Exploring the gut-brain axis in Attention-Deficit Hyperactivity Disorder

Sammanfattning: There is a bidirectional interaction between the gut and the brain, termed the gut-brain axis (GBA), involving e.g. the gut microbiota, and connecting the peripheral intestinal elements to the central nervous system (CNS). The GBA is increasingly recognized as a vital factor in the development and prognosis of neurological and psychiatric disorders. Of particular interest in this thesis is the signaling from gut microbiota to brain and back which is enabled through neural, endocrine, immune, and humoral pathways. Studies in germ-free and antibiotic-treated animal models have created a significant body of knowledge on the GBA and its role in regulating behavior. There are fewer studies using human subjects or with human materials. This thesis is focused on both clinical and biological aspects of GBA in attention-deficit hyperactivity disorder (ADHD). ADHD is a common childhood-onset psychiatric and neurodevelopmental disorder, which is characterized by having problems with paying attention and/or excessive activity and impulsivity, along with impairments on daily function and emotion regulation. ADHD is highly heritable and has high comorbidity with other psychiatric disorders and a higher-than-normal co-occurrence with some inflammatory disorders (e.g. asthma, eczema, rhinitis, celiac disease). However, ADHD is diagnosed primarily based on clinical observations of behaviors. Biomarkers for diagnosis and approaches for new therapy are lacking and the pathophysiology is poorly understood. This thesis consists of five studies attempting to explore some aspects of the GBA in neurodevelopmental and psychiatric disorders, with a focus on ADHD, utilizing a nationwide population-based cohort, a case-control design, a randomized controlled trial (RCT) and an in vitro model. It covers different aspects of the GBA, including gut microbiota and related antibiotic exposure, probiotic and prebiotic intervention, derived metabolites, inflammatory mediators, as well as the associations with clinical observations. Specifically, in Study I, we investigated whether exposure to antibiotic drugs, in utero and first two years after birth was associated with a risk for childhood-onset psychiatric disorders. Using Finnish nationwide registers, we found that antibiotic exposure was associated with a 10–50% increased risk for development of sleep disorders, ADHD, conduct disorders, mood disorders and anxiety disorders, which were accompanied by an increased risk for psychotropic drug use in childhood. The association with prenatal antibiotic exposure was neither explained completely by confounding factors related to family, nor by factors related to maternal infections. In Study II, we used a case-control design to study plasma levels of four inflammatory mediators: C-reactive protein (CRP), serum amyloid A (SAA), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) in ADHD and their associations with medication and clinical symptoms. We found that ADHD patients had higher levels of sICAM-1 and sVCAM-1 than healthy controls, especially in children currently on ADHD medication. Among adult participants with ADHD, sICAM-1 levels were positively associated with comorbid autism symptoms, and CRP levels were associated with GI symptoms and emotion dysregulation. In Study III, a double-blind RCT was conducted to determine whether a synbiotic (Synbiotic 2000), consisting of a mixture of three lactic acid bacteria and four fibers had effects on symptoms, daily functioning, and comorbid traits in ADHD patients. We found that Synbiotic 2000, compared to placebo, significantly reduced typical restricted, repetitive and stereotyped behaviors of autism symptoms in children, and alleviated problems with goal-directed behavior of emotion regulation in adults. The effects on autism symptoms and problems with emotion regulation were stronger in the subgroup with higher sVCAM-1 levels. In Study IV, using data from the same RCT as in study III, we investigated the effects of Synbiotic 2000 on plasma levels of immune activity markers and short-chain fatty acids (SCFAs) in ADHD. Adults with ADHD had at baseline higher levels of pro-inflammatory vascular adhesion molecules (sICAM-1, sVCAM-1) and lower levels of the anti-inflammatory interleukin (IL)-10 compared to controls. Synbiotic 2000, compared to placebo treatment, was associated with reduced the levels of IL-12/IL-23p40 in children on ADHD medication, and suggestively associated with reduced sICAM-1 levels and increased propionic acid levels in children. Moreover, in adult patients we found lower baseline levels compared to controls of formic acid and propionic acid. No obvious effects of Synbiotic 2000 were found on plasma levels of SCFAs. However, we found IL-10 levels correlating positively with formic acid and acetic acid at baseline for both children and adults with ADHD. In child patients, sVCAM-1 correlated negatively with acetic acid and propionic acid while sICAM-1 correlated negatively with acetic acid. In adult patients, a negative correlation was observed between sVCAM-1 and formic acid at baseline. In Study V, an in vitro model was used to explore the effects of three SCFAs: acetate, propionate and butyrate, on cell growth and cell death of early stage human neural progenitor cells (hNPCs). We found that acetate, propionate and butyrate at low μM levels, relevant to physiological levels, significantly increased the proliferation of hNPCs and induced more cells to undergo mitosis, while the SCFAs at high mM levels had toxic effects on hNPCs. In support of this, the SCFA exposure to hNPCs changed the expression of genes involved in neurogenesis, proliferation and apoptosis. These studies provide support of a role of components influenced by the GBA, including gut microbiota, immune activity markers and bacterial metabolites in clinical and biological aspects of ADHD. Finally, these studies contribute to a better understanding of the GBA in ADHD.