Ensemble-based design of tau to inhibit aggregation while preserving biological activity.

The microtubule-associated protein tau is implicated in neurodegenerative diseases characterized by amyloid formation. Mutations associated with frontotemporal dementia increase tau aggregation propensity and disrupt its endogenous microtubule-binding activity. The structural relationship between aggregation propensity and biological activity remains unclear. We employed a multi-disciplinary approach, including computational modeling, NMR, cross-linking mass spectrometry, and cell models to design tau sequences that stabilize its structural ensemble. Our findings reveal that substitutions near the conserved 'PGGG' beta-turn motif can modulate local conformation, more stably engaging in interactions with the 306VQIVYK311 amyloid motif to decrease aggregation in vitro and in cells. Designed tau sequences maintain microtubule binding and explain why 3R isoforms of tau exhibit reduced pathogenesis over 4R isoforms. We propose a simple mechanism to reduce the formation of pathogenic species while preserving biological function, offering insights for therapeutic strategies aimed at reducing protein misfolding in neurodegenerative diseases.

DnaJC7 specifically regulates tau seeding.

Neurodegenerative tauopathies are caused by accumulation of toxic tau protein assemblies. This appears to involve template-based seeding events, whereby tau monomer changes conformation and is recruited to a growing aggregate. Several large families of chaperone proteins, including Hsp70s and J domain proteins (JDPs), cooperate to regulate the folding of intracellular proteins such as tau, but the factors that coordinate this activity are not well known. The JDP DnaJC7 binds tau and reduces its intracellular aggregation. However, it is unknown whether this is specific to DnaJC7 or if other JDPs might be similarly involved. We used proteomics within a cell model to determine that DnaJC7 co-purified with insoluble tau and colocalized with intracellular aggregates. We individually knocked out every possible JDP and tested the effect on intracellular aggregation and seeding. DnaJC7 knockout decreased aggregate clearance and increased intracellular tau seeding. This depended on the ability of the J domain (JD) of DnaJC7 to stimulate Hsp70 ATPase activity, as JD mutations that block this interaction abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abolished its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.

DnaJC7 specifically regulates tau seeding.

Neurodegenerative tauopathies are caused by accumulation of toxic tau protein assemblies. This appears to involve template-based seeding events, whereby tau monomer changes conformation and is recruited to a growing aggregate. Several large families of chaperone proteins, including Hsp70s and J domain proteins (JDPs) cooperate to regulate the folding of intracellular proteins such as tau, but the factors that coordinate this activity are not well known. The JDP DnaJC7 binds tau and reduces its intracellular aggregation. However, it is unknown whether this is specific to DnaJC7 or if other JDPs might be similarly involved. We used proteomics within a cell model to determine that DnaJC7 co-purified with insoluble tau and colocalized with intracellular aggregates. We individually knocked out every possible JDP and tested the effect on intracellular aggregation and seeding. DnaJC7 knockout decreased aggregate clearance and increased intracellular tau seeding. This depended on the ability of the J domain (JD) of DnaJC7 to bind to Hsp70, as JD mutations that block binding to Hsp70 abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abrogated its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.

Ensemble-based design of tau to inhibit aggregation while preserving biological activity.

The microtubule-associated protein tau is implicated in neurodegenerative diseases characterized by amyloid formation. Mutations associated with frontotemporal dementia increase tau aggregation propensity and disrupt its endogenous microtubule-binding activity. The structural relationship between aggregation propensity and biological activity remains unclear. We employed a multi-disciplinary approach, including computational modeling, NMR, cross-linking mass spectrometry, and cell models to design tau sequences that stabilize its structural ensemble. Our findings reveal that substitutions near the conserved 'PGGG' beta-turn motif can modulate local conformation, more stably engaging in interactions with the 306 VQIVYK 311 amyloid motif to decrease aggregation in vitro and in cells. Designed tau sequences maintain microtubule binding and explain why 3R isoforms of tau exhibit reduced pathogenesis over 4R isoforms. We propose a simple mechanism to reduce the formation of pathogenic species while preserving biological function, offering insights for therapeutic strategies aimed at reducing protein misfolding in neurodegenerative diseases.

The dual fates of exogenous tau seeds: Lysosomal clearance versus cytoplasmic amplification.

