Research Article
Abstract
Parkinson disease (PD) is characterized by dopaminergic neurodegeneration and intracellular inclusions of α-synuclein amyloid fibers, which are stable and difficult to dissolve. Whether inclusions are neuroprotective or pathological remains controversial, because prefibrillar oligomers may be more toxic than amyloid inclusions. Thus, whether therapies should target inclusions, preamyloid oligomers, or both is a critically important issue. In yeast, the protein-remodeling factor Hsp104 cooperates with Hsp70 and Hsp40 to dissolve and reactivate aggregated proteins. Metazoans, however, have no Hsp104 ortholog. Here we introduced Hsp104 into a rat PD model. Remarkably, Hsp104 reduced formation of phosphorylated α-synuclein inclusions and prevented nigrostriatal dopaminergic neurodegeneration induced by PD-linked α-synuclein (A30P). An in vitro assay employing pure proteins revealed that Hsp104 prevented fibrillization of α-synuclein and PD-linked variants (A30P, A53T, E46K). Hsp104 coupled ATP hydrolysis to the disassembly of preamyloid oligomers and amyloid fibers composed of α-synuclein. Furthermore, the mammalian Hsp70 and Hsp40 chaperones, Hsc70 and Hdj2, enhanced α-synuclein fiber disassembly by Hsp104. Hsp104 likely protects dopaminergic neurons by antagonizing toxic α-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses.
Authors
Christophe Lo Bianco, James Shorter, Etienne Régulier, Hilal Lashuel, Takeshi Iwatsubo, Susan Lindquist, Patrick Aebischer
Abstract
Some cases of pre–B cell acute lymphoblastic leukemia (pre–B-ALL) are caused by the Philadelphia (Ph) chromosome–encoded BCR-ABL oncogene, and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL–mediated transformation in vitro; however, the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre–B-ALL, we found that deletion of both Pik3r1 and Pik3r2, genes encoding class IA PI3K regulatory isoforms, severely impaired transformation. BCR-ABL–dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes, but the cells were smaller, proliferated more slowly, and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However, they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR, PI-103, was more effective than rapamycin at suppressing proliferation of mouse pre–B-ALL and human CD19+CD34+ Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K, alone or together with mTOR, in the treatment of Ph+ ALL.
Authors
Michael G. Kharas, Matthew R. Janes, Vanessa M. Scarfone, Michael B. Lilly, Zachary A. Knight, Kevan M. Shokat, David A. Fruman
Abstract
β3-adrenergic receptor (β3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of β3-AR in the human atrium. Unexpectedly, β3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (ICa,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The ICa,L and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective β3-AR agonists, SR58611 and BRL37344, and a β3-AR partial agonist, CGP12177, stimulated ICa,L in HAMs with nanomolar potency and a 60%–90% efficacy compared with isoprenaline. The β3-AR agonists also increased contractility but with a much lower efficacy (~10%) than isoprenaline. The β3-AR antagonist L-748,337, β1-/β2-AR antagonist nadolol, and β1-/β2-/β3-AR antagonist bupranolol were used to confirm the involvement of β3-ARs (and not β1-/β2-ARs) in these effects. The β3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked ICa,L stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, β3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.
Authors
V. Arvydas Skeberdis, Vida Gendvilienė, Danguolė Zablockaitė, Rimantas Treinys, Regina Mačianskienė, Andrius Bogdelis, Jonas Jurevičius, Rodolphe Fischmeister
Abstract
Iatrogenic tumor cell implantation within surgical wounds can compromise curative cancer surgery. Adhesion of cancer cells, in particular colon cancer cells, is stimulated by exposure to increased extracellular pressure through a cytoskeleton-dependent signaling mechanism requiring FAK, Src, Akt, and paxillin. Mechanical stimuli during tumor resection may therefore negatively impact patient outcome. We hypothesized that perioperative administration of colchicine, which prevents microtubule polymerization, could disrupt pressure-stimulated tumor cell adhesion to surgical wounds and enhance tumor-free survival. Ex vivo treatment of Co26 and Co51 colon cancer cells with colchicine inhibited pressure-stimulated cell adhesion to murine surgical wounds and blocked pressure-induced FAK and Akt phosphorylation. Surgical wound contamination with pressure-activated Co26 and Co51 cells significantly reduced tumor-free survival compared with contamination with tumor cells under ambient pressure. Mice treated with pressure-activated Co26 and Co51 cells from tumors preoperatively treated with colchicine in vivo displayed reduced surgical site implantation and significantly increased tumor-free survival compared with mice exposed to pressure-activated cells from tumors not pretreated with colchicine. Our data suggest that pressure activation of malignant cells promotes tumor development and impairs tumor-free survival and that perioperative colchicine administration or similar interventions may inhibit this effect.
