Animal models of brain maldevelopment induced by cycad plant genotoxins

Cycads are long‐lived tropical and subtropical plants that contain azoxyglycosides (e.g., cycasin, macrozamin) and neurotoxic amino acids (notably β‐N‐methylamino‐l ‐alanine l ‐BMAA), toxins that have been implicated in the etiology of a disappearing neurodegenerative disease, amyotrophic lateral sclerosis and parkinsonism‐dementia complex that has been present in high incidence among three genetically distinct populations in the western Pacific. The neuropathology of amyotrophic lateral sclerosis/parkinsonism‐dementia complex includes features suggestive of brain maldevelopment, an experimentally proven property of cycasin attributable to the genotoxic action of its aglycone methylazoxymethanol (MAM). This property of MAM has been exploited by neurobiologists as a tool to study perturbations of brain development. Depending on the neurodevelopmental stage, MAM can induce features in laboratory animals that model certain characteristics of epilepsy, schizophrenia, or ataxia. Studies in DNA repair‐deficient mice show that MAM perturbs brain development through a DNA damage‐mediated mechanism. The brain DNA lesions produced by systemic MAM appear to modulate the expression of genes that regulate neurodevelopment and contribute to neurodegeneration. Epigenetic changes (histone lysine methylation) have also been detected in the underdeveloped brain after MAM administration. The DNA damage and epigenetic changes produced by MAM and, perhaps by chemically related substances (e.g., nitrosamines, nitrosoureas, hydrazines), might be an important mechanism by which early‐life exposure to genotoxicants can induce long‐term brain dysfunction. Birth Defects Research (Part C) 99:247–255, 2013. © 2013 Wiley Periodicals, Inc.     

Categorizing reactivity of bacteriorhodopsin cysteine mutants crosslinking to 4-bromoretinal.

The structure of bacteriorhodopsin (bR) has been probed by a large number of experimental methods. In earlier work distance constraints measured from the 1BRD Brookhaven structure (1, 2) were used to guide site-directed mutagenesis/affinity labeling experiments (3-5). In the present study we report on the use of limited molecular dynamics (MD) investigations of the same bR/affinity label system. We show here that the chiral center introduced when 4-bromo-all-trans retinal is synthesized produces variable impact on potential crosslinking. Our MD analysis suggests the following ranking of binding site mutants in order of reactivity: R118C > S118C > S121C > R141C > S141C > R121C, R138C, S138C. Chirality appears to have limited effect for the M118C mutants but shows more dramatic impact for the T121C and S141C mutants. These results are in excellent agreement with the experimental observations and offer encouragement that MD can be a useful component of experimental design with considerable predictive power.     

MAM3 catalyzes the formation of all aliphatic glucosinolate chain lengths in Arabidopsis

Chain elongated, methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana). The key enzymatic step in determining the length of the chain is the condensation of acetyl-coenzyme A with a series of omega-methylthio-2-oxoalkanoic acids, catalyzed by methylthioalkylmalate (MAM) synthases. The existence of two MAM synthases has been previously reported in the Arabidopsis ecotype Columbia: MAM1 and MAM3 (formerly known as MAM-L). Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. Underlining its broad substrate specificity, MAM3 also accepts a range of non-Met-derived 2-oxoacids, e.g. converting pyruvate to citramalate and 2-oxoisovalerate to isopropylmalate, a step in leucine biosynthesis. To investigate its role in vivo, we identified plant lines with mutations in MAM3 that resulted in a complete lack or greatly reduced levels of long-chain glucosinolates. This phenotype could be complemented by reintroduction of a MAM3 expression construct. Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. The localization of MAM3 in the chloroplast suggests that this organelle is the site of Met chain elongation.     

