RESUMEN
Mitochondrial defects are one of the common underlying causes of neuronal vulnerability in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), and TDP-43 pathology is the most commonly observed proteinopathy. Disrupted inner mitochondrial membrane (IMM) reported in the upper motor neurons (UMNs) of ALS patients with TDP-43 pathology is recapitulated in the UMNs of well-characterized hTDP-43 mouse model of ALS. The construct validity, such as shared and common cellular pathology in mice and human, offers a unique opportunity to test treatment strategies that may translate to patients. SBT-272 is a well-tolerated brain-penetrant small molecule that stabilizes cardiolipin, a phospholipid found in IMM, thereby restoring mitochondrial structure and respiratory function. We investigated whether SBT-272 can improve IMM structure and health in UMNs diseased with TDP-43 pathology in our well-characterized UMN reporter line for ALS. We found that SBT-272 significantly improved mitochondrial structural integrity and restored mitochondrial motility and function. This led to improved health of diseased UMNs in vitro. In comparison to edaravone and AMX0035, SBT-272 appeared more effective in restoring health of diseased UMNs. Chronic treatment of SBT-272 for sixty days starting at an early symptomatic stage of the disease in vivo led to a significant reduction in astrogliosis, microgliosis, and TDP-43 pathology in the ALS motor cortex. Our results underscore the therapeutic potential of SBT-272, especially within the context of TDP-43 pathology and mitochondrial dysfunction.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Ratones , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Neuronas Motoras/patología , Mitocondrias/patología , Proteínas de Unión al ADN/metabolismoRESUMEN
BACKGROUND: Low levels of insulin-like growth factor-1 (IGF-1) protein in preterm human infants are associated with bronchopulmonary dysplasia (BPD). We used our preterm lamb model of BPD to determine (1) dosage of recombinant human (rh) IGF-1 bound to binding protein-3 (IGFBP-3) to reach infant physiologic plasma levels; and (2) whether repletion of plasma IGF-1 improves pulmonary and cardiovascular outcomes. METHODS: Group 1: normal, unventilated lambs from 128 days gestation through postnatal age 5 months defined normal plasma levels of IGF-1. Group 2: continuous infusion of rhIGF-1/rhIGFBP-3 (0.5, 1.5, or 4.5 mg/kg/day; n = 2) for 3 days in mechanically ventilated (MV) preterm lambs determined that 1.5 mg/kg/day dosage attained physiologic plasma IGF-1 concentration of ~125 ng/mL, which was infused in four more MV preterm lambs. RESULTS: Group 1: plasma IGF-1 protein increased from ~75 ng/mL at 128 days gestation to ~220 ng/L at 5 months. Group 2: pilot study of the optimal dosage (1.5 mg/kg/day rhIGF-1/rhIGFBP-3) in six MV preterm lambs significantly improved some pulmonary and cardiovascular outcomes (p < 0.1) compared to six MV preterm controls. RhIGF-1/rhIGFBP-3 was not toxic to the liver, kidneys, or lungs. CONCLUSIONS: Three days of continuous iv infusion of rhIGF-1/rhIGFBP-3 at 1.5 mg/kg/day improved some pulmonary and cardiovascular outcomes without toxicity. IMPACT: Preterm birth is associated with rapid decreases in serum or plasma IGF-1 protein level. This decline adversely impacts the growth and development of the lung and cardiovascular system. For this pilot study, continuous infusion of optimal dosage of rhIGF-1/rhIGFBP-3 (1.5 mg/kg/day) to maintain physiologic plasma IGF-1 level of ~125 ng/mL during mechanical ventilation for 3 days statistically improved some structural and biochemical outcomes related to the alveolar formation that would favor improved gas exchange compared to vehicle-control. We conclude that 3 days of continuous iv infusion of rhIGF-1/rhIGFBP-3 improved some physiological, morphological, and biochemical outcomes, without toxicity, in mechanically ventilated preterm lambs.
