Human iN neuronal model of schizophrenia displays dysregulation of chromogranin B and related neuropeptide transmitter signatures.

Schizophrenia (SZ) is a serious mental illness and neuropsychiatric brain disorder with behavioral symptoms that include hallucinations, delusions, disorganized behavior, and cognitive impairment. Regulation of such behaviors requires utilization of neurotransmitters released to mediate cell-cell communication which are essential to brain functions in health and disease. We hypothesized that SZ may involve dysregulation of neurotransmitters secreted from neurons. To gain an understanding of human SZ, induced neurons (iNs) were derived from SZ patients and healthy control subjects to investigate peptide neurotransmitters, known as neuropeptides, which represent the major class of transmitters. The iNs were subjected to depolarization by high KCl in the culture medium and the secreted neuropeptides were identified and quantitated by nano-LC-MS/MS tandem mass spectrometry. Several neuropeptides were identified from schizophrenia patient-derived neurons, including chromogranin B (CHGB), neurotensin, and natriuretic peptide. Focusing on the main secreted CHGB neuropeptides, results revealed differences in SZ iNs compared to control iN neurons. Lower numbers of distinct CHGB peptides were found in the SZ secretion media compared to controls. Mapping of the peptides to the CHGB precursor revealed peptides unique to either SZ or control, and peptides common to both conditions. Also, the iNs secreted neuropeptides under both KCl and basal (no KCl) conditions. These findings are consistent with reports that chromogranin B levels are reduced in the cerebrospinal fluid and specific brain regions of SZ patients. These findings suggest that iNs derived from SZ patients can model the decreased CHGB neuropeptides observed in human SZ.

TFH-derived dopamine accelerates productive synapses in germinal centres.

Protective high-affinity antibody responses depend on competitive selection of B cells carrying somatically mutated B-cell receptors by follicular helper T (TFH) cells in germinal centres. The rapid T-B-cell interactions that occur during this process are reminiscent of neural synaptic transmission pathways. Here we show that a proportion of human TFH cells contain dense-core granules marked by chromogranin B, which are normally found in neuronal presynaptic terminals storing catecholamines such as dopamine. TFH cells produce high amounts of dopamine and release it upon cognate interaction with B cells. Dopamine causes rapid translocation of intracellular ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) to the B-cell surface, which enhances accumulation of CD40L and chromogranin B granules at the human TFH cell synapse and increases the synapse area. Mathematical modelling suggests that faster dopamine-induced T-B-cell interactions increase total germinal centre output and accelerate it by days. Delivery of neurotransmitters across the T-B-cell synapse may be advantageous in the face of infection.

The mechanisms of chromogranin B-regulated Cl- homeostasis.

Chloride is the most abundant inorganic anions in almost all cells and in human circulation systems. Its homeostasis is therefore important for systems physiology and normal cellular activities. This topic has been extensively studied with chloride loaders and extruders expressed in both cell surfaces and intracellular membranes. With the newly discovered, large-conductance, highly selective Cl- channel formed by membrane-bound chromogranin B (CHGB), which differs from all other known anion channels of conventional transmembrane topology, and is distributed in plasma membranes, endomembrane systems, endosomal, and endolysosomal compartments in cells expressing it, we will discuss the potential physiological importance of the CHGB channels to Cl- homeostasis, cellular excitability and volume control, and cation uptake or release at the cellular and subcellular levels. These considerations and CHGB's association with human diseases make the CHGB channel a possible druggable target for future molecular therapeutics.

Chromogranin B in blood serum of patients with neuroendocrine tumors.

Neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms from cells of the diffuse neuroendocrine system. Chromogranin B (CgB) is an acidic protein of the granin family, which can be used to detect the tumours of neuroendocrine nature. Analysis of levels and evaluation of the diagnostic efficiency of CgB in the blood serum of patients with NETs of various localizations. Patients with NETs (n=121) without specific treatment were examined. In the study were presented next localizations: 74 - pancreas, 20 - stomach, 12 - large intestine, 15 - other localizations (lungs, mammary gland, prostate gland, NETs with unidentified primary). 54 practically healthy donors were examined as control group. The determination of CgB in blood serum was performed with ELISA method on BEP 2000 analyzer using a standardized test system Human Chromogranin B (USCN, China). CgB levels in common NET group (median 18.9 ng/mL) were statistically significantly higher than in the control group (8.8 ng/mL). The highest median was obtained in group of intestinal NETs (21.2 ng/ml), which exceeded the median of the control group by more than 2.4 times. According to ROC analysis in the common NET group relative to the control group, the area under the curve AUC was 0.88 (95% CI 0.83-0.929). According to cut-off level of CgB - 15.8 ng/ml, the diagnostic sensitivity was 69.4%, with a specificity of 96.3%. The highest diagnostic sensitivity was in the group of the intestinal NETs (75.0%) and pancreas (71.2%). The study showed the significance of CgB as a potential biochemical marker of NETs with various localizations, alternative to CgA.

Chromogranin B regulates early-stage insulin granule trafficking from the Golgi in pancreatic islet ß-cells.

Chromogranin B (CgB, also known as CHGB) is abundantly expressed in dense core secretory granules of multiple endocrine tissues and has been suggested to regulate granule biogenesis in some cell types, including the pancreatic islet ß-cell, though the mechanisms are poorly understood. Here, we demonstrate a critical role for CgB in regulating secretory granule trafficking in the ß-cell. Loss of CgB impairs glucose-stimulated insulin secretion, impedes proinsulin processing to yield increased proinsulin content, and alters the density of insulin-containing granules. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, we show that loss of CgB impairs Golgi budding of proinsulin-containing secretory granules, resulting in a substantial delay in trafficking of nascent granules to the plasma membrane with an overall decrease in total plasma membrane-associated granules. These studies demonstrate that CgB is necessary for efficient trafficking of secretory proteins into the budding granule, which impacts the availability of insulin-containing secretory granules for exocytic release.This article has an associated First Person interview with the first author of the paper.

Neuropeptidomics of the Rat Habenular Nuclei.

Conserved across vertebrates, the habenular nuclei are a pair of small symmetrical structures in the epithalamus. The nuclei functionally link the forebrain and midbrain by receiving input from and projecting to several brain regions. Each habenular nucleus comprises two major asymmetrical subnuclei, the medial and lateral habenula. These subnuclei are associated with different physiological processes and disorders, such as depression, nicotine addiction, and encoding aversive stimuli or omitting expected rewarding stimuli. Elucidating the functions of the habenular nuclei at the molecular level requires knowledge of their neuropeptide complement. In this work, three mass spectrometry (MS) techniques-liquid chromatography (LC) coupled to Orbitrap tandem MS (MS/MS), LC coupled to Fourier transform (FT)-ion cyclotron resonance (ICR) MS/MS, and matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS-were used to uncover the neuropeptide profiles of the rodent medial and lateral habenula. With the assistance of tissue stabilization and bioinformatics, a total of 262 and 177 neuropeptides produced from 27 and 20 prohormones were detected and identified from the medial and lateral habenula regions, respectively. Among these neuropeptides, 136 were exclusively found in the medial habenula, and 51 were exclusively expressed in the lateral habenula. Additionally, novel sites of sulfation, a rare post-translational modification, on the secretogranin I prohormone are identified. The results demonstrate that these two small brain nuclei have a rich and differentiated peptide repertoire, with this information enabling a range of follow-up studies.

Dense-core vesicle biogenesis and exocytosis in neurons lacking chromogranins A and B.

