Cellular Physiology and Cell-to-Cell Propagation of Tau in Neurodegeneration : The Impact of Late-Onset Alzheimer's Disease Susceptibility Genes

Abnormal regulation of various posttranslational modifications (PTMs) of microtubule- associated protein tau induce its self-aggregation, which is a hallmark pathophysiological process of neurodegenerative diseases (NDDs) collectively called as tauopathies including Alzheimer s disease (AD) and frontotemporal dementia. Increased tau phosphorylation is a key PTM in conversion of tau into more toxic species in cells, which is regulated by interactions of various protein kinases and phosphatases. However, the exact mechanism(s) of how various combinatory PTMs affect aggregation and cell-to-cell propagation of tau are poorly understood. We developed a novel live cell reporter system based on protein-fragment complementation assay (PCA) and studied dynamic protein- protein interactions of tau in native cellular environment. The PCA was further validated on investigating cellular secretion and uptake of tau in live cells. A proof-of-concept screen was performed using PCA platform revealed several GABAA receptor activators that altered the interaction of tau-Pin1. Pin1 act as a critical facilitator of tau dephosphorylation by catalyzing the isomerization of cis/trans peptidyl-prolyl bond at phosphorylated Thr231-Pro motif of tau. Additionally, we showed that screen-identified GABAA receptor modulators increased tau phosphorylation at the AT8 phosphoepitope in cultures of mature primary cortical neurons and remained at elevated level 24 h after washout of the drugs. Mechanistic studies suggested that enhanced GABAA receptor- induced tau phosphorylation was associated with decreased interaction of tau and protein phosphatase 2A (PP2A) without any reduction in enzymatic activity of PP2A and involved CDK5 kinase. Furthermore, the assessment of expression and splicing status of late-onset AD (LOAD) susceptibility genes in our neuropathologically validated AD cohort of post mortem brain samples revealed increased expression of MS4A6A and decreased expression of FRMD4A in regards to increased AD-related neurofibrillary pathology according to Braak staging. Moreover, the expression level of FRMD4A was functionally associated with amyloidogenic APP processing and increased tau phosphorylation in vitro. FRMD4A expression levels also correlated with cellular tau secretion assessed by PCA-based assay platform using siRNA-mediated gene silencing. Subsequent mechanistic studies on secretion showed a more general involvement of cell polarity complex signaling including Par3/Par6/aPKCζ complex-induced activation of Arf6 via cytohesins. These novel connections of altered FRMD4A expression level in AD brain and its impact on cellular tau secretion further corroborate the suggested role of FRMD4A in LOAD pathogenesis and pathophysiology. Here, for the first time, we assessed a functional association between LOAD-related susceptibility gene and cell-to- cell propagation of tau, and also showed the decreased expression of FRMD4A related to increasing disease severity according to Braak staging.Erilaisten translaation jälkeisten mekanismien epänormaali säätely indusoi mikrotubulus-assosioituneen tau-proteiinin agregoitumista, joka on tunnusomainen patofysiologinen prosessi esimerkiksi Alzheimerin taudissa (AT) ja frontotemporaalisessa dementiassa, sekä useissa muissa yhteisesti tauopatioiksi kutsutuissa hermostorappeumasairauksissa. Lisääntynyt taun fosforylaatio, jota säätelee vuorovaikutus proteiinikinaasien ja -fosfataasien kanssa, on yksi tärkeimmistä translaation jälkeisistä mekanismeista taun muuttumisessa toksisempaan muotoon soluissa. Erilaisten translaation jälkeisten mekanismien yhteisvaikutuksien yksityiskohtaiset mekanismit taun agregaatioon ja solujen väliseen leviämiseen ovat kuitenkin huonosti tunnettuja. Kehitimme uuden, proteiinfragmentti komplementaatioon pohjautuvan reportteri assay-menetelmän (PKA), jonka avulla tutkimme dynaamisia proteiini-proteiini vuorovaikutuksia solujen luonnollisessa ympäristössä. PKA-menetelmä validoitiin lisäksi tutkiaksemme taun sekreetioa sekä soluunottoa elävissä soluissa. Kehittämämme menetelmän validointiseulonta (lääkeaineseulonta) paljasti useita GABAA -reseptoriaktivaattoreita, jotka muuttivat taun ja Pin1:n välistä vuorovaikutusta. Pin1 proteiini säätelee kriittisesti taun defosforylaatiota katalysoimalla cis/trans peptidyyli-prolyyli sidoksen isomerisaation taun ollessa treoniini231-proliini fosforyloitunut. Validointiseulonnassa identifioitujen GABAA -reseptorimodulaattoreiden havaittiin lisäksi lisäävän taun fosforylaatiota AT8-fosfoepitoopissa rotan aivokuoresta eristetyillä primäärineuroneilla, joka pysyi koholla ainakin 24 tuntia lääkeaineiden poistamisen jälkeen. Mekanistiset jatkotutkimukset viittasivat siihen, että lisääntynyt GABAA -reseptorien indusoima taun fosforylaatio liittyi sekä vähentyneeseen taun ja proteiinifosfataasi 2A:n (PP2A) väliseen vuorovaikutukseen kuitenkaan vaikuttamatta PP2A:n entsymaattiseen aktiivisuuteen, että proteiinikinaasi CDK5:n osallisuuteen prosessissa. Tutkittaessa myöhäisiän AT:n riskigeenien ilmentymistä ohimoaivokuorinäytteistä AT-potilasaineistossa, havaittiin lisääntynyt MS4A6A-geenin ja vähentynyt FRMD4A-geenin ilmentyminen suhteessa taudin etenemiseen liittyvään neurofibrillipatologiaan. In vitro-tutkimuksissa havaittiin lisäksi toiminnallinen yhteys FRMD4A-geenin ilmentymistason ja amyloidogeenisen APP:n prosessoinnin sekä lisääntyneen taun fosforylaation välillä. PKA-menetelmää ja siRNA-välitteistä geenien hiljentämistä käyttämällä havaittiin FRMD4A:n ilmentymistason korreloivan myös taun solusekreetioon. Tarkemmat mekanistiset tutkimukset paljastivat yleisemmän solun polaarisuuskompleksin osallistumisen taun solusekreetioon, johon kuului Par3/Par6/aPKCζ -kompleksin indusoima sytohesiini-välitteinen Arf6:n aktivaatio. Nämä uudet havaitut yhteydet muuttuneen FRMD4A:n ilmentymisen tason niin AT-aivoissa kuin sen vaikutukset taun solusekreetioon vahvistavat FRMD4A:n ehdotettua roolia myöhäisiän AT:n patogeneesissä ja patofysiologiassa. Tässä väitöskirjassa esitellään ensikertaa toiminnallinen yhteys myöhäisiän AT:n riskigeenin ja taun solujenvälisen leviämiseen välillä, sekä näytetään FRMD4A-geenin ilmentymisen vähentyminen suhteessa lisääntyneeseen neurofibrillipatologiaan taudin edetessä

