BACE1 activity regulates cell surface contactin-2 levels

Background: Although BACE1 is a major therapeutic target for Alzheimer’s disease (AD), potential side effects of BACE1 inhibition are not well characterized. BACE1 cleaves over 60 putative substrates, however the majority of these cleavages have not been characterized. Here we investigated BACE1-mediated cleavage of human contactin-2, a GPI-anchored cell adhesion molecule. Results: Our initial protein sequence analysis showed that contactin-2 harbors a strong putative BACE1 cleavage site close to its GPI membrane linker domain. When we overexpressed BACE1 in CHO cells stably transfected with human contactin-2, we found increased release of soluble contactin-2 in the conditioned media. Conversely, pharmacological inhibition of BACE1 in CHO cells expressing human contactin-2 and mouse primary neurons decreased soluble contactin-2 secretion. The BACE1 cleavage site mutation 1008MM/AA dramatically impaired soluble contactin-2 release. We then asked whether contactin-2 release induced by BACE1 expression would concomitantly decrease cell surface levels of contactin-2. Using immunofluorescence and surface-biotinylation assays, we showed that BACE1 activity tightly regulates contactin-2 surface levels in CHO cells as well as in mouse primary neurons. Finally, contactin-2 levels were decreased in Alzheimer’s disease brain samples correlating inversely with elevated BACE1 levels in the same samples. Conclusion: Our results clearly demonstrate that mouse and human contactin-2 are physiological substrates for BACE1. BACE1-mediated contactin-2 cleavage tightly regulates the surface expression of contactin-2 in neuronal cells. Given the role of contactin-2 in cell adhesion, neurite outgrowth and axon guidance, our data suggest that BACE1 may play an important role in these physiological processes by regulating contactin-2 surface levels

Human Neurospheroid Arrays for In Vitro Studies of Alzheimer’s Disease

Neurospheroids are commonly used for in vitro disease modeling and drug screening. However, the heterogeneity in size of the neurospheroids mixtures available through current methods limits their utility when employed for basic mechanistic studies of neurodegenerative diseases or screening for new interventions. Here, we generate neurospheroids from immortalized neural progenitor cells and human induced pluripotent stem cells that are uniform in size, into large-scale arrays. In proof of concept experiments, we validate the neurospheroids array as a sensitive and robust tool for screening compounds over extended time. We show that when suspended in three-dimensional extracellular matrix up to several weeks, the stem cell-derived neurospheroids display extensive neurite outgrowth and extend thick bundles of dendrites outward. We also cultivate genetically-engineered stem cell-derived neurospheroids with familial Alzheimer’s disease mutations for eight weeks in our microarray system. Interestingly, we observed robust accumulation of amyloid-β and phosphorylated tau, key hallmarks of Alzheimer’s disease. Overall, our in vitro model for engineering neurospheroid arrays is a valuable tool for studying complex neurodegenerative diseases and accelerating drug discovery

Synaptotagmins interact with APP and promote Aβ generation

Background: Accumulation of the β-amyloid peptide (Aβ) is a major pathological hallmark of Alzheimer’s disease (AD). Recent studies have shown that synaptic Aβ toxicity may directly impair synaptic function. However, proteins regulating Aβ generation at the synapse have not been characterized. Here, we sought to identify synaptic proteins that interact with the extracellular domain of APP and regulate Aβ generation. Results: Affinity purification-coupled mass spectrometry identified members of the Synaptotagmin (Syt) family as novel interacting proteins with the APP ectodomain in mouse brains. Syt-1, −2 and −9 interacted with APP in cells and in mouse brains in vivo. Using a GST pull-down approach, we have further demonstrated that the Syt interaction site lies in the 108 amino acids linker region between the E1 and KPI domains of APP. Stable overexpression of Syt-1 or Syt-9 with APP in CHO and rat pheochromocytoma cells (PC12) significantly increased APP-CTF and sAPP levels, with a 2 to 3 fold increase in secreted Aβ levels in PC12 cells. Moreover, using a stable knockdown approach to reduce the expression of endogenous Syt-1 in PC12 cells, we have observed a ~ 50 % reduction in secreted Aβ generation. APP processing also decreased in these cells, shown by lower CTF levels. Lentiviral-mediated knock down of endogenous Syt-1 in mouse primary neurons also led to a significant reduction in both Aβ40 and Aβ42 generation. As secreted sAPPβ levels were significantly reduced in PC12 cells lacking Syt-1 expression, our results suggest that Syt-1 regulates Aβ generation by modulating BACE1-mediated cleavage of APP. Conclusion: Altogether, our data identify the synaptic vesicle proteins Syt-1 and 9 as novel APP-interacting proteins that promote Aβ generation and thus may play an important role in the pathogenesis of AD. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0028-5) contains supplementary material, which is available to authorized users

COVID-19 in breast cancer patients: a subanalysis of the OnCovid registry

Background: cancer patients are at higher risk of COVID-19 complications and mortality than the rest of the population. Breast cancer patients seem to have better prognosis when infected by SARS-CoV-2 than other cancer patients. Methods: we report a subanalysis of the OnCovid study providing more detailed information in the breast cancer population. Results: we included 495 breast cancer patients with a SARS-CoV-2 infection. Mean age was 62.6 years; 31.5% presented more than one comorbidity. The most frequent breast cancer subtype was luminal-like (n = 245, 49.5%) and 177 (35.8%) had metastatic disease. A total of 332 (67.1%) patients were receiving active treatment, with radical intent in 232 (47.6%) of them. Hospitalization rate was 58.2% and all-cause mortality rate was 20.3%. One hundred twenty-nine (26.1%) patients developed one COVID-19 complication, being acute respiratory failure the most common (n = 74, 15.0%). In the multivariable analysis, age older than 70 years, presence of COVID-19 complications, and metastatic disease were factors correlated with worse outcomes, while ongoing anticancer therapy at time of COVID-19 diagnosis appeared to be a protective factor. No particular oncological treatment was related to higher risk of complications. In the context of SARS-CoV-2 infection, 73 (18.3%) patients had some kind of modification on their oncologic treatment. At the first oncological reassessment (median time: 46.9 days ± 36.7), 255 (51.6%) patients reported to be fully recovered from the infection. There were 39 patients (7.9%) with long-term SARS-CoV-2-related complications. Conclusion: in the context of COVID-19, our data confirm that breast cancer patients appear to have lower complications and mortality rate than expected in other cancer populations. Most breast cancer patients can be safely treated for their neoplasm during SARS-CoV-2 pandemic. Oncological treatment has no impact on the risk of SARS-CoV-2 complications, and, especially in the curative setting, the treatment should be modified as little as possible