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 beta-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by c-secretase to generate the beta-amyloid (Ab) found in senile plaques. In previous reports, we and others have shown that c-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 c-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.We thank Drs. Orian Shirihai and Marc Liesa (UCLA) for assistance with the Seahorse measurements, Dr. Huaxi Xu (Sanford Burnham Institute) for the APP-DKO MEFs and Dr. Mark Mattson (NIH) for the PS1 knock-in mice, Drs. Arancio and Teich for the APP-KO mice tissues used in these studies, Dr. Hua Yang (Columbia University) for mouse husbandry, and Drs. Marc Tambini, Ira Tabas, and Serge Przedborski for helpful comments. This work was supported by the Fundacion Alfonso Martin Escudero (to M.P.); the Alzheimer's Drug Discovery Foundation, the Ellison Medical Foundation, the Muscular Dystrophy Association, the U.S. Department of Defense W911NF-12-1-9159 and W911F-15-1-0169), and the J. Willard and Alice S. Marriott Foundation (to E.A.S.); the U.S. National Institutes of Health (P01-HD080642 and P01-HD032062 to E.A.S.; NS071571 and HD071593 to M.F.M.; R01-NS056049 and P50-AG008702 to G.D.P.; 1S10OD016214-01A1 to G.S.P. and F.P.M, and K01-AG045335 to E.A.-G.), the Lucien Cote Early Investigator Award in Clinical Genetics from the Parkinson's Disease Foundation (PDF-CEI-1364 and PDF-CEI-1240) to C.G.-L., and National Defense Science and Engineering Graduate Fellowship (FA9550-11-C-0028) to R.R.A.S

PID 6088 Estudio de la influencia de las hormonas tiroideas en el control de los sistemas de adhesión cadherinas-cateninas durante el desarrollo de vertebrados

El desarrollo de organismos pluricelulares depende en gran medida del establecimiento y mantenimiento de contactos adhesivos fuertes, pero a la vez dinámicos para posibilitar el remodelamiento tisular frente a señales específicas, tanto durante el desarrollo como en el estado adulto. Interesados en evaluar mecanismos de control hormonal de estos contactos, estudiamos si las hormonas tiroideas eran capaces de controlar el desarrollo animal a través de la regulación de la expresión de moléculas de adhesión celular (CAMs), las cuales influencian la adhesión celular y la morfología celular y tisular. Para ello, en una primera etapa, se implementaron bioensayos de bloqueo e inducción de la metamorfosis de Rhinella arenarum y se analizó por inmunohistoquímica cuantitativa el patrón de expresión de las CAMs cadherina E, b- y a-catenina en el intestino anterior o estómago larval de esta especie. En una segunda etapa se utilizaron técnicas de inmunofluorescencia y microscopia de desconvolución digital tridimensional cuantitativa para analizar la influencia de los niveles de fosforilación de las proteínas de los complejos de unión cadherina-catenina in vivo. En una tercera etapa, se analizaron los niveles de expresión de los ARNm de cadherina E y β-catenina y de sus proteínas para correlacionar los estudios morfométricos realizados con estudios moleculares, empleando retrotranscripción y amplificación de ADNcopia (ADNc) por reacción en cadena de la polimerasa (RT-PCR) y western blotting, para, respectivamente. Para ello, se realizaron análisis bioinformáticos de las secuencias y estructuras de las moléculas bajo estudio. Los resultados obtenidos permiten postular por primera vez en forma cuantitativa, un control positivo espacial y temporal de cadherina E, b- y a-catenina por la hormona T3 durante el desarrollo metamórfico del estómago larval de Rhinella arenarum. La alteración de los niveles de fosforilación de las proteínas de los complejos de unión cadherina E-β-catenina, produce una drástica pérdida de estas moléculas en los contactos célula-célula y el incremento citoplasmático y nuclear de β-catenina en las células epidérmicas, sugiriendo la activación de la ruta de señalización nuclear mediada por β-catenina. Sorprendentemente, no se detectan cambios en la forma celular o en la arquitectura de la piel, sugiriendo que la cadherina E epidérmica estaría involucrada en la señalización celular más que en el mantenimiento de los contactos intercelulares durante el mantenimiento de la arquitectura epitelial in vivo. Finalmente, se aislaron, secuenciaron y caracterizaron filogenéticamente secuencias de nucleótidos de cadherina E y de b-catenina de Rhinella arenarum, que resultaron estar altamente conservadas rentre 8 especies de vertebrados.

