EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) α-helix, and a C-terminal intracellular kinase domain. The TM helix forms a dimer, as seen in recent NMR studies. We have modeled the EphA1 TM dimer using a multiscale approach combining coarse-grain (CG) and atomistic molecular dynamics (MD) simulations. The one-dimensional potential of mean force (PMF) for this system, based on interhelix separation, has been calculated using CG MD simulations. This provides a view of the free energy landscape for helix-helix interactions of the TM dimer in a lipid bilayer. The resulting PMF profiles suggest two states, consistent with a rotation-coupled activation mechanism. The more stable state corresponds to a right-handed helix dimer interacting via an N-terminal glycine zipper motif, consistent with a recent NMR structure (2K1K). A second metastable state corresponds to a structure in which the glycine zipper motif is not involved. Analysis of unrestrained CG MD simulations based on representative models from the PMF calculations or on the NMR structure reveals possible pathways of interconversion between these two states, involving helix rotations about their long axes. This suggests that the interaction of TM helices in EphA1 dimers may be intrinsically dynamic. This provides a potential mechanism for signaling whereby extracellular events drive a shift in the repopulation of the underlying TM helix dimer energy landscape.

While the typical role of receptor tyrosine kinases is to receive and transmit signals at the cell surface, in some cellular contexts (particularly transformed cells) they may also act as nuclear proteins. Aberrant nuclear localization of receptor tyrosine kinases associated with transformation often enhances the transformed phenotype (i.e. nuclear ErbBs promote tumor progression in breast cancer). Rhabdomyosarcoma (RMS), the most common soft tissue tumor in children, develops to resemble immature skeletal muscle and has been proposed to derive from muscle stem/progenitor cells (satellite cells). It is an aggressive cancer with a 5-year survival rate of 33% if it has metastasized. Eph receptor tyrosine kinases have been implicated in the development and progression of many other tumor types, but there are only two published studies of Ephs localizing to the nucleus of any cell type and to date no nuclear RTKs have been identified in RMS. In a screen for protein expression of Ephs in canine RMS primary tumors as well as mouse and human RMS cell lines, we noted strong expression of EphA1 in the nucleus of interphase cells in tumors from all three species. This localization pattern changes in dividing cells, with EphA1 localizing to the nucleus or the cytoplasm depending on the phase of the cell cycle. These data represent the first case of a nuclear RTK in RMS, and the first time that EphA1 has been detected in the nucleus of any cell type.

Eph receptors are ubiquitous class of transmembrane receptors that mediate cell-cell communication, proliferation, differentiation, and migration. EphA1 receptors specifically play an important role in angiogenesis, fetal development, and cancer progression; however, studies of this receptor can be challenging as its ligand, ephrinA1, binds and activates several EphA receptors simultaneously. Optogenetic strategies could be applied to circumvent this requirement for ligand activation and enable selective activation of the EphA1 subtype. In this work, we designed and tested several iterations of an optogenetic EphA1 - Cryptochrome 2 (Cry2) fusion, investigating their capacity to mimic EphA1-dependent signaling in response to light activation. We then characterized the key cell signaling target of MAPK phosphorylation activated in response to light stimulation. The optogenetic regulation of Eph receptor RTK signaling without the need for external stimulus promises to be an effective means of controlling individual Eph receptor-mediated activities and creates a path forward for the identification of new Eph-dependent functions.

Deficiencies in epithelial barrier integrity are involved in the pathogenesis of chronic rhinosinusitis (CRS). This study aimed to investigate the role of ephrinA1/ephA2 signaling on sinonasal epithelial permeability and rhinovirus-induced epithelial permeability. This role in the process of epithelial permeability was evaluated by stimulating ephA2 with ephrinA1 and inactivating ephA2 with ephA2 siRNA or inhibitor in cells exposed to rhinovirus infection. EphrinA1 treatment increased epithelial permeability, which was associated with decreased expression of ZO-1, ZO-2, and occludin. These effects of ephrinA1 were attenuated by blocking the action of ephA2 with ephA2 siRNA or inhibitor. Furthermore, rhinovirus infection upregulated the expression levels of ephrinA1 and ephA2, increasing epithelial permeability, which was suppressed in ephA2-deficient cells. These results suggest a novel role of ephrinA1/ephA2 signaling in epithelial barrier integrity in the sinonasal epithelium, suggesting their participation in rhinovirus-induced epithelial dysfunction.

Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization.

The EPH receptor tyrosine kinases and their cell-bound ligands, the ephrins, have been shown to be associated with cancer development and progression. In this study, mRNA and protein expression of the receptors EPHA1 and EPHA2 as well as of their ligand EFNA1 and their prognostic relevance in clear cell renal cell carcinoma was evaluated. Gene expression was measured in 75 cryo-preserved primary tumors and matched non-malignant renal specimens by quantitative PCR. Protein expression was analyzed by immunohistochemistry on tissue microarrays comprising non-malignant, primary tumors and metastatic renal tissues of 241 patients. Gene and protein expression of all three factors was altered in tumor specimens with EPHA1 and EPHA2 being generally diminished in tumors compared to normal renal tissue, whereas EFNA1 was commonly elevated. A positive EPHA1 and EPHA2 protein staining as well as a low EFNA1 protein level were significantly linked to more aggressive tumor features, but only a positive EPHA1 immunoreactivity was significantly associated with poor survival. In subgroup analyses, EPHA1 and EPHA2 protein levels were significantly higher in metastatic than in primary lesions. Patients with EPHA1/EPHA2-positive tumors or with tumors with positive EPHA1 and low EFNA1 immunoreactivity had the shortest survival rates compared to the respective other combinations. In a multivariate model, EPHA1 was an independent prognostic marker for different survival endpoints. In conclusion, an impaired EPH-ephrin signaling could contribute to the pathogenesis and progression of clear cell renal cell carcinoma.

Much cell-to-cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK-protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase-deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well-studied RTKs, offers insights into the functions of less well-studied RTKs, and highlights RTK-RTK interactions and shared signaling pathways.

Objective To investigate whether lymphoedema in a Chinese family showed the hereditary and clinical characteristics of Milroy disease, an autosomal dominant form of congenital lymphoedema, typically characterized by chronic lower limb tissue swelling due to abnormal lymphatic vasculature development, and to perform mutational analyses of vascular endothelial growth factor receptor ( VEGFR)3. Methods Individuals from a three-generation family affected by congenital lymphoedema were clinically assessed for Milroy disease. Mutation analysis of VEGFR3 was performed using DNA from family members and healthy controls. Results Out of 20 family members, eight were diagnosed with hereditary lymphoedema. Mutation analyses revealed a novel mutation site for c.3163 G>A, resulting in a p.1055D>N mutation in the second tyrosine kinase domain of VEGFR3, which was present in affected individuals only (absent in all unaffected family members and 130 healthy controls). Computed functional analyses showed the mutation may lead to structural alterations with a probability of 0.99999 of being disease causing. Conclusion A novel mutation associated with Milroy disease was identified in a Chinese family, expanding our knowledge of VEGFR3 gene function and providing a potential molecular target for treating hereditary lymphoedema.

Bone morphogenetic protein (BMP) signaling plays important roles in glioblastoma multiforme (GBM), a lethal form of brain tumor. BMP reduces GBM tumorigenicity through its differentiation- and apoptosis-inducing effects on glioma-initiating cells (GIC). However, some GIC do not respond to the tumor suppressive effects of BMP. Using a phosphoreceptor tyrosine kinase array, we found that EPHA6 (erythropoietin-producing hepatocellular carcinoma receptor A6) phosphorylation was regulated by BMP-2 signaling in some GIC. Analysis of The Cancer Genome Atlas showed that EPHA6 expression was lower in patients with GBM than in the normal brain, and that high EPHA6 expression was correlated with better prognosis. EPHA6 receptor increased the susceptibility of both sensitive and resistant GIC to BMP-2-induced apoptosis. The cooperative effect on apoptosis induction depended on the kinase activity of BMP type I receptor but was independent of EPHA6 kinase function. Overexpression of the EPHA6 receptor in GIC resulted in the formation of a protein complex of EPHA6 receptor and the BMP type I receptor ALK-2, which was associated with BMP-induced apoptosis in GIC. Intracranial injection of GIC into nude mice showed that gain-of-function of EPHA6 together with BMP-2 pretreatment slowed GBM tumor progression in the mouse brain and promoted mouse survival. In summary, EPHA6 together with BMP-2 signaling led to apoptotic cell death in GIC, and thus is a putative tumor suppressor in GBM.

