Differential responses of primary neuron-secreted MCP-1 and IL-9 to type 2 diabetes and Alzheimer's disease-associated metabolites

Abstract

Type 2 diabetes (T2D) is implicated as a risk factor for Alzheimer's disease (AD), the most common form of dementia. In this work, we investigated neuroinflammatory responses of primary neurons to potentially circulating, blood-brain barrier (BBB) permeable metabolites associated with AD, T2D, or both. We identified nine metabolites associated with protective or detrimental properties of AD and T2D in literature (lauric acid, asparagine, fructose, arachidonic acid, aminoadipic acid, sorbitol, retinol, tryptophan, niacinamide) and stimulated primary mouse neuron cultures with each metabolite before quantifying cytokine secretion via Luminex. We employed unsupervised clustering, inferential statistics, and partial least squares discriminant analysis to identify relationships between cytokine concentration and disease-associations of metabolites. We identified MCP-1, a cytokine associated with monocyte recruitment, as differentially abundant between neurons stimulated by metabolites associated with protective and detrimental properties of AD and T2D. We also identified IL-9, a cytokine that promotes mast cell growth, to be differentially associated with T2D. Indeed, cytokines, such as MCP-1 and IL-9, released from neurons in response to BBB-permeable metabolites associated with T2D may contribute to AD development by downstream effects of neuroinflammation.

Figure 1

Unsupervised hierarchical clustering of cytokines released by primary neurons (a) Hierarchical clustering of the log₂ fold change of cytokine concentrations across the nine tested metabolites. Each metabolite is associated with a category of disease, with respective cells representing a quantified replicate. Significance was determined from a Kruskal–Wallis test corrected by the Benjamini–Hochberg method (FDR q value < 0.10). The direction of the arrows indicates disease (↑) and protective (↓) characteristics related to AD and T2D. (b) Principal component analysis of the log₂ fold changes categorized by individual metabolites and disease-associations. (c) The log₂ ratio of cytokine concentration to vehicles of MCP-1, IL-9, and MIP-1β. Mann–Whitney pair-wise testing was applied to each metabolite group based on disease association (p value denoted within the plot, with significance defined as p value < 0.05). The FDR q value from the corrected Kruskal–Wallis is displayed next to each respective cytokine.

Figure 2

Separation of different disease-associated metabolites is detected from the PLS-DA model. (a,b) Four-way disease associations; (c,d) AD-protective and AD associated classifications, and (e,f) T2D-protective and T2D associated classifications. Loading variables for each model (LV1 and LV2) with a VIP > 1 is labeled with a star, and the color of the loading bar represents the cytokine with the highest contribution to the specific class (metabolite grouping).

Figure 3

Hypothesized Pathway of the Shared Neuroinflammatory Response. (a) In a healthy state, few metabolites may cross the BBB through specialized transport. The stimulated neurons produce cytokines that may activate glia such as microglia, which will accumulate in the brain and promote clearance of amyloid-beta and other debris in the central nervous system. (b) In a diseased state, metabolites may cross the BBB in higher concentrations, and stimulate the neurons and glia cells. This chronic, low-grade stimulation of neuronal cells may result in the eventual breakdown of the BBB, leading to downstream migration of immune cells and more metabolites to enter the brain. The release of neuroinflammatory cytokines may generate a feed-forward loop of neuroinflammation, potentially leading to the development of eventual AD. (Created with BioRender.com).

Figure 4

Method in plating and culturing primary neurons derived from embryonic CD1 mice. The embryonic litters from pregnant CD-1 mice were decapitated, and the cortical regions were isolated for primary neuron culturing. On day 8, the metabolites and respective vehicles were used to stimulate the primary neurons. After 3 days, the neuron media samples were collected for the quantification of released cytokines using the Luminex platform. (Created with BioRender.com).