MAM (mitochondria-associated membranes) in mammalian cells: Lipids and beyond

One mechanism by which communication between the endoplasmic reticulum (ER) and mitochondria is achieved is by close juxtaposition between these organelles via mitochondria-associated membranes (MAM). The MAM consist of a region of the ER that is enriched in several lipid biosynthetic enzyme activities and becomes reversibly tethered to mitochondria. Specific proteins are localized, sometimes transiently, in the MAM. Several of these proteins have been implicated in tethering the MAM to mitochondria. In mammalian cells, formation of these contact sites between MAM and mitochondria appears to be required for key cellular events including the transport of calcium from the ER to mitochondria, the import of phosphatidylserine into mitochondria from the ER for decarboxylation to phosphatidylethanolamine, the formation of autophagosomes, regulation of the morphology, dynamics and functions of mitochondria, and cell survival. This review focuses on the functions proposed for MAM in mediating these events in mammalian cells. In light of the apparent involvement of MAM in multiple fundamental cellular processes, recent studies indicate that impaired contact between MAM and mitochondria might underlie the pathology of several human neurodegenerative diseases, including Alzheimer's disease. Moreover, MAM has been implicated in modulating glucose homeostasis and insulin resistance, as well as in some viral infections.     

C-S-R triangle theory: community-level predictions, tests, evaluation of criticisms, and relation to other theories

Grime's C - S - R triangle theory has been discussed in plant ecology for two decades, but it has rarely been tested, and not often dispassionately evaluated. We consider the theory from a community viewpoint, and attempt to develop and test predictions for plant communities. C - S - R assumes that in high-disturbance (ruderal, R ) patches or habitats, competition will be absent, or low in intensity. Testing this is problematic because of the difficulty of defining the intensity of competition, and we could find no rigorous evidence to support or refute the prediction. The theory also implies that in high-disturbance habitats there will be no difference in species composition between 'competition' and 'stress' sites, but from available evidence this does not seem to be true. C - S - R assumes that in stressful ( S ) habitats, competition will be low. This assumption is difficult to define or test, because of the overall difference in plant growth rate between habitats. A prediction from the theory is that in stressful habitats autosuccession should occur, i.e. that the climax species should regenerate directly, with no specialist secondary pioneer ( R ) species. There is some evidence that autosuccession occurs under the most extreme stresses of various types. Previous criticisms of C - S - R are evaluated. Only a few are considered valid, mainly those that emphasise that C - S - R theory is a considerable simplification of reality. Previous tests of C - S - R theory appear to be inconclusive. C - S - R theory is basically a combination of r / K theory and Leaf Amortisation theory. We conclude that there is limited support for the C - S - R theory. Whether the gain in generality that the theory offers justifies the loss via simplification that it involves is still an open question. As formulated, it has limited utility as a predictive model in community ecology. Yet, it is currently the most comprehensive and coherent theory for community ecology.     

Engagement of Toll-like receptors by mycoplasmal superantigen: downregulation of TLR2 by MAM/TLR4 interaction

Mycoplasma arthritidis mitogen (MAM) is a superantigen (SAg) from M. arthritidis, an agent of murine toxic shock syndrome and arthritis. We previously demonstrated that C3H/HeJ and C3H/HeSnJ mice that differ in expression of TLR4 differed in immune reactivity to MAM. We show here that MAM directly interacts with TLR2 and TLR4 by using monoclonal antibodies to TLR2 and TLR4 which inhibit cytokine responses of THP-1 cells to MAM. Also, using macrophages from C3H substrains and TLR2-deficient mice, we confirmed that both TLR2 and TLR4 are used by MAM. Levels of IL-6 in supernatants of MAM-challenged macrophages were higher in mice which expressed only TLR2, lesser with both TLR2 and TLR4, and absent in mice lacking both TLR2 and TLR4. In addition, expression of TLR2 and TLR4 was moderately upregulated in wild-type cells but cells lacking TLR4 showed a fivefold increase in TLR2 expression. Further, blockade of TLR4 on macrophages of C3H/HeN mice with antibody greatly increased expression of TLR2 and release of IL-12p40 in response to MAM. These results indicate that the SAg, MAM, interacts with both TLR2 and TLR4 and that TLR4 signalling might downregulate the MAM/TLR2 inflammatory response.     