Authors
David H. Craig, Cheri R. Owen, William C. Conway, Mary F. Walsh, Christina Downey, Marc D. Basson
Abstract
Pseudomonas aeruginosa uses quorum sensing, an interbacterial communication system, to regulate gene expression. The signaling molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is thought to play a central role in quorum sensing. Since 3OC12-HSL can be degraded by paraoxonase (PON) family members, we hypothesized that PONs regulate P. aeruginosa virulence in vivo. We chose Drosophila melanogaster as our model organism because it has been shown to be a tractable model for investigating host-pathogen interactions and lacks PONs. By using quorum-sensing–deficient P. aeruginosa, synthetic acyl-HSLs, and transgenic expression of human PON1, we investigated the role of 3OC12-HSL and PON1 on P. aeruginosa virulence. We found that P. aeruginosa virulence in flies was dependent upon 3OC12-HSL. PON1 transgenic flies expressed enzymatically active PON1 and thereby exhibited arylesterase activity and resistance to organophosphate toxicity. Moreover, PON1 flies were protected from P. aeruginosa lethality, and protection was dependent on the lactonase activity of PON1. Our findings show that PON1 can interfere with quorum sensing in vivo and provide insight into what we believe is a novel role for PON1 in the innate immune response to quorum-sensing–dependent pathogens. These results raise intriguing possibilities about human-pathogen interactions, including potential roles for PON1 as a modifier gene and for PON1 protein as a regulator of normal bacterial florae, a link between infection/inflammation and cardiovascular disease, and a potential therapeutic modality.
Authors
David A. Stoltz, Egon A. Ozer, Peter J. Taft, Marilyn Barry, Lei Liu, Peter J. Kiss, Thomas O. Moninger, Matthew R. Parsek, Joseph Zabner
Abstract
Neuronal ceroid lipofuscinoses represent the most common childhood neurodegenerative storage disorders. Infantile neuronal ceroid lipofuscinosis (INCL) is caused by palmitoyl protein thioesterase-1 (PPT1) deficiency. Although INCL patients show signs of abnormal neurotransmission, manifested by myoclonus and seizures, the molecular mechanisms by which PPT1 deficiency causes this abnormality remain obscure. Neurotransmission relies on repeated cycles of exo- and endocytosis of the synaptic vesicles (SVs), in which several palmitoylated proteins play critical roles. These proteins facilitate membrane fusion, which is required for neurotransmitter exocytosis, recycling of the fused SV membrane components, and regeneration of fresh vesicles. However, palmitoylated proteins require depalmitoylation for recycling. Using postmortem brain tissues from an INCL patient and tissue from the PPT1-knockout (PPT1-KO) mice that mimic INCL, we report here that PPT1 deficiency caused persistent membrane anchorage of the palmitoylated SV proteins, which hindered the recycling of the vesicle components that normally fuse with the presynaptic plasma membrane during SV exocytosis. Thus, the regeneration of fresh SVs, essential for maintaining the SV pool size at the synapses, was impaired, leading to a progressive loss of readily releasable SVs and abnormal neurotransmission. This abnormality may contribute to INCL neuropathology.
