Welcome!

The Central Roles of Non-coding RNAs in Neurodegenerative Disorders Contact Technical Support: [email protected] 1-800-362-7737 Webinar-related questions: [email protected]

Wei Cao, Ph.D. [email protected]

Sample to Insight Noncoding RNAs in Neurodegeneration

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Welcome to our 3-part webinar series on neurodegeneration Neurodegenerative disorders: molecular mechanisms and circulating biomarker discovery – a 3-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

Sample to Insight Molecular Mechanisms of Neurodegeneration

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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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Agenda  Molecular mechanisms in neurodegeneration 

Abnormal protein assemblies (protein misfolding)



Late-life cell death in adulthood



Oxidative stress



Inflammation - induced neurotoxicity

 Introduction to non-coding RNA 

Small non-coding RNA - microRNA



Long non-coding RNA (lncRNA)

 Non-coding RNAs in neural function and diseases  Study ncRNA: miRNA and lncRNA  Questions

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Molecular mechanisms of neurodegeneration Neurodegeneration is featured by progressive dysfunction and death of cells in selected areas in the nervous system. 1. 2. 3.

Alzheimer’s Disease (AD) Parkinson’s Disease (PD) Huntington’s disease (HD)

Common molecular mechanisms 

Abnormal protein assemblies (protein misfolding)



Late-life cell death in adulthood



Oxidative stress



Inflammation - induced neurotoxicity

Molecular basis for neurodegeneration 

Gene mutations and accumulation of abnormal proteins and inclusion bodies are hallmarks in most neurodegenerative diseases.

 ncRNAs and ncRNA-regulatory processes are important players in the pathogenesis of neurodegenerative diseases. Sample to Insight Noncoding RNAs in Neurodegeneration

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non-coding RNAs “Dark matter” of the genome – non-coding RNA  Over 90% of human genome are actively transcribed, and proteincoding genes constitute only ~2% of the genome  For a long time, people have no idea of the other part of genome’s function. They were called “dark matter” or “junk”.  non-coding RNAs: small non-coding RNA and long non-coding RNA

Proportion of non protein-coding sequence increase with eukaryotes complexity

Schwarzenbach, H. et. al. (2013) “Cell-free nucleic acids as biomarkers in cancer patients.” Nat. Rev. Cancer 11, 426. Rönnau, C.G.H. (2014) “Noncoding RNAs as novel biomarkers in prostate cancer.” Biomed. Res. Int. 2014; 591703: 17

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non-coding RNAs – miRNA and lncRNA

Short non-coding RNAs - microRNAs (miRNAs) 

miRNAs are ~21 nt in size, an abundant class of ncRNAs, and the most widely studied ncRNAs



miRNAs regulate a variety of cellular processes through the posttranscriptional repression of gene expression



miRNA expression is frequently deregulated in various diseases, therefore has potential to serve as biomarkers

Long non-coding RNAs (lncRNAs)  lncRNAs are a novel class of RNAs, with > 200 nucleotides in sizes  lncRNAs regulate protein-coding genes transcription in more complex ways than the miRNAs do  Changes in lncRNA can be correlated with a variety of human diseases

Schwarzenbach, H. et. al. (2013) “Cell-free nucleic acids as biomarkers in cancer patients.” Nat. Rev. Cancer 11, 426. Rönnau, C.G.H. (2014) “Noncoding RNAs as novel biomarkers in prostate cancer.” Biomed. Res. Int. 2014; 591703: 17

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The RNA-RNA interactions among different RNA species The central dogma and regulatory complexity  One gene gives rise to one RNA to produce one protein. RNA has more versatile functions than its protein synthesis role.  RNA regulates most cell processes, including epigenetic control, gene transcription, translation, RNA turnover, chromosomal organization, and genome defense, cellular developmental and proliferation programs.

 RNA always works through interactions with proteins, however, the RNA–RNA interactions, mediated by ncRNA add another layer of regulatory complexity. - ncRNAs regulate gene expression, for example, a single miRNA can affect the expression of over 100 transcripts - Control splicing through direct base pairing - Control translation - lncRNA–miRNA interactions: ncRNA regulates miRNA biogenesis, and miRNAs regulate lncRNAs

Guil. S. and Esteller, M. (2015) “RNA–RNA interactions in gene regulation: the coding and noncoding players.” Trends in Biochemical Sciences.

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miRNA biogenesis and regulatory mechanism MicroRNA (miRNA) biogenesis pathway  Transcribed by RNA Polymerase II as a long primary transcript (pri-miRNAs), which may contain more than one miRNA.  In the nucleus, pri-miRNAs are processed to hairpin-like pre-miRNAs by RNAse IIIlike enzyme Drosha.  Pre-miRNAs are then exported to the cytosol by exportin 5.  In the cytosol RNAse III-like Dicer processes these precursors to mature miRNAs.  These miRNAs are incorporated in RISC.  miRNAs with high homology to the target mRNA lead to mRNA cleavage.  miRNAs with imperfect base pairing to the target mRNA lead to translational repression and/or mRNA degradation.

