RESUMEN
Traumatic brain injury (TBI) from closed-head trauma is a leading cause of disability, with limited effective interventions. Many TBI models impact brain parenchyma directly, and are limited by the fact that these forces do not recapitulate clinically relevant closed head injury. However, applying clinically relevant injury mechanics to the intact skull may lead to variability and as a result, preclinical modeling TBI remains a challenge. Current models often do not explore sex differences in TBI, which is critically important for translation to clinical practice. We systematically investigated sources of variability in a murine model of closed-head TBI and developed a framework to reduce variability across severity and sex. We manipulated pressure, dwell time, and displacement to determine effects on motor coordination, spatial learning, and neuronal damage in 10-week-old male and female mice. Increasing pressure beyond 70 psi had a ceiling effect on cellular and behavioral outcomes, while manipulating dwell time only affected behavioral performance. Increasing displacement precisely graded injury severity in both sexes across all outcomes. Physical signs of trauma occurred more frequently at higher displacements. Stratifying severity based on day-1 rotarod performance retained histological relationships and separated both sexes into injury severity cohorts with distinct patterns of behavioral recovery. Utilizing this stratification strategy, within-group rotarod variability over 6 days post-injury was reduced by 50%. These results have important implications for translational research in TBI and provide a framework for using this clinically relevant translational injury model in both male and female mice.
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Lesiones Traumáticas del Encéfalo , Ratones , Femenino , Masculino , Animales , Modelos Animales de Enfermedad , Lesiones Traumáticas del Encéfalo/patología , Encéfalo/patología , Neuronas , CabezaRESUMEN
The treatment of traumatic brain injury (TBI) in military populations is hindered by underreporting and underdiagnosis. Clinical symptoms and outcomes may be mitigated with an effective pre-injury prophylaxis. This study evaluates whether CN-105, a 5-amino acid apolipoprotein E (ApoE) mimetic peptide previously shown to modify the post-traumatic neuroinflammatory response, would maintain its neuroprotective effects if administered prior to closed-head injury in a clinically relevant murine model. CN-105 was synthesized by Polypeptide Inc. (San Diego, CA) and administered to C57-BL/6 mice intravenously (IV) and/or by intraperitoneal (IP) injection at various time points prior to injury while vehicle treated animals received IV and/or IP normal saline. Animals were randomized following injury and behavioral observations were conducted by investigators blinded to treatment. Vestibulomotor function was assessed using an automated Rotarod (Ugo Basile, Comerio, Italy), and hippocampal microglial activation was assessed using F4/80 immunohistochemical staining in treated and untreated mice 7 days post-TBI. Separate, in vivo assessments of the pharmacokinetics was performed in healthy CD-1. IV CN-105 administered prior to head injury improved vestibulomotor function compared to vehicle control-treated animals. CN-105 co-administered by IP and IV dosing 6 h prior to injury also improved vestibulomotor function up to 28 days following injury. Microglia counted in CN-105 treated specimens were significantly fewer (P = 0.03) than in vehicle specimens. CN-105 improves functional outcomes and reduces hippocampal microglial activation when administered prior to injury and could be adapted as a pre-injury prophylaxis for soldiers at high risk for TBI.