Asunto(s)
Displasia Broncopulmonar , Nacimiento Prematuro , Lactante , Femenino , Humanos , Animales , Recién Nacido , Ovinos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Displasia Broncopulmonar/tratamiento farmacológico , Proyectos Piloto , Recien Nacido Prematuro , Proteínas Recombinantes/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Oveja DomésticaRESUMEN
OBJECTIVE: Elamipretide (SS31) is a mitochondria-targeted peptide that has reported functions of stabilizing mitochondrial cristae structure and improving mitochondrial bioenergetics. Several studies have documented cell protective features of this peptide, including impairment of intrinsic apoptosis by inhibiting the recruitment and activation of the pro-apoptotic BAX protein. We used live-cell imaging of ARPE-19 cells expressing fluorescently labeled BAX, cytochrome c, and a mitochondrial marker to investigate the effect of elamipretide on the kinetics of BAX recruitment, mitochondrial outer membrane permeabilization (as a function of cytochrome c release), and mitochondrial fragmentation, respectively. RESULT: In nucleofected and plated ARPE-19 cells, elamipretide accelerated the formation of larger mitochondria. In the presence of the apoptotic stimulator, staurosporine, cells treated with elamipretide exhibited moderately slower rates of BAX recruitment. Peptide treatment, however, did not significantly delay the onset of BAX recruitment or the final total amount of BAX that was recruited. Additionally, elamipretide showed no impairment or delay of cytochrome c release or mitochondrial fragmentation, two events associated with normal BAX activation during cell death. These results indicate that the protective effect of elamipretide is not at the level of BAX activity to induce pro-apoptotic mitochondrial dysfunction after the initiation of staurosporine-induced apoptosis.
Asunto(s)
Apoptosis , Mitocondrias , Oligopéptidos/farmacología , Proteína X Asociada a bcl-2RESUMEN
Duchenne muscular dystrophy is characterized by structural degeneration of muscle, which is exacerbated by localized functional ischemia due to loss of nitric oxide synthase-induced vasodilation. Treatment strategies aimed at increasing vascular perfusion have been proposed. Toward this end, we have developed monoclonal antibodies (mAbs) that bind to the vascular endothelial growth factor (VEGF) receptor VEGFR-1 (Flt-1) and its soluble splice variant isoform (sFlt-1) leading to increased levels of free VEGF and proangiogenic signaling. The lead chimeric mAb, 21B3, had high affinity and specificity for both human and mouse sFlt-1 and inhibited VEGF binding to sFlt-1 in a competitive manner. Proof-of-concept studies in the mdx mouse model of Duchenne muscular dystrophy showed that intravenous administration of 21B3 led to elevated VEGF levels, increased vascularization and blood flow to muscles, and decreased fibrosis after 6-12 weeks of treatment. Greater muscle strength was also observed after 4 weeks of treatment. A humanized form of the mAb, 27H6, was engineered and demonstrated a comparable pharmacologic effect. Overall, administration of anti-Flt-1 mAbs in mdx mice inhibited the VEGF:Flt-1 interaction, promoted angiogenesis, and improved muscle function. These studies suggest a potential therapeutic benefit of Flt-1 inhibition for patients with Duchenne muscular dystrophy.
RESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease in which the dystrophin coding for a membrane stabilizing protein is mutated. Recently, the vasculature has also shown to be perturbed in DMD and DMD model mdx mice. Recent DMD transcriptomics revealed the defects were correlated to a vascular endothelial growth factor (VEGF) signaling pathway. To reveal the relationship between DMD and VEGF signaling, mdx mice were crossed with constitutive (CAGCreERTM:Flt1LoxP/LoxP) and endothelial cell-specific conditional gene knockout mice (Cdh5CreERT2:Flt1LoxP/LoxP) for Flt1 (VEGFR1) which is a decoy receptor for VEGF. Here, we showed that while constitutive deletion of Flt1 is detrimental to the skeletal muscle function, endothelial cell-specific Flt1 deletion resulted in increased vascular density, increased satellite cell number and improvement in the DMD-associated phenotype in the mdx mice. These decreases in pathology, including improved muscle histology and function, were recapitulated in mdx mice given anti-FLT1 peptides or monoclonal antibodies, which blocked VEGF-FLT1 binding. The histological and functional improvement of dystrophic muscle by FLT1 blockade provides a novel pharmacological strategy for the potential treatment of DMD.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Distrofia Muscular de Duchenne/tratamiento farmacológico , Péptidos/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Técnicas de Inactivación de Genes , Masculino , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , Especificidad de Órganos , Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
