DataSheet1_Bioenergetic signatures of neurodevelopmental regression.docx

Background: Studies have linked autism spectrum disorder (ASD) to physiological abnormalities including mitochondrial dysfunction. Mitochondrial dysfunction may be linked to a subset of children with ASD who have neurodevelopmental regression (NDR). We have developed a cell model of ASD which demonstrates a unique mitochondrial profile with mitochondrial respiration higher than normal and sensitive to physiological stress. We have previously shown similar mitochondrial profiles in individuals with ASD and NDR.Methods: Twenty-six ASD individuals without a history of NDR (ASD-NoNDR) and 15 ASD individuals with a history of NDR (ASD-NDR) were recruited from 34 families. From these families, 30 mothers, 17 fathers and 5 typically developing (TD) siblings participated. Mitochondrial respiration was measured in peripheral blood mononuclear cells (PBMCs) with the Seahorse 96 XF Analyzer. PBMCs were exposed to various levels of physiological stress for 1 h prior to the assay using 2,3-dimethoxy-1,4-napthoquinone.Results: ASD-NDR children were found to have higher respiratory rates with mitochondria that were more sensitive to physiological stress as compared to ASD-NoNDR children, similar to our cellular model of NDR. Differences in mitochondrial respiration between ASD-NDR and TD siblings were similar to the differences between ASD-NDR and ASD-NoNDR children. Interesting, parents of children with ASD and NDR demonstrated patterns of mitochondrial respiration similar to their children such that parents of children with ASD and NDR demonstrated elevated respiratory rates with mitochondria that were more sensitive to physiological stress. In addition, sex differences were seen in ASD children and parents. Age effects in parents suggested that mitochondria of older parents were more sensitive to physiological stress.Conclusion: This study provides further evidence that children with ASD and NDR may have a unique type of mitochondrial physiology that may make them susceptible to physiological stressors. Identifying these children early in life before NDR occurs and providing treatment to protect mitochondrial physiology may protect children from experiencing NDR. The fact that parents also demonstrate mitochondrial respiration patterns similar to their children implies that this unique change in mitochondrial physiology may be a heritable factor (genetic or epigenetic), a result of shared environment, or both.</p

Mitochondrial dysfunction in the gastrointestinal mucosa of children with autism: A blinded case-control study

<div><p>Gastrointestinal (GI) symptoms are prevalent in autism spectrum disorder (ASD) but the pathophysiology is poorly understood. Imbalances in the enteric microbiome have been associated with ASD and can cause GI dysfunction potentially through disruption of mitochondrial function as microbiome metabolites modulate mitochondrial function and mitochondrial dysfunction is highly associated with GI symptoms. In this study, we compared mitochondrial function in rectal and cecum biopsies under the assumption that certain microbiome metabolites, such as butyrate and propionic acid, are more abundant in the cecum as compared to the rectum. Rectal and cecum mucosal biopsies were collected during elective diagnostic colonoscopy. Using a single-blind case-control design, complex I and IV and citrate synthase activities and complex I-V protein quantity from 10 children with ASD, 10 children with Crohn’s disease and 10 neurotypical children with nonspecific GI complaints were measured. The protein for all complexes, except complex II, in the cecum as compared to the rectum was significantly higher in ASD samples as compared to other groups. For both rectal and cecum biopsies, ASD samples demonstrated higher complex I activity, but not complex IV or citrate synthase activity, compared to other groups. Mitochondrial function in the gut mucosa from children with ASD was found to be significantly different than other groups who manifested similar GI symptomatology suggesting a unique pathophysiology for GI symptoms in children with ASD. Abnormalities localized to the cecum suggest a role for imbalances in the microbiome, potentially in the production of butyrate, in children with ASD.</p></div

Western Blots of Matched Groups from (A) Rectum and (B) Cecum.

<p>Notice that bands for several complexes, particularly complex I, III and IV are darker for the child with autism as compared to controls in the cecum but not the rectum.</p

Synthesis of findings of the study.

<p>Dysbiotic bacteria in the gastrointestinal tract of individuals with autism produce butyrate that drives the mitochondria to become overactive and very sensitive to oxidative stress. Xenobiotic agents (see previous review [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186377#pone.0186377.ref035" target="_blank">35</a>]) can increase oxidative stress through inflammation or by their intrinsic nature. This results in mitochondrial dysfunction that can contribute to gastrointestinal symptoms such as dysmotility (arrow symbol from mitochondrial dysfunction to colon).</p

Patient symptoms and gastrointestinal abnormalities.

<p>Patient symptoms and gastrointestinal abnormalities.</p

Mitochondrial pathways utilize short chain fatty acids as substrates.

