We built a molecular landscape of the overlap between AD and SID by analyzing and integrating the available GWAS data for these diseases and assessing the functions and interactions of the proteins encoded by the genes found to be relevant for both AD and SID. In addition, we investigated genetic sharing of AD/SID with the levels of 237 blood and 338 CSF metabolites. In total, 147 of the proteins encoded by the 219 input genes (67%) could be placed into the landscape. A number of functional themes play a role in the molecular landscape of the overlap between AD and SID. First, we found enrichment of estrogen (ESR1) signaling, insulin signaling and synaptic functioning in the input genes/proteins for the landscape. Furthermore, from the genetic sharing analyses, we observed energy metabolism (oxidative phosphorylation and mitochondrial functioning), lipid metabolism and tau signaling as possibly shared metabolic pathways. From the proteins interact in the molecular landscape, we further discuss five potential (novel) drug targets for AD, some of which could also be of relevance for SID given their molecular overlap with AD.

Estrogen receptor 1 (ESR1) signaling was identified as a major functional theme in the AD/SID landscape. ESR1 is a nuclear hormone receptor and transcription factor regulating many genes in different target tissues, including the insulin receptor [104]. Furthermore, estradiol (E2) treatment increases insulin sensitivity through FOXO1 inhibition (via the PI3K/AKT pathway) [103]. Estradiol and other estrogens binds to the estrogen receptors ESR1 and ESR2, which are mainly located in the nucleus and cytoplasm, after which the ESRs can dimerize [9, 26]. In the nucleus, ESR1 and ESR2 can bind to estrogen response elements at the promoter region of their target genes and exert different effects on transcription. In addition to directly binding to target DNA, there are also indirect transcriptional effects of ESR1 and ESR2 [9, 26]. With regard to the brain, estrogen signaling is important for normal brain functioning and reduced estrogen signaling has been thought to be involved in the etiology of AD [92]. Similarly, we observed reduced expression of ESR1 in AD versus controls for two AD-related brain areas (temporal cortex and parahippocampal gyrus). Furthermore, estrogen signaling regulates the expression of glucose transporter type 4 (GLUT4) [29] and possibly also of GLUT3 [18]. Important landscape proteins that are regulated by ESR1 include CKB (involved in energy metabolism), MAPT, the MHC class 1 complex, PHB1 (involved in mitochondrial functioning and transcription) and RTN2. In addition, ESR1 binds and interacts with other mitochondrial landscape proteins (such as MTCH2, TOMM22 and TOMM40), and the important lipogenic transcription factor SREBF1. Therefore, ESR1 also connects different functional themes in the landscape, including estrogen signaling, insulin signaling, lipid metabolism, tau signaling and mitochondrial functioning/energy metabolism.

Another important functional landscape theme is insulin signaling. Brain insulin resistance has been thought to be a key mechanism in AD [38, 47]. Insulin binds to the insulin receptor (INSR) and the insulin-INSR complex translocates from the cell membrane to the cytoplasm and nucleus. It is then either recycled back to the plasma membrane or sent to lysosomes for degradation [28, 39]. Insulin increases glucose uptake by muscle and adipose tissue through the insertion of GLUT4 in the cell membrane [27]. Furthermore, insulin inhibits hepatic gluconeogenesis and glycogenolysis, affects fatty acid and protein synthesis, and has effects on growth and development [7, 99]. As can also be seen from the landscape, insulin signaling is linked to both APP and MAPT, and, indeed, has been implicated in the formation of both amyloid plaques and neurofibrillary tangles, the two key pathological characteristics of AD [38]. Furthermore, brain insulin signaling plays roles in multiple processes including growth and maturation of neurons, synaptic functioning, and regulating overall glucose homeostasis through hypothalamic feedback on hepatic glucose release [10, 28],Martina, Ribeiro and Antunes, 2018; [78]. Modulation of synaptic plasticity by insulin may be related to its effects on dopamine, norepinephrine and serotonin signaling [10, 28]. Furthermore, while GLUT4 plays a key role in peripheral insulin signaling, in the brain, GLUT1 and GLUT3 play major roles in glucose uptake across the blood–brain-barrier and by neurons and glia cells [32, 45, 56]. Although GLUT1 and -3 have been considered as insulin-insensitive, evidence suggests that there might be (indirect) mechanisms through which insulin signaling may affect GLUT1/3-mediated glucose uptake in the brain [57].

