Parkinson´s disease is one of the major neurodegenerative diseases that afflict the increasing population of aged humans. Over 90% of patients with Parkinson´s disease derive from a non-inherited, environmentally caused type of the disease of unknown origin1. It is generally believed that decreased protection against noxious oxidative stress renders the dopaminergic neurons of the substantia nigra particularly vulnerable2. Loss of dopaminergic cells leads to deficits in locomotion abilities and facial expression, resulting finally in precocious death. Identification of target proteins dysregulated in the disease and functional characterisation of targeted proteins at the translational or posttranslational levels ameliorating the symptoms of Parkinson´s disease would constitute a major achievement in the cure of this devastating disease.
MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine) in-duces degeneration of dopaminergic neurons by specifically interfering with oxidative phosphorylation in the dopaminergic neurons of the substatia nigra3. We propose to use this well established mouse model of Parkinson´s disease to identify the target proteins involved in pathogenesis and in the disease amelioration.
We have discovered and characterised the neural cell adhesion molecule L14-6 and found that constitutive7 and conditional8 L1 deficient mutants share many features with human mutations of the L1 gene9. In addition, dopaminergic neurons have been identified to be abnormal in the L1 deficient mouse10. Furthermore, we have generated a transgenic mouse that expresses L1 ectopically in astrocytes11-13, thus preventing cell death of dopaminergic neurons. In the L1 overexpressing transgenic mice not only regeneration of severed axons is enhanced, but also learning and memory deficits seen in the constitutive and conditional L1 deficient mutants are converted positively in the sense that these transgenic mice learn and memorise better not only at young ages, but particularly at old ages, when they retain their ability to learn and memorize as well as in the juvenile state (unpublished observations). We have used these mice in the MPTP model of Parkinson´s disease14 and shown that neural stem cells transfected with the L1 adhesion molecule not only survive better in the MPTP lesioned animals, but also ameliorate the locomotory abnormalities induced by MPTP treatment at young and old ages, resulting in a sparing of dopaminergic neurons (Ourednik et al., submitted for publication).
Project Status
By using a proteomics approach we wish to identify proteins and their posttranslational modifications that are abnormally regulated at different time points after a noxious MPTP insult to the inbred C57BL/6J mouse strain. Furthermore we will treat transgenic L1 mice that show a robust resistance towards MPTP treatment. This transgenic mice line express the neuro-protective neural cell adhesion molecule L1 under the control of the glial fibrillary acid protein promoter in astrocytes that normally do not express this protein10. After MPTP treatment, these mice do not lose dopaminergic neurons to the extent that the wild type control littermates do (Ourednik et al., submitted for publication) and they also show less neurodegeneration at older ages as seen by behavioural experiments on learning and memory (unpublished observations). Thus, we wish to identify the target proteins preventing death or lose of dopaminergic neurons in these cured brains. We will not only analyse such target proteins in the intact tissue and afflicted brain regions, but also in cell culture of dopaminergic neurons, which succumb to treatment with MPP+, the toxic derivative of MPTP15. For this more defined determination of neuronal subpopulation-specific degeneration, dopaminergic neurons in enriched cultures will be studied under MPTP treatment and after the known beneficial treatment with purified L1 protein.
Outlook
Upon identifying target proteins that are affected in their expression in the MPTP mouse model of Parkinson´s disease these proteins will be compared to those dysregulated in the genetic mouse models of Parkinson´s disease, such as the mice constitutive deficient in synuclein and parkin, and to probes obtained from diseased human brain tissue of selected regions. In addition, these proteins will be compared to those found to be abnormally regulated in Alzheimer´s disease and in aged human and mouse brains specimens. These comparative investigations should help to identify protein targets affected in Parkinson´s and other neurodegenerative diseases and by age with the hope for therapeutic intervention in humans with Parkinson´s disease but also for other neurodegenerative disease, in particular in aged individuals. Since dopaminergic neurons are abnormal in L1- deficient mice and since previous work funded within NGFN-1 has identified genes that are dysregulated at the mRNA level and at the protein level in L1-deficient mice and are involved in signal transduction and cytoskeletal re-arrangements, a comparative analysis of proteins identified as being dysregulated in the MPTP mouse model of Parkinson´s disease and in the L1 deficient mouse will be carried out and could yield further insights in cell death of dopaminergic neurons.
Upon identifying target that are dysregulated under pathological conditions and/or should be reverted to normal levels after MPTP treatment or prevent death of dopaminergic neurons under L1 mediated neuroprotective conditions, e.g. in the transgenic mouse that overexpresses the neural recognition molecule L1. The identified beneficial target proteins will then be overexpressed in transgenic mice with a neuronal (synapsin I) and astroglial (glial fibrillary acid protein) promoters. These mice will be characterised with regard to their beneficial effects on survival of dopaminergic neurons and amelioration of the behavioural deficits of Parkinson´s disease and the proteins identified as distinctly regulated in neurodegenerative diseases will be compared to those identified in aged brains. The hope is that the identified proteins will be suitable for targeted intervention in the therapeutic prevention of Parkinson´s disease in humans.
Lit.: 1. Jost WH. Autonomic dysfunctions in idiopathic Parkinson's disease. J Neurol. 2003; 250:I28-30. 2. Jenner P. Oxidative stress in Parkinson's disease. Ann Neurol. 2003; 53:S26-38. 3. Jenner P. The contribution of the MPTP-treated primate model to the development of new treatment strategies for Parkinson's disease. Parkinsonism Relat Disord. 2003; 9:131-137. 4. Lindner J et al. L1 mono- and polyclonal antibodies modify cell migration in early postnatal mouse cerebellum. Nature. 1983; 305:427-430. 5. Moos M et al. Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature. 1988; 334:701-713. 6. Rathjen FG and Schachner, M. Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion. EMBO J. 1984; 3:1-10. 7. Dahme M et al. Disruption of the mouse L1 gene leads to malformations of the nervous system. Nat Genet. 1997; 17:346-349. 8. Law JW et al. Decreased anxiety, altered place learning, and increased CA1 basal excitatory synaptic transmission in mice with conditional ablation of the neural cell adhesion molecule L1. J Neurosci. 2003; 23:10419-10432. 9. Weller S and Gaertner J. Genetic and clinical aspects of X-linked hydrocephalus (L1 disease): Mutations in the L1CAM gene. Hum Mutat. 2001; 18:1-12. 10. Demyanenko GP et al. Altered distribution of dopaminergic neurons in the brain of L1 null mice. Brain Res Dev Brain Res. 2001; 126:21-30. 11. Mohajeri MH et al. Neurite outgrowth on non-permissive substrates in vitro is enhanced by ectopic expression of the neural adhesion molecule L1 by mouse astrocytes. Eur J Neurosci. 1996; 8: 1085-1097. 12. Wolfer DP et al. Increased flexibility and selectivity in spatial learning of transgenic mice ectopically expressing the neural cell adhesion molecule L1 in astrocytes. Eur J Neurosci. 1998; 10:708-717. 13. Luthi A et al. Reduction of hippocampal long-term potentiation in transgenic mice ectopically expressing the neural cell adhesion molecule L1 in astrocytes. J Neurosci Res. 1996; 46:1-6. 14. Ourednik et al. Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons. Nat Biotechnol. 2002; 20:1103-1110. 15. Hulley P et al. L1 neural cell adhesion molecule is a survival factor for fetal dopaminergic neurons. J Neurosci Res. 1998; 53:129-134.