RATIONALE: Pregnancies complicated by antenatal stress, including preeclampsia (PE) and chorioamnionitis (CA), increase the risk for bronchopulmonary dysplasia (BPD) in preterm infants, but biologic mechanisms linking prenatal factors with BPD are uncertain. Levels of sFlt-1 (soluble fms-like tyrosine kinase 1), an endogenous antagonist to VEGF (vascular endothelial growth factor), are increased in amniotic fluid and maternal blood in PE and associated with CA. OBJECTIVES: Because impaired VEGF signaling has been implicated in the pathogenesis of BPD, we hypothesized that fetal exposure to sFlt-1 decreases lung growth and causes abnormal lung structure and pulmonary hypertension during infancy. METHODS: To test this hypothesis, we studied the effects of anti-sFlt-1 monoclonal antibody (mAb) treatment on lung growth in two established antenatal models of BPD that mimic PE and CA induced by intraamniotic (i.a.) injections of sFlt-1 or endotoxin, respectively. In experimental PE, mAb was administered by three different approaches, including antenatal treatment by either i.a. instillation or maternal uterine artery infusion, or by postnatal intraperitoneal injections. RESULTS: With each strategy, mAb therapy improved infant lung structure as assessed by radial alveolar count, vessel density, right ventricular hypertrophy, and lung function. As found in the PE model, the adverse lung effects of i.a. endotoxin were also reduced by antenatal or postnatal mAb therapy. CONCLUSIONS: We conclude that treatment with anti-sFlt-1 mAb preserves lung structure and function and prevents right ventricular hypertrophy in two rat models of BPD of antenatal stress and speculate that early mAb therapy may provide a novel strategy for the prevention of BPD.
Asunto(s)
Displasia Broncopulmonar/fisiopatología , Endotelio Vascular/crecimiento & desarrollo , Pulmón/crecimiento & desarrollo , Alveolos Pulmonares/crecimiento & desarrollo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/uso terapéutico , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/embriología , Modelos Animales de Enfermedad , Endotelio Vascular/embriología , Femenino , Humanos , Pulmón/embriología , Embarazo , Alveolos Pulmonares/embriología , Ratas , Ratas Sprague-DawleyRESUMEN
Recently, medical research has seen a strong push toward translational research, or "bench to bedside" collaborations, that strive to enhance the utility of laboratory science for improving medical treatment. The success of that paradigm supports the potential application of the process to other fields, such as risk assessment. Close collaboration among academic, government, and industry scientists may enhance the translation of scientific findings to regulatory decision making. The National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), and U.S. Food and Drug Administration (FDA) developed a consortium-based research program to link more effectively academic and guideline-compliant research. An initial proof-of-concept collaboration, the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA), uses bisphenol A (BPA) as a test chemical. The CLARITY-BPA program combines a core perinatal guideline-compliant 2-year chronic toxicity study with mechanistic studies/endpoints conducted by academic investigators. Twelve extramural grantees were selected by NIEHS through an RFA-based initiative to participate in the overall study design and conduct disease-relevant investigations using tissues and animals from the core study. While the study is expected to contribute to our understanding of potential effects of BPA, it also has ramifications beyond this specific focus. Through CLARITY-BPA, NIEHS has established an unprecedented level of collaboration among extramural grantees and regulatory researchers. By drawing upon the strengths of academic and regulatory expertise and research approaches, CLARITY-BPA represents a potential new model for filling knowledge gaps, enhancing quality control, informing chemical risk assessment, and identifying new methods or endpoints for regulatory hazard assessments.
Asunto(s)
Compuestos de Bencidrilo/toxicidad , Estrógenos no Esteroides/toxicidad , Fenoles/toxicidad , Proyectos de Investigación/normas , Animales , Conducta Cooperativa , Adhesión a Directriz , Humanos , National Institute of Environmental Health Sciences (U.S.) , Medición de Riesgo/normas , Estados Unidos , United States Food and Drug AdministrationRESUMEN
How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.
