RESUMO
Neurovascular unit and barrier maturation rely on vascular basement membrane (vBM) composition. Laminins, a major vBM component, are crucial for these processes, yet the signaling pathway(s) that regulate their expression remain unknown. Here, we show that mural cells have active Wnt/ß-catenin signaling during central nervous system development in mice. Bulk RNA sequencing and validation using postnatal day 10 and 14 wild-type versus adenomatosis polyposis coli downregulated 1 (Apcdd1-/-) mouse retinas revealed that Lama2 mRNA and protein levels are increased in mutant vasculature with higher Wnt/ß-catenin signaling. Mural cells are the main source of Lama2, and Wnt/ß-catenin activation induces Lama2 expression in mural cells in vitro. Markers of mature astrocytes, including aquaporin 4 (a water channel in astrocyte endfeet) and integrin-α6 (a laminin receptor), are upregulated in Apcdd1-/- retinas with higher Lama2 vBM deposition. Thus, the Wnt/ß-catenin pathway regulates Lama2 expression in mural cells to promote neurovascular unit and barrier maturation.
Assuntos
Via de Sinalização Wnt , beta Catenina , Animais , Camundongos , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However, the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell.
Assuntos
Retículo Endoplasmático/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Ácidos Graxos/metabolismo , Células HeLa , HumanosRESUMO
Interactions among neuronal, glial and vascular components are crucial for retinal angiogenesis and blood-retinal barrier (BRB) maturation. Although synaptic dysfunction precedes vascular abnormalities in many retinal pathologies, how neuronal activity, specifically glutamatergic activity, regulates retinal angiogenesis and BRB maturation remains unclear. Using in vivo genetic studies in mice, single-cell RNA-sequencing and functional validation, we show that deep plexus angiogenesis and paracellular BRB maturation are delayed in Vglut1 -/- retinas where neurons fail to release glutamate. In contrast, deep plexus angiogenesis and paracellular BRB maturation are accelerated in Gnat1 -/- retinas where constitutively depolarized rods release excessive glutamate. Norrin expression and endothelial Norrin/ß-catenin signaling are downregulated in Vglut1 -/- retinas, and upregulated in Gnat1 -/- retinas. Pharmacological activation of endothelial Norrin/ß-catenin signaling in Vglut1 -/- retinas rescued defects in deep plexus angiogenesis and paracellular BRB maturation. Our findings demonstrate that glutamatergic neuronal activity regulates retinal angiogenesis and BRB maturation by modulating endothelial Norrin/ß-catenin signaling.
RESUMO
Interactions among neuronal, glial, and vascular components are crucial for retinal angiogenesis and blood-retinal barrier (BRB) maturation. Although synaptic dysfunction precedes vascular abnormalities in many retinal pathologies, how neuronal activity, specifically glutamatergic activity, regulates retinal angiogenesis and BRB maturation remains unclear. Using in vivo genetic studies in mice, single-cell RNA sequencing (scRNA-seq), and functional validation, we show that deep plexus angiogenesis and paracellular BRB maturation are delayed in Vglut1-/- retinas where neurons fail to release glutamate. By contrast, deep plexus angiogenesis and paracellular BRB maturation are accelerated in Gnat1-/- retinas, where constitutively depolarized rods release excessive glutamate. Norrin expression and endothelial Norrin/ß-catenin signaling are downregulated in Vglut1-/- retinas and upregulated in Gnat1-/- retinas. Pharmacological activation of endothelial Norrin/ß-catenin signaling in Vglut1-/- retinas rescues defects in deep plexus angiogenesis and paracellular BRB maturation. Our findings demonstrate that glutamatergic neuronal activity regulates retinal angiogenesis and BRB maturation by modulating endothelial Norrin/ß-catenin signaling.