Expression and characterization of a magnetosome-associated protein, TPR-containing MAM22, in Escherichia coli

A magnetosome-associated protein, MAM22, contains a TPR domain (five TPR motifs and one putative TPR motif) that has been known to mediate protein-protein interactions. We expressed the mam22 gene in Escherichia coli and found that the purified MAM22 was reversibly self-aggregated by NaCl. The structural model of MAM22 which has been proposed on the basis of the crystal structure of the N-terminal TPR domain of a human Ser/Thr protein phosphatase suggests the novel hydrophobic colloidal features of MAM22 with TPR motifs.     

S4 charges move close to residues in the pore domain during activation in a K channel.

Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET(+)). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 A) to a position close (8 A) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.     

Cell-wall regeneration by isolated tomato-fruit protoplasts

Summary Using both light microscopy and electron microscopy of thin sections, and surface replicas, it has been shown that isolated tomato fruit locule tissue protoplasts regenerate a new cell wall when maintained in suitable culture media.This work was supported by a special grant from the S.R.C. Dr. E. Pojnar was in receipt of an EMBO Fellowship.     

Early Presymptomatic Changes in the Proteome of Mitochondria-Associated Membrane in the APP/PS1 Mouse Model of Alzheimer’s Disease

Intracellular β-amyloid (Aβ) accumulation is an early event in Alzheimer’s disease (AD) progression. Recently, it has been uncovered that presenilins (PSs), the key components of the amyloid precursor protein (APP) processing and the β-amyloid producing γ-secretase complex, are highly enriched in a special sub-compartment of the endoplasmic reticulum (ER) functionally connected to mitochondria, called mitochondria-associated ER membrane (MAM). A current hypothesis of pathogenesis of Alzheimer’s diseases (AD) suggests that MAM is involved in the initial phase of AD. Since MAM supplies mitochondria with essential proteins, the increasing level of PSs and β-amyloid could lead to metabolic dysfunction because of the impairment of ER-mitochondrion crosstalk. To reveal the early molecular changes of this subcellular compartment in AD development MAM fraction was isolated from the cerebral cortex of 3 months old APP/PS1 mouse model of AD and age-matched C57BL/6 control mice, then mass spectrometry-based quantitative proteome analysis was performed. The enrichment and purity of MAM preparations were validated with EM, LC-MS/MS and protein enrichment analysis. Label-free LC-MS/MS was used to reveal the differences between the proteome of the transgenic and control mice. We obtained 77 increased and 49 decreased protein level changes in the range of ??6.365 to +?2.988, which have mitochondrial, ER or ribosomal localization according to Gene Ontology database. The highest degree of difference between the two groups was shown by the ATP-binding cassette G1 (Abcg1) which plays a crucial role in cholesterol metabolism and suppresses Aβ accumulation. Most of the other protein changes were associated with increased protein synthesis, endoplasmic-reticulum-associated protein degradation (ERAD), oxidative stress response, decreased mitochondrial protein transport and ATP production. The interaction network analysis revealed a strong relationship between the detected MAM protein changes and AD. Moreover, it explored several MAM proteins with hub position suggesting their importance in Aβ induced early MAM dysregulation. Our identified MAM protein changes precede the onset of dementia-like symptoms in the APP/PS1 model, suggesting their importance in the development of AD.     

TOM20-mediated transfer of Bcl2 from ER to MAM and mitochondria upon induction of apoptosis

In this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2–TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER–mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2–TOM20 interaction is thus an additional player in the control of apoptosis.Subject terms: Cancer, Apoptosis, Mitochondria     

Sigma-1 Receptor Chaperone at the ER-Mitochondrion Interface Mediates the Mitochondrion-ER-Nucleus Signaling for Cellular Survival

The membrane of the endoplasmic reticulum (ER) of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R) in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.     

