Capillary contact points determine beta cell polarity, control secretion and are disrupted in the db/db mouse model of diabetes.

AIMS/HYPOTHESIS: Almost all beta cells contact one capillary and insulin granule fusion is targeted to this region. However, there are reports of beta cells contacting more than one capillary. We therefore set out to determine the proportion of beta cells with multiple contacts and the impact of this on cell structure and function. METHODS: We used pancreatic slices in mice and humans to better maintain cell and islet structure than in isolated islets. Cell structure was assayed using immunofluorescence and 3D confocal microscopy. Live-cell two-photon microscopy was used to map granule fusion events in response to glucose stimulation. RESULTS: We found that 36% and 22% of beta cells in islets from mice and humans, respectively, have separate contact with two capillaries. These contacts establish a distinct form of cell polarity with multiple basal regions. Both capillary contact points are enriched in presynaptic scaffold proteins, and both are a target for insulin granule fusion. Cells with two capillary contact points have a greater capillary contact area and secrete more, with analysis showing that, independent of the number of contact points, increased contact area is correlated with increased granule fusion. Using db/db mice as a model for type 2 diabetes, we observed changes in islet capillary organisation that significantly reduced total islet capillary surface area, and reduced area of capillary contact in single beta cells. CONCLUSIONS/INTERPRETATION: Beta cells that contact two capillaries are a significant subpopulation of beta cells within the islet. They have a distinct form of cell polarity and both contact points are specialised for secretion. The larger capillary contact area of cells with two contact points is correlated with increased secretion. In the db/db mouse, changes in capillary structure impact beta cell capillary contact, implying that this is a new factor contributing to disease progression.

Structural and functional polarisation of human pancreatic beta cells in islets from organ donors with and without type 2 diabetes.

AIMS/HYPOTHESIS: We hypothesised that human beta cells are structurally and functional polarised with respect to the islet capillaries. We set out to test this using confocal microscopy to map the 3D spatial arrangement of key proteins and live-cell imaging to determine the distribution of insulin granule fusion around the cells. METHODS: Human pancreas samples were rapidly fixed and processed using the pancreatic slice technique, which maintains islet structure and architecture. Slices were stained using immunofluorescence for polarity markers (scribble, discs large [Dlg] and partitioning defective 3 homologue [Par3]) and presynaptic markers (liprin, Rab3-interacting protein [RIM2] and piccolo) and imaged using 3D confocal microscopy. Isolated human islets were dispersed and cultured on laminin-511-coated coverslips. Live 3D two-photon microscopy was used on cultured cells to image exocytic granule fusion events upon glucose stimulation. RESULTS: Assessment of the distribution of endocrine cells across human islets found that, despite distinct islet-to-islet complexity and variability, including multi-lobular islets, and intermixing of alpha and beta cells, there is still a striking enrichment of alpha cells at the islet mantle. Measures of cell position demonstrate that most beta cells contact islet capillaries. Subcellularly, beta cells consistently position polar determinants, such as Par3, Dlg and scribble, with a basal domain towards the capillaries and apical domain at the opposite face. The capillary interface/vascular face is enriched in presynaptic scaffold proteins, such as liprin, RIM2 and piccolo. Interestingly, enrichment of presynaptic scaffold proteins also occurs where the beta cells contact peri-islet capillaries, suggesting functional interactions. We also observed the same polarisation of synaptic scaffold proteins in islets from type 2 diabetic patients. Consistent with polarised function, isolated beta cells cultured onto laminin-coated coverslips target insulin granule fusion to the coverslip. CONCLUSIONS/INTERPRETATION: Structural and functional polarisation is a defining feature of human pancreatic beta cells and plays an important role in the control of insulin secretion.

Parkinson's disease-linked human PARK9/ATP13A2 maintains zinc homeostasis and promotes α-Synuclein externalization via exosomes.

α-Synuclein plays a central causative role in Parkinson's disease (PD). Increased expression of the P-type ATPase ion pump PARK9/ATP13A2 suppresses α-Synuclein toxicity in primary neurons. Our data indicate that ATP13A2 encodes a zinc pump; neurospheres from a compound heterozygous ATP13A2(-/-) patient and ATP13A2 knockdown cells are sensitive to zinc, whereas ATP13A2 over-expression in primary neurons confers zinc resistance. Reduced ATP13A2 expression significantly decreased vesicular zinc levels, indicating ATP13A2 facilitates transport of zinc into membrane-bound compartments or vesicles. Endogenous ATP13A2 localized to multi-vesicular bodies (MVBs), a late endosomal compartment located at the convergence point of the endosomal and autophagic pathways. Dysfunction in MVBs can cause a range of detrimental effects including lysosomal dysfunction and impaired delivery of endocytosed proteins/autophagy cargo to the lysosome, both of which have been observed in cells with reduced ATP13A2 function. MVBs also serve as the source of intra-luminal nanovesicles released extracellularly as exosomes that can contain a range of cargoes including α-Synuclein. Elevated ATP13A2 expression reduced intracellular α-Synuclein levels and increased α-Synuclein externalization in exosomes >3-fold whereas ATP13A2 knockdown decreased α-Synuclein externalization. An increased export of exosome-associated α-Synuclein may explain why surviving neurons of the substantia nigra pars compacta in sporadic PD patients were observed to over-express ATP13A2. We propose ATP13A2's modulation of zinc levels in MVBs can regulate the biogenesis of exosomes capable of containing α-Synuclein. Our data indicate that ATP13A2 is the first PD-associated gene involved in exosome biogenesis and indicates a potential neuroprotective role of exosomes in PD.

