Circulating Biomarkers for Alzheimer's Disease: Neurodegenerative Disorders Webinar Series Part 3

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Molecular Mechanisms of Neurodegeneration

Circulating Biomarkers for Alzheimer’s DiseaseAli Bierly, Ph.D.

[email protected]

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Welcome!

Contact Technical Support:

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Webinar-related questions: [email protected]

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Welcome to our three-part webinar series on neurodegeneration

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Neurodegenerative disorders: molecular mechanisms and circulating biomarker discovery – a three-part webinar series

Part 1: Molecular Mechanisms of Neurodegeneration

Part 2: The Central Roles of Non-coding RNAs in Neurodegenerative Disorders

Part 3: Circulating Biomarkers for Alzheimer’s Disease

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Legal disclaimer

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QIAGEN products shown here are intended for molecular biology

applications. These products are not intended for the diagnosis,

prevention or treatment of a disease.

For up-to-date licensing information and product-specific

disclaimers, see the respective QIAGEN kit handbook or user

manual. QIAGEN kit handbooks and user manuals are available

at www.QIAGEN.com or can be requested from QIAGEN

Technical Services or your local distributor.

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Recent Alzheimer’s disease miRNA biomarkers4

Agenda

Alzheimer‘s disease1

Biomarkers for detecting diseases2

miRNA as biomarker signatures3

QIAGEN tools for biomarker discovery5

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Alzheimer’s disease

Chronic neurodegenerative disease affecting 21–35 million people worldwide

Most common cause of dementia

Early symptoms often mistaken for normal aging

Four stages of AD:

Pre-dementia

MCI – Mild cognitive impairment (short-term memory loss, apathy)

Early

Increased difficulty with learning and memory, definitive diagnosis

Moderate

Paraphasia, loss of reading and writing, long-term memory problems, behavioral symptoms

Advanced

Eventual loss of speech, inability to perform basic tasks, apathy and exhaustion

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Molecular basis for AD

Mutations in APP, PSEN1, PSEN2 cause early-onset autosomal dominant AD

APOE4 e4 allele predisposes to sporadic AD, but other factors need to be investigated

Two major protein abnormalities: Amyloid plaques (amyloid-beta peptide, a

fragment of APP, and cellular material) Neurofibrillary tangles (hyperphosphorylated

Tau, a microtubule-associated protein)

Neurons and synapses are lost in the cerebral cortex and some subcortical regions leading to atrophy

Inflammation also appears to play a role

Two types of AD: Familial (less than 5%) and sporadic

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Agenda





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Why do we need early Alzheimer’s biomarkers?

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Current diagnosis combines neuropsychological evaluation, neuroimaging and Ab and Tau CSF levels, but sensitivity is only ~93% and specificity only ~55%

Evidence shows that treatment at earlier stages of AD can prevent cognitive decline, whereas treatment at later stages isn’t as effective. Experiments like the A4 study (http://www.adcs.org/Studies/A4.aspx) evaluate the efficacy of drugs before the onset of symptoms

Better biomarkers could provide an early sign before symptoms start to show, enabling more effective prevention and treatment

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Biomarkers could detect AD before irreversible decline

What types of biomarkers are being investigated in AD?

Plasma phospholipids (Mapstone, M. et al.

2014, Nat. Med.)

CSF proteins (Tau, amyloid beta) (Frankfort S.V. et al. 2008, Curr.

Clin. Pharmacol.)

Blood levels of Tau and amyloid beta (Frankfort S.V. et al. 2008,

Curr. Clin. Pharmacol.)

microRNA levels in the blood

Possible AD biomarkers

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Circulating biomarkers in neurodegenerative disease


Benefits of circulating biomarkers: Some samples, like CSF, can be painful to

collect. Blood samples are less invasive and can be drawn regularly.

Circulating biomarkers identified in neurodegenerative disorders: AD: amyloid beta, Tau, low mtDNA, p21, p53 MS: serum lactate PD: alpha-synuclein, HNF4A & PTBP1 mRNA,

SRRM2 (RNA splicing factor)

Biomarker

“A characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.”