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Lesiones Traumáticas del Encéfalo , Traumatismos Cerrados de la Cabeza , Fármacos Neuroprotectores , Animales , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Modelos Animales de Enfermedad , Traumatismos Cerrados de la Cabeza/complicaciones , Traumatismos Cerrados de la Cabeza/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Microglía , Fármacos Neuroprotectores/farmacologíaRESUMEN
BACKGROUND: Intracerebral hemorrhage (ICH) is a devastating form of cerebrovascular disease for which there are no approved pharmacological interventions that improve outcomes. Apolipoprotein E (apoE) has emerged as a promising therapeutic target given its isoform-specific neuroprotective properties and ability to modify neuroinflammatory responses. We developed a 5-amino acid peptide, CN-105, that mimics the polar face of the apoE helical domain involved in receptor interactions, readily crosses the blood-brain barrier, and improves outcomes in well-established preclinical ICH models. In the current study, we investigated the therapeutic potential of CN-105 in translational ICH models that account for hypertensive comorbidity, sex, species, and age. METHODS: In three separate experiments, we delivered three intravenous doses of CN-105 (up to 0.20 mg/kg) or vehicle to hypertensive male BPH/2 J mice, spontaneously hypertensive female rats, or 11-month-old male mice within 24-h of ICH. Neuropathological and neurobehavioral outcomes were determined over 3, 7, and 9 days, respectively. RESULTS: In spontaneously hypertensive male mice, there was a significant dose-dependent effect of CN-105 on vestibulomotor function at 0.05 and 0.20 mg/kg doses (p < 0.05; 95% CI: 0.91-153.70 and p < 0.001; 95% CI: 49.54-205.62), while 0.20 mg/kg also improved neuroseverity scores (p < 0.05; 95% CI: 0.27-11.00) and reduced ipsilateral brain edema (p < 0.05; 95% CI: - 0.037 to - 0.001). In spontaneously hypertensive female rats, CN-105 (0.05 mg/kg) had a significant effect on vestibulomotor function (p < 0.01; η2 = 0.093) and neuroseverity scores (p < 0.05; η2 = 0.083), and reduced contralateral edema expansion (p < 0.01; 95% CI: - 1.41 to - 0.39). In 11-month-old male mice, CN-105 had a significant effect on vestibulomotor function (p < 0.001; η2 = 0.111) but not neuroseverity scores (p > 0.05; η2 = 0.034). CONCLUSIONS: Acute treatment with CN-105 improves outcomes in translational ICH models independent of sex, species, age, or hypertensive comorbidity.
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Edema Encefálico , Hemorragia Cerebral , Animales , Apolipoproteínas E , Barrera Hematoencefálica , Hemorragia Cerebral/tratamiento farmacológico , Femenino , Inflamación , Masculino , Ratones , RatasRESUMEN
IRGM and its mouse orthologue Irgm1 are dynamin-like proteins that regulate vesicular remodeling, intracellular microbial killing, and pathogen immunity. IRGM dysfunction is linked to inflammatory bowel disease (IBD), and while it is thought that defective intracellular killing of microbes underscores IBD susceptibility, studies have yet to address how IRGM/Irgm1 regulates immunity to microbes relevant to intestinal inflammation. Here we find that loss of Irgm1 confers marked susceptibility to Citrobacter rodentium, a noninvasive intestinal pathogen that models inflammatory responses to intestinal bacteria. Irgm1-deficient mice fail to control C. rodentium outgrowth in the intestine, leading to systemic pathogen spread and host mortality. Surprisingly, susceptibility due to loss of Irgm1 function was not linked to defective intracellular killing of C. rodentium or exaggerated inflammation, but was instead linked to failure to remodel specific colon lamina propria (C-LP) myeloid cells that expand in response to C. rodentium infection and are essential for C. rodentium immunity. Defective immune remodeling was most striking in C-LP monocytes, which were successfully recruited to the infected C-LP, but subsequently underwent apoptosis. Apoptotic susceptibility was induced by C. rodentium infection and was specific to this setting of pathogen infection, and was not apparent in other settings of intestinal inflammation. These studies reveal a novel role for Irgm1 in host defense and suggest that deficiencies in survival and remodeling of C-LP myeloid cells that control inflammatory intestinal bacteria may underpin IBD pathogenesis linked to IRGM dysfunction.
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Citrobacter rodentium/inmunología , Colon/inmunología , Infecciones por Enterobacteriaceae/inmunología , Proteínas de Unión al GTP/deficiencia , Enfermedades Inflamatorias del Intestino/inmunología , Monocitos/inmunología , Animales , Colon/microbiología , Colon/patología , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/patología , Proteínas de Unión al GTP/inmunología , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/microbiología , Enfermedades Inflamatorias del Intestino/patología , Ratones , Ratones Noqueados , Monocitos/microbiología , Monocitos/patología , Membrana Mucosa/inmunología , Membrana Mucosa/microbiología , Membrana Mucosa/patologíaRESUMEN
Alzheimer's disease (AD) is the most common form of dementia and is characterized pathologically by Aß plaques. Current treatments are purely symptomatic despite decades of intensive research interest. Notably, patients with the APOE4 allele are at increased risk for developing AD. One hypothesis regarding the mechanism by which the APOE4 allele might increase AD risk is loss of adaptive function, raising the possibility that the exogenous administration of apoE mimetics would have therapeutic effects. In this study, we utilized a previously characterized murine model of AD containing human APP, PS1 and APOE4TR, the APP/PS1/APOETR mouse. We treated male APP/PS1/APOETR mice with the apoE mimetic CN-105 or vehicle for 40d, beginning either at 14-18 or 25-28 weeks of age. After termination of treatment we tested animals in both Morris water maze and contextual fear conditioning, and examined soluble Aß by biochemistery and Aß deposition in cortex by unbiased stereology. We found that transient treatment with CN-105 for 40d beginning at 14-18 weeks reduced Aß pathology and rescued memory deficits in male APP/PS1/APOETR mice. Notably, delaying treatment onset to 25-28 weeks did not produce as robust an effect. These results suggest CN-105 treatment in a mouse model of AD results in a reduction in AD pathology and improved behavioral outcomes when administered early in the course of disease. As CN-105 has an excellent safety profile and is already in clinical trials, these findings raise the possibility that CN-105 represents a novel and translatable therapeutic strategy for AD.