Asunto(s)
Endocitosis/inmunología , Endosomas/inmunología , Granzimas/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Cloruro de Amonio/farmacología , Animales , Apoptosis/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Citosol/inmunología , Citosol/metabolismo , Endosomas/metabolismo , Citometría de Flujo , Granzimas/metabolismo , Células HeLa , Humanos , Células Asesinas Naturales , Macrólidos/farmacología , Microscopía Confocal , Microscopía por Video , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Proteínas Citotóxicas Formadoras de Poros/metabolismo , RatasRESUMEN
SHG2210, a fusion protein containing the N-terminus of human nicotinic acetylcholine receptor α (AchR-α; aa1-210) and human transferrin (TF), was characterized as a potential therapeutic for myasthenia gravis (MG) caused predominately by α subunit autoantibodies. SHG2210 was shown to be able to bind to α subunit autoantibodies and the TF receptor (TFR). SHG2210 and SHG2210-anti-AchR antibody complex are internalized through TFR-mediated endocytosis. The SHG2210 and SHG2210-anti-AchR antibody complex is present in Lamp1-positive lysosomal compartments after internalization; however, neither SHG2210 nor SHG2210-antibody complex is present in Rab11-positive recycling endosomes. SHG2210 bound to α subunit of AChR autoantibodies may be cleared by the lysosome, resulting in short cellular half-life relative to SHG2210. SHG2210 is shown to have a protective effect on antigenic modulation of the AChR induced by serum from select patients with MG, suggesting that a fusion protein approach may be an effective therapeutic for treating MG.
Asunto(s)
Miastenia Gravis/inmunología , Receptores Nicotínicos/inmunología , Receptores de Transferrina/inmunología , Proteínas Recombinantes/farmacología , Transferrina/inmunología , Unión Competitiva/inmunología , Células HeLa , Humanos , Microscopía Confocal , Miastenia Gravis/tratamiento farmacológico , Receptores Nicotínicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Transferrina/genéticaRESUMEN
Cytotoxic T lymphocytes and natural killer cells destroy target cells via the polarized exocytosis of lytic effector proteins, perforin and granzymes, into the immunologic synapse. How these molecules enter target cells is not fully understood. It is debated whether granzymes enter via perforin pores formed at the plasma membrane or whether perforin and granzymes are first endocytosed and granzymes are then released from endosomes into the cytoplasm. We previously showed that perforin disruption of the plasma membrane induces a transient Ca(2+) flux into the target cell that triggers a wounded membrane repair response in which lysosomes and endosomes donate their membranes to reseal the damaged membrane. Here we show that perforin activates clathrin- and dynamin-dependent endocytosis, which removes perforin and granzymes from the plasma membrane to early endosomes, preserving outer membrane integrity. Inhibiting clathrin- or dynamin-dependent endocytosis shifts death by perforin and granzyme B from apoptosis to necrosis. Thus by activating endocytosis to preserve membrane integrity, perforin facilitates granzyme uptake and avoids the proinflammatory necrotic death of a membrane-damaged cell.
Asunto(s)
Apoptosis/inmunología , Membrana Celular/inmunología , Clatrina/inmunología , Dinaminas/inmunología , Endocitosis/inmunología , Granzimas/inmunología , Perforina/inmunología , Animales , Apoptosis/efectos de los fármacos , Membrana Celular/metabolismo , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/inmunología , Endosomas/metabolismo , Granzimas/farmacología , Células HeLa , Humanos , Perforina/metabolismo , RatasRESUMEN
BACKGROUND: Myasthenia gravis (MG) is an autoimmune disease affecting approximately 40,000 patients in the United States. One of the major mechanisms of disease pathology in MG is the binding, internalization, and eventual destruction of acetylcholine receptors (AChR) at the neuromuscular junction by cross-linking AChR-specific autoantibodies. This process, known as antigenic modulation, ultimately attenuates the ability of muscle cells to contract in response to signals from neurons, leading to muscle weakness and fatigue. For this reason, antigenic modulation of the AChR on cultured cells has become an important diagnostic tool for assessing the pathogenicity of AChR-specific autoantibodies. Traditionally, these assays have been done using radiolabeled AChR ligands such as (125)I alpha-bungarotoxin to determine relative AChR number. Here, we present a high-throughput immunofluorescent flow cytometry-based assay that can be used to quantify AChR levels on the cell surface and assess the efficacy of molecules designed to rescue antigenic modulation. METHODS: AChR levels were quantified on human muscle cells before and after treatment with AChR antibodies via immunofluorescent labeling with the AChR monoclonal antibodies, mAb210 and mAb B3, followed by flow cytometry of EDTA-treated cells. RESULTS: Using a novel, flow cytometry-based assay, antigenic modulation of the AChR was demonstrated on human cells using both AChR-specific monoclonal antibody and MG patient serum. The degree of antigenic modulation was dose responsive to antibody levels and could be reversed by preincubating antibodies with soluble AChR alpha subunit extracellular domain. SUMMARY: A rapid, nonradioactive assay was developed to determine the potential of AChR-specific antibodies in the serum of MG patients to bind and down-regulate the AChR. This assay can be used to assess the ability of putative therapeutics that rescue antigenic modulation and could be developed for the treatment of MG.