Andrés Norambuena

Amyloid-β signals through tau to drive ectopic neuronal cell cycle re-entry in Alzheimer's disease.

Summary Normally post-mitotic neurons that aberrantly re-enter the cell cycle without dividing account for a substantial fraction of the neurons that die in Alzheimers disease (AD). We now report that this ectopic cell cycle re-entry (CCR) requires soluble amyloid-&bgr; (A&bgr;) and tau, the respective building blocks of the insoluble plaques and tangles that accumulate in AD brain. Exposure of cultured wild type (WT) neurons to A&bgr; oligomers caused CCR and activation of the non-receptor tyrosine kinase, fyn, the cAMP-regulated protein kinase A and calcium-calmodulin kinase II, which respectively phosphorylated tau on Y18, S409 and S416. In tau knockout (KO) neurons, A&bgr; oligomers activated all three kinases, but failed to induce CCR. Expression of WT, but not Y18F, S409A or S416A tau restored CCR in tau KO neurons. Tau-dependent CCR was also observed in vivo in an AD mouse model. CCR, a seminal step in AD pathogenesis, therefore requires signaling from A&bgr; through tau independently of their incorporation into plaques and tangles.

RalA-Exocyst Complex Regulates Integrin-Dependent Membrane Raft Exocytosis and Growth Signaling

Anchorage dependence of cell growth is a key metastasis-suppression mechanism that is mediated by effects of integrins on growth signaling pathways. The small GTPase RalA is activated in metastatic cancers through multiple mechanisms and specifically induces anchorage independence. Loss of integrin-mediated adhesion triggers caveolin-dependent internalization of cholesterol- and sphingolipid-rich lipid raft microdomains to the recycling endosomes; these domains serve as platforms for many signaling pathways, and their clearance from the plasma membrane (PM) after cell detachment suppresses growth signaling. Conversely, readhesion triggers their return to the PM and restores growth signaling. Activation of Arf6 by integrins mediates exit of raft markers from the recycling endosomes but is not sufficient for return to the PM. We now show that RalA but not RalB mediates integrin-dependent membrane raft exocytosis through the exocyst complex. Constitutively active RalA restores membrane raft targeting to promote anchorage-independent growth signaling. Ras-transformed pancreatic cancer cells also show RalA-dependent constitutive PM raft targeting. These results identify RalA as a key determinant of integrin-dependent membrane raft trafficking and regulation of growth signaling. They therefore define a mechanism by which RalA regulates anchorage dependence and provide a new link between integrin signaling and cancer.

Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation

Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity.

Effects of integrin-mediated cell adhesion on plasma membrane lipid raft components and signaling

Loss of integrin-mediated cell adhesion is known to induce internalization of lipid rafts, which alters of the plasma membranes physical and signaling properties. Analysis of multiple proteins now shows a wide range of behaviors in which internalization and exit from the ordered to disordered domains are regulated separately.

Regulation of Rac1 translocation and activation by membrane domains and their boundaries.

ABSTRACT The activation of Rac1 and related Rho GTPases involves dissociation from Rho GDP-dissociation inhibitor proteins and translocation to membranes, where they bind effectors. Previous studies have suggested that the binding of Rac1 to membranes requires, and colocalizes with, cholesterol-rich liquid-ordered (lo) membrane domains (lipid rafts). Here, we have developed a fluorescence resonance energy transfer (FRET) assay that robustly detects Rac1 membrane targeting in living cells. Surprisingly, FRET with acceptor constructs that were targeted to either raft or non-raft areas indicated that Rac1 was present in both regions. Functional studies showed that Rac1 localization to non-raft regions decreased GTP loading as a result of inactivation by GTPase-activating proteins. In vitro, Rac1 translocation to supported lipid bilayers also required lo domains, yet Rac1 was concentrated in the liquid-disordered (ld) phase. Single-molecule analysis demonstrated that translocation occurred preferentially at lo–ld boundaries. These results, therefore, suggest that Rac1 translocates to the membrane at domain boundaries, then diffuses into raft and non-raft domains, which controls interactions. These findings resolve discrepancies in our understanding of Rac biology and identify novel mechanisms by which lipid rafts modulate Rho GTPase signaling.

Phosphatidic Acid Induces Ligand-independent Epidermal Growth Factor Receptor Endocytic Traffic through PDE4 Activation

Endocytic traffic can control cell surface versus intracellular distribution of empty/inactive EGFR, an thus its accessibility to external stimuli, through a pathway involving down regulation of PKA activity mediated by PA signaling towards PDE4. This novel control mechanism can trans-modulate EGFR function by heterologous stimuli of PLD.

