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Autism-Associated Variants of Syntaxin Binding Protein 5 (STXBP5) Disrupt Dendritic Morphology Via the Regulation of Rho Signaling
Autism is a neurological condition characterized by marked qualitative differences in communication and social interaction. Genetic studies have implicated numerous genes, including those encoding proteins important for synaptic development and function that may contribute to the diverse autism phenotypes. Deletion and mutation of syntaxin binding protein 5 (STXBP5) have been identified in individuals with autism. STXBP5 encodes the protein tomosyn which contains an N-terminal WD40 domain and a C-terminal SNARE motif. Tomosyn was first identified as a syntaxin binding protein that negatively regulates neurotransmitter release. However, tomosyn has also been shown to regulate neurite outgrowth in immature neurons via interaction with ROCK, the downstream effector of RhoA. Here we examined the mechanism by which tomosyn controls the structural stability of mature neurons and also evaluated the hypothesis that the autism-associated mutation of STXBP5/tomosyn disrupts dendritic morphology by altering the Rho signaling pathway.
Objectives: We first examined the effect of the loss of tomosyn in primary cultured neurons and investigated alterations in the Rho signaling pathway as a mechanism underlying the affected cellular phenotype. We then determined the functional domain of STXBP5/tomosyn that is responsible for regulating Rho activity. We further evaluated whether two variants of STXBP5 identified from individuals with autism resulted in the disruption of the Rho signaling pathway and compromised the structural stability of neurons.
Methods:
We used shRNA to knock down tomosyn in mouse primary hippocampal neurons and examined dendritic morphology using morphometric analysis. Miniature excitatory post-synaptic currents (mEPSC) were measured to determine the synaptic function using whole-cell patch-clamp electrophysiology. Rho activity was measured by an intramolecular Forster Resonance Energy Transfer (FRET) assay with co-expression of the RhoA biosensor. Domain deletions and autism-associated variants of STXBP5/tomosyn were engineered and the effects on Rho activity and dendritic morphology were measured using the approaches mentioned above.
Results:
Tomosyn knockdown neurons exhibited compromised dendrite arborization and reduced dendritic spine density. These neurons also showed decreased mEPSC amplitude and frequency. Increased Rho activity was identified in tomosyn knockdown neurons measured by Rho biosensor FRET. Inhibiting Rho activity with dominant negative RhoA or C3 transferase was sufficient to restore complete dendritic morphology. Neurons expressing the C-terminus of tomosyn (tomosyn-DN) showed increased Rho activity and reduction in dendrite length similar to the knockdown, suggesting the N-terminus of tomosyn is responsible for regulating Rho activity. Two STXBP5 variants (L412V and Y502C) found in individuals with autism exhibit single mutations at the N-terminal WD40 domain of tomosyn. The shRNA-resistant wildtype tomosyn, but not autism-associated variants, rescued the dendritic phenotype in tomosyn knockdown neurons.
Conclusions:
We showed that tomosyn controls dendritic morphology via regulation of Rho signaling. Two STXBP5 variants found in individuals with autism contain single mutations at the N-terminus that may contribute to cellular pathophysiology of autism by disrupting Rho regulation and the structural stability of neurons.