RESUMO
BACKGROUND: Microvascular complications are the major outcome of type 2 diabetes progression, and the underlying mechanism remains to be determined. METHODS: High-throughput RNA sequencing was performed using human monocyte samples from controls and diabetes. The transgenic mice expressing human CTSD (cathepsin D) in the monocytes was constructed using CD68 promoter. In vivo 2-photon imaging, behavioral tests, immunofluorescence, transmission electron microscopy, Western blot analysis, vascular leakage assay, and single-cell RNA sequencing were performed to clarify the phenotype and elucidate the molecular mechanism. RESULTS: Monocytes expressed high-level CTSD in patients with type 2 diabetes. The transgenic mice expressing human CTSD in the monocytes showed increased brain microvascular permeability resembling the diabetic microvascular phenotype, accompanied by cognitive deficit. Mechanistically, the monocytes release nonenzymatic pro-CTSD to upregulate caveolin expression in brain endothelium triggering caveolae-mediated transcytosis, without affecting the paracellular route of brain microvasculature. The circulating pro-CTSD activated the caveolae-mediated transcytosis in brain endothelial cells via its binding with low-density LRP1 (lipoprotein receptor-related protein 1). Importantly, genetic ablation of CTSD in the monocytes exhibited a protective effect against the diabetes-enhanced brain microvascular transcytosis and the diabetes-induced cognitive impairment. CONCLUSIONS: These findings uncover the novel role of circulatory pro-CTSD from monocytes in the pathogenesis of cerebral microvascular lesions in diabetes. The circulatory pro-CTSD is a potential target for the intervention of microvascular complications in diabetes.
Assuntos
Catepsina D , Diabetes Mellitus Tipo 2 , Monócitos , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Catepsina D/metabolismo , Catepsina D/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Precursores Enzimáticos , Camundongos Transgênicos , Monócitos/metabolismo , Transcitose/fisiologiaRESUMO
BACKGROUND: It is well known that high-fat diet (HFD)-induced metabolic syndrome plays a crucial role in cognitive decline and brain-blood barrier (BBB) breakdown. However, whether the bone-brain axis participates in this pathological process remains unknown. Here, we report that platelet-derived growth factor-BB (PDGF-BB) secretion by preosteoclasts in the bone accelerates neuroinflammation. The expression of alkaline phosphatase (ALPL), a nonspecific transcytosis marker, was upregulated during HFD challenge. MAIN BODY: Preosteoclast-specific Pdgfb transgenic mice with high PDGF-BB concentrations in the circulation recapitulated the HFD-induced neuroinflammation and transcytosis shift. Preosteoclast-specific Pdgfb knockout mice were partially rescued from hippocampal neuroinflammation and transcytosis shifts in HFD-challenged mice. HFD-induced PDGF-BB elevation aggravated microglia-associated neuroinflammation and interleukin-1ß (IL-1ß) secretion, which increased ALPL expression and transcytosis shift through enhancing protein 1 (SP1) translocation in endothelial cells. CONCLUSION: Our findings confirm the role of bone-secreted PDGF-BB in neuroinflammation and the transcytosis shift in the hippocampal region during HFD challenge and identify a novel mechanism of microglia-endothelial crosstalk in HFD-induced metabolic syndrome.
