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Supplementary Figure from mTOR Inhibition and T-DM1 in HER2-Positive Breast Cancer

Supplementary Figure 1 shows generation and cell-lineage characterization of primary CAF cultures derived from HER2-positive breast cancer patient biopsies. Moreover, it also shows NRG1 staining in two FFPE-associated patient samples from which primary CAFs were derived.

Supplementary Figure 3 shows how trastuzumab plus pertuzumab, but not trastuzumab alone, overcome NRG1 enhanced pro-survival signaling in HER2-positive breast cancer cells and in ex vivo tumors.

Effects of T-DM1 on 750 cyclin B1 (CCNB1) mRNA expression in T751 DM1 sensitive and resistant breast cancer cells.

Supplementary Figure legend

Supplementary Table 1 shows main clinical-pathological characteristics of HER2-positive breast cancer patients whom primary CAF cultures were derived from.

Purpose: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance.Experimental Design: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2-positive breast cancer explants.Results: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation.Conclusions: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer. Clin Cancer Res; 23(22); 7006–19. ©2017 AACR.

Supplementary Figure 2 shows how NRG1/CAFs, but not normal mammary fibroblasts, mediate trastuzumab resistance in HER2-positive breast cancer cells.

Representative immunohistochemistry images of control and 765 T-DM1 treated explant obtained from a liver metastasis.

Supplementary Figure 4 shows that NRG1 enhances pro-migratory capacity of breast cancer cells as well as the induction of early markers from the EMT process. Moreover, it also shows that trastuzumab plus pertuzumab, but not trastuzumab alone, are able to prevent NRG1-derived effects.

Supplementary Table 2 shows main clinical-pathological characteristics of HER2-positive breast cancer patient's cohort treated with neoadjuvant-based therapy and the association of NRG1 protein expression with the diverse clinical parameters analysed.

Purpose of review The purpose of this brief review is to gain an understanding on the multiple roles that lipids exert on the brain, and to highlight new ideas in the impact of lipid homeostasis in the regulation of synaptic transmission. Recent findings Recent data underline the crucial function of lipid homeostasis in maintaining neuronal function and synaptic plasticity. Moreover, new advances in analytical approaches to study lipid classes and species is opening a new door to understand and monitor how alterations in lipid pathways could shed new light into the pathogenesis of neurodegeneration. Summary Lipids are one of the most essential elements of the brain. However, our understanding of the role of lipids within the central nervous system is still largely unknown. Identifying the molecular mechanism (s) by which lipids can regulate neuronal transmission represents the next frontier in neuroscience, and a new challenge in our understanding of the brain and the mechanism(s) behind neurological disorders.

In patients with trastuzumab-resistant HER2-positive breast cancer, the combination of everolimus (mTORC1 inhibitor) with trastuzumab failed to show a clinically significant benefit. However, the combination of mTOR inhibition and the antibody-drug conjugate (ADC) trastuzumab-emtansine (T-DM1) remains unexplored. We tested T-DM1 plus everolimus in a broad panel of HER2-positive breast cancer cell lines. The combination was superior to T-DM1 alone in four cell lines (HCC1954, SKBR3, EFM192A, and MDA-MB-36) and in two cultures from primary tumor cells derived from HER2-positive patient-derived xenografts (PDX), but not in BT474 cells. In the trastuzumab-resistant HCC1954 cell line, we characterized the effects of the combination using TAK-228 (mTORC1 and -2 inhibitor) and knockdown of the different mTOR complex components. T-DM1 did not affect mTOR downstream signaling nor induct autophagy. Importantly, mTOR inhibition increased intracellular T-DM1levels, leading to increased lysosomal accumulation of the compound. The increased efficacy of mTOR inhibition plus T-DM1 was abrogated by lysosome inhibitors (chloroquine and bafilomycin A1). Our experiments suggest that BT474 are less sensitive to T-DM1 due to lack of optimal lysosomal processing and intrinsic resistance to the DM1 moiety. Finally, we performed several in vivo experiments that corroborated the superior activity of T-DM1 and everolimus in HCC1954 and PDXderived mouse models. In summary, everolimus in combination with T-DM1 showed strong antitumor effects in HER2-positive breast cancer, both in vitro and in vivo. This effect might be related, at least partially, to mTOR-dependent lysosomal processing of T-DM1, a finding that might apply to other ADCs that require lysosomal processing., This work was supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219), Generalitat de Catalunya (2017 SGR 507). MINECO through gBFU2015-71371-R grant and the CRIS Cancer Foundation supported work in AP lab. D. Casadevall was supported by ISCIII (Rio Hortega Research Contract CM16/00023 and Juan Rodés Research Contract JR18/00003). F.J. Sánchez-Martín and S. Menéndez were supported by Department de Salut Generalitat de Catalunya (PERIS SLT002/16/00008 and PERIS SLT006/17/00040). M. Qin received financial support from the China Scholarship Council (CSC) for her doctoral fellowship. Work carried out in our laboratories receives support from the European Community through the Regional Development Funding Program (FEDER).

