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The Morris K. Udall Parkinson's Disease Research Center of Excellence at Columbia University

Progress Report: August 2004 to June 2007

Project Title: Mechanisms of dopamine neuron degeneration

Principal Investigator: Robert E. Burke, MD

Date Submitted: June 6, 2007

History of the Udall Center at Columbia University

A Morris K. Udall Parkinson's Disease Research Center of Excellence was first awarded to Columbia University in 1999, under the Directorship of Dr Stanley Fahn, Head of the Movement Disorder Division of the Department of Neurology. The original Center consisted of six projects. Four were laboratory projects devoted to the pathogenesis of dopamine neuron degeneration, under the direction of Drs. Serge Przedborski, David Sulzer, R. Burke and Lloyd Greene. A fifth project, under the direction of Dr David Eidelberg, investigated the neural imaging correlates of Parkinson's Disease (PD) progression, and a sixth, under the direction of Dr Karen Marder, studied the epidemiology of PD in Blacks and women. In 2003, the Directorship was transferred to Robert E. Burke, the Director of Laboratories for Research in Parkinson's Disease and Related Disorders in the Department of Neurology.

The Center was competitively renewed in 2004 with a sharpened focus, and consisted of the four integrated laboratory projects dedicated to a single integrating theme: to understand the molecular and cellular mechanisms of neuron degeneration in Parkinson's disease. In Project 1, Dr. Serge Przedborski focused his efforts on understanding the role of non-cell autonomous, inflammatory mediators in the pathogenesis of neurodegeneration. He specifically proposed to examine the role of cyclooxygenase type-2 (COX-2) in the mediation of dopamine neuron degeneration in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of parkinsonism. In Project 2, Dr. David Sulzer aimed to examine the role of the autophagic/lysosomal pathways in the degradation of proteins implicated in PD, and the influence of alterations in their degradation on cytosolic dopamine homoeostasis. This work is being performed in collaboration with Dr Ana Maria Cuervo, Albert Einstein College of Medicine. Project 3, directed by Dr. Robert Burke, proposed to explore the role of the MAP kinase signaling cascade in programmed cell death of dopamine neurons, and its regulation by the survival signaling kinase, Akt. Dr. Lloyd Greene, in Project 4, proposed to further evaluate the functional role of ER stress in mediating programmed cell death, and he planned to assess the role of death-related gene families identified in the original funding period by Serial Analysis of Gene Expression (SAGE).

Progress Report: August 2004 to June 2007

Project 1, Dr. Serge Przedborski: An Experimental Model of Nigral Neuron Degeneration. Efforts in this Project have been devoted to assessing the role of COX-2 in the MPTP model in knockout and transgenic mice. Work has confirmed that the lack of COX-2 is associated with a greater resistance of the nigrostriatal dopaminergic neurons to MPTP, whereas increasing the expression of COX-2, in transgenic mice, is associated with enhanced susceptibility. Interestingly, in transgenic COX-2 mice, there was a larger number of dying dopaminergic neurons compared to their non-transgenic littermates, as evidenced by quantification of FluoroJade-positive, degenerating neuronal profiles. In contrast, at 4 days post MPTP injections, there was no difference in the number of apoptotic profiles between the two groups of mice. This finding suggests that the increased susceptibility to MPTP in the overexpressing transgenic mice may not be linked to a more pronounced recruitment of a caspase, particularly caspase 3, dependent pathway. Work in this Project has also demonstrated that ablation of cPLA2, which is the main producer of the COX-2 substrate, and the EP3 receptor, which is the main PGE2 receptor in the substantia nigra, fail to modulate MPTP neurotoxicity.

Project 2, Drs. David Sulzer and Ana Maria Cuervo: Roles for cytosolic dopamine in PD pathogenesis.

