Levels of Alzheimer Beta-Amyloid Precursor Protein (APP) in Children With Severely Autistic Behavior and Aggression

Deborah K. Sokol PhD, MD; Demao Chen PhD; Martin R.Farlow MD; David W. Dunn MD; Bryan Maloney BA; Jennifer A. Zimmer MD; Debomoy K. Lahiri PhD


J Child Neurol. 2006;21(6):444-449. 

In This Article

Experimental Design and Methods

The study group consisted of 10 volunteer children with autism (9 boys, ages 4 to 17 years), as determined by Diagnostic and Statistical Manual of Mental Disorders-IV criteria, who already were found to show an elevation in choline as determined by hydrogen ion magnetic resonance spectroscopy.[15] These children were neurology clinic outpatients. In addition to autism, two of these patients were diagnosed with fragile X syndrome; three other children had seizures, and one child had the sickle cell trait ( Table 1 ). Eleven age- and gender-matched volunteers (10 boys, ages 4 to 18 years), seen in our neurology outpatient clinic for conditions other than autism (see Table 1 ), served as a medical control group. This study was approved by the Human Subjects Committee-Institutional Review Board at the Indiana University School of Medicine. All parents signed informed consent for their child's participation.

Each child's caretaker was administered the Childhood Autism Rating Scale (CARS). This parent questionnaire is a 15-item behavioral rating scale developed to identify children with mild to moderate autism (raw score 30-36.5) and severe autism (scores above 37). Validity and reliability for the Childhood Autism Rating Scale have been established.[16]

Serum acetylcholinesterase was measured via the Mayo Clinic laboratory acetylcholinesterase activity assay. The results of red blood cell acetylcholinesterase were expressed as U/g hemoglobin.

Levels of secreted beta-amyloid precursor protein a were determined by a sensitive sandwich enzyme-linked immunosorbent assay using 6E10 monoclonal antibody and normalized to the concentration of the total protein in the sample. Levels of secreted beta-amyloid precursor protein a were expressed as nanogram/milligram of plasma protein. The relative density of total secreted beta-amyloid precursor protein bands was measured by Western blotting using 22 C11 monoclonal antibody as described previously.[17]

A sensitive enzyme-linked immunosorbent assay for measuring the plasma levels of amyloid beta 40 was performed as briefly described below from our previous report.[18] A solid-phase sandwich enzyme-linked immunosorbent assay was used with two kinds of highly specific antibodies (capture and detection IgG) to detect amyloid-beta 40 species. First, affinity-purified antiamyloid-beta (against residue 35-40) rabbit IgG was used as a capture antibody, and, second, affinity-purified horseradish peroxidase conjugated anti-amyloid beta (against residue 11-28) rabbit IgG Fab was used as a detection antibody. Given that an antibody against amyloid-beta (11-28) was used as a secondary conjugated antibody, amyloid-beta 40 variants that cleaved at N-terminus sites were also detectable in our assay. In this assay, 3,3',5,5'-tetramethylbenzidine was used as a coloring agent (chromogen). The strength of coloring was in proportion to the quantities of amyloid beta 40.

To measure the longer amyloid-beta species in human plasma samples, the affinity-purified anti-amyloid beta (against residue 38-42) rabbit IgG was used as a capture antibody. The rest of the procedure was the same as for the shorter species, and the values were expressed as picogram/milligram of protein as described previously.[17]

Waller-Duncan multiple-range tests were used to compare mean values for acetylcholinesterase and each of the neuronal proteins by autistic symptoms (mild versus severe) and age (under age 7 years and over 10 years). Pearson's correlation was performed to determine the association between the severity of autism, age, the presence of a seizure disorder, acetylcholinesterase, and the plasma protein markers. Differences between groups were assessed by one-way analysis of variance. The acceptance level of significance was P < .05 using a two-tailed distribution.


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