[Purpose]: To characterize expression of neuregulin-1 (NRG1), an HER3 ligand, in HER2-positive breast cancer and its relation with the efficacy of trastuzumab with or without pertuzumab., [Experimental Design]: Characterization of NRG1 expression in tumor cell lines, in tumor specimens, and in cancer-associated fibroblasts (CAFs). Patient-derived CAFs were used to investigate NRG1 impact on the activity of trastuzumab with or without pertuzumab in HER2-positive breast cancer cells. The relationship between NRG1 expression and pathologic response to anti-HER2–based neoadjuvant therapy was assessed in a retrospective patient cohort and in the NeoSphere trial., [Results]: NRG1 was expressed in HER2-positive breast cancer–derived fibroblasts at significantly higher levels than in cancer cells. NRG1 and the conditioned media (CM) from CAFs phosphorylated HER3 and AKT in cancer cells and mediated trastuzumab resistance. Stable genetic depletion of NRG1 from CAFs overcame trastuzumab resistance. Pertuzumab effectively suppressed trastuzumab resistance mediated by either NRG1 or CAF's CM. NRG1 engaged an epithelial-to-mesenchymal transition that was prevented by trastuzumab and pertuzumab. In clinical samples, stromal and/or tumor cell expression of NRG1 determined by immunohistochemistry was uncommon (13.2%) yet significantly linked with residual disease following trastuzumab-based neoadjuvant therapy. In the NeoSphere trial, the magnitude of the difference of pathologic complete response rates favoring the pertuzumab arm was higher in the NRG1-high group., [Conclusions]: CAF-derived NRG1 mediates trastuzumab resistance through HER3/AKT, which might be reverted by pertuzumab. In patients with HER2-positive breast cancer, high expression of NRG1 was associated to poor response to trastuzumab, but not in combination with pertuzumab., This work is supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219 and by Generalitat de Catalunya (2017 SGR 507). S. Menendez is supported by Department de Salut, Generalitat de Catalunya (PERIS SLT006/17/00040). MARBiobanc is supported by ISCiii/FEDER (PT17/0015/0011) and by “Xarxa de Bancs de tumors” sponsored by Pla Director d’ Oncologia de Catalunya (XBTC) and Fundacion Jimenez Díaz Biobanks Platform by PT13/0010/0012 grant. Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R) and the CRIS Cancer Foundation provides support to A. Pandiella. Work carried out in our laboratories receive support from the European Community through the Regional Development Funding Program (FEDER). J.C. Montero is funded by the ISCIII through a Miguel Servet program (CPII17/00015) and receives research support from the same institution (PI18/00796). J. Albanell is supported by Breast Cancer Research Foundation (BCRF20-08), Instituto de Salud Carlos III Project Reference number AC15/00062 and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociacion Espanola Contra el Cáncer (AECC).

The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP), and how this relationship relates to Alzheimer's disease (AD) pathogenesis, is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cellular cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol levels resides. The intracellular transport of cholesterol from the PM to the ER is believed to be activated by a lipid‐sensing peptide(s) in the ER that can cluster PM‐derived cholesterol into transient detergent‐resistant membrane domains (DRMs) within the ER, also called the ER regulatory pool of cholesterol. When formed, these cholesterol‐rich domains in the ER maintain cellular homeostasis by inducing cholesterol esterification as a mechanism of detoxification while attenuating its de novo synthesis. In this manuscript, we propose that the 99‐aa C‐terminal fragment of APP (C99), when delivered to the ER for cleavage by γ‐secretase, acts as a lipid‐sensing peptide that forms regulatory DRMs in the ER, called mitochondria‐associated ER membranes (MAM). Our data in cellular AD models indicates that increased levels of uncleaved C99 in the ER, an early phenotype of the disease, upregulates the formation of these transient DRMs by inducing the internalization of extracellular cholesterol and its trafficking from the PM to the ER. These results suggest a novel role for C99 as a mediator of cholesterol disturbances in AD, potentially explaining early hallmarks of the disease., When delivered to the ER, the C‐terminal BACE1 cleavage product of amyloid precursor protein APP acts as lipid‐sensing peptide, facilitating formation of detergent‐resistant mitochondria‐associated ER membranes (MAM) via internalization and retrograde transport of cholesterol.

