefalia , Ratones , Animales , Hidrocefalia/complicaciones , Proteínas del Sistema Complemento , Hemorragia Cerebral/complicaciones , Inflamación/complicaciones , Complejo de Ataque a Membrana del Sistema Complemento , Hierro , Proteínas Recombinantes de FusiónRESUMEN
BACKGROUND: Germinal matrix hemorrhage is a devastating disease of pre-term infancy commonly resulting in post-hemorrhagic hydrocephalus, periventricular leukomalacia, and subsequent neurocognitive deficits. We demonstrate vascular expression of the adhesion molecule P-selectin after GMH and investigate a strategy to specifically target complement inhibition to sites of P-selectin expression to mitigate the pathological sequelae of GMH. METHODS: We prepared two fusion proteins consisting of different anti-P-selectin single chain antibodies (scFv's) linked to the complement inhibitor Crry. One scFv targeting vehicle (2.12scFv) blocked the binding of P-selectin to its PSGL-1 ligand expressed on leukocytes, whereas the other targeting vehicle (2.3scFv) bound P-selectin without blocking ligand binding. Post-natal C57BL/6 J mice on day 4 (P4) were subjected to collagenase induced-intraventricular hemorrhage and treated with 2.3Psel-Crry, 2.12Psel-Crry, or vehicle. RESULTS: Compared to vehicle treatment, 2.3Psel-Crry treatment after induction of GMH resulted in reduced lesion size and mortality, reduced hydrocephalus development, and improved neurological deficit measurements in adolescence. In contrast, 2.12Psel-Crry treatment resulted in worse outcomes compared to vehicle. Improved outcomes with 2.3Psel-Crry were accompanied by decreased P-selectin expression, and decreased complement activation and microgliosis. Microglia from 2.3Psel-Crry treated mice displayed a ramified morphology, similar to naïve mice, whereas microglia in vehicle treated animals displayed a more ameboid morphology that is associated with a more activated status. Consistent with these morphological characteristics, there was increased microglial internalization of complement deposits in vehicle compared to 2.3Psel-Crry treated animals, reminiscent of aberrant C3-dependent microglial phagocytosis that occurs in other (adult) types of brain injury. In addition, following systemic injection, 2.3Psel-Crry specifically targeted to the post-GMH brain. Likely accounting for the unexpected finding that 2.12Psel-Crry worsens outcome following GMH was the finding that this construct interfered with coagulation in this hemorrhagic condition, and specifically with heterotypic platelet-leukocyte aggregation, which express P-selectin and PSGL-1, respectively. CONCLUSIONS: GMH induces expression of P-selectin, the targeting of which with a complement inhibitor protects against pathogenic sequelae of GMH. A dual functioning construct with both P-selectin and complement blocking activity interferes with coagulation and worsens outcomes following GMH, but has potential for treatment of conditions that incorporate pathological thrombotic events, such as ischemic stroke.