1998, NIH Biomarkers Definitions Working Group

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Agenda





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microRNAs in gene regulation


miRNAs

Small non-coding RNAs, 18–25 nt

Found in animals, plants and some viruses

Base-pair with complementary mRNA sequences in the 3’ UTR, resulting in silencing by either degradation of the strand or prevention of translation

A single miRNA can regulate up to 200 mRNAs, and several miRNAs can target the same mRNA

Involved in normal biological processes like inflammation, apoptosis and development

Implicated in numerous disease states, including cancer, heart disease and nervous system disorders

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microRNAs in circulation

Shielded in exosomes or bound to Argonaute2 or HDL, miRNAs can be stable in blood and, therefore, a suitable candidate for circulating biomarkers

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microRNAs as biomarkers in CNS disorders

Small molecules with a substantial impact

Several miRNAs are involved in normal neurological development, including miR-124-3p, miR-125b-5p, miR-132-3p, miR-134, miR-138-5p and miR-9-5p. miRNAs are highly expressed in brain and spinal fluid

Circulating miRNA biomarker signatures are being investigated in many neurodegenerative diseases, such as:

Alzheimer’s disease: (to be discussed later in the presentation)

Parkinson’s disease: miR-331-5p, -1826, -450b-3p, -626, -505

Amyotrophic lateral sclerosis (ALS): in leukocytes, miR-451, miR-1275, miR-328 and 5 others were identified as potential candidates

Huntington’s disease: possibly miR-34b

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Techniques for detecting microRNA in blood


qPCR Focused profiling of pathway-related miRNAs Whole miRNome profiling of all known miRNAs Individual assays

Next-generation sequencing Can detect currently-unknown miRNAs Results can then be verified with qPCR or microarray

Microarray

Basic structure of a microRNA biomarker discovery study:

Screen for miRNAsdifferentially expressed between disease and control

Verification of signature by another technique or in another cohort

Determine which genes and pathways are being targeted

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Agenda





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Identifying miRNA signatures in AD

Leidinger et al. (2013) A blood based 12-miRNA signature of Alzheimer disease patients. Genome Biology 14, R78. Identified a new AD miRNA biomarker signature by NGS and verified with miScript

Primer Assays

Kumar et al. (2013) Circulating miRNA biomarkers for Alzheimer’s disease. PLOS One 8, e69807. Used Nanostring technology to identify a circulating miRNA biomarker signature

and elucidated the neurology-related pathways involving genes targeted by these miRNAs using Ingenuity Pathway Analysis (IPA) software

Literature examples

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Study 1: a 12-miRNA AD signature in blood


Leidinger et al., 2013

Goal: to develop a miRNA expression signature specific to Alzheimer’s disease that could be translated for use in combination with other non-invasive diagnostic techniques like amyloid load imaging

Approach: Used next-generation sequencing to analyze blood from AD patients and healthy

age-matched controls Classified AD and control samples to determine an effective signature for AD Verified the signature using qPCR (the miScript system) and non-AD patients with

other neurological disorders Predicted miRNA targets using miRDB

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Study 1: a 12-miRNA AD signature in blood


Results

NGS results Found 140 unique mature miRNAs that were significantly altered between AD

patients and normal controls (58 downregulated, 82 upregulated) Found 15 novel miRNAs upregulated in AD (brain miRNAs) Families heavily represented: miR-30 (5 miRNAs upregulated), let-7 (9

downregulated) Selected 12 miRNAs specific to AD to form the final signature

qPCR verification Ten of the 12 showed dysregulation in the same direction by NGS and qPCR No stage dependence – mild and moderate AD groups were both equally identified

using the signature The signature also distinguished major depression, schizophrenia and bipolar

patients from controls, but was only about 75% accurate at distinguishing AD from Parkinson’s, MS and the psychological disorders, so it may need to be refined for that purpose

Target prediction Target genes were enriched in nervous system development and neuron projection

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Study 2: a 7-miRNA signature for AD


Kumar et al., 2013

Goal: develop a signature that could someday translate to the clinic not only in diagnostics, but also in patient stratification for drug trials and monitoring patient response to the treatment.