The purpose of this brief review is to gain an understanding on the multiple roles that lipids exert on the brain, and to highlight new ideas in the impact of lipid homeostasis in the regulation of synaptic transmission.Recent data underline the crucial function of lipid homeostasis in maintaining neuronal function and synaptic plasticity. Moreover, new advances in analytical approaches to study lipid classes and species is opening a new door to understand and monitor how alterations in lipid pathways could shed new light into the pathogenesis of neurodegeneration.Lipids are one of the most essential elements of the brain. However, our understanding of the role of lipids within the central nervous system is still largely unknown. Identifying the molecular mechanism (s) by which lipids can regulate neuronal transmission represents the next frontier in neuroscience, and a new challenge in our understanding of the brain and the mechanism(s) behind neurological disorders.

SUMMARY Along with Parkin, PINK1 plays a critical role in maintaining mitochondrial quality control. Although PINK1 is expressed constitutively, its level is kept low in healthy mitochondria by polyubiquitination and ensuing proteasomal degradation of its mature, 52 kDa, form. We show here that the target of PINK1 polyubiquitination is the mature form and is mediated by ubiquitination of a conserved lysine at position 137. Notably, the full-length protein also contains Lys-137 but is not ubiquitinated. On the basis of our data, we propose that cleavage of full-length PINK1 at Phe-104 disrupts the major hydrophobic membrane-spanning domain in the protein, inducing a conformation change in the resultant mature form that exposes Lys-137 to the cytosol for subsequent modification by the ubiquitination machinery. Thus, the balance between the full-length and mature PINK1 allows its levels to be regulated via ubiquitination of the mature form and ensures that PINK1 functions as a mitochondrial quality control factor., In Brief PINK1 mutations cause Parkinson’s disease. PINK1 is cleaved into a shorter 52-kDa form at the mitochondrial membrane, but regulation of the turnover of cleaved PINK1 is unknown. Liu et al. show that polyubiquitination of cleaved PINK1 regulates its degradation via the proteasome.

The link between cholesterol homeostasis and the cleavage of the amyloid precursor protein (APP), and their relationship to the pathogenesis of Alzheimer’s disease (AD) is still unknown. Cellular cholesterol levels are regulated by a crosstalk between the plasma membrane (PM), where most of the cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol resides. This crosstalk between PM and ER is believed to be regulated by lipid-sensing peptide(s) that can modulate the internalization of extracellular cholesterol and/or itsde novosynthesis in the ER. Our data here indicates that the 99-aa C-terminal fragment of APP (C99), a cholesterol-binding peptide, regulates cholesterol trafficking between the PM and the ER. In AD models, increases in C99 provoke the upregulation of cholesterol internalization and its delivery to the ER, which in turn result into the loss of lipid homeostasis and the appearance of AD signatures, such as higher production of longer forms of amyloid β. Our data suggest a novel role of C99 as mediator of cholesterol disturbances in AD, and as a potential early hallmark of the disease.

Mitochondrial respiratory deficiencies have been observed in numerous neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. For decades, these reductions in oxidative phosphorylation (OxPhos) have been presumed to trigger an overall bioenergetic crisis in the neuron, resulting in cell death. While the connection between respiratory defects and neuronal death has never been proven, this hypothesis has been supported by the detection of nonspecific mitochondrial DNA mutations in these disorders. These findings led to the notion that mitochondrial respiratory defects could be initiators of these common neurodegenerative disorders, instead of being consequences of a prior insult, a theory we believe to be misconstrued. Herein, we review the roots of this mitochondrial hypothesis and offer a new perspective wherein mitochondria are analyzed not only from the OxPhos point of view, but also as a complex organelle residing at the epicenter of many metabolic pathways.

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., U.S. Department of Defense (W911F-15-1-0169 to E.A.S.); J. Willard and Alice S. Marriott Foundation (to E.A.S.); U.S. National Institutes of Health (P01-HD032062 to E.A.S. and K01-AG045335 to E.A.-G.); American Parkinson Disease Association (to C.G.-L.); Keith B Hayes Foundation and the Kassab Family (to H.O.A.); CARIPARO Starting Grant 2016 AIFbiol (to M.G.), DeBio Departmental Research Project PRID Seed 2017 (to M.G.) and Unipd STARS Consolidator (‘Featuring a new class of Interface Regulators: the mitochondria–ER spacers’ to M.G.); Wellcome Centre for Mitochondrial Research (109915/Z/15/Z and 203105/Z/16/Z to R.H.); MRC (UK; MR/N025431/1 to R.H.); European Research Council (309548 to R.H.); Wellcome Trust Pathfinder Scheme (201064/Z/16/Z to R.H.); Newton Fund (UK/Turkey, MR/N027302/1 to R.H.). M.S. is supported in part by the Inherited Neuropathies Consortium (U54NS065712) that is a part of the NIH National Center for Advancing Translational Sciences (NCATS) Rare Diseases Clinical Research Network (RDCRN).

