Immunophenotype of Activated Astrocytes Associated with Brain-Region-Specific Neuronal Migration Abnormalities in Idiopathic Autism and Autism Caused By Chromosome 15q11.2-q13 Duplications

Background: The majority of postmortem brain studies in autism are focused on neuron pathology as a direct cause of functional abnormalities. However, recent studies reveal that neurogenesis, neuron migration, cortical lamination, and synaptogenesis are regulated by astrocytes (Barres et al. 2008). Neuropathological studies of subjects diagnosed with autism demonstrate almost all neuronal abnormalities (Wegiel et al. 2012, Casanova et al. 2002, Hutsler and Zhang, 2010) known to be regulated by astrocytes. Immunocytochemical studies demonstrating reactive astrocytosis in the cerebral cortex and white matter (Vargas et al, 2005) and western blots demonstrating increased GFAP levels in the cerebral cortex and cerebellum of autistic brains (Laurence and Fatemi, 2005) may be associated with neuronal alterations. Based on our preliminary studies, we hypothesized that in autism, abnormal neuronal migrations, including ectopic and heterotopic neurons, and focal microdysplasia, are associated with the enhanced focal proliferation of astrocytes with an immature immunophenotype (strong expression of FMRP and minimal GFAP expression).  

Objectives: 1. To demonstrate that the pattern of astrocytosis in autism is not random but brain region specific and enhanced in regions enriched in heterotopic neurons and microdysplastic areas. 2. To verify the hypothesis that the neuronal developmental abnormalities are associated with a similar pattern of astrocytosis regardless of autism etiology.  

Methods: One formalin-fixed brain hemisphere from each of 7 subjects with idiopathic autism, 7 with dup15/autism 5 to 39-years of age, and 7 age-matched control subjects was dehydrated, embedded in polyethylene glycol (PEG) and cut into serial hemispheric 50-µm-thick sections. Global mapping and characterization of astrocyte immunophenotype was performed by the application of antibodies detecting glial fibrillary acidic protein (GFAP; pAb G9269) and mouse mAb MMS-5231 (BioLegend, Dedham, MA) which recognizes the 340-355 aa region of human FMRP (LaFauci et al. 2013) and labels cytoplasmic and nuclear FMRP in neurons, including subcortical ectopic or heterotopic neurons, as well as astrocytes typical for the early postnatal period of life.  

Results: In contrast to the reported decrease/loss of astrocyte FMRP shortly after birth, hemispheric sections immunostained with mAb 6B8 revealed prominent FMRP immunoreactivity in astrocytes in several brain areas in subjects diagnosed with idiopathic autism and dup(15)/autism. The most common and prominent astrocytosis was found in the border between the cerebral cortex and white matter, and in the cortical molecular layer, both enriched in ectopic neurons. Clusters of FMRP+ astrocytes were detected in the deep layers of the cerebral cortex, in cerebellar white matter and cortex, as well as in the amygdala and thalamus. Strong FMRP immunolabeling was detected in astrocytes in the ventricular subependymal zone and adjacent white matter, as well as in the glia limitans both in control and autistic subjects. Double staining revealed that the majority of subcortical and cortical FMRP+ astrocytes expressed only traces of GFAP and resembled early postnatal phenotype.  

Conclusions: This postmortem study demonstrates that FMRP+ astrocytosis is associated with cerebral and cerebellar neuronal developmental abnormalities in autistic subjects of unknown and known etiology. FMRP immunostaining reveals new properties and function of astrocytes in the brain of subjects with autism.