SOD1 mediates lysosome-to-mitochondria communication and its dysregulation by amyloid-ß oligomers.

Altered mitochondrial DNA (mtDNA) occurs in neurodegenerative disorders like Alzheimer's disease (AD); how mtDNA synthesis is linked to neurodegeneration is poorly understood. We previously discovered Nutrient-induced Mitochondrial Activity (NiMA), an inter-organelle signaling pathway where nutrient-stimulated lysosomal mTORC1 activity regulates mtDNA replication in neurons by a mechanism sensitive to amyloid-ß oligomers (AßOs), a primary factor in AD pathogenesis (Norambuena et al., 2018). Using 5-ethynyl-2'-deoxyuridine (EdU) incorporation into mtDNA of cultured neurons, along with photoacoustic and mitochondrial metabolic imaging of cultured neurons and mouse brains, we show these effects being mediated by mTORC1-catalyzed T40 phosphorylation of superoxide dismutase 1 (SOD1). Mechanistically, tau, another key factor in AD pathogenesis and other tauopathies, reduced the lysosomal content of the tuberous sclerosis complex (TSC), thereby increasing NiMA and suppressing SOD1 activity and mtDNA synthesis. AßOs inhibited these actions. Dysregulation of mtDNA synthesis was observed in fibroblasts derived from tuberous sclerosis (TS) patients, who lack functional TSC and elevated SOD1 activity was also observed in human AD brain. Together, these findings imply that tau and SOD1 couple nutrient availability to mtDNA replication, linking mitochondrial dysfunction to AD.

Aberrant Neuronal Cell Cycle Re-Entry: The Pathological Confluence of Alzheimer's Disease and Brain Insulin Resistance, and Its Relation to Cancer.

Aberrant neuronal cell cycle re-entry (CCR) is a phenomenon that precedes and may mechanistically lead to a majority of the neuronal loss observed in Alzheimer's disease (AD). Recent developments concerning the regulation of aberrant neuronal CCR in AD suggest that there are potential intracellular signaling "hotspots" in AD, cancer, and brain insulin resistance, the latter of which is characteristically associated with AD. Critically, these common signaling nodes across different human diseases may represent currently untapped therapeutic opportunities for AD. Specifically, repurposing of existing US Food and Drug Administration-approved pharmacological agents, including experimental therapeutics that target the cell cycle in cancer, may be an innovative avenue for future AD-directed drug discovery and development. In this review we discuss overlapping aspects of AD, cancer, and brain insulin resistance from the perspective of neuronal CCR, and consider strategies to exploit them for prevention or therapeutic intervention of AD.

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

The mechanisms of mitochondrial dysfunction in Alzheimer's 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-ß oligomers, which are precursors of amyloid plaques in Alzheimer's 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 Alzheimer's 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 Alzheimer's disease by a mechanism dependent on the soluble building blocks of the poorly soluble plaques and tangles.

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.

Epidermal growth factor receptor endocytic traffic perturbation by phosphatidate phosphohydrolase inhibition: new strategy against cancer.

Epidermal growth factor receptor (EGFR) exaggerated (oncogenic) function is currently targeted in cancer treatment with drugs that block receptor ligand binding or tyrosine kinase activity. Because endocytic trafficking is a crucial regulator of EGFR function, its pharmacological perturbation might provide a new anti-tumoral strategy. Inhibition of phosphatidic acid (PA) phosphohydrolase (PAP) activity has been shown to trigger PA signaling towards type 4 phosphodiesterase (PDE4) activation and protein kinase A inhibition, leading to internalization of empty/inactive EGFR. Here, we used propranolol, its l- and d- isomers and desipramine as PAP inhibitors to further explore the effects of PAP inhibition on EGFR endocytic trafficking and its consequences on EGFR-dependent cancer cell line models. PAP inhibition not only made EGFR inaccessible to stimuli but also prolonged the signaling lifetime of ligand-activated EGFR in recycling endosomes. Strikingly, such endocytic perturbations applied in acute/intermittent PAP inhibitor treatments selectively impaired cell proliferation/viability sustained by an exaggerated EGFR function. Phospholipase D inhibition with FIPI (5-fluoro-2-indolyl des-chlorohalopemide) and PDE4 inhibition with rolipram abrogated both the anti-tumoral and endocytic effects of PAP inhibition. Prolonged treatments with a low concentration of PAP inhibitors, although without detectable endocytic effects, still counteracted cell proliferation, induced apoptosis and decreased anchorage-independent growth of cells bearing EGFR oncogenic influences. Overall, our results show that PAP inhibitors can counteract EGFR oncogenic traits, including receptor overexpression or activating mutations resistant to current tyrosine kinase inhibitors, perturbing EGFR endocytic trafficking and perhaps other as yet unknown processes, depending on treatment conditions. This puts PAP activity forward as a new suitable target against EGFR-driven malignancy.