Maintaining a pool of functional mitochondria requires degradation of damaged ones within the cell. PINK1 is critical in this quality-control process: loss of mitochondrial membrane potential causes PINK1 to accumulate on the mitochondrial surface, triggering mitophagy. However, little is known about how PINK1 is regulated. Recently, we showed that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation of its mature form via a mechanism inconsistent with the proposed N-end rule process. Using both human female and monkey cell lines, we now demonstrate that once generated within the mitochondria, 52 kDa PINK1 adopts a mitochondrial topology most consistent with it being at the mitochondrial–endoplasmic reticulum (ER) interface. From this particular submitochondrial location, PINK1 interacts with components of the ER-associated degradation pathway, such as the E3 ligases gp78 and HRD1, which cooperate to catalyze PINK1 ubiquitination. The valosin-containing protein and its cofactor, UFD1, then target ubiquitinated PINK1 for proteasomal degradation. Our data show that PINK1 in healthy mitochondria is negatively regulated via an interplay between mitochondria and ER, and shed light on how this mitochondrial protein gains access to the proteasome. SIGNIFICANCE STATEMENT Regulation of mitochondrial content of PINK1, a contributor to mitophagy, is an important area of research. Recently, we found that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation. We now extend and refine this novel finding by showing that PINK1 localizes at the mitochondrial–endoplasmic reticulum (ER) interface, from where it interacts with the ER-associated degradation machinery, which catalyzes its ubiquitination and transfer to the proteasome. Thus, these data show that PINK1 in healthy mitochondria is negatively regulated via a mitochondria and ER interplay, and how this mitochondrial protein gains access to the proteasome.

In addition to the appearance of senile plaques and neurofibrillary tangles, Alzheimer's disease (AD) is characterized by aberrant lipid metabolism and early mitochondrial dysfunction. We recently showed that there was increased functionality of mitochondria‐associated endoplasmic reticulum (ER) membranes (MAM), a subdomain of the ER involved in lipid and cholesterol homeostasis, in presenilin‐deficient cells and in fibroblasts from familial and sporadic AD patients. Individuals carrying the ε4 allele of apolipoprotein E (ApoE4) are at increased risk for developing AD compared to those carrying ApoE3. While the reason for this increased risk is unknown, we hypothesized that it might be associated with elevated MAM function. Using an astrocyte‐conditioned media (ACM) model, we now show that ER–mitochondrial communication and MAM function—as measured by the synthesis of phospholipids and of cholesteryl esters, respectively—are increased significantly in cells treated with ApoE4‐containing ACM as compared to those treated with ApoE3‐containing ACM. Notably, this effect was seen with lipoprotein‐enriched preparations, but not with lipid‐free ApoE protein. These data are consistent with a role of upregulated MAM function in the pathogenesis of AD and may help explain, in part, the contribution of ApoE4 as a risk factor in the disease.

Familial cases of Parkinson's disease (PD) can be associated with overexpression or mutation of α-synuclein, a synaptic protein reported to be localized mainly in the cytosol and mitochondria. We recently showed that wild-type α-synuclein is not present in mitochondria, as previously thought, but rather is located in mitochondrial-associated endoplasmic reticulum membranes. Remarkably, we also found that PD-related mutated α-synuclein results in its reduced association with mitochondria-associated membranes, coincident with a lower degree of apposition of endoplasmic reticulum with mitochondria and an increase in mitochondrial fragmentation, as compared with wild-type. This new subcellular localization of α-synuclein raises fundamental questions regarding the relationship of α-synuclein to mitochondria-associated membranes function, in both normal and pathological states. In this article, we attempt to relate aspects of PD pathogenesis to what is known about mitochondria-associated membranes' behavior and function. We hypothesize that early events occurring in dopaminergic neurons at the level of the mitochondria-associated membranes could cause long-term disturbances that lead to PD.