Chromogranin A and B (Cgs) are considered to be master regulators of cargo sorting for the regulated secretory pathway (RSP) and dense-core vesicle (DCV) biogenesis. To test this, we analyzed the release of neuropeptide Y (NPY)-pHluorin, a live RSP reporter, and the distribution, number, and appearance of DCVs, in mouse hippocampal neurons lacking expression of CHGA and CHGB genes. qRT-PCR analysis showed that expression of other granin family members was not significantly altered in CgA/B-/- neurons. As synaptic maturation of developing neurons depends on secretion of trophic factors in the RSP, we first analyzed neuronal development in standardized neuronal cultures. Surprisingly, dendritic and axonal length, arborization, synapse density, and synaptic vesicle accumulation in synapses were all normal in CgA/B-/- neurons. Moreover, the number of DCVs outside the soma, stained with endogenous marker Secretogranin II, the number of NPY-pHluorin puncta, and the total amount of reporter in secretory compartments, as indicated by pH-sensitive NPY-pHluorin fluorescence, were all normal in CgA/B-/- neurons. Electron microscopy revealed that synapses contained a normal number of DCVs, with a normal diameter, in CgA/B-/- neurons. In contrast, CgA/B-/- chromaffin cells contained fewer and smaller secretory vesicles with a smaller core size, as previously reported. Finally, live-cell imaging at single vesicle resolution revealed a normal number of fusion events upon bursts of action potentials in CgA/B-/- neurons. These events had normal kinetics and onset relative to the start of stimulation. Taken together, these data indicate that the two chromogranins are dispensable for cargo sorting in the RSP and DCV biogenesis in mouse hippocampal neurons.

Sex-dependent effects of chromogranin B P413L allelic variant as disease modifier in amyotrophic lateral sclerosis.

Recent genetic studies yielded conflicting results regarding a role for the variant chromogranin B (CHGB)P413L allele as a disease modifier in ALS. Moreover, potential deleterious effects of the CHGBP413L variant in ALS pathology have not been investigated. Here we report that in transfected cultured cells, the variant CHGBL413 protein exhibited aberrant properties including mislocalization, failure to interact with mutant superoxide dismutase 1 (SOD1) and defective secretion. The CHGBL413 transgene in SOD1G37R mice precipitated disease onset and pathological changes related to misfolded SOD1 specifically in female mice. However, the CHGBL413 variant also slowed down disease progression in SOD1G37R mice, which is in line with a very slow disease progression that we report for a Swedish woman with ALS who is carrier of two mutant SOD1D90A alleles and two variant CHGBP413L and CHGBR458Q alleles. In contrast, overexpression of the common CHGBP413 allele in SOD1G37R mice did not affect disease onset but significantly accelerated disease progression and pathological changes. As in transgenic mice, the CHGBP413L allele conferred an earlier ALS disease onset in women of Japanese and French Canadian origins with less effect in men. Evidence is presented that the sex-dependent effects of CHGBL413 allelic variant in ALS may arise from enhanced neuronal expression of CHGB in females because of a sex-determining region Y element in the gene promoter. Thus, our results suggest that CHGB variants may act as modifiers of onset and progression in some ALS populations and especially in females because of higher expression levels compared to males.

Regulation of circulating chromogranin B levels in heart failure.

BACKGROUND: Chromogranin B (CGB) regulates B-type natriuretic peptide (BNP) production. Circulating CGB levels are elevated in heart failure (HF) animal models and HF patients, but also increase in healthy individuals in response to physical activity. Therefore, CGB seems to integrate information from myocardial stress and systemic neuro-endocrine activation. Substantial gaps remain in our understanding of CGB regulation in HF. METHODS AND RESULTS: We conducted a retrospective registry study including 372 patients. CGB and N-terminal pro-BNP (NT-proBNP) plasma levels were assessed in acute HF and chronic valvular HF patients and controls. CGB levels were significantly increased in acute HF and chronic valvular HF, but significantly higher in the latter. Patients in chronic valvular HF with severe mitral regurgitation (cHF-MR) showed significantly higher CGB levels than patients in chronic valvular HF with severe aortic stenosis. CGB levels progressively increased with worsening NYHA functional status and were moderately correlated to NT-proBNP, but independent of left ventricular (LV) ejection fraction (LVEF), LV mass, age and body weight. Finally, cHF-MR patients showed significant reductions of CGB levels after interventional mitral valve repair. CONCLUSION: CGB is a promising emerging biomarker in HF patients with unique potential to integrate information from myocardial stress and neuro-endocrine activation.

