Autism is a severe neuro-developmental disorder of childhood characterized by impairments in social interaction, deficits in verbal and non-verbal communication, and restricted repetitive and stereotyped patterns of behavior and interests. Susceptibility to autism is clearly attributable to genetic factors, but the etiology of this disorder is unknown, and no biomarkers have yet been proven to be characteristic of autism. Recently, a number of studies have shown that inflammatory cytokines are elevated in the serum, cerebrospinal fluid and brain of autistic subjects. Several studies also implicate the apoptosis-related protein Bcl-2 and p53 as being involved in autism. In addition, it has been suggested that the apoptosis induced by cytokines TNF-α and IFN-γ may be mediated by cathepsin D, which is the predominant lysosomal aspartic acid protease abundantly expressed in the brain.
Objectives: The aim of this study is to determine whether inflammatory cytokines and apoptosis related protein Bcl2 are abnormally regualted in the lymphoblast of autistic subjects and whether cathepsin D is involved in the modulation of the inflammatory and apoptotic processes in autism.
Methods: Lymphoblasts of 6 autistic subjects (mean age 8.4 ±0.27 yrs) and 6 age-matched normal controls (mean age 7.0 ±0.91 yrs) were included in this study. Subjects fit the diagnostic criteria for autism of the Diagnostic and Statistical Manual-IV, as confirmed by the Autism Diagnostic Interview-Revised. Participants were excluded from the study if they had a diagnosis of fragile X syndrome, epileptic seizures, obsessive–compulsive disorder, affective disorders, or any additional psychiatric or neurological diagnoses. This study was approved by the Institutional Review Board of the NY State Institute of Basic Research. In this study, Invitrogen's Multiplex Bead Immunoassays was used to determine the levels of the different cytokines in lymphoblast. Western Blot Analyses were used to detect the protein expression levels of Bcl2 and cathepsin D. Northern Blot analysis and EM Immuno-gold labeling were used to detect the RNA and protein levels of cathepsin D in lymphoblast.
Results:
We found that cytokines TNF-α and IL-6 were significantly elevated in the lymphoblasts of autistic subjects as compared to controls, while anti-apoptotic Bcl2 protein expression was significantly reduced. In addition, our study shows that cathepsin D mRNA and protein expression levels were significantly increased in lymphoblasts of autistic subjects as compared to controls.
Conclusions:
These findings strongly suggest that there is increased inflammation and apoptosis present in autistic lymphoblasts. The elevated activation of cathepsin D in autistic lymphoblasts indicates that cathepsin D may be involved in the regulation of cytokine induced apoptosis in autism.