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Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/psicología , Apolipoproteínas E/administración & dosificación , Encéfalo/efectos de los fármacos , Encéfalo/patología , Memoria/efectos de los fármacos , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Conducta Animal , Modelos Animales de Enfermedad , Masculino , Ratones Transgénicos , Agregación Patológica de Proteínas/prevención & controlRESUMEN
Toxoplasma gondii secretes rhoptry (ROP) and dense granule (GRA) effector proteins to evade host immune clearance mediated by interferon gamma (IFN-γ), immunity-related GTPase (IRG) effectors, and CD8+ T cells. Here, we investigated the role of parasite-secreted effectors in regulating host access to parasitophorous vacuole (PV) localized parasite antigens and their presentation to CD8+ T cells by the major histocompatibility class I (MHC-I) pathway. Antigen presentation of PV localized parasite antigens by MHC-I was significantly increased in macrophages and/or dendritic cells infected with mutant parasites that lacked expression of secreted GRA (GRA2, GRA3, GRA4, GRA5, GRA7, GRA12) or ROP (ROP5, ROP18) effectors. The ability of various secreted GRA or ROP effectors to suppress antigen presentation by MHC-I was dependent on cell type, expression of IFN-γ, or host IRG effectors. The suppression of antigen presentation by ROP5, ROP18, and GRA7 correlated with a role for these molecules in preventing PV disruption by IFN-γ-activated host IRG effectors. However, GRA2 mediated suppression of antigen presentation was not correlated with PV disruption. In addition, the GRA2 antigen presentation phenotypes were strictly co-dependent on the expression of the GRA6 protein. These results show that MHC-I antigen presentation of PV localized parasite antigens was controlled by mechanisms that were dependent or independent of IRG effector mediated PV disruption. Our findings suggest that the GRA6 protein underpins an important mechanism that enhances CD8+ T cell recognition of parasite-infected cells with damaged or ruptured PV membranes. However, in intact PVs, parasite secreted effector proteins that associate with the PV membrane or the intravacuolar network membranes play important roles to actively suppress antigen presentation by MHC-I to reduce CD8+ T cell recognition and clearance of Toxoplasma gondii infected host cells.
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Presentación de Antígeno/inmunología , Antígenos de Protozoos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Toxoplasmosis Animal/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Vacuolas/inmunologíaRESUMEN
Toxoplasma gondii evades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)- and intravacuolar network (IVN) membrane-localized dense granule (GRA) proteins in the development of acute and chronic Toxoplasma infection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic-stage cysts in vivo without affecting the parasite growth rate or the ability to differentiate into cysts in vitro Acute virulence of the PruΔgra2, PruΔgra3, and PruΔgra4 mutants was reduced but not abolished. In contrast, the PruΔgra12 mutant was avirulent in mice and PruΔgra12 parasites failed to establish a chronic infection. High-virulence type I strain RHΔgra12 parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-γ)-activated macrophages, type I RHΔgra12 and type II PruΔgra12 parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RHΔku80 and type II PruΔku80 strains, respectively. Despite this resistance, Δgra12 PVs ultimately succumbed to IFN-γ-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-γ and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection.IMPORTANCEToxoplasma gondii cysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated dense granule (GRA) proteins orchestrate the development of chronic cysts in vivo A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-γ). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection.
