Correction: Palmitoylated APP Forms Dimers, Cleaved by BACE1.

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

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.

Synaptotagmins interact with APP and promote Aß generation.

BACKGROUND: Accumulation of the ß-amyloid peptide (Aß) is a major pathological hallmark of Alzheimer's disease (AD). Recent studies have shown that synaptic Aß toxicity may directly impair synaptic function. However, proteins regulating Aß 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 Aß 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 Aß 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 Aß 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 Aß40 and Aß42 generation. As secreted sAPPß levels were significantly reduced in PC12 cells lacking Syt-1 expression, our results suggest that Syt-1 regulates Aß 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 Aß generation and thus may play an important role in the pathogenesis of AD.

γ-Secretase modulators reduce endogenous amyloid ß42 levels in human neural progenitor cells without altering neuronal differentiation.

Soluble γ-secretase modulators (SGSMs) selectively decrease toxic amyloid ß (Aß) peptides (Aß42). However, their effect on the physiologic functions of γ-secretase has not been tested in human model systems. γ-Secretase regulates fate determination of neural progenitor cells. Thus, we studied the impact of SGSMs on the neuronal differentiation of ReNcell VM (ReN) human neural progenitor cells (hNPCs). Quantitative PCR analysis showed that treatment of neurosphere-like ReN cell aggregate cultures with γ-secretase inhibitors (GSIs), but not SGSMs, induced a 2- to 4-fold increase in the expression of the neuronal markers Tuj1 and doublecortin. GSI treatment also induced neuronal marker protein expression, as shown by Western blot analysis. In the same conditions, SGSM treatment selectively reduced endogenous Aß42 levels by ∼80%. Mechanistically, we found that Notch target gene expressions were selectively inhibited by a GSI, not by SGSM treatment. We can assert, for the first time, that SGSMs do not affect the neuronal differentiation of hNPCs while selectively decreasing endogenous Aß42 levels in the same conditions. Our results suggest that our hNPC differentiation system can serve as a useful model to test the impact of GSIs and SGSMs on both endogenous Aß levels and γ-secretase physiologic functions including endogenous Notch signaling.

A three-dimensional human neural cell culture model of Alzheimer's disease.

Alzheimer's disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-ß plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer's disease posits that the excessive accumulation of amyloid-ß peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer's disease (FAD) mutations exhibit amyloid-ß-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer's disease, including distinct neurofibrillary tangle pathology. Human neurons derived from Alzheimer's disease patients have shown elevated levels of toxic amyloid-ß species and phosphorylated tau but did not demonstrate amyloid-ß plaques or neurofibrillary tangles. Here we report that FAD mutations in ß-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-ß, including amyloid-ß 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 phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-ß generation with ß- or γ-secretase inhibitors not only decreased amyloid-ß pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-ß-mediated tau phosphorylation. We have successfully recapitulated amyloid-ß and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer's disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.

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.

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.

BACE1 and presenilin/γ-secretase regulate proteolytic processing of KCNE1 and 2, auxiliary subunits of voltage-gated potassium channels.

BACE1 and presenilin (PS)/γ-secretase play a major role in Alzheimer's disease pathogenesis by regulating amyloid-ß peptide generation. We recently showed that these secretases also regulate the processing of voltage-gated sodium channel auxiliary ß-subunits and thereby modulate membrane excitability. Here, we report that KCNE1 and KCNE2, auxiliary subunits of voltage-gated potassium channels, undergo sequential cleavage mediated by either α-secretase and PS/γ-secretase or BACE1 and PS/γ-secretase in cells. Elevated α-secretase or BACE1 activities increased C-terminal fragment (CTF) levels of KCNE1 and 2 in human embryonic kidney (HEK293T) and rat neuroblastoma (B104) cells. KCNE-CTFs were then further processed by PS/γ-secretase to KCNE intracellular domains. These KCNE cleavages were specifically blocked by chemical inhibitors of the secretases in the same cell models. We also verified our results in mouse cardiomyocytes and cultured primary neurons. Endogenous KCNE1- and KCNE2-CTF levels increased by 2- to 4-fold on PS/γ-secretase inhibition or BACE1 overexpression in these cells. Furthermore, the elevated BACE1 activity increased KCNE1 processing and shifted KCNE1/KCNQ1 channel activation curve to more positive potentials in HEK cells. KCNE1/KCNQ1 channel is a cardiac potassium channel complex, and the positive shift would lead to a decrease in membrane repolarization during cardiac action potential. Together, these results clearly showed that KCNE1 and KCNE2 cleavages are regulated by BACE1 and PS/γ-secretase activities under physiological conditions. Our results also suggest a functional role of KCNE cleavage in regulating voltage-gated potassium channels.