Platelets play a crucial role in cancer and thrombosis. However, the receptor-ligand repertoire mediating prostate cancer (PCa) cell-platelet interactions and ensuing consequences have not been fully elucidated. Microvilli emanating from the plasma membrane of PCa cell lines (RC77 T/E, MDA PCa 2b) directly contacted individual platelets and platelet aggregates. PCa cell-platelet interactions were associated with calcium mobilization in platelets, and translocation of P-selectin and integrin αIIbβ3 onto the platelet surface. PCa cell-platelet interactions reciprocally promoted PCa cell invasion and apoptotic resistance, and these events were insensitive to androgen receptor blockade by bicalutamide. PCa cells were exceedingly sensitive to activation by platelets in vitro, occurring at a PCa cell:platelet coculture ratio as low as 1:10 (whereas PCa patient blood contains 1:2,000,000 per ml). Conditioned medium from cocultures stimulated PCa cell invasion but not apoptotic resistance nor platelet aggregation. Candidate transmembrane signaling proteins responsible for PCa cell-platelet oncogenic events were identified by RNA-Seq and broadly divided into 4 major categories: (1) integrin-ligand, (2) EPH receptor-ephrin, (3) immune checkpoint receptor-ligand, and (4) miscellaneous receptor-ligand interactions. Based on antibody neutralization and small molecule inhibitor assays, PCa cell-stimulated calcium mobilization in platelets was found to be mediated by a fibronectin1 (FN1)-αIIbβ3 signaling axis. Platelet-stimulated PCa cell invasion was facilitated by a CD55-adhesion G protein coupled receptor E5 (ADGRE5) axis, with contribution from platelet cytokines CCL3L1 and IL32. Platelet-stimulated PCa cell apoptotic resistance relied on ephrin-EPH receptor and lysophosphatidic acid (LPA)-LPA receptor (LPAR) signaling. Of participating signaling partners, FN1 and LPAR3 overexpression was observed in PCa specimens compared to normal prostate, while high expression of CCR1 (CCL3L1 receptor), EPHA1 and LPAR5 in PCa was associated with poor patient survival. These findings emphasize that non-overlapping receptor-ligand pairs participate in oncogenesis and thrombosis, highlighting the complexity of any contemplated clinical intervention strategy.

The activities of the ephrin family in breast cancer (BrCa) are complex. Family A receptors (EPHA) and ligands (EFNA) can act as oncogenes or tumor suppressors and are implicated in chemoresistance. Here, we examined the expression pattern and prognostic value of the EPHA/EFNA family in patients with breast cancer, including patients with different subtypes or different chemotherapy cohorts. In the UALCAN database, the mRNA expression of EPHA1, EPHA10, EFNA1, EFNA3, and EFNA4 was significantly higher, whereas that of EPHA2, EPHA4, EPHA5, and EFNA5 was significantly lower in breast cancer tissues than in paracancerous tissues. The transcriptional levels of EPHA/EFNA family members were correlated with intrinsic subclasses of breast cancer. The relationship between EPHA/EFNA and the clinicopathological parameters of BrCa was analyzed using bc-GenExMiner V4.5. EPHA1, EPHA2, EPHA4, EPHA7, EFNA3, EFNA4, and EFNA5 were upregulated in estrogen receptor- (ER-) and progesterone receptor- (PR-) negative tumors, whereas EPHA3, EPHA6, and EFNA1 were upregulated in ER- and PR-positive tumors. EPHA1, EPHA2, EFNA3, and EFNA4 mRNA expression was significantly higher in human epidermal growth factor receptor 2- (HER2-) positive tumors than in HER2-negative tumors. Triple-negative status was positively correlated with EPHA1, EPHA2, EPHA4, EPHA7, EFNA3, EFNA4, and EFNA5 and negatively correlated with EPHA3 and EPHA10 mRNA expression. Genetic alterations of EPHA/EFNA in breast cancer varied from 1.1% to 10% for individual genes, as determined by the cBioPortal database. The Kaplan-Meier plotter indicated that high EphA7 mRNA expression was associated with poor overall survival (OS) and recurrence-free survival (RFS), especially in the HER2 and luminal A subtypes. EFNA4 was predicted to have poor OS and RFS in breast cancers, especially in luminal B, basal-like subtype, and patients treated with adjuvant chemotherapy. High EPHA3 expression was significantly associated with better OS and RFS, especially in the luminal A subtype, but with poor RFS in BrCa patients receiving chemotherapy. Our findings systematically elucidate the expression pattern and prognostic value of the EPHA/EFNA family in BrCa, which might provide potential prognostic factors and novel targets in BrCa patients, including those with different subtypes or treated with chemotherapy.

