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.

We have tested whether the lack of chromogranins (Cgs) A and B could provoke CNS disorders when combined with an excess of dopamine. We chronically treated (over 6 months) mice lacking both chromogranins A and B (Cgs-KO) with a low oral dosage of L-DOPA/benserazide (10/2.5 mg/kg). Motor performance in the rota-rod test, open field activity, and metabolic cages indicated a progressive impairment in motor coordination in these mice, and an increase in rearing behavior, which was accompanied by an increase in DA within the substantia nigra. We conclude that mild chronic L-DOPA treatment does not produce nigro-striatal toxicity that could be associated with parkinsonism, neither in control nor Cgs-KO mice. Rather, Cgs-KO mice exhibit behaviors compatible with an amphetamine-like effect, probably caused by the excess of catecholamines in the CNS.

Altered fecal levels of chromogranins (Cg) and secretogranins (Sg) are demonstrated in irritable bowel syndrome (IBS), but their role in IBS pathophysiology remains unknown. This study aimed to determine if granins are associated with bacterial composition, immune activation and IBS symptoms. Protein levels of fecal granins (CgA, CgB, SgII and SgIII) were analysed with immunoassays. Mucosal mRNA expression of granins, TPH1 and immune markers were evaluated with RT-qPCR. 16S rRNA gene sequencing was performed on fecal and mucosal bacteria. The intestinal granin profile, based on fecal protein levels and mucosal mRNA expression, could not discriminate between IBS patients (n = 88) and healthy subjects (HS, n = 33). IBS patients dominated by high fecal or mucosal granin levels, respectively, did not differ in symptom or immune profiles. Fecal-dominated and mucosal-dominated granin clusters of IBS patients and HS, demonstrated separate fecal and mucosal bacterial profiles and high fecal abundance of granins were associated with a less diverse bacterial composition and the Bacteroides enterotype. The intestinal granin profiles of IBS patients and HS are linked to the intestinal bacterial composition, diversity and enterotypes. These findings suggest that granins may be one of several host-produced factors regulating the microbiota composition of the intestine.

Paraneurons are receptosecretory cells producing substances of neurons. Like neurons, they are characterized by membrane-bound granules and vesicles which contain bioactive (and inactive) peptides, amines or other classic messengers, adenine nucleotides and acidic carrier proteins including chromogranins. The cell-biological significance of the carrier proteins in the secretory granules and the utility of immunohistochemical detection of chromogranins as reliable markers of paraneurons are emphasized. Cells "ectopically" producing bioactive peptides and/or amines, including ANP producing muscle cells of the heart, do not contain chromogranin immunoreactivity. The boundary between the neuron and paraneuron and that of the paraneuron are unclear because the cells comprise gradational spectra from typical ones to atypical ones. It is arbitrary whether to include the mast cell in the paraneurons, although it seems reasonable to exclude it by defining a paraneuron as being epithelial in nature.

Chromogranin A, chromogranin B and secretogranin II belong to the chromogranin family which consists of large protein molecules that are found in large dense core vesicles. Chromogranins are endoproteolytically processed to smaller peptides. This study was designed to elucidate the regulation of chromgranin expression by acute and subchronic phencyclidine administration. The behavioral syndrome produced by phencyclidine represents a pharmacological model for some aspects of schizophrenia [Jentsch and Roth (1999) Neuropsychopharmacology 20, 201-225]. Tissue concentrations of chromogranins were measured with specific radioimmunoassays. Alterations in secretogranin II gene expression were investigated by in situ hybridization. A single dose of phencyclidine (10mg/kg) led to a transient decrease in secretoneurin tissue levels in the prefrontal cortex after 4h followed by an increase in secretoneurin tissue levels after 12h. Repeated phencyclidine treatment (10mg/kg/day) for five days resulted in elevated secretoneurin levels in cortical areas whereas chromogranin A and chromogranin B tissue levels were unchanged. After the same treatment, a significant increase in the number of secretoneurin containing neurons was found in cortical layers II-III, and V-VI as revealed by immunocytochemistry. The increases in secretoneurin levels were paralleled by an increased number of secretogranin II messenger RNA containing neurons as well as by an increased expression of secretogranin II by individual neurons. The present study shows that secretoneurin II tissue concentration and secretogranin II messenger RNA expression is distinctly altered after acute and subchronic phencyclidine application. From these results we suggest that phencyclidine may induce synaptic alterations in specific brain areas and may contribute to a better understanding of synaptic dysfunction which may also occur in schizophrenia.

The release of monoamine neurotransmitters from cell bodies and dendrites has an important role in behavior, but the mechanism (vesicular or non vesicular) has remained unclear. Because the location of vesicular monoamine transporter 2 (VMAT2) defines the secretory vesicles capable of monoamine release, we have studied its trafficking to assess the potential for monoamine release by exocytosis. In neuroendocrine PC12 cells, VMAT2 localizes exclusively to large dense-core vesicles (LDCVs), and we now show that cytoplasmic signals target VMAT2 directly to LDCVs within the biosynthetic pathway. In neurons, VMAT2 localizes to a population of vesicles that we now find undergo regulated exocytosis in dendrites. Although hippocampal neurons do not express typical LDCV proteins, transfected chromogranins A, B, and brain-derived neurotrophic factor (BDNF) colocalize with VMAT2. VMAT2 thus defines a population of secretory vesicles that mediate the activity-dependent somatodendritic release of multiple retrograde signals involved in synaptic function, growth, and plasticity.

