Sex-divergent effects on the NAD+-dependent deacetylase sirtuin signaling across the olfactory-entorhinal-amygdaloid axis in Alzheimer's and Parkinson's diseases

Abstract

Background: Smell impairment is one of the earliest features in Alzheimer's (AD) and Parkinson's diseases (PD). Due to sex differences exist in terms of smell and olfactory structures as well as in the prevalence and manifestation of both neurological syndromes, we have applied olfactory proteomics to favor the discovery of novel sex-biased physio-pathological mechanisms and potential therapeutic targets associated with olfactory dysfunction.

Methods: SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) and bioinformatic workflows were applied in 57 post-mortem olfactory tracts (OT) derived from controls with no known neurological history (n = 6F/11M), AD (n = 4F/13M) and PD (n = 7F/16M) subjects. Complementary molecular analyses by Western-blotting were performed in the olfactory bulb (OB), entorhinal cortex (EC) and amygdala areas.

Results: 327 and 151 OT differentially expressed proteins (DEPs) were observed in AD women and AD men, respectively (35 DEPs in common). With respect to PD, 198 DEPs were identified in PD women, whereas 95 DEPs were detected in PD men (20 DEPs in common). This proteome dyshomeostasis induced a disruption in OT protein interaction networks and widespread sex-dependent pathway perturbations in a disease-specific manner, among them Sirtuin (SIRT) signaling. SIRT1, SIRT2, SIRT3 and SIRT5 protein levels unveiled a tangled expression profile across the olfactory-entorhinal-amygdaloid axis, evidencing disease-, sex- and brain structure-dependent changes in olfactory protein acetylation.

Conclusions: Alteration in the OT proteostasis was more severe in AD than in PD. Moreover, protein expression changes were more abundant in women than men independent of the neurological syndrome. Mechanistically, the tangled SIRT profile observed across the olfactory pathway-associated brain regions in AD and PD indicates differential NAD (+)-dependent deacetylase mechanisms between women and men. All these data shed new light on differential olfactory mechanisms across AD and PD, pointing out that the evaluation of the feasibility of emerging sirtuin-based therapies against neurodegenerative diseases should be considered with caution, including further sex dimension analyses in vivo and in clinical studies.

Keywords: Alzheimer; Olfaction; Parkinson; Proteomics; Sexual dimorphism; Sirtuin.

Fig. 1

Sex differences in the OT proteome in AD and PD. A Heatmap representing the differential OT proteotyping in AD across sexes. B Sex-independent protein clusters specifically modulated in AD. C Sex-dependent protein clusters modulated in controls or in AD subjects. D Heatmap representing the differential OT proteotyping in PD across sexes. E Sex-independent protein clusters specifically modulated in PD. F Sex-dependent protein clusters modulated in controls or in PD subjects. G Circos-plot representing the OT deregulated proteome shared between AD and PD across sexes. On the inside, dark orange color represents the proteins that appear in multiple data sets and light orange color represents unique deregulated proteins specific of each experimental group. Purple lines indicate the proteome that is shared across biological conditions. H Functional clustering of proteins commonly deregulated in AD/PD men or AD/PD women. I Synaptic ontology analysis (subcellular distribution) of OT deregulated proteomes. J Synaptic ontology analysis (molecular function) of OT deregulated proteomes (m: men; w: women)

Fig. 2

Functional impact of the deregulated OT proteostasis in AD and PD across both sexes. A Number of common and dissimilar deregulated proteins between men and women in each neurological disorder. B Integrative analysis of our OT proteome data derived from AD subjects with the brain region-specific omics data sets from AlzData database (http://www.alzdata.org/) [34]. C Functional mapping of disrupted OT proteome in AD women and men. D Functional mapping of disrupted OT proteome in PD women and men. Black triangles indicate biological processes commonly altered in AD and PD between both sexes. E Mass spectrometry-based quantitation of astrogliosis and axonal damage markers (m: men; w: women)

Fig. 3

Sex influences on the composition of the OT protein complexes differentially modulated in AD and PD. Protein complexes embedded in OT proteomics outputs were automatically extracted by the MCODE algorithm [35]. Through Metascape tool [32], three most significantly enriched ontology terms were combined to annotate putative biological roles for each MCODE complex (left). Protein components of each complex differentially modulated in AD and PD considered in our survey (lower) (m: men; w: women)

Fig. 4

Sex-dependent changes in the constitutive interactome of neuropathological substrates in AD and PD. Experimentally demonstrated protein interactors of human APP, Tau and α-synuclein were obtained from Biogrid database [36]. Mass spectrometry-based protein intensity of deregulated APP interactors (A), Tau interactors (B), shared APP and Tau interactors (C) and α-synuclein interactors (D)

Fig. 5

Differential impact of sex in OT protein networks and signaling dynamics in AD and PD. A Functional networks associated with AD women (A), PD women (B), AD men (C) and PD men (D). AKT, FAK and NFkB activation state in the OT derived from AD (E) and PD (F) subjects

Fig. 6

Predictive activation profile of pathways, biofunctions and upstream regulators at the level of OT in AD and PD. Based on OT proteomic data sets, Systems Biology analysis were performed through the Ingenuity Pathway Analysis software [100]. Activation prediction of significantly altered pathways and neuronal functions (A, B) as well as upstream regulators (C–H). The activation z-score is calculated as previously described [100]. It makes predictions about potential regulators using information about the direction of protein regulation and comparing with a model that assigns random regulation directions. Blue and orange squares indicate inhibition and activation directionality, respectively. Black triangles refer to processes/molecules with an activation score exclusively associated with AD. Pink and blue triangles indicate processes/molecules with an activation profile specifically associated with women or men, respectively. Red: up-regulation; green: down-regulation; m: men; w: women)

Fig. 7

Differential acetylome across the olfactory axis in AD and PD. A Deregulated OT protein expression profile related to SIRT signaling pathway (red: up-regulation; green: down-regulation; m: men; w: women). B Western-blotting against Lys-acetylated proteins at the level of OB, OT, EC and amygdala in AD and PD women. Sample pooling (n = 5/group) was used in all biological conditions. Equal loading of the gels was assessed by stain-free digitalization. Right panels indicate relative intensity levels for multiple bands (w.1 to w.9 indicated on the blot) normalized by total protein in each gel lane across OB, OT, EC and amygdala protein extracts. C Western-blotting against Lys-acetylated proteins at the level of OB, OT, EC and amygdala in AD and PD men. Sample pooling (n = 5/group) was used in all biological conditions. Equal loading of the gels was assessed by stain-free digitalization. Right panels indicate relative intensity levels for multiple bands (m.1 to m.7 indicated on the blot) normalized by total protein in each gel lane across OB, OT, EC and amygdala protein extracts

Fig. 8

Differential sex-associated changes in Sirtuin (SIRT) signaling across the olfactory bulb, olfactory tract, entorhinal cortex and amygdala in AD and PD. A Protein expression of SIRT family (SIRT1, 2, 3 and 5) across the olfactory axis in AD. B Protein expression of SIRT family (SIRT1, 2, 3 and 5) across the olfactory axis in PD. Western-blotting were performed in n = 2–6/group/structure (Additional file 1: Table S1). Representative images are shown. Equal loading of the gels was assessed by stain-free digitalization. Panels show histograms of band densities. Data are presented as mean ± SEM. *P < 0.05 vs. control group; **P < 0.01 vs. control group (a.u: arbitrary units)