Drs Sulzer and Cuervo have shown that synuclein and its mutant forms are degraded by chaperone-mediated autophagy (CMA). Both overexpression of synuclein and the mutant forms inhibit chaperone-mediated autophagy and thereby interfere with the degradation of other cellular proteins by this system. Thus their observations may provide a mechanism for understanding how human PD due either to overexpression of synuclein or to the mutations can result from disturbance in cellular protein degradation. Their work was performed in collaboration with Dr Peter Lansbury, of the Udall Center of Brigham and Women's Hospital of Harvard University, and published in Science, 2004. Drs Sulzer and Cuervo also find that dopamine adducts of synuclein (kindly provided by Harry Ischiropolous, University of Pennsylvania) act similarly to mutant a-SYN in that they bind to the lysosomal CMA receptor, but they are not transported. The phosphorylated mutants, oxidized, and aggregated, and nitrated forms are each poor CMA substrates, but they do not block CMA: only the DA modified and pathogenic mutants do this. Thus, these findings could provide an explanation for the relative vulnerability of the dopamine and noradrenergic neurons in PD: the presence in the cytosol of dopamine could be conducive to the formation of proteins that block CMA.

Project 3, Dr. Robert Burke: The Molecular Basis Of Programmed Cell Death In Dopamine Neurons.

In spite of promising preclinical studies, neurotrophic factors have not yet achieved an established role in the treatment of PD. One impediment has been the difficulty in providing these macromolecules in sufficient quantity at affected sites. An alternative approach is to directly activate, by viral vector transduction, intracellular signaling pathways that mediate neurotrophic effects. We have evaluated this approach in dopamine neurons of the substantia nigra, by AAV1 transduction with a gene encoding a myristoylated, constitutively active form of the oncoprotein Akt/PKB. AAV Myr-Akt has pronounced trophic effects on dopamine neurons of adult and aged mice, including increases in neuron size and sprouting. Transduction confers almost complete protection against apoptotic cell death in the 6OHDA model. Interestingly, Myr-Akt not only protected SN dopamine neuron cell bodies, but it also had a protective effect on their striatal axonal projections. Furthermore, we have found that Myr-Akt not only protects SN dopamine neurons and their axons when the AAV is administered before 6OHDA, but also when it is administered 3 weeks after 6OHDA. We conclude that activation of intracellular neurotrophic signaling pathways by vector transfer is a feasible approach to neuroprotection and restorative treatment of PD. We have also successfully produced AAV1 viral vectors to achieve expression of three dominant negative forms of the MLKs: DN-MLK3 (K144R), DN-DLK (K152A), DN-DLK-LZ. We find that two dominant negative forms of dual leucine zipper kinase (DLK) inhibit apoptosis and enhance long term survival of dopamine neurons, but a dominant negative of MLK3 does not. Interestingly, the kinase dead form of DLK not only blocks apoptosis, but also has trophic effects on dopamine neurons.

Project 4, Dr. Lloyd Greene: Gene Regulation in Parkinson's Disease.

Since the original funding of the Columbia Udall Center in 1999, the goal of this Project has been to carry out serial analysis of gene expression (SAGE) to identify genes regulated in a cellular model of Parkinson disease (PD). Dr Greene and his colleagues have identified over 500 named genes that are up-regulated in PC12 cells after exposure to the PD mimetic 6-OHDA. Selection of genes for further study has been based on consideration of the potential roles of these genes in PD and possible therapeutic intervention in the disease. Their studies have identified both genes that might be implicated in neuron cell death as well as those that possess the potential to protect neurons from death. Among the genes regulated was PUMA, a pro-apoptotic member of the Bcl2 family. PUMA protein up-regulation was verified in the cell culture model. Studies also demonstrated that PUMA is in turn regulated by the transcription factor p53. Down-regulation of PUMA by siRNA was highly protective, as was blockade of p53 function.

The most highly induced gene in the SAGE study was one designated as RTP801. A previous report showed that over-expression of this molecule can cause death of neuronal cells. Studies verified that the protein as well as transcript is elevated in the model. Other PD mimetics including MPP+ and rotenone also induce the protein. To determine the potential relevance of this finding to PD itself, immunohistochemistry was performed on PD postmortem brain tissue, and high expression was observed only in substantial nigral neurons of PD patients. Suppression of RTP801 expression by siRNAs was highly protective from neuron death. Further studies support a mechanism in which RTP801 suppresses mTor activity and that this in turn depresses intracellular activity of the neuronal survival kinase AKT to promote death. RTP801 over-expression as well as exposure to PD mimetics lead to loss of neuronal phospho-AKT. Moreover, expression of constitutively active AKT protects from such mimetics as well as from RTP801 over-expression. A second major finding was that RTP801 induction by PD mimetics is blocked by the drug rapamycin. A third finding has been that RTP801 can suppress neurite outgrowth under conditions in which it does not kill neurons. This suggests that in addition to having lethal effects, over-expression of the protein in PD could have negative effects of interaction of dopaminergic neurons with their targets.

COLUMBIA UDALL CENTER
PEER-REVIEWED PUBLICATIONS
2004-JUNE 2007

  1. Benner EJ, Mosley RL, Destache CJ, Lewis TB, Jackson-Lewis V, Gorantla S, Nemachek C, Green SR, Przedborski S, Gendelman HE. Therapeutic immunization protects dopaminergic neurons in a mouse model of Parkinson's disease. Proc Natl Acad Sci U S A 2004;101:9435-9440.
  2. Chuhma N, Zhang H, Masson J, Zhuang X, Sulzer D, Hen R, Rayport S. Dopamine neurons mediate a fast excitatory signal via their glutamatergic synapses. J Neurosci 2004;24:972-981.
  3. Cuervo AM, Stefanis L, Fredenburg R, Lansbury PT, Sulzer D. Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science 2004;305:1292-1295.
  4. Ganguly A, Oo TF, Rzhetskaya M, Pratt R, Yarygina O, Momoi T, Kholodilov N, Burke RE. CEP11004, a novel inhibitor of the mixed lineage kinases, suppresses apoptotic death in dopamine neurons of the substantia nigra induced by 6-hydroxydopamine. J Neurochem 2004;88:469-480.
  5. Hayley S, Crocker SJ, Smith PD, Shree T, Jackson-Lewis V, Przedborski S, Mount M, Slack R, Anisman H, Park DS. Regulation of dopaminergic loss by Fas in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease. J Neurosci 2004;24:2045-2053.
  6. Hunot S, Vila M, Teismann P, Davis RJ, Hirsch EC, Przedborski S, Rakic P, Flavell RA. JNK-mediated induction of cyclooxygenase 2 is required for neurodegeneration in a mouse model of Parkinson's disease. Proc Natl Acad Sci U S A 2004;101:665-670.
  7. Tieu K, Perier C, Vila M, Caspersen C, Zhang HP, Teismann P, Jackson-Lewis V, Stern DM, Yan SD, Przedborski S. L-3-hydroxyacyl-CoA dehydrogenase II protects in a model of Parkinson's disease. Ann Neurol 2004;56:51-60.
  8. Zucca FA, Giaveri G, Gallorini M, Albertini A, Toscani M, Pezzoli G, Lucius R, Wilms H, Sulzer D, Ito S, Wakamatsu K, Zecca L. The neuromelanin of human substantia nigra: physiological and pathogenic aspects. Pigment Cell Res 2004;17:610-617.
  9. Biswas SC, Ryu E, Park C, Malagelada C, Greene LA. Puma and p53 play required roles in death evoked in a cellular model of Parkinson disease. Neurochem Res 2005;30:839-845.
  10. Giraudo CG, Hu C, You D, Slovic AM, Mosharov EV, Sulzer D, Melia TJ, Rothman JE. SNAREs can promote complete fusion and hemifusion as alternative outcomes. J Cell Biol 2005;170:249-260.