The Eph family receptor-interacting (ephrin) ligands and erythropoietin-producing hepatocellular carcinoma (Eph) receptors constitute the largest known family of receptor tyrosine kinases. Ephrin ligands and their receptors form an important cell communication system with widespread roles in normal physiology and disease pathogenesis. In order to investigate potential roles of the ephrin-Eph system during palatogenesis and tongue development, we have characterized the cellular mRNA expression of family members EphrinA1-A3, EphA1-A8, and EphrinB2, EphB1, EphB4 during murine embryogenesis between embryonic day 13.5-16.5 using radioactive in situ hybridization. With the exception of EphA6 and ephrinA3, all genes were regionally expressed during the process of palatogenesis, with restricted and often overlapping domains. Transcripts were identified in the palate epithelium, localized at the tip of the palatal shelves, in the mesenchyme and also confined to the medial epithelium seam. Numerous Eph transcripts were also identified during tongue development. In particular, EphA1 and EphA2 demonstrated a highly restricted and specific expression in the tongue epithelium at all stages examined, whereas EphA3 was strongly expressed in the lateral tongue mesenchyme. These results suggest regulatory roles for ephrin-EphA signaling in development of the murine palate and tongue.

The purpose of this study was to investigate the role of intramyocardial administration of chimeric ephrinA1-Fc in modulating the extent of injury and inflammation in non reperfused myocardial infarction (MI). Our results show that intramyocardial injection of 6 μg ephrinA1-Fc into the border zone immediately after permanent coronary artery ligation in B6129s mice resulted in 50% reduction of infarct size, 64% less necrosis, 35% less chamber dilatation and 32% less left ventricular free wall thinning at 4 days post-MI. In the infarct zone, Ly6G+ neutrophil density was 57% reduced and CD45+ leukocyte density was 21% reduced. Myocyte damage was also reduced in ephrinA1-Fc-treated hearts, as evidenced by 54% reduced serum cardiac troponin I. Further, we observed decreased cleaved PARP, increased BAG-1 protein expression, increased phosphorylated AKT/total AKT protein, and reduced NF-κB protein with ephrinA1-Fc administration, indicating improved cellular survival. Of the eight EphA receptors known to be expressed in mice (A1–A8), RT-PCR revealed that A1–A4, A6 and A7 were expressed in the uninjured adult myocardium. Expression of EphA1–A3 and EphA7 were significantly increased following MI while EphA6 expression decreased. Treatment with ephrinA1-Fc further increased EphA1 and EphA2 gene expression and resulted in a 2-fold increase in EphA4. Upregulation and combinatorial activation of these receptors may promote tissue survival. We have identified a novel, beneficial role for ephrinA1-Fc administration at the time of MI, and propose this as a promising new target for infarct salvage in non reperfused MI. More experiments are in progress to identify receptor-expressing cell types as well as the functional implications of receptor activation.

The EphA2 receptor tyrosine kinase is overexpressed in a variety of human epithelial cancers and is a determinant of malignant cellular behavior in pancreatic adenocarcinoma cells. Moreover, it is expressed in tumor endothelium and its activation promotes angiogenesis. To better clarify the therapeutic potential of monoclonal antibodies (mAbs) directed to the EphA2 receptor, we generated a large number of mAbs by differential screening of phage-Ab libraries by oligonucleotide microarray technology and implemented a strategy for the rapid identification of antibodies with the desired properties. We selected two high-affinity and highly specific EphA2 monoclonal antibodies with different in vitro properties on the human pancreatic tumor cell line MiaPaCa2. One is a potent EphA2-agonistic antibody, IgG25, that promotes receptor endocytosis and subsequent degradation, and the second is a ligand antagonist, IgG28, that blocks the binding to ephrin A1 and is cross-reactive with the mouse EphA2 receptor. We measured the effect of antibody treatment on the growth of MiaPaCa2 cells orthotopically transplanted in nude mice. Both IgG25 and IgG28 had strong antitumor and antimetastatic efficacy. In vivo treatment with IgG25 determined the reduction of the EphA2 protein levels in the tumor and the phosphorylation of FAK on Tyr576 while administration of IgG28 caused a decrease in tumor vascularization as measured by immunohistochemical analysis of CD31 in tumor sections. These data show that in a pancreatic cancer model comparable therapeutic efficacy is obtained either by promoting receptor degradation or by blocking receptor activation.