Assuntos
Barreira Hematorretiniana , Proteínas do Olho , Ácido Glutâmico , Proteínas do Tecido Nervoso , Transdução de Sinais , beta Catenina , Animais , Barreira Hematorretiniana/metabolismo , beta Catenina/metabolismo , Camundongos , Ácido Glutâmico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Transdução de Sinais/fisiologia , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Neurônios/metabolismo , Camundongos Knockout , Neovascularização Retiniana/metabolismo , Retina/metabolismo , Camundongos Endogâmicos C57BL , AngiogêneseRESUMO
Mucolipidosis IV (MLIV) is a rare, autosomal recessive, lysosomal disease characterized by intellectual disability, motor deficits, and progressive vision loss. Using adeno-associated vector 9 (AAV9) and AAV-PHP.B as delivery vectors, we previously demonstrated the feasibility of modifying disease course in a mouse model of MLIV by the human MCOLN1 gene transfer. Here, using a primate-enabling capsid AAV.CPP.16 (CPP16), we constructed a new, clinic-oriented MCOLN1 gene expression vector and demonstrated its efficacy in the preclinical model of MLIV. Systemic administration of CPP16-MCOLN1 in adult symptomatic Mcoln1 -/- mice at a dose of 1e12 vg per mouse resulted in MCOLN1 expression in the brain and peripheral tissues, alleviated brain pathology, rescued neuromotor function, and completely prevented paralysis. Notable expression of MCOLN1 transcripts was also detected in the retina of the mouse, which had exhibited significant degeneration at the time of the treatment. However, no increase in retinal thickness was observed after gene therapy treatment. Our results suggest a new AAV-based systemic gene replacement therapy for the treatment of MLIV that could be translated into clinical studies.
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Modulation of the cervix by steroid hormones and commensal microbiome play a central role in the health of the female reproductive tract. Here we describe organ-on-a-chip (Organ Chip) models that recreate the human cervical epithelial-stromal interface with a functional epithelial barrier and production of mucus with biochemical and hormone-responsive properties similar to living cervix. When Cervix Chips are populated with optimal healthy versus dysbiotic microbial communities (dominated by Lactobacillus crispatus and Gardnerella vaginalis, respectively), significant differences in tissue innate immune responses, barrier function, cell viability, proteome, and mucus composition are observed that are similar to those seen in vivo. Thus, human Cervix Organ Chips represent physiologically relevant in vitro models to study cervix physiology and host-microbiome interactions, and hence may be used as a preclinical testbed for development of therapeutic interventions to enhance women's health.
Assuntos
Colo do Útero , Interações entre Hospedeiro e Microrganismos , Imunidade Inata , Microbiota , Humanos , Feminino , Colo do Útero/microbiologia , Colo do Útero/imunologia , Microbiota/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Gardnerella vaginalis/imunologia , Lactobacillus crispatus/imunologia , Muco/imunologia , Muco/microbiologia , Muco/metabolismo , Dispositivos Lab-On-A-ChipRESUMO
Mucolipidosis IV (MLIV) is an ultra-rare, recessively inherited lysosomal disorder resulting from inactivating mutations in MCOLN1, the gene encoding the lysosomal cation channel TRPML1. The disease primarily affects the central nervous system (CNS) and manifests in the first year with cognitive and motor developmental delay, followed by a gradual decline in neurological function across the second decade of life, blindness, and premature death in third or fourth decades. Brain pathology manifestations in MLIV are consistent with hypomyelinating leukodystrophy with brain iron accumulation. Presently, there are no approved or investigational therapies for MLIV, and pathogenic mechanisms remain largely unknown. The MLIV mouse model, Mcoln1-/- mice, recapitulates all major manifestations of the human disease. Here, to better understand the pathological mechanisms in the MLIV brain, we performed cell type specific LC-MS/MS proteomics analysis in the MLIV mouse model and reconstituted molecular signatures of the disease in either freshly isolated populations of neurons, astrocytes, oligodendrocytes, and neural stem cells, or whole tissue cortical homogenates from young adult symptomatic Mcoln1-/- mice. Our analysis confirmed on the molecular level major histopathological hallmarks of MLIV universally present in Mcoln1-/- tissue and brain cells, such as hypomyelination, lysosomal dysregulation, and impaired metabolism of lipids and polysaccharides. Importantly, pathway analysis in brain cells revealed mitochondria-related alterations in all Mcoln1-/- brain cells, except oligodendrocytes, that was not possible to resolve in whole tissue. We also report unique proteome signatures and dysregulated pathways for each brain cell population used in this study. These data shed new light on cell-intrinsic mechanisms of MLIV and provide new insights for biomarker discovery and validation to advance translational studies for this disease.
