RESUMO
Understanding the vesicular trafficking of receptors and receptor ligands in the brain capillary endothelium is essential for the development of the next generations of biologics targeting neurodegenerative diseases. Such complex biological questions are often approached by in vitro models in combination with various techniques. Here, we present the development of a stem cell-based human in vitro blood-brain barrier model composed of induced brain microvascular endothelial cells (iBMECs) on the modular µSiM (a microdevice featuring a silicon nitride membrane) platform. The µSiM was equipped with a 100 nm thick nanoporous silicon nitride membrane with glass-like imaging quality that allowed the use of high-resolution in situ imaging to study the intracellular trafficking. As a proof-of-concept experiment, we investigated the trafficking of two monoclonal antibodies (mAb): an anti-human transferrin receptor mAb (15G11) and an anti-basigin mAb (#52) using the µSiM-iBMEC-human astrocyte model. Our results demonstrated effective endothelial uptake of the selected antibodies; however, no significant transcytosis was observed when the barrier was tight. In contrast, when the iBMECs did not form a confluent barrier on the µSiM, the antibodies accumulated inside both the iBMECs and astrocytes, demonstrating that the cells have an active endocytic and subcellular sorting machinery and that the µSiM itself does not hinder antibody transport. In conclusion, our µSiM-iBMEC-human astrocyte model provides a tight barrier with endothelial-like cells, which can be used for high-resolution in situ imaging and for studying receptor-mediated transport and transcytosis in a physiological barrier.
Assuntos
Barreira Hematoencefálica , Células Endoteliais , Humanos , Barreira Hematoencefálica/metabolismo , Técnicas de Cocultura , Células Endoteliais/metabolismo , Encéfalo/metabolismo , Anticorpos/metabolismo , Dispositivos Lab-On-A-ChipRESUMO
BACKGROUND AND AIM: Interleukin-21 (IL-21) is primarily a T cell-derived cytokine; it is upregulated in patients with Crohn's Disease (CD) and could be a potential new therapeutic target in CD. METHODS: In human material, IL-21 and IL-21R expression was investigated by in situ hybridization (ISH) and immunohistochemistry (IHC) in noninflammatory bowel disease (non-IBD) controls and patients with CD. The pathologic role of IL-21 was examined in murine models of T cell-dependent and T cell-independent colitis, either with a neutralizing monoclonal antibody against IL-21 or with the transfer of CD4+CD45RBhighIL-21R-/- T cells. Colonic pathology was examined by endoscopy, histopathology, IHC, ELISA, and Luminex. RESULTS: In the human intestine, IL-21 and IL-21R mRNA and protein-expressing cells were observed in the mucosa, in lymphoid aggregates of submucosa in non-IBD controls, and in lymphoid aggregates of muscularis externa in patients with CD. IL-21 expression was most abundant in germinal centers (GCs) of the lymphoid aggregates, and IL-21R expression assessed semiquantitatively, was significantly higher in patients with CD compared to non-IBD controls. Following prophylactic and interventive anti-IL-21 mAb treatment in the adoptive transfer (AdTr) model, clinical and pathological parameters were significantly reduced. The most persistent finding was a reduction in colonic infiltrating neutrophils. As well, Rag2-/- mice receiving CD4+CD45RBhighIL-21R-/- T cells developed less severe colitis compared to Rag2-/- mice receiving CD4+CD45RBhighIL-21R+/+ T cells. No effect of reduced IL-21 signalling was observed in T cell-independent colitis. CONCLUSION: Our study shows that patients with CD have significant expression of IL-21 and IL-21R in the gut. As well, we show that neutralization of IL-21 in experimental T cell-driven colitis is associated with a reduction in clinical and pathological findings. This amelioration seems to be associated with a reduction in colon-infiltrating neutrophils.