Inhibition of Homophilic Interactions and Ligand Binding of the Receptor for Advanced Glycation End Products by Heparin and Heparin-Related Carbohydrate Structures

Background: Heparin and heparin-related sulphated carbohydrates inhibit ligand binding of the receptor for advanced glycation end products (RAGE). Here, we have studied the ability of heparin to inhibit homophilic interactions of RAGE in living cells and studied how heparin related structures interfere with RAGE–ligand interactions. Methods: Homophilic interactions of RAGE were studied with bead aggregation and living cell protein-fragment complementation assays. Ligand binding was analyzed with microwell binding and chromatographic assays. Cell surface advanced glycation end product binding to RAGE was studied using PC3 cell adhesion assay. Results: Homophilic binding of RAGE was mediated by V1- and modulated by C2-domain in bead aggregation assay. Dimerisation of RAGE on the living cell surface was inhibited by heparin. Sulphated K5 carbohydrate fragments inhibited RAGE binding to amyloid β-peptide and HMGB1. The inhibition was dependent on the level of sulfation and the length of the carbohydrate backbone. α-d-Glucopyranosiduronic acid (glycyrrhizin) inhibited RAGE binding to advanced glycation end products in PC3 cell adhesion and protein binding assays. Further, glycyrrhizin inhibited HMGB1 and HMGB1 A-box binding to heparin. Conclusions: Our results show that K5 polysaccharides and glycyrrhizin are promising candidates for RAGE targeting drug development.Peer reviewe

Comprehensive miRNome-wide profiling in a neuronal cell model of synucleinopathy implies involvement of cell cycle genes

Growing evidence suggests that epigenetic mechanisms like microRNA-mediated transcriptional regulation contribute to the pathogenesis of parkinsonism. In order to study the influence of microRNAs (miRNAs), we analyzed the miRNome 2 days prior to major cell death in α-synuclein-overexpressing Lund human mesencephalic neurons, a well-established cell model of Parkinson\u27s disease (PD), by next-generation sequencing. The expression levels of 23 miRNAs were significantly altered in α-synuclein-overexpressing cells, 11 were down- and 12 upregulated

Proteome wide association studies of LRRK2 variants identify novel causal and druggable proteins for Parkinson\u27s disease