Asunto(s)
Hemorragia Cerebral , Hidrocefalia , Animales , Ratones , Hemorragia Cerebral/patología , Inactivadores del Complemento , Proteínas del Sistema Complemento , Ligandos , Ratones Endogámicos C57BL , Selectina-P/metabolismoRESUMEN
Background Germinal Matrix Hemorrhage is a devastating disease of pre-term infancy commonly resulting in post-hemorrhagic hydrocephalus, periventricular leukomalacia, and subsequent neurocognitive deficits. We demonstrate vascular expression of the adhesion molecule P-selectin after GMH and investigate a strategy to specifically target complement inhibition to sites of P-selectin expression to mitigate the pathological sequelae of GMH. Methods We prepared two fusion proteins consisting of different anti-P-selectin single chain antibodies (scFv's) linked to the complement inhibitor Crry. One scFv targeting vehicle (2.12scFv) blocked the binding of P-selectin to its PSGL-1 ligand expressed on leukocytes, whereas the other targeting vehicle (2.3scFv) bound P-selectin without blocking ligand binding. Post-natal mice on day 4 (P4) were subjected to collagenase induced-intraventricular hemorrhage and treated with 2.3Psel-Crry, 2.12Psel-Crry, or vehicle. Results Compared to vehicle treatment, 2.3Psel-Crry treatment after induction of GMH resulted in reduced lesion size and mortality, reduced hydrocephalus development, and improved neurological deficit measurements in adolescence. In contrast, 2.12Psel-Crry treatment resulted in worse outcomes compared to vehicle. Improved outcomes with 2.3Psel-Crry were accompanied by decreased P-selectin expression, and decreased complement activation and microgliosis. Microglia from 2.3Psel-Crry treated mice displayed a ramified morphology, similar to naïve mice, whereas microglia in vehicle treated animals displayed a more ameboid morphology that is associated with a more activated status. Consistent with these morphological characteristics, there was increased microglial internalization of complement deposits in vehicle compared to 2.3Psel-Crry treated animals, reminiscent of aberrant C3-dependent microglial phagocytosis that occurs in other (adult) types of brain injury. Also, following systemic injection, 2.3Psel-Crry specifically targeted to the post-GMH brain. Likely accounting for the unexpected finding that 2.12Psel-Crry worsens outcome following GMH was the finding that this construct interfered with coagulation in this hemorrhagic condition, and specifically with heterotypic platelet-leukocyte aggregation, which express P-selectin and PSGL-1, respectively. Conclusion GMH induces expression of P-selectin, the targeting of which with a complement inhibitor protects against pathogenic sequelae of GMH. A dual functioning construct with both P-selectin and complement blocking activity interferes with coagulation and worsens outcomes following GMH, but has potential for treatment of conditions that incorporate pathological thrombotic events, such as ischemic stroke.
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Traumatic brain injury (TBI) represents one of the major public health concerns worldwide due to the increase in TBI incidence as a result of injuries from daily life accidents such as sports and motor vehicle transportation as well as military-related practices. This type of central nervous system trauma is known to predispose patients to several neurological disorders such as Parkinson's disease, Alzheimer's disease, chronic trauamatic encephalopathy, and age-related Dementia. Recently, several proteomic and lipidomic platforms have been applied on different TBI studies to investigate TBI-related mechanisms that have broadened our understanding of its distinct neuropathological complications. In this study, we provide an updated comprehensive overview of the current knowledge and novel perspectives of the spatially resolved microproteomics and microlipidomics approaches guided by mass spectrometry imaging used in TBI studies and its applications in the neurotrauma field. In this regard, we will discuss the use of the spatially resolved microproteomics and assess the different microproteomic sampling methods such as laser capture microdissection, parafilm assisted microdissection, and liquid microjunction extraction as accurate and precise techniques in the field of neuroproteomics. Additionally, we will highlight lipid profiling applications and their prospective potentials in characterizing molecular processes involved in the field of TBI. Specifically, we will discuss the phospholipid metabolism acting as a precursor for proinflammatory molecules such as eicosanoids. Finally, we will survey the current state of spatial neuroproteomics and microproteomics applications and present the various studies highlighting their findings in these fields.
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Enfermedad de Alzheimer , Lesiones Traumáticas del Encéfalo , Humanos , Espectrometría de Masas , Proteómica/métodos , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/metabolismoRESUMEN
BACKGROUND AND AIMS: The role of inflammatory immune responses in colorectal cancer (CRC) development and response to therapy is a matter of intense debate. While inflammation is a known driver of CRC, inflammatory immune infiltrates are a positive prognostic factor in CRC and predispose to response to immune checkpoint blockade (ICB) therapy. Unfortunately, over 85% of CRC cases are primarily unresponsive to ICB due to the absence of an immune infiltrate, and even the cases that show an initial immune infiltration can become refractory to ICB. The identification of therapy supportive immune responses in the field has been partially hindered by the sparsity of suitable mouse models to recapitulate the human disease. In this study, we aimed to understand how the dysregulation of the complement anaphylatoxin C3a receptor (C3aR), observed in subsets of patients with CRC, affects the immune responses, the development of CRC, and response to ICB therapy. METHODS: We use a comprehensive approach encompassing analysis of publicly available human CRC datasets, inflammation-driven and newly generated spontaneous mouse models of CRC, and multiplatform high-dimensional analysis of immune responses using microbiota sequencing, RNA sequencing, and mass cytometry. RESULTS: We found that patients' regulation of the complement C3aR is associated with epigenetic modifications. Specifically, downregulation of C3ar1 in human CRC promotes a tumor microenvironment characterized by the accumulation of innate and adaptive immune cells that support antitumor immunity. In addition, in vivo studies in our newly generated mouse model revealed that the lack of C3a in the colon activates a microbiota-mediated proinflammatory program which promotes the development of tumors with an immune signature that renders them responsive to the ICB therapy. CONCLUSIONS: Our findings reveal that C3aR may act as a previously unrecognized checkpoint to enhance antitumor immunity in CRC. C3aR can thus be exploited to overcome ICB resistance in a larger group of patients with CRC.
