A pathophysiological mechanism and treatment strategy for autism
Duffney, Lara Jean
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Autism spectrum disorders (ASDs) are characterized by deficits in sociability and communication, as well as restricted and repetitive behaviors, which can be associated to deficits in glutamatergic transmission in the prefrontal cortex (PFC). Various molecular and genetic studies have implicated SHANK3, a synaptic scaffolding protein in the postsynaptic density, as a causal factor for autism spectrum disorders. Although the role of the Shank3 protein in promoting proper spine morphology and synapse formation is well-known, the molecular mechanisms underlying this regulation of synaptic proteins, as well as transmission and plasticity, are not well understood. This research aimed to reveal the function of Shank3 in regulating proper glutamatergic function in the PFC. Specifically, this work examined (1) the mechanism by which Shank3 regulates the proper functioning of NMDA receptors and (2) in vivo behavioral deficits in a Shank3 mouse model and how these deficits can be ameliorated using molecular intervention to restore NMDA function in the PFC. (1) In order to elucidate the role of Shank3 in proper glutamatergic functioning in the PFC, a small interfering RNA (siRNA) was utilized to knock down Shank3 expression. This caused a significant reduction of NMDAR-mediated ionic or synaptic current, as well as the surface expression of NR1 subunits, in rat cortical cultures. The effect of Shank3 siRNA on NMDAR currents was blocked by an actin stabilizer, and was occluded by an actin destabilizer, suggesting the involvement of actin cytoskeleton. Since actin dynamics is regulated by the GTPase Rac1 and downstream effector p21-activated kinase (PAK), Shank3 regulation of NMDARs when Rac1 or PAK was manipulated was further examined. It was found that the reducing effect of Shank3 siRNA on NMDAR currents was mimicked and occluded by specific inhibitors for Rac1 or PAK, and was blocked by constitutively active Rac1 or PAK. Immunocytochemical data showed a strong reduction of F-actin clusters after Shank3 knockdown, which was occluded by a PAK inhibitor. Inhibiting cofilin, the primary downstream target of PAK and a major actin depolymerizing factor, prevented Shank3 siRNA from reducing NMDAR currents and F-actin clusters. Together, these results suggest that Shank3 deficiency induces NMDAR hypofunction by interfering with the Rac1/PAK/cofilin/actin signaling, leading to the loss of NMDAR surface expression. It provides a potential mechanism for the role of Shank3 in cognitive deficit in autism. (2) This study examined the role of Shank3 in vivo by examining deficient male mice, found to exhibit autistic-like behaviors. Additionally, they showed significantly diminished NMDAR-mediated synaptic responses and NMDAR surface and synaptic expression in prefrontal cortex. Shank3-deficient mice had a strong loss of cortical actin filaments, which was associated with the reduced activity of Rac1 and PAK, and increased activity of the major actin depolymerizing factor cofilin. The social behavioral deficits and NMDAR hypofunction were rescued by inhibiting cofilin or activating Rac1 in Shank3-deficient mice, and were induced by inhibiting PAK or Rac1 in wild-type mice. It suggests that the aberrant actin regulators, actin dynamics and ensuing dysregulation of NMDA receptors contribute to the manifestation of autistic-like phenotypes, providing a mechanism-oriented drug discovery strategy for autism treatment.