Cellular fates and secretion ofAlzheimer’s disease-related proteins APP and tau

Neurodegenerative disorders are progressive, age-dependent, devastating conditions with only symptomatic treatment available. The progressive accumulation and spread of misfolded proteins in the nervous system is the common attribute of multiple neurodegenerative diseases. In Alzheimer’s disease (AD), two types of aggregates accumulate and spread through the brain: extracellular amyloid plaques composed of β-amyloid peptide and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein. β-amyloid peptide originates from the pathological processing of amyloid precursor protein (APP). APP processing is susceptible to various stimuli and controlled by multiple proteins and interactions with lipids. The regulation of APP processing, however, is not fully understood. Tau is one of the major microtubule-associated proteins in neurons. In AD and other tauopathies, however, tau becomes hyperphosphorylated, detaches from microtubules, and first forms small soluble oligomers and then larger, insoluble aggregates. Like several other neurodegeneration-related proteins, pathological tau spreads through the brain via cell-to-cell transmission, which involves secretion and internalization stages, and can initiate templated misfolding of normal tau in recipient cells. Unfortunately, the mechanisms of cell-to-cell transfer and templated misfolding of tau are rather elusive. The aim of this thesis is to (1) develop novel assays to advance the understanding of the regulation of processing and trafficking of neurodegenerative-related proteins and (2) investigate the molecular mechanisms of tau secretion. In this thesis, two novel in vitro live-cell assays were developed based on protein-fragment complementation to study APP, tau, and other neurodegeneration-associated proteins. The first assay can generate multiple readouts, reflecting cellular fates of APP: total cellular APP level, total secreted sAPP level in the media, APP-BACE1 interaction in cells, and in culture media. The second assay can monitor protein localization to dynamic nanoscale cholesterol/sphingomyelin-rich microdomains at the plasma membrane, usually called lipid rafts. This assay may be beneficial for neurodegenerative disease research, as many misfolded proteins associate with lipid rafts, including APP and tau. Additionally, this thesis addressed the molecular mechanisms of tau secretion. In N2A cells overexpressing human tau as well as in primary neurons, tau secretion to the extracellular space was shown to occur via an unconventional, vesicle-free mechanism. Imaging studies have revealed that tau clusters at the plasma membrane in the discrete microdomains and does not localize to membranous intracellular organelles. Instead, tau secretion depended on the lipid composition of the plasma membrane, particularly on lipid-organizing lipid rafts, such as cholesterol and sphingolipids. Tau secretion was also shown to depend on its oligomerization state and heparan sulfate proteoglycans at the cell surface. The data collectively suggest that tau secretion happens via translocation through the plasma membrane, which likely occurs in lipid rafts. In summary, the studies included in this thesis provide both methodological and conceptual insights in the field of neurodegeneration.Ikääntymiseen liittyvät hermorappeumasairaudet ovat kroonisia eteneviä sairauksia, joihin on saatavilla vain oireita lievittäviä hoitoja. Tyypillistä näille sairauksille on hitaasti etenevä neuropatologia, johon liittyy väärin laskostuneiden proteiinien sakkakertymät aivoissa. Alzheimerin taudissa aivoihin kertyy kahden tyyppisiä proteiinisakkoja: pääosin solunulkoisesta tilasta löytyviä β-amyloidipeptidistä koostuvia amyloidiplakkeja sekä pääosin hermosolujen sisäisiä, Tau-proteiinista koostuvia hermosäiekimppuja. β-amyloidipeptidiä syntyy, kun solut pilkkovat suurempaa APP-esiasteproteiinia. Tämä prosessi on herkkä erilaisille muutoksille solussa ja sitä säätelevät lukuisat eri proteiinit ja rasva-aineet. APP:n pilkkomismekanismi patologiseksi β-amyloidipeptidiksi tunnetaan vielä puutteellisesti. Alzheimerin taudissa ja muissa tauopatioiksi kutsuissa sairauksissa Tau-proteiini irtoaa solutukirangan mikrotubuleista hyperfosforyloitumisen seurauksena, johtaen pienten Tau-oligomeerien kautta laajempien liukenemattomien proteiinisakkojen muodostumiseen. Patologiset Tau-proteiinin muodot kykenevät siirtymään aivoissa yhdestä sairastuneesta solusta toiseen levittäen Tau-patologiaa uusille aivoalueille sairauden edetessä. Nämä patologisen Tau-proteiinin leviämismekanismit tunnetaan vielä puutteellisesti ja ovat viime vuosina olleet laajan mielenkiinnon kohteena. Tämän väitöskirjatyön tavoitteet olivat: (1) kehittää uusia menetelmiä hermorappeumasairauksissa sakkaantuvien proteiinien prosessointiin ja solunsisäiseen kuljetukseen liittyen, ja (2) tutkia molekyylitason mekanismeja jotka säätelevät Taun proteiinin eritystä soluista. Väitöskirjatyössä kehitettiin kaksi uutta proteiinifragmenttikomplementaatioon perustuvaa solupohjaista testimenetelmää APP:n ja Taun tutkimukseen. Ensimmäisessä menetelmässä seurataan neljää eri päätemuuttujaa jotka liityvät APP:n kuljetukseen ja prosessointiin solussa. Nämä päätemuuttujat ovat APP:n kokonaismäärä solussa, eritetty APP:n solunulkoinen fragmentti ja APP:n vuorovaikutus β-amyloidipeptidin tuotannon kannalta keskeisen BACE1-entsyymin kanssa sekä soluissa että elatusaineessa. Toinen menetelmä mittaa elävissä soluissa tutkittavan proteiinin lokalisaatiota solukalvon pieniin dynaamisiin kolesteroli/sfingomyeliini-rikkaisiin lipidilauttoihin. Tällä menetelmällä on lukuisia käyttösovelluksia koska monet esimerkiksi hermorappeumasairauksissa keskeiset valkuaisaineet kuten APP ja Tau ovat vuorovaikutuksessa lipidilauttojen kanssa. Lisäksi väitöskirjatyössä tuotettiin uutta tutkimustietoa Taun eritysmekanismeihin liittyen. Erilaisia soluviljelymenetelmiä käyttäen osoitettiin, että Taun eritys soluista solunulkoiseen tilaan tapahtuu epätyypillisen vesikkelivapaan mekanismin välityksellä. Erilaisia kuvantamismenetelmiä käyttäen Tau-proteiiniryppäitä havaittiin solukalvon välittömässä läheisyydessä mutta ei juurikaan solunsisäisissä vesikkelirakenteissa tai soluelimissä. Solukalvon rasva-ainekoostumus vaikutti merkittävästi Taun eritykseen siten, että erityisesti lipidilauttoihin rikastuvat kolesteroli ja sfingomyeliini olivat keskeisessä asemassa Taun erityksen kannalta. Myös Taun oligomerisaatioaste ja solun pinnalla sijaitsevat heparaanisulfaattiproteoglykaanit vaikuttivat Taun eritykseen. Näihin havaintoihin perustuen näyttäisi siltä, että Tau erittyy solusta läpäisemällä solukalvon suoraan, mahdollisesti lipidilauttojen välityksellä. Yhteenvetona tämä väitöskirjatyö tuotti sekä uusia menetelmiä että tieteellisiä konsepteja hermorappeumasairauksien solu- ja molekyylitason mekanismien tutkimukseen

