Laura A. MacKenzie Ingano

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.

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.

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)

Role of acyl-coenzyme a: cholesterol acyltransferase activity in the processing of the amyloid precursor protein.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory deficit, cognitive impairment, and personality changes accompanied by specific structural abnormalities in the brain. Deposition of amyloid-β (Aβ) peptide into senile plaques is a consistent feature of the brains of patients affected by AD. Studies with both animal and cellular models of AD have shown that cholesterol homeostasis and distribution regulate Aβ generation. We have provided genetic, biochemical, and metabolic evidence that implicates intracellular cholesterol distribution, rather than total cholesterol levels, in the regulation of Aβ generation. This minireview focuses on the role of acyl-coenzyme A: cholesterol acyltransferase activity (ACAT) in Aβ generation. In genetically mutant cell lines that overproduce cholesterol but cannot synthesize cholesteryl esters (CEs) because of deficient ACAT activity, Aβ production is almost completely inhibited. Acyl-coenzyme A: cholesterol acyltransferase activity (ACAT) inhibitors, currently being developed for the treatment and prevention of atherosclerosis, reduce CE levels and Aβ generation by up to 50% in cell culture models of AD. Future mechanistic and transgenic animal studies are needed to evaluate the potential use of ACAT inhibitors in the therapeutic treatment or prevention of AD.

Novel N-terminal Cleavage of APP Precludes Aβ 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 β-peptide (Aβ) 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 Aβ 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 α and the amyloidogenic β cleavages of APP. This alternative pathway involves a novel cleavage of APP holoprotein at Glu281, which correlates with reduced ACAT activity and Aβ 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 Aβ 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.

Presenilin 1 forms aggresomal deposits in response to heat shock

Aggresomes have been described as cytoplasmic membrane protein aggregates that are induced by proteasome inhibition or overexpression of certain proteins. Here, we characterized aggresomes formed by the Alzheimers disease-associated presenilin 1 (PS1) protein. Proteasome inhibition induced accumulation of PS1 in the endoplasmic reticulum (ER) and retrotranslocation of the protein from the ER membrane into the cytoplasm. Aggresomes formed by PS1 modified the ER structure whereas proteasomes were inhibited. Therefore, clear visual identification of PS1 aggresomes required removal of the proteasome inhibitor followed by hours of recovery to redistribute the ER throughout the cells. Aggresomes formed by PS1 did not potentiate or attenuate apoptotic cell death induced by staurosporine treatment. Selective presence of the heat-shock proteins Hsp70 and HDJ-2/HSDJ, but not Hsp90, in aggresomes suggested chaperone-mediated transport of PS1 into these structures. Because proteasome inhibition and heat shock are both known to induce expression of heat shock proteins, we also demonstrated that heat shock alone was sufficient to induce PS1 aggresome formation and Hsp70 expression. These results indicate that aggresome formation by PS1 is chaperone-mediated and can be induced in response to heat-shock stress, a common cellular event in neurodegenerative diseases. Malfunctioning of the proteasome or heat-shock stress response in the brains of patients affected by Alzheimers disease may lead to the accumulation of stable aggresomes of PS1, perhaps contributing to neurodegeneration.

Salmonella Typhi Colonization Provokes Extensive Transcriptional Changes Aimed at Evading Host Mucosal Immune Defense During Early Infection of Human Intestinal Tissue

Commensal microorganisms influence a variety of host functions in the gut, including immune response, glucose homeostasis, metabolic pathways and oxidative stress, among others. This study describes how Salmonella Typhi, the pathogen responsible for typhoid fever, uses similar strategies to escape immune defense responses and survive within its human host. To elucidate the early mechanisms of typhoid fever, we performed studies using healthy human intestinal tissue samples and “mini-guts,” organoids grown from intestinal tissue taken from biopsy specimens. We analyzed gene expression changes in human intestinal specimens and bacterial cells both separately and after colonization. Our results showed mechanistic strategies that S. Typhi uses to rearrange the cellular machinery of the host cytoskeleton to successfully invade the intestinal epithelium, promote polarized cytokine release and evade immune system activation by downregulating genes involved in antigen sampling and presentation during infection. This work adds novel information regarding S. Typhi infection pathogenesis in humans, by replicating work shown in traditional cell models, and providing new data that can be applied to future vaccine development strategies.

Human Fetal-Derived Enterospheres Provide Insights on Intestinal Development and a Novel Model to Study Necrotizing Enterocolitis (NEC)

Background & Aims Untreated necrotizing enterocolitis (NEC) can lead to massive inflammation resulting in intestinal necrosis with a high mortality rate in preterm infants. Limited access to human samples and relevant experimental models have hampered progress in NEC pathogenesis. Earlier evidence has suggested that bacterial colonization of an immature and developing intestine can lead to an abnormally high inflammatory response to bacterial bioproducts. The aim of our study was to use human fetal organoids to gain insights into NEC pathogenesis. Methods RNA sequencing analysis was performed to compare patterns of gene expression in human fetal-derived enterospheres (FEnS) and adult-derived enterospheres (AEnS). Differentially expressed genes were analyzed using computational techniques for dimensional reduction, clustering, and gene set enrichment. Unsupervised cluster analysis, Gene Ontology, and gene pathway analysis were used to predict differences between gene expression of samples. Cell monolayers derived from FEnS and AEnS were evaluated for epithelium function and responsiveness to lipopolysaccharide and commensal bacteria. Results Based on gene expression patterns, FEnS clustered according to their developmental age in 2 distinct groups: early and late FEnS, with the latter more closely resembling AEnS. Genes involved in maturation, gut barrier function, and innate immunity were responsible for these differences. FEnS-derived monolayers exposed to either lipopolysaccharide or commensal Escherichia coli showed that late FEnS activated gene expression of key inflammatory cytokines, whereas early FEnS monolayers did not, owing to decreased expression of nuclear factor-κB–associated machinery. Conclusions Our results provide insights into processes underlying human intestinal development and support the use of FEnS as a relevant human preclinical model for NEC. Accession number of repository for expression data: GSE101531.