Bioinformatic and Microarray Strategies to Identify Peripheral Biomarkers for Parkinson

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Bioinformatic and Microarray Strategies to
Identify Peripheral Biomarkers for Parkinsons
Disease

• Bruce Chase
• University of Nebraska - Omaha

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Identifying Peripheral Biomarkers for PD

• Parkinsons Disease (PD) as a complex syndrome
• How might peripheral biomarkers be useful?
• Is there evidence for peripheral biomarkers?
• Bioinformatic/Microarray approaches
• Proof of concept

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Parkinsons Disease Is A Complex Syndrome

• Cardinal Features
• Resting tremor
• Rigidity
• Bradykinesia
• Postural instability
• Positive and long-lasting response to levodopa

• Parkinsons Plus Syndromes
• poor or short-lived response to levodopa
• autonomic dysfunction
• dementia
• ophthalmoplegia
• amyotrophy
• dystonia
• depression
• ataxia

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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

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Lewy bodies
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Clinical Spectrum of Lewy Body Disorders
Modified from Arch Neurol 2001 58186
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Genetic Complexity In Parkinsons Disease

• Common Idiopathic Forms
• Unknown cause
• Environmental ( Genetic?)
• Less Common Monogenic Forms
• ?-synuclein (PARK1)
• Parkin (PARK2)
• UCH-L1 (PARK5)
• Tau
• gt4 others

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Molecular Complexity ?-Synuclein

• Main component of intracellular fibrillar protein
  deposits in affected brain regions in multiple
  neurodegenerative disorders
• Parkinsons disease (Lewy bodies)
• Alzheimer disease (plaques)
• Multiple system atrophy
• Amyotrophic lateral sclerosis
• Mutations in the coding region and gene
  triplications only cause familial Parkinsons
  disease

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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

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Identifying Peripheral Biomarkers for PD

• Parkinsons Disease (PD) as a complex syndrome
• How might peripheral biomarkers be useful?
• Is there evidence for peripheral biomarkers?
• Bioinformatic/Microarray approaches
• Proof of concept

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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

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Identifying Peripheral Biomarkers for PD

• Parkinsons Disease (PD) as a complex syndrome
• How might peripheral biomarkers be useful?
• Is there evidence for peripheral biomarkers?
• Bioinformatic/Microarray approaches
• Proof of Concept

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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

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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
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Mutant Alleles Show Reduced Expression In
Late-Stage Familial Parkinsons Disease
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Identifying Peripheral Biomarkers for PD

• Parkinsons Disease (PD) as a complex syndrome
• How might peripheral biomarkers be useful?
• Is there evidence for peripheral biomarkers?
• Bioinformatic/Microarray approaches
• Proof of concept

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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

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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 Parkinsons Disease Rating
  Scale) to score disease severity
• Compare first-degree relatives who are
• Symptomatic gene-positive vs. gene-negative
• Symptomatic vs. asymptomatic gene-positive

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Identifying Peripheral Biomarkers for PD

• Parkinsons Disease (PD) as a complex syndrome
• How might peripheral biomarkers be useful?
• Is there evidence for peripheral biomarkers?
• Bioinformatic/Microarray approaches
• Proof of concept

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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

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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

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Summary

• Parkinsons 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

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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