RESUMEN
Pancreatic islets are endocrine organs that depend on their microvasculature to function. Along with endothelial cells, pericytes comprise the islet microvascular network. These mural cells are crucial for microvascular stability and function, but it is not known if/how they are affected during the development of type 1 diabetes (T1D). Here, we investigate islet pericyte density, phenotype, and function using living pancreas slices from donors without diabetes, donors with a single T1D-associated autoantibody (GADA+), and recent onset T1D cases. Our data show that islet pericyte and capillary responses to vasoactive stimuli are impaired early on in T1D. Microvascular dysfunction is associated with a switch in the phenotype of islet pericytes toward myofibroblasts. Using publicly available RNA sequencing (RNA-seq) data, we further found that transcriptional alterations related to endothelin-1 signaling and vascular and extracellular matrix (ECM) remodeling are hallmarks of single autoantibody (Aab)+ donor pancreata. Our data show that microvascular dysfunction is present at early stages of islet autoimmunity.
Asunto(s)
Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Humanos , Diabetes Mellitus Tipo 1/patología , Pericitos/patología , Células Endoteliales/patología , Islotes Pancreáticos/irrigación sanguínea , AutoanticuerposRESUMEN
Brain-derived neurotrophic factor (Bdnf) expression is tightly controlled at the transcriptional and post-transcriptional levels. Previously, we showed that inhibition of noncoding Bdnf antisense (Bdnf-AS) RNA upregulates Bdnf protein. Here, we generated a Bdnf-antisense knockout (Bdnf-AS KO) mouse model by deleting 6 kilobases upstream of Bdnf-AS. After verifying suppression of Bdnf-AS, baseline behavioral tests indicated no significant difference in knockout and wild type mice, except for enhanced cognitive function in the knockout mice in the Y-maze. Following acute involuntary exercise, Bdnf-AS KO mice were re-assessed and a significant increase in Bdnf mRNA and protein were observed. Following long-term involuntary exercise, we observed a significant increase in nonspatial and spatial memory in novel object recognition and Barnes maze tests in young and aged Bdnf-AS KO mice. Our data provides evidence for the beneficial effects of endogenous Bdnf upregulation and the synergistic effect of Bdnf-AS knockout on exercise and memory retention.
RESUMEN
At the time of Ivan Pavlov, pancreatic innervation was studied by looking at pancreas secretions in response to electrical stimulation of nerves. Nowadays we have ways to visualize neuronal activity in real time thanks to advances in fluorescent reporters and imaging techniques. We also have very precise optogenetic and pharmacogenetic approaches that allow neuronal manipulations in a very specific manner. These technological advances have been extensively employed for studying the central nervous system and are just beginning to be incorporated for studying visceral innervation. Pancreatic innervation is complex, and the role it plays in physiology and pathophysiology of the organ is still not fully understood. In this review we highlight anatomical aspects of pancreatic innervation, techniques for pancreatic neuronal labeling, and approaches for imaging pancreatic innervation in vitro and in vivo.
Asunto(s)
Neuronas/fisiología , Imagen Óptica/métodos , Páncreas/inervación , Animales , Humanos , Páncreas/fisiopatologíaRESUMEN
BACKGROUND AND AIMS: Destroying visceral sensory nerves impacts pancreatic islet function, glucose metabolism, and diabetes onset, but how islet endocrine cells interact with sensory neurons has not been studied. METHODS: We characterized the anatomical pattern of pancreatic sensory innervation by combining viral tracing, immunohistochemistry, and reporter mouse models. To assess the functional interactions of ß-cells with vagal sensory neurons, we recorded Ca2+ responses in individual nodose neurons in vivo while selectively stimulating ß-cells with chemogenetic and pharmacologic approaches. RESULTS: We found that pancreatic islets are innervated by vagal sensory axons expressing Phox2b, substance P, calcitonin-gene related peptide, and the serotonin receptor 5-HT3R. Centrally, vagal neurons projecting to the pancreas terminate in the commissural nucleus of the solitary tract. Nodose neurons responded in vivo to chemogenetic stimulation of ß-cells and to pancreas infusion with serotonin, but were not sensitive to insulin. Responses to chemogenetic and pharmacologic stimulation of ß-cells were blocked by a 5-HT3R antagonist and were enhanced by increasing serotonin levels in ß-cells. We further confirmed directly in living pancreas slices that sensory terminals in the islet were sensitive to serotonin. CONCLUSIONS: Our study establishes that pancreatic ß-cells communicate with vagal sensory neurons, likely using serotonin signaling as a transduction mechanism. Serotonin is coreleased with insulin and may therefore convey information about the secretory state of ß-cells via vagal afferent nerves.