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Neoplasias Colorrectales , Inhibidores de Puntos de Control Inmunológico , Anafilatoxinas , Animales , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Regulación hacia Abajo , Humanos , Factores Inmunológicos , Inmunoterapia/métodos , Inflamación/patología , Ratones , Microambiente TumoralRESUMEN
The Early-Career Complementologists (ECCO) is a task force that was established, in close collaboration with the European Complement Network (ECN) and the International Complement Society (ICS), with the specific mission to support and connect early-career researchers (ECRs) in the complement field. ECRs are junior scientists at the early stages of their training which include undergraduate as well as graduate students, Ph.D. graduates, and post-doctoral fellows. This unique population within the scientific community represents the next generation of scientific leaders. However, ECRs are faced with key challenges and the COVID-19 pandemic has disproportionately impacted them. In this paper, we provide further insight into specific needs and challenges of ECRs in the complement field. We surveyed 52 ECRs in the complement field and assessed their perceptions of 1) mentor and peer support, 2) working conditions as well as 3) career interests and prospects. Furthermore, we review the various activities carried out by ECCO over the past years such as our social media presence, social events, and newly-created awards. We also discuss the future activities and events to be carried out by ECCO. Through these initiatives and activities, ECCO strives to boost collaborations between ECRs, provide recognition, and improve the visibility of their work. In addition, continuous joint efforts must also be made by the scientific community, research institutes, and funding organizations to nurture and invest in ECRs.
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COVID-19 , Pandemias , Humanos , Investigadores/educaciónRESUMEN
Germinal matrix hemorrhage (GMH) is a devastating disease of infancy that results in intraventricular hemorrhage, post-hemorrhagic hydrocephalus (PHH), periventricular leukomalacia, and neurocognitive deficits. There are no curative treatments and limited surgical options. We developed and characterized a mouse model of GMH based on the injection of collagenase into the subventricular zone of post-natal pups and utilized the model to investigate the role of complement in PHH development. The site-targeted complement inhibitor CR2Crry, which binds deposited C3 complement activation products, localized specifically in the brain following its systemic administration after GMH. Compared to vehicle, CR2Crry treatment reduced PHH and lesion size, which was accompanied by decreased perilesional complement deposition, decreased astrocytosis and microgliosis, and the preservation of dendritic and neuronal density. Complement inhibition also improved survival and weight gain, and it improved motor performance and cognitive outcomes measured in adolescence. The progression to PHH, neuronal loss, and associated behavioral deficits was linked to the microglial phagocytosis of complement opsonized neurons, which was reversed with CR2Crry treatment. Thus, complement plays an important role in the pathological sequelae of GMH, and complement inhibition represents a novel therapeutic approach to reduce the disease progression of a condition for which there is currently no treatment outside of surgical intervention.