Tau assembly movement from the extracellular to intracellular space may underlie transcellular propagation of neurodegenerative tauopathies. This begins with tau binding to cell surface heparan sulfate proteoglycans, which triggers macropinocytosis. Pathological tau assemblies are proposed then to exit the vesicular compartment as "seeds" for replication in the cytoplasm. Tau uptake is highly efficient, but only ∼1 to 10% of cells that endocytose aggregates exhibit seeding. Consequently, we studied fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells or "biosensor" cells expressing FL tau or repeat domain. FL tau fibrils bound tubulin. Seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but fluorescence imaging revealed a small percentage that steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure, and most seeded cells had no evidence of endolysosome rupture. However, live-cell imaging indicated that cells with pre-existing Gal3-positive puncta were seeded at a slightly higher rate than the general population, suggesting a potential predisposing role for vesicle instability. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. The lysosome/autophagy inhibitor bafilomycin inhibited vesicular clearance, whereas the proteasome inhibitor MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds amplify, and are cleared by the proteasome.

The NMD Pathway Regulates GABARAPL1 mRNA during the EMT.

EMT is a reversible cellular process that is linked to gene expression reprogramming, which allows for epithelial cells to undergo a phenotypic switch to acquire mesenchymal properties. EMT is associated with cancer progression and cancer therapeutic resistance and it is known that, during the EMT, many stress response pathways, such as autophagy and NMD, are dysregulated. Therefore, our goal was to study the regulation of ATG8 family members (GABARAP, GABARAPL1, LC3B) by the NMD and to identify molecular links between these two cellular processes that are involved in tumor development and metastasis formation. IHC experiments, which were conducted in a cohort of patients presenting lung adenocarcinomas, showed high GABARAPL1 and low UPF1 levels in EMT+ tumors. We observed increased levels of GABARAPL1 correlated with decreased levels of NMD factors in A549 cells in vitro. We then confirmed that GABARAPL1 mRNA was indeed targeted by the NMD in a 3'UTR-dependent manner and we identified four overlapping binding sites for UPF1 and eIF4A3 that are potentially involved in the recognition of this transcript by the NMD pathway. Our study suggests that 3'UTR-dependent NMD might be an important mechanism that is involved in the induction of autophagy and could represent a promising target in the development of new anti-cancer therapies.

GABARAPL1 Inhibits EMT Signaling through SMAD-Tageted Negative Feedback.

The pathway of selective autophagy, leading to a targeted elimination of specific intracellular components, is mediated by the ATG8 proteins, and has been previously suggested to be involved in the regulation of the Epithelial-mesenchymal transition (EMT) during cancer's etiology. However, the molecular factors and steps of selective autophagy occurring during EMT remain unclear. We therefore analyzed a cohort of lung adenocarcinoma tumors using transcriptome analysis and immunohistochemistry, and found that the expression of ATG8 genes is correlated with that of EMT-related genes, and that GABARAPL1 protein levels are increased in EMT+ tumors compared to EMT- ones. Similarly, the induction of EMT in the A549 lung adenocarcinoma cell line using TGF-ß/TNF-α led to a high increase in GABARAPL1 expression mediated by the EMT-related transcription factors of the SMAD family, whereas the other ATG8 genes were less modified. To determine the role of GABARAPL1 during EMT, we used the CRISPR/Cas9 technology in A549 and ACHN kidney adenocarcinoma cell lines to deplete GABARAPL1. We then observed that GABARAPL1 knockout induced EMT linked to a defect of GABARAPL1-mediated degradation of the SMAD proteins. These findings suggest that, during EMT, GABARAPL1 might intervene in an EMT-regulatory loop. Indeed, induction of EMT led to an increase in GABARAPL1 levels through the activation of the SMAD signaling pathway, and then GABARAPL1 induced the autophagy-selective degradation of SMAD proteins, leading to EMT inhibition.

DnaJC7 binds natively folded structural elements in tau to inhibit amyloid formation.

Molecular chaperones, including Hsp70/J-domain protein (JDP) families, play central roles in binding substrates to prevent their aggregation. How JDPs select different conformations of substrates remains poorly understood. Here, we report an interaction between the JDP DnaJC7 and tau that efficiently suppresses tau aggregation in vitro and in cells. DnaJC7 binds preferentially to natively folded wild-type tau, but disease-associated mutants in tau reduce chaperone binding affinity. We identify that DnaJC7 uses a single TPR domain to recognize a ß-turn structural element in tau that contains the 275VQIINK280 amyloid motif. Wild-type tau, but not mutant, ß-turn structural elements can block full-length tau binding to DnaJC7. These data suggest DnaJC7 preferentially binds and stabilizes natively folded conformations of tau to prevent tau conversion into amyloids. Our work identifies a novel mechanism of tau aggregation regulation that can be exploited as both a diagnostic and a therapeutic intervention.