Asunto(s)
Vías Aferentes/fisiología , Comunicación Celular , Células Secretoras de Insulina/fisiología , Ganglio Nudoso/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Femenino , Insulina/metabolismo , Microscopía Intravital , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Modelos Animales , Ganglio Nudoso/citología , Serotonina/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Neuromodulation is a promising new therapeutic avenue to treat chronic diseases. A paper that appeared recently in Nature Biotechnology (Guyot et al., 2019) now provides a roadmap for how to establish electrostimulation of nerves to delay or even prevent type 1 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1 , Terapia por Estimulación Eléctrica , HumanosRESUMEN
AIMS/HYPOTHESIS: Tissue-resident macrophages sense the microenvironment and respond by producing signals that act locally to maintain a stable tissue state. It is now known that pancreatic islets contain their own unique resident macrophages, which have been shown to promote proliferation of the insulin-secreting beta cell. However, it is unclear how beta cells communicate with islet-resident macrophages. Here we hypothesised that islet macrophages sense changes in islet activity by detecting signals derived from beta cells. METHODS: To investigate how islet-resident macrophages respond to cues from the microenvironment, we generated mice expressing a genetically encoded Ca2+ indicator in myeloid cells. We produced living pancreatic slices from these mice and used them to monitor macrophage responses to stimulation of acinar, neural and endocrine cells. RESULTS: Islet-resident macrophages expressed functional purinergic receptors, making them exquisite sensors of interstitial ATP levels. Indeed, islet-resident macrophages responded selectively to ATP released locally from beta cells that were physiologically activated with high levels of glucose. Because ATP is co-released with insulin and is exclusively secreted by beta cells, the activation of purinergic receptors on resident macrophages facilitates their awareness of beta cell secretory activity. CONCLUSIONS/INTERPRETATION: Our results indicate that islet macrophages detect ATP as a proxy signal for the activation state of beta cells. Sensing beta cell activity may allow macrophages to adjust the secretion of factors to promote a stable islet composition and size.
Asunto(s)
Adenosina Trifosfato/metabolismo , Macrófagos/metabolismo , Páncreas/citología , Páncreas/metabolismo , Animales , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , RatonesRESUMEN
Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 µm of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Exocitosis/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Microscopía Confocal/métodos , Neuropéptido Y/metabolismo , Animales , Humanos , Secreción de Insulina , Islotes Pancreáticos/citología , RatonesRESUMEN
BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by welldefined neuropathological brain changes including amyloid plaques, neurofibrillary tangles and the presence of chronic neuroinflammation. OBJECTIVE: The brain penetrant BET bromodomain inhibitor JQ1 has been shown to regulate inflammation responses in vitro and in vivo, but its therapeutic potential in AD is currently unknown. METHOD: Three-month-old 3xTg mice were injected once a day with JQ1 (50 mg/kg) or vehicle for 15 weeks. At the end of the treatment learning and memory was assessed using the modified Barnes maze and the Y maze behavioral tests. Tissue from the brain and other organs was collected for molecular evaluation of neuroinflammation tau pathology and amyloid ß. RESULTS: JQ1 treatment reduced splenomegaly and neuroinflammation in the brain of treated mice where we observed a reduction in the expression of the pro-inflammatory modulators Il-1b, Il-6, Tnfa, Ccl2, Nos2 and Ptgs2. Additionally, JQ1-treated mice showed a reduction of tau phosphorylation at Ser396 in the hippocampus and frontal cortex while total levels of tau remained unaffected. On the other hand, JQ1 did not ameliorate learning and memory deficits in 7-month-old 3xTg mice. CONCLUSION: Taken together, our data suggest that BET bromodomain inhibitors hold the promise to be used for the treatment of neurological disorders characterized by neuroinflammation.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Azepinas/farmacología , Encéfalo/efectos de los fármacos , Inflamación/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Triazoles/farmacología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/psicología , Péptidos beta-Amiloides/metabolismo , Animales , Antiinflamatorios/farmacología , Encéfalo/inmunología , Encéfalo/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Inflamación/metabolismo , Inflamación/patología , Aprendizaje/efectos de los fármacos , Masculino , Memoria/efectos de los fármacos , Ratones Transgénicos , Tamaño de los Órganos , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patologíaRESUMEN
The underlying genetic variations of late-onset Alzheimer's disease (LOAD) cases remain largely unknown. A combination of genetic variations with variable penetrance and lifetime epigenetic factors may converge on transcriptomic alterations that drive LOAD pathological process. Transcriptome profiling using deep sequencing technology offers insight into common altered pathways regardless of underpinning genetic or epigenetic factors and thus represents an ideal tool to investigate molecular mechanisms related to the pathophysiology of LOAD. We performed directional RNA sequencing on high quality RNA samples extracted from hippocampi of LOAD and age-matched controls. We further validated our data using qRT-PCR on a larger set of postmortem brain tissues, confirming downregulation of the gene encoding substance P (TAC1) and upregulation of the gene encoding the plasminogen activator inhibitor-1 (SERPINE1). Pathway analysis indicates dysregulation in neural communication, cerebral vasculature, and amyloid-ß clearance. Beside protein coding genes, we identified several annotated and non-annotated long noncoding RNAs that are differentially expressed in LOAD brain tissues, three of them are activity-dependent regulated and one is induced by Aß(1-42) exposure of human neural cells. Our data provide a comprehensive list of transcriptomics alterations in LOAD hippocampi and warrant holistic approach including both coding and non-coding RNAs in functional studies aimed to understand the pathophysiology of LOAD.