RESUMO
Neurodegeneration is the main cause for permanent disability in multiple sclerosis. The effect of current immunomodulatory treatments on neurodegeneration is insufficient. Therefore, direct neuroprotection and myeloprotection remain an important therapeutic goal. Targeting acid-sensing ion channel 1 (encoded by the ASIC1 gene), which contributes to the excessive intracellular accumulation of injurious Na(+) and Ca(2+) and is over-expressed in acute multiple sclerosis lesions, appears to be a viable strategy to limit cellular injury that is the substrate of neurodegeneration. While blockade of ASIC1 through amiloride, a potassium sparing diuretic that is currently licensed for hypertension and congestive cardiac failure, showed neuroprotective and myeloprotective effects in experimental models of multiple sclerosis, this strategy remains untested in patients with multiple sclerosis. In this translational study, we tested the neuroprotective effects of amiloride in patients with primary progressive multiple sclerosis. First, we assessed ASIC1 expression in chronic brain lesions from post-mortem of patients with progressive multiple sclerosis to identify the target process for neuroprotection. Second, we tested the neuroprotective effect of amiloride in a cohort of 14 patients with primary progressive multiple sclerosis using magnetic resonance imaging markers of neurodegeneration as outcome measures of neuroprotection. Patients with primary progressive multiple sclerosis underwent serial magnetic resonance imaging scans before (pretreatment phase) and during (treatment phase) amiloride treatment for a period of 3 years. Whole-brain volume and tissue integrity were measured with high-resolution T(1)-weighted and diffusion tensor imaging. In chronic brain lesions of patients with progressive multiple sclerosis, we demonstrate an increased expression of ASIC1 in axons and an association with injury markers within chronic inactive lesions. In patients with primary progressive multiple sclerosis, we observed a significant reduction in normalized annual rate of whole-brain volume during the treatment phase, compared with the pretreatment phase (P = 0.018, corrected). Consistent with this reduction, we showed that changes in diffusion indices of tissue damage within major clinically relevant white matter (corpus callosum and corticospinal tract) and deep grey matter (thalamus) structures were significantly reduced during the treatment phase (P = 0.02, corrected). Our results extend evidence of the contribution of ASIC1 to neurodegeneration in multiple sclerosis and suggest that amiloride may exert neuroprotective effects in patients with progressive multiple sclerosis. This pilot study is the first translational study on neuroprotection targeting ASIC1 and supports future randomized controlled trials measuring neuroprotection with amiloride in patients with multiple sclerosis.
Assuntos
Bloqueadores do Canal Iônico Sensível a Ácido/uso terapêutico , Canais Iônicos Sensíveis a Ácido/genética , Amilorida/uso terapêutico , Encéfalo/efeitos dos fármacos , Esclerose Múltipla Crônica Progressiva/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Canais Iônicos Sensíveis a Ácido/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Encéfalo/patologia , Imagem de Tensor de Difusão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Crônica Progressiva/genética , Esclerose Múltipla Crônica Progressiva/metabolismo , Esclerose Múltipla Crônica Progressiva/patologia , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/patologia , Projetos Piloto , Resultado do TratamentoRESUMO
Although there is growing evidence for a role of excess intracellular cations, particularly calcium ions, in neuronal and glial cell injury in multiple sclerosis, as well as in non-inflammatory neurological conditions, the molecular mechanisms involved are not fully determined. We previously showed that the acid-sensing ion channel 1 which, when activated under the acidotic tissue conditions found in inflammatory lesions opens to allow influx of sodium and calcium ions, contributes to axonal injury in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, the extent and cellular distribution of acid-sensing ion channel 1 expression in neurons and glia in inflammatory lesions is unknown and, crucially, acid-sensing ion channel 1 expression has not been determined in multiple sclerosis lesions. Here we studied acute and chronic experimental autoimmune encephalomyelitis and multiple sclerosis spinal cord and optic nerve tissues to describe in detail the distribution of acid-sensing ion channel 1 and its relationship with neuronal and glial damage. We also tested the effects of amiloride treatment on tissue damage in the mouse models. We found that acid-sensing ion channel 1 was upregulated in axons and oligodendrocytes within lesions from mice with acute experimental autoimmune encephalomyelitis and from patients with active multiple sclerosis. The expression of acid-sensing ion channel 1 was associated with axonal damage as indicated by co-localization with the axonal injury marker beta amyloid precursor protein. Moreover, blocking acid-sensing ion channel 1 with amiloride protected both myelin and neurons from damage in the acute model, and when given either at disease onset or, more clinically relevant, at first relapse, ameliorated disability in mice with chronic-relapsing experimental autoimmune encephalomyelitis. Together these findings suggest that blockade of acid-sensing ion channel 1 has the potential to provide both neuro- and myelo-protective benefits in multiple sclerosis.
