Correction: Palmitoylated APP Forms Dimers, Cleaved by BACE1.

[This corrects the article DOI: 10.1371/journal.pone.0166400.].

The neuronal-specific isoform of BIN1 regulates ß-secretase cleavage of APP and Aß generation in a RIN3-dependent manner.

Genome-wide association studies have identified BIN1 (Bridging integrator 1) and RIN3 (Ras and Rab interactor 3) as genetic risk factors for late-onset Alzheimer's disease (LOAD). The neuronal isoform of BIN1 (BIN1V1), but not the non-neuronal isoform (BIN1V9), has been shown to regulate tau-pathology and Aß generation via RAB5-mediated endocytosis in neurons. BIN1 directly interacts with RIN3 to initiate RAB5-mediated endocytosis, which is essential for ß-secretase (BACE1)-mediated ß-secretase cleavage of ß-amyloid precursor protein (APP) to generate Amyloid-ß (Aß), the key component of senile plaques in AD. Understanding the regulatory roles of BIN1 (neuronal BIN1V1) and RIN3 in ß-secretase mediated cleavage of APP and Aß generation is key to developing novel therapeutics to delay or prevent AD progression. Neuronal and non-neuronal isoforms of BIN1 (BIN1V1 and BIN1V9, respectively) were introduced with RIN3 into an in vitro cell-based system to test RIN3-dependent effects of neuronal BIN1V1 and non-neuronal BIN1V9 on ß-secretase-mediated cleavage of APP and Aß generation. Confocal microscopy was performed to examine RIN3-dependent subcellular localization of BIN1V1 and BIN1V9. Western blot analysis was performed to assess the effects of RIN3 and BIN1V1/BIN1V9 on ß-secretase mediated processing of APP. We enriched cells expressing BIN1V1 without or with RIN3 via FACS to measure Aß generation using Aß ELISA assay, and to evaluate APP internalization by chasing biotinylated or antibody-labeled cell surface APP. Neuronal BIN1V1 containing the CLAP domain and non-neuronal BIN1V9 lacking the CLAP domain are the major isoforms present in the brain. Employing confocal microscopy, we showed that RIN3 differentially regulates the recruitment of both BIN1V1 and BIN1V9 into RAB5-endosomes. We further showed that BIN1V1, but not BIN1V9, downregulates ß-secretase (BACE1)-mediated processing of APP in a RIN3-dependent manner. Overexpression of BIN1V1 also attenuated Aß generation in a RIN3-dependent manner. Using cell-based internalization assays, we show BIN1V1, but not BIN1V9, delays the endocytosis of APP, but not of BACE1, into early endosomes, thereby spatially and temporally separating these two proteins into different cellular compartments, resulting in reduced cleavage of APP by BACE1 and reduced Aß generation-all in a RIN3-dependent manner. Finally, we show that RIN3 sequesters BIN1V1 in RAB5-positive early endosomes, likely via the CLAP-domain, resulting in attenuated ß-secretase processing of APP and Aß generation by delaying endocytosis of APP. Our findings provide new mechanistic data on how two AD-associated molecules, RIN3 and BIN1 (neuronal BIN1V1), interact to govern Aß production, implicating these two proteins as potential therapeutic targets for the prevention and treatment of AD.

Microfluidic separation of axonal and somal compartments of neural progenitor cells differentiated in a 3D matrix.

This protocol describes the differentiation of human neural progenitor cells (hNPCs) in a microfluidic device containing a thin 3D matrix with two separate chambers, enabling a cleaner separation between axons and soma/bulk neurons. We have used this technique to study how mitochondria-associated ER membranes (MAMs) regulate the generation of somal and axonal amyloid ß (Aß) in FAD hNPCs, a cellular model of Alzheimer's disease. This protocol also details the quantification of Aß molecules and isolation of pure axons via axotomy. For complete details on the use and execution of this profile, please refer to Bhattacharyya et al. (2021).

Axonal generation of amyloid-ß from palmitoylated APP in mitochondria-associated endoplasmic reticulum membranes.

Axonal generation of Alzheimer's disease (AD)-associated amyloid-ß (Aß) plays a key role in AD neuropathology, but the cellular mechanisms involved in its release have remained elusive. We previously reported that palmitoylated APP (palAPP) partitions to lipid rafts where it serves as a preferred substrate for ß-secretase. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are cholesterol-rich lipid rafts that are upregulated in AD. Here, we show that downregulating MAM assembly by either RNA silencing or pharmacological modulation of the MAM-resident sigma1 receptor (S1R) leads to attenuated ß-secretase cleavage of palAPP. Upregulation of MAMs promotes trafficking of palAPP to the cell surface, ß-secretase cleavage, and Aß generation. We develop a microfluidic device and use it to show that MAM levels alter Aß generation specifically in neuronal processes and axons, but not in cell bodies. These data suggest therapeutic strategies for reducing axonal release of Aß and attenuating ß-amyloid pathology in AD.

