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Abstract: The hallmarks of Alzheimer’s disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aβ-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network.
Author Summary: More than 20 years ago, the amyloid precursor protein (APP) was identified as the precursor protein of the Aβ peptide, the main component of senile plaques in brains affected by Alzheimer’s disease. However, little is known about the physiological function of amyloid precursor protein. Allocating APP to the proteome of the structurally and functionally dynamic presynaptic active zone highlights APP as a hitherto unknown player within the presynaptic network. The hippocampus is the most prominent brain region for learning and memory consolidation, and a vulnerable target for neurodegenerative disease, e. g. Alzheimer’s disease. Therefore, our experimental design is focused on the hippocampal neurotransmitter release site. Currently, the underlying mechanism of how APP acts within presynaptic networks is still elusive. Within the scope of this research article, we constructed a network of APP within the presynaptic active zone and how deletion of APP affects these individual networks. We combine bioinformatics tools and biochemical approaches to address the dataset provided by proteomics. Furthermore, we could unravel that APP executes regulatory functions within the synaptic vesicle cycle, cytoskeletal rearrangements and Ca2+-homeostasis. Taken together, our findings offer a new perspective on the physiological function of APP in the central nervous system and may provide a molecular link to the pathogenesis of Alzheimer’s disease.
Synaptic release sites are characterized by exocytosis-competent synaptic vesicles tightly anchored to the presynaptic active zone (PAZ) whose proteome orchestrates the fast signaling events involved in synaptic vesicle cycle and plasticity. Allocation of the amyloid precursor protein (APP) to the PAZ proteome implicated a functional impact of APP in neuronal communication. In this study, we combined state-of-the-art proteomics, electrophysiology and bioinformatics to address protein abundance and functional changes at the native hippocampal PAZ in young and old APP-KO mice. We evaluated if APP deletion has an impact on the metabolic activity of presynaptic mitochondria. Furthermore, we quantified differences in the phosphorylation status after long-term-potentiation (LTP) induction at the purified native PAZ. We observed an increase in the phosphorylation of the signaling enzyme calmodulin-dependent kinase II (CaMKII) only in old APP-KO mice. During aging APP deletion is accompanied by a severe decrease in metabolic activity and hyperphosphorylation of CaMKII. This attributes an essential functional role to APP at hippocampal PAZ and putative molecular mechanisms underlying the age-dependent impairments in learning and memory in APP-KO mice.
Membrane receptors are central to cell-cell communication. Receptor clustering at the plasma membrane modulates physiological responses, and mesoscale receptor organization is critical for downstream signaling. Spatially restricted cluster formation of the neuropeptide Y2 hormone receptor (Y2R) was observed in vivo; however, the relevance of this confinement is not fully understood. Here, we controlled Y2R clustering in situ by a chelator nanotool. Due to the multivalent interaction, we observed a dynamic exchange in the microscale confined regions. Fast Y2R enrichment in clustered areas triggered a ligand-independent downstream signaling determined by an increase in cytosolic calcium, cell spreading, and migration. We revealed that the cell response to ligand-induced activation was amplified when cells were pre-clustered by the nanotool. Ligand-independent signaling by clustering differed from ligand-induced activation in the binding of arrestin-3 as downstream effector, which was recruited to the confined regions only in the presence of the ligand. This approach enables in situ clustering of membrane receptors and raises the possibility to explore different modalities of receptor activation.
Background & Aims: Liver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis.
Methods; Intracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor β (TGFβ) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model.
Results: In our study, we show that TGFβ and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets’ expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease.
Conclusions: We provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.
Introduction: Merkel cell carcinoma (MCC) is linked to the presence of clonally integrated Merkel cell polyomavirus (MCPyV) in up to 80% of the cases. The aim of the study was to determine the prognostic value of baseline MCPyV viral load and lymphocytic infiltration.
Methods: MCPyV DNA prevalence, integration status and viral load were determined by specific quantitative real-time PCR in surgical specimens obtained from 49 patients with MCC treated with (n = 22, 45%) or without postoperative radiotherapy (RT). CD8+ tumor infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) status were assessed using immunohistochemistry. MCPyV characteristics and immune marker expression were correlated with clinicopathological factors and overall survival (OS).
Results: Median age at diagnosis was 74 (range, 42–100); 51% of the patients were female. One-, three, and five-year OS rates were 83.8, 58.6, and 47.1%, respectively. A positive MCPyV status was associated with female gender (p = 0.042). Tumor localization (head/arms vs. trunk) positively correlated with PD-L1 status (p = 0.011) and combined CD8/PD-L1 expression (p = 0.038). Overall CD8+ infiltration was inversely associated with N-stage (p = 0.048). Stromal TILs correlated significantly with both PD-L1 expression (p = 0.010) and N-stage (p = 0.037). A high viral load (>median) was significantly associated with worse OS (p = 0.029) and high intratumoral CD8+ infiltration with improved OS for the entire cohort (p = 0.045).
