Proteomic Analysis of Rhesus Macaque Brain Explants Treated With Borrelia burgdorferi Identifies Host GAP-43 as a Potential Factor Associated With Lyme Neuroborreliosis

Abstract

Background: Lyme neuroborreliosis (LNB) is one of the most dangerous manifestations of Lyme disease, but the pathogenesis and inflammatory mechanisms are not fully understood.

Methods: Cultured explants from the frontal cortex of rhesus monkey brain (n=3) were treated with live Borrelia burgdorferi (Bb) or phosphate-buffered saline (PBS) for 6, 12, and 24 h. Total protein was collected for sequencing and bioinformatics analysis. In addition, changes in protein expression in the explants over time following Bb treatment were screened.

Results: We identified 1237 differentially expressed proteins (DEPs; fold change ≥1.5 or ≤0.67, P-value ≤0.05). One of these, growth-associated protein 43 (GAP-43), was highly expressed at all time points in the explants. The results of the protein-protein interaction network analysis of DEPs suggested that GAP-43 plays a role in the neuroinflammation associated with LNB. In HMC3 cells incubated with live Bb or PBS for 6, 12, and 24 h, real-time PCR and western blot analyses confirmed the increase of GAP-43 mRNA and protein, respectively.

Conclusions: Elevated GAP-43 expression is a potential marker for LNB that may be useful for diagnosis or treatment.

Keywords: Borrelia burgdorferi; GAP-43; HMC3; lyme neuroborreliosis; neuroinflammation; proteomic analysis.

Figure 1

Schematic illustration of the experimental design contains the experiment with explants from the frontal cortex of rhesus macaque brains co-cultured with live Bb and validation experiment using the human microglia HMC3 line.

Figure 2

Screening of DEPs in this study. (A) The number of increased and decreased DEPs in different groups. (B) Heatmap of DEPs identified by protein sequencing showing the hierarchical clustering of the relative expression of a portion of the DEPs in each group for brevity. High and low abundance of protein expression is shown in red and blue, respectively. (C) Analysis of our target protein, GAP-43, by PPI network to find its related proteins for the following study. Our target protein has been marked with red pentagrams in the figure.

Figure 3

Significant alteration of GAP-43 mRNA and protein levels in explants from the frontal cortex of rhesus macaque brains at different time points after Bb treatment. (A) Protein expression of GAP-43 in explants from the frontal cortex of rhesus macaque brains co-cultured with live Bb and the controls at 6, 12, and 24 h, validated by western blotting. (B) Quantitative analysis of GAP-43 protein expression levels. (C) qPCR analysis of GAP-43 mRNA expression comparing explants from the frontal cortex of rhesus macaque brains co-cultured with live Bb with the controls at 6, 12, and 24 h. **P < 0.01, *P < 0.05. Analyzed with two-way ANOVA and data were expressed as the means ± SD.

Figure 4

Significant alteration of GAP-43 mRNA and protein levels after Bb treatment with HMC3. (A) Protein expression of GAP-43 in HMC3 cell lines, co-cultured with live Bb and controls at 6, 12 and 24 h, was verified by western blotting. (B) Quantitative analysis of GAP-43 protein expression levels. (C) qPCR analysis of GAP-43 expression in HMC3 cell lines, comparing co-cultured with live Bb and controls at 6, 12 and 24 h. **P < 0.01. Our data were analyzed with two-way ANOVA and expressed as the means ± SD.