Palmitoylated APP Forms Dimers, Cleaved by BACE1.

A major rate-limiting step for Aß generation and deposition in Alzheimer's disease brains is BACE1-mediated cleavage (ß-cleavage) of the amyloid precursor protein (APP). We previously reported that APP undergoes palmitoylation at two cysteine residues (Cys186 and Cys187) in the E1-ectodomain. 8-10% of total APP is palmitoylated in vitro and in vivo. Palmitoylated APP (palAPP) shows greater preference for ß-cleavage than total APP in detergent resistant lipid rafts. Protein palmitoylation is known to promote protein dimerization. Since dimerization of APP at its E1-ectodomain results in elevated BACE1-mediated cleavage of APP, we have now investigated whether palmitoylation of APP affects its dimerization and whether this leads to elevated ß-cleavage of the protein. Here we report that over 90% of palAPP is dimerized while only ~20% of total APP forms dimers. PalAPP-dimers are predominantly cis-oriented while total APP dimerizes in both cis- and trans-orientation. PalAPP forms dimers 4.5-times more efficiently than total APP. Overexpression of the palmitoylating enzymes DHHC7 and DHHC21 that increase palAPP levels and Aß release, also increased APP dimerization in cells. Conversely, inhibition of APP palmitoylation by pharmacological inhibitors reduced APP-dimerization in coimmunoprecipitation and FLIM/FRET assays. Finally, in vitro BACE1-activity assays demonstrate that palmitoylation-dependent dimerization of APP promotes ß-cleavage of APP in lipid-rich detergent resistant cell membranes (DRMs), when compared to total APP. Most importantly, generation of sAPPß-sAPPß dimers is dependent on APP-palmitoylation while total sAPPß generation is not. Since BACE1 shows preference for palAPP dimers over total APP, palAPP dimers may serve as novel targets for effective ß-cleavage inhibitors of APP as opposed to BACE1 inhibitors.

The E280A presenilin mutation reduces voltage-gated sodium channel levels in neuronal cells.

BACKGROUND: Familial Alzheimer's disease (FAD) mutations in presenilin (PS) modulate PS/γ-secretase activity and therefore contribute to AD pathogenesis. Previously, we found that PS/γ-secretase cleaves voltage-gated sodium channel ß2-subunits (Navß2), releases the intracellular domain of Navß2 (ß2-ICD), and thereby, increases intracellular sodium channel α-subunit Nav1.1 levels. Here, we tested whether FAD-linked PS1 mutations modulate Navß2 cleavages and Nav1.1 levels. OBJECTIVE: It was the aim of this study to analyze the effects of PS1-linked FAD mutations on Navß2 processing and Nav1.1 levels in neuronal cells. METHODS: We first generated B104 rat neuroblastoma cells stably expressing Navß2 and wild-type PS1 (wtPS1), PS1 with one of three FAD mutations (E280A, M146L or ΔE9), or PS1 with a non-FAD mutation (D333G). Navß2 processing and Nav1.1 protein and mRNA levels were then analyzed by Western blot and real-time RT-PCR, respectively. RESULTS: The FAD-linked E280A mutation significantly decreased PS/γ-secretase-mediated processing of Navß2 as compared to wtPS1 controls, both in cells and in a cell-free system. Nav1.1 mRNA and protein levels, as well as the surface levels of Nav channel α-subunits, were also significantly reduced in PS1(E280A) cells. CONCLUSION: Our data indicate that the FAD-linked PS1(E280A) mutation decreases Nav channel levels by partially inhibiting the PS/γ-secretase-mediated cleavage of Navß2 in neuronal cells.

Palmitoylation of amyloid precursor protein regulates amyloidogenic processing in lipid rafts.

Brains of patients affected by Alzheimer's disease (AD) contain large deposits of aggregated amyloid ß-protein (Aß). Only a small fraction of the amyloid precursor protein (APP) gives rise to Aß. Here, we report that ∼10% of APP undergoes a post-translational lipid modification called palmitoylation. We identified the palmitoylation sites in APP at Cys¹86 and Cys¹87. Surprisingly, point mutations introduced into these cysteines caused nearly complete ER retention of APP. Thus, either APP palmitoylation or disulfide bridges involving these Cys residues appear to be required for ER exit of APP. In later compartments, palmitoylated APP (palAPP) was specifically enriched in lipid rafts. In vitro BACE1 cleavage assays using cell or mouse brain lipid rafts showed that APP palmitoylation enhanced BACE1-mediated processing of APP. Interestingly, we detected an age-dependent increase in endogenous mouse brain palAPP levels. Overexpression of selected DHHC palmitoyl acyltransferases increased palmitoylation of APP and doubled Aß production, while two palmitoylation inhibitors reduced palAPP levels and APP processing. We have found previously that acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition led to impaired APP processing. Here we demonstrate that pharmacological inhibition or genetic inactivation of ACAT decrease lipid raft palAPP levels by up to 76%, likely resulting in impaired APP processing. Together, our results indicate that APP palmitoylation enhances amyloidogenic processing by targeting APP to lipid rafts and enhancing its BACE1-mediated cleavage. Thus, inhibition of palAPP formation by ACAT or specific palmitoylation inhibitors would appear to be a valid strategy for prevention and/or treatment of AD.

Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit.

BACKGROUND: The voltage-gated sodium channel ß2 subunit (Navß2) is a physiological substrate of BACE1 (ß-site APP cleaving enzyme) and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navß2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navß2. 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 Navß2 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 Navß2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navß2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navß2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navß2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navß2 cleavage products decreased by ~50% in cells expressing Navß2 (147LM/VI) and ~75% in cells expressing Navß2 (147LM/AA) as compared to cells expressing wildtype Navß2. CONCLUSION: We identified a major (147-148 L↓M) and a minor (144-145 L↓Q) BACE1 cleavage site in human Navß2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navß2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.

ACAT inhibition and amyloid beta reduction.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder. Accumulation and deposition of the beta-amyloid (Abeta) peptide generated from its larger amyloid precursor protein (APP) is one of the pathophysiological hallmarks of AD. Intracellular cholesterol was shown to regulate Abeta production. Recent genetic and biochemical studies indicate that not only the amount, but also the distribution of intracellular cholesterol is critical to regulate Abeta generation. Acyl-coenzyme A: cholesterol acyl-transferase (ACAT) is a family of enzymes that regulates the cellular distribution of cholesterol by converting membrane cholesterol into hydrophobic cholesteryl esters for cholesterol storage and transport. Using pharmacological inhibitors and transgenic animal models, we and others have identified ACAT1 as a potential therapeutic target to lower Abeta generation and accumulation. Here we discuss data focusing on ACAT inhibition as an effective strategy for the prevention and treatment of AD.

Inhibition of acyl-coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway.

Amyloid beta-peptide (Abeta) has a central role in the pathogenesis of Alzheimer's disease (AD). Cellular cholesterol homeostasis regulates endoproteolytic generation of Abeta from the amyloid precursor protein (APP). Previous studies have identified acyl-coenzyme A: cholesterol acyltransferase (ACAT), an enzyme that regulates subcellular cholesterol distribution, as a potential therapeutic target for AD. Inhibition of ACAT activity decreases Abeta generation in cell- and animal-based models of AD through an unknown mechanism. Here we show that ACAT inhibition retains a fraction of APP molecules in the early secretory pathway, limiting the availability of APP for secretase-mediated proteolytic processing. ACAT inhibitors delayed the trafficking of immature APP molecules from the endoplasmic reticulum (ER) as shown by metabolic labeling and live-cell imaging. This resulted in partial ER retention of APP and enhanced ER-associated degradation of APP by the proteasome, without activation of the unfolded protein response pathway. The ratio of mature APP to immature APP was reduced in brains of mice treated with ACAT inhibitors, and strongly correlated with reduced brain APP-C99 and cerebrospinal fluid Abeta levels in individual animals. Our results identify a novel ACAT-dependent mechanism that regulates secretory trafficking of APP, likely contributing to decreased Abeta generation in vivo.

Differences in Galpha12- and Galpha13-mediated plasma membrane recruitment of p115-RhoGEF.

Regulator of G protein signaling domain-containing Rho guanine-nucleotide exchange factors (RGS-RhoGEFs) directly links activated forms of the G12 family of heterotrimeric G protein alpha subunits to the small GTPase Rho. Stimulation of G(12/13)-coupled GPCRs or expression of constitutively activated forms of alpha(12) and alpha(13) has been shown to induce the translocation of the RGS-RhoGEF, p115-RhoGEF, from the cytoplasm to the plasma membrane (PM). However, little is known regarding the functional importance and mechanisms of this regulated PM recruitment, and thus PM recruitment of p115-RhoGEF is the focus of this report. A constitutively PM-localized mutant of p115-RhoGEF shows a much greater activity compared to wild type p115-RhoGEF in promoting Rho-dependent neurite retraction of NGF-differentiated PC12 cells, providing the first evidence that PM localization can activate p115-RhoGEF signaling. Next, we uncovered the unexpected finding that Rho is required for alpha(13)-induced PM translocation of p115-RhoGEF. However, inhibition of Rho did not prevent alpha(12)-induced PM translocation of p115-RhoGEF. Additional differences between alpha(13) and alpha(12) in promoting PM recruitment of p115-RhoGEF were revealed by analyzing RGS domain mutants of p115-RhoGEF. Activated alpha(12) effectively recruits the isolated RGS domain of p115-RhoGEF to the PM, whereas alpha(13) only weakly does. On the other hand, alpha(13) strongly recruits to the PM a p115-RhoGEF mutant containing amino acid substitutions in an acidic region at the N-terminus of the RGS domain; however, alpha(12) is unable to recruit this p115-RhoGEF mutant to the PM. These studies provide new insight into the function and mechanisms of alpha(12/13)-mediated PM recruitment of p115-RhoGEF.