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Hemorragia Cerebral , Hidrocefalia , Animales , Animales Recién Nacidos , Hemorragia Cerebral/metabolismo , Progresión de la Enfermedad , Hidrocefalia/metabolismo , Ratones , Ratas , Ratas Sprague-DawleyRESUMEN
Traumatic Brain Injury (TBI) is one of the leading causes of death and disability worldwide. Aspirin (ASA) and clopidogrel (CLOP) are antiplatelet agents that inhibit platelet aggregation. They are implicated in worsening the intracerebral haemorrhage (ICH) risk post-TBI. However, antiplatelet drugs may also exert a neuroprotective effect post-injury. We determined the impact of ASA and CLOP treatment, alone or in combination, on ICH and brain damage in an experimental rat TBI model. We assessed changes in platelet aggregation and measured serum thromboxane by enzyme immune assay. We also explored a panel of brain damage and apoptosis biomarkers by immunoblotting. Rats were treated with ASA and/or CLOP for 48 h prior to TBI and sacrificed 48 h post-injury. In rats treated with antiplatelet agents prior to TBI, platelet aggregation was completely inhibited, and serum thromboxane was significantly decreased, compared to the TBI group without treatment. TBI increases UCHL-1 and GFAP, but decreases hexokinase expression compared to the non-injured controls. All groups treated with antiplatelet drugs prior to TBI had decreased UCH-L1 and GFAP serum levels compared to the TBI untreated group. Furthermore, the ASA and CLOP single treatments increased the hexokinase serum levels. We confirmed that αII-spectrin cleavage increased post-TBI, with the highest cleavage detected in CLOP-treated rats. Aspirin and/or CLOP treatment prior to TBI is a double-edged sword that exerts a dual effect post-injury. On one hand, ASA and CLOP single treatments increase the post-TBI ICH risk, with a further detrimental effect from the ASA + CLOP treatment. On the other hand, ASA and/or CLOP treatments are neuroprotective and result in a favourable profile of TBI injury markers. The ICH risk and the neuroprotection benefits from antiplatelet therapy should be weighed against each other to ameliorate the management of TBI patients.
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Lesiones Traumáticas del Encéfalo , Lesiones Encefálicas , Animales , Aspirina/farmacología , Aspirina/uso terapéutico , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Clopidogrel/farmacología , Humanos , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , RatasRESUMEN
Research on the complement system, like most areas of immunology, has seen tremendous progress over the last decades. Further advances in the complement field will rely on the next generation of scientific leaders, which are today's early-career researchers (ECRs). ECRs are emerging scientists who obtained their PhD degree within the past five years. They represent a distinct population within the scientific community, and accordingly have unique needs. Unfortunately, ECRs are faced with significant challenges that require customized solutions. The current paper provides a snapshot of the major obstacles ECRs face, such as an unhealthy work-life balance, lack of mentor and peer support, and uncertain career prospects. Efforts must consequently be taken to ensure stability and success of ECRs, not only to benefit these researchers in the early stages of their career, but the entire field of complement research. The Early-Career Complementologists (ECCO) was, therefore, launched as a new Task Force to support ECRs in the complement field. This new initiative aims to support and connect ECRs in the complement field worldwide. Furthermore, ECCO is supported by both the International Complement Society (ICS) and the European Complement Network (ECN); two professional societies led by scientists investigating the complement system.
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Proteínas del Sistema Complemento/inmunología , Humanos , InvestigadoresRESUMEN
Stroke is a major cause of mortality worldwide, and survivors often have major life-changing disabilities. Annually in the United States, an estimated 795,000 people experience a new or recurrent stroke. All types of stroke involve an inflammatory reaction that follows the initial phase of incidence. However, investigations into any links between inflammatory markers and recovery processes in the context of post-stroke rehabilitation are lacking. In this systematic review, we searched the literature in PubMed, SCOPUS, and CINAHL databases to gather information on inflammatory biomarkers related to stroke and their association with rehabilitation outcomes, according to PRISMA guidelines. Eleven articles (n=1.773 stroke patients) were selected. Immune markers (interleukin 6 [IL-6], C-reactive protein, IL-1α, tumor necrosis factor α, soluble intercellular adhesion molecule 1) and functional status assessments (Modified Rankin Score, National Institutes of Health Stroke Scale, Functional Independence Measure, etc.) were the primary measures used in the reviewed studies. We found preliminary evidence for the evaluation of inflammatory biomarkers post-stroke, including the role of inflammation in functional recovery and the influence of rehabilitation on inflammation. This is the first systematic review of the topic. The review identifies several gaps in the literature that are critical for understanding the potential use of inflammatory markers to improve post-stroke outcomes.