Chromogranins were reported to interact specifically with mutant forms of superoxide dismutase that are linked to amyotrophic lateral sclerosis (ALS). Particularly, a variation c.1238C>T (p.Pro413Leu) in the chromogranin B gene, CHGB, has been associated with an earlier age at onset in both familial and sporadic ALS in French/French-Canadian populations studied. The aim of our study was to evaluate the P413L chromogranin variation in Italian patients with sporadic ALS. The study included 366 Italian patients with sporadic ALS and 382 control subjects. Genotyping of the polymorphism P413L in the CHGB gene was performed and the clinical characteristics of patients were analyzed in relation to their genotype. Our study on a cohort of Italian patients with SALS and controls failed to confirm an increased frequency of the 413L variant in SALS patients. Furthermore, we did not confirm the previous observation of a difference of age at onset between T-allele carriers and non-carriers (median age of onset 58.5 vs. 60.2years of age, respectively). Our findings do not support the 413L variant as a risk factor for sporadic ALS in the Italian population.

Synaptic disturbances may play a key role in the pathophysiology of Alzheimer's disease. To characterize differential synaptic alterations in the brains of Alzheimer patients, chromogranin A, chromogranin B and secretoneurin were applied as soluble constituents for large dense core vesicles, synaptophysin as a vesicle membrane marker and calbindin as a cytosolic protein. In controls, chromogranin B and secretogranin are largely co-contained in interneurons, whereas chromogranin A is mostly found in pyramidal neurons. In Alzheimer's disease, about 30% of beta-amyloid plaques co-labelled with chromogranin A, 20% with secretoneurin and 15% with chromogranin B. Less than 5% of beta-amyloid plaques contained synaptophysin or calbindin, respectively. Semiquantitative immunohistochemistry revealed a significant loss for chromogranin B- and secretoneurin-like immunoreactivity in the dorsolateral, the entorhinal, and orbitofrontal cortex. Chromogranin A displayed more complex changes. It was the only chromogranin peptide to be expressed in glial fibrillary acidic protein containing cells. About 40% of chromogranin A immunopositive plaques and extracellular deposits were surrounded and pervaded by activated microglia. The present study demonstrates a loss of presynaptic proteins involved in distinct steps of exocytosis. An imbalanced availability of chromogranins may be responsible for impaired neurotransmission and a reduced functioning of dense core vesicles. Chromogranin A is likely to be a mediator between neuronal, glial and inflammatory mechanisms found in Alzheimer disease.

Delirium is a common cause and complication of hospitalization in older people, being associated with higher risk of future dementia and progression of existing dementia. However relatively little data are available on which biochemical pathways are dysregulated in the brain during delirium episodes, whether there are protein expression changes common among delirium subjects and whether there are any changes which correlate with the severity of delirium. We now present the first proteomic analysis of delirium cerebrospinal fluid (CSF), and one of few studies exploring protein expression changes in delirium. More than 270 proteins were identified in two delirium cohorts, 16 of which were dysregulated in at least 8 of 17 delirium subjects compared with a mild Alzheimer's disease neurological control group, and 31 proteins were significantly correlated with cognitive scores (mini-mental state exam and acute physiology and chronic health evaluation III). Bioinformatics analyses revealed expression changes in several protein family groups, including apolipoproteins, secretogranins/chromogranins, clotting/fibrinolysis factors, serine protease inhibitors and acute-phase response elements. These data not only provide confirmatory evidence that the inflammatory response is a component of delirium, but also reveal dysregulation of protein expression in a number of novel and unexpected clusters of proteins, in particular the granins. Another surprising outcome of this work is the level of similarity of CSF protein profiles in delirium patients, given the diversity of causes of this syndrome. These data provide additional elements for consideration in the pathophysiology of delirium as well as potential biomarker candidates for delirium diagnosis.

Candida species (Candida spp.) are important fungal pathogens, which cause numerous clinical diseases associated with significant mortality and morbidity in healthcare settings. In our previous study, we identified a recombinant peptide, chromogranin A (CGA)-N46, corresponding to the N-terminal Pro31-Gln76 sequence of human CGA, that exhibited antifungal activity against Candida albicans. The present study investigated the antifungal activity of CGA-N46, and its underlying mechanism, against numerous Candida spp. CGA-N46 inhibited the growth of all of the tested Candida spp., of which Candida krusei exhibited the greatest sensitivity. CGA-N46 was able to disrupt the stability of the phospholipid monolayer without damaging the integrity and permeability of the outer membrane of C. krusei cells, and induced cytoplasm vacuolization and mitochondrial damage. In addition, treatment of C. krusei with CGA-N46 was associated with decreased levels of intracellular reactive oxygen species, a reduction in the mitochondrial membrane potential, and DNA synthesis inhibition. The results of the present study suggested that CGA-N46 was able to pass through the cell membrane of Candida spp. by temporarily destabilizing the phospholipid membrane, which in turn led to mitochondrial dysfunction and inhibition of DNA synthesis. Therefore, CGA-N46 may be considered a novel antifungal compound for the treatment of patients with C. krusei infections.