Molecular approaches to malaria: seeking the whole picture

This year, Australia hosted its first major international conference on malaria - Molecular Approaches to Malaria in Lorne, Victoria, 2-5 February 2000 (MAM2000). The worldwide research effort toward a better understanding of the pathogenesis and control of malaria in the post-genomic era was discussed and debated by over 250 researchers from 18 countries during four days packed with molecular biology, cell biology, genomics, vaccines and pathogenic mechanisms. This special malaria edition of Parasitology Today is an attempt to capture and summarize the quality and breadth of work presented at the conference and place this in the context of the current global malaria research effort; eight of the nine Reviews in this issue have been written by session chairs or presenters at MAM2000.     

Three Novel Mutant Arylsulfatase A Alleles Causing Metachromatic Leukodystrophy

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. This leads to the accumulation of 3-O-sulfogalactosylceramide, which results in severe demyelination. Here we describe a novel non-sense mutation W124ter and two disease-causing missense mutations E382Q and C500F in arylsulfatase A gene. Another so far unknown allele harbors three sequence alterations: two polymorphisms (N350S, R496H) and a missense mutation (R288H). The R288H substitution and the N350S polymorphism have previously been found on one allele together with a polymorphism in a polyadenylation signal characteristic for the arylsulfatase A pseudodeficiency allele. The R496H has been shown to occur on another allele. The presence of the R288H, N350S, and R496H substitution on one allele in the absence of the polyadenylation site polymorphism shows that this allele has probably arisen by recombination between the nucleotides of codon 350 and 496.     

The human cytomegalovirus protein UL37 exon 1 associates with internal lipid rafts

The human cytomegalovirus (HCMV) protein UL37 exon 1 (pUL37x1), also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), sequentially traffics from the endoplasmic reticulum (ER) through mitochondrion-associated membranes (MAMs) to the outer mitochondrial membrane (OMM), where it robustly inhibits apoptosis. Here, we report the association of pUL37x1/vMIA with internal lipid rafts (LRs) in the ER/MAM. The MAM, which serves as a site for lipid transfer and calcium signaling to mitochondria, is enriched in detergent-resistant membrane (DRM)-forming lipids, including cholesterol and ceramide, which are found in lower concentrations in the bulk ER. Sigma 1 receptor (Sig-1R), a MAM chaperone affecting calcium signaling to mitochondria, is anchored in the MAM by its LR association. Because of its trafficking through the MAM and partial colocalization with Sig-1R, we tested whether pUL37x1/vMIA associates with MAM LRs. Extraction with methyl-β-cyclodextrin (MβCD) removed pUL37x1/vMIA from lysed but not intact cells, indicating its association with internal LRs. Furthermore, the isolation of DRMs from purified intracellular organelles independently verified the localization of pUL37x1/vMIA within ER/MAM LRs. However, pUL37x1/vMIA was not detected in DRMs from mitochondria. pUL37x1/vMIA associated with LRs during all temporal phases of HCMV infection, indicating the likely importance of this location for HCMV growth. Although detected during its sequential trafficking to the OMM, the pUL37x1/vMIA LR association was independent of its mitochondrial targeting signals. Rather, it was dependent upon cholesterol binding. These studies suggest a conserved ability of UL37 proteins to interact with cholesterol and LRs, which is functionally distinguishable from their sequential trafficking to mitochondria.     

TLR2 and TLR4 differentially regulate B7-1 resulting in distinct cytokine responses to the mycoplasma superantigen MAM as well as to disease induced by Mycoplasma arthritidis

