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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

Slides for Centering the Margins: Land Acknowledgement Discussion held virtually for the UMass Chan community on September 15, 2021. The presentation covers the importance of a land acknowledgement, examples of land acknowledgements, and a brief history of the Nipmuc Nation.

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

In the interest of making data findable, accessible, interoperable, and reusable (FAIR), the National Institutes of Health (NIH) will institute a new Research Data Management and Sharing Policy in January 2023. This policy will require researchers applying for NIH funding to submit a Data Management and Sharing Plan. As 63% of grant dollars received by UMass Chan Medical School researchers comes from the NIH, we explored whether UMass Chan researchers are currently sharing data associated with their published research. PubMed was searched for articles published in 2019 with a UMass Chan researcher as either the first or last author. These articles were then examined for evidence of original or reused data, the type of data, whether the article stated that data was available, and where and how to find that data. Of the 713 articles found with a UMass Chan first and/or last author, 535 were research articles that produced original data or reused existing data. Of those articles with data, 57.4% (307) were NIH funded, 17.2% (92) had a data availability statement, and 10.8% had data deposited in a repository. This poster will highlight how the results of the study will inform our library services for researchers and provide tips on making UMass Chan data FAIR.

Supplemental material, CEP877662 Supplemental Material for CGRP-dependent and independent mechanisms of acute and persistent post-traumatic headache following mild traumatic brain injury in mice by Edita Navratilova, Jill Rau, Janice Oyarzo, Jason Tien, Kimberly Mackenzie, Jennifer Stratton, Bethany Remeniuk, Todd Schwedt, Trent Anderson, David Dodick and Frank Porreca 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 regulation of divalent metal ion transporter DMT1, the primary non-heme iron importer in mammals, is critical for maintaining iron homeostasis. Previously we identified ubiquitin-dependent regulation of DMT1 involving the Nedd4 family of ubiquitin ligases and the Ndfip1 and Ndfip2 adaptors. We also established the in vivo function of Ndfip1 in the regulation of DMT1 in the duodenum of mice. Here we have studied the function of Ndfip2 using Ndfip2-deficient mice. The DMT1 protein levels in the duodenum were comparable in wild type and Ndfip2−/− mice, as was the transport activity of isolated enterocytes. A complete blood examination showed no significant differences between wild type and Ndfip2−/− mice in any of the hematological parameters measured. However, when fed a low iron diet, female Ndfip2−/− mice showed a decrease in liver iron content, although they maintained normal serum iron levels and transferrin saturation, compared to wild type female mice that showed a reduction in serum iron and transferrin saturation. Ndfip2−/− female mice also showed an increase in DMT1 expression in the liver, with no change in male mice. We suggest that Ndfip2 controls DMT1 in the liver with female mice showing a greater response to altered dietary iron than the male mice.

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