Meza-Sosa KF, et al. “Role of microRNAs in central nervous system development and pathology”, J Neurosci Res, 2012; 90:1

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miRNA biogenesis and regulatory mechanism MicroRNA (miRNA) biogenesis pathway •

miRNAs are highly expressed in CNS including the brain and spinal cord

•

Key modulators of both CNS development and plasticity

•

Proteins implicated in neurodegenerative diseases are involved in multiple steps of the miRNA biogenesis pathway

•

Represent a novel class of therapeutic targets for neurodegenerative disorders

FUS

TDP-43

Atx2

Meza-Sosa KF, et al. “Role of microRNAs in central nervous system development and pathology”, J Neurosci Res, 2012; 90:1

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miRNAs in neurodegenerative disorders miRNAs have been implicated in neurodegenerative disorders

miRNAs

miRNAs as mediators of brain development and neuronal differentiation Play roles in neuronal longevity and survival Regulate neurodegenerative disease-associated pathways miRNA dysfunction downstream of disease-linked TDP-43 alterations could represent an important pathogenic mechanism in neurodegenerative disease

Szafranski, K., et al. “Non-codingRNAinneuralfunction,disease,andaging”, Frontiers in genetics , March 2015

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Introduction to long non-coding RNAs (lncRNAs)  lncRNAs are non-protein coding transcripts longer than 200 nucleotides in size

 Most of lncRNAs are localized in the nucleus, whereas some lncRNAs are found in the cytoplasm

 Many lncRNAs are molecularly indistinguishable from mRNAs

 Abundance: Some lncRNAs, such as MALAT1, are highly abundant transcripts, many lncRNAs do show low count. Low transcription levels do not necessarily reflect lack of functionality

 May contain a Poly-A tail like mRNA or may be absent

 Normally, lncRNAs tend to be less conserved across species and often show low expression levels and high tissue specificity. Schwarzenbach, H. et. al. (2013) “Cell-free nucleic acids as biomarkers in cancer patients.” Nat. Rev. Cancer 11, 426. Rönnau, C.G.H. (2014) “Noncoding RNAs as novel biomarkers in prostate cancer.” Biomed. Res. Int. 2014; 591703: 17

Sample to Insight Noncoding RNAs in Neurodegeneration

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lncRNA classification and subgroup lncRNAs can exceed 100,000 nucleotides and cover a wide range of gene positions lncRNAs can be divided into three big groups: • Transcribe relative to the host PCG • Transcribed from gene regulatory regions • Transcripted from the specific chromosomal regions

Classified based on their relative position to PCG (protein-coding genes)

Intergenic Intronic Exonic Overlapping Sense Antisense

Wu P. et al. “Roles of long noncoding RNAs in brain development, functional diversification and neurodegenerative diseases”, (2013). Brain Research Bulletin 97: 69 Sample to Insight Noncoding RNAs in Neurodegeneration

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The molecular functions of lncRNAs

Martin L, Chang HY. (2012) “Uncovering the role of genomic "dark matter" in human disease”, J Clin Invest, 122 1589

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lncRNAs in the central nervous system (CNS) The complex functions of lncRNA coincides with the diversity and elaborate nature of CNS lncRNAs in brain development

lncRNAs in neural differentiation and maintenance

lncRNAs in synaptic plasticity, cognitive function and Memory

lncRNAs in aged brain and neurodegenerative disorders

Wu P. et al. “Roles of long noncoding RNAs in brain development, functional diversification and neurodegenerative diseases”, (2013). Brain Research Bulletin 97: 69 Sample to Insight Noncoding RNAs in Neurodegeneration

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Dysregulated lncRNAs in neurodegenerative diseases lncRNAs

Disease associated

Biological function

BACE1-AS

AD

Increase BACE1 mRNA stability through a post-transcriptional feed-forward mechanism

NAT-Rad18

AD

Down-regulate DNA repair protein Rad18, causing the neuron more sensitive to apoptosis

17A

AD

Impair GABAB signaling pathway by decreasing GABAB R2 transcription

GDNFOS

AD

Modulate the expression of endogenous GDNF in human brain

Sox2OT 1810014B01Rik

AD, PD AD, PD

Regulate co-transcribed Sox2 gene expression to down neurogenesis Not known yet

BC200

AD, PD

Modulate local proteins in postsynaptic dendritic microdomains to maintain long-term synaptic plasticity

naPINK1

PD

Stabilize the svPINK1 resulting in disturbed mitochondrial respiratory chain, increase the sensitivity to apoptosis

