In silico Gene Set and Pathway Enrichment Analyses Highlight Involvement of Ion Transport in Cholinergic Pathways in Autism: Rationale for Nutritional Intervention

Abstract

Food is the primary human source of choline, an essential precursor to the neurotransmitter acetylcholine, which has a central role in signaling pathways that govern sensorimotor functions. Most Americans do not consume their recommended amount of dietary choline, and populations with neurodevelopmental conditions like autism spectrum disorder (ASD) may be particularly vulnerable to consequences of choline deficiency. This study aimed to identify a relationship between ASD and cholinergic signaling through gene set enrichment analysis and interrogation of existing database evidence to produce a systems biology model. In gene set enrichment analysis, two gene ontologies were identified as overlapping for autism-related and for cholinergic pathways-related functions, both involving ion transport regulation. Subsequent modeling of ion transport intensive cholinergic signaling pathways highlighted the importance of two genes with autism-associated variants: GABBR1, which codes for the gamma aminobutyric acid receptor (GABA_{B 1}), and KCNN2, which codes for calcium-activated, potassium ion transporting SK2 channels responsible for membrane repolarization after cholinergic binding/signal transmission events. Cholinergic signal transmission pathways related to these proteins were examined in the Pathway Studio environment. The ion transport ontological associations indicated feasibility of a dietary choline support as a low-risk therapeutic intervention capable of modulating cholinergic sensory signaling in autism. Further research at the intersection of dietary status and sensory function in autism is warranted.

Keywords: acetylcholine; autism; cholinergic; dietary choline; gene set enrichment analysis; sensory processing.

FIGURE 1

Gene ontology overlap between autism and cholinergic metabolism gene sets. Overlap between an autism-associated genome-wide associated studies gene set (Wu et al., 2020) and cholinergic pathways gene set (MSigDB v7.1) was identified using Venny 2.1. The two shared ontologies were (1) regulation of ion transport and (2) positive regulation of ion transport, identifying ion transport functionality as a critical area of enrichment for further analysis.

FIGURE 2

Sensory processing is associated with autism functionally, in the context of ion transport and cholinergic signaling. Acetylcholine regulates both the ion transport and the GABA signaling. A deficiency in its precursor, dietary choline, may impact ion transport, GABA⇒GABBR1 signaling, and KCNN2/SK2 channel function, either or all of which may result in altered sensory processing function.

FIGURE 3

Overview of acetylcholinergic signaling. Acetylcholine (ACh), sourced from precursors choline and acetyl-CoA, leaves the presynaptic neuron’s axon terminal through vesicle-mediated exocytosis. Acetylcholine molecules diffuse through the synapse, until binding with cholinergic receptors (CR) in the membrane of the postsynaptic signal-recipient neuron’s dendrite. This binding event permits influx of calcium ions into the dendrite. Ionic membrane depolarization activates an action potential, causing an electrical signal to be propagated down the length of the axon in a series of ion transport events, until the signal reaches the axon terminal and is translated again into neurotransmitter signals destined to reach the next neuron’s dendrite.

FIGURE 4

Clearance of acetylcholine from the synapse by acetylcholinesterase. Acetylcholine (ACh) is delivered to the synapse as a result of vesicle exocytosis from pre-synaptic neuronal signaling and a release of the recycled acetylcholine from the postsynaptic neuron’s cholinergic receptors. In the synaptic cleft, acetylcholinesterase (ACHE) then degrades acetylcholine. Resulting choline is then taken back by the presynaptic neuron to be recycled in order to make more acetylcholine for future signaling. This figure was extracted from Pathway Studio’s curated pathways and was edited according to the needs of this study.

FIGURE 5

GABBR1 may influence acetylcholinesterase regulation. GABBR1 is an autism-associated gene that codes for a membrane-bound protein (GABA_B1) (Wu et al., 2020). The binding of the neurotransmitter GABA to GABA_B1 has several implications for cholinergic signaling, sensory signal transduction, and ion transport, across multiple cascades. As depicted here, a typical GABA/GABBR1 binding cascade inhibits adenylyl cyclases (ADCY), thereby preventing positive upregulation of acetylcholinesterase (ACHE). This figure was extracted from Pathway Studio’s curated pathways and was edited according to the needs of this study.

FIGURE 6

GABBR1/ACHE regulation. (A) In the presence of GABBR1 variant. The autism-associated GABBR1 variant may affect levels of acetylcholinesterase (ACHE) in the synaptic cleft by decreasing either total levels of GABBR1 expression and/or function of its gene product, the GABA_B1 receptor. Under this condition, the cascade that normally downregulates acetylcholinesterase expression may be suppressed. Because of that, larger amounts of the enzyme are produced, and the acetylcholine degrades at an elevated rate, resulting in less acetylcholine in the synaptic cleft and less signaling through the cholinergic receptor. (B) In the presence of GABBR1 variant and a dietary choline deficiency. When choline levels are deficient, less acetylcholine (ACh) is available in the neurons for signaling, and a decrease in cholinergic binding/signaling attributed to GABBR1 variant would be exacerbated.

FIGURE 7

KCNN2/SK2 impact on cholinergic signal modulation. (A) SK2 channel operation. SK2 channels operate in tandem with a propagation of cholinergic signal as they counter the membrane depolarization brought about by calcium influx through cholinergic receptors. The calcium influx activates the calmodulin domains on SK2 channels, permitting passage of potassium back into the synapse to restore membrane hyperpolarization. Thus, SK2 channels serve a critical role in helping a neuron to recover and re-hyperpolarize before receiving the next cholinergic signal. (B) In the presence of KCNN2/SK2 variant. When SK2 channel binding activity and/or functionality is altered, the sensory signaling modulation could either be too efficient—that is, the membrane repolarizes too soon due to overflow of K + ions back into the synapse, or not efficient enough, with membrane taking too long to repolarize, leaving the neuron less ready to process any subsequent signals transmitted from the presynaptic neuron. In either of these two conditions, the neuron’s capacity to modulate sensory signaling through membrane repolarization may be diminished. (C) In the presence of KCNN2/SK2 variant and a dietary choline deficiency. When choline levels are deficient, less acetylcholine (ACh) is available in the neurons for signaling, and a post-signal repolarization may be less amenable to modulation by SK2 channels. When these channels are working in a suboptimal regimen due to the presence of KCNN2 variant, modulation leeway may be curtailed even further.