[Purpose]: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance. [Experimental Design]: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2-positive breast cancer explants. [Results]: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation. [Conclusions]: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer., This work was supported by ISCiii (CIBERONC CB16/12/00481, RD12/0036/0051, RD12/0036/0070, RD12/0036/0003, PIE15/00008, PI13/00864, PI15/00146, PI15/00934, PI15/01617, PT13/0010/0005), Generalitat de Catalunya (2014 SGR 740), and the >Xarxa de Bancs de tumors sponsored by Pla Director d'Oncologia de Catalunya (XBTC). MINECO through BFU2015-71371-R grant supported work in A. Pandiella's laboratory. Our work was supported by the EU through the regional funding development program (FEDER). P. González-Alonso was supported by Fundación Conchita Rábago de Jiménez Díaz grant.

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. Sí

Autosomal-dominant Alzheimer's disease (ADAD) is a genetic disorder caused by mutations in Amyloid Precursor Protein (APP) or Presenilin (PSEN) genes. Studying the mechanisms underlying these mutations can provide insight into the pathways that lead to AD pathology. The majority of biochemical studies on APP mutations to-date have focused on comparing mechanisms between mutations at different codons. It has been assumed that amino acid position is a major determinant of protein dysfunction and clinical phenotype. However, the differential effect of mutations at the same codon has not been sufficiently addressed. In the present study we compared the effects of the aggressive ADAD-associated APP I716F mutation with I716V and I716T on APP processing in human neuroglioma and CHO-K1 cells. All APP I716 mutations increased the ratio of Aβ42/40 and changed the product line preference of γ-secretase towards Aβ38 production. In addition, the APP I716F mutation impaired the ε-cleavage and the fourth cleavage of γ-secretase and led to abnormal APP β-CTF accumulation at the plasma membrane. Taken together, these data indicate that APP mutations at the same codon can induce diverse abnormalities in APP processing, some resembling PSEN1 mutations. These differential effects could explain the clinical differences observed among ADAD patients bearing different APP mutations at the same position. The amyloid precursor protein (APP) I716F mutation is associated with autosomal dominant Alzheimer's disease with the youngest age-at-onset for the APP locus. Here, we describe that this mutation, when compared to two other familial Alzheimer's disease mutations at the same codon (I716V and I716T), interfered distinctly with γ-secretase cleavage. While all three mutations direct γ-secretase cleavage towards the 48→38 production line, the APP I716F mutation also impaired the ε-cleavage and the fourth cleavage of γ-secretase, resembling a PSEN1 mutation. These features may contribute to the aggressiveness of this mutation.

Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by γ-secretase, via an unknown mechanism. We recently showed that presenilin-1 and -2, the catalytic components of γ-secretase, and γ-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER–mitochondrial communication—as measured by cholesteryl ester and phospholipid synthesis, respectively—are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and γ-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER–mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD.

We report the clinical, pathologic, and biochemical characteristics of the recently described amyloid precursor protein (APP) I716F mutation. We present the clinical findings of individuals carrying the APP I716F mutation and the neuropathologic examination of the proband. The mutation was found in a patient with Alzheimer disease with onset at the age of 31 years and death at age 36 years and who had a positive family history of early-onset Alzheimer disease. Neuropathologic examination showed abundant diffuse amyloid plaques mainly composed of amyloid-beta42 and widespread neurofibrillary pathology. Lewy bodies were found in the amygdala. Chinese hamster ovary cells transfected with this mutation showed a marked increase in the amyloid-beta42/40 ratio and APP C-terminal fragments and a decrease in APP intracellular domain production, suggesting reduced APP proteolysis by gamma-secretase. Taken together, these findings indicate that the APP I716F mutation is associated with the youngest age of onset for this locus and strengthen the inverse association between amyloid-beta42/40 ratio and age of onset. The mutation leads to a protein that is poorly processed by gamma-secretase. This loss of function may be an additional mechanism by which some mutations around the gamma-secretase cleavage site lead to familial Alzheimer disease.

Activity-dependent gene expression mediating changes of synaptic efficacy is important for memory storage, but the mechanisms underlying gene transcriptional changes in age-related memory disorders are poorly understood. In this study, we report that gene transcription mediated by the cAMP-response element binding protein (CREB)-regulated transcription coactivator CRTC1 is impaired in neurons and brain from an Alzheimer's disease (A beta) transgenic mouse expressing the human beta-amyloid precursor protein (APP(Sw,Ind)). Suppression of CRTC1-dependent gene transcription by beta-amyloid (A beta) in response to cAMP and Ca2+ signals is mediated by reduced calcium influx and disruption of PP2B/calcineurin-dependentCRTC1dephosphorylation at Ser151. Consistently, expression of CRTC1 or active CRTC1 S151A and calcineurin mutants reverse the deficits on CRTC1 transcriptional activity in APPSw, Ind neurons. Inhibition of calcium influx by pharmacological blockade of L-type voltage-gated calcium channels (VGCCs), but not by blocking NMDA or AMPA receptors, mimics the decrease on CRTC1 transcriptional activity observed in APP(Sw,Ind) neurons, whereas agonists of L-type VGCCs reverse efficiently these deficits. Consistent with a role of CRTC1 on A beta-induced synaptic and memory dysfunction, we demonstrate a selective reduction of CRTC1-dependent genes related to memory (Bdnf, c-fos, and Nr4a2) coinciding with hippocampal-dependent spatial memory deficits in APP(Sw,Ind) mice. These findings suggest that CRTC1 plays a key role in coupling synaptic activity to gene transcription required for hippocampal-dependent memory, and that A beta could disrupt cognition by affecting CRTC1 function.

