Accumulation of Amyloid-Beta Peptide Species In Four Brain Structures In Children with Autism

Background: Secreted beta-amyloid precursor protein (APP) has been detected in elevated levels in the plasma of about 60% of autistic children. Particularly high plasma levels of APP were detected in children with severe autism and aggression. It has been hypothesized that increased APP processing in the alpha-secretase pathway takes place in autism, and that plasma levels of sAPP-α can be an early biomarker of at least a subgroup of children with autism. Our preliminary study of autopsy brains revealed amyloid-beta peptide (Aβ) in intracellular deposits in neurons of various brain structures. This study is focused on frontal and temporal lobes, and on cerebellum in which developmental alterations of cytoarchitectonics and size have been described in autism.

Objectives: To detect and characterize amyloid-β accumulation in brain cortex in the subjects with autism and in cerebellum in children with autism.

Methods: Formalin-fixed and frozen autopsy brain samples of individuals with autism (n=9, aged from 9 to 56 years) in which diagnosis was confirmed by ADI-R score, and controls (n=12, aged from 13 to 59 years) were used in the study. Aβ was detected by immunocytochemical staining in sections of formalin-fixed brains using mAbs 4G8 and 6E10 by the immunoperoxidase method. Aβ was detected by immunoblotting in samples of frozen frontal and temporal cortex, cerebellar cortex and dentate nucleus. Aβ: monomeric and bound in complexes was detected with antibodies specific for the Aβ species with C-terminal aa 40 and 42.

Results: Neurons in the frontal and temporal cortex, Purkinje cells and neurons in the dentate nucleus were immunopositive in all autistic cases. Similar immunoreactivity was observed in control brains, however, in majority of autistic subjects the reaction was stronger and present in more cells, including astrocytes.  Intracellular Aβ was aminoterminally truncated, i.e. reactive with mAb 4G8 but not mAb 6E10. Biochemical characterization of frontal and temporal cortex samples revealed the Aβ40 and Aβ42 species as monomers, dimers, and several distinct complexes/oligomers of the molecular sizes between 24 and 52 kD. Brain samples from autism contained significantly more of Aβ42 in the 30-34kD and 50-52 kD protein bands. Biochemical characterization of cerebellum revealed a pattern of Aβ40- and Aβ42-immunoreactive protein bands different than in cortex, indicating formation of distinct Aβ complexes. The levels of Aβ42 bound to the proteins of the molecular sizes between 30 and 34 kD were significantly higher in dentate nucleus in autism. Sequential centrifugation revealed that only a subset of Aβ42 complexes, of the molecular sizes between 17 and 34 kD, was accumulated within the large cellular deposits.

Conclusions: Brain neurons in cortex and cerebellar cortex and dentate nucleus in autism in the post-developmental period of life accumulate intracellular N-terminally truncated Aβ42. This may result from excessive processing of APP in the non-amyloidogenic pathway or defective degradation of N-terminally truncated Aβ42.