Surface trafficking of sodium channels in cells and in hippocampal slices.

The voltage-gated sodium channel (Nav1) plays an important role in initiating and propagating action potentials in neuronal cells. We and others have recently found that the Alzheimer's disease-related secretases BACE1 and presenilin (PS)/γ-secretase regulate Nav1 function by cleaving auxiliary subunits of the channel complex. We have also shown that elevated BACE1 activity significantly decreases sodium current densities in neuroblastoma cells and acutely dissociated adult hippocampal neurons. For detailed molecular studies of sodium channel regulation, biochemical methods are now complementing classical electrophysiology. To understand how BACE1 regulates sodium current densities in our studies, we setup conditions to analyze surface levels of the pore-forming Nav1 α-subunits. By using a cell surface biotinylation protocol, we found that elevated BACE1 activity significantly decreases surface Nav1 α-subunit levels in both neuroblastoma cells and acutely prepared hippocampal slices. This finding would explain the decreased sodium currents shown by standard electrophysiological methods. The biochemical methods used in our studies would be applicable to analyses of surface expression levels of other ion channels as well as Nav1 in cells and adult hippocampal neurons.

The many substrates of presenilin/γ-secretase.

The Alzheimer's disease (AD)-associated amyloid-ß protein precursor (AßPP) is cleaved by α-, ß-, and presenilin (PS)/γ-secretases through sequential regulated proteolysis. These proteolytic events control the generation of the pathogenic amyloid-ß (Aß) peptide, which excessively accumulates in the brains of individuals afflicted by AD. A growing number of additional proteins cleaved by PS/γ-secretase continue to be discovered. Similarly to AßPP, most of these proteins are type-I transmembrane proteins involved in vital signaling functions regulating cell fate, adhesion, migration, neurite outgrowth, or synaptogenesis. All the identified proteins share common structural features, which are typical for their proteolysis. The consequences of the PS/γ-secretase-mediated cleavage on the function of many of these proteins are largely unknown. Here, we review the current literature on the proteolytic processing mediated by the versatile PS/γ-secretase complex. We begin by discussing the steps of AßPP processing and PS/γ-secretase complex composition and localization, which give clues to how and where the processing of other PS/γ-secretase substrates may take place. Then we summarize the typical features of PS/γ-secretase-mediated protein processing. Finally, we recapitulate the current knowledge on the possible physiological function of PS/γ-secretase-mediated cleavage of specific substrate proteins.

Reduced sodium channel Na(v)1.1 levels in BACE1-null mice.

