A developmental gene (Tolloid/BMP-1) is regulated in Aplysia neurons by treatments that induce long-term sensitization

Abstract

Long-term sensitization training, or procedures that mimic the training, produces long-term facilitation of sensory-motor neuron synapses in Aplysia. The long-term effects of these procedures require mRNA and protein synthesis (Montarolo et al., 1986; Castellucci et al., 1989). Using the techniques of differential display reverse transcription PCR (DDRT-PCR) and ribonuclease protection assays (RPA), we identified a cDNA whose mRNA level was increased significantly in sensory neurons by treatments of isolated pleural-pedal ganglia with serotonin for 1.5 hr or by long-term behavioral training of Aplysia. The effects of serotonin and behavioral training on this mRNA were mimicked by treatments that elevate cAMP. The aplysia mRNA increased by serotonin and behavioral training was 41-45% identical to a developmentally regulated gene family which includes Drosophila tolloid and human bone morphogenetic protein-1 (BMP-1). Both tolloid and BMP-1 encode metalloproteases that might activate TGF-beta (transforming growth factor beta)-like molecules or process procollagens. Aplysia tolloid/BMP-1-like protein (apTBL-1) might regulate the morphology and efficacy of synaptic connections between sensory and motor neurons, which are associated with long-term sensitization.

Fig. 1.

Differential display reverse transcription-PCR (DDRT-PCR). Total RNAs extracted from pleural-pedal ganglia treated with 5 μm 5-HT for 1.5 hr (E) or without treatment (C) were differentially displayed with an anchored oligo-dT (T₁₂MG) and five arbitrary 10-mers (AGCCAGCGAA, GACCGCTTGT, AGGTGACCGT, GGTACTCCAC, and GTTGCGATCC). The bands (E) that appeared to be affected by 5-HT treatments are marked by arrows andnumbers.

Fig. 2.

Ribonuclease protection assays of clone 2.A1, Effects of 5-HT on pleural-pedal ganglia. Experimental pleural-pedal ganglia were treated with 5 μm5-HT for 1.5 hr, whereas matched contralateral control ganglia were untreated. Total RNAs (2 μg) from experimental (E) or control (C) pleural-pedal ganglia were hybridized with riboprobes of clone 2 and HSC70 (heat shock cognate protein).A2, Effects of 5-HT on sensory neurons. Total RNAs were extracted from sensory neurons of pleural-pedal ganglia treated with 5 μm 5-HT for 1.5 hr (E) or without treatment (C). A3, Effects of behavioral training on sensory neurons. Total RNAs were extracted from sensory neurons of pleural-pedal ganglia from the stimulated side (E) or unstimulated side (C) of animals. The size and purity of the probes are shown in A4.B, The time course of clone 2 mRNA change. Pleural-pedal ganglia were treated with 5 μm 5-HT for 0.75 and 1.5 hr, and sensory clusters were isolated and processed for RPA as described in Materials and Methods. After 1.5 hr, 5-HT was removed by washing with BFSW and ganglia were kept in BFSW. At 1.5 and 22.5 hr after removing 5-HT (time 3 and 24 hr), sensory clusters were isolated and processed for RPA.

Fig. 3.

Nucleotide and deduced amino acid sequences of apTBL-1 cDNA. apTBL-1 cDNA contains two possible translation initiation methionines (circled). The deduced amino acid sequence encodes a potential signal peptide at the N terminus (underlined with broken lines). Also, the deduced amino acid sequence contains the sequence homologous to the crayfish astacin family of metalloproteases (boxed), two 40-amino-acid repeats with EGF-like sequences (thick underlines), and seven potential glycosylation sites (thin underlined). The four cysteine residues for each CUB (complement subcomponents C1r/C1s, Uegf, BMP-1) repeat are enclosed in stippled or open boxes for alternate CUB repeats. The poly[A] signal sequences and RNA destabilization signal sequences are underlined with thin dashed lines. The nucleotide sequence of apTBL-1 cDNA has been submitted to GenBank under accession number U57369.

Fig. 4.

