Person:

Kovacs, Dora

Background: The voltage-gated sodium channel β2 subunit (Navβ2) is a physiological substrate of BACE1 (β-site APP cleaving enzyme) and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2. Results: We found a major (147-148 L↓M, where ↓ indicates the cleavage site) and a minor (144145 L↓Q) BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI) and ~75% in cells expressing Navβ2 (147LM/AA) as compared to cells expressing wildtype Navβ2. Conclusion: We identified a major (147-148 L↓M) and a minor (144-145 L↓Q) BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.

Alzheimer’s disease (AD) is the most common form of dementia, characterized by two pathological hallmarks: β-amyloid plaques and neurofibrillary tangles1. The amyloid hypothesis of AD posits that excessive accumulation of β-amyloid peptide (Aβ) leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau2,3. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. AD mouse models with familial AD (FAD) mutations exhibited Aβ-induced synaptic and memory deficits but they were not able to fully recapitulate other key pathological events of AD including clear neurofibrillary tangle pathology4,5. AD patient-derived human neurons showed elevated levels of toxic Aβ species and phosphor-tau (p-tau) but they also could not replicate β-amyloid plaques or neurofibrillary tangles6-11. Here we show that FAD mutations in the amyloid-β precursor protein (APP) and presenilin (PS) 1 genes are able to induce robust extracellular deposition of Aβ, including β-amyloid plaques, in a human neural stem cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of p-tau in the soma and neurites. Immunoelectron microscopy also demonstrated the presence of filamentous tau, only in detergent-resistant fractions from 3D-cultured cells expressing FAD mutations. Inhibition of Aβ generation with β- or γ-secretase inhibitors not only decreased Aβ pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated Aβ-mediated tau phosphorylation. In summary, we have successfully recapitulated Aβ and tau pathology in a single 3D human neural cell culture system for the first time. Our unique strategy for recapitulating AD pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models for other neurodegenerative disorders.

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.