| abstract |
Autism Spectrum Disorder (ASD) is disorder associated with difficulties in social skills,
communication, sensory information, and repetitive behaviors. A small subset of patients have
mutations in the pre and post synaptic transmembrane synaptic adhesion molecules, neurexin
(NRX) and neuroligin (NLG). In humans, there are five forms of NLGN proteins and three forms
of NRXN. Genetically liked autism may be associated with mutations in NLGN3 or NLGN4.
Caenorhabditis elegans (C. elegans) are widely used as model organisms, due to their
relatively short life cycle of approximately three days. They have a total of 302 neurons and have
homologues to NLGN and NRXN. The nine different isoforms of worm neuroligin do not
currently have predicted crystal structures. Utilizing the online resources SWISS Model, the
Zhanglab I-TASSER program and PyMol, the structures were predicted. Exonal differences were
also determined; isoform E contains all sixteen exons. Seven of nine isoforms are missing exon
14. Two other isoforms begin at the second start found within exon 8. The protein structure of
the nlg-1 knockout strain VC228, was also predicted. It was hypothesized that it is unable to
form dimers; nor is it capable of binding to nrx-1 following computational modeling.
To better understand the role of neuroligin in C. elegans, a complete knockout of the
gene by CRISPR/Cas9 was designed. A rescue null knockout plasmid was constructed utilizing
PCR and Gibson assemblies. Sanger sequencing determined multiple mutations within coding
regions of the final construct (pSW13): thus, a restriction digest method was utilized to complete
the construction. Components were isolated from pSW13 and pDD282 (commercial plasmid
used as part of pSW13 construction) a combinations of different restriction enzymes. Site-
directed mutagenesis was used to correct one PCR mutation. Additional rounds of restriction
fragment replacement were used to correct remaining mutations. Completion of the rescue
plasmid is ongoing. Once transgenic C. elegans are created, the knockout mutant can be used to
explore functions of individual isoforms, mutations, and even human variations of neuroligin.
As such computational modeling of the C. elegans nlg-1 protein showed that the wild
type isoforms should all be capable of forming dimers, however the knockout strain, VC228
cannot, thereby making its less drastic phenotype even more confusing. Thus, creating a
complete knockout will allow for a greater understanding of the protein.
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