López-Murcia FJ et al. (2015), SPARC triggers a cell-autonomous program of syn...

XB-ART-51274

Proc Natl Acad Sci U S A 2015 Oct 27;11243:13366-71. doi: 10.1073/pnas.1512202112.

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SPARC triggers a cell-autonomous program of synapse elimination.

López-Murcia FJ , Terni B , Llobet A .

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Elimination of the excess synaptic contacts established in the early stages of neuronal development is required to refine the function of neuronal circuits. Here we investigate whether secreted protein acidic and rich in cysteine (SPARC), a molecule produced by glial cells, is involved in synapse removal. SPARC production peaks when innervation of the rat superior cervical ganglion and the tail of Xenopus tropicalis tadpoles are remodeled. The formation of new cholinergic synapses in autaptic single-cell microcultures is inhibited by SPARC. The effect resides in the C-terminal domain, which is also responsible for triggering a concentration- and time-dependent disassembly of stable cholinergic synapses. The loss of synaptic contacts is associated with the formation of retracted axon terminals containing multivesicular bodies and secondary lysosomes. The biological relevance of in vitro results was supported by injecting the tail of Xenopus tropicalis tadpoles with peptide 4.2, a 20-aa sequence derived from SPARC that mimics full-length protein effects. Swimming was severely impaired at ∼5 h after peptide application, caused by the massive elimination of neuromuscular junctions and pruning of axonal branches. Effects revert by 6 d after injection, as motor innervation reforms. In conclusion, SPARC triggers a cell-autonomous program of synapse elimination in cholinergic neurons that likely occurs when protein production peaks during normal development.