Familial cases of Parkinson's disease (PD) can be associated with overexpression or mutation of α-synuclein, a synaptic protein reported to be localized mainly in the cytosol and mitochondria. We recently showed that wild-type α-synuclein is not present in mitochondria, as previously thought, but rather is located in mitochondrial-associated endoplasmic reticulum membranes. Remarkably, we also found that PD-related mutated α-synuclein results in its reduced association with mitochondria-associated membranes, coincident with a lower degree of apposition of endoplasmic reticulum with mitochondria and an increase in mitochondrial fragmentation, as compared with wild-type. This new subcellular localization of α-synuclein raises fundamental questions regarding the relationship of α-synuclein to mitochondria-associated membranes function, in both normal and pathological states. In this article, we attempt to relate aspects of PD pathogenesis to what is known about mitochondria-associated membranes' behavior and function. We hypothesize that early events occurring in dopaminergic neurons at the level of the mitochondria-associated membranes could cause long-term disturbances that lead to PD.

Background and Purpose— It has been proposed that the deposition of the β-amyloid peptide (Aβ) in the brain parenchyma and brain blood vessels has deleterious effects. We tested the hypothesis that the levels of plasma Aβ are related to the outcome in patients with intracerebral hemorrhage. Methods— In a multicenter study, we prospectively included patients with spontaneous intracerebral hemorrhage within the first 24 hours after onset. At admission, we measured plasma Aβ40 and Aβ42 levels using ELISA techniques. Also, we recorded age, sex, vascular risk factors, National Institutes of Health Stroke Scale score, presence of intraventricular hemorrhage, localization, cause, and volume of the hematoma. We obtained the modified Rankin scale and defined a unfavorable outcome as modified Rankin scale >2 at 3 months. Bivariate and multivariate regression analyses were performed. Results— We studied 160 patients (mean age, 73.8±11.3 years; 59.4% of them were men). A favorable outcome was observed in 64 (40%) of the patients. In the bivariate analyses, unfavorable outcome was associated with high age, female sex, diabetes mellitus, presence of intraventricular hemorrhage, high blood glucose, high National Institutes of Health Stroke Scale score, high volume, and high plasma levels of Aβ42 and Aβ40. The multivariate analysis showed that increased age (odds ratio, 1.07; 95% confidence interval, 1.035–1.21; P P P =0.02), and Aβ42 levels >9.7 pg/mL (odds ratio, 4.11; 95% confidence interval, 1.65–10.1; P =0.02) were independently associated with an increased likelihood of an unfavorable outcome. Conclusions— High levels of plasma Aβ42 in patients with acute intracerebral hemorrhage are associated with a poor functional prognosis.

Familial Parkinson disease is associated with mutations in α-synuclein (α-syn), a presynaptic protein that has been localized not only to the cytosol, but also to mitochondria. We report here that wild-type α-syn from cell lines, and brain tissue from humans and mice, is present not in mitochondria but rather in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a structurally and functionally distinct subdomain of the ER. Remarkably, we found that pathogenic point mutations in human α-syn result in its reduced association with MAM, coincident with a lower degree of apposition of ER with mitochondria, a decrease in MAM function, and an increase in mitochondrial fragmentation compared with wild-type. Although overexpression of wild-type α-syn in mutant α-syn-expressing cells reverted the fragmentation phenotype, neither overexpression of the mitochondrial fusion/MAM-tethering protein MFN2 nor inhibition/ablation of the mitochondrial fission protein DRP1 was able to do so, implying that α-syn operates downstream of the mitochondrial fusion/fission machinery. These novel results indicate that wild-type α-syn localizes to the MAM and modulates mitochondrial morphology, and that these behaviors are impaired by pathogenic mutations in α-syn. We believe that our results have far-reaching implications for both our understanding of α-syn biology and the treatment of synucleinopathies.