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Rehabilitación de Accidente Cerebrovascular , Accidente Cerebrovascular , Biomarcadores , Humanos , Inflamación/complicaciones , Recuperación de la Función , Accidente Cerebrovascular/complicacionesRESUMEN
Traumatic brain injury (TBI) can result in progressive cognitive decline occurring for years after the initial insult, and for which there is currently no pharmacological treatment. An ongoing chronic inflammatory response after TBI is thought to be an important factor in driving this cognitive decline. Here, we investigate the role of complement in neuroinflammation and cognitive decline for up to 6 months after murine TBI. Male C57BL/6 mice were subjected to open head injury using a controlled cortical impact device. At 2 months post TBI, mice were moved to large cages with an enriched environment to simulate rehabilitation therapy, and assigned to one of three treatment groups: 1. vehicle (PBS), 2. CR2Crry (3 doses over 1 week), 3. CR2Crry (continuous weekly dose until the end of the study). The study was terminated at 6 months post-TBI for all groups. Motor and cognitive function was analyzed, with histopathological analysis of brain tissue. Measured at 6 months after TBI, neither of the complement inhibition paradigms improved motor performance. However, mice receiving continuous CR2Crry treatment showed improved spatial learning and memory compared to both mice receiving only 3 doses and to mice receiving vehicle control. Analysis of brain sections at 6 months after injury revealed ongoing complement activation in the control group, with reduced complement activation and C3 deposition in the continuous CR2Crry treatment group. The ipsilateral hemisphere of continuously treated animals also showed a decrease in microglia/macrophage and astrocyte activation compared to vehicle. There was also increased astrocytosis in the contralateral hippocampus of vehicle treated vs. naïve mice, which was reduced in mice continuously treated with CR2Crry. This study demonstrates continued complement mediated neuroinflammation at extended chronic time points after TBI, and extends the potential treatment window for complement inhibition, which has previously been shown to improve outcomes after murine TBI.
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Lesiones Traumáticas del Encéfalo/metabolismo , Disfunción Cognitiva/metabolismo , Activación de Complemento/fisiología , Proteínas del Sistema Complemento/metabolismo , Mediadores de Inflamación/metabolismo , Animales , Lesiones Traumáticas del Encéfalo/patología , Disfunción Cognitiva/patología , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
Cognitive deficits following traumatic brain injury (TBI) remain a major cause of disability and early-onset dementia, and there is increasing evidence that chronic neuroinflammation occurring after TBI plays an important role in this process. However, little is known about the molecular mechanisms responsible for triggering and maintaining chronic inflammation after TBI. Here, we identify complement, and specifically complement-mediated microglial phagocytosis of synapses, as a pathophysiological link between acute insult and a chronic neurodegenerative response that is associated with cognitive decline. Three months after an initial insult, there is ongoing complement activation in the injured brain of male C57BL/6 mice, which drives a robust chronic neuroinflammatory response extending to both hemispheres. This chronic neuroinflammatory response promotes synaptic degeneration and predicts progressive cognitive decline. Synaptic degeneration was driven by microglial phagocytosis of complement-opsonized synapses in both the ipsilateral and contralateral brain, and complement inhibition interrupted the degenerative neuroinflammatory response and reversed cognitive decline, even when therapy was delayed until 2 months after TBI. These findings provide new insight into our understanding of TBI pathology and its management; and whereas previous therapeutic investigations have focused almost exclusively on acute treatments, we show that all phases of TBI, including at chronic time points after TBI, may be amenable to therapeutic interventions, and specifically to complement inhibition.SIGNIFICANCE STATEMENT There is increasing evidence of a chronic neuroinflammatory response after traumatic brain injury (TBI), but little is known about the molecular mechanisms responsible for triggering and maintaining chronic inflammation. We identify complement, and specifically complement-mediated microglial phagocytosis of synapses, as a pathophysiological link between acute insult and a chronic neurodegenerative response, and further that this response is associated with cognitive decline. Complement inhibition interrupted this response and reversed cognitive decline, even when therapy was delayed until 2 months after injury. The data further support the concept that TBI should be considered a chronic rather than an acute disease condition, and have implications for the management of TBI in the chronic phase of injury, specifically with regard to the therapeutic application of complement inhibition.