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Interacciones Huésped-Patógeno , Evasión Inmune , Interferón gamma/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Vacuolas/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Membranas Intracelulares/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Teóricos , Proteínas Protozoarias/genética , Análisis de Supervivencia , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/parasitología , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Crohn's disease (CD) represents a chronic inflammatory disorder of the intestinal tract. Several susceptibility genes have been linked to CD, though their precise role in the pathogenesis of this disorder remains unclear. Immunity-related GTPase M (IRGM) is an established risk allele in CD. We have shown previously that conventionally raised (CV) mice lacking the IRGM ortholog, Irgm1 exhibit abnormal Paneth cells (PCs) and increased susceptibility to intestinal injury. In the present study, we sought to utilize this model system to determine if environmental conditions impact these phenotypes, as is thought to be the case in human CD. To accomplish this, wild-type and Irgm1-/- mice were rederived into specific pathogen-free (SPF) and germ-free (GF) conditions. We next assessed how these differential housing environments influenced intestinal injury patterns, and epithelial cell morphology and function in wild-type and Irgm1-/- mice. Remarkably, in contrast to CV mice, SPF Irgm1-/- mice showed only a slight increase in susceptibility to dextran sodium sulfate-induced inflammation. SPF Irgm1-/- mice also displayed minimal abnormalities in PC number and morphology, and in antimicrobial peptide expression. Goblet cell numbers and epithelial proliferation were also unaffected by Irgm1 in SPF conditions. No microbial differences were observed between wild-type and Irgm1-/- mice, but gut bacterial communities differed profoundly between CV and SPF mice. Specifically, Helicobacter sequences were significantly increased in CV mice; however, inoculating SPF Irgm1-/- mice with Helicobacter hepaticus was not sufficient to transmit a pro-inflammatory phenotype. In summary, our findings suggest the impact of Irgm1-deficiency on susceptibility to intestinal inflammation and epithelial function is critically dependent on environmental influences. This work establishes the importance of Irgm1-/- mice as a model to elucidate host-environment interactions that regulate mucosal homeostasis and intestinal inflammatory responses. Defining such interactions will be essential for developing novel preventative and therapeutic strategies for human CD.
Asunto(s)
Ambiente , Proteínas de Unión al GTP/deficiencia , Inflamación/patología , Intestinos/patología , Células de Paneth/patología , Animales , Biodiversidad , Proliferación Celular , Colitis/microbiología , Colitis/patología , Sulfato de Dextran , Susceptibilidad a Enfermedades , Células Epiteliales/patología , Proteínas de Unión al GTP/metabolismo , Microbioma Gastrointestinal , Genotipo , Células Caliciformes/patología , Helicobacter/fisiología , Inflamación/microbiología , Intestinos/microbiología , Ratones Noqueados , Células de Paneth/metabolismo , Fenotipo , Organismos Libres de Patógenos EspecíficosRESUMEN
OBJECTIVE: At present, the absence of a pharmacological neuroprotectant represents an important unmet clinical need in the treatment of ischemic and traumatic brain injury. Recent evidence suggests that administration of apolipoprotein E mimetic therapies represent a viable therapeutic strategy in this setting. We investigate the neuroprotective and anti-inflammatory properties of the apolipoprotein E mimetic pentapeptide, CN-105, in a microglial cell line and murine model of ischemic stroke. METHODS: Ten to 13-week-old male C57/BL6 mice underwent transient middle cerebral artery occlusion and were randomly selected to receive CN-105 (0.1 mg/kg) in 100 µL volume or vehicle via tail vein injection at various time points. Survival, motor-sensory functional outcomes using rotarod test and 4-limb wire hanging test, infarct volume assessment using 2,3,5-Triphenyltetrazolium chloride staining method, and microglial activation in the contralateral hippocampus using F4/80 immunostaining were assessed at various time points. In vitro assessment of tumor necrosis factor-alpha secretion in a microglial cell line was performed, and phosphoproteomic analysis conducted to explore early mechanistic pathways of CN-105 in ischemic stroke. RESULTS: Mice receiving CN-105 demonstrated improved survival, improved functional outcomes, reduced infarct volume, and reduced microglial activation. CN-105 also suppressed inflammatory cytokines secretion in microglial cells in vitro. Phosphoproteomic signals suggest that CN-105 reduces proinflammatory pathways and lower oxidative stress. INTERPRETATION: CN-105 improves functional and histological outcomes in a murine model of ischemic stroke via modulation of neuroinflammatory pathways. Recent clinical trial of this compound has demonstrated favorable pharmacokinetic and safety profile, suggesting that CN-105 represents an attractive candidate for clinical translation.
