Dora M. Kovacs

Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid β-peptide

The pathogenic event common to all forms of Alzheimers disease is the abnormal accumulation of the amyloid β-peptide (Aβ). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Aβ. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Aβ production. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Aβ through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Aβ generation, indicating their potential for use in the treatment of Alzheimers disease.

The β-Secretase Enzyme BACE in Health and Alzheimer's Disease: Regulation, Cell Biology, Function, and Therapeutic Potential

The β-amyloid (Aβ) peptide is the major constituent of amyloid plaques in Alzheimers disease (AD) brain and is likely to play a central role in the pathogenesis of this devastating neurodegenerative disorder. The β-secretase, β-site amyloid precursor protein cleaving enzyme (BACE1; also called Asp2, memapsin 2), is the enzyme responsible for initiating Aβ generation. Thus, BACE is a prime drug target for the therapeutic inhibition of Aβ 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.

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-β plaques and neurofibrillary tangles. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β 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-β-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-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β 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-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β 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 regulates voltage-gated sodium channels and neuronal activity

BACE1 activity is significantly increased in the brains of Alzheimers disease patients, potentially contributing to neurodegeneration. The voltage-gated sodium channel (Nav1) β2-subunit (β2), a type I membrane protein that covalently binds to Nav1 α-subunits, is a substrate for BACE1 and γ-secretase. Here, we find that BACE1–γ-secretase cleavages release the intracellular domain of β2, which increases mRNA and protein levels of the pore-forming Nav1.1 α-subunit in neuroblastoma cells. Similarly, endogenous β2 processing and Nav1.1 protein levels are elevated in brains of BACE1-transgenic mice and Alzheimers disease patients with high BACE1 levels. However, Nav1.1 is retained inside the cells and cell surface expression of the Nav1 α-subunits and sodium current densities are markedly reduced in both neuroblastoma cells and adult hippocampal neurons from BACE1-transgenic mice. BACE1, by cleaving β2, thus regulates Nav1 α-subunit levels and controls cell-surface sodium current densities. BACE1 inhibitors may normalize membrane excitability in Alzheimers disease patients with elevated BACE1 activity.

Nectin-1α, an Immunoglobulin-like Receptor Involved in the Formation of Synapses, Is a Substrate for Presenilin/γ-Secretase-like Cleavage

Nectin-1 is a member of the immunoglobulin superfamily and a Ca2+-independent adherens junction protein involved in synapse formation. Here we show that nectin-1α undergoes intramembrane proteolytic processing analogous to that of the Alzheimers disease amyloid precursor protein, mediated by a presenilin (PS)-dependent γ-secretase-like activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of Chinese hamster ovary cells activated a first proteolytic event, resulting in ectodomain shedding of nectin-1α. Subsequent cleavage of the remaining 26-kDa membrane-anchored C-terminal fragment (CTF) was inhibited independently by three specific γ−secretase inhibitors and by expression of the dominant negative form of PS1. The PS/γ-secretase-like cleavage product was detected in vivofollowing proteasome inhibitor treatment of cells. An in vitro γ-secretase assay confirmed the generation of a 24-kDa nectin-1α intracellular domain, peripherally associated with the membrane fraction. We also found nectin-1α to interact with the N-terminal fragment of PS1. Finally, γ-secretase inhibition resulted in β-catenin release from cell junctions, concomitantly with the accumulation of the 26-kDa nectin-1α CTF, suggesting that high levels of nectin-1α CTF interfere with TPA-induced remodeling of cell-cell junctions. Our results are consistent with a previously reported role for PS/γ-secretase in adherens junction function involving cleavage of cadherins. Similar to nectin-1, other members of the immunoglobulin superfamily involved in synapse formation may also serve as substrates for PS/γ-secretase-like intramembrane proteolytic activity.

Presenilin/γ-Secretase-mediated Cleavage of the Voltage-gated Sodium Channel β2-Subunit Regulates Cell Adhesion and Migration

The voltage-gated sodium channel β2-subunit (β2) is a member of the IgCAM superfamily and serves as both an adhesion molecule and an auxiliary subunit of the voltage-gated sodium channel. Here we found that β2 undergoes ectodomain shedding followed by presenilin (PS)-dependent γ-secretase-mediated cleavage. 12-O-Tetradecanoylphorbol-13-acetate treatment or expression of an α-secretase enzyme, ADAM10, resulted in ectodomain cleavage of β2 in Chinese hamster ovary cells. Subsequent cleavage of the remaining 15-kDa C-terminal fragment (β2-CTF) was independently inhibited by three specific γ-secretase inhibitors, expression of the dominant negative form of PS1, and in PS1/PS2 knock-out cells. γ-Secretase inhibitor treatment also increased endogenous β2-CTF levels in neuroblastoma cells and mouse primary neuronal cultures. In a cell-free γ-secretase assay, we detected γ-secretase activity-dependent generation of a 12 kDa β2 intracellular domain (ICD), which was loosely associated with the membrane fraction. To assess the functional role of β2 processing by γ-secretase, we tested whether N-[N-(3,5-difluorophenylacetyl-l-alanyl)]-S-phenylglycine t-butylester (DAPT), a specific γ-secretase inhibitor, would alter β2-mediated cell adhesion and migration. We found that DAPT inhibited cell-cell aggregation and migration in a wound healing assay carried out with Chinese hamster ovary cells expressing β2. DAPT also reduced migration of neuroblastoma cells in a modified Boyden chamber assay. Since DAPT treatment resulted in increased β2-CTF levels, we also tested whether β2-CTFs or β2-ICDs would directly affect cell migration by overexpressing recombinant proteins. Interestingly, elevated levels of β2-CTFs, but not ICDs, also blocked cell migration by 81 to 93%. Together, our findings show for the first time that β2 is a PS/γ-secretase substrate and γ-secretase mediated cleavage of β2-CTF is required for cell-cell adhesion and migration of β2-expressing cells.

Alzheimer disease β-amyloid activity mimics cholesterol oxidase

The abnormal accumulation of amyloid beta-peptide (Abeta) in the form of senile (or amyloid) plaques is one of the main characteristics of Alzheimer disease (AD). Both cholesterol and Cu2+ have been implicated in AD pathogenesis and plaque formation. Abeta binds Cu2+ with very high affinity, forming a redox-active complex that catalyzes H2O2 production from O2 and cholesterol. Here we show that Abeta:Cu2+ complexes oxidize cholesterol selectively at the C-3 hydroxyl group, catalytically producing 4-cholesten-3-one and therefore mimicking the activity of cholesterol oxidase, which is implicated in cardiovascular disease. Abeta toxicity in neuronal cultures correlated with this activity, which was inhibited by Cu2+ chelators including clioquinol. Cell death induced by staurosporine or H2O2 did not elevate 4-cholesten-3-one levels. Brain tissue from AD subjects had 98% more 4-cholesten-3-one than tissue from age-matched control subjects. We observed a similar increase in the brains of Tg2576 transgenic mice compared with nontransgenic littermates; the increase was inhibited by in vivo treatment with clioquinol, which suggests that brain Abeta accumulation elevates 4-cholesten-3-one levels in AD. Cu2+-mediated oxidation of cholesterol may be a pathogenic mechanism common to atherosclerosis and AD.

The Many Substrates of Presenilin/γ-Secretase

The Alzheimers disease (AD)-associated amyloid-β protein precursor (AβPP) is cleaved by α-, β-, and presenilin (PS)/γ-secretases through sequential regulated proteolysis. These proteolytic events control the generation of the pathogenic amyloid-β (Aβ) 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 AβPP, 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 AβPP 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.

Palmitoylation of Amyloid Precursor Protein Regulates Amyloidogenic Processing in Lipid Rafts

Brains of patients affected by Alzheimers disease (AD) contain large deposits of aggregated amyloid β-protein (Aβ). Only a small fraction of the amyloid precursor protein (APP) gives rise to Aβ. 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 Aβ 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.

HtrA2 Regulates β-Amyloid Precursor Protein (APP) Metabolism through Endoplasmic Reticulum-associated Degradation

Alzheimer disease-associated β-amyloid peptide is generated from its precursor protein APP. By using the yeast two-hybrid assay, here we identified HtrA2/Omi, a stress-responsive chaperone-protease as a protein binding to the N-terminal cysteinerich region of APP. HtrA2 coimmunoprecipitates exclusively with immature APP from cell lysates as well as mouse brain extracts and degrades APP in vitro. A subpopulation of HtrA2 localizes to the cytosolic side of the endoplasmic reticulum (ER) membrane where it contributes to ER-associated degradation of APP together with the proteasome. Inhibition of the proteasome results in accumulation of retrotranslocated forms of APP and increased association of APP with HtrA2 and Derlin-1 in microsomal membranes. In cells lacking HtrA2, APP holoprotein is stabilized and accumulates in the early secretory pathway correlating with elevated levels of APP C-terminal fragments and increased Aβ secretion. Inhibition of ER-associated degradation (either HtrA2 or proteasome) promotes binding of APP to the COPII protein Sec23 suggesting enhanced trafficking of APP out of the ER. Based on these results we suggest a novel function for HtrA2 as a regulator of APP metabolism through ER-associated degradation.