Distribution of apTBL-1 in various tissues ofAplysia. Total RNA (2.5 μg for pleural sensory neurons, 4 μg for other tissues) from Aplysia tissues was isolated and analyzed by RPA using the ³²P-labeled apTBL-1 riboprobe discussed in Materials and Methods. The sources of RNA were as follows: PD, pedal ganglia;SN, pleural sensory neurons; CNS, central nervous system; GL, gill; HT, heart;KN, kidney; BW, body wall;HP, hepatopancreas; PN, penis;OT, ovotestis.

Fig. 5.

In vitro translation of apTBL-1.In vitro translation products with (B) or without (A) capped cRNA of full-length apTBL-1 cDNA in the presence of [³⁵S]methionine. The Perfect Protein Marker (Novagen) was used as a molecular weight marker.

Fig. 6.

Immunoblot analysis of apTBL-1 proteins in pleural-pedal ganglia. Pleural-pedal ganglia of Aplysia californica were isolated and homogenized in 20 mmTris-HCl, pH 7.5, containing 1 μm leupeptin, 1 μm chymostatin, 1 μm pepstatin, 1 μm bestatin, 5 mm EGTA, 5 mmEDTA, and 1 mm PMSF. The homogenate was centrifuged at 800 × g for 5 min. Immunoblot of the supernatant was performed as described in Materials and Methods. Ten micrograms of total protein (A) and 20 μg of total protein (B) were used for SDS-PAGE. Prestained molecular weight markers (Amersham)—myosin (M_r = 200,000), phosphorylase b (M_r= 97,400), bovine serum albumin (M_r = 66,000), and ovalbumin (M_r = 46,000)—were used.

Fig. 7.

Immunolocalization of apTBL-1 in the pleural ganglion. The antibody directed against apTBL-1 protein produced punctuate staining in numerous cell bodies throughout the pleural ganglion, including sensory neurons (arrows). However, not all neurons in the sensory cluster were labeled (arrowheads). There was also staining in the neuropil (NP) and processes passing through the neuropil. In these relatively thick sections, it was not possible to identify stained structures in the neuropil.

Fig. 8.

Comparison of the domain structures of tolloid/BMP-1-like proteins. The tolloid/BMP-1 gene family includes Aplysia TBL-1, Drosophila tolloid (Shimell et al., 1991),tolloid-related-1 (Nguyen et al., 1994), mouse BMP-1 (Fukagawa et al., 1994), human BMP-1 (Wozney et al., 1988), sea urchin BMP-1 (Hwang et al., 1994), and Xenopus BMP-1 (Maeno et al., 1993). The potential signal peptide is represented by ablack box, and propeptides are represented byhatched boxes. The metalloprotease domain, CUB repeats, and EGF-like repeats are marked accordingly. The C-terminal nonhomologous sequences are represented by open boxes. The group of apTBL-1, tolloid,tolloid-related-1, and muBMP-1 contains five CUB repeats and two EGF-like repeats, and the group of huBMP-1, suBMP-1, and xeBMP-1 contains three CUB repeats and one EGF-like repeat. The length of the signal peptide and propeptide at the N terminus is different among the members of the family.

Fig. 9.

Model of possible roles of apTBL-1 in long-term presynaptic facilitation. A sensory neuron, motor neuron, and glial cell are represented schematically. The growth processes of sensory neurons and motor neurons are drawn with dotted lines. 5-HT increases the transcription of the apTBL-1 gene. apTBL-1 protein might remain in the cytoplasm by alternative translation and might play a role as a protease to modify the cytoskeleton structure in the growth process within the sensory neuron. apTBL-1 also might be secreted to modify the extracellular matrix (procollagen) or activate TGF-β-like growth factors. The activated growth factors could bind to Ser/Thr kinase receptors and trigger the signal transduction cascade, leading to the regulation of cell growth. The activated growth factors also might modify the motor neurons to complement the morphological changes in the sensory neurons, or they might activate glial cells to secrete extracellular matrix components that might then stabilize the morphological changes. Some of the same events elicited by the activation of TGF-β also could be caused by modification of the extracellular matrix component collagen.