Assuntos
Becaplermina , Dieta Hiperlipídica , Células Endoteliais , Hipocampo , Síndrome Metabólica , Microglia , Transcitose , Animais , Camundongos , Becaplermina/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Transcitose/fisiologia , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Microglia/metabolismo , Microglia/patologia , Dieta Hiperlipídica/efeitos adversos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Masculino , Osso e Ossos/metabolismo , Osso e Ossos/patologiaRESUMO
Elevated transport of Caveolin-1 (CAV-1) vesicles within vascular endothelial cells constitutes a significant secondary pathogenic event contributing to the compromise of the blood-brain barrier (BBB) post-traumatic brain injury (TBI). While Wnt/ß-catenin signaling is recognized for its critical involvement in angiogenesis and the maintenance of BBB integrity, its influence on vascular endothelial transcytosis in the aftermath of TBI is not well-defined. This study aims to elucidate the impact of Wnt/ß-catenin signaling on cerebrovascular vesicular transcytosis following TBI. In this experiment, adult male wild-type (WT) C57BL/6 mice underwent various interventions. TBI was induced utilizing the controlled cortical impact technique. Post-TBI, mice were administered either an inhibitor or an agonist of Wnt signaling via intraperitoneal injection. Recombinant adeno-associated virus (rAAV) was administered intracerebroventricularly to modulate the expression of the CAV-1 inhibitory protein, Major facilitator superfamily domain-containing 2a (Mfsd2a). This research utilized Evans blue assay, Western blot analysis, immunofluorescence, transmission electron microscopy, and neurobehavioral assessments. Post-TBI observations revealed substantial increases in macromolecule (Evans blue and albumin) leakage, CAV-1 transport vesicle count, astrocyte end-feet edema, and augmented aquaporin-4 (AQP4) expression, culminating in BBB disruption. The findings indicate that Wnt signaling pathway inhibition escalates CAV-1 transport vesicle activity and aggravates BBB compromise. Conversely, activating this pathway could alleviate BBB damage by curtailing CAV-1 vesicle presence. Post-TBI, there is a diminution in Mfsd2a expression, which is directly influenced by the modulation of WNT signals. Employing a viral approach to regulate Mfsd2a, we established that its down-regulation undermines the protective benefits derived from reducing CAV-1 transport vesicles through WNT signal enhancement. Moreover, we verified that the WNT signaling agonist LiCl notably ameliorates neurological deficits following TBI in mice. Collectively, our data imply that Wnt/ß-catenin signaling presents a potential therapeutic target for safeguarding against BBB damage and enhancing neurological function after TBI.
Assuntos
Barreira Hematoencefálica , Lesões Encefálicas Traumáticas , Caveolina 1 , Camundongos Endogâmicos C57BL , Transcitose , Via de Sinalização Wnt , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/efeitos dos fármacos , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Camundongos , Masculino , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Transcitose/efeitos dos fármacos , Transcitose/fisiologia , Caveolina 1/metabolismo , SimportadoresRESUMO
BACKGROUND: Group B Streptococcus (GBS) is the leading cause of neonatal meningitis responsible for a substantial cause of death and disability worldwide. The vast majority of GBS neonatal meningitis cases are due to the CC17 hypervirulent clone. However, the cellular and molecular pathways involved in brain invasion by GBS CC17 isolates remain largely elusive. Here, we studied the specific interaction of the CC17 clone with the choroid plexus, the main component of the blood-cerebrospinal fluid (CSF) barrier. METHODS: The interaction of GBS CC17 or non-CC17 strains with choroid plexus cells was studied using an in vivo mouse model of meningitis and in vitro models of primary and transformed rodent choroid plexus epithelial cells (CPEC and Z310). In vivo interaction of GBS with the choroid plexus was assessed by microscopy. Bacterial invasion and cell barrier penetration were examined in vitro, as well as chemokines and cytokines in response to infection. RESULTS: GBS CC17 was found associated with the choroid plexus of the lateral, 3rd and 4th ventricles. Infection of choroid plexus epithelial cells revealed an efficient internalization of the bacteria into the cells with GBS CC17 displaying a greater ability to invade these cells than a non-CC17 strain. Internalization of the GBS CC17 strain involved the CC17-specific HvgA adhesin and occurred via a clathrin-dependent mechanism leading to transcellular transcytosis across the choroid plexus epithelial monolayer. CPEC infection resulted in the secretion of several chemokines, including CCL2, CCL3, CCL20, CX3CL1, and the matrix metalloproteinase MMP3, as well as immune cell infiltration. CONCLUSION: Our findings reveal a GBS strain-specific ability to infect the blood-CSF barrier, which appears to be an important site of bacterial entry and an active site of immune cell trafficking in response to infection.
Assuntos
Plexo Corióideo , Streptococcus agalactiae , Plexo Corióideo/metabolismo , Plexo Corióideo/microbiologia , Plexo Corióideo/imunologia , Animais , Streptococcus agalactiae/patogenicidade , Camundongos , Adesinas Bacterianas/metabolismo , Virulência , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Barreira Hematoencefálica/microbiologia , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/imunologia , Camundongos Endogâmicos C57BL , Transcitose/fisiologia , FemininoRESUMO
Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.