In the landscape, insulin signaling plays an important role. The landscape protein CELF1, strongly associated with both AD and SID in the latest available GWASs, is a splicing regulator that causes skipping of exon 11 of the INSR, resulting in the neuronal, A-isoform [72, 75]. On the contrary, peripheral INSRs are mainly the B-isoform, which includes exon 11 [28, 56]. The INSR A-isoform can bind both insulin and IGF2, and also plays a role in fetal growth [75, 99] and cancer [73]. The INSR regulates the expression of numerous important landscape proteins, including SREBF1 [85], PEMT [51] (converts phosphatidylethanolamine to phosphatidylcholine, of which blood/CSF-levels shows genetic sharing with AD/SID), the transcription factor POU5F1 (OCT4)(Kee Keong [37]) and RTN2 [11] (regulated by ESR1 and regulates APP, GLUT4 and MAPT [74]). In addition, GRN – an important upstream regulator in the landscape with downstream targets APOC1, APOE, APP and the MHC (class 1 and 2) complex and known for its link with frontotemporal dementia [80] – is also regulated by the INSR. Therefore, in summary, insulin signaling plays a central role and links different themes in the molecular landscape, including estrogen signaling, lipid metabolism, synaptic functioning and tau signaling.

In addition to estrogen and insulin signaling, energy metabolism emerged as a functional theme from the landscape and genetic sharing analyses. In the mitochondria, ATP – as a cellular energy source – is generated through oxidative phosphorylation. To this end, the mitochondrial electron transport chain consists of a series of protein complexes that are located in the mitochondrial membrane. Electrons are passing through this chain of protein complexes through a number of redox reactions. This electron transport creates a proton gradient across the mitochondrial membrane, which subsequently drives the synthesis of ATP from ADP by the final complex of the chain (ATP synthase). The Krebs, tricarboxylic acid or citric acid cycle is a critical step in cellular respiration, by providing the electron carriers NADH and FADH2 to the electron transport chain. NADH and FADH2 are converted back to their oxidized forms, NAD + and FAD—by the electron transport chain—that are then used again in the Krebs cycle and in glycolysis [25, 64]. Interestingly, in the genetic sharing analyses, we found that genetic variants/SNPs that increase AD risk also contribute to lower CSF levels of pyruvate, whereas SNPs that increase MES risk also contribute to lower CSF-levels of isocitrate and increased blood levels of fumarate. These metabolites can be mapped to the Krebs cycle as a potentially shared metabolic pathway between AD and SID (MES). In addition, a number of mitochondrial proteins from the landscape – such as CKB, DNAJC11, LACTB, NDUFS2, NDUFS3, PHB1, TOMM22 and TOMM40 – are involved in energy metabolism or other mitochondrial functions such as membrane organization, mitochondrial dynamics and protein import. Further, different interactions of CELF1, ESR1, MAPT and SREBF1 with (the aforementioned) mitochondrial landscape proteins point to the link of mitochondrial energy production and functioning with other functional themes in the landscape, including insulin signaling, estrogen signaling, lipid metabolism and tau signaling.

We found that there is genetic sharing between AD as well as SID and the blood levels of eight specific lipids (mainly triglycerides (TAGs), a diglyceride (DAG 34:2) and a lysophospholipid (LPE 18:0)). The direction of genetic sharing for AD and SID (DM2 and MES) was positive with regard to these lipid species, whereas for OBS the direction of genetic sharing was negative. In addition, we identified genetic sharing between AD/SID and the blood levels of several other TAGs, DAGs and some lysophospholipids. DAGs may play a role in (peripheral) insulin resistance through disrupted fatty acid processing and consequent activation of certain protein kinase C isoforms [21, 71]. TAGs are strongly related to and serve as biomarkers for insulin resistance, both in peripheral tissues and in the brain [6, 86]. In addition, there is genetic sharing between AD/SID and the blood and CSF levels of different other lipid species such as lysophosholipids and phosphatidylcholines (PCs). PCs are important for normal lipid metabolism [49] and increased breakdown of PCs has been linked to AD [12]. Thus, lipid metabolism emerges as an important functional theme from the molecular landscape, and is closely connected with (brain) insulin signaling. In addition, the expression of genes related to cholesterol synthesis has been linked to tau pathology [91]. Lastly, as can also be seen in the landscape, the lipid-transport protein encoded by APOE, and in particular by the AD risk factor APOE4, increases the phosphorylation of MAPT and is (consequently) involved in tau hyperphosphorylation [31, 88]. Importantly, APOE4 has also been shown to impair brain insulin signaling through trapping of the INSR in endosomes, which, in turn adversely affects insulin-dependent energy metabolism in the mitochondria [106]. These effects can be accelerated through increased dietary intake of fatty acids [106].