Cooperative regulation of p70S6 kinase by receptor tyrosine kinases and G protein-coupled receptors augments airway smooth muscle growth.

We have previously demonstrated that concomitant activation of receptor tyrosine kinases and certain G protein-coupled receptors (GPCRs) can promote a synergistic increase in the rate of airway smooth muscle cell (ASM) proliferation. Here we clarify the role of p70S6 kinase (p70S6K) as an integrator of receptor tyrosine kinase and GPCR signaling that augments ASM DNA synthesis by demonstrating that specific p70S6K phosphorylation sites receive distinct regulatory input from GPCRs that promotes sustained kinase activity critical to mitogenesis. Prolonged stimulation of ASM cells with EGF and thrombin induced a greater than additive effect in levels of p70S6K phosphorylated at residue T389, whereas a significant but more modest increase in the level of T229 and T421/S424 phosphorylation was also observed. The augmenting effects of thrombin could be dissociated from p42/p44 MAPK activation, as selective inhibition of thrombin-stimulated p42/p44 failed to alter the profile of cooperative p70S6K T389 phosphorylation, p70S6K kinase activity, or ASM [(3)H]thymidine incorporation. Thrombin stimulated a sustained increase in the level of Akt phosphorylation and also augmented EGF-stimulated Akt phosphorylation. The cooperative effects of thrombin on Akt/p70S6K phosphorylation and [(3)H]thymidine incorporation were all attenuated by heterologous expression of Gbetagamma sequestrants. These data suggest that PI3K-dependent T389/T229 phosphorylation is limiting in late-phase p70S6K activation by EGF and contributes to the cooperative effect of GPCRs on p70S6K activity and cell growth.

Mutation of an N-terminal acidic-rich region of p115-RhoGEF dissociates alpha13 binding and alpha13-promoted plasma membrane recruitment.

The Ras homology (Rho) guanine nucleotide exchange factor p115-RhoGEF couples the alpha(13) heterotrimeric guanine nucleotide binding protein (G protein) subunit to Rho GTPase. Alpha(13) binds to a regulator of G protein signaling (RGS) domain in p115-RhoGEF, but the mechanism of alpha(13) activation of p115-RhoGEF is poorly understood. In this report, we demonstrate in cell-based assays that the acidic-rich N-terminus, adjacent to the RGS domain, is required for binding to activated alpha(13), and refine the importance of this region by showing that mutation of glutamic acids 27 and 29 in full-length p115-RhoGEF is sufficient to prevent interaction with activated alpha(13). However, alpha(13)-interacting deficient N-terminal mutants of p115-RhoGEF retain alpha(13)-dependent plasma membrane recruitment. Overall, these findings demonstrate a critical role for the N-terminal extension of p115-RhoGEF in mediating binding to alpha(13) and dissociate two activities of p115-RhoGEF: binding to activated alpha(13) and translocation to the PM in response to activated alpha(13).

Characterization of G alpha 13-dependent plasma membrane recruitment of p115RhoGEF.

The Ras homology (Rho) guanine nucleotide exchange factor (GEF), p115RhoGEF, provides a direct link between the G-protein alpha subunit, alpha(13), and the small GTPase Rho. In the present study, we demonstrate that activated mutants of alpha(13) or alpha(12), but not alpha(q), promote the redistribution of p115RhoGEF from the cytoplasm to the plasma membrane (PM). We also show that the PM translocation of p115RhoGEF is promoted by stimulation of thromboxane A(2) receptors. Furthermore, we define domains of p115RhoGEF required for its regulated PM recruitment. The RhoGEF RGS (regulators of G-protein signalling) domain of p115RhoGEF is required for PM recruitment, but it is not sufficient for strong alpha(13)-promoted PM recruitment, even though it strongly interacts with activated alpha(13). We also identify the pleckstrin homology domain as essential for alpha(13)-mediated PM recruitment. An amino acid substitution of lysine to proline at position 677 in the pleckstrin homology domain of p115RhoGEF inhibits Rho-mediated gene transcription, but this mutation does not affect alpha(13)-mediated PM translocation of p115RhoGEF. The results suggest a mechanism whereby multiple signals contribute to regulated PM localization of p115RhoGEF.