Seven practices for pursuing equity through learning health systems: Notes from the field.

INTRODUCTION: Despite learning health systems' focus on improvement in health outcomes, inequities in outcomes remain deep and persistent. To achieve and sustain health equity, it is critical that learning health systems (LHS) adapt and function in ways that directly prioritize equity. METHODS: We present guidance, including seven core practices, borne from theory, evidence, and experience, for actors within LHS pursuing equity. RESULTS: We provide a foundational definition of equity. We then offer seven core practices for how LHS may effectively pursue equity in health: establish principle, measure for equity, lead from lived experience, co-produce, redistribute power, practice a growth mindset, and engage beyond the healthcare system. We include three use cases that illustrate ways in which we have begun to center equity in the work of our own LHS. CONCLUSION: The achievement of equity requires real transformation at individual, institutional, and structural levels and requires sustained and persistent effort.

Cultivating social relationships and disrupting social isolation in low-income, high-disparity neighbourhoods in Ohio, USA.

Social isolation undermines health. Inequities in social networks exist due to historical and contemporary practices of socioeconomic and racial segregation. Thus, lower income and minority families are less likely to have the number, strength, and variety of social connections as higher income and white families. Therefore, social isolation may contribute to inequities in health and well-being across socioeconomic and racial groups. Disrupting social isolation by strengthening social networks may be a meaningful way to equitably improve population health. In this study we aimed to better understand the factors that influence the formation and sustainment of social connections in neighbourhoods experiencing a disproportionate burden of social needs and poor health outcomes. Participants were recruited through our community-academic partnership, Healthy Homes (HH). Healthy Homes serves families with pregnant women and/or children <6 years in two low-income, high-morbidity neighbourhoods, focusing on supporting families' needs and hopes. Between October 2016 and April 2017, we conducted in-depth qualitative interviews (n = 20) with English-speaking mothers and grandmothers of children under <6 years. Interviews were audio-recorded, transcribed verbatim and independently coded. After applying an a priori code list, we conducted emergent coding to identify additional themes. Themes focused on the social environment, including social connections and social isolation, among vulnerable populations in included neighbourhoods. Families want connection to one another and to resources but look to others to facilitate those connections. Families may want or need social connections but do not engage if it means sacrificing their values or sense of self-worth. These findings provide a deeper understanding of the factors that might allow us to disrupt social isolation by building relationships in communities that face social and health inequities.

Description of the unusual digestive tract of Platax orbicularis and the potential impact of Tenacibaculum maritimum infection.

BACKGROUND: Ephippidae fish are characterized by a discoid shape with a very small visceral cavity. Among them Platax orbicularis has a high economic potential due to its flesh quality and flesh to carcass ratio. Nonetheless, the development of its aquaculture is limited by high mortality rates, especially due to Tenacibaculum maritimum infection, occurring one to three weeks after the transfer of fishes from bio-secure land-based aquaculture system to the lagoon cages for growth. Among the lines of defense against this microbial infection, the gastrointestinal tract (GIT) is less studied. The knowledge about the morphofunctional anatomy of this organ in P. orbicularis is still scarce. Therefore, the aims of this study are to characterize the GIT in non-infected P. orbicularis juveniles to then investigate the impact of T. maritimum on this multifunctional organ. METHODS: In the first place, the morpho-anatomy of the GIT in non-infected individuals was characterized using various histological techniques. Then, infected individuals, experimentally challenged by T. maritimum were analysed and compared to the previously established GIT reference. RESULTS: The overlapped shape of the GIT of P. orbicularis is probably due to its constrained compaction in a narrow visceral cavity. Firstly, the GIT was divided into 10 sections, from the esophagus to the rectum. For each section, the structure of the walls was characterized, with a focus on mucus secretions and the presence of the Na+/K+ ATPase pump. An identification key allowing the characterization of the GIT sections using in toto histology is given. Secondly, individuals challenged with T. maritimum exhibited differences in mucus type and proportion and, modifications in the mucosal and muscle layers. These changes could induce an imbalance in the trade-off between the GIT functions which may be in favour of protection and immunity to the disadvantage of nutrition capacities.

Accompagner la professionnalisation des étudiants en soins infirmiers par l'approche du corps.