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Lesiones Traumáticas del Encéfalo/patología , Disfunción Cognitiva/patología , Activación de Complemento/fisiología , Sinapsis/patología , Animales , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/inmunología , Disfunción Cognitiva/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/inmunología , Fagocitosis/inmunologíaRESUMEN
Neurological disorders are major contributors to death and disability worldwide. The pathology of injuries and disease processes includes a cascade of events that often involve molecular and cellular components of the immune system and their interaction with cells and structures within the central nervous system. Because of this, there has been great interest in developing neuroprotective therapeutic approaches that target neuroinflammatory pathways. Several neuroprotective anti-inflammatory agents have been investigated in clinical trials for a variety of neurological diseases and injuries, but to date the results from the great majority of these trials has been disappointing. There nevertheless remains great interest in the development of neuroprotective strategies in this arena. With this in mind, the complement system is being increasingly discussed as an attractive therapeutic target for treating brain injury and neurodegenerative conditions, due to emerging data supporting a pivotal role for complement in promoting multiple downstream activities that promote neuroinflammation and degeneration. As we move forward in testing additional neuroprotective and immune-modulating agents, we believe it will be useful to review past trials and discuss potential factors that may have contributed to failure, which will assist with future agent selection and trial design, including for complement inhibitors. In this context, we also discuss inhibition of the complement system as a potential neuroprotective strategy for neuropathologies of the central nervous system.
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Antiinflamatorios/uso terapéutico , Lesiones Traumáticas del Encéfalo/terapia , Enfermedades del Sistema Nervioso Central/terapia , Sistema Nervioso Central/patología , Fármacos Neuroprotectores/uso terapéutico , Animales , Ensayos Clínicos como Asunto , HumanosRESUMEN
The focus of this review is the role of complement-mediated phagocytosis in retinal and neurological diseases affecting the visual system. Complement activation products opsonize synaptic material on neurons for phagocytic removal, which is a normal physiological process during development, but a pathological process in several neurodegenerative diseases and conditions. We discuss the role of complement in the refinement and elimination of synapses in the retina and lateral geniculate nucleus, both during development and in disease states. How complement and aberrant phagocytosis promotes injury to the visual system is discussed primarily in the context of multiple sclerosis, where it has been extensively studied, although the role of complement in visual dysfunction in other diseases such as stroke and traumatic brain injury is also highlighted. Retinal diseases are also covered, with a focus on glaucoma and age-related macular degeneration. Finally, we discuss the potential of complement inhibitory strategies to treat diseases affecting the visual system.
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Proteínas del Sistema Complemento/inmunología , Microglía/inmunología , Enfermedades del Sistema Nervioso/inmunología , Enfermedades de la Retina/inmunología , Animales , Inactivadores del Complemento/uso terapéutico , Humanos , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Fagocitosis , Retina/inmunología , Enfermedades de la Retina/tratamiento farmacológicoRESUMEN
Combining proteomics and systems biology approaches, we demonstrate that neonatal microglial cells derived from two different CNS locations, cortex and spinal cord, and cultured in vitro displayed different phenotypes upon different physiological or pathological conditions. These cells also exhibited greater variability in terms of cellular and small extracellular vesicles (sEVs) protein content and levels. Bioinformatic data analysis showed that cortical microglia exerted anti-inflammatory and neurogenesis/tumorigenesis properties, while the spinal cord microglia were more inflammatory. Interestingly, while both sEVs microglia sources enhanced growth of DRGs processes, only the spinal cord-derived sEVs microglia under LPS stimulation significantly attenuated glioma proliferation. These results were confirmed using the neurite outgrowth assay on DRGs cells and glioma proliferation analysis in 3D spheroid cultures. Results from these in vitro assays suggest that the microglia localized at different CNS regions can ensure different biological functions. Together, this study indicates that neonatal microglia locations regulate their physiological and pathological functional fates and could affect the high prevalence of brain vs spinal cord gliomas in adults.