P2X1 receptors localized in lipid rafts mediate ATP motor responses in the human vas deferens longitudinal muscles.

To assess the role of the P2X1 receptors (P2X1R) in the longitudinal and circular layers of the human vas deferens, ex vivo-isolated strips or rings were prepared from tissue biopsies to record isometric contractions. To ascertain its membrane distribution, tissue extracts were analyzed by immunoblotting following sucrose gradient ultracentrifugation. ATP, alpha,beta-methylene ATP, or electrical field stimulation elicited robust contractions of the longitudinal layer but not of the circular layer which demonstrated inconsistent responses. Alpha,beta-methylene ATP generated stronger and more robust contractions than ATP. In parallel, prostatic segments of the rat vas deferens were examined. The motor responses in both species were not sustained but decayed within the first minute, showing desensitization to additional applications. Cross-desensitization was established between alpha,beta-methylene ATP or ATP-evoked contractions and electrical field stimulation-induced contractions. Full recovery of the desensitized motor responses required more than 30 min and showed a similar pattern in human and rat tissues. Immunoblot analysis of the human vas deferens extracts revealed a P2X1R oligomer of approximately 200 kDa under nonreducing conditions, whereas dithiothreitol-treated extracts showed a single band of approximately 70 kDa. The P2X1R was identified in ultracentrifugation fractions containing 15%-29% sucrose; the receptor localized in the same fractions as flotillin-1, indicating that it regionalized into smooth muscle lipid rafts. In conclusion, ATP plays a key role in human vas deferens contractile responses of the longitudinal smooth muscle layer, an effect mediated through P2X1Rs.

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

Anchorage dependence of cell growth, which is mediated by multiple integrin-regulated signaling pathways, is a key defense against cancer metastasis. Detachment of cells from the extracellular matrix triggers caveolin-1-dependent internalization of lipid raft components, which mediates suppression of Rho GTPases, Erk, and phosphatidylinositol 3-kinase in suspended cells. Elevation of cyclic adenosine monophosphate (cAMP) following cell detachment is also implicated in termination of growth signaling in suspended cells. Studies of integrins and lipid rafts, however, examined mainly ganglioside GM1 and glycosylphosphatidylinositol-linked proteins as lipid raft markers. In this study, we examine a wider range of lipid raft components. Whereas many raft components internalized with GM1 following cell detachment, flotillin2, connexin43, and Gα(s) remained in the plasma membrane. Loss of cell adhesion caused movement of many components from the lipid raft to the nonraft fractions on sucrose gradients, although flotillin2, connexin43, and H-Ras were resistant. Gα(s) lost its raft association, concomitant with cAMP production. Modification of the lipid tail of Gα(s) to increase its association with ordered domains blocked the detachment-induced increase in cAMP. These data define the effects of that integrin-mediated adhesion on the localization and behavior of a variety of lipid raft components and reveal the mechanism of the previously described elevation of cAMP after cell detachment.

Phosphatidic acid induces ligand-independent epidermal growth factor receptor endocytic traffic through PDE4 activation.

