A single Aplysia neurotrophin mediates synaptic facilitation via differentially processed isoforms

Abstract

Neurotrophins control the development and adult plasticity of the vertebrate nervous system. Failure to identify invertebrate neurotrophin orthologs, however, has precluded studies in invertebrate models, limiting our understanding of fundamental aspects of neurotrophin biology and function. We identified a neurotrophin (ApNT) and Trk receptor (ApTrk) in the mollusk Aplysia and found that they play a central role in learning-related synaptic plasticity. Blocking ApTrk signaling impairs long-term facilitation, whereas augmenting ApNT expression enhances it and induces the growth of new synaptic varicosities at the monosynaptic connection between sensory and motor neurons of the gill-withdrawal reflex. Unlike vertebrate neurotrophins, ApNT has multiple coding exons and exerts distinct synaptic effects through differentially processed and secreted splice isoforms. Our findings demonstrate the existence of bona fide neurotrophin signaling in invertebrates and reveal a posttranscriptional mechanism that regulates neurotrophin processing and the release of proneurotrophins and mature neurotrophins that differentially modulate synaptic plasticity.

Figure 1. Cloning and Characterization of ApTrk

A. Conserved domain architecture and sequence features of ApTrk. Top: schematic representation of ApTrk structural domains: L- Leader/signal peptide; C-Cysteine; LRR-Leucine Rich Repeat; IgG-C2 – IgG like; TM – trans membrane; TK- tyrosine kinase. Bottom: Amino acid sequence of the ApTrk ORF. Domains within sequence are color coded corresponding to the schematic representation on top. Arrow shows position of cleavage of the signal peptide. Notable intracellular catalytic and tyrosine signaling residues are shown in black background with their surrounding consensus sequence highlighted in grey. Note the presence of the Shc site (NPNY) in the juxtamembrane region and Sh2 site (YLPI) at the C-terminus responsible in vertebrates for activating the Erk and PLCγ signaling pathways respectively. B. ApTrk belongs to the Trk family receptors. Clustal W analysis with the conserved tyrosine kinase domains of representative members of the Trk, Trk like (Trkl), ROR and RET families of RTKs. Percent identity to ApTrk is noted next to each sequence. See also Figure S1. C. RT-PCR analysis of ApTrk with cDNA from cultured Aplysia sensory or L7 motor neurons. Sensorin is a sensory neuron marker and 18S RNA is a loading control. D. Western blot analysis with ApTrk10 antibody against the ApTrk ectodomain on Aplysia CNS extract. A 300kDa band representing an SDS resistant receptor dimer is marked with an asterisk. A minor band at 110 kDa which is not consistently detected and might correspond to a truncated form of the receptor lacking a tyrosine kinase domain is marked with an arrow. See also Figure S2. E. Subcellular localization of ApTrk in cultured motor neurons. Immunostaining with the ApTrk10 antibody. Top: Cell body and initial segment, arrows point to ApTrk localization at the plasma membrane and concentration in puncta. Bottom: distal processes of the same cell showing patchy/punctuate ApTrk appearance. Scale bar: 10 microns.

Figure 2. Cloning and Characterization of ApNT

A. Multiple exon structure and sequence of ApNT. Top: Schematic representation of a single exon vertebrate neurotrophin and the multi-exon structure and features of ApNT. Exon 1 (purple) encodes the cleavable signal peptide. Exons 2 and 3 (red) encode the pro region. Exon 4 (blue) is a short miniexon encoding the first half site of the consensus furin enzyme processing site (RKKR). Exons 5 and 6 (green) encode the conserved mature domain of ApNT. Bottom: ApNT amino acid sequence color coded as above. Arrow points to the cleavage position of the furin processing site (underlined). Vertical bar shows exon boundary. Boxed exons 2 and 4 are alternatively spliced. Cysteine residues forming the cysteine knot structure are in black. Asparagines predicted to be N-glycosylated in the mature domain are in bold and underlined. B. Clustal W phylogenetic analysis of the mature portions of ApNT with vertebrate and putative invertebrate neurotrophins. Percent identity to ApNT is noted next to each sequence. See also Figure S3. C. 3-D structural models of ApNT and mouse NGF generated by the Swiss-Model comparative protein modeling server (http://swissmodel.expasy.org/workspace/). D. RT-PCR analysis of ApNT expression in sensory and L7 motor neurons as in 1C. E. Western blot analysis of ApNT in Aplysia CNS extract (soluble fraction) using pro region specific (P47) and mature region specific (M50) antibodies. Positions of intact precursor and processed pro and mature peptides are shown with arrows. See also Figure S4. F. C-terminally hexahistidine tagged ApNT purified from conditioned medium of ApNT(+) expressing HEK293 cells was mock treated or treated with a mix of deglycosylating enzymes for 3 hours at 37 °C and analyzed on SDS-PAGE and silver stained. G. ApNT exists as dimer in solution. Purified ApNT (50ug/ml) was crosslinked with 0.5, 1 or 2 mM bis(sulfosuccinimidyl)suberate (BS³) for 30 min. at room temperature or mock treated and the reactions analyzed by SDS-PAGE and western blotting with M50 antibody.