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The stability of tight junctions (TJs) between endothelial cells (ECs) is essential to maintain blood-brain barrier (BBB) function in the healthy brain. Following ischemic stroke, TJ strand dismantlement due to protein degradation leads to BBB dysfunction, yet the mechanisms driving this process are poorly understood. Here, we show that endothelial-specific ablation of Rab7a, a small GTPase that regulates endolysosomal protein degradation, reduces stroke-induced TJ strand disassembly resulting in decreased paracellular BBB permeability and improved neuronal outcomes. Two pro-inflammatory cytokines, TNFα and IL1ß, but not glucose and oxygen deprivation, induce Rab7a activation via Ccz1 in brain ECs in vitro, leading to increased TJ protein degradation and impaired paracellular barrier function. Silencing Rab7a in brain ECs in vitro reduces cytokine-driven endothelial barrier dysfunction by suppressing degradation of a key BBB TJ protein, Claudin-5. Thus, Rab7a activation by inflammatory cytokines promotes degradation of select TJ proteins leading to BBB dysfunction after ischemic stroke.
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Enteric symptoms are hallmarks of prodromal Parkinson's disease (PD) that appear decades before the onset of motor symptoms and diagnosis. PD patients possess circulating T cells that recognize specific α-synuclein (α-syn)-derived epitopes. One epitope, α-syn32-46, binds with strong affinity to the HLA-DRB1∗15:01 allele implicated in autoimmune diseases. We report that α-syn32-46 immunization in a mouse expressing human HLA-DRB1∗15:01 triggers intestinal inflammation, leading to loss of enteric neurons, damaged enteric dopaminergic neurons, constipation, and weight loss. α-Syn32-46 immunization activates innate and adaptive immune gene signatures in the gut and induces changes in the CD4+ TH1/TH17 transcriptome that resemble tissue-resident memory (TRM) cells found in mucosal barriers during inflammation. Depletion of CD4+, but not CD8+, T cells partially rescues enteric neurodegeneration. Therefore, interaction of α-syn32-46 and HLA-DRB1∗15:0 is critical for gut inflammation and CD4+ T cell-mediated loss of enteric neurons in humanized mice, suggesting mechanisms that may underlie prodromal enteric PD.
Assuntos
Doença de Parkinson , Camundongos , Humanos , Animais , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Cadeias HLA-DRB1/genética , Epitopos , Neurônios Dopaminérgicos/metabolismo , InflamaçãoRESUMO
Defects in endolysosomal and autophagic functions are increasingly viewed as key pathological features of neurodegenerative disorders. A master regulator of these functions is phosphatidylinositol-3-phosphate (PI3P), a phospholipid synthesized primarily by class III PI 3-kinase Vps34. Here we report that disruption of neuronal Vps34 function in vitro and in vivo impairs autophagy, lysosomal degradation as well as lipid metabolism, causing endolysosomal membrane damage. PI3P deficiency also promotes secretion of unique exosomes enriched for undigested lysosomal substrates, including amyloid precursor protein C-terminal fragments (APP-CTFs), specific sphingolipids, and the phospholipid bis(monoacylglycero)phosphate (BMP), which normally resides in the internal vesicles of endolysosomes. Secretion of these exosomes requires neutral sphingomyelinase 2 and sphingolipid synthesis. Our results reveal a homeostatic response counteracting lysosomal dysfunction via secretion of atypical exosomes eliminating lysosomal waste and define exosomal APP-CTFs and BMP as candidate biomarkers for endolysosomal dysfunction associated with neurodegenerative disorders.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Exossomos/metabolismo , Lipídeos/análise , Lisossomos/metabolismo , Neurônios/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Células HEK293 , Humanos , Lisofosfolipídeos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoglicerídeos/metabolismo , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismoRESUMO
Coordinating angiogenesis with acquisition of tissue-specific properties in endothelial cells is essential for vascular function. In the retina, endothelial cells form a blood-retina barrier by virtue of tight junctions and low transcytosis. While the canonical Norrin/Fz4/Lrp5/6 pathway is essential for angiogenesis, vascular remodeling, and barrier maturation, how these diverse processes are coordinated remains poorly understood. Here we demonstrate that Apcdd1, a negative regulator of Wnt/ß-catenin signaling, is expressed in retinal endothelial cells during angiogenesis and barrier formation. Apcdd1-deficient mice exhibit a transient increase in vessel density at ages P10-P12 due to delayed vessel pruning. Moreover, Apcdd1 mutant endothelial cells precociously form the paracellular component of the barrier. Conversely, mice that overexpress Apcdd1 in retina endothelial cells have reduced vessel density but increased paracellular barrier permeability. Apcdd1 thus serves to precisely modulate Wnt/Norrin signaling activity in the retinal endothelium and coordinate the timing of both vascular pruning and barrier maturation.