Endocytosis modulates EGFR function by compartmentalizing and attenuating or enhancing its ligand-induced signaling. Here we show that it can also control the cell surface versus intracellular distribution of empty/inactive EGFR. Our previous observation that PKA inhibitors induce EGFR internalization prompted us to test phosphatidic acid (PA) generated by phospholipase D (PLD) as an endogenous down-regulator of PKA activity, which activates rolipram-sensitive type 4 phosphodiesterases (PDE4) that degrade cAMP. We found that inhibition of PA hydrolysis by propranolol, in the absence of ligand, provokes internalization of inactive (neither tyrosine-phosphorylated nor ubiquitinated) EGFR, accompanied by a transient increase in PA levels and PDE4s activity. This EGFR internalization is mimicked by PA micelles and is strongly counteracted by PLD2 silencing, rolipram or forskolin treatment, and PKA overexpression. Accelerated EGFR endocytosis seems to be mediated by clathrin-dependent and -independent pathways, leading to receptor accumulation in juxtanuclear recycling endosomes, also due to a decreased recycling. Internalized EGFR can remain intracellular without degradation for several hours or return rapidly to the cell surface upon discontinuation of the stimulus. This novel regulatory mechanism of EGFR, also novel function of signaling PA, can transmodulate receptor accessibility in response to heterologous stimuli.

UTP controls cell surface distribution and vasomotor activity of the human P2Y2 receptor through an epidermal growth factor receptor-transregulated mechanism.

Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y(2) receptor (P2Y(2)R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-beta-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1-10 mum UTP, a selective P2Y(2)R agonist, displaced the P2Y(2)R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y(2)R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y(2)R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y(2)R in intact human blood vessels.

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.

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

The nucleotide P2Y(1) receptor (P2Y(1)R) 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 P2Y(1)R 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 P2Y(1)R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl beta-cyclodextrin reduced the raft partitioning of the P2Y(1)R and obliterated the P2Y(1)R-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 P2Y(1)R agonist, not only displaced within 4 min the P2Y(1)R localization out of membrane rafts but also induced its subsequent internalization. 2'-Deoxy-N(6)-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y(1)R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y(1)R 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 P2Y(1)R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y(1)R to membrane rafts, highlighting the role of this microdomain in P2Y(1)R signaling.

Sorting competition with membrane-permeable peptides in intact epithelial cells revealed discrimination of transmembrane proteins not only at the trans-Golgi network but also at pre-Golgi stages.

Transmembrane proteins destined to the basolateral cell surface of epithelial cells contain in their cytosolic domain at least two classes of sorting signals: one class promotes exit from the endoplasmic reticulum (ER) and transport to the Golgi complex, and the other class operates at the trans-Golgi network (TGN) specifying segregation into basolateral exocytic pathways. Both kinds of addressing motifs are quite diverse among different proteins. It is unclear to what extent this feature reflects alternative decoding mechanisms or variations in motifs recognized by the same sorting factor. Here we applied a novel strategy based on permeable peptide technology and temperature-sensitive model proteins to study competition between cytosolic sorting motifs in the context of mammalian living cells. We used the transduction domain of HIV-1 Tat protein to make a membrane-permeable peptide of the cytosolic tail of GtsO45, which contains a well characterized ER exit di-acidic (DIE) motif and a tyrosine-based basolateral sorting signal (YTDI). This peptide added to the media inhibited transport of GtsO45 from both ER-to-Golgi and TGN-to-basolateral cell surface in transfected Madin-Darby canine kidney cells. Instead, it did not affect the exocytic trafficking of a GtsO45-derived chimeric protein bearing 30 juxtamembrane residues from the cytosolic domain of the epidermal growth factor receptor that contains a variant ER exit motif (ERE) and an unconventional proline-based basolateral sorting signal. These results not only proved the feasibility of competing for sorting events in intact cells but also showed that distinct plasma membrane proteins can be discriminated at pre-TGN stages, and that basolateral sorting involves different recognition elements for tyrosine-based motifs and an unconventional basolateral motif.