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

• Published in: Journal of neurochemistry 2018-02, Vol.144 (3), p.241-254
• Main Authors: Dominguez, Natalia, Weering, Jan R. T., Borges, Ricardo, Toonen, Ruud F. G., Verhage, Matthijs
• Format: Article
• Language: English
• Subjects: Animals; Biosynthesis; Chromaffin cells; Chromogranin A - genetics; Chromogranin A - physiology; Chromogranin B - genetics; Chromogranin B - physiology; Chromogranins; Compartments; Electron microscopy; Exocytosis; Female; Fluorescence; Gene expression; Hippocampus; Hippocampus - physiology; Hippocampus - ultrastructure; Kinetics; live‐cell imaging; Lymphocytes B; Male; Mice, Inbred C57BL; Mice, Knockout; neuromodulation; Neurons; Neurons - physiology; Neurons - ultrastructure; Neuropeptide Y; neuropeptides; neurotransmission; Organelle Biogenesis; Original; ORIGINAL ARTICLES; pH effects; Primary Cell Culture; Regulators; Rodents; Secretion; Secretory vesicles; Secretory Vesicles - physiology; Secretory Vesicles - ultrastructure; Synapses; Synapses - ultrastructure; Synaptic density; Trophic factors
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/jnc.14263

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. Chromogranin A and B (Cgs) have been implicated in the biogenesis and release of Dense‐Core Vesicles (DCVs) in chromaffin cells. Here, we demonstrate that despite the reduction in the number and size of DCVs in chromaffin cells, this phenotype is not reproduced in hippocampal neurons. We conclude that Cgs are not essential for DCVs biogenesis, cargo sorting, and release in hippocampal neurons.