HID-1 controls formation of large dense core vesicles by influencing cargo sorting and trans-Golgi network acidification

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.</jats:p

Author Correction: The CaMKII/NMDA receptor complex controls hippocampal synaptic transmission by kinase-dependent and independent mechanisms.

The originally published version of this Article contained errors in Figure 5, for which we apologise. In panel c, the scatter graph was inadvertently replaced with a scatter graph comprising a subset of data points from panel d. Furthermore, the legends to Figures 5c and 5d were inverted. These errors have now been corrected in both the PDF and HTML versions of the Article, and the incorrect version of Fig. 5c is presented in the Author Correction associated with this Article

The CaMKII/NMDA receptor complex controls hippocampal synaptic transmission by kinase-dependent and independent mechanisms.

CaMKII is one of the most studied synaptic proteins, but many critical issues regarding its role in synaptic function remain unresolved. Using a CRISPR-based system to delete CaMKII and replace it with mutated forms in single neurons, we have rigorously addressed its various synaptic roles. In brief, basal AMPAR and NMDAR synaptic transmission both require CaMKIIα, but not CaMKIIβ, indicating that, even in the adult, synaptic transmission is determined by the ongoing action of CaMKIIα. While AMPAR transmission requires kinase activity, NMDAR transmission does not, implying a scaffolding role for the CaMKII protein instead. LTP is abolished in the absence of CaMKIIα and/or CaMKIIβ and with an autophosphorylation impaired CaMKIIα (T286A). With the exception of NMDAR synaptic currents, all aspects of CaMKIIα signaling examined require binding to the NMDAR, emphasizing the essential role of this receptor as a master synaptic signaling hub

Efficient, Complete Deletion of Synaptic Proteins using CRISPR

One of the most powerful ways to test the function of a protein is to characterize the consequences of its deletion. In the past, this has involved inactivation of the gene by homologous recombination either in the germline or later through conditional deletion. RNA interference (RNAi) provides an alternative way to knock down proteins, but both of these approaches have their limitations. Recently, the CRISPR/Cas9 system has suggested another way to selectively inactivate genes. We have now tested this system in postmitotic neurons by targeting two well-characterized synaptic proteins, the obligatory GluN1 subunit of the NMDA receptor and the GluA2&nbsp;subunit of the AMPA receptor. Expression of CRISPR/Cas9 in hippocampal slice cultures completely eliminated NMDA receptor and GluA2 function. CRISPR/Cas9 thus provides a powerful tool to study the function of synaptic proteins

Identification of the functional domain of the dense core vesicle biogenesis factor HID-1.

Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. HID-1 is a trans-Golgi network (TGN) localized peripheral membrane protein contributing to LDCV formation. There is no information about HID-1 structure or domain architecture, and thus it remains unknown how HID-1 binds to the TGN and performs its function. We report that the N-terminus of HID-1 mediates membrane binding through a myristoyl group with a polybasic amino acid patch but lacks specificity for the TGN. In addition, we show that the C-terminus serves as the functional domain. Indeed, this isolated domain, when tethered to the TGN, can rescue the neuroendocrine secretion and sorting defects observed in HID-1 KO cells. Finally, we report that a point mutation within that domain, identified in patients with endocrine and neurological deficits, leads to loss of function

Artificial tethering of the HID-1 C-terminus domain to the TGN is sufficient to rescue the HID-1 KO phenotype.

(A-C) HID-1 KO PC12 cells were transiently co-transfected with GFP-TGN38, GFP-TGN38-HID-1-C-terminus or HID-1-GFP and NPY-sfCherry3. (A) Anti-GFP immunoblot showing expression level of the fusion proteins. (B-C) Cells were washed and incubated in Tyrode’s buffer containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated) for 30 min at 37°C. The amount of cellular and secreted NPY-sfCherry3 was determined using a plate reader (Tecan). *, p D) HID-1 KO PC12 cells were transiently co-transfected with GFP, GFP-TGN38-HID-1-C-terminus, immunostained for SgII and the amount of fluorescence in GFP positive cells was quantified and normalized to WT PC12 cells. ***, p<0.001 relative to HID-1-KO by one-way ANOVA (n = 226 cells for WT, n = 217 cells for KO, n = 186 cells for GFP, n = 185 cells for GFP-TGN38-Cterm from three independent experiments). The bar graphs indicate mean ± s.e.</p

HID-1 myristoylation is not required for function.

(A-C) HID-1 KO PC12 cells transduced with indicated HID-1-HA lentivirus were washed and incubated for 30 minutes in Tyrode’s solution containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated). Cellular and secreted SgII were measured by quantitative fluorescent immunoblotting (A), with the secreted SgII normalized to tubulin and expressed as percent of basal secretion in the KO (B), and the cellular SgII normalized to tubulin (C). *, p < 0.05 relative to HID-1 KO by one-way ANOVA (n = 4). The bar graphs indicate mean ± s.e.</p

S2 Fig -

(A) Genomic DNA sequence of rat HID-1. The predicted Cas9 cleavage site is indicated. (B) Indels (shown in red) detected in INS-1 HID-1 KO cells. (C) Indels (shown in red) detected in PC12 HID-1 KO cells. (D-E) HID-1 KO PC12 and INS-1 immunostained for endogenous HID-1. Scale bars indicate 10μm. (TIF)</p