The sterile alpha motif (SAM) for protein-protein interactions is encountered in over 200 proteins, but the structural basis for its interactions is just becoming clear. Here we solved the structure of the EphA2-SHIP2 SAM:SAM heterodimeric complex by use of NMR restraints from chemical shift perturbations, NOE and RDC experiments. Specific contacts between the protein surfaces differ significantly from a previous model and other SAM:SAM complexes. Molecular dynamics and docking simulations indicate fluctuations in the complex toward alternate, higher energy conformations. The interface suggests that EphA family members bind to SHIP2 SAM, whereas EphB members may not; correspondingly, we demonstrate binding of EphA1, but not of EphB2, to SHIP2. A variant of EphB2 SAM was designed that binds SHIP2. Functional characterization of a mutant EphA2 compromised in SHIP2 binding reveals two previously unrecognized functions of SHIP2 in suppressing ligand-induced activation of EphA2 and in promoting receptor coordinated chemotactic cell migration.

Gastric cancer is a leading cause of mortality due to neoplastic disease. Although early detection of gastric cancers can decrease the mortality rate, it remains a diagnostic challenge because of the lack of effective biomarkers. In this study, fifteen gastric cancer patients and ten healthy subjects were recruited to assess novel serum biomarkers for gastric cancer using antibody microarray technology. ELISA was utilized to validate the antibody array results. As a result, compared to the controls, eleven cytokines were found to be significantly increased in gastric cancer, including interferon gamma receptor 1 (IFNGR1), neurogenic locus notch homolog protein 3 (Notch-3), tumor necrosis factor receptor superfamily member 19L (TNFRSF19L), growth hormone receptor (GHR), signaling lymphocytic activation molecule family 8 (SLAMF8), folate receptor beta (FR-beta), integrin alpha 5, galectin-8, erythropoietin-producing hepatocellular A1 (EphA1), epiregulin, and fibroblast growth factor 12 (FGF-12) with P < 0.05. ELISA validation supported the results of the antibody array. More importantly, most of these eleven cytokines, including IFNGR1, TNFRSF19L, GHR, SLAMF8, FR-beta, and integrin alpha 5 were discovered to be elevated in gastric cancer serum samples for the first time in this study, suggesting that these proteins may serve as novel biomarkers for the early diagnosis and prognosis determination of gastric cancer.

EphA2 is a receptor tyrosine kinase (RTK) that triggers keratinocyte differentiation upon activation and subsequent downregulation by ephrin-A1 ligand. The objective of this study was to determine whether the EphA2/ephrin-A1 signaling axis was altered in psoriasis, an inflammatory skin condition in which keratinocyte differentiation is abnormal. Microarray analysis of skin biopsies from psoriasis patients revealed increased mRNA transcripts for several members of this RTK family in plaques, including the EphA1, EphA2, and EphA4 subtypes prominently expressed by keratinocytes. Of these, EphA2 showed the greatest upregulation, a finding that was confirmed by quantitative reverse-transcriptase-PCR, immunohistochemistry (IHC), and ELISA. In contrast, psoriatic lesions exhibited reduced ephrin-A ligand immunoreactivity. Exposure of primary keratinocytes induced to differentiate in high calcium or a three-dimensional (3D) raft culture of human epidermis to a combination of growth factors and cytokines elevated in psoriasis increased EphA2 mRNA and protein expression while inducing S100A7 and disrupting differentiation. Pharmacological delivery of a soluble ephrin-A1 peptidomimetic ligand led to a reduction in EphA2 expression and ameliorated proliferation and differentiation in raft cultures exposed to EGF and IL-1α. These findings suggest that ephrin-A1-mediated downregulation of EphA2 supports keratinocyte differentiation in the context of cytokine perturbation.