Supporting the professionalisation of student nurses through the approach of the body. When student nurses begin their training, their approach to others' bodies is shaped by social norms. This relationship changes through their studies and their practice placements, until they become qualified professionals. Becoming a caregiver requires them to change their relationship with the body. How does the training support students in the transformations of their representations, enabling them to be competent in the nurse-patient relationship within the other person's private sphere?

Differential basal expression of immune genes confers Crassostrea gigas resistance to Pacific oyster mortality syndrome.

BACKGROUND: As a major threat to the oyster industry, Pacific Oyster Mortality Syndrome (POMS) is a polymicrobial disease affecting the main oyster species farmed across the world. POMS affects oyster juveniles and became panzootic this last decade, but POMS resistance in some oyster genotypes has emerged. While we know some genetic loci associated with resistance, the underlying mechanisms remained uncharacterized. So, we developed a comparative transcriptomic approach using basal gene expression profiles between different oyster biparental families with contrasted phenotypes when confronted to POMS (resistant or susceptible). RESULTS: We showed that POMS resistant oysters show differential expression of genes involved in stress responses, protein modifications, maintenance of DNA integrity and repair, and immune and antiviral pathways. We found similarities and clear differences among different molecular pathways in the different resistant families. These results suggest that the resistance process is polygenic and partially varies according to the oyster genotype. CONCLUSIONS: We found differences in basal expression levels of genes related to TLR-NFκB, JAK-STAT and STING-RLR pathways. These differences could explain the best antiviral response, as well as the robustness of resistant oysters when confronted to POMS. As some of these genes represent valuable candidates for selective breeding, we propose future studies should further examine their function.

Tau local structure shields an amyloid-forming motif and controls aggregation propensity.

Tauopathies are neurodegenerative diseases characterized by intracellular amyloid deposits of tau protein. Missense mutations in the tau gene (MAPT) correlate with aggregation propensity and cause dominantly inherited tauopathies, but their biophysical mechanism driving amyloid formation is poorly understood. Many disease-associated mutations localize within tau's repeat domain at inter-repeat interfaces proximal to amyloidogenic sequences, such as 306VQIVYK311. We use cross-linking mass spectrometry, recombinant protein and synthetic peptide systems, in silico modeling, and cell models to conclude that the aggregation-prone 306VQIVYK311 motif forms metastable compact structures with its upstream sequence that modulates aggregation propensity. We report that disease-associated mutations, isomerization of a critical proline, or alternative splicing are all sufficient to destabilize this local structure and trigger spontaneous aggregation. These findings provide a biophysical framework to explain the basis of early conformational changes that may underlie genetic and sporadic tau pathogenesis.

ATG9A Is Overexpressed in Triple Negative Breast Cancer and Its In Vitro Extinction Leads to the Inhibition of Pro-Cancer Phenotypes.

Early detection and targeted treatments have led to a significant decrease in mortality linked to breast cancer (BC), however, important issues need to be addressed in the future. One of them will be to find new triple negative breast cancer (TNBC) therapeutic strategies, since none are currently efficiently targeting this subtype of BC. Since numerous studies have reported the possibility of targeting the autophagy pathway to treat or limit cancer progression, we analyzed the expression of six autophagy genes (ATG9A, ATG9B, BECLIN1, LC3B, NIX and P62/SQSTM1) in breast cancer tissue, and compared their expression with healthy adjacent tissue. In our study, we observed an increase in ATG9A mRNA expression in TNBC samples from our breast cancer cohort. We also showed that this increase of the transcript was confirmed at the protein level on paraffin-embedded tissues. To corroborate these in vivo data, we designed shRNA- and CRISPR/Cas9-driven inhibition of ATG9A expression in the triple negative breast cancer cell line MDA-MB-436, in order to determine its role in the regulation of cancer phenotypes. We found that ATG9A inhibition led to an inhibition of in vitro cancer features, suggesting that ATG9A can be considered as a new marker of TNBC and might be considered in the future as a target to develop new specific TNBC therapies.

Improved Sensitivity in Ultrasound Molecular Imaging With Coherence-Based Beamforming.