Figure 3. Functional Interaction between ApNT and ApTrk in PC12 cells

A. ApNT functionally interacts with ApTrk and activates Erk, Akt and PLCγ. PC12nnr5 cells were transfected with ApTrkGFP or GFP and treated for 5 min with 100 ng/ml of purified ApNT or with control vector transfected purified media (-) and then lysed and analyzed by western blotting with the indicated antibodies. B. BDNF cross-reacts with ApTrk. Cells were transfected with Trk receptors or GFP as indicated and treated for 5 min with indicated concentrations of purified ApNT or recombinant BDNF or NGF lysed and analyzed as in Figure 3A. C. ApNT-ApTrk induced neurite outgrowth in transfected PC12nnr5 cells. Cells were transfected with GFP alone or co-transfected with GFP and the indicated receptor and stimulated with 20 ng/ml of the indicated ligand and then imaged after 4 days. D. Quantification of percentage of cells exhibiting neurite outgrowth in Figure 3C.

Figure 4. Differential Processing, Trafficking and Localization of ApNT Splice Isoforms

A. Schematic representation of ApNT splice isoforms. B. Denaturing PAGE analysis of RT-PCR reactions of Aplysia CNS cDNA with primers flanking the 9 nucleotides miniexon. Plasmids containing (+) or lacking (-) the miniexon were used for control reactions with the same primer set. C. Processing and secretion of ApNT-GFP isoforms from transfected HEK293 cells. Western blot analysis of cell extracts and media collected 48h after transfection. See also Figure S5 D. Net charge distribution across ApNT functional domains (top) and net charge of the ApNT processing products. E. Differential processing expression and secretion pattern of ApNT isoforms in sensory neurons. Confocal imaging of ApNT(+)GFP (top panel), ApNT(-)GFP (middle panel) and ApNTS(-) (bottom panel) expressing sensory neurons. Top row - cell bodies, bottom row – distal neurites. Columns as labeled from left: DIC; GFP fluorescence (green); P47 staining (red); merged; zoomed detail of boxed area from merged. Scale bar: 5 microns

Figure 5. Endogenous ApNT-ApTrk Signaling Mediates LTF

A. ApTrkDN-GFP fusion protein is properly expressed, trafficked and targeted in sensory neurons co-cultured with motor neurons. Top: localization in soma and initial axon segment, arrows point to plasma membrane localization. Bottom: localization in distal SN processes in contact with the motor neuron initial segment (synaptic area). Scale bar: 10 microns. B. Presynaptic ApTrk signaling is not required for STF. Overexpression of ApTrkDN in sensory neurons did not inhibit the EPSP increase measured 5 minutes after single pulse of 5-HT (10 μM, 5 min). Data is presented as mean +/- SEM. C. ApTrk signaling is required for LTF induction. Overexpression of the ApTrkDN in sensory neurons significantly reduced the EPSP increase measured 24 hour after induction by five pulses of 5-HT (10 μM, 5 min). Data is presented as mean +/- SEM.

Figure 6. ApNT Signaling Induces Synaptic Enhancement and Growth in Sensory-motor co-cultures

A. Recombinant ApNT induces modest synaptic enhancement. EPSPs in sensory-motor co-cultures were recorded before or 24 hour after purified ApNT (50ng/ml) or control BSA protein was added to the media. Data is presented as mean +/- SEM. B. ApNT over-expression in sensory neurons induces significant synaptic enhancement. EPSPs were recorded before and 48h after injection of ApNT isoforms as labeled. Data is presented as mean +/- SEM. C. ApNT over-expression in sensory neurons of sensory-motor co-cultures induces growth of new synaptic varicosities. ApNT(+)GFP and RFP (top) or RFP alone (bottom) were injected in sensory neurons and confocal images collected 12 and 48 hours after injection. Arrows point to synaptic varicosities in apposition to the motor neuron receptive surface. D. Quantification of percent varicosity change in sensory cells injected as described in Figure 6C. at 48h. Data is presented as mean +/- SEM.

Figure 7. ApNT Over-expression Facilitates 5-HT Induced LTF and Counteracts FMRFamide induced LTI

A. ApNT expression facilitates LTF. 48h after injection of ApNTGFP initial EPSPs were recorded and cells were treated with one (STF) or five (LTF) 5-HT pulses (10 μM, 5 min) or mock treated and EPSPs recorded again after 5 min (STF) or 24h (LTF). Data is presented as mean +/- SEM. B. Same as A. but cultures were treated with five FMRFamide pulses (1 μM, 5 min) or mock treated and EPSPs recorded again after 24h. Data is presented as mean +/- SEM.