Ephrin receptors (Ephs) are receptor tyrosine kinases (RTKs) implicated in tissue development and homeostasis, and they are aberrantly expressed in tumors. Here, immunohistochemical Eph type-A and -B expression in thymic epithelial tumors (TETs) was assessed and correlated with clinicopathological parameters. Tissue microarrays from 98 TETs were stained for EphA1, -A2, -A4 -A6, -B1, -B2, -B4 and -B6. The relationship between neoplastic and lymphoid cell immunoreactivity score (H-score), histopathological parameters (Pearson's test) and survival of 35 patients (Mantel-Cox model) was explored. Epithelial-rich subtypes showed higher EphA6 cytoplasmic H-score (B2/B3, carcinoma) (p < 0.001) and stronger EphA4 H-score (B3, carcinoma) (p = 0.011). The immature T-cells, especially in subtypes AB/B1, had higher EphB6 H-score than carcinoma-associated mature lymphocytes (p < 0.001); carcinomas had higher lymphocytic EphB1 H-score (p = 0.026). Higher lymphocytic and lower epithelial EphB6 H-score correlated with Masaoka stage ≤II (p = 0.043, p = 0.010, respectively). All cases showed variable epithelial and lymphocytic EphA2 expression, but clinicopathological associations were not reached. Our study confirmed that Eph type-A and -B expression in TETs is associated with established prognostic parameters, i.e., tumor subtype and Masaoka stage, although correlation with patient survival was not reached. Such findings suggest involvement of these RTKs in thymic neoplasia, as well as their potential utility as treatment targets.

Alzheimer's disease (AD) is characterized by a long pre-clinical phase (20-30 years), during which significant brain pathology manifests itself. Disease mechanisms associated with pathological hallmarks remain elusive. Most processes associated with AD pathogenesis, such as inflammation, synaptic dysfunction, and hyper-phosphorylation of tau are dependent on protein kinase activity. The objective of this study was to determine the involvement of protein kinases in AD pathogenesis. Protein kinase activity was determined in postmortem hippocampal brain tissue of 60 patients at various stages of AD and 40 non-demented controls (Braak stages 0-VI) using a peptide-based microarray platform. We observed an overall decrease of protein kinase activity that correlated with disease progression. The phosphorylation of 96.7% of the serine/threonine peptides and 37.5% of the tyrosine peptides on the microarray decreased significantly with increased Braak stage (p-value <0.01). Decreased activity was evident at pre-clinical stages of AD pathology (Braak I-II). Increased phosphorylation was not observed for any peptide. STRING analysis in combination with pathway analysis and identification of kinases responsible for peptide phosphorylation showed the interactions between well-known proteins in AD pathology, including the Ephrin-receptor A1 (EphA1), a risk gene for AD, and sarcoma tyrosine kinase (Src), which is involved in memory formation. Additionally, kinases that have not previously been associated with AD were identified, e.g., protein tyrosine kinase 6 (PTK6/BRK), feline sarcoma oncogene kinase (FES), and fyn-associated tyrosine kinase (FRK). The identified protein kinases are new biomarkers and potential drug targets for early (pre-clinical) intervention.

Several genome-wide association studies (GWAS) have been carried out with late-onset Alzheimer's disease (LOAD), mainly in European and Asian populations. Different polymorphisms were associated, but several of them without a functional explanation. GWAS are fundamental for identifying loci associated with diseases, although they often do not point to causal polymorphisms. In this sense, functional investigations are a fundamental tool for discovering causality, although the failure of this validation does not necessarily indicate a non-causality. Furthermore, the allele frequency of associated genetic variants may vary widely between populations, requiring replication of these associations in other ethnicities. In this sense, our study sought to replicate in 150 AD patients and 114 elderly controls from the South Brazilian population 18 single-nucleotide polymorphisms (SNPs) associated with AD in European GWAS, with further functional investigation using bioinformatic tools for the associated SNPs. Of the 18 SNPs investigated, only four were associated in our population: rs769449 (APOE), rs10838725 (CELF1), rs6733839, and rs744373 (BIN1-CYP27C1). We identified 54 variants in linkage disequilibrium (LD) with the associated SNPs, most of which act as expression or splicing quantitative trait loci (eQTLs/sQTLs) in genes previously associated with AD or with a possible functional role in the disease, such as CELF1, MADD, MYBPC3, NR1H3, NUP160, SPI1, and TOMM40. Interestingly, eight of these variants are located within long non-coding RNA (lncRNA) genes that have not been previously investigated regarding AD. Some of these polymorphisms can result in changes in these lncRNAs' secondary structures, leading to either loss or gain of microRNA (miRNA)-binding sites, deregulating downstream pathways. Our pioneering work not only replicated LOAD association with polymorphisms not yet associated in the Brazilian population but also identified six possible lncRNAs that may interfere in LOAD development. The results lead us to emphasize the importance of functional exploration of associations found in large-scale association studies in different populations to base personalized and inclusive medicine in the future.