Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. They form at the trans-Golgi network (TGN), where their soluble content aggregates to form a dense core, but the mechanisms controlling biogenesis are still not completely understood. Recent studies have implicated the peripheral membrane protein HID-1 in neuropeptide sorting and insulin secretion. Using CRISPR/Cas9, we generated HID-1 KO rat neuroendocrine cells, and we show that the absence of HID-1 results in specific defects in peptide hormone and monoamine storage and regulated secretion. Loss of HID-1 causes a reduction in the number of LDCVs and affects their morphology and biochemical properties, due to impaired cargo sorting and dense core formation. HID-1 KO cells also exhibit defects in TGN acidification together with mislocalization of the Golgi-enriched vacuolar H + -ATPase subunit isoform a2. We propose that HID-1 influences early steps in LDCV formation by controlling dense core formation at the TGN.

The regulated release of peptide hormones depends on their packaging into dense-core vesicles (DCVs). Two models have been proposed for DCV cargo sorting. The "sorting by entry" model proposes that DCV cargos selectively enter nascent DCVs at the trans-Golgi network (TGN). The "sorting by exit" model proposes that sorting occurs by the post-TGN removal of non-DCV cargos and retention of mature DCV cargos. Here we show that the coiled-coil protein CCDC186 controls sorting by exit. Ccdc186 KO insulinoma cells secrete less insulin, fail to retain insulin and carboxypeptidase E in mature DCVs at the cell periphery, and fail to remove carboxypeptidase D from immature DCVs. A mutation affecting the endosome-associated recycling protein (EARP) complex causes similar defects in DCV cargo retention and removal.CCDC186 and EARP may act together to control the post-Golgi retention of cargos in mature DCVs.cargos in mature DCVs in axons, but it is unclear how RAB-2 and CCCP-1/CCDC186 are connected to EARP. In both C. elegans and in the rat insulinoma 832/13 cell line, RAB-2 and CCCP-1/CCDC186 colocalize near the TGN where the early steps of DCV biogenesis take place (Ailion et al., 2014; Cattin-Ortolá et al., 2017). EARP is localized to two distinct compartments in 832/13 cells, a recycling endosome compartment and a compartment near the TGN where it colocalizes with CCDC186 (Topalidou, Cattin-Ortolá, et al., 2018). Additionally, Eipr1 knockout 832/13 cells have reduced insulin secretion and an altered distribution of insulin in the cell, with relatively more insulin near the TGN and less at the cell periphery (Topalidou, Cattin-Ortolá, et al., 2018). Thus, EARP and EIPR1 may be important for acting near the TGN to ensure the retention of processed cargos in mature DCVs.Here we investigate the role of CCDC186 in DCV biogenesis, maturation, and cargo sorting in 832/13 cells. Our results suggest that CCDC186 acts together with EARP to control several aspects of DCV cargo sorting, including the post-Golgi removal of some non-DCV cargos and retention of mature DCV cargos.

Formation of secretory granules (SGs) occurs at the trans-Golgi network (TGN). Here we show that transmembrane SG cargoes (phogrin and VMAT2) do not sort directly onto SGs during budding, but rather exit the TGN into nonregulated vesicles to get incorporated to SGs at a later step, suggesting a more complex model of SG biogenesis than anticipated.

Dense-core vesicles (DCVs) are secretory vesicles that package and secrete cargoes like insulin, but how cargo is sorted to DCVs is poorly understood. Here, it is shown that the EARP complex-interacting protein EIPR1 controls insulin secretion and localization of DCV cargo in insulin-secreting cells.

Objective: Precursors of peptide hormones undergo posttranslational modifications within the trans-Golgi network (TGN). Dysfunction of proteins involved at different steps of this process cause several complex syndromes affecting the central nervous system (CNS). We aimed to clarify the genetic cause in a group of patients characterized by hypopituitarism in combination with brain atrophy, thin corpus callosum, severe developmental delay, visual impairment, and epilepsy. Methods: Whole exome sequencing was performed in seven individuals of six unrelated families with these features. Postmortem histopathological and HID1 expression analysis of brain tissue and pituitary gland were conducted in one patient. Functional consequences of the homozygous HID1 variant p.R433W were investigated by Seahorse XF Assay in fibroblasts of two patients. Results: Bi-allelic variants in the gene HID1 domain-containing protein 1 ( HID1 ) were identified in all patients. Postmortem examination confirmed cerebral atrophy with enlarged lateral ventricles. Markedly reduced expression of pituitary hormones was found in pituitary gland tissue. Colocalization of HID1 protein with the TGN was not altered in fibroblasts of patients compared to controls, while the extracellular acidification rate upon stimulation with potassium chloride was significantly reduced in patient fibroblasts compared to controls. Interpretation: Our findings indicate that mutations in HID1 cause an early infantile encephalopathy with hypopituitarism as the leading presentation, and expand the list of syndromic CNS diseases caused by interference of TGN function.