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.

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.

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 regulates voltage-gated sodium channels and neuronal activity.

BACE1 activity is significantly increased in the brains of Alzheimer's disease patients, potentially contributing to neurodegeneration. The voltage-gated sodium channel (Na(v)1) beta2-subunit (beta2), a type I membrane protein that covalently binds to Na(v)1 alpha-subunits, is a substrate for BACE1 and gamma-secretase. Here, we find that BACE1-gamma-secretase cleavages release the intracellular domain of beta2, which increases mRNA and protein levels of the pore-forming Na(v)1.1 alpha-subunit in neuroblastoma cells. Similarly, endogenous beta2 processing and Na(v)1.1 protein levels are elevated in brains of BACE1-transgenic mice and Alzheimer's disease patients with high BACE1 levels. However, Na(v)1.1 is retained inside the cells and cell surface expression of the Na(v)1 alpha-subunits and sodium current densities are markedly reduced in both neuroblastoma cells and adult hippocampal neurons from BACE1-transgenic mice. BACE1, by cleaving beta2, thus regulates Na(v)1 alpha-subunit levels and controls cell-surface sodium current densities. BACE1 inhibitors may normalize membrane excitability in Alzheimer's disease patients with elevated BACE1 activity.

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.

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.

Correction: Palmitoylated APP Forms Dimers, Cleaved by BACE1.

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

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.

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).

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.

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.

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.

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.

Novel N-terminal cleavage of APP precludes Abeta generation in ACAT-defective AC29 cells.

A common pathogenic event that occurs in all forms of Alzheimer's disease is the progressive accumulation of amyloid beta-peptide (Abeta) in brain regions responsible for higher cognitive functions. Inhibition of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which generates intracellular cholesteryl esters from free cholesterol and fatty acids, reduces the biogenesis of the Abeta from the amyloid precursor protein (APP). Here we have used AC29 cells, defective in ACAT activity, to show that ACAT activity steers APP either toward or away from a novel proteolytic pathway that replaces both alpha and the amyloidogenic beta cleavages of APP. This alternative pathway involves a novel cleavage of APP holoprotein at Glu281, which correlates with reduced ACAT activity and Abeta generation in AC29 cells. This sterol-dependent cleavage of APP occurs in the endosomal compartment after internalization of cell surface APP. The resulting novel C-terminal fragment APP-C470 is destined to proteasomal degradation limiting the availability of APP for the Abeta generating system. The proportion of APP molecules that are directed to the novel cleavage pathway is regulated by the ratio of free cholesterol and cholesteryl esters in cells. These results suggest that subcellular cholesterol distribution may be an important regulator of the cellular fate of APP holoprotein and that there may exist several competing proteolytic systems responsible for APP processing within the endosomal compartment.

The ACAT inhibitor CP-113,818 markedly reduces amyloid pathology in a mouse model of Alzheimer's disease.

Amyloid beta-peptide (Abeta) accumulation in specific brain regions is a pathological hallmark of Alzheimer's disease (AD). We have previously reported that a well-characterized acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor, CP-113,818, inhibits Abeta production in cell-based experiments. Here, we assessed the efficacy of CP-113,818 in reducing AD-like pathology in the brains of transgenic mice expressing human APP(751) containing the London (V717I) and Swedish (K670M/N671L) mutations. Two months of treatment with CP-113,818 reduced the accumulation of amyloid plaques by 88%-99% and membrane/insoluble Abeta levels by 83%-96%, while also decreasing brain cholesteryl-esters by 86%. Additionally, soluble Abeta(42) was reduced by 34% in brain homogenates. Spatial learning was slightly improved and correlated with decreased Abeta levels. In nontransgenic littermates, CP-113,818 also reduced ectodomain shedding of endogenous APP in the brain. Our results suggest that ACAT inhibition may be effective in the prevention and treatment of AD by inhibiting generation of the Abeta peptide.

Alzheimer disease beta-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.

Presenilin/gamma-secretase and alpha-secretase-like peptidases cleave human MHC Class I proteins.

HLA (human leucocyte antigen)-A2 is an MHC Class I protein with primary functions in T-cell development and initi-ation of immune cell responses. MHC I proteins also play roles in intercellular adhesion, apoptosis, cell proliferation and neuronal plasticity. By utilizing a sequence comparison analysis, we recently identified HLA-A2 as a potential substrate for the Alzheimer's disease-associated PS1 (presenilin 1)/gamma-secretase. alpha-Secretase-like membrane metalloproteinases are responsible for an initial shedding event, partially mediated by ADAM (a disinteg-rin and metalloproteinase)-10. Accordingly, activation or inhibition of alpha-secretase-like membrane metalloproteinases directly modulated levels of a 14 kDa HLA-A2 CTF (C-terminal frag-ment) in CHO (Chinese-hamster ovary) cells. To show that the HLA-A2 CTF is subsequently cleaved by PS1/gamma-secretase, we re-duced its activity in cell lines stably expressing HLA-A2 and in Jurkat T-cells expressing endogenous MHC I. Treatment with specific PS1/gamma-secretase inhibitors or expression of a dominant-negative construct led to a significant accumulation of HLA-A2 CTFs. We also identified the PS1/gamma-secretase cleavage product of HLA-A2 CTF, termed HLA-A2 intracellular domain, in cell-free and cell-based experiments. In the absence of proteasome inhibitors, HLA-A2 intracellular domain underwent rapid degrad-ation. These data indicate that MHC I proteins undergo extra-cellular domain cleavage mediated by alpha-secretases and the cleavage product is subsequently cleaved by PS1/gamma-secretase.

ACAT as a drug target for Alzheimer's disease.

Accumulation of beta-amyloid peptide (Abeta) in the brain regions responsible for memory and cognitive functions is a neuropathological hallmark of Alzheimer's disease. Cholesterol may be involved in many aspects of Abeta metabolism. It affects generation, aggregation and clearance of Abeta in the brain. Not only the amount but also the distribution of cholesterol within cells appears to modulate Abeta biogenesis. ACAT is an enzyme that regulates subcellular cholesterol distribution by converting membrane cholesterol to cholesteryl esters for storage and transport. We have used various cell- and animal based models to show that inhibition of ACAT strongly reduces Abeta generation and protects from amyloid pathology. Here, we discuss data supporting ACAT inhibition as a strategy to treat Alzheimer's disease.

Alzheimer's disease: the cholesterol connection.

A hallmark of all forms of Alzheimer's disease (AD) is an abnormal accumulation of the beta-amyloid protein (Abeta) in specific brain regions. Both the generation and clearance of Abeta are regulated by cholesterol. Elevated cholesterol levels increase Abeta in cellular and most animals models of AD, and drugs that inhibit cholesterol synthesis lower Abeta in these models. Recent studies show that not only the total amount, but also the distribution of cholesterol within neurons, impacts Abeta biogenesis. The identification of a variant of the apolipoprotein E (APOE) gene as a major genetic risk factor for AD is also consistent with a role for cholesterol in the pathogenesis of AD. Clinical trials have recently been initiated to test whether lowering plasma and/or neuronal cholesterol levels is a viable strategy for treating and preventing AD. In this review, we describe recent findings concerning the molecular mechanisms underlying the cholesterol-AD connection.