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Supplemental material, sj-pdf-5-cep-10.1177_0333102420983282 for Migraine therapeutics differentially modulate the CGRP pathway by Minoti Bhakta, Trang Vuong, Tetsuya Taura, David S Wilson, Jennifer R Stratton and Kimberly D Mackenzie in Cephalalgia

Supplemental material, sj-pdf-3-cep-10.1177_0333102420983282 for Migraine therapeutics differentially modulate the CGRP pathway by Minoti Bhakta, Trang Vuong, Tetsuya Taura, David S Wilson, Jennifer R Stratton and Kimberly D Mackenzie in Cephalalgia

Supplemental material, sj-pdf-2-cep-10.1177_0333102420983282 for Migraine therapeutics differentially modulate the CGRP pathway by Minoti Bhakta, Trang Vuong, Tetsuya Taura, David S Wilson, Jennifer R Stratton and Kimberly D Mackenzie in Cephalalgia

Supplemental material, sj-pdf-6-cep-10.1177_0333102420983282 for Migraine therapeutics differentially modulate the CGRP pathway by Minoti Bhakta, Trang Vuong, Tetsuya Taura, David S Wilson, Jennifer R Stratton and Kimberly D Mackenzie in Cephalalgia

Supplemental material, sj-pdf-1-cep-10.1177_0333102420983282 for Migraine therapeutics differentially modulate the CGRP pathway by Minoti Bhakta, Trang Vuong, Tetsuya Taura, David S Wilson, Jennifer R Stratton and Kimberly D Mackenzie in Cephalalgia

Supplemental material, sj-pdf-4-cep-10.1177_0333102420983282 for Migraine therapeutics differentially modulate the CGRP pathway by Minoti Bhakta, Trang Vuong, Tetsuya Taura, David S Wilson, Jennifer R Stratton and Kimberly D Mackenzie in Cephalalgia

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1−/− cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Refereed/Peer-reviewed

The release of extracellular vesicles (EVs) is important for both normal physiology and disease. However, a basic understanding of the targeting of EV cargoes, composition and mechanism of release is lacking. Here we present evidence that the divalent metal ion transporter (DMT1) is unexpectedly regulated through release in EVs. This process involves the Nedd4-2 ubiquitin ligase, and the adaptor proteins Arrdc1 and Arrdc4 via different budding mechanisms. We show that mouse gut explants release endogenous DMT1 in EVs. Although we observed no change in the relative amount of DMT1 released in EVs from gut explants in Arrdc1 or Arrdc4 deficient mice, the extent of EVs released was significantly reduced indicating an adaptor role in biogenesis. Furthermore, using Arrdc1 or Arrdc4 knockout mouse embryonic fibroblasts, we show that both Arrdc1 and Arrdc4 are non-redundant positive regulators of EV release. Our results suggest that DMT1 release from the plasma membrane into EVs may represent a novel mechanism for the maintenance of iron homeostasis, which may also be important for the regulation of other membrane proteins.

Huntingtin-associated protein-1 (HAP1) is involved in intracellular trafficking, vesicle transport, and membrane receptor endocytosis. However, despite such diverse functions, the role of HAP1 in the synaptic vesicle (SV) cycle in nerve terminals remains unclear. Here, we report that HAP1 functions in SV exocytosis, controls total SV turnover and the speed of vesicle fusion in nerve terminals and regulates glutamate release in cortical brain slices. We found that HAP1 interacts with synapsin I, an abundant neuronal phosphoprotein that associates with SVs during neurotransmitter release and regulates synaptic plasticity and neuronal development. The interaction between HAP1 with synapsin I was confirmed by reciprocal co-immunoprecipitation of the endogenous proteins. Furthermore, HAP1 co-localizes with synapsin I in cortical neurons as discrete puncta. Interestingly, we find that synapsin I localization is specifically altered in Hap1−/− cortical neurons without an effect on the localization of other SV proteins. This effect on synapsin I localization was not because of changes in the levels of synapsin I or its phosphorylation status in Hap1−/− brains. Furthermore, fluorescence recovery after photobleaching in transfected neurons expressing enhanced green fluorescent protein-synapsin Ia demonstrates that loss of HAP1 protein inhibits synapsin I transport. Thus, we demonstrate that HAP1 regulates SV exocytosis and may do so through binding to synapsin I. (Figure presented.) The Proposed mechanism of synapsin I transport mediated by HAP1 in neurons. HAP1 interacts with synapsin I, regulating the trafficking of synapsin I containing vesicles and/or transport packets, possibly through its engagement of microtubule motors. The absence of HAP1 reduces synapsin I transport and neuronal exocytosis. These findings provide insights into the processes of neuronal trafficking and synaptic signaling. Refereed/Peer-reviewed