Identification of proteins that bind to the neuroprotective agent neoechinulin A.

Neoechinulin A is an indole alkaloid with several biological activities. We previously reported that this compound protects neuronal PC12 cells from cytotoxicity induced by the peroxynitrite generator 3-morpholinosydnonimine (SIN-1), but the target proteins and precise mechanism of action of neoechinulin A were unclear. Here, we employed a phage display screen to identify proteins that bind directly with neoechinulin A. Our findings identified two proteins, chromogranin B and glutaredoxin 3, as candidate target binding partners for the alkaloid. QCM analyses revealed that neoechinulin A displays high affinity for both chromogranin B and glutaredoxin 3. RNA interference-mediated depletion of chromogranin B decreased the sensitivity of PC12 cells against SIN-1. Our results suggested chromogranin B is a plausible target of neoechinulin A.

Circulating chromogranin B levels in patients with acute respiratory failure: data from the FINNALI Study.

PURPOSE: Circulating chromogranin B (CgB) levels are increased in situations characterized by systemic and myocardial stress, but whether CgB provides prognostic information in patients with acute respiratory failure (ARF) is unknown. METHODS: We included 584 patients with ARF, defined as ventilatory support >6 h, and with blood samples available on Intensive Care Unit (ICU) admission and day 3 (n = 479). CgB levels were measured by radioimmunoassay and follow-up was 90 days. RESULTS: One-hundred-sixty-nine patients (29%) died during follow-up. Admission CgB levels separated non-survivors from survivors: median 1234 (Q1-3 989-1742) vs. 917 (753-1224) pmol/L, respectively, p < 0.001. CgB levels on ICU admission (logarithmically transformed) were associated with time to death after adjustment for established risk indices available on ICU admission, including N-terminal pro-B-type natriuretic levels: HR 2.62 (95%C.I. 1.82-3.77), p < 0.001. Admission CgB levels also improved prognostication on top of SOFA and SAPS II scores as assessed by Cox regression analyses and the category-free net reclassification index. The area under the curve (AUC) for admission CgB levels to separate survivors and non-survivors was 0.72 (95%CI 0.67-0.76), while the AUC on day 3 was 0.60 (0.54-0.66). CONCLUSIONS: CgB levels measured on ICU admission provided additional prognostic information to established risk indices in ARF patients.

Utility of chromogranin B compared with chromogranin A as a biomarker in Japanese patients with pancreatic neuroendocrine tumors.

OBJECTIVE: Currently, serum chromogranin A is a well-established biomarker for pancreatic neuroendocrine tumors; however, other pancreatic diseases, oral use of a proton pump inhibitor and renal impairment can affect chromogranin A. Meanwhile, chromogranin B, belonging to the same granin family as chromogranin A, is not fully examined in these conditions. The present study aimed to evaluate the utility of chromogranin B as a pancreatic neuroendocrine tumor biomarker. METHODS: Serum chromogranin B levels were determined by radioimmunoassay and serum chromogranin A levels by enzyme-linked immunosorbent assay in pancreatic neuroendocrine tumor (n = 91) and other pancreatic conditions, and in healthy people (n = 104), to assess the relationships with clinical features. RESULTS: The diagnostic ability of chromogranin B was as good as chromogranin A. The area under the curve was 0.79 for chromogranin B (sensitivity/specificity: 72%/77%), and 0.78 for chromogranin A (sensitivity/specificity: 79%/64%). Chromogranin B was not affected by proton pump inhibitor use and age, which affected chromogranin A. The number of cases without liver metastases was larger in pancreatic neuroendocrine tumor patients with positive chromogranin B and negative chromogranin A. Though chromogranin A significantly elevated cases with proton pump inhibitor treatment and had positive correlation with age, chromogranin B did not have the tendencies. However, both chromogranin B and chromogranin A elevated in the case with renal impairment. In addition, the logistic regression analysis showed that chromogranin B was superior to chromogranin A in differentiation of pancreatic neuroendocrine tumor from other pancreatic diseases. CONCLUSIONS: Compared with chromogranin A, chromogranin B may be more useful during proton pump inhibitor treatment and can detect tumors without liver metastases. In addition, chromogranin B may be an excellent biomarker when differentiation of pancreatic neuroendocrine tumor from other pancreatic diseases is required.

Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans.

Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.

Routine measurement of plasma chromogranin B has limited clinical utility in the management of patients with neuroendocrine tumours.

OBJECTIVE: Chromogranin A (CgA) and B (CgB) are markers for monitoring disease status in patients with gastroenteropancreatic neuroendocrine tumours (NETs). These are specialized diagnostic tests often necessitating referral of specimens to a supraregional assay service (SAS) laboratory for analysis. The aim of this audit was to assess whether measurement of either plasma CgA or CgB alone provides sufficient clinical information in comparison with the current practice of measuring both markers together. DESIGN: A retrospective analysis was undertaken for all chromogranin tests requested for patients with a known NET diagnosis. Results were categorized based on whether plasma concentrations were elevated for one or both CgA and CgB. RESULTS: A total of 325 sequential patients with a NET diagnosis had plasma chromogranin levels measured during the period of review. Baseline CgA was elevated in 60·9% of patients. Isolated elevations in CgA (with normal CgB) were found in 44·9% of patients, whilst combined elevations in both CgA and CgB were found in 16% of patients. Combined CgA and CgB concentrations within the normal range were observed for 38·5% of patients. Only two patients (0·6%) had an isolated elevation in CgB at baseline. Both patients had a diagnosis of pancreatic NET and were radiologically stable. Plasma CgA and CgB corresponded with disease stage (localized vs metastatic). CgB in addition to CgA did not provide any significant improvement in diagnostic performance for identification of metastatic disease compared to CgA alone. CONCLUSIONS: Based on this NET population and specific assay performance characteristics, CgA alone provides sufficient information for the management of NET patients; the routine estimation of CgB in all patients is not informative in clinical practice.

Deciphering the biochemistry and identifying biomarkers to multiple sclerosis.

Multiple sclerosis is an idiopathic demyelinating disease of the CNS. Despite being extensively studied during the last decades, many molecular aspects of the disease are still to be elucidated. Moreover, biomarkers for treatment and early diagnosis are major issues to be tackled. In this edition of Kroksveen et al. (Proteomics 2015, 15, 3361-3369) present biomarker candidates for the early detection of multiple sclerosis. Despite the need for validation in larger sets of samples, this dataset contributes to resolve open questions associated to multiple sclerosis.

Quantitative proteomics suggests decrease in the secretogranin-1 cerebrospinal fluid levels during the disease course of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS with unknown cause. Proteins with different abundance in the cerebrospinal fluid (CSF) from relapsing-remitting MS (RRMS) patients and neurological controls could give novel insight to the MS pathogenesis and be used to improve diagnosis, predict prognosis and disease course, and guide in therapy decisions. We combined iTRAQ labeling and Orbitrap mass spectrometry to discover proteins with different CSF abundance between six RRMS patients and 18 neurological disease controls. From 777 quantified proteins seven were selected as biomarker candidates, namely chitinase-3-like protein 1, secretogranin-1 (Sg1), cerebellin-1, neuroserpin, cell surface glycoprotein MUC18, testican-2 and glutamate receptor 4. An independent sample set of 13 early-MS patients, 13 RRMS patients and 13 neurological controls was used in a multiple reaction monitoring verification study. We found the intracellular calcium binding protein Sg1 to be increased in early-MS patients compared to RRMS and neurological controls. Sg1 should be included in further studies to elucidate its role in the early phases of MS pathogenesis and its potential as a biomarker for this disease.

Large-scale identification of endogenous secretory peptides using electron transfer dissociation mass spectrometry.