Enhanced structure and function of human pluripotent stem cell-derived beta-cells cultured on extracellular matrix.

The differentiation of human stem cells into insulin secreting beta-like cells holds great promise to treat diabetes. Current protocols drive stem cells through stages of directed differentiation and maturation and produce cells that secrete insulin in response to glucose. Further refinements are now needed to faithfully phenocopy the responses of normal beta cells. A critical factor in normal beta cell behavior is the islet microenvironment which plays a central role in beta cell survival, proliferation, gene expression and secretion. One important influence on native cell responses is the capillary basement membrane. In adult islets, each beta cell makes a point of contact with basement membrane protein secreted by vascular endothelial cells resulting in structural and functional polarization. Interaction with basement membrane proteins triggers local activation of focal adhesions, cell orientation, and targeting of insulin secretion. This study aims to identifying the role of basement membrane proteins on the structure and function of human embryonic stem cell and induced pluripotent stem cell-derived beta cells. Here, we show that differentiated human stem cells-derived spheroids do contain basement membrane proteins as a diffuse web-like structure. However, the beta-like cells within the spheroid do not polarize in response to this basement membrane. We demonstrate that 2D culture of the differentiated beta cells on to basement membrane proteins enforces cell polarity and favorably alters glucose dependent insulin secretion.

Machine Learning Algorithms, Applied to Intact Islets of Langerhans, Demonstrate Significantly Enhanced Insulin Staining at the Capillary Interface of Human Pancreatic ß Cells.

Pancreatic ß cells secrete the hormone insulin into the bloodstream and are critical in the control of blood glucose concentrations. ß cells are clustered in the micro-organs of the islets of Langerhans, which have a rich capillary network. Recent work has highlighted the intimate spatial connections between ß cells and these capillaries, which lead to the targeting of insulin secretion to the region where the ß cells contact the capillary basement membrane. In addition, ß cells orientate with respect to the capillary contact point and many proteins are differentially distributed at the capillary interface compared with the rest of the cell. Here, we set out to develop an automated image analysis approach to identify individual ß cells within intact islets and to determine if the distribution of insulin across the cells was polarised. Our results show that a U-Net machine learning algorithm correctly identified ß cells and their orientation with respect to the capillaries. Using this information, we then quantified insulin distribution across the ß cells to show enrichment at the capillary interface. We conclude that machine learning is a useful analytical tool to interrogate large image datasets and analyse sub-cellular organisation.

Local Integrin Activation in Pancreatic ß Cells Targets Insulin Secretion to the Vasculature.

The extracellular matrix (ECM) critically affects ß cell functions via integrin activation. But whether these ECM actions drive the spatial organization of ß cells, as they do in epithelial cells, is unknown. Here, we show that within islets of Langerhans, focal adhesion activation in ß cells occurs exclusively where they contact the capillary ECM (vascular face). In cultured ß cells, 3D mapping shows enriched insulin granule fusion where the cells contact ECM-coated coverslips, which depends on ß1 integrin receptor activation. Culture on micro-contact printed stripes of E-cadherin and fibronectin shows that ß cell contact at the fibronectin stripe selectively activates focal adhesions and enriches exocytic machinery and insulin granule fusion. Culture of cells in high glucose, as a model of glucotoxicity, abolishes granule targeting. We conclude that local integrin activation targets insulin secretion to the islet capillaries. This mechanism might be important for islet function and may change in disease.

ATP13A2 (PARK9) protein levels are reduced in brain tissue of cases with Lewy bodies.

BACKGROUND: ATP13A2 (PARK9) loss of function mutations are a genetic cause of an early-onset form of Parkinson's disease (PD), with in vitro studies showing that ATP13A2 deficits lead to lysosomal and mitochondrial dysfunction and α-synuclein accumulation, while elevated ATP13A2 expression reduces α-synuclein toxicity. The three human brain tissue studies assessing changes in ATP13A2 expression in PD produced divergent results; mRNA is increased while protein levels were observed to be either increased or decreased. This apparent conflict in protein levels might have arisen from examining Lewy body disease cases with coexisting Alzheimer-type pathologies.To assess whether ATP13A2 levels in Lewy body disease are modified by Alzheimer-type ß-amyloid deposition, we evaluated cases of pure PD and pure dementia with Lewy bodies (DLB) for changes in ATP13A2, α-synuclein and ß-amyloid protein levels in cortical regions with and without Lewy bodies. RESULTS: In all Lewy body disease cases, we identified decreased ATP13A2 protein levels that correlated with increases in both α-synuclein and ß-amyloid. Partial colocalization was observed between ATP13A2 and α-synuclein in Lewy bodies, whereas ATP13A2 did not colocalize with pathological ß-amyloid deposition. CONCLUSIONS: Our data show that patients with Lewy body diseases have an overall deficit in ATP13A2 protein levels, with the remaining protein being more insoluble and partially redistributing towards Lewy bodies. This supports the concept that increasing ATP13A2 levels may offer potential therapeutic benefits to patients with Lewy body diseases.