Syntaxin Binding Protein 5 Regulates Neurite Differentiation in an Autism-Related Model

Thursday, May 12, 2016: 11:30 AM-1:30 PM
Hall A (Baltimore Convention Center)
W. Shen, J. A. Frei and Y. C. Lin, Laboratory of Neuronal Connectivity, Hussman Institute for Autism, Baltimore, MD

Autism is a neurological condition that features marked qualitative differences in communication and social interaction. It has been estimated that as many as 1/3 of individuals with autism spectrum condition also have epilepsy. Consistent with the extremely heterogeneous presentation of autism, genetic studies have implicated numerous genes that may contribute to the autism phenotype. Deletion and mutations of syntaxin binding protein 5 (STXBP5, also known as tomosyn) are identified in association with autism and epilepsy. STXBP5 contains three distinct domains: C-terminal region containing an R-SNARE, synaptobrevin-like coiled-coil domain, and an N-terminal region enriched with WD40 repeats.  STXBP5 has a presynaptic role that negatively regulates neurotransmitter release by forming syntaxin-SNAP25-tomosyn complex. STXBP5 is also detected in the postsynaptic domain in cerebellum. However, the postsynaptic role of STXBP5 has not been well elucidated. Interestingly, WD40 as scaffolding domains, interact with diverse proteins, peptides or nucleic acids using multiple surfaces. It has been found that STXBP5 is homologous to the L(2)gl, which is instrumental in establishing cell polarity. Therefore, we hypothesize that STXBP5 may be involved in regulating cell growth and differentiation. 


To knock down STXBP5 and generate mutations of STXBP5 in cell lines and primary mouse neurons to reveal the postsynaptic function of STXBP5 and determine how mutations of STXBP5 found in autism may contribute to the phenotype. 


Several shRNAs were created to knock down murine STXBP5. Knockdown efficiency is determined by Western blot analysis and the most efficient one is selected to knock down STXBP5 in Neuro-2A cells. We use retinoic acid (RA) to induce Neuro-2A differentiation and determine whether STXBP5 plays a role in this process. Neurite outgrowth is measured by live cell imaging with IncuCyte. We also use a mutagenesis method to generate several STXBP5 mutations that are found in individuals with autism. Ongoing experiments are using primary neuron cultures as a model system to investigate the function of STXBP5 for synapse and dendrite stability.


RA-induced Neuro-2A cells exhibit a characteristic bipolar-like shape, suggesting retinoic acid stimulates neurite outgrowth. shRNA of STXBP5 shows 57% knockdown efficiency in Neuro-2A cells. Suppression of STXBP5 by shRNA remarkably inhibits neurite outgrowth induced by retinoic acid. 


STXBP5 plays a role in regulating neurite outgrowth. Mutations of STXBP5 found in individuals with autism may play a key role in altering neuronal differentiation including neurite outgrowth potentially disrupting normal developmental processes.