The Alzheimer BACE1 enzyme cleaves numerous substrates, with largely unknown physiological consequences. We have previously identified the contribution of elevated BACE1 activity to voltage-gated sodium channel Na(v)1.1 density and neuronal function. Here, we analyzed physiological changes in sodium channel metabolism in BACE1-null mice. Mechanistically, we first confirmed that endogenous BACE1 requires its substrate, the ß-subunit Na(v)ß(2), to regulate levels of the pore-forming α-subunit Na(v)1.1 in cultured primary neurons. Next, we analyzed sodium channel α-subunit levels in brains of BACE1-null mice at 1 and 3 months of age. At both ages, we found that Na(v)1.1 protein levels were significantly decreased in BACE1-null versus wild-type mouse brains, remaining unchanged in BACE1-heterozygous mouse brains. Interestingly, levels of Na(v)1.2 and Na(v)1.6 α-subunits also decreased in 1-month-old BACE1-null mice. In the hippocampus of BACE1-null mice, we found a robust 57% decrease of Na(v)1.1 levels. Next, we performed surface biotinylation studies in acutely dissociated hippocampal slices from BACE1-null mice. Hippocampal surface Na(v)1.1 levels were significantly decreased, but Na(v)1.2 surface levels were increased in BACE1-null mice perhaps as a compensatory mechanism for reduced surface Na(v)1.1. We also found that Na(v)ß(2) processing and Na(v)1.1 mRNA levels were significantly decreased in brains of BACE1-null mice. This suggests a mechanism consistent with BACE1 activity regulating mRNA levels of the α-subunit Na(v)1.1 via cleavage of cell-surface Na(v)ß(2). Together, our data show that endogenous BACE1 activity regulates total and surface levels of voltage-gated sodium channels in mouse brains. Both decreased Na(v)1.1 and elevated surface Na(v)1.2 may result in a seizure phenotype. Our data caution that therapeutic BACE1 activity inhibition in Alzheimer disease patients may affect Na(v)1 metabolism and alter neuronal membrane excitability in Alzheimer disease patients.

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.

Alzheimer's secretases regulate voltage-gated sodium channels.

BACE1 and presenilin (PS)/γ-secretase are primary proteolytic enzymes responsible for the generation of pathogenic amyloid ß-peptides (Aß) in Alzheimer's disease. We and others have found that ß-subunits of the voltage-gated sodium channel (Na(v)ßs) also undergo sequential proteolytic cleavages mediated by BACE1 and PS/γ-secretase. In a follow-up study, we reported that elevated BACE1 activity regulates total and surface expression of voltage-gated sodium channels (Na(v)1 channels) and thereby modulates sodium currents in neuronal cells and mouse brains. In this review, we focus on the molecular mechanism of how BACE1 and PS/γ-secretase regulate Na(v)1 channels in neuronal cells. We will also discuss potential physiological and pathological roles of BACE1- and PS/γ-secretase-mediated processing of Na(v)ßs in relation to Na(v)1 channel function.

The acyl-coenzyme A: cholesterol acyltransferase inhibitor CI-1011 reverses diffuse brain amyloid pathology in aged amyloid precursor protein transgenic mice.

Cerebral accumulation of amyloid-beta (Abeta) is characteristic of Alzheimer disease and of amyloid precursor protein (APP) transgenic mice. Here, we assessed the efficacy of CI-1011, an inhibitor of acyl-coenzyme A:cholesterol acyltransferase, which is suitable for clinical use, in reducing amyloid pathology in both young (6.5 months old) and aged (16 months old) human APP transgenic mice. Treatment of young animals with CI-1011 decreased amyloid plaque load in the cortex and hippocampus and reduced the levels of insoluble Abeta40 and Abeta42 and C-terminal fragments of APP in brain extracts. In aged mice, CI-1011 specifically reduced diffuse amyloid plaques with a minor effect on thioflavin S-positive dense-core plaques. Reduced diffusible amyloid was accompanied by suppression of astrogliosis and enhanced microglial activation. Collectively, these data suggest that CI-1011 treatment reduces amyloid burden in human APP mice by limiting generation and increasing clearance of diffusible Abeta.

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.

The beta-secretase enzyme BACE in health and Alzheimer's disease: regulation, cell biology, function, and therapeutic potential.

The beta-amyloid (Abeta) peptide is the major constituent of amyloid plaques in Alzheimer's disease (AD) brain and is likely to play a central role in the pathogenesis of this devastating neurodegenerative disorder. The beta-secretase, beta-site amyloid precursor protein cleaving enzyme (BACE1; also called Asp2, memapsin 2), is the enzyme responsible for initiating Abeta generation. Thus, BACE is a prime drug target for the therapeutic inhibition of Abeta production in AD. Since its discovery 10 years ago, much has been learned about BACE. This review summarizes BACE properties, describes BACE translation dysregulation in AD, and discusses BACE physiological functions in sodium current, synaptic transmission, myelination, and schizophrenia. The therapeutic potential of BACE will also be considered. This is a summary of topics covered at a symposium held at the 39th annual meeting of the Society for Neuroscience and is not meant to be a comprehensive review of BACE.