RESUMEN
At present, there are no proven pharmacological treatments demonstrated to improve long term functional outcomes following traumatic brain injury(TBI). In the setting of non-penetrating TBI, sterile brain inflammatory responses are associated with the development of cerebral edema, intracranial hypertension, and secondary neuronal injury. There is increasing evidence that endogenous apolipoprotein E(apoE) modifies the neuroinflammatory response through its role in downregulating glial activation, however, the intact apoE holoprotein does not cross the blood-brain barrier due to its size. To address this limitation, we developed a small 5 amino acid apoE mimetic peptide(CN-105) that mimics the polar face of the apoE helical domain involved in receptor interactions. The goal of this study was to investigate the therapeutic potential of CN-105 in a murine model of closed head injury. Treatment with CN-105 was associated with a durable improvement in functional outcomes as assessed by Rotarod and Morris Water Maze and a reduction in positive Fluoro-Jade B stained injured neurons and microglial activation. Administration of CN-105 was also associated with reduction in mRNA expression of a subset of inflammatory and immune-related genes.
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Apolipoproteínas E/uso terapéutico , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Fragmentos de Péptidos/uso terapéutico , Recuperación de la Función/efectos de los fármacos , Animales , Apolipoproteínas E/farmacología , Hipocampo/efectos de los fármacos , Ratones , Modelos Animales , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/farmacologíaRESUMEN
Crohn's disease (CD) is a chronic, immune-mediated, inflammatory disorder of the intestine that has been linked to numerous susceptibility genes, including the immunity-related GTPase (IRG) M (IRGM). IRGs comprise a family of proteins known to confer resistance to intracellular infections through various mechanisms, including regulation of phagosome processing, cell motility, and autophagy. However, despite its association with CD, the role of IRGM and other IRGs in regulating intestinal inflammation is unclear. We investigated the involvement of Irgm1, an ortholog of IRGM, in the genesis of murine intestinal inflammation. After dextran sodium sulfate exposure, Irgm1-deficient [Irgm1 knockout (KO)] mice showed increased acute inflammation in the colon and ileum, with worsened clinical responses. Marked alterations of Paneth cell location and granule morphology were present in Irgm1 KO mice, even without dextran sodium sulfate exposure, and were associated with impaired mitophagy and autophagy in Irgm1 KO intestinal cells (including Paneth cells). This was manifested by frequent tubular and swollen mitochondria and increased LC3-positive autophagic structures. Interestingly, these LC3-positive structures often contained Paneth cell granules. These results suggest that Irgm1 modulates acute inflammatory responses in the mouse intestine, putatively through the regulation of gut autophagic processes, that may be pivotal for proper Paneth cell functioning.
Asunto(s)
Colitis/inducido químicamente , Proteínas de Unión al GTP/metabolismo , Células de Paneth/patología , Animales , Autofagia , Colitis/metabolismo , Sulfato de Dextran/toxicidad , Femenino , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica/fisiología , Ileítis/inducido químicamente , Ileítis/metabolismo , Inflamación/genética , Inflamación/metabolismo , Masculino , Ratones , Ratones Noqueados , Mitofagia , Células de Paneth/metabolismoRESUMEN
BACKGROUND: Microglial inhibition may reduce secondary tissue injury and improve functional outcome following acute brain injury. Utilizing clinically relevant murine models of traumatic brain injury and intracerebral hemorrhage, neuroinflammatory responses and functional outcome were examined in the presence of a potential microglial inhibitor, TT-301. METHODS: TT-301 or saline was administered following traumatic brain injury or intracerebral hemorrhage, and then for four subsequent days. The effect of TT-301 on neuroinflammatory responses and neuronal viability was assessed, as well as short-term vestibulomotor deficit (Rotorod) and long-term neurocognitive impairment (Morris water maze). Finally differential gene expression profiles of mice treated with TT-301 were compared with those of vehicle. RESULTS: Reduction in F4/80+ staining was demonstrated at 1 and 10 days, but not 28 days, after injury in mice treated with TT-301 (n = 6). These histologic findings were associated with improved neurologic function as assessed by Rotorod, which improved by 52.7% in the treated group by day 7, and Morris water maze latencies, which improved by 232.5% as a function of treatment (n = 12; P < 0.05). Similar benefit was demonstrated following intracerebral hemorrhage, in which treatment with TT-301 was associated with functional neurologic improvement of 39.6% improvement in Rotorod and a reduction in cerebral edema that was independent of hematoma volume (n = 12; P < 0.05). Differential gene expression was evaluated following treatment with TT-301, and hierarchical cluster analysis implicated involvement of the Janus kinase-Signal Transducer and Activator of Transcription pathway after administration of TT-301 (n = 3/group). CONCLUSIONS: Modulation of neuroinflammatory responses through TT-301 administration improved histologic and functional parameters in murine models of acute neurologic injury.