Common and rare variants in the LRRK2 locus are associated with Parkinson\u27s disease (PD) risk, but the downstream effects of these variants on protein levels remain unknown. We performed comprehensive proteogenomic analyses using the largest aptamer-based CSF proteomics study to date (7006 aptamers (6138 unique proteins) in 3107 individuals). The dataset comprised six different and independent cohorts (five using the SomaScan7K (ADNI, DIAN, MAP, Barcelona-1 (Pau), and Fundació ACE (Ruiz)) and the PPMI cohort using the SomaScan5K panel). We identified eleven independent SNPs in the LRRK2 locus associated with the levels of 25 proteins as well as PD risk. Of these, only eleven proteins have been previously associated with PD risk (e.g., GRN or GPNMB). Proteome-wide association study (PWAS) analyses suggested that the levels of ten of those proteins were genetically correlated with PD risk, and seven were validated in the PPMI cohort. Mendelian randomization analyses identified GPNMB, LCT, and CD68 causal for PD and nominate one more (ITGB2). These 25 proteins were enriched for microglia-specific proteins and trafficking pathways (both lysosome and intracellular). This study not only demonstrates that protein phenome-wide association studies (PheWAS) and trans-protein quantitative trail loci (pQTL) analyses are powerful for identifying novel protein interactions in an unbiased manner, but also that LRRK2 is linked with the regulation of PD-associated proteins that are enriched in microglial cells and specific lysosomal pathways

Multiplex assay for simultaneous live-cell detection of APP-BACE1 interaction and proteolytic cleavage of APP.

<p>(A) Graphical presentation of the multiplex PCA reporter constructs. Alkaline phosphatase (AP) with a signal peptide was placed in the N-terminus of APP-GLuc2. AP reporter is depicted in beige and GLuc reporter in red color. The same colors are used in column graphs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098619#pone-0098619-g003" target="_blank">Fig. 3B–F</a> for corresponding reporter data. (B) Normal proteolytic processing of the AP/APP-hGLuc fusion protein in N2A cells. Cells were transiently transfected with indicated combinations of various APP constructs: APP-GLuc2 and AP/APP-GLuc2 and BACE1-GLuc1. Western blots were probed with APP C-terminal antibody (A8717), dNGluc (BACE1) and GAPDH as a loading control. (C) Sensitivity and linearity of secreted alkaline phosphatase-sAPP (SEAP) assay from conditioned media. N2A cells were transiently transfected with BACE1-GLuc1 and AP/APP-GLuc2. Cells were incubated in serum-free media for up to 30 hours (inset graph shows data up to 60 h). AP activity in cell-free conditioned media was detected using a chemiluminescent SEAP assay. Normalization of cell numbers and transfection efficiency was done with an internal vector control (using a plasmid expressing β-galactosidase). The values are normalized chemiluminescence signals recorded from expressed pair of constructs. The number of replicate wells was four. Linearity of data was evaluated by regression analysis; correlation coefficient (R²) was 0.98806. Error bars represent the SEM. (D) Effects of brefeldin A (BFA) in the multiplex assay (PCA+AP data). N2A cells were transiently transfected with BACE1-GLuc1 and AP-APP-GLuc2, and treated with indicated concentration of BFA for 24 h before measurement of PCA and SEAP signals (48 h after transfection). The average values are displayed as percentage of change as compared to vehicle-treated control cells. (E) Effects of BACE inhibitor IV in the multiplex assay (PCA+AP data). N2A cells were transfected as in D, and treated with indicated concentration of BACE inhibitor IV for 6 h before measurement PCA and SEAP signals (48 h after transfection). The average values are displayed as percentage of change as compared to vehicle-treated control cells. (F) Effects of dynole 34-2 in the multiplex assay (PCA+AP data). N2A cells were transfected as in D, and treated with indicated concentration of dynole 34-2 for 6 h before measurement PCA and SEAP signals (48 h after transfection). The average values are displayed as percentage of change as compared to vehicle-treated control cells. Error bars represent the SEM, and statistical significance was assessed using Student's t test (four replicate wells/experiment, four independent experiments). * p<0.05, ** p<0.01, *** p<0.001.</p

Exosomal secretion of AP/APP and BACE1 PCA reporter proteins.

