Bioinformatic and Microarray Strategies to

Identify Peripheral Biomarkers for Parkinson’s

Disease

Bruce Chase University of Nebraska - Omaha

Identifying Peripheral Biomarkers for PD

Parkinson’s Disease (PD) as a complex syndromeHow might peripheral biomarkers be useful?Is there evidence for peripheral biomarkers?Bioinformatic/Microarray approachesProof of concept

Parkinson’s Disease Is A Complex Syndrome

Cardinal FeaturesResting tremorRigidityBradykinesiaPostural instabilityPositive and long-lasting response to levodopa

Parkinson’s Plus Syndromes

poor or short-lived response to levodopaautonomic dysfunctiondementiaophthalmoplegiaamyotrophydystoniadepressionataxia

Neuronal Complexity in PDNeurodegeneration

Progressive loss of dopaminergic neurons in the substantia nigraFormation of Lewy bodies

Impacts multiple neurochemical pathways

dopaminenorepinephrineserotoninacetylcholineGABAglutamate

Lewy bodies

Clinical Spectrum of Lewy Body Disorders

Modified from Arch Neurol 2001; 58:186

DLB

Visual Hallucinations

Behavioral Abnormalities

PD PD WithDementia

LB VariantOf AD

AD

Extrapyramidal Disorder

Memory Disorder

Genetic Complexity In Parkinson’s Disease

Common Idiopathic FormsUnknown causeEnvironmental (+ Genetic?)

Less Common Monogenic Forms-synuclein (PARK1)Parkin (PARK2)UCH-L1 (PARK5)Tau>4 others

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 atrophyAmyotrophic lateral sclerosis

Mutations in the coding region and gene triplications only cause familial Parkinson’s disease

Molecular Complexity: -Synuclein

-Synuclein interactions-amyloidtauparkinphospholipase D2transcription factor Elk-1dopamine transportertyrosine hydroxylaselipids

Biophysical propertiesCan exist in multiple conformations

Affected by environment and mutationsCan form protofibrils and fibrils

Affected by lipid binding

Identifying Peripheral Biomarkers for PD

Parkinson’s Disease (PD) as a complex syndromeHow might peripheral biomarkers be useful?Is there evidence for peripheral biomarkers?Bioinformatic/Microarray approachesProof of concept

How Might Peripheral Biomarkers Be Useful?

Clinical Issues in PD

Etiology of PD is largely unknownBiomarkers 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 stagePeripheral 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

Identifying Peripheral Biomarkers for PD

Parkinson’s Disease (PD) as a complex syndromeHow might peripheral biomarkers be useful?Is there evidence for peripheral biomarkers?Bioinformatic/Microarray approachesProof of Concept

Test Case: Do -Synuclein Expression Levels Serve as a Biomarker?

-Synuclein expression in lymphocytesLow levels: RT-PCR

Lanes 1-4: lymphocyte RNALanes 5-7: Lymphoblastoid cell linesLanes 8-9: Negative controls

Do levels vary with disease status?Assess levels of mutant and normal gene products as a function of disease status

Assess -Synuclein Expression Levels In Kindreds Transmitting -Synuclein Mutations

Autosomal dominant mutationsVariable expressivity

Age of onsetDisease severity/durationPresence of dementiaPathological findingsWithin & between kindreds

G209A exon 4

G88C exon 3

G209A exon 4

Mutant Alleles Show Reduced Expression In Late-Stage Familial Parkinson’s DiseaseDirect sequencing

of RT-PCR products

G209A G88C

RFLPRT-PCR

G209A G88C

qRT-PCR

Identifying Peripheral Biomarkers for PD

Parkinson’s Disease (PD) as a complex syndromeHow might peripheral biomarkers be useful?Is there evidence for peripheral biomarkers?Bioinformatic/Microarray approachesProof of concept

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 PDAlternative: 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 severityDisease symptoms vary widely in idiopathic diseaseGenetic/environmental background effects (noise) could be as large as disease effects (signal)

Statistical evaluationRelevance to neuronal function

Kindred Members As “Biological” Replicates

G209A exon 4

G88C exon 3

Pseudosolution:Reduce genetic (and possibly environmental) variationCompare profiles obtained from nuclear families transmitting a dominant mutationUse UPDRS (Unified Parkinson’s Disease Rating Scale) to score disease severityCompare first-degree relatives who are

Symptomatic gene-positive vs. gene-negativeSymptomatic vs. asymptomatic gene-positive

Identifying Peripheral Biomarkers for PD

Parkinson’s Disease (PD) as a complex syndromeHow might peripheral biomarkers be useful?Is there evidence for peripheral biomarkers?Bioinformatic/Microarray approachesProof of concept

Trial Design

Sample Label Gene Status Symptoms

1 Cy3 G209A/+ Severe

2 Cy5 G209A/+ Mild

3 Cy5 G209A/+ None

Extract RNA from G209A/ + heterozygotesLabel RNA from a severely symptomatic individual with Cy5Label RNA from mildly symptomatic and asymptomatic individuals with Cy3Probe cDNA spotted arrays; Affymetrix chips

Multiple Processes Appear Affected

Energy/metabolismATP synthase, ATPasecytochrome C oxidaseNADH dehydrogenase

NeurotransmissionGABA-A receptor subunits, associated proteins DOPA decarboxylase Catechol-O-methyltransferase Chloride channel

Neurodegeneration / protein degradation / apoptosisalpha-Synuclein interacting protein (synphilin)Huntingtin interacting protein C Tumor necrosis factor receptor superfamily, membersE3 ubiquitin ligaseApoptosis-inducing serine-threonine kinase

Transcriptional regulation / DevelopmentHeterogeneous nuclear ribonucleoprotein H1Bicaudal

TranslationEukaryotic translation initiation and elongation factors

Gene Expression In Cell Lines Established From G209A Heterozygotes Differing in Disease Status

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Gene

Rat

io o

f M

edia

ns

#2 (Mild) : #1 (Severe) #3 (Asymptomatic) : #1 (Severe)

SummaryParkinson’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

Acknowledgments

CollaboratorsKaterina Markopoulou, UNMC, OmahaZbigniew Wszolek, Mayo Clinic, JacksonvilleLola Katechalidou, ELPIS Hospital, AthensNobu Hattori, Juntendo Medical School, Tokyo

Microarray consultantsJim Eudy, UNMC, OmahaDan Bosinov, UNMC, Omaha

FundingNIH/NINDSNE-BRIN