Bioinformatic and Microarray Strategies to Identify Peripheral Biomarkers for Parkinson’s Disease

1. Bioinformatic and Microarray Strategies to Identify Peripheral Biomarkers for Parkinson’s Disease Bruce Chase University of Nebraska - Omaha

2. Identifying Peripheral Biomarkers for PD • Parkinson’s Disease (PD) as a complex syndrome • How might peripheral biomarkers be useful? • Is there evidence for peripheral biomarkers? • Bioinformatic/Microarray approaches • Proof of concept

3. Parkinson’s Disease Is A Complex Syndrome • Cardinal Features • Resting tremor • Rigidity • Bradykinesia • Postural instability • Positive and long-lasting response to levodopa • Parkinson’s Plus Syndromes • poor or short-lived response to levodopa • autonomic dysfunction • dementia • ophthalmoplegia • amyotrophy • dystonia • depression • ataxia

4. Neuronal Complexity in PD • Neurodegeneration • Progressive loss of dopaminergic neurons in the substantia nigra • Formation of Lewy bodies • Impacts multiple neurochemical pathways • dopamine • norepinephrine • serotonin • acetylcholine • GABA • glutamate

5. Lewy bodies

6. Behavioral Abnormalities DLB Memory Disorder Extrapyramidal Disorder PD PD With Dementia LB Variant Of AD AD Visual Hallucinations Clinical Spectrum of Lewy Body Disorders Modified from Arch Neurol 2001; 58:186

7. Genetic Complexity In Parkinson’s Disease • Common Idiopathic Forms • Unknown cause • Environmental (+ Genetic?) • Less Common Monogenic Forms • -synuclein (PARK1) • Parkin (PARK2) • UCH-L1 (PARK5) • Tau • >4 others

8. Molecular Complexity: -Synuclein • Main component of intracellular fibrillar protein deposits in affected brain regions in multiple neurodegenerative disorders • Parkinson’s disease (Lewy bodies) • Alzheimer disease (plaques) • Multiple system atrophy • Amyotrophic lateral sclerosis • Mutations in the coding region and gene triplications only cause familial Parkinson’s disease

9. Molecular Complexity: -Synuclein • -Synuclein interactions • b-amyloid • tau • parkin • phospholipase D2 • transcription factor Elk-1 • dopamine transporter • tyrosine hydroxylase • lipids • Biophysical properties • Can exist in multiple conformations • Affected by environment and mutations • Can form protofibrils and fibrils • Affected by lipid binding

10. Identifying Peripheral Biomarkers for PD • Parkinson’s Disease (PD) as a complex syndrome • How might peripheral biomarkers be useful? • Is there evidence for peripheral biomarkers? • Bioinformatic/Microarray approaches • Proof of concept

11. How Might Peripheral Biomarkers Be Useful? • Clinical Issues in PD • Etiology of PD is largely unknown • Biomarkers could aid in understanding PD etiology • PD is a chronic, progressive and complex syndrome where diagnosis is subjective, confirmable only at autospy, and disease progression is variable • Biomarkers could discriminate between forms of PD, support early diagnosis, document stage • Peripheral biomarkers are evaluated using relatively noninvasive methods • Therapy is based solely on symptoms, and requires periodic adjustment • Biomarkers could aid in design/implementation of optimal therapeutic regimens

12. Identifying Peripheral Biomarkers for PD • Parkinson’s Disease (PD) as a complex syndrome • How might peripheral biomarkers be useful? • Is there evidence for peripheral biomarkers? • Bioinformatic/Microarray approaches • Proof of Concept

13. Test Case: Do -Synuclein Expression Levels Serve as a Biomarker? • -Synuclein expression in lymphocytes • Low levels: RT-PCR • Lanes 1-4: lymphocyte RNA • Lanes 5-7: Lymphoblastoid cell lines • Lanes 8-9: Negative controls • Do levels vary with disease status? • Assess levels of mutant and normal gene products as a function of disease status