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Identifying new lipid markers linked to traumatic brain injury (TBI) is of major importance in characterizing their central role in the regeneration process and inflammatory response in such an injury model. In the present study, an advanced lipidomics analysis using high spectral resolution matrix-assisted laser desorption/ionization-mass spectrometry imaging was performed on different brain regions in an experimental rat model of moderate controlled cortical impact (CCI) while considering different time points (1 day, 3 days, 7 days, and 10 days) assessing the acute and subacute phase after injury. Our results revealed a new family of lipids, the acylcarnitines, as TBI-lipid related markers, with maximum expression at 3 days after impact and main colocalization within resident microglia of the brain. Furthermore, our experiments highlighted the upregulation of these acylcarnitine lipids, secreted by microglia, in the ipsilateral substantia nigra, the main region in the brain affected in Parkinson's disease (PD).
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Lesiones Traumáticas del Encéfalo/metabolismo , Encéfalo/metabolismo , Carnitina/análogos & derivados , Lípidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Carnitina/análisis , Modelos Animales de Enfermedad , Procesamiento de Imagen Asistido por Computador , Metabolismo de los Lípidos , Masculino , Microglía/metabolismo , Microglía/patología , Palmitoilcarnitina/análisis , Palmitoilcarnitina/metabolismo , Ratas Sprague-Dawley , Espectrometría de Masas en TándemRESUMEN
Traumatic brain injury (TBI) represents a major health concerns with no clinically-approved FDA drug available for therapeutic intervention. Several genomics and neuroproteomics studies have been employed to decipher the underlying pathological mechanisms involved that can serve as potential neurotherapeutic targets and unveil a possible underlying relation of TBI to other secondary neurological disorders. In this work, we present a novel high throughput systems biology approach using a spatially resolved microproteomics platform conducted on different brain regions in an experimental rat model of moderate of controlled cortical injury (CCI) at a temporal pattern postinjury (1 day, 3 days, 7 days, and 10 days). Mapping the spatiotemporal landscape of signature markers in TBI revealed an overexpression of major protein families known to be implicated in Parkinson's disease (PD) such as GPR158, HGMB1, synaptotagmin and glutamate decarboxylase in the ipsilateral substantia nigra. In silico bioinformatics docking experiments indicated the potential correlation between TBI and PD through alpha-synuclein. In an in vitro model, stimulation with palmitoylcarnitine triggered an inflammatory response in macrophages and a regeneration processes in astrocytes which also further confirmed the in vivo TBI proteomics data. Taken together, this is the first study to assess the microproteomics landscape in TBI, mainly in the substantia nigra, thus revealing a potential predisposition for PD or Parkinsonism post-TBI.
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Lesiones Traumáticas del Encéfalo/metabolismo , Enfermedad de Parkinson/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Masculino , Proteómica , Ratas Sprague-DawleyRESUMEN
Traumatic brain injury (TBI) is a major cause of death and disability in children and young adults worldwide according to the World Health Organization (WHO). The emergence of mass-spectrometry-based techniques, such as MALDI-MSI, has allowed the monitoring and visualization of changes post injury, providing a global picture of the impact of TBI on different classes of molecules in a single study. In this work, we show the ability to track lipid changes post-TBI by three-dimensional matrix-assisted laser desorption/ionization-mass-spectrometry imaging (MALDI-MSI). Controlled cortical impact (CCI) was induced on adult male rats resulting in direct mechanical injury to the cortical tissue on the right ipsilateral hemisphere of the brain. Images of lipid distribution in coronally sectioned injured brains were acquired using a high-resolution mass spectrometer (MALDI-LTQ-Orbitrap-XL). Results reveal unique lipid signatures for the injured cortical tissue, which further segregate into two subgroups of injury (lesion interior and lesion exterior). Although both subgroups show different profiles from that of the noninjured cortical tissue, the lesion interior is more similar to the ventricular system than the lesion exterior. For example, m/ z 725.56 showed expression in both injured tissue and the ventricular system, whereas m/ z 856.59 (phosphatidylcholine 42:9) is uniquely expressed in injured tissue. On the other hand, m/ z 797.59 (also a phosphatidylcholine) showed unique expression to the ventricular system and not to the injured cortical tissue. Our results can help in further monitoring and identifying lesion-specific lipids in a 3D manner to obtain a better understanding and visualization of molecular and cellular events occurring post-TBI.