Conclusion: These data provide important insight on the role of MCPy DNA viral load and TILs in the context of PD-L1 in patients with Merkel cell carcinoma. Future clinical studies should aim to explore the effect of PD-1/PD-L1 immune-checkpoint inhibitors in combination with existing radiotherapy approaches.
Membrane receptor clustering is fundamental to cell–cell communication; however, the physiological function of receptor clustering in cell signaling remains enigmatic. Here, we developed a dynamic platform to induce cluster formation of neuropeptide Y2 hormone receptors (Y2R) in situ by a chelator nanotool. The multivalent interaction enabled a dynamic exchange of histidine-tagged Y2R within the clusters. Fast Y2R enrichment in clustered areas triggered ligand-independent signaling as determined by an increase in cytosolic calcium and cell migration. Notably, the calcium and motility response to ligand-induced activation was amplified in preclustered cells, suggesting a key role of receptor clustering in sensitizing the dose response to lower ligand concentrations. Ligand-independent versus ligand-induced signaling differed in the binding of arrestin-3 as a downstream effector, which was recruited to the clusters only in the presence of the ligand. This approach allows in situ receptor clustering, raising the possibility to explore different receptor activation modalities.
Background: Juvenile dermatomyositis (JDM) is the most common inflammatory myopathy in childhood and a major cause of morbidity among children with pediatric rheumatic diseases. The management of JDM is very heterogeneous. The JDM working group of the Society for Pediatric Rheumatology (GKJR) aims to define consensus- and practice-based strategies in order to harmonize diagnosis, treatment and monitoring of JDM.
Methods: The JDM working group was established in 2015 consisting of 23 pediatric rheumatologists, pediatric neurologists and dermatologists with expertise in the management of JDM. Current practice patterns of management in JDM had previously been identified via an online survey among pediatric rheumatologists and neurologists. Using a consensus process consisting of online surveys and a face-to-face consensus conference statements were defined regarding the diagnosis, treatment and monitoring of JDM. During the conference consensus was achieved via nominal group technique. Voting took place using an electronic audience response system, and at least 80% consensus was required for individual statements.
Results: Overall 10 individual statements were developed, finally reaching a consensus of 92 to 100% regarding (1) establishing a diagnosis, (2) case definitions for the application of the strategies (moderate and severe JDM), (3) initial diagnostic testing, (4) monitoring and documentation, (5) treatment targets within the context of a treat-to-target strategy, (6) supportive therapies, (7) explicit definition of a treat-to-target strategy, (8) various glucocorticoid regimens, including intermittent intravenous methylprednisolone pulse and high-dose oral glucocorticoid therapies with tapering, (9) initial glucocorticoid-sparing therapy and (10) management of refractory disease.
Conclusion: Using a consensus process among JDM experts, statements regarding the management of JDM were defined. These statements and the strategies aid in the management of patients with moderate and severe JDM.
Introduction: Evidence from a number of open-label, uncontrolled studies has suggested that rituximab may benefit patients with autoimmune diseases who are refractory to standard-of-care. The objective of this study was to evaluate the safety and clinical outcomes of rituximab in several standard-of-care-refractory autoimmune diseases (within rheumatology, nephrology, dermatology and neurology) other than rheumatoid arthritis or non-Hodgkin's lymphoma in a real-life clinical setting.
Methods: Patients who received rituximab having shown an inadequate response to standard-of-care had their safety and clinical outcomes data retrospectively analysed as part of the German Registry of Autoimmune Diseases. The main outcome measures were safety and clinical response, as judged at the discretion of the investigators.
Results: A total of 370 patients (299 patient-years) with various autoimmune diseases (23.0% with systemic lupus erythematosus, 15.7% antineutrophil cytoplasmic antibody-associated granulomatous vasculitides, 15.1% multiple sclerosis and 10.0% pemphigus) from 42 centres received a mean dose of 2,440 mg of rituximab over a median (range) of 194 (180 to 1,407) days. The overall rate of serious infections was 5.3 per 100 patient-years during rituximab therapy. Opportunistic infections were infrequent across the whole study population, and mostly occurred in patients with systemic lupus erythematosus. There were 11 deaths (3.0% of patients) after rituximab treatment (mean 11.6 months after first infusion, range 0.8 to 31.3 months), with most of the deaths caused by infections. Overall (n = 293), 13.3% of patients showed no response, 45.1% showed a partial response and 41.6% showed a complete response. Responses were also reflected by reduced use of glucocorticoids and various immunosuppressives during rituximab therapy and follow-up compared with before rituximab. Rituximab generally had a positive effect on patient well-being (physician's visual analogue scale; mean improvement from baseline of 12.1 mm).
Conclusions: Data from this registry indicate that rituximab is a commonly employed, well-tolerated therapy with potential beneficial effects in standard of care-refractory autoimmune diseases, and support the results from other open-label, uncontrolled studies.