Approach: Profiled 654 miRNAs from 11 AD patients and 20 controls using NanoString Verified the miRNA signature using qPCR Verified signature in a different cohort with 20 AD and 17 control samples Analyzed targets to identify related biological pathways using Ingenuity Pathway

Analysis (looked at mRNAs targeted by two or more of the signature miRNAs)

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Study 2: circulating biomarkers for AD


Results

NanoString results: Identified 12 microRNAs with differential expression in AD samples of at least

1.5-fold let-7d-5p, let-7g-5p, miR-15b-5p, miR-142-3p, miR-191-5p, miR-301a-3p, miR-323b-

5p, miR-545-3p, miR-563, miR-600, miR-1274a, miR-1975

qPCR verification: Seven of the 12 miRNAs identified by NanoString also showed differential

expression in AD by qPCR assays (let-7d-5p, let-7g-5p, miR-15b-5p, miR-142-3p, miR-191-5p, miR-301a-3p, miR-545-3p)

Independent cohort verification: Combination of miR-545-3p, let-7g-5p, and miR-15b-5p gave 94.1% specificity and

95% sensitivity. Best standalone biomarkers were miR-191-5p, miR-15b-5p and let-7d-5p

Target analysis Axonal guidance signaling, ephrin receptor signaling, actin cytoskeleton signaling,

rhoA signaling, clathrin-mediated endocytosis and others were targeted

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Agenda





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QIAGEN tools for circulating miRNA biomarker discovery


miRNeasy Serum / Plasma Kit – purifies RNA from 18 nt upwards (both miRNA and mRNA)

Employs a spike-in control for normalization (a C. elegans miR-39 mimic)

Automatable on the QIAcube

Ingenuity Pathway Analysis (IPA) – software for analysis, integration and understanding of data from gene expression, miRNA and SNP microarrays, as well as metabolomics, proteomics and RNA-seq experiments

miScript Primer Assays – quantify any mature human, dog, rat or mouse miRNA in miRBase

miScript miRNA PCR Arrays – arrays of related miRNA assays arranged by pathway or disease. miRNome arrays updated through miRBase version 21

miScript PreAMP PCR Kit – preamplifies up to 400 targets in one reaction for limited samples

Isolation Data interpretationDetection (qPCR)

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miRNA expression – miScript miRNA PCR Arrays

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miRNome Human: miRBase v21, covers 2,402 primer assays Mouse: miRBase v21, covers 1,765 primer assays Rat: 653 primer assays Dog: 277 primer assays Rhesus macaque: 469 primer assays Cow: 744 primer assays

Pathway-focused arrays (over 20 arrays) miFinder Neurological development and disease Neuropathic and inflammatory pain Apoptosis Cell development and differentiation Brain cancers Serum and plasma miRNAs

miScript PreAMP Kit Optional step for small or precious samples Full miRNome profiling from as little as 1 ng RNA

http://www.qiagen.com/products/catalog/assay-technologies/mirna/miscript-mirna-pcr-arrays

Pre-formatted, single-use PCR arrays with wet lab-verified assays

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Ingenuity Pathway Analysis (IPA)

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Features numerous tools to help interpret complex miRNA data: miRNA Target Filter: gives microRNA-mRNA pairings and biological effects using

experimentally verified interactions from TarBase and miRecords, and predicted miRNA-mRNA interactions from TargetScan

Pathway analysis, canonical pathways, overlapping pathways, pathway import and scoring: Helps you determine the most significantly affected pathways

Network analysis: Build miRNA–mRNA networks

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Thank you for attending

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Thank you for attending today’s webinar!Contact QIAGENCall: 1-800-426-8157

Email: [email protected]

Questions?

Ali Bierly, Ph.D.

[email protected]

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.

Circulating Biomarkers for Alzheimer's Disease: Neurodegenerative Disorders Webinar Series Part 3

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Transcript of Circulating Biomarkers for Alzheimer's Disease: Neurodegenerative Disorders Webinar Series Part 3