Asunto(s)
Lesiones Traumáticas del Encéfalo/metabolismo , Lípidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Lesiones Traumáticas del Encéfalo/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
PACAP has opposing roles ranging from activation to inhibition of tumor growth and PACAP agonists/antagonists could be used in tumor therapy. In this study, the effect of PACAP stimulation on signaling pathways was investigated in MCF-7 human adenocarcinoma breast cancer cells. Results showed that MCF-7 cells express VPAC1 and VPAC2, but not PAC1, receptors. In addition, PACAP increased the phosphorylation levels of STAT1, Src and Raf within seconds, confirming their involvement in early stages of PACAP signaling whereas maximal phosphorylation of AKT, ERK and p38 was reached 10 to 20â¯min later. Moreover, selective inhibition of Src or PI3K resulted in a significant decrease in the phosphorylation of ERK and AKT, but not p38, demonstrating that PACAP signaling follows Src/Raf/ERK and PI3K/AKT pathways. On the other hand, selective inhibition of PLC or PKA resulted in a significant decrease in the phosphorylation of p38, but not AKT or ERK, indicating that PACAP signaling also follows the PLC and PKA/cAMP pathways. Furthermore, PACAP induced ROS through H2O2 production whereas pretreatment with NAC inhibitor decreased AKT and ERK phosphorylation, but not p38. Selective NOX2 inhibition affected Src/Raf/Erk and PI3K/Akt pathways, without affecting the p38/PLC/PKA pathway whereas other inhibitors (ML171, VAS2870) had no effect on PACAP induced ROS generation. On the other hand, PACAP induced calcium release, which was decreased by pretreatment with PLC inhibitor. Finally, PACAP stimulation promoted apoptosis by increasing Bax and decreasing Bcl2 expression. In conclusion, we demonstrated that PACAP signaling in MCF-7 cells follows the Src/Raf/ERK and PI3K/AKT pathways and is VPAC1 dependent in a ROS dependent manner, whereas it follows PLC and PKA/cAMP pathways and is VPAC2 dependent through p38 MAP kinase activation involving calcium.
Asunto(s)
Neoplasias de la Mama/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Transducción de Señal/fisiología , Apoptosis/fisiología , Calcio/metabolismo , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Células MCF-7 , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The therapeutic use of RhoA inhibitors (RhoAi) has been experimentally tested in spinal cord injury (SCI). In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomic-systems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed. The data obtained allow a better understanding of regeneration/degeneration induced above and below the lesion site. Results notably showed a time-dependent alteration of the transcription factors profile along with the synthesis of growth cone-related factors (receptors, ligands, and signaling pathways) in RhoAi treated DRG cells. Furthermore, we assessed in a rat SCI model the in vivo impact of RhoAi treatment administered in situ via alginate scaffold that was combined with FK506 delivery. The improved recovery of locomotion was detected only at the early postinjury time points, whereas after overall survival a dramatic increase of synaptic contacts on outgrowing neurites in affected segments was observed. We validate these results by in vivo proteomic studies along the spinal cord segments from tissue and secreted media analyses, confirming the increase of the synaptogenesis expression factors under RhoAi treatment. Taken together, we demonstrate that RhoAi treatment seems to be useful to stimulate neurite outgrowth in both in vitro as well in vivo environments. However, for in vivo experiments there is a need for sustained delivery regiment to facilitate axon regeneration and promote synaptic reconnections with appropriate target neurons also at chronic phase, which in turn may lead to higher assumption for functional improvement.