Neuronal Secretory Pathway Research Articles

Dual Modes of Endoplasmic Reticulum-to-Golgi Transport in Dendrites Revealed by Live-Cell Imaging

Organelles of the neuronal secretory pathway are critical for the addition of membrane that accompanies neuronal development, as well as for the proper localization of plasma membrane proteins necessary for polarity, synaptic transmission, and plasticity. Here, we demonstrate that two organizations of the secretory pathway exist in neurons: one requiring processing of membrane and lipids in the Golgi complex of the cell body and one in which endoplasmic reticulum (ER)-to-Golgi trafficking is localized to dendrites. Using time-lapse imaging of green fluorescent protein-tagged cargo proteins and compartment markers, we show that organelles of the secretory pathway, including ER, ER exit sites, and Golgi, are present and engage in trafficking in neuronal dendrites. We find that ER-to-Golgi trafficking involves highly mobile vesicular carriers that traffic in both the anterograde and retrograde directions throughout the dendritic arbor. Dendritic Golgi outposts, which appear developmentally during the phase of process outgrowth, are involved in the trafficking of both integral membrane proteins and the secreted neuronal growth factor BDNF. This distributed dendritic Golgi represents an organization of the secretory pathway unique among mammalian cells.

The G-protein inhibitor, pertussis toxin, inhibits the secretion of brain-derived neurotrophic factor

Secretion of neurotrophins is critical for the delivery of neurotrophic support. Brain-derived neurotrophic factor is targeted to a regulated secretory pathway in neurons as well as the neurosecretory AtT-20 cells. Here, we show that pertussis toxin, which inactivates Gi and Go G proteins, inhibits up to 50% of the regulated release of brain derived neurotrophic factor by AtT-20 cells. To determine whether pertussis toxin-sensitive G proteins may regulate brain-derived neurotrophic factor release in vivo, the effect of intraocular pertussis toxin was assessed on the isthmo-optic nucleus in the developing chick visual system. The isthmo-optic nucleus projects axons from the midbrain to innervate retinal amacrine cells and depends on target-derived brain-derived neurotrophic factor between embryonic days 13 and 17 (E13–17). During this period approximately 50% of isthmo-optic neurons are eliminated by programmed cell death. Intraocular pertussis toxin administered at E13 increased cell death of isthmo-optic neurons by 42%, whereas injections at E19 had no effect. Co-injection of brain-derived neurotrophic factor with pertussis toxin rescued approximately 50% of isthmo-optic neurons from enhanced cell death, although overall retinal brain derived neurotrophic factor protein levels were unaffected by pertussis toxin. Retrograde transport of exogenous 125I-labeled brain derived neurotrophic factor from the retina to the midbrain was increased by co-administration of pertussis toxin, possibly owing to diminished competition from endogenously released brain-derived neurotrophic factors for the receptors that mediate retrograde axonal transport. These data suggest that the release of a major fraction of brain-derived neurotrophic factor in the secretory pathway in vitro and in vivo is regulated by the activity of pertussis toxin-sensitive G proteins.

Cultured sympathetic neurons express functional interleukin-1 receptors

Since recent work has identified an apoptotic pathway in sympathetic neurons that is mediated by autocrine interleukin-1 (IL-1), we investigated whether cultured sympathetic neurons possess functional IL-1 receptors. Cultured sympathetic neurons express levels of IL-1RI and IL-1RAcP mRNAs consistent with signal transduction. Neurons stimulated with IL-1 demonstrate enhanced p65 NF-κB nuclear translocation and enhanced NF-κB DNA binding activity, with at least p65 and p50 subunits participating in the DNA binding activity. RNA differential display identified several neuronal mRNAs regulated by IL-1, including a member of the reticulon family. We conclude that IL-1 stimulates a potential component of a neuronal secretory pathway.

Novel Proteins That Interact with the COOH-terminal Cytosolic Routing Determinants of an Integral Membrane Peptide-processing Enzyme

The steady state distribution of membrane forms of peptidylglycine alpha-amidating monooxygenase (PAM) in the secretory pathway of neurons and endocrine cells depends on signals in its cytosolic COOH-terminal domain (CD). Mutagenesis studies yielded catalytically active PAM proteins that are not properly localized or internalized. Employing the yeast two-hybrid system, we isolated two distinct cDNAs whose protein products showed a strong interaction with the CD of PAM. The interaction of these novel PAM COOH-terminal interactor proteins (P-CIPs) did not occur with a misrouted CD mutant as bait in the yeast system. Both proteins, P-CIP2 and P-CIP10, were expressed as fusion proteins that interacted in vitro with solubilized integral membrane PAM. P-CIP2 was homologous to several serine/threonine and dual specificity protein kinases, while P-CIP10 contained spectrin-like repeats. Endogenous P-CIP2 was localized to the Golgi region of AtT-20 corticotrope tumor cells, and expression of integral membrane PAM disrupted the distribution of endogenous P-CIP2. Both P-CIP2 and P-CIP10 mRNAs were found to be expressed in rat brain neurons also expressing PAM proteins. P-CIP2 and P-CIP10 may be members of a family of cytosolic proteins involved in the routing of membrane proteins that function in the regulated secretory pathway.

Peptide Processing and Targeting in the Neuronal Secretory Pathway

The abdominal ganglion of the marine mollusk Aplysia contains a pair of identified neuronal clusters, the bag cells, which control egg laying by means of a number of unique regulatory mechanisms. Each neuron in the bag cell clusters synthesizes several peptides derived from a single prohormone and packages them into separate vesicles. These vesicles are then differentially localized in specific neuronal processes, thus segregating peptides destined for autocrine and hormonal release sites. Therefore in this system, protein trafficking through the secretory pathway organizes multiple peptide neurochemical messengers to efficiently regulate simple behaviors.