Expression Profiling of TLR Signaling Pathway Genes in Brain Tissue from Patients with Autism

Background: The pathogenesis of autism has been associated with genetic and environmental factors that influence brain development and neuronal function. As part of the genetic and environmental interactions, immune factors and neuroimmune responses appear to play pathophysiological roles in autism. Innate immune mechanisms are involved in regulating synaptic/dendritic organization as well as neuronal dysfunction. Neuroimmune responses such as microglial/astroglial activation are part of the spectrum of neurobiological abnormalities that occur in individuals with autism (Vargas et al 2005). Toll-like receptors (TLRs) and their signaling pathways are associated with initiation and regulation of the innate immune response to diverse pathogen-associated molecular challenges. At least 10 TLRs have been demonstrated in humans and some of them are expressed in neurons, astrocytes, and microglia in the central nervous system (CNS).

Objectives: To assess the role of TLR signaling pathways the brain of patients with autism and to examine the patterns of expression within the CNS. 

Methods: Profiles of TLR signaling pathway gene expression was assessed by genomic techniques using PCR gene arrays. Brain tissues from 4 regions, midfrontal (MFG), midtemporal (MTG), anterior cingulate (ACG) and Occipital (OG) gyri from 7 patients with autism (age range 5-42 years, mean 14.6 years) and 10 control patients (age range 5-20 years, mean 12.8 years) were studied. Expression of selected TLRs was validated by immunoblot analysis. Cellular localization of TLRs in the CNS was assessed by immunocytochemistry and laser confocal microscopy.

Results:  Although no significant statistical differences were observed in expression of TLR genes with the exception of TLR-2, the study revealed significant differences in at least 13 genes involved in TLR signaling pathways. The MFG and ACG regions revealed most of the changes in expression of TLR signaling genes. Most of the increases in gene expression occurred in the ACG, where genes such as TLR-2, IL-2, IRAK1, NFKB1 and TNFRSF1A, were significantly up-regulated (p<0.05).  Interestingly, gene expression changes in the MFG showed down-regulation of different set of genes including SARM1 (p=0.01) and UBE2N (p=0.01). Immunocytochemical studies demonstrated that TLR-2 was localized in astrocytes and microglia cells, a pattern that was almost identical to TLR-3. The expression of TLR-4 was observed mostly in microglial cells.

Conclusions:   The major focus of TLR signaling gene expression changes occurred in ACG, an area of important interest for social/behavioral changes in autism. The gene expression changes in ACG reflect the status of neuroimmune activation in selected regions of the cerebral cortex and concurs with previous observations of increased innate neuroimmune and neuroglia responses in autism.  Two of the up-regulated genes, IL-2 and TLR-2, play roles in modulation of innate immunity and inflammation. The immunolocalization of TLR-2 in microglia and astrocytes support the view that TLR pathways are associated with neuroglia activation in patients with autism. Interestingly, two of the down-regulated genes in the MFG (SARM1 and UBE2N) are also associated with regulation of the number of neurons. This  finding may suggests their involvement in neuropathological abnormalities such as the excess of neurons observed in some brain regions in autism.