Lastly, tau (or MAPT) is thought to play a key role in AD. Hyperphosphorylation of tau decreases its binding to microtubules and its aggregation results in neurofibrillary tangles inside affected neurons in AD and other ‘tauopathies’ [15, 35]. Tau signaling also emerges as an important functional theme in the landscape. Specifically, tau has many interactions with other landscape proteins such as APOE(4), CRHR1, FKBP5, MARK4 and NDUFS3, and it is connected to insulin signaling, estrogen signaling and APP. Moreover, knockout of the neuronal INSR increases tau phosphorylation, thereby (indeed) pointing to a role for brain insulin resistance in AD [74]. Therefore, Tau signaling plays an important role in the overlap between AD and SID, both through its link with insulin signaling and other functional themes in the landscape. In addition, tau interacts with a number of potential novel drug targets for AD (see below).

MAP/microtubule affinity-regulating kinase 4 (MARK4, localized to the cytoplasm) is a serine/threonine-protein kinase that phosphorylates MAPT [87]. MARK4 shows regional specificity for AD, as it is differentially expressed (downregulated) in the hippocampus and parahippocampal gyrus from AD patients versus controls (Supplementary Table 6). Furthermore, MARK4 is associated with early tau phosphorylation in AD granulovacuolar degeneration bodies [52], pointing to its temporal specificity for AD. Dl-3-n-butylphthalide inhibits MARK4 and reduces cognitive deficits, synaptic loss and tau phosphorylation (in tau transgenic mice) [17]. Inactivation of MARK4 leads to increased insulin sensitivity (in mice) [84]. This shows that MARK4 also has symptomatic specificity, in that it is involved in different clinical signs/symptoms of AD (and SID). In the molecular landscape of AD-SID overlap, MARK4 interacts with multiple other important landscape proteins, including tau, constituting its molecular specificity. Importantly, inhibition of MARK4 could be beneficial for AD and SID through possible effects on tau phosphorylation, neuroinflammation, and insulin resistance [76, 77, 84], reflecting its modulatory specificity. MARK4 is also inhibited by acetylcholinesterase (AChE) inhibitors (e.g. donepezil, rivastigmine) – that are among the only approved drugs for AD [67] – antidiabetics (e.g. metformin, linagliptin), and a number of other compounds (including serotonin, irisin). Taken together, as it adheres to all five aspects of target specificity, we would submit that MARK4 would be an excellent drug target for AD.

Another putative drug target from the landscape of AD-SID overlap is insulin-like growth factor-binding protein 3 receptor (IGFBP3R, other name:TMEM219), a cell membrane receptor specific for IGFBP3. TMEM219 is highly expressed in the brain, including in the hippocampus [89]. In addition, binding of IGFBP3 to TMEM219 leads to pancreatic beta cell loss and dysfunction [20]. These findings show the regional specificity of TMEM219 for the brain and pancreas. In addition, decreased blood levels of the TMEM219-ligand IGFBP3 are linked with increased age and decreased cognitive skills in AD patients [34], and higher Aβ42 CSF levels (a robust biomarker of AD) [36], constituting the temporal and symptomatic specificity of TMEM219 in AD. Nevertheless, the decrease in IGFBP3 blood levels with age may be a confounding factor here [34] and increased IGFBP3 blood levels have also been linked to AD [36]. Furthermore, TMEM219 interacts with multiple other (important) landscape proteins such as the MHC class II complex and the acetylcholine receptor, suggesting its molecular specificity for AD. Moreover, inhibition of TMEM219/IGFBP3 signaling has been suggested to be beneficial for both AD and SID (DM), by decreasing the loss/dysfunction of TMEM219-expressing cells affected in these diseases [63]. However, TMEM219 has been found to be suppressed in different types of cancers, which may be a contra-indication for TMEM219-antagonism as a treatment for AD [63]. Summarizing, TMEM219 could be a potential drug target for AD that warrants further investigation.