Alzheimer’s-associated upregulation of mitochondria-associated ER membranes after traumatic brain injury

23 p.-5 fig.-3 tab.-1 graph. abst.Traumatic brain injury (TBI) can lead to neurodegenerative diseases such as Alzheimer’s disease (AD) through mechanisms that remain incompletely characterized. Similar to AD, TBI models present with cellular metabolic alterations and modulated cleavage of amyloid precursor protein (APP). Specifically, AD and TBI tissues display increases in amyloid-β as well as its precursor, the APP C-terminal fragment of 99 a.a. (C99). Our recent data in cell models of AD indicate that C99, due to its affinity for cholesterol, induces the formation of transient lipid raft domains in the ER known as mitochondria-associated endoplasmic reticulum (ER) membranes (“MAM” domains). The formation of these domains recruits and activates specific lipid metabolic enzymes that regulate cellular cholesterol trafficking and sphingolipid turnover. Increased C99 levels in AD cell models promote MAM formation and significantly modulate cellular lipid homeostasis. Here, these phenotypes were recapitulated in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, the injured cortex and hippocampus displayed significant increases in C99 and MAM activity, as measured by phospholipid synthesis, sphingomyelinase activity and cholesterol turnover. In addition, our cell type-specific lipidomics analyses revealed significant changes in microglial lipid composition that are consistent with the observed alterations in MAM-resident enzymes. Altogether, we propose that alterations in the regulation of MAM and relevant lipid metabolic pathways could contribute to the epidemiological connection between TBI and AD.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by the U.S. National Institutes of Health (T32-DK007647 to RRA; R21NS125395 to LS; S10-OD016214 and P30-CA013330 to FPM; R01-EB029523 to WM; R01-NS095803 to SGK; R01-NS088197 to RJD; R01-AG056387 to EA-G) and the U.S. Department of Defense (National Defense Science and Engineering Graduate Fellowship, FA9550-11-C-0028, to RRA).Peer reviewe

MFN2 mutations in Charcot-Marie-Tooth disease alter mitochondria-associated ER membrane function but do not impair bioenergetics.

Charcot-Marie-Tooth disease (CMT) type 2A is a form of peripheral neuropathy, due almost exclusively to dominant mutations in the nuclear gene encoding the mitochondrial protein mitofusin-2 (MFN2). However, there is no understanding of the relationship of clinical phenotype to genotype. MFN2 has two functions: it promotes inter-mitochondrial fusion and mediates endoplasmic reticulum (ER)-mitochondrial tethering at mitochondria-associated ER membranes (MAM). MAM regulates a number of key cellular functions, including lipid and calcium homeostasis, and mitochondrial behavior. To date, no studies have been performed to address whether mutations in MFN2 in CMT2A patient cells affect MAM function, which might provide insight into pathogenesis. Using fibroblasts from three CMT2AMFN2 patients with different mutations in MFN2, we found that some, but not all, examined aspects of ER-mitochondrial connectivity and of MAM function were indeed altered, and correlated with disease severity. Notably, however, respiratory chain function in those cells was unimpaired. Our results suggest that CMT2AMFN2 is a MAM-related disorder but is not a respiratory chain-deficiency disease. The alterations in MAM function described here could also provide insight into the pathogenesis of other forms of CMT

Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB

Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening?closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA

The molecular interface between the type IV secretion systems and their substrates