<p>(A) Detection of AP/APP-BACE1 PCA signal in cleared, cell- and debris-free conditioned media. N2A cells were transiently transfected with BACE1-GLuc1 and AP/APP-GLuc2. Cells were incubated in serum-free media for up to 18 hours. PCA signals in cell-free conditioned media were detected at indicated time points. Normalization of cell numbers and transfection efficiency was done with an internal vector control. The values are normalized bioluminescence signals recorded from expressed pair of constructs. The number of replicate wells was four. The linearity was evaluated by regression analysis, correlation coefficient (R²) is 0.9971. Error bars represent the SEM. (B) N2A cells were transfected with AP/APP-GLuc2 and BACE1-GLuc1 reporter plasmids. Exosomes were isolated from 30-h conditioned media (inset graph shows data up to 60 h) by ultracentrifugation and the presence of AP/APP-GLuc2 and BACE1-GLuc1 reporters in exosomes was analyzed by Western blotting using antibodies for APP (C-terminal antibody A8717), dNGluc (BACE1) and Alix as an exosomal marker. Total cell extracts were analyzed in parallel with the isolated exosome fraction. (C) Pharmacological modulation of ceramide levels alters exosomal secretion of AP/APP-BACE1 PCA reporters. N2A cells were transiently transfected with BACE1-GLuc1 and AP/APP-GLuc2, and treated with 10 µM of GW-4869, the neutral sphingomyelinase (nSMase) inhibitor, for 24 h. PCA signal in cell-free conditioned media was measured at 48 h after transfection. Normalization of cell numbers and transfection efficiency was done with an internal vector control. The average values are displayed as percentage of change as compared to vehicle-treated control cells. Error bars represent the SEM, and statistical significance was assessed using Student's t test (four replicate wells/experiment, four independent experiments). *** p<0.001.</p

Functional assay validation of GLuc PCA for APP-BACE1 interaction.

<p>For genetic assay validation of BACE1-APP PCA, plasmids expressing GGA3 and VPS35 shRNA were cotransfected to N2A cells with plasmids encoding BACE1-GLuc1 and APP-GLuc2 reporters. (A) PCA signal was measured at 48 h post-transfection. (B) Aβ₄₀ and Aβ₄₂ in conditioned media were determined by sandwich ELISA. The number of replicate wells for PCA was four (96-well plate) and for Aβ ELISA two (6-well plate). Error bars represent the SEM, and statistical significance was assessed using ANOVA. * p<0.05, ** p<0.01, *** p<0.001.</p

Protein-fragment complementation assay-based detection of APP and BACE1 interaction in live cells.

<p>(A) Graphical presentation of PCA reporter constructs of APP and BACE1. hGLuc, humanized <i>Gaussia</i> luciferase fragment; SP, signal peptide; TM, transmembrane domain. (B) Normal expression and proteolytic processing of APP-GLuc2 and BACE1-GLuc1 fusion proteins in N2A neuroblastoma cells. Cells were transiently transfected with indicated combinations of expression constructs and analyzed for APP fragments and BACE1 protein in cell lysates. Western blots were probed with APP C-terminal antibody (A8717), dNGluc antibody (detects the GLuc1 fragment) and GAPDH as a loading control. (C) Validation of GLuc-based PCA for detection of APP-BACE1 interaction in N2A cells. Cells were transiently transfected with indicated combinations of expression constructs. Luminescence signal was measured 48 h post-transfection in live cells. Normalization of cell numbers and transfection efficiency was done with an internal vector control (using a plasmid expressing β-galactosidase). Control plasmids were empty GLuc1/2 plasmids expressing the indicated GLuc fragment alone. The values are normalized bioluminescence signals recorded from expressed pairs of reporter constructs. Error bars represent the SEM, and statistical significance was assessed using ANOVA. *** p<0.001.</p

Proteome wide association studies of LRRK2 variants identify novel causal and druggable proteins for Parkinson’s disease

Abstract Common and rare variants in the LRRK2 locus are associated with Parkinson’s disease (PD) risk, but the downstream effects of these variants on protein levels remain unknown. We performed comprehensive proteogenomic analyses using the largest aptamer-based CSF proteomics study to date (7006 aptamers (6138 unique proteins) in 3107 individuals). The dataset comprised six different and independent cohorts (five using the SomaScan7K (ADNI, DIAN, MAP, Barcelona-1 (Pau), and Fundació ACE (Ruiz)) and the PPMI cohort using the SomaScan5K panel). We identified eleven independent SNPs in the LRRK2 locus associated with the levels of 25 proteins as well as PD risk. Of these, only eleven proteins have been previously associated with PD risk (e.g., GRN or GPNMB). Proteome-wide association study (PWAS) analyses suggested that the levels of ten of those proteins were genetically correlated with PD risk, and seven were validated in the PPMI cohort. Mendelian randomization analyses identified GPNMB, LCT, and CD68 causal for PD and nominate one more (ITGB2). These 25 proteins were enriched for microglia-specific proteins and trafficking pathways (both lysosome and intracellular). This study not only demonstrates that protein phenome-wide association studies (PheWAS) and trans-protein quantitative trail loci (pQTL) analyses are powerful for identifying novel protein interactions in an unbiased manner, but also that LRRK2 is linked with the regulation of PD-associated proteins that are enriched in microglial cells and specific lysosomal pathways