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

Chromogranin B: intra- and extra-cellular mechanisms to regulate catecholamine storage and release, in catecholaminergic cells and organisms.

Chromogranin B (CHGB) is the major matrix protein in human catecholamine storage vesicles. CHGB genetic variation alters catecholamine secretion and blood pressure. Here, effective Chgb protein under-expression was achieved by siRNA in PC12 cells, resulting in ~ 48% fewer secretory granules on electron microscopy, diminished capacity for catecholamine uptake (by ~ 79%), and a ~ 73% decline in stores available for nicotinic cholinergic-stimulated secretion. In vivo, loss of Chgb in knockout mice resulted in a ~ 35% decline in chromaffin granule abundance and ~ 44% decline in granule diameter, accompanied by unregulated catecholamine release into plasma. Over-expression of CHGB was achieved by transduction of a CHGB-expressing lentivirus, resulting in ~ 127% elevation in CHGB protein, with ~ 122% greater abundance of secretory granules, but only ~ 14% increased uptake of catecholamines, and no effect on nicotinic-triggered secretion. Human CHGB protein and its proteolytic fragments inhibited nicotinic-stimulated catecholamine release by ~ 72%. One conserved-region CHGB peptide inhibited nicotinic-triggered secretion by up to ~ 41%, with partial blockade of cationic signal transduction. We conclude that bi-directional quantitative derangements in CHGB abundance result in profound changes in vesicular storage and release of catecholamines. When processed and released extra-cellularly, CHGB proteolytic fragments exert a feedback effect to inhibit catecholamine secretion, especially during nicotinic cholinergic stimulation.

The role of chromogranin B in an animal model of multiple sclerosis.

Chromogranin B (CGB) is a high capacity, low affinity calcium binding protein in the endoplasmic reticulum (ER) that binds to the inositol 1,4,5 trisphosphate receptor (InsP3R) and amplifies calcium release from ER stores. Recently, it was discovered that levels of CGB-derived peptides are decreased in the cerebrospinal fluid of multiple sclerosis (MS) patients. One of the mechanisms by which neurodegeneration in MS is thought to occur is through increased levels of intra-axonal calcium. The combination of excess intracellular calcium and dysregulated levels of CGB in MS led us to hypothesize that CGB may be involved in MS pathophysiology. Here, we show in a mouse model of MS that CGB levels are elevated in neurons prior to onset of symptoms. Once symptoms develop, CGB protein levels increase with disease severity. Additionally, we show that elevated levels of CGB may have a role in the pathophysiology of MS and suggest that the initial elevation of CGB, prior to symptom onset, is due to inflammatory processes. Upon development of symptoms, CGB accumulation in neurons results from decreased ubiquitination and decreased secretion. Furthermore, we show that calpain activity is increased and levels of InsP3R are decreased. From these results, we suggest that the elevated levels of CGB and altered InsP3R levels may contribute to the axonal/neuronal damage and dysregulated calcium homeostasis observed in MS. Additionally, we propose that CGB can be a biomarker that predicts the onset and severity of disease in patients with MS.

Chromogranins A and B are key proteins in amine accumulation, but the catecholamine secretory pathway is conserved without them.

Chromogranins are the main soluble proteins in the large dense core secretory vesicles (LDCVs) found in aminergic neurons and chromaffin cells. We recently demonstrated that chromogranins A and B each regulate the concentration of adrenaline in chromaffin granules and its exocytosis. Here we have further studied the role played by these proteins by generating mice lacking both chromogranins. Surprisingly, these animals are both viable and fertile. Although chromogranins are thought to be essential for their biogenesis, LDCVs were evident in these mice. These vesicles do have a somewhat atypical appearance and larger size. Despite their increased size, single-cell amperometry recordings from chromaffin cells showed that the amine content in these vesicles is reduced by half. These data demonstrate that although chromogranins regulate the amine concentration in LDCVs, they are not completely essential, and other proteins unrelated to neurosecretion, such as fibrinogen, might compensate for their loss to ensure that vesicles are generated and the secretory pathway conserved.