Mycoplasma arthritidis mitogen (MAM) is a superantigen secreted by M. arthritidis, an agent of murine arthritis and toxicity. We previously demonstrated that C3H mouse sub-strains differing in expression of Toll-like receptor 4 (TLR4), differed in immune reactivity to MAM due to differential engagement of TLR2 and TLR4. Here we examine the role of B7 co-stimulatory molecules in immune outcome and disease manifestations resulting from these different MAM/TLR2 and MAM/TLR4 interactions. Injections of MAM into C3H/HeJ mice upregulated expression of B7-1 but not B7-2 on peritoneal adherent cells, whereas B7-1 expression was lower on cells from C3H/HeSnJ mice. Anti-B7-1 antibody but not anti-B7-2, injected in vivo, changed the type 1 cytokines in MAM-injected C3H/HeJ mice to a type 2 cytokines and, conversely, the type 2 response in C3H/HeSnJ mice injected with anti-B7-1 shifted to a type 1 pattern. Whereas anti-B7-2 exerted no effect on disease in either mouse strain, anti-B7-1 significantly delayed the lethal toxicity of M. arthritidis in C3H/HeJ mice but enhanced arthritis in C3H/HeSnJ mice. Thus, TLR-mediated regulation of B7-1 results in diverse cytokine profiles in C3H sub-strains, and that the interaction of MAM with different TLR(s) may differentially affect cytokine responses and ultimately, M. arthritidis disease.     

Stereospecificity of the products of the fatty acid oxygenases derived from psoriatic scales

The principal in vivo oxygenase products of arachidonic acid and linoleic acid in psoriatic skin scales are 12-hydroxyeicosatetraenoic acid (R/S ratio = 5.7), 13-hydroxyoctadecadienoic acid (S/R = 1.9), and 9-hydroxyoctadecadienoic acid (R/S = 2.4). Definition of the enzymatic origin of these fatty acid derivatives is an important step in assessing their possible role in the pathogenesis of psoriasis. Psoriatic skin scales were incubated with radiolabeled arachidonic acid and linoleic acid and the monohydroxylated derivatives produced in vitro were characterized. The products of incubation with [3H]arachidonic acid were an enantiopure 15(S)-[3H]hydroxyeicosatetraenoic acid and a nonracemic mixture of the 12-[3H]hydroxyeicosatetraenoic acid steroisomers (R/S ratio = 4.5). An enantiopure 13(S)-[14C]hydroxyoctadecadienoic acid was produced from [14C]linoleic acid. No radiolabeled products were derived from incubations with heat-denatured scales. These results provide evidence for two distinct oxygenase activities that are preserved in psoriatic skin scales. One is that of an omega-6 oxygenase with strict (S) stereospecificity, consistent with the activity of a lipoxygenase. This enzyme activity appears to be similar to that of the 15-lipoxygenase which has been described in cultured human keratinocytes. The second activity is that of an arachidonic acid 12(R)-oxygenase that has not been observed in normal human epidermis but which appears to be expressed in psoriatic epidermis.     

A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor

The primary structure of a 61-amino-acid residue peptide from the pancreas of the European eel (Anguilla anguilla) has been established as E E K S G(5)L Y R K P(10)S C G E M(15)S A M H A(20)C P M N F(25)A P V C G(30)T D G N T(35)Y P N E C(40)S L C F Q(45)R Q N T K(50)T D I L I(55)T K D D R(60)C. There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin-releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1-P1' reactive site lysine-isoleucine (or arginine-isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine-asparagine bond. This region does, however, show limited homology to the reactive centre of human alpha 1-protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.     

NMR evidence for syn-anti interconversion of a trans opened (10R)-dA adduct of benzo[a]pyrene (7S,8R)-diol (9R,10S)-epoxide in a DNA duplex

2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12))-d(T(13)G(14)C( 15)C(16)C(17)T(18)T(19)G(20)A(21)C(22)T(23)A(24)), containing a 10R adduct at dA(7) that corresponds to trans addition of the N(6)-amino group of dA(7) to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5'-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S, R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.     

Increased localization of APP‐C99 in mitochondria‐associated ER membranes causes mitochondrial dysfunction in Alzheimer disease

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β‐secretase to generate a 99‐aa C‐terminal fragment (C99) that is then cleaved by γ‐secretase to generate the β‐amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ‐secretase activity is enriched in mitochondria‐associated endoplasmic reticulum (ER) membranes (MAM) and that ER–mitochondrial connectivity and MAM function are upregulated in AD. We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ‐secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.