Alterations of Mitochondrial Biology in the Oral Mucosa of Chilean Children with Autism Spectrum Disorder (ASD)

Abstract

Autistic Spectrum Disorder (ASD) is characterized by the impairment of socio-communicative skills and the presence of restricted and stereotyped behavior patterns. Recent researches have revealed the influence of mitochondrial physiology on the development of ASD. Several research groups have identified defects in respiratory complexes, coenzyme-Q10 deficiency, increased oxidative damage, decreased of superoxide dismutase (SOD2). A study on the influence of mitochondrial physiology on the development of ASD can provide new alternatives and challenges. That is why we set ourselves the general objective to initiate studies of mitochondrial physiology in Chilean children with ASD. A sample of oral mucosa was collected in a group of 12 children diagnosed with ASD and 12 children without ASD. In children with ASD, we found a significant increase in mitochondrial DNA levels. Likewise, in these children, an increase in the protein oxidation was observed. Finally, a downward trend in the expression of the HIGD2A and SOD2 genes was observed, while DRP1, FIS1, MFN1, MFN2, and OPA1 gene expression show an upward trend. The increment of mitochondrial DNA, high oxidative stress, and high expression of the MFN2 gene could help as a scanner of the mitochondrial function in children with ASD.

Keywords: ASD; autism; gene expression; mitochondrial DNA; oral mucosa; oxidative stress.

Figure 1

The relative levels of mitochondrial DNA (mtDNA) in children with Autistic Spectrum Disorder (ASD) and Healthy Controls (HC). (a) Total DNA was isolated from oral mucose cells, total DNA was used for the analysis of mitochondrial DNA. (b) For mitochondrial DNA quantification, the mitochondrial gene tRNA- Leu (UUU) was amplified by qPCR, and the nuclear gene B2-microglobulim amplification was used for the normalization. Each bar graph represents the mean ± SEM, n = 12 ASD and 12 HC children, analyzed by unpaired t-test (P < 0.05) and producing a P value = 0.0006 (***).

Figure 2

Oral mucosa total protein oxidation levels in Chilean children with ASD. (a) Total proteins were isolated from oral mucosa cells by TRIzol™ reagent (Invitrogen) and fractionated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Protein carbonylation was detected with the Oxyblot Protein Detection Kit. αDNP: Anti-2,4-dinitrophenylhydrazone antibody. (b) SDS-PAGE Coomassie blue staining of SDS-PAGE of derivatized proteins. (c) Densitometry quantification of protein carbonylation was made with the ImageJ software. Carbonylation of proteins was normalized by Coomassie blue staining. Each bar represents the mean ± SEM, analyzed by unpaired t-test. Healthy controls (HC), n = 11 and ASD children, n = 11.

Figure 3

Protein expression analysis of mitochondrial respiratory complexes. (a) Oxidative Phosphorylation (OXPHOS) protein expression total proteins were obtained from oral mucosa and fractionated by SDS-PAGE. Complex I (CI), complex II (CII), complex III (CIII), complex IV (CIV) and ATP synthase (CV) were detected by immunoblots. (b) SDS-PAGE Coomassie blue staining was used as a loading control. n = 6 ASD and 5 HC children.

Figure 4

The presence of the Ala16val-superoxide dismutase (Ala16val-SOD2) polymorphism in Chilean children with ASD and in HCs. (a) Representative genotypes analyzed in an agarose gel electrophoresis. (b) Scheme of genotypes. (c) The observed genotypes were analyzed with a Chi-squared (X²) test with an alpha <0.05 statistical significance, obtaining a P value = 0.2231 n/s (non-significant). (d) The distribution of Ala16val-SOD2 polymorphism in the ASD severity level was analyzed with a Chi-squared (X²) test with an alpha <0.05 statistical significance, obtaining a P value = 0.1897 n/s (non-significant).

Figure 5

Relative expression of genes in children with Autism Spectrum Disorder (ASD) and Healthy Controls (HC). (a) SOD2, (b) HIGD2A, (c) DRP1, (d) FIS1, (e) MFN1, (f) MFN2, (g) OPA1. The PPIA gene was taken as a reference during the qPCR procedure. Each bar graph represents the mean ± SEM, n = 10 ASD and 11 HC children, analyzed by unpaired t-test (** P < 0.05).

Figure 6

Prediction of gene association with the genetic bases of ASD. (a) Network of SOD2 genetic associations, ranked #12001 among all genes in the human genome based on the prediction of association with ASD. (b) Network of HIGD2A genetic associations, rank #21384. (c) Network of DRP1 (DNM1L) genetic associations, rank #2558. (d) Network of FIS1 genetic associations, rank #2822. (e) Network of MFN1 genetic associations, rank #22827. (f) Network of MFN2 genetic associations, rank #11607. (g) Network of OPA1 genetic associations, rank #3271 [50].