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Autoanticuerpos/inmunología , Regulación hacia Abajo , Citometría de Flujo/métodos , Fluorescencia , Receptores Colinérgicos/análisis , Receptores Colinérgicos/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Reacciones Antígeno-Anticuerpo/inmunología , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Humanos , Miastenia Gravis/inmunología , Miastenia Gravis/metabolismo , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Granzyme A (GzmA) induces caspase-independent cell death with morphological features of apoptosis. Here, we show that GzmA at nanomolar concentrations cleaves Ku70, a key double-strand break repair (DSBR) protein, in target cells. Ku70 is cut after Arg(301), disrupting Ku complex binding to DNA. Cleaving Ku70 facilitates GzmA-mediated cell death, as silencing Ku70 by RNA interference increases DNA damage and cell death by GzmB cluster-deficient cytotoxic T lymphocytes or by GzmA and perforin, whereas Ku70 overexpression has the opposite effect. Ku70 has two known antiapoptotic effects-facilitating DSBR and sequestering bax to prevent its translocation to mitochondria. However, GzmA triggers single-stranded, not double-stranded, DNA damage, and GzmA-induced cell death does not involve bax. Therefore, Ku70 has other antiapoptotic functions in GzmA-induced cell death, which are blocked when GzmA proteolyses Ku70.
Asunto(s)
Antígenos Nucleares/metabolismo , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Antígenos Nucleares/genética , Arginina/genética , Arginina/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , ADN/genética , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Granzimas , Humanos , Autoantígeno Ku , Glicoproteínas de Membrana/farmacología , Ratones , Perforina , Proteínas Citotóxicas Formadoras de Poros , Inhibidores de Proteasas/farmacología , Unión Proteica , Serina Endopeptidasas/farmacología , Linfocitos T Citotóxicos/metabolismoRESUMEN
Perforin delivers granzymes to induce target-cell apoptosis. At high concentrations, perforin multimerizes in the plasma membrane to form pores. However, whether granzymes enter target cells via membrane pores is uncertain. Here we find that perforin at physiologically relevant concentrations and during cell-mediated lysis creates pores in the target-cell membrane, transiently allowing Ca(2+) and small dyes into the cell. The Ca(2+) flux triggers a wounded membrane-repair response in which internal vesicles, including lysosomes and endosomes, donate their membranes to reseal the damaged membrane. Perforin also triggers the rapid endocytosis of granzymes into large EEA-1-staining vesicles. The restoration of target-cell membrane integrity by triggering the repair response is necessary for target cells subjected to cytotoxic T lymphocyte attack to avoid necrosis and undergo the slower process of programmed cell death. Thus, the target cell actively participates in determining its own fate during cell-mediated death.
Asunto(s)
Apoptosis/inmunología , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfocitos T Citotóxicos/inmunología , Calcio/metabolismo , Membrana Celular/inmunología , Endocitosis/fisiología , Granzimas , Humanos , Lisosomas/inmunología , Lisosomas/metabolismo , Glicoproteínas de Membrana/inmunología , Microscopía Confocal , Perforina , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidasas/metabolismo , Células U937RESUMEN
Perforin (PFN) delivery of granzymes (Gzm) into the target cell at the immunological synapse is the major pathway for inducing apoptosis of virus-infected cells and tumors. A validated model for how PFN delivers Gzm into the cytosol is still lacking. PFN was originally thought to work by forming pores in the target cell plasma membrane that allow Gzm entry. This model was questioned when it was shown that GzmB is endocytosed without PFN. Moreover, apoptosis could be triggered by adding PFN to washed cells that have previously endocytosed GzmB. In this study, we show that GzmB binds to the plasma membrane mostly via nonspecific charge interactions. Washing in saline does not remove bound Gzm. However, if externally bound GzmB is completely removed, subsequent addition of PFN does not release previously endocytosed GzmB and does not trigger apoptosis. Therefore, PFN must be coendocytosed with GzmB to deliver it into the cytosol.