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Sistema Nervioso Central/lesiones , Activación de Macrófagos/efectos de los fármacos , Microglía/efectos de los fármacos , Piperazinas/farmacología , Piridazinas/farmacología , Piridinas/farmacología , Animales , Análisis de los Gases de la Sangre , Glucemia/metabolismo , Agua Corporal/metabolismo , Química Encefálica , Lesiones Encefálicas/tratamiento farmacológico , Recuento de Células , Línea Celular , Hemorragia Cerebral/inducido químicamente , Hemorragia Cerebral/tratamiento farmacológico , Citocinas/biosíntesis , Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Inflamación/tratamiento farmacológico , Inflamación/patología , Lipopolisacáridos/farmacología , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Equilibrio Postural/efectos de los fármacos , Desempeño Psicomotor/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Resultado del TratamientoRESUMEN
The immunity-related GTPases (IRGs) are a family of proteins induced by interferon-γ that play a crucial role in innate resistance to intracellular pathogens. The M subfamily of IRG proteins (IRGM) plays a profound role in this context, in part because of the ability of its members to regulate the localization and expression of other IRG proteins. We present here evidence that IRGM proteins affect the localization of the guanylate-binding proteins (GBPs), a second family of interferon-induced GTP-binding proteins that also function in innate immunity. Absence of Irgm1 or Irgm3 led to accumulation of Gbp2 in intracellular compartments that were positive for both the macroautophagy (hereafter referred to as autophagy) marker LC3 and the autophagic adapter molecule p62/Sqstm1. Gbp2 was similarly relocalized in cells in which autophagy was impaired because of the absence of Atg5. Both in Atg5- and IRGM-deficient cells, the IRG protein Irga6 relocalized to the same compartments as Gbp2, raising the possibility of a common regulatory mechanism. However, other data indicated that Irga6, but not Gbp2, was ubiquitinated in IRGM-deficient cells. Similarly, coimmunoprecipitation studies indicated that although Irgm3 did interact directly with Irgb6, it did not interact with Gbp2. Collectively, these data suggest that IRGM proteins indirectly modulate the localization of GBPs through a distinct mechanism from that through which they regulate IRG protein localization. Further, these results suggest that a core function of IRGM proteins is to regulate autophagic flux, which influences the localization of GBPs and possibly other factors that instruct cell-autonomous immune resistance.
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Autofagia , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Células 3T3 , Animales , Flavonoles , Glicósidos , Inmunoprecipitación , Interferones/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Fagosomas/metabolismo , Unión Proteica , Ubiquitina/metabolismoRESUMEN
Intracellular tau deposits are characteristic of several neurodegenerative disorders called tauopathies. The tau protein regulates the stability and assembly of microtubules by binding to microtubules through three or four microtubule-binding repeats (3R and 4R). The number of microtubule-binding repeats is determined by the inclusion or exclusion of the second microtubule-binding repeat encoded by exon 10 of the TAU gene. TAU gene mutations that alter the inclusion of exon 10, and hence the 4R:3R ratio, are causal in the tauopathy frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). A mutation located in exon 10 has been identified in several FTDP-17 families that present with increased exon 10 inclusion in both mRNA and protein, parkinsonism, movement disorders, and dementia. We have engineered a human tau minigene construct that was designed to allow alternative splicing of the tau exon 10. Here we demonstrate that transgenic mice expressing human tau protein with this mutation develop neurodegeneration as result of aberrant splicing. The mice recapitulate many of the disease hallmarks that are seen in patients with this mutation, including increased tau exon 10 inclusion in both mRNA and protein, motor and behavioral deficits, and tau protein accumulation in neurons and tufted astrocytes. Furthermore, these mice present with degeneration of the nigrostriatal dopaminergic pathway, suggesting a possible mechanism for parkinsonism in FTDP-17. Additionally, activated caspase-3 immunoreactivity in both neurons and astrocytes implicates the involvement of the apoptotic pathway in the pathology of these mice.
