Alzheimer Disease: Thinakaran G

Thinakaran G, Koo EH. · Department of Neurobiology, The University of Chicago, Chicago, Illinois 60637, USA. · J Biol Chem. · Pubmed #18650430 No free full text.

Abstract: Intracellular trafficking and proteolytic processing of amyloid precursor protein (APP) have been the focus of numerous investigations over the past two decades. APP is the precursor to the amyloid beta-protein (Abeta), the 38-43-amino acid residue peptide that is at the heart of the amyloid cascade hypothesis of Alzheimer disease (AD). Tremendous progress has been made since the initial identification of Abeta as the principal component of brain senile plaques of individuals with AD. Specifically, molecular characterization of the secretases involved in Abeta production has facilitated cell biological investigations on APP processing and advanced efforts to model AD pathogenesis in animal models. This minireview summarizes salient features of APP trafficking and amyloidogenic processing and discusses the putative biological functions of APP.

Cheng H, Vetrivel KS, Gong P, Meckler X, Parent A, Thinakaran G. · University of Chicago, IL, USA. · Nat Clin Pract Neurol. · Pubmed #17611486 No free full text.

Abstract: Alzheimer's disease (AD) is the most common cause of age-related dementia. Pathologically, AD is characterized by the deposition in the brain of amyloid-beta peptides derived from proteolysis of amyloid precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) and gamma-secretase. A growing body of evidence implicates cholesterol and cholesterol-rich membrane microdomains in amyloidogenic processing of APP. Here, we review recent findings regarding the association of BACE1, gamma-secretase and APP in lipid rafts, and discuss potential therapeutic strategies for AD that are based on knowledge gleaned from the membrane environment that fosters APP processing.

Vetrivel KS, Thinakaran G. · Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, IL 60637, USA. · Neurology. · Pubmed #16432149 No free full text.

Abstract: Trafficking and proteolytic processing of amyloid precursor protein (APP) have been the focus of numerous investigations in the past two decades, since the identification of Abeta as the principal component of brain senile plaques and the cloning of APP cDNA. Tremendous progress has been made in the recent past toward the characterization of beta- and gamma-secretases. Here, we review the salient features of Alzheimer disease amyloidogenesis, and discuss the current knowledge on APP trafficking and amyloidogenic processing of APP in intracellular membrane compartments and microdomains.

Thinakaran G, Parent AT. · Departments of Neurobiology, Pharmacology and Physiology, University of Chicago, Knapp R212, 924 East 57th Street, Chicago, IL 60637, USA. · Pharmacol Res. · Pubmed #15304238 No free full text.

Abstract: Mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2) account for the majority of the cases of familial early-onset Alzheimer's disease (FAD). Presenilins (PSs) facilitate the intramembraneous cleavage of amyloid precursor protein (APP), coined gamma-secretase cleavage, which generates beta-amyloid peptides (A beta). Considerable evidence suggests that FAD-linked PS variants exert their pathogenic influence by selectively elevating the levels of highly fibrillogenic A beta 42 peptides. In addition, numerous other functions have been ascribed to PSs based on subcellular localization, protein interactions, loss of function studies, and intramembraneous gamma-secretase cleavage of growing number of substrates. This review summarizes the diverse physiological functions that are regulated by PSs beyond APP metabolism.

Thinakaran G. · Department of Neurobiology, Pharmacology and Physiology, Center for Molecular Neurobiology, The University of Chicago, IL 60637, USA. · J Mol Neurosci. · Pubmed #11816791 No free full text.

Abstract: Understanding mechanisms involved in the production of Abeta has long been the central focus of cell biologists engaged in molecular AD research. The discovery of two genes that encode homologous polytopic membrane proteins termed Presenilins (PS), has lead to several exciting recent findings on the proteolytic processes responsible for generating the COOH-terminus of Abeta. What we now know is that PS proteins play an important role in Abeta production and are considered one of the therapeutic targets. Here I have reviewed the vast literature on the biology of PS, especially focusing on PS endoproteolysis and the accumulation of stable PS derivatives that are likely the functional units.

Thinakaran G. · Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Knapp R212, 924 East 57th Street, Chicago, Illinois 60637, USA. · J Clin Invest. · Pubmed #10562290 links to  free full text

This publication has no abstract.