Parkinson’s disease (PD) is a common neurodegenerative disorder caused by genetic and environmental factors that results in degeneration of the nigrostriatal dopaminergic pathway in the brain. We analyzed neural cells generated from induced pluripotent stem cells (iPSCs) derived from PD patients and presymptomatic individuals carrying mutations in the PINK1 (PTEN-induced putative kinase 1 )a ndLRRK2 (leucine-rich repeat kinase 2) genes, and compared them to those of healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage, and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial dysfunction in iPSC-derived neural cells from familial PD patients and at-risk individuals could be rescued with coenzyme Q10, rapamycin, or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSCderived neural cells from PD patients carrying different mutations provides insight into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in this neurodegenerative disease.

Inherited familial Alzheimer's disease (AD) is characterized by small increases in the ratio of Aβ42 versus Aβ40 peptide which is thought to drive the amyloid plaque formation in the brain of these patients. Little is known however whether ageing, the major risk factor for sporadic AD, affects amyloid beta-peptide (Aβ) generation as well. Here we demonstrate that the secretion of Aβ is enhanced in an in vitro model of neuronal ageing, correlating with an increase in γ-secretase complex formation. Moreover we found that peroxynitrite (ONOO -), produced by the reaction of superoxide anion with nitric oxide, promoted the nitrotyrosination of presenilin 1 (PS1), the catalytic subunit of γ-secretase. This was associated with an increased association of the two PS1 fragments, PS1-CTF and PS1-NTF, which constitute the active catalytic centre. Furthermore, we found that peroxynitrite shifted the production of Aβ towards Aβ 42 and increased the Aβ 42/Aβ 40 ratio. Our work identifies nitrosative stress as a potential mechanistic link between ageing and AD. © 2012 EMBO Molecular Medicine., Fund for Scientific Research, Flanders; the K.U.Leuven; the VIB, Methusalem (K.U.Leuven and the Flemisch government); the Foundation for Alzheimer Research (SAO/FRMA); European Research council; NIH AG15379 (OB); Spanish Ministry of Science and Innovation SAF 2010-14906; Consolider 2010-00045 (CGD); Spanish Ministery of Health (Fondo de Investigación Sanitaria-PI10/00587 and Red HERACLES RD06/0009); The European FEDER Fundings; and Fundació La Marató de TV3 (Catalonia; Spain; no. 100310)

Autosomal-dominant Alzheimer disease (ADAD) is a genetic disorder caused by mutations in Amyloid Precursor Protein (APP) or Presenilin (PSEN) genes. Studies from families with ADAD have been critical to support the amyloid cascade hypothesis of Alzheimer disease (AD), the basis for the current development of amyloid-based disease-modifying therapies in sporadic AD (SAD). However, whether the pathological changes in APP processing in the CNS in ADAD are similar to those observed in SAD remains unclear. In this study, we measured β-site APP-cleaving enzyme (BACE) protein levels and activity, APP and APP C-terminal fragments in brain samples from subjects with ADAD carrying APP or PSEN1 mutations (n = 18), patients with SAD (n = 27) and age-matched controls (n = 22). We also measured sAPPβ and BACE protein levels, as well as BACE activity, in CSF from individuals carrying PSEN1 mutations (10 mutation carriers and 7 non-carrier controls), patients with SAD (n = 32) and age-matched controls (n = 11). We found that in the brain, the pattern in ADAD was characterized by an increase in APP β-C-terminal fragment (β-CTF) levels despite no changes in BACE protein levels or activity. In contrast, the pattern in SAD in the brain was mainly characterized by an increase in BACE levels and activity, with less APP β-CTF accumulation than ADAD. In the CSF, no differences were found between groups in BACE activity or expression or sAPPβ levels. Taken together, these data suggest that the physiopathological events underlying the chronic Aβ production/clearance imbalance in SAD and ADAD are different. These differences should be considered in the design of intervention trials in AD. Electronic supplementary material The online version of this article (doi:10.1007/s00401-012-1062-9) contains supplementary material, which is available to authorized users.

BackgroundThe simultaneous accumulation of different misfolded proteins in the central nervous system is a common feature in many neurodegenerative diseases. In most cases, co-occurrence of abnormal deposited proteins is observed in different brain regions and cell populations, but, in some instances, the proteins can be found in the same cellular aggregates. Co-occurrence of tau and α-synuclein (α-syn) aggregates has been described in neurodegenerative disorders with primary deposition of α-syn, such as Parkinson's disease and dementia with Lewy bodies. Although it is known that tau and α-syn have pathological synergistic effects on their mutual fibrillization, the underlying biological effects remain unclear.Methodology/principal findingsWe used different cell models of synucleinopathy to investigate the effects of tau on α-syn aggregation. Using confocal microscopy and FRET-based techniques we observed that tau colocalized and interacted with α-syn aggregates. We also found that tau overexpression changed the pattern of α-syn aggregation, reducing the size and increasing the number of aggregates. This shift was accompanied by an increase in the levels of insoluble α-syn. Furthermore, co-transfection of tau increased secreted α-syn and cytotoxicity.Conclusions/significanceOur data suggest that tau enhances α-syn aggregation and toxicity and disrupts α-syn inclusion formation. This pathological synergistic effect between tau and α-syn may amplify the deleterious process and spread the damage in neurodegenerative diseases that show co-occurrence of both pathologies.