Huntingtin-associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localisation and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon-fibre amperometry we measured single vesicle exocytosis in chromaffin cells obtained from HAP1-/- and HAP1+/+ littermate mice. The number of Ca2+-dependent and Ca2+-independent full fusion events in HAP1-/- cells is significantly decreased compared to HAP1+/+ cells. We observe no change in the frequency of 'kiss-and-run' fusion events or in Ca2+ entry. While release per full fusion event is unchanged in HAP1-/- cells, early fusion pore duration is prolonged, as indicated by increased duration of pre-spike foot (PSF) signals. Kiss-and-run events have a shorter duration, indicating opposing roles for HAP1 in the stabilisation of the fusion pore during full fusion and transient fusion. We further demonstrate with electron microscopy a reduction in the number of vesicles docked at the plasma membrane of HAP1-/- cells and membrane capacitance measurements reveal a reduced size of the readily releasable pool of vesicles. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation and the early stages of fusion pore formation. Refereed/Peer-reviewed

Author version made available in accordance with the publisher's policy., We have previously shown that Regulator of Calcineurin 1 (RCAN1) regulates multiple stages of vesicle exocytosis. However, the mechanisms by which RCAN1 affects secretory vesicle exocytosis and quantal release kinetics remain unknown. Here we use carbon fiber amperometry to detect exocytosis from chromaffin cells and identify these underlying mechanisms. We observe reduced exocytosis with repeated stimulations in chromaffin cells overexpressing RCAN1 (RCAN1ox), but not in wild type (WT) cells, indicating a negative effect of RCAN1 on vesicle recycling and endocytosis. Acute exposure to calcineurin inhibitors, cyclosporine A and FK-506, replicates this effect in WT cells but has no additional effect in RCAN1ox cells. When we chronically expose WT cells to cyclosporine A and FK-506 we find that catecholamine release per vesicle and pre-spike foot (PSF) signal parameters are decreased, similar to that in RCAN1ox cells. Inhibiting calcineurin activity in RCAN1ox cells has no additional effect on the amount of catecholamine release per vesicle but further reduces PSF signal parameters. Electron microscopy studies indicate these changes are not due to altered vesicle number or distribution in RCAN1ox cells but reduced vesicle release may be cause by decreased vesicle and dense core size in RCAN1ox cells. Thus, our results indicate that RCAN1 may negatively affects vesicle recycling and quantal release kinetics via the inhibition of calcineurin activity.

RCAN1 is a chromosome 21 gene that controls secretion in endocrine cells, regulates mitochondrial function, and is sensitive to oxidative stress. Regulator of calcineurin 1 (RCAN1) is also an endogenous inhibitor of the protein phosphatase calcineurin, the inhibition of which leads to hypoinsulinemia and diabetes in humans and mice. However, the presence or the role of RCAN1 in insulin-secreting β-cells and its potential role in the pathogenesis of diabetes is unknown. Hence, the aim of this study is to investigate the presence of RCAN1 in β-cells and identify its role in β-cell function. RCAN1 is expressed in mouse islets and in the cytosol of pancreatic β-cells. We find RCAN1 is a glucose-responsive gene with a 1.5-fold increase in expression observed in pancreatic islets in response to chronic hyperglycemia. The overexpression of the human RCAN1.1 isoform in mice under the regulation of its endogenous promoter causes diabetes, age-associated hyperglycemia, reduced glucose tolerance, hypoinsulinemia, loss of β-cells, reduced β-cell insulin secretion, aberrant mitochondrial reactive oxygen species production, and the down-regulation of key β-cell genes. Our data therefore identifies a novel molecular link between the overexpression of RCAN1 and β-cell dysfunction. The glucose-responsive nature of RCAN1 provides a potential mechanism of action associated with the β-cell dysfunction observed in diabetes., This work was supported by a BioInnovation SA Fellowship, Australian Research Council Future Fellowship, National Health and Medical Research Council Project Grant, and Diabetes Australia Research Trust Grant.

How fusion pore formation during exocytosis affects the subsequent release of vesicle contents remains incompletely understood. It is unclear if the amount released per vesicle is dependent upon the nature of the developing fusion pore and whether full fusion and transient kiss and run exocytosis are regulated by similar mechanisms. We hypothesise that if consistent relationships exist between these aspects of exocytosis then they will remain constant across any age. Using amperometry in mouse chromaff in cells we measured catecholamine efflux during single exocytotic events at P0, 1 month and6 months. At all ages we observed full fusion (amperometric spike only), full fusion preceded by fusion pore flickering (prespikefoot (PSF) signal followed by a spike) and pure ''kiss and run'' exocytosis (represented by stand alone foot (SAF) signals). We observe age-associated increases in the size of all 3 modes of fusion but these increases occur at different ages. The release probability of PSF signals or full spikes alone doesn't alter across any age in comparison with an age-dependent increase in the incidence of ''kiss and run'' type events. However, the most striking changes we observe are age-associated changes in the relationship between vesicle size and the membrane bending energy required for exocytosis. Our data illustrates that vesicle size does not regulate release probability, as has been suggested, that membrane elasticity or flexural rigidity change with age and that the mechanisms controlling full fusion may differ from those controlling ''kiss and run'' fusion. Refereed/Peer-reviewed