Sisodia SS, Kim SH, Thinakaran G. · Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Chicago, IL, USA uchicago.edu · Am J Hum Genet. · Pubmed #10364510 links to  free full text

This publication has no abstract.

Vetrivel KS, Meckler X, Chen Y, Nguyen PD, Seidah NG, Vassar R, Wong PC, Fukata M, Kounnas MZ, Thinakaran G. · Department of Neurobiology, Neurology, and Pathology, The University of Chicago, Chicago, Illinois 60637, USA. · J Biol Chem. · Pubmed #19074428 No free full text.

Abstract: Alzheimer disease beta-amyloid (Abeta) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and gamma-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Abeta production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Abeta. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.

Cheng H, Vetrivel KS, Drisdel RC, Meckler X, Gong P, Leem JY, Li T, Carter M, Chen Y, Nguyen P, Iwatsubo T, Tomita T, Wong PC, Green WN, Kounnas MZ, Thinakaran G. · Department of Neurobiology, The University of Chicago, Chicago, Illinois 60637, USA. · J Biol Chem. · Pubmed #19028695 No free full text.

Abstract: Proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases generates beta-amyloid (Abeta) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Abeta production. Previously, we and others reported that the four integral subunits of the gamma-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of gamma-secretase subunits. We report that in cultured cells and in brain the gamma-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys(689), and APH-1 is S-palmitoylated at Cys(182) and Cys(245). S-Palmitoylation-defective nicastrin and APH-1 form stable gamma-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for gamma-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Abeta40, Abeta42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate gamma-secretase processing of APP and other substrates.

Vetrivel KS, Kodam A, Gong P, Chen Y, Parent AT, Kar S, Thinakaran G. · Department of Neurobiology, The University of Chicago, Chicago, IL 60637, USA. · Neurobiol Dis. · Pubmed #18652896 No free full text.

Abstract: Sequential processing of amyloid precursor protein by beta- and gamma-secretases generates Alzheimer's disease (AD)-associated beta-amyloid peptides. Recently it was reported that the transmembrane protein p23/TMP21 associates with gamma-secretase, and negatively regulates beta-amyloid production. Despite the link between p23 function and AD pathogenesis, the expression of p23 has not been examined in the brain. Here, we describe the detailed immunohistochemical characterization of p23 expression in rodent and human brain. We report that p23 is co-expressed with gamma-secretase subunits in select neuronal cell populations in rodent brain. Interestingly, the steady-state level of p23 in the brain is high during embryonic development and then declines after birth. Furthermore, the steady-state p23 levels are reduced in the brains of individuals with AD. We conclude that p23 is expressed in neurons throughout the brain and the decline in p23 expression during postnatal development may significantly contribute to enhanced beta-amyloid production in the adult brain.

Vetrivel KS, Zhang X, Meckler X, Cheng H, Lee S, Gong P, Lopes KO, Chen Y, Iwata N, Yin KJ, Lee JM, Parent AT, Saido TC, Li YM, Sisodia SS, Thinakaran G. · Department of Neurobiology and Neurology, The University of Chicago, Chicago, Illinois 60637, USA. · J Biol Chem. · Pubmed #18456655 links to  free full text

Abstract: Cerebral deposition of beta-amyloid (Abeta) peptides is a pathological hallmark of Alzheimer disease. Intramembranous proteolysis of amyloid precursor protein by a multiprotein gamma-secretase complex generates Abeta. Previously, it was reported that CD147, a glycoprotein that stimulates production of matrix metalloproteinases (MMPs), is a subunit of gamma-secretase and that the levels of secreted Abeta inversely correlate with CD147 expression. Here, we show that the levels and localization of CD147 in fibroblasts, as well as postnatal expression and distribution in brain, are distinct from those of integral gamma-secretase subunits. Notably, we show that although depletion of CD147 increased extracellular Abeta levels in intact cells, membranes isolated from CD147-depleted cells failed to elevate Abeta production in an in vitro gamma-secretase assay. Consistent with an extracellular source that modulates Abeta metabolism, synthetic Abeta was degraded more rapidly in the conditioned medium of cells overexpressing CD147. Moreover, modulation of CD147 expression had no effect on epsilon-site cleavage of amyloid precursor protein and Notch1 receptor. Collectively, our results demonstrate that CD147 modulates Abeta levels not by regulating gamma-secretase activity, but by stimulating extracellular degradation of Abeta. In view of the known function of CD147 in MMP production, we postulate that CD147 expression influences Abeta levels by an indirect mechanism involving MMPs that can degrade extracellular Abeta.