14. G88C exon 3 G209A exon 4 Assess -Synuclein Expression Levels In Kindreds Transmitting -Synuclein Mutations • Autosomal dominant mutations • Variable expressivity • Age of onset • Disease severity/duration • Presence of dementia • Pathological findings • Within & between kindreds G209A exon 4

15. G209A G88C Direct sequencing of RT-PCR products qRT-PCR G209A G88C RFLP RT-PCR Mutant Alleles Show Reduced Expression In Late-Stage Familial Parkinson’s Disease

16. Identifying Peripheral Biomarkers for PD • Parkinson’s Disease (PD) as a complex syndrome • How might peripheral biomarkers be useful? • Is there evidence for peripheral biomarkers? • Bioinformatic/Microarray approaches • Proof of concept

17. Bioinformatic/Microarray Approaches • Evaluate gene expression profiles to identify a molecular signature associated with PD stages/forms • Targets identified using bioinformatic approach: all genes in pathways previously suggested relevant to PD • Alternative: Assess all genes without an initial bias • Concerns: • Power: What constitutes a biological replicate in RNA samples? • What are normal levels of variation? • Are parkinsonian individuals more variable? • Affected individuals fluctuate in disease severity • Disease symptoms vary widely in idiopathic disease • Genetic/environmental background effects (noise) could be as large as disease effects (signal) • Statistical evaluation • Relevance to neuronal function

18. G209A exon 4 G88C exon 3 Kindred Members As “Biological” Replicates • Pseudosolution: • Reduce genetic (and possibly environmental) variation • Compare profiles obtained from nuclear families transmitting a dominant mutation • Use UPDRS (Unified Parkinson’s Disease Rating Scale) to score disease severity • Compare first-degree relatives who are • Symptomatic gene-positive vs. gene-negative • Symptomatic vs. asymptomatic gene-positive

19. Identifying Peripheral Biomarkers for PD • Parkinson’s Disease (PD) as a complex syndrome • How might peripheral biomarkers be useful? • Is there evidence for peripheral biomarkers? • Bioinformatic/Microarray approaches • Proof of concept

20. Trial Design • Extract RNA from G209A/ + heterozygotes • Label RNA from a severely symptomatic individual with Cy5 • Label RNA from mildly symptomatic and asymptomatic individuals with Cy3 • Probe cDNA spotted arrays; Affymetrix chips

21. Multiple Processes Appear Affected • Energy/metabolism • ATP synthase, ATPase • cytochrome C oxidase • NADH dehydrogenase • Neurotransmission • GABA-A receptor subunits, associated proteins • DOPA decarboxylase • Catechol-O-methyltransferase • Chloride channel • Neurodegeneration / protein degradation / apoptosis • alpha-Synuclein interacting protein (synphilin) • Huntingtin interacting protein C • Tumor necrosis factor receptor superfamily, members • E3 ubiquitin ligase • Apoptosis-inducing serine-threonine kinase • Transcriptional regulation / Development • Heterogeneous nuclear ribonucleoprotein H1 • Bicaudal • Translation • Eukaryotic translation initiation and elongation factors

23. Summary • Parkinson’s Disease is a complex syndrome • Biomarkers hold promise for aiding diagnosis and implementing treatment regimens • Peripheral biomarkers are likely to exist • Microarray-based approaches hold promise for peripheral biomarker development • Comparisons between nuclear family members in FPD kindreds may serve to increase power and reduce environmental and genetic effects in the initial identification of peripheral biomarkers

24. Acknowledgments • Collaborators • Katerina Markopoulou, UNMC, Omaha • Zbigniew Wszolek, Mayo Clinic, Jacksonville • Lola Katechalidou, ELPIS Hospital, Athens • Nobu Hattori, Juntendo Medical School, Tokyo • Microarray consultants • Jim Eudy, UNMC, Omaha • Dan Bosinov, UNMC, Omaha • Funding • NIH/NINDS • NE-BRIN