Proteomic Characterization of the Olfactory Molecular Imbalance in Dementia with Lewy Bodies

Abstract

Olfactory dysfunction is one of the prodromal symptoms in dementia with Lewy bodies (DLB). However, the molecular pathogenesis associated with decreased smell function remains largely undeciphered. We generated quantitative proteome maps to detect molecular alterations in olfactory bulbs (OB) derived from DLB subjects compared to neurologically intact controls. A total of 3214 olfactory proteins were quantified, and 99 proteins showed significant alterations in DLB cases. Protein interaction networks disrupted in DLB indicated an imbalance in translation and the synaptic vesicle cycle. These alterations were accompanied by alterations in AKT/MAPK/SEK1/p38 MAPK signaling pathways that showed a distinct expression profile across the OB-olfactory tract (OT) axis. Taken together, our data partially reflect the missing links in the biochemical understanding of olfactory dysfunction in DLB.

Keywords: Lewy bodies; dementia; olfaction; olfactory bulb; proteomics.

Figure 1

(A) An overview of the workflow used for the molecular characterization of the olfactory bulbs (OB) derived from dementia with Lewy bodies (DLB) subjects. (B) Quantified proteome distribution across OB cell layers. (C) Cluster-enriched genes in specific OB cell layers that are differentially expressed at the level of the OB in DLB subjects. (D) OB protein expression changes of ALDH1B1 and PI16 in DLB subjects by Western blotting. Data are presented as mean ± SEM. * p < 0.05 vs. control group; *** p < 0.001 vs. control group (a.u: arbitrary units; DEPs: differential expressed proteins; EPL: external plexiform layer; OSN: olfactory sensory neuron).

Figure 2

Impairment of OB protein translation and synaptic function in DLB. (A) Interactome network for OB deregulated proteome using STRING (Search Tool for the Retrieval of Interacting Genes). Proteins are represented with nodes and the physical/functional interactions with continuous lines. Interactions tagged as “high confidence” (>0.7) in the STRING database were exclusively considered. K means clustering was used. (B) Western-blotting of P70 S6K protein levels across controls and DLB subjects. Data are presented as mean ± SEM. * p < 0.05 vs. control group. In this case, equal loading of the gel was assessed using stain-free imaging technology, and protein normalization was performed by measuring total protein directly on the gels (a.u; arbitrary units). (C) Significantly enriched synapsis-related pathways in the OB differential proteome using the STRING tool.

Figure 3

Disruption of OB signaling routes in DLB. (A) Visual representation of protein interactome maps for OB differentially expressed proteins in DLB. Upregulated proteins in red and downregulated proteins in green. Continuous lines represent direct interactions, while discontinuous lines correspond to indirect functional interactions. See complete legend and high resolution images in Figures S3–S5. (B) Activation state and protein measurements of AKT and Bcl2-associated agonist of cell death (BAD) across controls and DLB subjects by Western-blotting. (C) OB ERK1/2, SEK1 and p38 MAPK levels at the level of OB. Data are presented as mean ± SEM. * p < 0.05 vs. control group; *** p < 0.001 vs. control group. Equal loading of the gels was assessed using stain-free imaging technology, and protein normalization was performed by measuring total protein directly on the gels (a.u; arbitrary units).

Figure 4

Disruption of olfactory tract (OT) signaling routes in DLB. Steady-state levels of AKT, MEK, PKAc, SEK1 and p38 MAPK in the OTs derived from controls and DLB subjects. Data are presented as mean ± SEM. * p < 0.05 vs. control group; *** p < 0.001 vs. control group. Equal loading of the gels was assessed using stain-free imaging technology, and protein normalization was performed by measuring total protein directly on the gels (a.u; arbitrary units).