Doo Yeon Kim

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.

ADAM10 Missense Mutations Potentiate β-Amyloid Accumulation by Impairing Prodomain Chaperone Function

The generation of Aβ, the main component of senile plaques in Alzheimers disease (AD), is precluded by α-secretase cleavage within the Aβ domain of the amyloid precursor protein (APP). We identified two rare mutations (Q170H and R181G) in the prodomain of the metalloprotease, ADAM10, that cosegregate with late-onset AD (LOAD). Here, we addressed the pathogenicity of these mutations in transgenic mice expressing human ADAM10 in brain. In Tg2576 AD mice, both mutations attenuated α-secretase activity of ADAM10 and shifted APP processing toward β-secretase-mediated cleavage, while enhancing Aβ plaque load and reactive gliosis. We also demonstrated ADAM10 expression potentiates adult hippocampal neurogenesis, which is reduced by the LOAD mutations. Mechanistically, both LOAD mutations impaired the molecular chaperone activity of ADAM10 prodomain. Collectively, these findings suggest that diminished α-secretase activity, owing to LOAD ADAM10 prodomain mutations, leads to AD-related pathology, strongly supporting ADAM10 as a promising therapeutic target for this devastating disease.

A 3D human neural cell culture system for modeling Alzheimer’s disease

Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimers disease (AD), because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel 3D culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of amyloid-β (Aβ) and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix and the analysis of AD pathogenesis. The 3D culture generation takes 1–2 d. The aggregation of Aβ is observed after 6 weeks of differentiation, followed by robust tau pathology after 10–14 weeks.

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.

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 Nav1.1 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 β-subunit Navβ2, to regulate levels of the pore-forming α-subunit Nav1.1 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 Nav1.1 protein levels were significantly decreased in BACE1-null versus wild-type mouse brains, remaining unchanged in BACE1-heterozygous mouse brains. Interestingly, levels of Nav1.2 and Nav1.6 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 Nav1.1 levels. Next, we performed surface biotinylation studies in acutely dissociated hippocampal slices from BACE1-null mice. Hippocampal surface Nav1.1 levels were significantly decreased, but Nav1.2 surface levels were increased in BACE1-null mice perhaps as a compensatory mechanism for reduced surface Nav1.1. We also found that Navβ2 processing and Nav1.1 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 Nav1.1 via cleavage of cell-surface Navβ2. Together, our data show that endogenous BACE1 activity regulates total and surface levels of voltage-gated sodium channels in mouse brains. Both decreased Nav1.1 and elevated surface Nav1.2 may result in a seizure phenotype. Our data caution that therapeutic BACE1 activity inhibition in Alzheimer disease patients may affect Nav1 metabolism and alter neuronal membrane excitability in Alzheimer disease patients.

Presenilin/γ-Secretase-mediated Cleavage Regulates Association of Leukocyte-Common Antigen-related (LAR) Receptor Tyrosine Phosphatase with β-Catenin

Leukocyte-common antigen-related (LAR) receptor tyrosine phosphatase regulates cell adhesion and formation of functional synapses and neuronal networks. Here we report that LAR is sequentially cleaved by α- and presenilin (PS)/γ-secretases, which also affect signaling and/or degradation of type-I membrane proteins including the Alzheimer disease-related β-amyloid precursor protein. Similar to the previously characterized PS/γ-secretase substrates, inhibition of γ-secretase activity resulted in elevated LAR C-terminal fragment (LAR-CTF) levels in stably LAR-overexpressing Chinese hamster ovary (CHO) cells, human neuroglioma cells, and mouse cortical neurons endogenously expressing LAR. Furthermore, LAR-CTF levels increased in cells lacking functional PS, indicating that γ-secretase-mediated cleavage of LAR was PS-dependent. Inhibition of α-secretase activity by TAPI-1 treatment blocked LAR-CTF accumulation, demonstrating that prior ectodomain shedding was prerequisite for PS/γ-secretase-mediated cleavage of LAR. Moreover, we identified the product of PS/γ-secretase cleavage, LAR intracellular domain (LICD), both in vitro and in cells overexpressing full-length (FL) LAR or LAR-CTFs. LAR localizes to cadherin-β-catenin-based cellular junctions. Assembly and disassembly of these junctions are regulated by tyrosine phosphorylation. We found that endogenous tyrosine-phosphorylated β-catenin coimmunoprecipitated with LAR in CHO cells. However, when PS/γ-secretase activity was inhibited, the association between LAR and β-catenin significantly diminished. In addition to cell adhesion, β-catenin is involved in transcriptional regulation. We observed that LICD significantly decreased transcription of cyclin D1, one of the β-catenin target genes. Thus, our results show that PS/γ-secretase-mediated cleavage of LAR controls LAR-β-catenin interaction, suggesting an essential role for PS/γ-secretase in the regulation of LAR signaling.

Presenilin/γ-secretase activity regulates protein clearance from the endocytic recycling compartment

The presenilin (PS)/γ‐secretase complex proteolytically cleaves more than 20 different proteins in addition to the amyloid precursor protein (APP). These substrates are almost exclusively type I membrane proteins. Many undergo internalization from the cell surface followed by degradation or recycling back to the plasma membrane through the endo‐cytic recycling compartment (ERC). Evidence shows that the PSs also regulate intracellular trafficking of APP and its C‐terminal fragments (CTFs). To investigate whether PS/γ‐secretase activity is required for normal endosomal recycling, we performed live cell imaging experiments with fluorescently labeled transferrin, reported to specifically traffic through the ERC. By using pharmacological γ‐secretase inhibitors or cell lines lacking functional PS/γ‐secretase, here we show that PS/γ‐secretase activity is required for clearance of transferrin from the ERC. Interestingly, lack of PS/γ‐secretase function also resulted in the accumulation of APP and APP‐CTFs in the ERC in addition to the cell surface. Familial Alzheimers disease mutations in APP‐CTFs did not affect endocytic recycling of these proteins. Our results suggest that PS/γ‐secretase activity is required for normal endosomal recycling of soluble and membrane‐associated proteins through the ERC and propose a new mechanism by which impaired PS/γ‐secretase function may eventually contribute to neurodegeneration.—Zhang, M., Haapasalo, A., Kim, D. Y., MacKenzie Ingano, L. A., Pettingell, W. H., and Kovacs, D. M. Presenilin/γ‐secretase activity regulates protein clearance from the endocytic recycling com‐partaient. FASEB J. 20, E271‐E280 (2006)