Assuntos
Axônios/metabolismo , Doenças Desmielinizantes/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Canais de Sódio/biossíntese , Canais Iônicos Sensíveis a Ácido , Idoso , Amilorida/farmacologia , Amilorida/uso terapêutico , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Técnicas de Cultura de Células , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/prevenção & controle , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Oligodendroglia/metabolismo , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Regulação para CimaRESUMO
An abnormal accumulation of cytosolic dopamine resulting in reactive oxygen species and dopamine-quinone products may play an important role in the rather selective degeneration of substantia nigra pars compacta (SNc) dopaminergic neurons in Parkinson's disease. The neuronal-specific vesicular monoamine transporter (VMAT2), responsible for uptake of dopamine into vesicles, has been shown to play a central role both in intracellular dopamine homeostasis and sequestration of dopaminergic neurotoxins. Direct or indirect enhancement of VMAT2 activity could therefore have neuroprotective effects by decreasing cytosolic dopamine levels. Here, we demonstrate that transfection of VMAT2 in the dopaminergic cell line, PC12, increases intracellular dopamine content, augments potassium-induced dopamine release and attenuates cell death induced by the cytosolic dopamine enhancer, methamphetamine, suggesting an enhancement in vesicular dopamine storage. In rat ventral mesencephalic cultures highly enriched for dopaminergic neurons, lentiviral delivery of recombinant VMAT2 using a neuronal-specific promoter also resulted in elevated intracellular dopamine content and neurotransmitter release after depolarization. The opposite was seen after downregulation of VMAT2 using virally delivered shRNAs. Furthermore, using this VMAT2 knockdown model, we are the first to report a direct link between enhanced cytoplasmic dopamine levels, measured following mild permeabilization of the plasma membrane using digitonin, and neurite degeneration in primary dopaminergic neurons. In conclusion, our data support the hypothesis that an increase in vesicular sequestration of dopamine by modulation of VMAT2 activity could restore neuronal function and enhance dopaminergic cell survival in conditions of dysregulated dopamine homeostasis such as Parkinson's disease.
Assuntos
Citosol/metabolismo , Dopamina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/fisiologia , Inibidores da Captação Adrenérgica/farmacologia , Análise de Variância , Animais , Proliferação de Células , Citosol/efeitos dos fármacos , Interações Medicamentosas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , L-Lactato Desidrogenase/metabolismo , Metanfetamina/farmacologia , Células PC12/citologia , Cloreto de Potássio/farmacologia , Ratos , Reserpina/farmacologia , Sais de Tetrazólio , Tiazóis , Fatores de Tempo , Transfecção , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Multiple sclerosis is a neuroinflammatory disease associated with axonal degeneration. The neuronally expressed, proton-gated acid-sensing ion channel-1 (ASIC1) is permeable to Na+ and Ca2+, and excessive accumulation of these ions is associated with axonal degeneration. We tested the hypothesis that ASIC1 contributes to axonal degeneration in inflammatory lesions of the central nervous system (CNS). After induction of experimental autoimmune encephalomyelitis (EAE), Asic1-/- mice showed both a markedly reduced clinical deficit and reduced axonal degeneration compared to wild-type mice. Consistently with acidosis-mediated injury, pH measurements in the spinal cord of EAE mice showed tissue acidosis sufficient to open ASIC1. The acidosis-related protective effect of Asic1 disruption was also observed in nerve explants in vitro. Amiloride, a licensed and clinically safe blocker of ASICs, was equally neuroprotective in nerve explants and in EAE. Although ASICs are also expressed by immune cells, this expression is unlikely to explain the neuroprotective effect of Asic1 inactivation, as CNS inflammation was similar in wild-type and Asic1-/- mice. In addition, adoptive transfer of T cells from wild-type mice did not affect the protection mediated by Asic1 disruption. These results suggest that ASIC1 blockers could provide neuroprotection in multiple sclerosis.
Assuntos
Autoimunidade , Axônios/patologia , Sistema Nervoso Central/patologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Canais de Sódio/fisiologia , Canais Iônicos Sensíveis a Ácido , Amilorida/farmacologia , Animais , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Inflamação/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Degeneração Neural , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/farmacologia , Canais de Sódio/metabolismo , Medula Espinal/patologiaRESUMO
Gephyrin is required for the formation of clusters of the glycine receptor (GlyR) in the neuronal postsynaptic membrane. It can make trimers and dimers through its N- and C-terminal G and E domains, respectively. Gephyrin oligomerization could thus create a submembrane lattice providing GlyR-binding sites. We investigated the relationships between the stability of cell surface GlyR and the ability of gephyrin splice variants to form oligomers. Using truncated and full-length gephyrins we found that the 13-amino acid sequence (cassette 5) prevents G domain trimerization. Moreover, E domain dimerization is inhibited by the gephyrin central L domain. All of the gephyrin variants bind GlyR beta subunit cytoplasmic loop with high affinity regardless of their cassette composition. Coexpression experiments in COS-7 cells demonstrated that GlyR bound to gephyrin harboring cassette 5 cannot be stabilized at the cell surface. This gephyrin variant was found to deplete synapses from both GlyR and gephyrin in transfected neurons. These data suggest that the relative expression level of cellular variants influence the overall oligomerization pattern of gephyrin and thus the turnover of synaptic GlyR.