Warren H. Pettingell

Candidate gene for the chromosome 1 familial Alzheimer's disease locus

A candidate gene for the chromosome 1 Alzheimers disease (AD) locus was identified (STM2). The predicted amino acid sequence for STM2 is homologous to that of the recently cloned chromosome 14 AD gene (S182). A point mutation in STM2, resulting in the substitution of an isoleucine for an asparagine (N141l), was identified in affected people from Volga German AD kindreds. This N141l mutation occurs at an amino acid residue that is conserved in human S182 and in the mouse S182 homolog. The presence of missense mutations in AD subjects in two highly similar genes strongly supports the hypothesis that mutations in both are pathogenic.

Endoproteolytic Cleavage and Proteasomal Degradation of Presenilin 2 in Transfected Cells

Mutations in the presenilin genes, PS1 and PS2, cause a major portion of early onset familial Alzheimers disease (FAD). The biological roles of the presenilins and how their pathological mutations confer FAD are unknown. In this study, we set out to examine the processing and degradation pathways of PS2. For regulated expression of PS2, we have established inducible cell lines expressing PS2 under the tight control of the tetracycline-responsive transactivator. Western blot analysis revealed that PS2 was detected as an ∼53-55-kDa polypeptide (54-kDa PS2) as well as a high molecular mass form (HMW-PS2). Using a stably transfected, inducible cell system, we have found that PS2 is proteolytically cleaved into two stable cellular polypeptides including an ∼20-kDa C-terminal fragment and an ∼34-kDa N-terminal fragment. PS2 is polyubiquitinated in vivo, and the degradation of PS2 is inhibited by proteasome inhibitors, N-acetyl-L-leucinal-L-norleucinal and lactacystin. Our studies suggest that PS2 normally undergoes endoproteolytic cleavage and is degraded via the proteasome pathway.

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.

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-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)

Inhibition of acyl-coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway

Amyloid β‐peptide (Aβ) has a central role in the pathogenesis of Alzheimers disease (AD). Cellular cholesterol homeostasis regulates endoproteo‐ lytic generation of Aβ from the amyloid precursor protein (APP). Previous studies have identified acyl‐ coenzyme A: cholesterol acyltransferase (ACAT)’ an enzyme that regulates subcellular cholesterol distribu‐tion, as a potential therapeutic target for AD. Inhibition of ACAT activity decreases Aβ 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 deg‐radation 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 Aβ levels in individual animals. Our results identify a novel ACAT‐dependent mechanism that regulates secretory trafficking of APP, likely contributing to decreased Aβ generation in vivo.—Huttunen, H. J., Peach, C., Bhattacharyya, R., Barren, C., Pettingell, W., Hutter‐Paier, B., Windisch, M., Berezovska, O., Kovacs, D. M. Inhibition of acyl‐coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway. FASEB J. 23, 3819‐3828 (2009). www.fasebj.org

Genetic heterogeneity of gene defects responsible for familial Alzheimer disease.

Inherited Alzheimers disease is a genetically heterogeneous disorder that involves gene defects on at least five chromosomal loci. Three of these loci have been found by genetic linkage studies to reside on chromosomes 21, 19, and 14. On chromosomes 21, the gene encoding the precursor protein of Alzheimerassociated amyloid (APP) has been shown to contain several mutations in exons 16 and 17 which account for roughly 2–3% of familial Alzheimers disease (FAD). The other loci include what appears to be a susceptibility gene on chromosome 19 associated with late-onset (>65 years) FAD, and a major early-onset FAD gene defect on the long arm of chromosome 14. In other early-and late-onset FAD kindreds, the gene defects involved do not appear to be linked to any of these three loci, indicating the existence of additional and as of yet unlocalized FAD genes. This review provides a historical perspective of the search for FAD gene defects and summarizes the progress made in world-wide attempts to isolate and characterize the genes responsible for this disorder.

The Diverse Molecular Nature of Inherited Alzheimer’s Disease

Alzheimer’s diseases (AD) is a major health problem which will continue to intensify in magnitude as the elderly in the population continue to increase in number. The age at which AD strikes is variable, ranging from the fourth to tenth decades, with the greatest proportion of cases occurring in the seventh and eighth decades. A genetic component of this disorder has been strongly indicated by family and survey studies, as well as life table analyses (reviewed in St George-Hyslop et al. 1989). Genetic linkage and association studies of kindreds displaying evidence for familial AD (FAD) have led to the localization of gene defects responsible for this genetically heterogeneous disorder on chromosomes 14, 19 and 21. In a small set of FAD kindreds, mutations have been found in the amyloid beta protein precursor (APP) gene. Yet, the available data indicate that the identity of the genes responsible for the majority of late-onset (> 65 years) as well as early-onset inherited AD remain unknown. Powerful and novel advances in the methodology available for performing genetic linkage analyses on genetically complex disorders have made it feasible to scan the entire human genome in a relatively fast and easy manner for the purpose of localizing the genes responsible for, or predisposing to, inherited AD. Here we describe progress on attempts to further localize and identify various FAD gene defects throughout the genome, with special emphasis on the major early-onset gene defect residing on the long arm of chromosome 14.