Ag, a novel protein secreted fromAplysia glia

Abstract

We have isolated a cDNA (ag for Aplysia glial) corresponding to an mRNA specific to the nervous system of Aplysia californica. In this study, we characterized the ag cDNA sequence and the distribution of ag mRNA and protein in the Aplysia nervous system. The ag cDNA contains an open reading frame that encodes a novel 29 kD protein. In situ hybridizations demonstrate that ag mRNA is conspicuously absent from the cell bodies of the large neurons constituting the external layer of the ganglia. Instead, it is largely confined to a subset of small, apparently non‐neuronal cells surrounding the neurons at the border of the neuropil, is sparsely scattered within the neuropil, and is widespread within the connective nerves, a pattern consistent with glial localization. Polyclonal anti‐ag antiserum recognizes a protein between 27 and 29 kD that is more broadly distributed, especially within the neuropil. The distributions of ag mRNA and protein, together with the presence of a putative signal peptide, suggest that ag protein is secreted. Two findings support this hypothesis: first, ag protein is detectable by western blot in Aplysia hemolymph. Second, full length ag protein expressed in COS cells is secreted, but ag lacking the putative signal peptide is not. Secretion from glia raises the possibility that this abundant protein may affect neighboring neurons. © 1996 John Wiley & Sons, Inc.

Arterial cells and CNS sheath cells from Aplysia californica produce factors that enhance neurite outgrowth in co-cultured neurons

“…Inevitably, some debris and support cells were present in the aliquots of dispersed neurons, and the former were found on the side of the coverslips not containing PVC/AA cells. However, debris and support cells (for example, adsomatic glia; Lockhart et al 1996) present in our aliquots of dispersed neurons had no overt effect on neurite growth in control conditions (i.e., SL-15 alone; see Results and Discussion). We also tested for the presence of diffusible factors in sheath cell-neuron cocultures by gently agitating the culture dishes.…”

“…When Aplysia neurons are dissociated for cell culture, some Ag-positive glia remain attached to isolated somata (Lockhart et al 1996). It is possible that some adaxonal glia expressing Ag were dissociated from the axonal core of the PVCs during our trituration protocol.…”

Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in Aplysia

“…3A). Immunoblots for the Aplysia glial-specific protein Ag were performed (31) to determine the presence of glia and immunoblots for synaptophysin were used to assess the presence of nerve terminals (30). Densitometric analysis indicated that Ag was 10-fold enriched in P2 (cellular fraction pellet) relative to P3 (synaptosomal fraction pellet) (Fig.…”

“…The enrichment of synaptosomes was verified via immunoblot of synaptophysin, a nerve terminal protein (30), and Ag, a glial protein (31). Whole fractions and their pellets were solubilized in 1% SDS and diluted in a 3X loading dye (Bio-Rad).…”

“…Ag is a secreted glial protein in Aplysia (31). To ensure that the immunoblotted Ag was contained within glia or glial fragments, all fractions were exposed to proteinase K [Sigma; 0.125 mg͞ml in artificial seawater: 395 mM NaCl͞28 mM Na 2 SO 4 ͞10 mM KCl͞50 mM MgCl 2 ͞10 mM CaCl 2 ͞10 mM Tris⅐HCl (pH 8)] to eliminate extracellular protein.…”