Cerebrin prohormone processing, distribution and action in Aplysia californica

Abstract

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17-residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic puri®cation guided by matrix-assisted laser desorption/ionization time-of-¯ight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F-and C-clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral-buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command-like neuron CBI-2, dramatically shortening the duration of the radula protraction in a concentration-dependent manner, mimicking the motor-pattern alterations observed in food induced arousal states. These ®ndings suggest that cerebrin may contribute to food-induced arousal in the animal. Cerebrin-like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin-like peptides may be widespread throughout gastropoda. Keywords: Aplysia californica, cDNA cloning, in situ hybridization, immunocytochemistry, Lymnaea stagnalis, MALDI MS. Invertebrate model systems are used extensively to gain insight into neural mechanisms that are responsible for the expression and plasticity of behavior. In these systems, characterization of behavior-mediating circuits is greatly facilitated by the fact that neural circuits contain a limited number of neurons. Consequently, in many invertebrates, the neural circuits that mediate a variety of behaviors have been extensively characterized. However, in order to understand the neural basis of behavior it is also necessary to have fairly complete knowledge of signaling molecules that affect the circuits from within and without. Neuropeptides represent the largest and most diverse group of signaling molecules in the nervous system. Advantageous features of invertebrate nervous systems have greatly facilitated identi®cation of neuropeptides that are involved in generating and modifying a number of behaviors. Several important insights into the role of various neuropeptides have been obtained and the functional consequence of the large peptide diversity in several well-de®ned neuronal networks is actively being investigated (Marder et al. 1995;Brezina and Weiss 19...

Identification of a New Neuropeptide Precursor Reveals a Novel Source of Extrinsic Modulation in the Feeding System ofAplysia

“…Antibodies to several of the peptides that were predicted from the MMG2 precursor were produced in rats as described previously (Fujisawa et al, 1999;Furukawa et al, 2001;Li et al, 2001;Sweedler et al, 2002). Briefly, the antigen was prepared by coupling synthetic peptide (SynPep, Dublin, CA) to BSA (catalog #A0281; Sigma-Aldrich) using 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC; catalog #E7750; Sigma-Aldrich).…”

A Newly Identified Buccal Interneuron Initiates and Modulates Feeding Motor Programs inAplysia

“…In response to seaweed application to the inner lips of the animal, CBI-2 can fire Ͼ10 Hz during each protraction phase, for several cycles of activity (Rosen et al 1991). When CBI-2 is stimulated at similar frequencies (8 -15 Hz) during the protraction phase, a single cycle of patterned activity (single-cycle motor program) is elicited Weiss 2001, 2002;Li et al 2001). Stimulating CBI-2 for longer periods (beyond a single cycle of patterned activity) triggers multiple motor program cycles (Church and Lloyd 1994;Hurwitz et al 2003;Morgan et al 2000;Rosen et al 1991).…”

Improved identification and quantitation of mature endogenous peptides in the rodent hypothalamus using a rapid conductive sample heating system

“…They are derived from larger precursor proteins, prohormones, after enzymatic cleavage by prohormone convertases at enzyme-specific sequences, with these expected cleavage sites referred to as conventional cleavage sites. 1–3 Neuropeptides participate in a broad range of physiological and pathological processes—pain sensation, food intake, circadian rhythm, depression, tissue regeneration, and drug addiction—by acting as neuromodulators, neurotransmitters, and hormones. 4–11 Significant advances in the study of neuropeptides have been achieved in the past three decades, in part due to rapid progress in mass spectrometry (MS) instrumentation and software tools for data analysis.…”

“…However, they did not distinguish between full-length neuropeptides, intermediate processing and/or linker peptides derived from the prohormones and, most importantly, numerous truncated versions of neuropeptides often resulting from sequential one amino acid loss that is commonly observed in peptidomics measurements. 25 The distinction is important as full-length peptides are produced, accumulated, and released from cells in-vivo , 1–3 and are more likely to be biologically active, whereas randomly truncated forms often observed in the MS measurements could be sampling artifacts caused by the loss of the sample cellular integrity and release of lysosomal proteases, or by chemical degradation under the extreme pH conditions typical for peptide extract preparation. Ladder sequence truncation, as evidenced in the supplemental data set by Secher et al 42 for example, is a well-known form of peptide chemical degradation under extreme pH that had been used for peptide sequencing before the advent of tandem MS (MS/MS)-capable instrumentation.…”