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

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

Mutations in HID1 Cause Syndromic Infantile Encephalopathy and Hypopituitarism.

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. ANN NEUROL 2021

Reticular synthesis of porous molecular 1D nanotubes and 3D networks

Synthetic control over pore size and pore connectivity is the crowning achievement for porous metal–organic frameworks (MOFs). The same level of control has not been achieved for molecular crystals, which are not defined by strong, directional intermolecular coordination bonds. Hence, molecular crystallization is inherently less controllable than framework crystallization, and there are fewer examples of ‘reticular synthesis’, in which multiple building blocks can be assembled according to a common assembly motif. Here we apply a chiral recognition strategy to a new family of tubular covalent cages to create both 1D porous nanotubes and 3D diamondoid pillared porous networks. The diamondoid networks are analogous to MOFs prepared from tetrahedral metal nodes and linear ditopic organic linkers. The crystal structures can be rationalized by computational lattice-energy searches, which provide an in silico screening method to evaluate candidate molecular building blocks. These results are a blueprint for applying the ‘node and strut’ principles of reticular synthesis to molecular crystals

Sorting of Cargo Proteins Within the Regulated Secretory Pathway: the Peripheral Membrane Protein HID-1 as a Sorting and Vesicle Biogenesis Factor

Large dense core vesicles (LDCVs) form at the trans-Golgi network (TGN) and mediate the regulated release of neuropeptides and peptide hormones. Despite their central role to physiology, the mechanisms controlling biogenesis and sorting to LDCVs is not well understood. Optimizing the retention using selective hooks (RUSH) method in neuroendocrine cells, we show it is possible to visualize sorting to the constitutive and regulated secretory pathways in real-time and that the bulk of transmembrane LDCV cargoes do not sort directly onto LDCVs, but exit the TGN into non-regulated vesicles to be incorporated to LDCVs at a later step. Additionally, we characterize the TGN peripheral membrane protein, HID-1, as a LDCV biogenesis factor. We show that ablation of HID-1 results in defects in cargo storage and regulated secretion, a reduction in the number of LDCVs with perturbed morphology and biochemical properties, and results in defects in TGN acidification. Despite the importance of HID-1 to regulated secretion, little is known about HID-1 domain architecture and it is unclear how HID-1 associates to the TGN. We report that the N-terminus of HID-1 mediates membrane binding through a myristoyl group in combination with an unidentified binding motif within the C-terminus, and an interaction with the Golgi protein, Golga5, allows for targeting to the TGN. Finally, we identify the C-terminus as the functional domain of HID-1, capable of rescuing the secretion and sorting defects. A point mutation within that domain, identified in patients with endocrine and neurological deficits, is not functional. We propose that HID-1 N-terminal and C-terminal domains, in cooperation with Golga5, ensure specific binding to Golgi membranes and functionality of HID-1

Aging Changes The Neural Circuit Underlying Extinction of Fearful Memories

Over 16 million people in the United States are living with some form of aging-related cognitive impairment. Understanding changes in brain function during aging is important in finding novel ways to combat aging-related cognitive decline, including impairments in cognitive flexibility. We use extinction of fear conditioning as an example of cognitive flexibility; during extinction animals learn that a neutral tone that was previously paired with a shock is no longer predictive of a shock. We have demonstrated that fear extinction is impaired in aging rodents (Kaczorowski et al, 2012). Since fear extinction is dependent on the activity of neurons in specific regions of the medial prefrontal cortex (mPFC), we hypothesize that aging-related changes in neuronal activity in these regions may be linked to impaired fear extinction. In the present study we measured neuronal activation by investigating changes in the expression of Zif-268 and c-fos proteins, markers used to label recently active neurons. Young, middle-aged, and aged rats were separated into four experimental groups; naïve, unpaired, trace fear conditioned (TRACE), and trace fear extinction groups (EXT). On day 1, TRACE and EXT rats were exposed to 10 tone-shock pairings where the tone was followed by the shock 30s later. Unpaired rats were presented 10 tones and shocks but these were not explicitly paired. On days 2 and 3, EXT and unpaired rats were given 10 tone presentations to extinguish the tone-shock association. On the 4th day, TRACE, EXT, and unpaired rats were given 2 tone presentations to obtain an index of fear learning and fear extinction. Brains were subsequently removed and prepared for immunohistochemical staining for Zif-268 and c-fos expression. Our data suggest that within the prelimbic and infralimbic subregions of the mPFC, Zif-268 and c-fos expression was increased in unpaired, TRACE and EXT groups. This effect was more robust in young compared to aging rats. Moreover, despite differences in Zif-268 and c-fos expression, middle-aged, and aged rats were able to acquire trace fear conditioning, as well as extinction. Therefore, fear learning may engage mPFC differentially in young and aged rats. These data suggest that there may be significant changes in the brain regions normally engaged during successful acquisition and extinction of trace fear conditioning, which may be important in development of neurotherapeutic strategies for improving cognitive function in aged animals, including humans

Implicit Self-Esteem, Explicit Self-Concept and Personality Traits Discrepancy

The present study investigated implicit self-esteem and its relationship to explicit self-concept and Big Five personality traits, using Greenwald and Farnham’s (2000) Implicit Association Test (IAT) for self-concept. Eighty undergraduate psychology students, age ranging from 18 to 55 years (M=22.53, SD=7.00), participated in the study at the University of Wisconsin-Milwaukee. Participant’s implicit self-esteem was assessed through an IAT, measuring reaction time (RT) to self-other and positive-negative words. Explicit self-concept was measured using the Rosenberg Self-Concept Scale (RSCS) and personality was assessed using the Revised NEO Personality Inventory (NEO-PI-R). RT of the pairings “self-positive” and “other-negative” as well as “self-negative” and “other-positive” were first added respectively and then subtracted to obtain an implicit self-esteem score. The latter scores were converted into Z-scores and subtracted from the RSCS scores to obtain the overall self-esteem discrepancy score between both implicit and explicit measures. The relationship between the IAT and RSCS as well as the overall self-esteem discrepancy (as measured by the IAT and RSCS) and the neuroticism factor (as measured by the NEO-PI-R) was investigated using Pearson product-moment correlation coefficient. A discrepancy between implicit and explicit aspects of self-concept were expected to show a positive relationship with NEO-PI-R Neuroticism factor and its six facets, but a negative relationship with the NEO-PI-R Extraversion factor. Correlations showed a strong positive correlation between NEO-PI-R Neuroticism and the discrepancy score (r=.51) and a positive medium-sized correlations with the NEO-PI-R Neuroticism facets: Depression (r=.48); Angry Hostility (r=.34); and Self-Consciousness (r=.44). As predicted, there was also a medium-sized negative relationship between the discrepancy score and the NEO-PI-R Extraversion factor (r=-.41). Further research is needed to replicate these results and to explore further relationships between neuroticism and implicit self-concept measures

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