Synaptogenesis Is Modulated by Heparan Sulfate in Caenorhabditis elegans

Abstract

The nervous system regulates complex behaviors through a network of neurons interconnected by synapses. How specific synaptic connections are genetically determined is still unclear. Male mating is the most complex behavior in Caenorhabditis elegans It is composed of sequential steps that are governed by > 3000 chemical connections. Here, we show that heparan sulfates (HS) play a role in the formation and function of the male neural network. HS, sulfated in position 3 by the HS modification enzyme HST-3.1/HS 3-O-sulfotransferase and attached to the HS proteoglycan glypicans LON-2/glypican and GPN-1/glypican, functions cell-autonomously and nonautonomously for response to hermaphrodite contact during mating. Loss of 3-O sulfation resulted in the presynaptic accumulation of RAB-3, a molecule that localizes to synaptic vesicles, and disrupted the formation of synapses in a component of the mating circuits. We also show that the neural cell adhesion protein NRX-1/neurexin promotes and the neural cell adhesion protein NLG-1/neuroligin inhibits the formation of the same set of synapses in a parallel pathway. Thus, neural cell adhesion proteins and extracellular matrix components act together in the formation of synaptic connections.

Keywords: C. elegans; Proteoglycans; heparan sulfate; neurexin; neuroligin; synapse formation.

Figure 1

Heparan sulfate modification enzymes and heparan sulfate proteoglycans are required for the response to hermaphrodite contact during male mating behavior. (A) Schematic of the steps of male mating behavior. (B and D) Quantification of the response to hermaphrodite contact during male mating behavior in the genotypes indicated. Error bars denote the SEM; statistical significance is shown as follows: * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.00005. ns, not significant. The data for control are identical (B–D) and shown for comparison only. (C) Quantification of a hst-3.1-containing fosmid rescue of the response to hermaphrodite contact during male mating behavior in the hst-3.1(tm734) mutant. Error bars denote the SEM; statistical significance is shown as follows: * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.00005. ns, not significant. The data for control and hst-3.1 are identical to (B) and shown for comparison only.

Figure 2

Heterologous transgenic rescue experiments. (A–C) Tissue-specific rescue of response to hermaphrodite contact during male mating behavior in hst-3.1(tm734) mutants with hst-3.1 cDNA under heterologous promoters as indicated. Rescue was defined as restoration of response to hermaphrodite contact during male mating in transgenic animals (darker shade) and had to be statistically significant (P < 0.05) compared to nontransgenic siblings (lighter shade) (n ≥ 12). (D) Main postsynaptic partners of RnB neurons. The arrows and numbers represent the weight of the synaptic input from RnBs to the other neurons. (E–L) Cell-specific rescue of response to hermaphrodite contact during male mating behavior in hst-3.1(tm734) mutants with hst-3.1 cDNA under heterologous promoters as indicated. Rescue was defined as restoration of response to hermaphrodite contact during male mating in transgenic animals (darker shade) and had to be statistically significant (P < 0.05) compared to nontransgenic siblings (lighter shade) (n ≥ 12). GABA, γ-aminobutyric acid.

Figure 3

HST-3.1/HS 3-O-sulfotransferase is not required for axon guidance of B-type ray neurons. (A) Ventral views with schematics (i–vi) of adult male animals showing the B-type ray neurons. B-type ray neurons were visualized with bxIs14 (Is[Ppkd-2::GFP]). Red arrows represent missing commissures. Anterior is to the left. (B and C) Quantification of B-type ray neurons anteroposterior and dorsoventral axon guidance in the genotypes indicated. Error bars denote the SEM; statistical significance is shown as follows: * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.00005. ns, not significant.

Figure 4

HST-3.1/HS 3-O-sulfotransferase regulates presynaptic organization and synapse formation of B-type ray neurons. (A) Schematic of a ventral view showing the imaged region containing the synaptic ring in the preanal ganglion. (B) Confocal ventral views of the presynaptic densities as labeled with mCherry::RAB-3 of adult male animals in control and hst-3.1 mutant. The B-type ray neuron presynaptic distribution is not affected in hst-3.1/HS 3-O-sulfotransferase mutants. (C) Ventral views of the RnB axonal processes in the synaptic ring located in the preanal ganglion with cytosolic GFP and its corresponding presynaptic densities as labeled with mCherry::RAB-3 of adult hst-3.1(tm734) single mutants and lon-2gpn-1 double-mutant male worms. B-type ray neurons were visualized with bxIs30 that contains the cytosolic GFP (Is[Ppkd-2::GFP]) and the presynaptic marker (Is[Ppkd-2::mCherry::RAB-3]). Anterior is to the left. (D) Quantification of mCherry::RAB-3 fluorescence in the preanal ganglion synaptic ring in the genotypes indicated. Error bars denote the SEM; statistical significance is shown as follows: * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.00005. ns, not significant. The data presented is a ratio of mCherry::RAB-3 to GFP and control value. (E) Ventral views of trans-synaptic biotinylation labeling (iBLINC) of RnB → EFs synapses in puncta in hst-3.1(tm734) single mutants and lon-2gpn-1 double mutants. (F) The data presented is the normalized red fluorescence protein density in the synaptic area. Error bars denote the SEM; statistical significance is shown as follows: * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.00005. ns, not significant.

Figure 5

nrx-1/neurexin and nlg-1/neuroligin interacts genetically with hst-3.1/HS 3-O-sulfotransferase for response to hermaphrodite contact and synaptic function. (A) Quantification of response to hermaphrodite contact during male mating behavior in the genotypes indicated. Error bars denote the SEM; statistical significance is shown as follows: * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.00005. ns, not significant. The data for control and hst-3.1/ HS 3-O-sulfotransferase are identical to Figure 1 and shown for comparison only. (B) Quantification of mCherry::RAB-3 fluorescence in the preanal ganglion synaptic ring in the genotypes indicated. The data presented is a ratio of mCherry::RAB-3 to GFP and control (Norm) value. Error bars denote the SEM; statistical significance is shown as follows: * P < 0.05, ** P < 0.005, *** P < 0.0005, and **** P < 0.00005. ns, not significant. The data for control and hst-3.1/HS 3-O-sulfotransferase are identical to Figure 4 and shown for comparison only.

Figure 6

Proposed working model for the role of heparan sulfate (HS) 3-O sulfation in synapse formation. 3-O sulfation of HS chains located in LON-2 and GPN-1 mediates synapse formation between RnB neurons and EF interneurons, in parallel to NRX-1 and NLG-1, most likely by regulating the interaction between unidentified synaptic molecules. The absence of NRX-1 promotes synapse formation between RnB neurons and EF interneurons, while NLG-1 acts as a synaptic inhibitor. Disruption of this synaptic connection causes the accumulation of synaptic vesicles in the B-type ray neurons and behavioral defects in response to hermaphrodite contact during male mating. However, further biochemical experiments are needed to validate this model.