Authors
Sung-Jo Kim, Zhongjian Zhang, Chinmoy Sarkar, Pei-Chih Tsai, Yi-Ching Lee, Louis Dye, Anil B. Mukherjee
Abstract
Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe–/– mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1–/– mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe–/– mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.
Authors
Kazuyoshi Toyama, Heike Wulff, K. George Chandy, Philippe Azam, Girija Raman, Takashi Saito, Yoshimasa Fujiwara, David L. Mattson, Satarupa Das, James E. Melvin, Phillip F. Pratt, Ossama A. Hatoum, David D. Gutterman, David R. Harder, Hiroto Miura
Abstract
Previously, several individuals with X-linked SCID (SCID-X1) were treated by gene therapy to restore the missing IL-2 receptor γ (IL2RG) gene to CD34+ BM precursor cells using gammaretroviral vectors. While 9 of 10 patients were successfully treated, 4 of the 9 developed T cell leukemia 31–68 months after gene therapy. In 2 of these cases, blast cells contained activating vector insertions near the LIM domain–only 2 (LMO2) proto-oncogene. Here, we report data on the 2 most recent adverse events, which occurred in patients 7 and 10. In patient 10, blast cells contained an integrated vector near LMO2 and a second integrated vector near the proto-oncogene BMI1. In patient 7, blast cells contained an integrated vector near a third proto-oncogene,CCND2. Additional genetic abnormalities in the patients’ blast cells included chromosomal translocations, gain-of-function mutations activating NOTCH1, and copy number changes, including deletion of tumor suppressor gene CDKN2A, 6q interstitial losses, and SIL-TAL1 rearrangement. These findings functionally specify a genetic network that controls growth in T cell progenitors. Chemotherapy led to sustained remission in 3 of the 4 cases of T cell leukemia, but failed in the fourth. Successful chemotherapy was associated with restoration of polyclonal transduced T cell populations. As a result, the treated patients continued to benefit from therapeutic gene transfer.
Authors
Salima Hacein-Bey-Abina, Alexandrine Garrigue, Gary P. Wang, Jean Soulier, Annick Lim, Estelle Morillon, Emmanuelle Clappier, Laure Caccavelli, Eric Delabesse, Kheira Beldjord, Vahid Asnafi, Elizabeth MacIntyre, Liliane Dal Cortivo, Isabelle Radford, Nicole Brousse, François Sigaux, Despina Moshous, Julia Hauer, Arndt Borkhardt, Bernd H. Belohradsky, Uwe Wintergerst, Maria C. Velez, Lily Leiva, Ricardo Sorensen, Nicolas Wulffraat, Stéphane Blanche, Frederic D. Bushman, Alain Fischer, Marina Cavazzana-Calvo
Abstract
PKC isoforms t, α, and β play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.
Authors
Hans Skvara, Markus Dawid, Elise Kleyn, Barbara Wolff, Josef G. Meingassner, Hilary Knight, Thomas Dumortier, Tamara Kopp, Nasanin Fallahi, Georg Stary, Christoph Burkhart, Olivier Grenet, Juergen Wagner, Youssef Hijazi, Randall E. Morris, Claire McGeown, Christiane Rordorf, Christopher E.M. Griffiths, Georg Stingl, Thomas Jung
Abstract
X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-β region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.
Authors
Steven J. Howe, Marc R. Mansour, Kerstin Schwarzwaelder, Cynthia Bartholomae, Michael Hubank, Helena Kempski, Martijn H. Brugman, Karin Pike-Overzet, Stephen J. Chatters, Dick de Ridder, Kimberly C. Gilmour, Stuart Adams, Susannah I. Thornhill, Kathryn L. Parsley, Frank J.T. Staal, Rosemary E. Gale, David C. Linch, Jinhua Bayford, Lucie Brown, Michelle Quaye, Christine Kinnon, Philip Ancliff, David K. Webb, Manfred Schmidt, Christof von Kalle, H. Bobby Gaspar, Adrian J. Thrasher
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ISSN: 0021-9738 (print), 1558-8238 (online)