The Cell Biology of Tau Secretion

The progressive accumulation and spread of misfolded tau protein in the nervous system is the hallmark of tauopathies, progressive neurodegenerative diseases with only symptomatic treatments available. A growing body of evidence suggests that spreading of tau pathology can occurviacell-to-cell transfer involving secretion and internalization of pathological forms of tau protein followed by templated misfolding of normal tau in recipient cells. Several studies have addressed the cell biological mechanisms of tau secretion. It now appears that instead of a single mechanism, cells can secrete tauviathree coexisting pathways: (1) translocation through the plasma membrane; (2) membranous organelles-based secretion; and (3) ectosomal shedding. The relative importance of these pathways in the secretion of normal and pathological tau is still elusive, though. Moreover, glial cells contribute to tau propagation, and the involvement of different cell types, as well as different secretion pathways, complicates the understanding of prion-like propagation of tauopathy. One of the important regulators of tau secretion in neuronal activity, but its mechanistic connection to tau secretion remains unclear and may involve all three secretion pathways of tau. This review article summarizes recent advancements in the field of tau secretion with an emphasis on cell biological aspects of the secretion process and discusses the role of neuronal activity and glial cells in the spread of pathological forms of tau.Peer reviewe

Live-cell monitoring of protein localization to membrane rafts using protein-fragment complementation

The plasma membrane consists of a variety of discrete domains differing from the surrounding membrane in composition and properties. Selective partitioning of protein to these microdomains is essential for membrane functioning and integrity. Studying the nanoscale size and dynamic nature of the membrane microdomains requires advanced imaging approaches with a high spatiotemporal resolution and, consequently, expensive and specialized equipment, unavailable for most researchers and unsuited for large-scale studies. Thus, understanding of protein partitioning to the membrane microdomains in health and disease is still hampered by the lack of inexpensive live-cell approaches with an appropriate spatial resolution. Here, we have developed a novel approach based on Gaussia princeps luciferase protein-fragment complementation assay to quantitively investigate protein partitioning to cholesterol and sphingomyelin-rich domains, sometimes called 'lipid rafts', in intact living cells with a high-spatial resolution. In the assay, the reporter construct, carrying one half of the luciferase protein, is targeted to lipid microdomains through the fused acetylation motif from Src-family kinase Fyn. A protein of interest carries the second half of the luciferase protein. Together, this serves as a reversible real-time sensor of raft recruitment for the studied protein. We demonstrated that the assay can efficiently detect the dynamic alterations in raft localization of two disease-associated proteins: Akt and APP. Importantly, this method can be used in high-throughput screenings and other large-scale studies in living cells. This inexpensive, and easy to implement raft localization assay will benefit all researchers interested in protein partitioning in rafts.Peer reviewe