<p><b>(A)</b> The electron transport chain has two distinct starting points, Complex I and Complex II, each of which have a predominant fuel source. Complexes III, IV and IV are common to both of these pathways. (B) Butyrate and Propionic Acid enter mitochondrial metabolism through two slightly overlapping pathways. Butyrate enters the citric acid cycle through its key substrate Acetyl-CoA, similar to glucose. The citric acid cycle predominantly produces NADH that is the substrate for Complex I. Propionic acid can be metabolized through two different pathways, both of which result in a relatively greater production of FADH₂ that is the substrate for Complex II. Propionic acid can produce fatty acids that are then the substrates for fatty acid oxidation. Propionic acid can be metabolized through several enzymes resulting in bypassing the first half of the citric acid cycle and using up Acetyl-CoA.</p

Electron transport chain normalized complex protein quantity.

<p>Cecum protein quantity is greater in the autism group as compared to the two control groups for (A) Complex I, (C) Complex III and (E) Complex V. In the cecum, relative to the rectum, protein content is greater in the autism group as compared to the control groups both separately and combined for (F) Complex I, (H) Complex III, (I) Complex IV and (J) Complex V. Error bars represent standard error. The protein quantity values do not have any units because they are normalized. ASD = Autism Spectrum Disorder.</p

The microbiome in the lower gastrointestinal (GI) tract of healthy (A) and individuals with autism (B).

<p>(For review of difference in the microbiome between children with autism and neurotypical children please see our recent reviews [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186377#pone.0186377.ref035" target="_blank">35</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186377#pone.0186377.ref036" target="_blank">36</a>]). For both neurotypical and autistic children, we obtained biopsy samples from the cecum and rectum. It is believed that the GI tract of individuals with autism have a greater amount of dysbiotic bacteria as compared to commensal bacterial. While the microbiome of the healthy GI tract provides positive immune and metabolic regulation, the imbalance in bacteria in the GI tract of individuals with autism results in oxidative stress, inflammation and mitochondrial dysfunction. Since the cecum is one of the most metabolically active regions of the GI tract for the microbiome, we are particularly interested in measuring mitochondrial function in the cecum. We compared mitochondrial function in the cecum to the rectum since the rectum has a much less metabolically activity microbiome. To understand if mitochondrial abnormalities are unique to children with ASD, we compared measurement of mitochondrial function in children with ASD to two groups of children without ASD, those with Crohn’s disease and those with non-specific GI complaints.</p

The AD LCLs cluster into two subgroups.

<p>The difference in baseline reserve capacity between control and AD pairs was plotted against the difference in the change in reserve capacity (from 0 to 10 µM DMNQ) between control and AD pairs. The AD-A subgroup (green diamonds) exhibited greater differences in baseline reserve capacity and change in reserve capacity as compared to the paired control LCLs, whereas the AD-N subgroup (orange circles) exhibited reserve capacity parameters more similar to the paired control LCLs.</p

Mitochondrial respiratory parameters and responses to DMNQ differ in two AD LCL subgroups.

<p>Overall, the AD-N subgroup (A–D) demonstrates similar mitochondrial responses as the control LCLs while the AD-A subgroup (E–H) parallels the differences between the AD and control LCLs found in the overall analysis. For the AD-N subgroup (<b>A</b>) ATP-linked respiration and (<b>D</b>) reserve capacity were overall slightly but significantly lower in the AD-N LCLs while (<b>B</b>) proton leak respiration was overall slightly but significantly higher in the AD-N LCLs, and (<b>C</b>) maximal respiratory capacity was not different in the AD-N LCLs as compared to controls. For the AD-A subgroup, (<b>E</b>) ATP-linked respiration, (<b>F</b>) proton leak respiration and (<b>G</b>) maximal respiratory capacity were overall markedly higher for AD-A LCLs as compared to control LCLs. (<b>H</b>) Reserve capacity was significantly greater for the AD-A LCLs as compared to control LCLs at baseline but decreased such that it was significantly lower than controls at 10–15 µM DMNQ. (<b>I</b>) ATP-linked respiration was overall markedly higher for AD-A LCLs as compared to AD-N LCLs. (<b>J</b>) Proton leak respiration was significantly higher in the AD-A LCLs as compared to the AD-N LCLs at 5–15 µM DMNQ. (<b>K</b>) Maximal respiratory capacity was significantly higher for AD-A LCLs as compared to AD-N LCLs at baseline and 5 µM DMNQ. (<b>L</b>) Reserve capacity was significantly greater for the AD-A LCLs at baseline but decreased so that it was significantly lower for the AD-A LCLs as compared to the AD-N LCLs at 12.5 and 15 µM DMNQ. *p<0.001; **p<0.0001; # p<0.01; p<0.05; ↕ indicates an overall statistical difference between LCL groups.</p