Ultrasound molecular imaging (USMI) is accomplished by detecting microbubble (MB) contrast agents that have bound to specific biomarkers, and can be used for a variety of imaging applications, such as the early detection of cancer. USMI has been widely utilized in preclinical imaging in mice; however, USMI in humans can be challenging because of the low concentration of bound MBs and the signal degradation caused by the presence of heterogenous soft tissue between the transducer and the lesion. Short-lag spatial coherence (SLSC) beamforming has been proposed as a robust technique that is less affected by poor signal quality than standard delay-and-sum (DAS) beamforming. In this paper, USMI performance was assessed using contrast-enhanced ultrasound imaging combined with DAS (conventional CEUS) and with SLSC (SLSC-CEUS). Each method was characterized by flow channel phantom experiments. In a USMI-mimicking phantom, SLSC-CEUS was found to be more robust to high levels of additive thermal noise than DAS, with a 6dB SNR improvement when the thermal noise level was +6dB or higher. However, SLSC-CEUS was also found to be insensitive to increases in MB concentration, making it a poor choice for perfusion imaging. USMI performance was also measured in vivo using VEGFR2-targeted MBs in mice with subcutaneous human hepatocellular carcinoma tumors, with clinical imaging conditions mimicked using a porcine tissue layer between the tumor and the transducer. SLSC-CEUS improved the SNR in each of ten tumors by an average of 41%, corresponding to 3.0dB SNR. These results indicate that the SLSC beamformer is well-suited for USMI applications because of its high sensitivity and robust properties under challenging imaging conditions.

Multimodal assessment of SERS nanoparticle biodistribution post ingestion reveals new potential for clinical translation of Raman imaging.

Despite extensive research and development, new nano-based diagnostic contrast agents have faced major barriers in gaining regulatory approval due to their potential systemic toxicity and prolonged retention in vital organs. Here we use five independent biodistribution techniques to demonstrate that oral ingestion of one such agent, gold-silica Raman nanoparticles, results in complete clearance with no systemic toxicity in living mice. The oral delivery mimics topical administration to the oral cavity and gastrointestinal (GI) tract as an alternative to intravenous injection. Biodistribution and clearance profiles of orally (OR) vs. intravenously (IV) administered Raman nanoparticles were assayed over the course of 48 h. Mice given either an IV or oral dose of Raman nanoparticles radiolabeled with approximately 100 µCi (3.7MBq) of 64Cu were imaged with dynamic microPET immediately post nanoparticle administration. Static microPET images were also acquired at 2 h, 5 h, 24 h and 48 h. Mice were sacrificed post imaging and various analyses were performed on the excised organs to determine nanoparticle localization. The results from microPET imaging, gamma counting, Raman imaging, ICP-MS, and hyperspectral imaging of tissue sections all correlated to reveal no evidence of systemic distribution of Raman nanoparticles after oral administration and complete clearance from the GI tract within 24 h. Paired with the unique signals and multiplexing potential of Raman nanoparticles, this approach holds great promise for realizing targeted imaging of tumors and dysplastic tissues within the oral cavity and GI-tract. Moreover, these results suggest a viable path for the first translation of high-sensitivity Raman contrast imaging into clinical practice.

The autophagy GABARAPL1 gene is epigenetically regulated in breast cancer models.

BACKGROUND: The GABARAP family members (GABARAP, GABARAPL1/GEC1 and GABARAPL2 /GATE-16) are involved in the intracellular transport of receptors and the autophagy pathway. We previously reported that GABARAPL1 expression was frequently downregulated in cancer cells while a high GABARAPL1 expression is a good prognosis marker for patients with lymph node-positive breast cancer. METHODS: In this study, we asked using qRT-PCR, western blotting and epigenetic quantification whether the expression of the GABARAP family was regulated in breast cancer by epigenetic modifications. RESULTS: Our data demonstrated that a specific decrease of GABARAPL1 expression in breast cancers was associated with both DNA methylation and histone deacetylation and that CREB-1 recruitment on GABARAPL1 promoter was required for GABARAPL1 expression. CONCLUSIONS: Our work strongly suggests that epigenetic inhibitors and CREB-1 modulators may be used in the future to regulate autophagy in breast cancer cells.

New melatonin (MT1/MT2) ligands: design and synthesis of (8,9-dihydro-7H-furo[3,2-f]chromen-1-yl) derivatives.

Herein we describe the synthesis of novel tricyclic analogues issued from the rigidification of the methoxy group of the benzofuranic analogue of melatonin as MT1 and MT2 ligands. Most of the synthesized compounds displayed high binding affinities at MT1 and MT2 receptors subtypes. Compound 6b (MT1, Ki=0.07nM; MT2, Ki=0.08nM) exhibited with the vinyl 6c and allyl 6d the most interesting derivatives of this series. Functional activity of these compounds showed full agonist activity with EC50 in the nanomolar range. Compounds 6a (EC50=0.8nM and Emax=98%) and 6b (EC50=0.2nM and Emax=121%) exhibited good pharmacological profiles.