Inflammation has been associated with the two classic lesions in the Alzheimer's (AD) brain, amyloid deposits and neurofibrillary tangles. Recent data suggest that Triflusal, a compound with potent anti-inflammatory effects in the central nervous system in vivo, might delay the conversion from amnestic mild cognitive impairment to a fully established clinical picture of dementia. In the present study, we investigated the effect of Triflusal on brain A beta accumulation, neuroinflammation, axonal curvature and cognition in an AD transgenic mouse model (Tg2576). Triflusal treatment did not alter the total brain A beta accumulation but significantly reduced dense-cored plaque load and associated glial cell proliferation, proinflammatory cytokine levels and abnormal axonal curvature, and rescued cognitive deficits in Tg2576 mice. Behavioral benefit was found to involve increased expression of c-fos and BDNF, two of the genes regulated by CREB, as part of the signal transduction cascade underlying the molecular basis of long-term potentiation. These results add preclinical evidence of a potentially beneficial effect of Triflusal in AD. (C) 2010 Elsevier Inc. All rights reserved.

It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and gamma-secretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Abeta(40) and Abeta(42) in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP-Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP-PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of gamma-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts.

Amyloid deposits, neurofibrillary tangles, and neuronal cell death in selectively vulnerable brain regions are the chief hallmarks in Alzheimer's (AD) brains. Glycogen synthase kinase-3 (GSK-3) is one of the key kinases required for AD-type abnormal hyperphosphorylation of tau, which is believed to be a critical event in neurofibrillary tangle formation. GSK-3 has also been recently implicated in amyloid precursor protein (APP) processing/Aβ production, apoptotic cell death, and learning and memory. Thus, GSK-3 inhibition represents a very attractive drug target in AD and other neurodegenerative disorders. To investigate whether GSK-3 inhibition can reduce amyloid and tau pathologies, neuronal cell death and memory deficits in vivo, double transgenic mice coexpressing human mutant APP and tau were treated with a novel non-ATP competitive GSK-3β inhibitor, NP12. Treatment with this thiadiazolidinone compound resulted in lower levels of tau phosphorylation, decreased amyloid deposition and plaque-associated astrocytic proliferation, protection of neurons in the entorhinal cortex and CA1 hippocampal subfield against cell death, and prevention of memory deficits in this transgenic mouse model. These results show that this novel GSK-3 inhibitor has a dual impact on amyloid and tau alterations and, perhaps even more important, on neuronal survival in vivo further suggesting that GSK-3 is a relevant therapeutic target in AD., This work was funded by SAF2004-07802, FIS PI 061582 and CIBERNED.

We describe a Spanish family in which 3 of 4 siblings had dementia with Lewy bodies, 2 of them starting at age 26 years and the other at 29 years. The father has recently been diagnosed with Lewy body disease, with onset at 77 years. Neuropathological examination of the brain of the index patient disclosed unusual features characterized by diffuse Lewy body disease and generalized neurofibrillary tangle pathology but with not amyloid deposits in any region. Moreover, Lewy body pathology colocalized with neurofibrillary tangles in most affected neurons. Mutation screening that included all coding exons of presenilin 1 (PSEN1), presenilin 2 (PSEN2), alpha-synuclein (SNCA), beta-synuclein (SNCB), microtubule-associated protein tau (MAPT) leucine-rich repeat kinase 2 (LRRK2), glucocerebrosidase (GBA), and exons 16 and 17 of the amyloid precursor protein (APP) genes did not identify any mutation. Genome-wide single nucleotide polymorphism was performed in 4 family members and ruled out any pathogenic duplication or deletion in the entire genome. In summary. we report a unique family with pathologically confirmed early-onset dementia with Lewy, bodies with widespread tau and alpha-synuclein deposition. The absence Of Mutations in genes known to cause Lewy body disease suggests that a novel locus or loci are implicated in this neurodegenerative disease.

Ajuts: Beca bianual de la Fundació Olga Torres 2008-2009 Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells.