RESUMO
Traumatic brain injury has faced numerous challenges in drug development, primarily due to the difficulty of effectively delivering drugs to the brain. However, there is a potential solution in targeted drug delivery methods involving antibody-drug conjugates or nanocarriers conjugated with targeting antibodies. Following a TBI, the blood-brain barrier (BBB) becomes permeable, which can last for years and allow the leakage of harmful plasma proteins. Consequently, an appealing approach for TBI treatment involves using drug delivery systems that utilize targeting antibodies and nanocarriers to help restore BBB integrity. In our investigation of this strategy, we examined the efficacy of free antibodies and nanocarriers targeting a specific endothelial surface marker called vascular cell adhesion molecule-1 (VCAM-1), which is known to be upregulated during inflammation. In a mouse model of TBI utilizing central fluid percussion injury, free VCAM-1 antibody did not demonstrate superior targeting when comparing sham vs. TBI brain. However, the administration of VCAM-1-targeted nanocarriers (liposomes) exhibited a 10-fold higher targeting specificity in TBI brain than in sham control. Flow cytometry and confocal microscopy analysis confirmed that VCAM-1 liposomes were primarily taken up by brain endothelial cells post-TBI. Consequently, VCAM-1 liposomes represent a promising platform for the targeted delivery of therapeutics to the brain following traumatic brain injury.
Assuntos
Barreira Hematoencefálica , Lesões Encefálicas Traumáticas , Nanopartículas , Molécula 1 de Adesão de Célula Vascular , Animais , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Camundongos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Nanopartículas/química , Lipossomos , Masculino , Sistemas de Liberação de Medicamentos , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacosRESUMO
The extracellular accumulation of amyloid ß (Aß) fragments of amyloid precursor protein (APP) in brain parenchyma is a pathological hallmark of Alzheimer's disease (AD). APP can be cleaved into Aß on late endosomes/multivesicular bodies (MVBs). E3 ubiquitin ligases have been linked to Aß production, but specific E3 ligases associated with APP ubiquitination that may affect targeting of APP to endosomes have not yet been described. Using cultured cortical neurons isolated from rat pups, we reconstituted APP movement into the internal vesicles (ILVs) of MVBs. Loss of endosomal sorting complexes required for transport (ESCRT) components inhibited APP movement into ILVs and increased endosomal Aß42 generation, implying a requirement for APP ubiquitination. We identified an ESCRT-binding and APP-interacting endosomal E3 ubiquitin ligase, ubiquitination factor E4B (UBE4B) that regulates APP ubiquitination. Depleting UBE4B in neurons inhibited APP ubiquitination and internalization into MVBs, resulting in increased endosomal Aß42 levels and increased neuronal secretion of Aß42. When we examined AD brains, we found levels of the UBE4B-interacting ESCRT component, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), were significantly decreased in AD brains. These data suggest that ESCRT components critical for membrane protein sorting in the endocytic pathway are altered in AD. These results indicate that the molecular machinery underlying endosomal trafficking of APP, including the ubiquitin ligase UBE4B, regulates Aß levels and may play an essential role in AD progression.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Endossomos/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Ubiquitinação , Animais , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Transporte Proteico , Ratos , Vesículas Secretórias/metabolismoRESUMO
Regulating the residence time of membrane proteins on the cell surface can modify their response to extracellular cues and allow for cellular adaptation in response to changing environmental conditions. The fate of membrane proteins that are internalized from the plasma membrane and arrive at the limiting membrane of the late endosome/multivesicular body (MVB) is dictated by whether they remain on the limiting membrane, bud into internal MVB vesicles, or bud outwardly from the membrane. The molecular details underlying the disposition of membrane proteins that transit this pathway and the mechanisms regulating these trafficking events are unclear. We established a cell-free system that reconstitutes budding of membrane protein cargo into internal MVB vesicles and onto vesicles that bud outwardly from the MVB membrane. Both budding reactions are cytosol-dependent and supported by Saccharomyces cerevisiae (yeast) cytosol. We observed that inward and outward budding from the MVB membrane are mechanistically distinct but may be linked, such that inhibition of inward budding triggers a re-routing of cargo from inward to outward budding vesicles, without affecting the number of vesicles that bud outwardly from MVBs.
Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/química , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Regulação da Expressão Gênica , Células HeLa , Humanos , Membranas Intracelulares/ultraestrutura , Lisossomos/ultraestrutura , Corpos Multivesiculares/ultraestrutura , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Outcomes for children with high-risk neuroblastoma are poor, and improved understanding of the mechanisms underlying neuroblastoma pathogenesis, recurrence, and treatment resistance will lead to improved outcomes. Aberrant growth factor receptor expression and receptor tyrosine kinase signaling are associated with the pathogenesis of many malignancies. A germline polymorphism in the FGFR4 gene is associated with increased receptor expression and activity and with decreased survival, treatment resistance, and aggressive disease for many malignancies. We therefore investigated the role of this FGFR4 polymorphism in neuroblastoma pathogenesis. MATERIALS AND METHODS: Germline DNA from neuroblastoma patients and matched controls was assessed for the FGFR4 Gly/Arg388 polymorphism by RT-PCR. Allele frequencies were assessed for association with neuroblastoma patient outcomes and prognostic features. Degradation rates of the FGFR4 Arg388 and Gly388 receptors and rates of receptor internalization into the late endosomal compartment were measured. RESULTS: Frequency of the FGFR4 AA genotype and the prevalence of the A allele were significantly higher in patients with neuroblastoma than in matched controls. The Arg388 receptor demonstrated slower degradation than the Gly388 receptor in neuroblastoma cells and reduced internalization into multivesicular bodies. CONCLUSIONS: The FGFR4 Arg388 polymorphism is associated with an increased prevalence of neuroblastoma in children, and this association may be linked to differences in FGFR4 degradation rates. Our study provides the first evidence of a role for FGFR4 in neuroblastoma, suggesting that FGFR4 genotype and the pathways regulating FGFR4 trafficking and degradation may be relevant for neuroblastoma pathogenesis.
Assuntos
Predisposição Genética para Doença/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Polimorfismo de Fragmento de Restrição , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Western Blotting , Criança , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Masculino , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
Chloroquine (CQ) resistance (CQR) in Plasmodium falciparum originated from at least six foci in South America, Asia, and Oceania. Malaria parasites from these locations exhibit contrasting resistance phenotypes that are distinguished by point mutations and microsatellite polymorphisms in and near the CQR transporter gene, pfcrt, and the multidrug resistance transporter gene, pfmdr1. Amodiaquine (AQ), a 4-aminoquinoline related to CQ, is recommended and often used successfully against CQ-resistant P. falciparum in Africa, but it is largely ineffective across large regions of South America. The relationship of different pfcrt and pfmdr1 combinations to these drug-resistant phenotypes has been unclear. In two P. falciparum genetic crosses, particular pfcrt and pfmdr1 alleles from South America interact to yield greater levels of resistance to monodesethylamodiaquine (MDAQ; the active metabolite of AQ) than to CQ, whereas a pfcrt allele from Southeast Asia and Africa is linked to greater CQ than MDAQ resistance with all partner pfmdr1 alleles. These results, together with (i) available haplotype data from other parasites; (ii) evidence for an emerging focus of AQ resistance in Tanzania; and (iii) the persistence of 4-aminoquinoline-resistant parasites in South America, where CQ and AQ use is largely discontinued, suggest that different histories of drug use on the two continents have driven the selection of distinct suites of pfcrt and pfmdr1 mutations. Increasing use of AQ in Africa poses the threat of a selective sweep of highly AQ-resistant, CQ-resistant parasites with pfcrt and pfmdr1 mutations that are as advantaged and persistent as in South America.