  11. Li H, Waites CL, Staal RG, Dobryy Y, Park J, Sulzer DL, Edwards RH. Sorting of vesicular monoamine transporter 2 to the regulated secretory pathway confers the somatodendritic exocytosis of monoamines. Neuron 2005;48:619-633.
  12. Mosharov EV, Sulzer D. Analysis of exocytotic events recorded by amperometry. Nat Methods 2005;2:651-658.
  13. Paterlini M, Zakharenko SS, Lai WS, Qin J, Zhang H, Mukai J, Westphal KG, Olivier B, Sulzer D, Pavlidis P, Siegelbaum SA, Karayiorgou M, Gogos JA. Transcriptional and behavioral interaction between 22q11.2 orthologs modulates schizophrenia-related phenotypes in mice. Nat Neurosci 2005;8:1586-1594.
  14. Ryu EJ, Angelastro JM, Greene LA. Analysis of gene expression changes in a cellular model of Parkinson disease. Neurobiol Dis 2005;18:54-74.
  15. Silva RM, Ries V, Oo TF, Yarygina O, Jackson-Lewis V, Ryu EJ, Lu PD, Marciniak SJ, Ron D, Przedborski S, Kholodilov N, Greene LA, Burke RE. CHOP/GADD153 is a mediator of apoptotic death in substantia nigra dopamine neurons in an in vivo neurotoxin model of parkinsonism. J Neurochem 2005; 95:974-986.
  16. Son JH, Kawamata H, Yoo MS, Kim DJ, Lee YK, Kim S, Dawson TM, Zhang H, Sulzer D, Yang L, Beal MF, Degiorgio LA, Chun HS, Baker H, Peng C. Neurotoxicity and behavioral deficits associated with Septin 5 accumulation in dopaminergic neurons. J Neurochem 2005;94:1040-1053.
  17. Xu Z, Kukekov NV, Greene LA. Regulation of apoptotic c-Jun N-terminal kinase signaling by a stabilization-based feed-forward loop. Mol Cell Biol 2005;25:9949-9959.
  18. Kukekov NV, Xu Z, Greene LA. Direct interaction of the molecular scaffolds POSH and JIP is required for apoptotic activation of JNKs. J Biol Chem 2006;281:15517-15524.
  19. Larsen KE, Benn SC, Ay I, Chian RJ, Celia SA, Remington MP, Bejarano M, Liu M, Ross J, Carmillo P, Sah D, Phillips KA, Sulzer D, Pepinsky RB, Fishman PS, Brown RH, Jr., Francis JW. A glial cell line-derived neurotrophic factor (GDNF):tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice. Brain Res 2006;1120:1-12.
  20. Larsen KE, Schmitz Y, Troyer MD, Mosharov E, Dietrich P, Quazi AZ, Savalle M, Nemani V, Chaudhry FA, Edwards RH, Stefanis L, Sulzer D. Alpha-synuclein overexpression in PC12 and chromaffin cells impairs catecholamine release by interfering with a late step in exocytosis. J Neurosci 2006; 26:11915-11922.
  21. Malagelada C, Ryu EJ, Biswas SC, Jackson-Lewis V, Greene LA. RTP801 is elevated in Parkinson brain substantia nigral neurons and mediates death in cellular models of Parkinson's disease by a mechanism involving mammalian target of rapamycin inactivation. J Neurosci 2006;26:9996-10005.
  22. Mosharov EV, Staal RG, Bove J, Prou D, Hananiya A, Markov D, Poulsen N, Larsen KE, Moore CM, Troyer MD, Edwards RH, Przedborski S, Sulzer D. Alpha-synuclein overexpression increases cytosolic catecholamine concentration. J Neurosci 2006;26:9304-9311.
  23. Ries V, Henchcliffe C, Kareva T, Rzhetskaya M, Bland R, During MJ, Kholodilov N, Burke RE. Oncoprotein Akt/PKB induces trophic effects in murine models of Parkinson's disease. Proc Natl Acad Sci U S A 2006;103:18757-18762.
  24. Xu Z, Sproul A, Wang W, Kukekov N, Greene LA. Siah1 interacts with the scaffold protein POSH to promote JNK activation and apoptosis. J Biol Chem 2006;281:303-312.
  25. Xu Z, Greene LA. Activation of the apoptotic JNK pathway through the Rac1-binding scaffold protein POSH. Methods Enzymol 2006;406:479-489.
  26. Wilhelm M, Xu Z, Kukekov NV, Gire S, Greene LA. Proapoptotic Nix activates the JNK pathway by interacting with POSH and mediates death in a Parkinson disease model. J Biol Chem 2007;282:1288-1295.

Last updated August 29, 2008