Autism spectrum condition (ASC) is a life-long neurodevelopmental condition characterized by a triad of impairments in social skills, verbal communication, and behavior (Rapin, 1997
; Lord et al., 2000
). Cognitively, ASC is described as a disorder involving fundamental deficits in central coherence (Frith, 1989
), executive function (Ornitz et al., 1993
), theory of mind (Baron-Cohen et al., 1985
), and empathizing (Baron-Cohen, 2002
). Continuing investigations for a neurobiological basis for ASC support the view that genetic, environmental, neurological, and immunological factors contribute to its etiology (Neuhaus et al., 2010
). In particular, there is evidence to suggest an association between ASC and neuroinflammation in anterior regions of the neocortex (Pardo et al., 2005
; Vargas et al., 2005
; Zimmerman et al., 2005
), and areas relating to cognitive function appear to be affected by inflammation resulting from activation of microglia and astrocytes (Anderson et al., 2008
). In vivo
measurements of structural brain changes with magnetic resonance imaging have detected gray matter loss in the orbitofrontal cortex (Hardan et al., 2006
; Girgis et al., 2007
) and impairment of cognitive functions mediated by the orbitofrontal–amygdala circuit (Loveland et al., 2008
) in patients with ASC. Furthermore, markers of oxidative stress are elevated in the orbitofrontal cortex in post-mortem samples of ASC patients (Sajdel-Sulkowska et al., 2011
). This region is thus a likely candidate for an underlying cellular mechanism.
Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a protein that controls transcription of DNA and is found in almost all cell types (Perkins, 2004
). It mediates cellular response to external stressors and is central to the regulation of immune responses by inducing the expression of inflammatory cytokines and chemokines and, in turn, being induced by them (Pahl, 1999
; Perkins, 2004
). This establishes a positive feedback mechanism (Larsson et al., 2005
) which, when NF-κB becomes aberrantly active, has the potential to produce chronic or excessive inflammation associated with several inflammatory diseases (Pahl, 1999
). Furthermore, post-mortem studies suggest that NF-κB plays a key role in Alzheimer's disease (Akiyama et al., 1994
) and its possible treatment (Paris et al., 2007
), Parkinson's disease (Block and Hong, 2005
) and multiple sclerosis (Glass et al., 2010
Microglia mediate the immune responses of the central nervous system acting to remove extracellular debris with a similar function to macrophages. Microglial cells have been associated with brain inflammation (Liu and Hong, 2003
; Barger, 2005
; Kim and Joh, 2006
) and pro-inflammatory treatments of microglia acidify cellular lysosomes to a pH ~5 (Majumdar et al., 2007
). Preliminary characterization of this mechanism implicates activation of protein kinase A (PKA) and the activity of chloride channels (Majumdar et al., 2007
). The transcriptional activity of NF-κB is stimulated upon phosphorylation of its p65 subunit on serine 276 by PKA (Zhong et al., 1998
) and in turn PKA is a downstream target of the transcription factor (Kaltschmidt et al., 2006
). With this in mind we postulated that an association may exist between the transcription factor and lysosomal acidity.
This article describes measurement of NF-κB p65 expression levels and pH in post-mortem samples of orbitofrontal cortex from patients with a diagnosis of ASC and control samples from people healthy at the time of death. We hypothesized that concentrations of NF-κB would be elevated in patients and pH would be concomitantly reduced (i.e., acidification), providing evidence for a neuroinflammatory component to ASC.
This hypothesis was initially tested by Western immunodetection of post-mortem brain tissue to measure overall, nuclear and cytosolic NF-κB expression.
Investigations were then focused upon microglial cells due to their role in pro-inflammatory response, as these most strongly mediate aberrant expression of NF-κB. Antigen retrieval and immunofluorescence techniques were used to identify the differential concentrations of intracellular NF-κB in neurons, astrocytes, microglia, and highly activated (i.e., mature or functional) microglia. Immunoreactivity measurements were initially carried out to determine the concentration of NF-κB in the cytoplasm of each cell type as an indication of the availability of inactive NF-κB, and thus its potential for nuclear translocation. The expression of active NF-κB translocated to the cell nuclei was then measured directly, where it binds to DNA and transcribes proteins that result in the production of cytokines as part of an inflammatory
Confirmation of the immunodetection and immunofluorescence results in neurons and mature microglia was sought from an independent source of micro-array tissue slides donated from ASC patient and control groups.
Finally, pH was measured in homogenized tissue and compared to the corresponding intracellular NF-κB p65 expression from Western immunodetection. Acridine orange staining allowed measurements of pH localized to lysosomes.