Further, peptidyl-prolyl cis–trans isomerase (FKBP5, localized to the cytoplasm) is a peptidyl prolyl isomerase chaperone that is involved in multiple functions, including protein folding, activation and degradation [87]. FKBP5 has regional specificity for AD, since it is differentially expressed (upregulated) in AD cases versus controls in the hippocampus (Supplementary Table 6). Furthermore, FKBP5 is a biomarker of metabolic dysfunction [81] and is linked with Aβ-induced memory impairment [3] and insulin resistance [83], pointing to its temporal and symptomatic specificity for AD and SID. In the molecular landscape, FKBP5 interacts with many other (important) landscape proteins, such as tau and AKT, reflecting its molecular specificity. In addition, Apelin-13 inhibits FKBP5 and protects against Aβ-induced memory impairment [3], while increased expression of FKBP5 is linked to insulin resistance [79]. This suggests that inhibition of FKBP5 could be beneficial for AD and SID, constituting its modulatory specificity. In this respect, existing FKBP5-inhibitors include Rapamycin, Apelin-13 and selective serotonin reuptake inhibitors class antidepressants (e.g. fluoxetine, citalopram, sertraline). To summarize, FKBP5 is a potential novel drug target for AD – with already existing modulating compounds – that could be further studied/developed.

NADH dehydrogenase (ubiquinone) iron-sulfur protein 3 (NDUFS3, localized to the mitochondrion), is a core subunit of mitochondrial electron transport chain Complex 1 (NADH dehydrogenase) [87]. NDUFS3 expression is downregulated in the hippocampus and parahippocampal gyrus from AD patients versus controls (Supplementary Table 6), pointing to its regional specificity. In addition, plasma neuroexosomal NDUFS3 is increased at the early clinical stages of AD in people with SID (DM2) and may serve as an early prognostic and diagnostic biomarker of AD (onset) [19], thereby constituting its temporal specificity. Furthermore, inhibition of Complex 1 improves cognitive function, synaptic plasticity, phosphorylated tau (p-tau) and Aβ levels, neuroinflammation, insulin resistance, and neurodegeneration [90], reflecting the symptomatic specificity of NDUFS3/Complex 1 and suggesting that inhibition of Complex 1 could be beneficial for AD. Furthermore, NDUFS3 is regulated by tau and interacts with other mitochondrial landscape protein (such as prohibitin), pointing to its molecular specificity in AD/SID. Moreover, different Complex 1 inhibitors already exist, and especially a small molecule tricyclic pyrone compound (CP2) appears to be promising. CP2 showed good pharmacological properties, low toxicity and good efficacy in animal studies, including improvement in brain and peripheral energy homeostasis (including insulin resistance), synaptic activity and long-term potentiation, dendritic spine maturation, cognitive function, as well as reducing p-tau and Aβ levels and brain and peripheral inflammation, reflecting the modulatory specificity for NDUFS3/Complex 1 [90]. Therefore, we consider NDUFS3 (and mitochondrial Complex 1) as a putative novel drug target for AD that should be further investigated.

Lastly, interleukin-34 (IL34, localized extracellular) is a pro-inflammatory cytokine that promotes proliferation and differentiation of monocytes and macrophages [87]. IL34 expression is downregulated in the hippocampus from AD patients versus controls (Supplementary Table 6), constituting its regional specificity. Furthermore, by promoting microglial proliferation and thereby possibly neurodegeneration [65], IL34 may contribute to AD disease progression over time, and it has been associated with cognitive decline in vascular dementia [96]. In addition, IL34 impairs the ability of macrophages to ‘clear’ pathological amyloid beta [107] and plasma IL34 levels correlate positively with insulin resistance [60]. All these findings point to the temporal and symptomatic specificity of IL34 in AD. Further, IL34 interacts with one other landscape protein (HEY2) and is involved in neuroprotective signaling in neurodegeneration [53], constituting its molecular specificity. As for its modulatory specificity, inhibition of IL34 reduces microglial proliferation (and hence conveys a protective effect in AD) [65], although at the same time, IL34 was found to enhance the neuroprotective effects of microglia to attenuate amyloid beta neurotoxicity [58]. Therefore, with regard to AD, both IL34 inhibition and activation could have beneficial effects, so maintaining an optimal IL34 level may be recommended. With regard to SID (DM), it appears that IL34 inhibition may be beneficial, given the positive association of IL34 levels with insulin resistance, obesity [16], and beta cell dysfunction and apoptosis [