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Species referenced: Xenopus tropicalis
Genes referenced: sparc syp tff3.7 tuba4b vamp2
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	Fig. 1. Variations in SPARC production during refinement of cholinergic innervation in two different vertebrate structures. (A) Section of a rat superior cervical ganglion at postnatal day 20 showing SPARC accumulates around putative presynaptic terminals labeled with VAMP2 (arrows). (B) Plot profile of VAMP2 and SPARC fluorescence intensities measured from the dotted line shown in A). (C) Relative SPARC expression in the rat superior cervical ganglion at the indicated postnatal days. (D) Relative changes in SPARC levels in the tails of X. tropicalis during normal development. The relationship of Nieuwkoop– Faber stages to days postfertilization is indicated. Images show the characteristic morphology of tadpoles at the stages of embryogenesis (Emb), premetamorphosis (PreM), prometamorphosis (ProM), metamorphosis climax, and postmetamorphosis (PostM). Data in C and D are mean ± SEM.
	Fig. 2. Identification and mapping of the functional SPARC region inhibiting synaptic development. (A) Schematic diagram of the experimental procedure used to investigate the inhibitory role of SPARC throughout the whole period of synapse formation and maturation. The time course of in vitro synaptic development is indicated. (B) Effect of SPARC on evoked autaptic currents and the density of presynaptic terminals, visualized as bassoon puncta. (C) Amino acid sequence and location of p2.1 and p4.2 in the full-length protein. (D) Average EPSC of neurons developed in the presence of 5 nM SPARC (n = 24), 5 nM SPARC + 50 nM p2.1 (n = 8), and 5 nM SPARC + 50 nM p4.2 (n = 29). (E) Relative decay in EPSC amplitude as a function of SPARC or p4.2. Sigmoidal fits of averaged data (from 6–24 independent observations) revealed [SPARC]50 and [p4.2]50 of 13 nM and 14 nM, respectively. The absence of inhibition by 50 nM p2.1 (n = 6) is also indicated. (F) Summary of SPARC-derived peptides on the density of bassoon puncta (control, n = 14; 5 nM SPARC, n = 10; 25 nM SPARC, n = 6; 50 nM p2.1, n = 5; 10 nM p4.2, n = 12; 25 nM p4.2, n = 7; 50 nM p4.2, n = 17; 100 nM p4.2, n = 6). All averages are presented as mean ± SEM. P < 0.01; *P < 0.001.
	Fig. 3. SPARC p4.2 triggers the disassembly of mature cholinergic synapses. (A) Experimental protocol used to investigate the role of SPARC-derived peptides on the activity of stable autapses. (B) Average EPSCs of mature neurons acutely exposed to p2.1 (n = 8) or p4.2 (n = 33). (C) p4.2 decreased the number of functional presynaptic terminals measured as bassoon puncta. (D) The amplitude of postsynaptic responses decreased as a function of p4.2. Exposure to p2.1 did not modify EPSC amplitude. All peptides were incubated for 4–6 h before recording. Note that the effect of p4.2 was inhibited by jasplakinolide, but remained unchanged when firing of action potentials was blocked by TTX (200 nM p2.1, n = 13; 50 nM p4.2, n = 18; 100 nM p4.2, n = 6; 200 nM p4.2, n = 16, 1 μM jasplakinolide, n = 11; 1 μM jasplakinolide + 200 nM p4.2, n = 12; 20 nM TTX, n = 6; 20 nM TTX + 200 nM p4.2, n = 8). (E) Image of a control presynaptic terminal from a recorded autaptic neuron (Inset). (F) Image from a presynaptic terminal acutely treated with p4.2. Arrows indicate membrane invaginations near an active zone. (G–J) Images from four different synapses treated with p4.2. Note the presence of presynaptic and postsynaptic endocytic profiles (arrows). (J) A presynaptic terminal containing a multivesicular body (asterisk). All averages are presented as mean ± SEM. P < 0.01; *P < 0.001.
	Fig. 4. SPARC p4.2 activates the formation of secondary lysosomes in mature presynaptic terminals. (A) Lysosomes of neurons treated for 6 h with 200 nM p4.2 (n = 49) are larger than those found after acute exposure to 200 nM p2.1 (n = 40). (B) A secondary lysosome located in a presynaptic terminal treated with p4.2 (arrow). (C) A secondary lysosome formed by fusion to a multivesicular body. (D) A lysosome found in a retracting axon terminal. Averages are presented as mean ± SEM. **P < 0.001.
	Fig. 5. SPARC p4.2 induces retraction of motor neuron axons and impairs tadpole swimming. (A) Images from tadpole tails showing how the characteristic ∼3-Hz tail beat is blocked by local injection with p4.2 but is unaffected by p2.1. (B) Swimming activity integrated over 30 s. Motor behavior is transiently affected by p4.2, shown by tracking movements of four different tadpoles (squares) at three consecutive time points: 5 h, 3 d, and 6 d. Swimming is unaffected by p2.1 (Movies S1–S4). (C) Distribution and mean average speed (mean ± SEM; P < 0.01) of tadpoles at 5 h after injection with p2.1 (n = 80) or p4.2 (n = 80). (D) Same as C but at 6 d after injection with p2.1 (n = 76) or p4.2 (n = 78; P = 0.16). (E) Staining of X. tropicalis tadpole tails for acetylated tubulin and synaptophysin to indicate the location of axons and presynaptic terminals, respectively. Note how neuromuscular junctions virtually disappear at 5 h after p4.2 injection but become evident within 6 d.
	Fig. S5. Estimated p4.2 clearance from X. tropicalis tadpole tail obtained by injecting peptide conjugated to FITC. (A) Diffusion to distal tail regions was observed within 30 min of injection. (B) p4.2-FITC fluorescence collected from a 4 × 3-mm region around the injection site, covering ∼35% of the whole tail. Images were obtained using an ImageEM camera (Hamamatsu). Fluorescence decreased following double-exponential kinetics, indicating that p4.2 concentration was reduced by 80% at the injection site after ∼5 h, when impairment of motility is maximal. This time window supports an acute effect of the peptide in vivo and shows that [p4.2] is negligible at 6 d after injection, when recovery of swimming is observed.
	Fig. S6. p4.2 disrupts axonal arborizations of motor neurons. (A) Acetylated tubulin staining of a X. tropicalis tadpole tail injected with p2.1. An estimate of the width of axon branches was obtained by measuring interceptions with four lines covering the whole image (yellow). The image is a 620 × 620 μm Z-stack maximum projection, acquired with a 25×/0.95W objective (Leica). (B) Example of the profile measured from the line indicated by arrows in A. In this particular example, 52 putative axon branches were identified, and the corresponding FWHM was calculated using individual Gaussian fits. Axon thickness was above resolution, given the FWHM of 100-nm latex beads of 460 nm. (C) Apparent thickness of axonal branches crossing line profiles in the four experimental groups. Three different animals were used in each condition. Bars indicate mean ± SEM. **P < 0.001.

References [+] :

Aguayo, Neonatal neuronal loss in rat superior cervical ganglia: retrograde effects on developing preganglionic axons and Schwann cells. 1976, Pubmed