Vetrivel KS, Zhang YW, Xu H, Thinakaran G. · Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, IL 60637, USA. · Mol Neurodegener. · Pubmed #16930451 links to  free full text

Abstract: Mutations in PSEN1 and PSEN2 genes account for the majority of cases of early-onset familial Alzheimer disease. Since the first prediction of a genetic link between PSEN1 and PSEN2 with Alzheimer's disease, many research groups from both academia and pharmaceutical industry have sought to unravel how pathogenic mutations in PSEN cause presenile dementia. PSEN genes encode polytopic membrane proteins termed presenilins (PS1 and PS2), which function as the catalytic subunit of gamma-secretase, an intramembrane protease that has a wide spectrum of type I membrane protein substrates. Sequential cleavage of amyloid precursor protein by BACE and gamma-secretase releases highly fibrillogenic beta-amyloid peptides, which accumulate in the brains of aged individuals and patients with Alzheimer's disease. Familial Alzheimer's disease-associated presenilin variants are thought to exert their pathogenic function by selectively elevating the levels of highly amyloidogenic Abeta42 peptides. In addition to Alzheimer's disease, several recent studies have linked PSEN1 to familiar frontotemporal dementia. Here, we review the biology of PS1, its role in gamma-secretase activity, and discuss recent developments in the cell biology of PS1 with respect to Alzheimer's disease pathogenesis.

Wang H, Luo WJ, Zhang YW, Li YM, Thinakaran G, Greengard P, Xu H. · The Fisher Center for Alzheimer's Disease Research and the Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, USA. · J Biol Chem. · Pubmed #15247291 links to  free full text

Abstract: The intramembranous cleavage of Alzheimer beta-amyloid precursor protein and the signaling receptor Notch is mediated by the presenilin (PS, PS1/PS2)-gamma-secretase complex, the components of which also include nicastrin, APH-1, and PEN-2. In addition to its essential role in gamma-secretase activity, we and others have reported that PS1 plays a role in intracellular trafficking of select membrane proteins including nicastrin. Here we examined the fate of PEN-2 in the absence of PS expression or gamma-secretase activity. We found that PEN-2 is retained in the endoplasmic reticulum and has a much shorter half-life in PS-deficient cells than in wild type cells, suggesting that PSs are required for maintaining the stability and proper subcellular trafficking of PEN-2. However, the function of PS in PEN-2 trafficking is distinct from its contribution to gamma-secretase activity because inhibition of gamma-secretase activity by gamma-secretase inhibitors did not affect the PEN-2 level or its egress from the endoplasmic reticulum. Instead, membrane-permeable gamma-secretase inhibitors, but not a membrane-impermeable derivative, markedly increased the cell surface levels of PS1 and PEN-2 without affecting that of nicastrin. In support of its role in PEN-2 trafficking, PS1 was also required for the gamma-secretase inhibitor-induced plasma membrane accumulation of PEN-2. We further showed that gamma-secretase inhibitors specifically accelerated the Golgi to the cell surface transport of PS1 and PEN-2. Taken together, we demonstrate an essential role for PSs in intracellular trafficking of the gamma-secretase components, and that selective gamma-secretase inhibitors differentially affect the trafficking of the gamma-secretase components, which may contribute to an inactivation of gamma-secretase.

Thinakaran G, Bowen JW, Ito D, Leem JY, Veselits ML, Sato N. · Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, IL, USA. · Methods Mol Biol. · Pubmed #12840551 No free full text.

This publication has no abstract.

Takasugi N, Tomita T, Hayashi I, Tsuruoka M, Niimura M, Takahashi Y, Thinakaran G, Iwatsubo T. · Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113-0033, Japan. · Nature. · Pubmed #12660785 No free full text.