En los sistemas conjugativos, la proteína acopladora (T4CP) representa la interfase molecular entre el canal formado por el Sistema de Secreción Tipo IV (T4SS) y el sustrato transferido, un complejo nucleoproteico (relaxasa-DNA). En este trabajo hemos estudiado la relación estructural entre la T4CP del plásmido R388 TrwB con los demás componentes del T4SS de R388. También estudiamos los determinantes moleculares implicados en el reconocimiento específico y transferencia del sustrato conjugativo localizados en TrwB y en TrwC (relaxasa). Por otra parte, queríamos conocer hasta dónde podría llegar la versatilidad del T4SS respecto a la célula de destino, usando diferentes receptores de la conjugación como levaduras y mitocondrias. Los resultados de esta tesis indicaron que TrwB es un tercer módulo funcional de la maquinaria conjugativa estructuralmente independiente del complejo multiproteico formado por el T4SS. Del estudio de los determinantes moleculares de reconocimiento específico, encontramos que los residuos cargados positivamente localizados en el canal interno del hexámero de TrwB son importantes para la transferencia del sustrato, y que la señal de translocación de TrwC por el T4SS de R388 se encuentra en el tercio terminal de la proteína. Los datos obtenidos para la transferencia de DNA desde bacterias a levaduras o mitocondrias son prometedores, aunque no concluyentes. Los conocimientos adquiridos en este trabajo aportan aspectos claves de las proteínas acopladoras y su relación con los demás componentes de la maquinaria conjugativa, y sientan las bases para la manipulación de la especificidad de las proteínas acopladoras, así como el intercambio de sustratos por diferentes T4SS

Structural independence of conjugative coupling protein TrwB from its Type IV secretion machinery

The stability of components of multiprotein complexes often relies on the presence of the functional complex. To assess structural dependence among the components of the R388 Type IV secretion system (T4SS), the steady-state level of several Trw proteins was determined in the absence of other Trw components. While several Trw proteins were affected by the lack of others, we found that the coupling protein TrwB is not affected by the absence of other T4SS components, nor did its absence alter significantly the levels of integral components of the complex, underscoring the independent role of the coupling protein on the T4SS architecture. The cytoplasmic ATPases TrwK (VirB4) and TrwD (VirB11) were affected by the absence of several core complex components, while the pilus component TrwJ (VirB5) required the presence of all other Trw proteins (except for TrwB) to be detectable. Overall, the results delineate a possible assembly pathway for the T4SS of R388. We have also tested structural complementation of TrwD (VirB11) and TrwJ (VirB5) by their homologues in the highly related Trw system of Bartonella tribocorum (Bt). The results reveal a correlation with the functional complementation data previously reported. © 2013 Elsevier Inc.This work was supported by Grant BIO2010-11623-E to ML. Work in FC lab was supported by Spanish Ministry of Education (BFU2011-26608), and European VII Framework Program Grants no. 248919/FP7-ICT-2009-4 and 282004/FP7-HEALTH.2011.2.3.1-2. DL was a recipient of a JAE-PRE predoctoral fellowship from the CSIC (Spain).Peer Reviewe

Mutational analysis of conjugative coupling protein TrwB

The coupling protein TrwB is a component of the conjugal machinery that is responsible for connecting the substrate (the DNA strand covalently linked to a pilot protein) to the T4SS during conjugative DNA transfer of plasmid R388. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA after export of the pilot protein through the T4SS, working as a molecular motor. The 3D structure of TrwBΔN70, the cytoplasmic domain of TrwB, revealed a hexameric structure similar to other molecular motor. Based on this 3D structure, a series of point mutants were constructed. We have analyzed in vivo and in vitro properties of TrwB mutants which were only transfer deficient in standard conjugation assays under TrwB limiting condition. We have focused on lysine residues which protrude into the internal channel of the TrwB hexamer because this is the candidate region for DNA interaction, since the transferred DNA is expected to travel through the interior of the hexamer. The results show that lysines protruding into the internal channel have a deregulated ATPase activity; the enhancing effect of ssDNA and TrwA in ATPase activity was also affected in these mutants.Peer Reviewe

Functional Dissection of the Conjugative Coupling Protein TrwB▿

The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwBΔN70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus

Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with β-cell failure.

ObjectiveDiabetes is characterized by pancreatic β-cell dedifferentiation. Dedifferentiating β cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the β-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear.MethodsHere we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to β-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and nicotinamide actin dysfunction (NAD)/reduced nicotinamide actin dysfunction (NADH) ratios.ResultsThe expression of Cyb5r3 is decreased in FoxO1-deficient β cells. Mice with β-cell-specific deletion of Cyb5r3 have impaired insulin secretion, resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient β cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient β cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability.ConclusionsThe findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate β-cell failure