Up-Regulation of Ras/Raf/ERK1/2 Signaling In the Frontal Cortex of Autistic Subjects and BTBR Mice

Background:  Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors.  BTBR mice are a model for understanding mechanisms that may be responsible for the pathogenesis of autism, since they exhibit many autism-like behaviors. A number of studies have shown that the Ras/Raf/ERK1/2 signaling pathway plays important roles in the genesis of neural progenitors, learning, and memory. Ras/Raf/ERK1/2 and ERK5 have also been shown to have death-promoting apoptotic roles in neural cells. Recent studies have demonstrated a possible association between neural cell death and autism.  In addition, two recent studies reported that a deletion of a locus on chromosome 16, which included the MAPK3 gene that encodes ERK1, is associated with autism. We thus hypothesized that Ras/Raf/ERK1/2 signaling and ERK5 could be abnormally regulated in the brain of autistic subjects and the abnormality could be modeled in BTBR mice.

Objectives:  The aim of this study is to determine how Ras/Raf/ERK1/2 signaling pathway is regulated in the brain of both autistic subjects and BTBR mice.  

Methods:  

Frozen human brain tissues of six autistic subjects (mean age 8.3 ± 3.8 years) and six age-matched normal subjects (mean age 8 ± 3.7 years) were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders. Donors with autism fit the diagnostic criteria of the Diagnostic and Statistical Manual-IV, as confirmed by the Autism Diagnostic Interview-Revised.  Six female BTBR T+tfJ (BTBR) and six B6 were obtained from Jackson Laboratories (Bar Harbor, ME).  All procedures were conducted in compliance with the NIH Guidelines for the Care and Use of Laboratory Animals.  In this study, Western Blot Analyses were used to detect the protein expression and activity levels of Ras, A-Raf, B-Raf, C-Raf, MEK1/2 and ERK1/2. Immunohistochemistry studies were carried out to determine the protein expression and phosphorylation of MEK1/2. Enzyme-linked Immunosorbent Assay (Elisa) was carried out to determine the activity level of ERK1/2.

Results:  

We show that expression of Ras protein was significantly elevated in the frontal cortex of autistic subjects and BTBR mice. C-Raf phosphorylation, as well as both B-Raf and C-Raf phosphorylation were increased correspondently in autistic subjects and BTBR mice.  ERK1/2 and ERK5 protein expression were significantly up-regulated in autistic frontal cortex, and the kinase activities of MEK1/2 and ERK1/2 were found to be increased in the frontal cortex of BTBR mice. These results suggest that Ras/Raf/ERK1/2 signaling activities are up-regulated in the frontal cortex of autistic subjects and BTBR mice as compared with their controls.  Furthermore, we also examined the Ras/Raf/ERK1/2 pathway in the cerebellum of autistic subjects and BTBR mice. We did not detect significant changes in the protein expression and activities of MEK1/2 and ERK1/2 in autistic cerebellum, as well as in the cerebellum of BTBR mice, which indicates that this pathways is not abnormally regulated in the cerebellum of autistic subjects and BTBR mice.

Conclusions:  

Our findings suggest that up-regulated Ras/Raf/ERK1/2 signaling and ERK5 activities in the frontal cortex could be one of the cellular mechanisms responsible for the autism phenotype.