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Asparagina/genética , Cromosomas Humanos Par 17/genética , Demencia/genética , Lisina/genética , Trastornos Parkinsonianos/genética , Empalme del ARN/genética , Proteínas tau/genética , Factores de Edad , Análisis de Varianza , Animales , Conducta Animal , Sistema Nervioso Central/patología , Embrión de Mamíferos , Exones/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Transgénicos , Modelos Moleculares , Actividad Motora/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismoRESUMEN
We previously reported in guinea pig tracheal smooth muscle that maximal shortening velocity decreases from 3 weeks of age to adulthood. It is not known whether myosin light chain kinase (MLCK), a key enzyme determining the velocity of smooth muscle contraction, undergoes maturational changes. In the present work, we investigated MLCK protein content and mRNA expression in 1-week-old, 3-week-old, and adult guinea pigs. We extracted either proteins or RNA from isolated tracheal smooth muscle. The content of MLCK was assessed by Western immunoblots. MLCK mRNA was evaluated by Northern analysis and by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR). The content of MLCK increased 3-fold at 3 weeks of age and then decreased in adults, being 0.116 +/- 0.042, 0.330 +/- 0.125 (P < 0.05), and 0.153 +/- 0.054 microg/mg of total protein, respectively, in 1-week, 3-week, and adult animals. Quantitative RT-PCR revealed that MLCK mRNA increased with age to 135 +/- 35% and 177 +/- 23% (P < 0.01) in 3-week and adult animals, respectively, compared to 1-week animals. The transient increase of MLCK content in juvenile guinea pig tracheal smooth muscle may contribute to the increased shortening velocity at this age. We suggest that this increased content of MLCK is one of the mechanisms leading to maturation of airway smooth muscle contractility, which in turn contributes to the airway hyperresponsiveness reported in children and young animals.
Asunto(s)
Músculo Liso/enzimología , Quinasa de Cadena Ligera de Miosina/metabolismo , Factores de Edad , Animales , Western Blotting , Femenino , Cobayas , Masculino , Contracción Muscular/fisiología , Músculo Liso/crecimiento & desarrollo , Quinasa de Cadena Ligera de Miosina/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tráquea/enzimología , Tráquea/crecimiento & desarrolloRESUMEN
The formation and bipolar translocation of an ectoplasmic cytoskeleton of rings and meridional bands was studied in interphase zygotes of the glossiphoniid leech Theromyzon trizonare. Zygotes consisted of a peripheral organelle-rich ectoplasm and an internal yolk-rich endoplasm. After microinjection of labeled tubulin and/or actin, zygotes were examined by time-lapse video imaging, immunofluorescence and confocal microscopy. The rings and meridional bands were formed by condensation of a network of moving cytasters that represented ectoplasmic secondary centers of microtubule and actin filament nucleation. In some cases the network of cytasters persisted between the rings. The cytoskeleton had an outer actin layer and an inner microtubule layer that merged at the irregularly-shaped boundary zone. Bipolar translocation of the rings, meridional bands, or the network of cytasters led to accumulation of the cytoskeleton at both zygote poles. Translocation of the cytoskeleton was slowed or arrested by microinjected taxol or phalloidin, in a dose-dependent fashion. Results of drug treatment probably indicate differences in the degree and speed at which the cytoskeleton becomes stabilized. Moreover, drugs that selectively stabilized either microtubules or actin filaments stabilized and impaired movement of the entire cytoskeleton. Microtubule poisons and latrunculin-B failed to disrupt the cytoskeleton. It is concluded that the microtubule and actin cytoskeletons are dynamic, presumably cross-linked and resistant to depolymerizing drugs. They probably move along each other by a sliding mechanism that depends on the instability of microtubules and actin filaments.