Transgenic Expression of Mammalian Neuroligin Rescues the Oxidative Stress Phenotype of C. Elegans Neuroligin-Deficient Mutants

Background: Neuroligins are postsynaptic adhesion proteins originally identified by their binding to presynaptic neurexins.  Recent studies suggest that neuroligins function primarily in the maturation and/or maintenance of synapses.  There are four neuroligin genes in humans, and mutations in the genes encoding neuroligin-3 and neuroligin-4 are associated with autism spectrum disorders (ASDs) in some families.  We had previously examined the expression, localization and biological functions of neuroligin in a simple model organism, the nematode Caenorhabditis elegans.  C. elegans has a single neuroligin gene (nlg-1), and we had shown that nlg-1 null mutants are viable and are not deficient in any major motor functions.  However, they are defective in a subset of sensory behaviors and sensory processing.  nlg-1 mutants are also hypersensitive to oxidative stress (i.e., exposure to paraquat); this was an unexpected phenotype for a synaptic mutant.  Like many other stress-sensitive mutants, nlg-1 mutants also have a reduced lifespan and an increased level of oxidative protein damage.

Objectives: To extend our previous data on the properties and function of C. elegans neuroligin, and determine the extent to which transgenic expression of mammalian neuroligin could "rescue" the mutant phenotypes associated with a C. elegans neuroligin knockout mutant. 

Methods: We obtained cDNAs for human neuroligin-4 (hNLGN4) and rat neuroligin-1 (rNLGN1) from Thomas Südhof (Stanford).  Using standard methods, we replaced the mammalian signal sequences and 3'-UTRs with their nematode counterparts, and expressed the modified cDNAs as stable transgenes (driven by the C. elegans nlg-1 promoter) in nlg-1 mutants.  We then compared the behaviors and toxin sensitivity of animals expressing the mammalian transgenes to wild-type animals, nlg-1 null mutants, and nlg-1 mutants expressing a nematode neuroligin transgene.

Results: (1) In the presence of a temperature gradient, wild-type C. elegans will accumulate at the temperature at which they were grown, but nlg-1 mutants are insensitive or indifferent to temperature, and distribute uniformly across the gradient.  However, transgenic expression of hNLGN4, rNLGN1, or C. elegans NLG-1 restores the wild-type behavior.  (2) Wild-type C. elegans are strongly repelled by dilute n-octanol, but nlg-1 mutants are indifferent to the compound.  However, nlg-1 mutants expressing hNLGN4, rNLGN1, or C. elegans NLG-1 transgenes are strongly repelled by dilute n-octanol.  (3) Wild-type animals and nlg-1 mutants are comparably attracted to diacetyl and repelled by cupric acetate.  However, when presented with these two compounds simultaneously (i.e., with a cupric acetate barrier between the animals and the attractant), nlg-1 mutants are far more likely than wild-type animals to cross the barrier.  We found that transgenic expression of hNLGN4, rNLGN1, or C. elegans NLG-1 restored the behavior to wild-type values.  (4) nlg-1 mutants are hypersensitive to paraquat toxicity, but transgenic expression of either hNLGN4 or C. elegans NLG-1 restored normal paraquat sensitivity. 

Conclusions: The nematode and mammalian neuroligins appear to be functionally equivalent (including the ability to prevent or counteract oxidative stress).  This raises the possibility that neuroligin may play a role in the prevention of oxidative stress in mammals.