Mechanisms of secretion and spreading of pathological tau protein

Accumulation of misfolded and aggregated forms of tau protein in the brain is a neuropathological hallmark of tauopathies, such as Alzheimer’s disease and frontotemporal lobar degeneration. Tau aggregates have the ability to transfer from one cell to another and to induce templated misfolding and aggregation of healthy tau molecules in previously healthy cells, thereby propagating tau pathology across different brain areas in a prion-like manner. The molecular mechanisms involved in cell-to-cell transfer of tau aggregates are diverse, not mutually exclusive and only partially understood. Intracellular accumulation of misfolded tau induces several mechanisms that aim to reduce the cellular burden of aggregated proteins and also promote secretion of tau aggregates. However, tau may also be released from cells physiologically unrelated to protein aggregation. Tau secretion involves multiple vesicular and non-vesicle-mediated pathways, including secretion directly through the plasma membrane. Consequently, extracellular tau can be found in various forms, both as a free protein and in vesicles, such as exosomes and ectosomes. Once in the extracellular space, tau aggregates can be internalized by neighboring cells, both neurons and glial cells, via endocytic, pinocytic and phagocytic mechanisms. Importantly, accumulating evidence suggests that prion-like propagation of misfolding protein pathology could provide a general mechanism for disease progression in tauopathies and other related neurodegenerative diseases. Here, we review the recent literature on cellular mechanisms involved in cell-to-cell transfer of tau, with a particular focus in tau secretion.Peer reviewe

Multiplex Assay for Live-Cell Monitoring of Cellular Fates of Amyloid-beta Precursor Protein (APP)

Peer reviewe

Secretion of Tau via an Unconventional Non-vesicular Mechanism

Tauopathies are characterized by cerebral accumulation of Tau protein aggregates that appear to spread throughout the brain via a cell-to-cell transmission process that includes secretion and uptake of pathological Tau, followed by templated misfolding of normal Tau in recipient cells. Here, we show that phosphorylated, oligomeric Tau clusters at the plasma membrane in N2A cells and is secreted in vesicle-free form in an unconventional process sensitive to changes in membrane properties, particularly cholesterol and sphingomyelin content. Cell surface heparan sulfate proteoglycans support Tau secretion, possibly by facilitating its release after membrane penetration. Notably, secretion of endogenous Tau from primary cortical neurons is mediated, at least partially, by a similar mechanism. We suggest that Tau is released from cells by an unconventional secretory mechanism that involves its phosphorylation and oligomerization and that membrane interaction may help Tau to acquire properties that allow its escape from cells directly through the plasma membrane.Peer reviewe

Evaluation of a novel surface-coating formulation with time-extended antimicrobial activity for healthcare environment disinfection

Abstract Background The importance of environmental contamination in the transmission of pathogens among hospitalized patients is universally recognized, and disinfection of surfaces is a widely accepted modality for reducing healthcare-associated infections. Nevertheless, hospital disinfection is still suboptimal. In this study, we evaluated the sustained effects of the novel formulation OxiLast™ which extends the antimicrobial effects of chlorine-based disinfectants. Methods In an experimental lab phase, PVC surfaces were coated with OxiLast™ and then inoculated with representative Gram-positive and Gram-negative pathogenic bacteria. Cells were recovered at different contact times (5, 15, 30 min) to assess the reduction in bacterial counts compared to uncoated surfaces and also subject to various challenges to assess robustness. A similar methodology was then applied in an unoccupied hospital room to evaluate the sustained effect of OxiLast™ on high-touch surfaces. Results OxiLast™ demonstrated notable activity against the range of bacterial strains tested with ≥ 4 log10 reduction in bacterial counts observed for up to seven days following one surface application, for various strains and contact times. Similar results were observed following challenges such as simulated abrasion of coated surfaces, organic contamination or successive inoculations. The results were confirmed in a simulated patient care environment. Conclusions The addition of OxiLast™ to common chlorine-based disinfectants has shown a substantial and sustained reduction in bacterial pathogen counts for up to 7 days following one application. The consistent results in the laboratory and hospital are promising and should be tested in a real-life clinical scenario

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

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