P2Y1 receptor activation elicits its partition out of membrane rafts and its rapid internalization from human blood vessels: implications for receptor signaling.

The nucleotide P2Y1 receptor (P2Y1R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y1R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y1R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl β-cyclodextrin reduced the raft partitioning of the P2Y1R and obliterated the P2Y1R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y1R agonist, not only displaced within 4 min the P2Y1R localization out of membrane rafts but also induced its subsequent internalization. 2′-Deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y1R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y1R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y1R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y1R to membrane rafts, highlighting the role of this microdomain in P2Y1R signaling.

mTOR and neuronal cell cycle reentry: How impaired brain insulin signaling promotes Alzheimer's disease.

A major obstacle to presymptomatic diagnosis and disease‐modifying therapy for Alzheimers disease (AD) is inadequate understanding of molecular mechanisms of AD pathogenesis. For example, impaired brain insulin signaling is an AD hallmark, but whether and how it might contribute to the synaptic dysfunction and neuron death that underlie memory and cognitive impairment has been mysterious. Neuron death in AD is often caused by cell cycle reentry (CCR) mediated by amyloid‐β oligomers (AβOs) and tau, the precursors of plaques and tangles. We now report that CCR results from AβO‐induced activation of the protein kinase complex, mTORC1, at the plasma membrane and mTORC1‐dependent tau phosphorylation, and that CCR can be prevented by insulin‐stimulated activation of lysosomal mTORC1. AβOs were also shown previously to reduce neuronal insulin signaling. Our data therefore indicate that the decreased insulin signaling provoked by AβOs unleashes their toxic potential to cause neuronal CCR, and by extension, neuron death.

A Small Molecule Screen Exposes mTOR Signaling Pathway Involvement in Radiation-Induced Apoptosis

Individuals are at risk of exposure to acute ionizing radiation (IR) from a nuclear accident or terrorism, but we lack effective therapies to mitigate the lethal IR effects. In the current study, we exploited an optimized, cell-based, high throughput screening assay to interrogate a small molecule library comprising 3437 known pharmacologically active compounds for mitigation against IR-induced apoptosis. Thirty-three library compounds significantly reduced apoptosis when administered 1 h after 4 Gy IR. Two- or three-dimensional computational structural analyses of the compounds indicated only one or two chemical clusters with most of the compounds being unique structures. The mechanistic target of rapamycin complex 1 (mTORC1) inhibitor, rapamycin, was the most potent compound, and it mitigated apoptosis by 50% at 200 ± 50 pM. Other mTOR inhibitors, namely everolimus, AZD8055, and torin 1, also suppressed apoptosis, providing additional pharmacological evidence for mTOR pathway involvement in regulating cell death after IR. Everolimus and torin 1 treatment after IR decreased the S phase population and enforced both G1 and G2 phase arrest. This prorogation of cell cycle progression was accompanied by decreased IR-induced DNA damage measured by γH2AX phosphorylation at Ser139. RNA interference-mediated knockdown of the respective mTORC1 and mTORC2 subunits, Raptor or Rictor, also mitigated IR-induced apoptosis. Collectively, this study suggests a central role for the mTOR signaling in the cytotoxic response to IR and offers a useful platform to probe for additional agents.

A novel lysosome-to-mitochondria signaling pathway disrupted by amyloid-β oligomers.

The mechanisms of mitochondrial dysfunction in Alzheimers disease are incompletely understood. Using two‐photon fluorescence lifetime microscopy of the coenzymes, NADH and NADPH, and tracking brain oxygen metabolism with multi‐parametric photoacoustic microscopy, we show that activation of lysosomal mechanistic target of rapamycin complex 1 (mTORC1) by insulin or amino acids stimulates mitochondrial activity and regulates mitochondrial DNA synthesis in neurons. Amyloid‐β oligomers, which are precursors of amyloid plaques in Alzheimers disease brain and stimulate mTORC1 protein kinase activity at the plasma membrane but not at lysosomes, block this Nutrient‐induced Mitochondrial Activity (NiMA) by a mechanism dependent on tau, which forms neurofibrillary tangles in Alzheimers disease brain. NiMA was also disrupted in fibroblasts derived from two patients with tuberous sclerosis complex, a genetic disorder that causes dysregulation of lysosomal mTORC1. Thus, lysosomal mTORC1 couples nutrient availability to mitochondrial activity and links mitochondrial dysfunction to Alzheimers disease by a mechanism dependent on the soluble building blocks of the poorly soluble plaques and tangles.