Abstract: Mutations in presenilin genes account for the majority of the cases of the familial form of Alzheimer's disease (FAD). Presenilin is essential for gamma-secretase activity, a proteolytic activity involved in intramembrane cleavage of Notch and beta-amyloid precursor protein (betaAPP). Cleavage of betaAPP by FAD mutant presenilin results in the overproduction of highly amyloidogenic amyloid beta42 peptides. gamma-Secretase activity requires the formation of a stable, high-molecular-mass protein complex that, in addition to the endoproteolysed fragmented form of presenilin, contains essential cofactors including nicastrin, APH-1 (refs 15-18) and PEN-2 (refs 16, 19). However, the role of each protein in complex formation and the generation of enzymatic activity is unclear. Here we show that Drosophila APH-1 (Aph-1) increases the stability of Drosophila presenilin (Psn) holoprotein in the complex. Depletion of PEN-2 by RNA interference prevents endoproteolysis of presenilin and promotes stabilization of the holoprotein in both Drosophila and mammalian cells, including primary neurons. Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. Thus, APH-1 stabilizes the presenilin holoprotein in the complex, whereas PEN-2 is required for endoproteolytic processing of presenilin and conferring gamma-secretase activity to the complex.

Siman R, Flood DG, Thinakaran G, Neumar RW. · University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. · J Biol Chem. · Pubmed #11574534 links to  free full text

Abstract: Endoplasmic reticulum (ER) stress elicits protective responses of chaperone induction and translational suppression and, when unimpeded, leads to caspase-mediated apoptosis. Alzheimer's disease-linked mutations in presenilin-1 (PS-1) reportedly impair ER stress-mediated protective responses and enhance vulnerability to degeneration. We used cleavage site-specific antibodies to characterize the cysteine protease activation responses of primary mouse cortical neurons to ER stress and evaluate the influence of a PS-1 knock-in mutation on these and other stress responses. Two different ER stressors lead to processing of the ER-resident protease procaspase-12, activation of calpain, caspase-3, and caspase-6, and degradation of ER and non-ER protein substrates. Immunocytochemical localization of activated caspase-3 and a cleaved substrate of caspase-6 confirms that caspase activation extends into the cytosol and nucleus. ER stress-induced proteolysis is unchanged in cortical neurons derived from the PS-1 P264L knock-in mouse. Furthermore, the PS-1 genotype does not influence stress-induced increases in chaperones Grp78/BiP and Grp94 or apoptotic neurodegeneration. A similar lack of effect of the PS-1 P264L mutation on the activation of caspases and induction of chaperones is observed in fibroblasts. Finally, the PS-1 knock-in mutation does not alter activation of the protein kinase PKR-like ER kinase (PERK), a trigger for stress-induced translational suppression. These data demonstrate that ER stress in cortical neurons leads to activation of several cysteine proteases within diverse neuronal compartments and indicate that Alzheimer's disease-linked PS-1 mutations do not invariably alter the proteolytic, chaperone induction, translational suppression, and apoptotic responses to ER stress.

Sato N, Urano F, Yoon Leem J, Kim SH, Li M, Donoviel D, Bernstein A, Lee AS, Ron D, Veselits ML, Sisodia SS, Thinakaran G. · Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Knapp R212, 924 East 57th street, Chicago, Illinois, 60637, USA. · Nat Cell Biol. · Pubmed #11146649 No free full text.

Abstract: Presenilin 1 (PS1), a polytopic membrane protein, has a critical role in the trafficking and proteolysis of a selected set of transmembrane proteins. The vast majority of individuals affected with early onset familial Alzheimer's disease (FAD) carry missense mutations in PS1. Two studies have suggested that loss of PS1 function, or expression of FAD-linked PS1 variants, compromises the mammalian unfolded-protein response (UPR), and we sought to evaluate the potential role of PS1 in the mammalian UPR. Here we show that that neither the endoplasmic reticulum (ER) stress-induced accumulation of BiP and CHOP messenger RNA, nor the activation of ER stress kinases IRE1alpha and PERK, is compromised in cells lacking both PS1 and PS2 or in cells expressing FAD-linked PS1 variants. We also show that the levels of BiP are not significantly different in the brains of individuals with sporadic Alzheimer's disease or PS1-mediated FAD to levels in control brains. Our findings provide evidence that neither loss of PS1 and PS2 function, nor expression of PS1 variants, has a discernable impact on ER stress-mediated induction of the several established 'readouts' of the UPR pathway.

Takahashi M, Doré S, Ferris CD, Tomita T, Sawa A, Wolosker H, Borchelt DR, Iwatsubo T, Kim SH, Thinakaran G, Sisodia SS, Snyder SH. · Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. · Neuron. · Pubmed #11144356 No free full text.

Abstract: Amyloid precursor protein (APP) generates the beta-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimer's disease. We report that APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimer's disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP-HO interactions may contribute to neuronal cell death in Alzheimer's disease.

Kim SH, Lah JJ, Thinakaran G, Levey A, Sisodia SS. · Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois, 60637, USA. · Neurobiol Dis. · Pubmed #10783294 No free full text.

Abstract: We have investigated the subcellular distribution of presenilin-1 (PS1) and presenilin-2 (PS2) in a variety of mammalian cell lines. In Iodixanol-based density gradients, PS1 derivatives show a biphasic distribution, cofractionating with membranes containing ER-resident proteins and an additional population of membranes with low buoyant density that do not contain markers of the Golgi complex, ERGIC, COP II vesicles, ER exit compartment, COP II receptor, Golgi SNARE, trans-Golgi network, caveolar membranes, or endocytic vesicles. Confocal immunofluorescence and immunoelectron microscopy studies fully supported the fractionation studies. These data suggest that PS1 fragments accumulate in a unique subcompartment(s) of the ER or ER to Golgi trafficking intermediates. Interestingly, the FAD-linked PS1 variants show a marked redistribution toward the heavier region of the gradient. Finally, and in contrast to PS1, PS2 fragments are detected preponderantly in more densely sedimenting membranes, suggesting that the subcellular compartments in which these molecules accumulate are distinct.

Saura CA, Tomita T, Soriano S, Takahashi M, Leem JY, Honda T, Koo EH, Iwatsubo T, Thinakaran G. · Department of Neurobiology, Pharmacology, and Physiology, University of Chicago, Chicago, Illinois 60637, USA. · J Biol Chem. · Pubmed #10748144 links to  free full text

Abstract: Presenilin 1 (PS1) and presenilin 2 (PS2) are polytopic membrane proteins that are mutated in the majority of early onset familial Alzheimer's disease (FAD) cases. Two lines of evidence establish a critical role for PS in the production of beta-amyloid peptides (Abeta). FAD-linked PS mutations elevate the levels of highly amyloidogenic Abeta ending at residue 42 (Abeta42), and cells with ablated PS1 alleles secrete low levels of Abeta. Several recent reports have shown that the hydrophilic loop (HL) domain, located between transmembrane domains 6 and 7, contains sites for phosphorylation, caspase cleavage, and sequences that bind several PS-interacting proteins. In the present report, we examined the metabolism of PS polypeptides lacking the HL domain and the influence of these molecules on Abeta production. We report that the deletion of the HL domain does not have a deleterious effect on the regulated endoproteolysis of PS, saturable accumulation of PS fragments, or the self-association of PS fragments. Abeta production was not significantly altered in cells expressing HL-deleted PS polypeptides compared with cells expressing full-length PS. Importantly, deletion of the HL domain did not affect FAD mutation-mediated elevation in the production of Abeta42. Furthermore, the deletion of the HL domain did not impair the role of PS1 or PS2 in facilitating Notch processing. Thus, our results argue against a biologically or pathologically relevant role for the HL domain phosphorylation and caspase cleavage and the association of PS HL domain-interacting proteins, in amyloid precursor protein metabolism and Abeta production or Notch cleavage.

Li XF, Thinakaran G, Sisodia SS, Yu FS. · Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA. · J Biol Chem. · Pubmed #10480944 links to  free full text

Abstract: Previous studies have established that in response to wounding, the expression of amyloid precursor-like protein 2 (APLP2) in the basal cells of migrating corneal epithelium is greatly up-regulated. To further our understanding of the functional significance of APLP2 in wound healing, we have measured the migratory response of transfected Chinese hamster ovary (CHO) cells expressing APLP2 isoforms to a variety of extracellular matrix components including laminin, collagen types I, IV, and VII, fibronectin, and heparan sulfate proteoglycans (HSPGs). CHO cells overexpressing either of two APLP2 variants, differing in chondroitin sulfate (CS) attachment, exhibit a marked increase in chemotaxis toward type IV collagen and fibronectin but not to laminin, collagen types I and VII, and HSPGs. Cells overexpressing APLP2-751 (CS-modified) exhibited a greater migratory response to fibronectin and type IV collagen than their non-CS-attached counterparts (APLP2-763), suggesting that CS modification enhanced APLP2 effects on cell migration. Moreover, in the presence of chondroitin sulfate, transfectants overexpressing APLP2-751 failed to exhibit this enhanced migration toward fibronectin. The APLP2-ECM interactions were also explored by solid phase adhesion assays. While overexpression of APLP2 isoforms moderately enhanced CHO adhesion to laminin, collagen types I and VII, and HSPGs lines, especially those overexpressing APLP2-751, exhibited greatly increased adhesion to type IV collagen and fibronectin. These observations suggest that APLP2 contributes to re-epithelialization during wound healing by supporting epithelial cell adhesion to fibronectin and collagen IV, thus influencing their capacity to migrate over the wound bed. Furthermore, APLP2 interactions with fibronectin and collagen IV appear to be potentiated by the addition of a CS chain to the core proteins.

Saura CA, Tomita T, Davenport F, Harris CL, Iwatsubo T, Thinakaran G. · Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA. · J Biol Chem. · Pubmed #10318786 links to  free full text

Abstract: Mutations in genes encoding presenilins (PS1 and PS2) cosegregate with the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are polytopic membrane proteins that undergo endoproteolytic cleavage to generate stable NH2- and COOH-terminal derivatives (NTF and CTF, respectively). Several lines of evidence suggest that the endoproteolytic derivatives are likely the functional units of PS in vivo. In the present report, we examine the disposition of PS NTF and CTF assemblies in stable mouse N2a neuroblastoma cell lines expressing human PS polypeptides. We show that exogenous expression of PS1 NTFs neither assemble with endogenous CTF nor exhibit dominant negative inhibitory effects on the endogenous PS1 cleavage and the accumulation of derivatives. In cells co-expressing PS1 and PS2, PS1- and PS2-derived fragments do not form mixed assemblies. In contrast, cells expressing a chimeric PS1/PS2 polypeptide form stable PS1 NTF-PS2 CTF assemblies. Moreover, expression of chimeric PS1/PS2 polypeptides harboring a familial early onset AD-linked mutation (M146L) elevates the production of Abeta42 peptides. Our results provide evidence that assembly of structural domains contained within NH2- and COOH-terminal regions of PS occur prior to endoproteolytic cleavage.

Greenfield JP, Tsai J, Gouras GK, Hai B, Thinakaran G, Checler F, Sisodia SS, Greengard P, Xu H. · Laboratory of Molecular and Cellular Neuroscience, and Fisher Center for Research on Alzheimer Disease, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. · Proc Natl Acad Sci U S A. · Pubmed #9892704 links to  free full text

Abstract: The excessive generation and accumulation of 40- and 42-aa beta-amyloid peptides (Abeta40/Abeta42) in selectively vulnerable brain regions is a major neuropathological feature of Alzheimer's disease. Abeta, derived by proteolytic cleavage from the beta-amyloid precursor protein (betaAPP), is normally secreted. However, recent evidence suggests that significant levels of Abeta also may remain inside cells. Here, we have investigated the subcellular compartments within which distinct amyloid species are generated and the compartments from which they are secreted. Three experimental approaches were used: (i) immunofluorescence performed in intact cortical neurons; (ii) sucrose gradient fractionation performed with mouse neuroblastoma cells stably expressing wild-type betaAPP695 (N2a695); and (iii) cell-free reconstitution of Abeta generation and trafficking from N2a695 cells. These studies demonstrate that: (i) Abeta40 (Abeta1-40 plus Abetax-40, where x is an NH2-terminal truncation) is generated exclusively within the trans-Golgi Network (TGN) and packaged into post-TGN secretory vesicles; (ii) Abetax-42 is made and retained within the endoplasmic reticulum in an insoluble state; (iii) Abeta42 (Abeta1-42 plus Abetax-42) is made in the TGN and packaged into secretory vesicles; and (iv) the amyloid peptides formed in the TGN consist of two pools (a soluble population extractable with detergents and a detergent-insoluble form). The identification of the organelles in which distinct forms of Abeta are generated and from which they are secreted should facilitate the identification of the proteolytic enzymes responsible for their formation.

Neet KE, Thinakaran G. · Department of Biochemistry and Molecular Biology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064, USA. · J Biol Chem. · Pubmed #18650429 No free full text.

This publication has no abstract.

Thinakaran G, Sisodia SS. · No affiliation provided · Nat Neurosci. · Pubmed #17066063 No free full text.

This publication has no abstract.