Aberrant NF-kappaB expression in autism spectrum condition: a mechanism for neuroinflammation

Abstract

Autism spectrum condition (ASC) is recognized as having an inflammatory component. Post-mortem brain samples from patients with ASC display neuroglial activation and inflammatory markers in cerebrospinal fluid, although little is known about the underlying molecular mechanisms. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a protein found in almost all cell types and mediates regulation of immune response by inducing the expression of inflammatory cytokines and chemokines, establishing a feedback mechanism that can produce chronic or excessive inflammation. This article describes immunodetection and immunofluorescence measurements of NF-κB in human post-mortem samples of orbitofrontal cortex tissue donated to two independent centers: London Brain Bank, Kings College London, UK (ASC: n = 3, controls: n = 4) and Autism Tissue Program, Harvard Brain Bank, USA (ASC: n = 6, controls: n = 5). The hypothesis was that concentrations of NF-κB would be elevated, especially in activated microglia in ASC, and pH would be concomitantly reduced (i.e., acidification). Neurons, astrocytes, and microglia all demonstrated increased extranuclear and nuclear translocated NF-κB p65 expression in brain tissue from ASC donors relative to samples from matched controls. These between-groups differences were increased in astrocytes and microglia relative to neurons, but particularly pronounced for highly mature microglia. Measurement of pH in homogenized samples demonstrated a 0.98-unit difference in means and a strong (F = 98.3; p = 0.00018) linear relationship to the expression of nuclear translocated NF-κB in mature microglia. Acridine orange staining localized pH reductions to lysosomal compartments. In summary, NF-κB is aberrantly expressed in orbitofrontal cortex in patients with ASC, as part of a putative molecular cascade leading to inflammation, especially of resident immune cells in brain regions associated with the behavioral and clinical symptoms of ASC.

Keywords: NF-κB; autism spectrum condition; brain; inflammation; orbitofrontal cortex; pH.

Figure 1

NF-κB in post-mortem tissue from the orbitofrontal cortex from ASC and control donors. (A) Image of Western blot probed with the anti-NF-κB p65 and anti-β-actin antibodies (loading control). Molecular mass markers are shown in kDa. (B) Relative expression of NF-κB p65 subunit, normalized to the lowest value recorded (Study ID C01). (C) Image of fractioned samples probed with anti-NF-κB p65. Sample IDs suffixed “/N” are the nuclear component, and suffixed “/C” the cytosolic component. Sample IDs prefixed “A” are from ASC donors, prefixed “C” from control donors.

Figure 2

NF-κB p65 expression in neurons. (A) A representative image of a neuron stained with anti-β-III Tubulin (green), anti-p65 (red), and DAPI (blue). Scale bar = 10 μm. (B) Percentage of neurons with anti-p65 nuclear staining for each sample. Sample IDs prefixed “A” are from ASC donors, prefixed “C” from control donors.

Figure 3

NF-κB p65 expression in astrocytes and microglia. (A) Percentage of GFAP stained astrocytes with anti-p65 nuclear staining. (B) Percentage of CD11b stained microglia with anti-p65 nuclear staining. (C) Percentage of CD11c stained (highly activated) microglia with anti-p65 nuclear staining. (D) A representative microglial cell with nuclear NF-κB p65 staining with anti-CD11c (green), anti-p65 (red), and DAPI (blue). Scale bar = 20 μm. (E) The number of CD11c positive microglia found in 20 fields of random sampling. Blue bars represent samples from the UK cohort, red bars samples from the US cohort. Sample IDs prefixed “A” are from ASC donors, prefixed “C” from control donors.

Figure 4

Measurement of pH. (A) Graph of pH of homogenized tissue samples. (B) Plot of normalized overall expression of NF-κB p65 from quantification of Western blot vs. pH of homogenized samples with a superimposed line of linear regression. Filled circles are samples from ASC donors, open circles samples from control donors. (C) Immunofluorescent image from a tissue sample from control donor (×63 objective, scale bar = 20 μm) and (D) from a sample from ASC donor (×100 objective, scale bar = 20 μm). Images are pseudocolored and show the ratio of acridine orange/Lysotracker Red fluorescence. Green hues represent low lysomic pH, and red hues represent high lysomic pH.