HAR1F

HD

Aberrant nuclear-cytoplasmic REST/NRSF trafficking caused by mutated huntingtin resulting in the aberrant expression of HAR1in striatum

HTTAS

HD

HTTAS v1 specifically reduces endogenous HTT transcript levels

DGCR5

HD

DGCR5 is downstream target of REST in HD disease

NEAT1

HD

Essential for the integrity of the nuclear paraspeckle substructure

Wu P. et al. “Roles of long noncoding RNAs in brain development, functional diversification and neurodegenerative diseases”, (2013). Brain Research Bulletin 97: 69 Sample to Insight Noncoding RNAs in Neurodegeneration

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lncRNAs are essential for brain development lncRNA Peril−/− mice have reduced viability and die shortly after birth

Sauvageau M, 2013. Elife. 2:e01749

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874104/ Sample to Insight Noncoding RNAs in Neurodegeneration

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lncRNAs in Alzheimer’s disease (AD) 

AD is the most common neurodegenerative disorder.



The pathologic process of AD is not well understood, one of the main reasons is the amyloid plaques caused by increased levels



of Aβ42. A series of aberrant lncRNAs have been found in AD patients.

BACE1-AS, a lncRNA transcribed from the antisense protein-coding BACE 1 gene, is highly expressed in AD patients, and directly implicated in the increased abundance of Aβ42 in AD

BACE1-AS increases BACE1 mRNA stability, then generate additional Aβ42 through a post-transcriptional feed-forward mechanism Faghihi MA, et al (2008) Expression of a noncoding RNA is elevated in Alzheimer's disease and drives rapid feed-forward regulation of betasecretase. Nat Med. 14(7):723 Modarresi F, et al. (2011) “Knockdown of BACE1-AS Nonprotein-Coding Transcript Modulates Beta-Amyloid-Related Hippocampal Neurogenesis.” Int J Alzheimers Dis. 2011 Sample to Insight Noncoding RNAs in Neurodegeneration

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lncRNAs in Parkinson’s disease (PD) 

PD is the most frequent motor disorder, and Deep-brainstimulation (DBS) treatment alleviates the symptom.



RNA-Seq experiment: PD patients’ leukocytes pre- and postDBS treatment, and compared to healthy controls.



Results: identified 13 lncRNAs (out of 6,000 lncRNAs) with reduced expression level in PD patients.



Four lncRNAs, RP4-705O1.1, RP11-533O10.2, RP11-425I13.3 and RP11-79P5.3 were inversely altered in post-DBS

Sample to Insight Noncoding RNAs in Neurodegeneration

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lncRNAs alteration in Huntington's disease (HD) brains HD is caused by an expansion of a CAG triplet repeat stretch within the Huntington gene REST (RE1 Silencing Transcription Factor) is one of targets of mutated Huntington gene Many lncRNAs are direct targets of and regulated by REST

Possible mechanism of lncRNAs in HD

Rory Johnson. “Long non-coding RNAs in Huntington's disease neurodegeneration”, 2012, Neurobiology of Disease. 46:245

Sample to Insight Noncoding RNAs in Neurodegeneration

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Agenda  Molecular mechanisms in neurodegeneration 

Abnormal protein assemblies (protein misfolding)



Late-life cell death in adulthood



Oxidative stress



Inflammation - induced neurotoxicity

 Introduction to non-coding RNA 

Small non-coding RNA - microRNA



Long non-coding RNA (lncRNA)

 Non-coding RNAs in neural function and diseases  Study non-coding RNAs – miRNA and lncRNA  Questions Sample to Insight Noncoding RNAs in Neurodegeneration

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The current lncRNA quantification approaches 

RNA-seq (Whole transcriptome sequencing) - Discover new RNAs and splicing variants



Microarrays - Use data analysis approaches to identify lncRNAs



Real-time PCR based approaches - Sensitive and quantitative for low expressing RNAs and small gene changes

Sample Isolation

Amplification

miRNeasy exoRNeasy Serum/Plasma Kit

qPCR

miScript miRNA PreAMP PCR Kit

miScript miRNA PCR Array

RT² lncRNA PreAMP PCR Kit

RT2 lncRNA PCR System

Data Analysis & Interpretation

Free data analysis tool

Test and verify your hypothesis with miScript miRNA PCR Array and RT2 lncRNA qPCR Assays or Custom PCR Arrays

Sample to Insight Noncoding RNAs in Neurodegeneration

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Real-time PCR quantification of non-coding RNAs

 Has been the gold standard for gene quantification  Is the method of choice to confirm next-generation sequencing and microarray results  Simple and easy to carry out  High sensitivity, specificity  High throughput compatible, automatable  Needs very low amounts of template

Sample to Insight Noncoding RNAs in Neurodegeneration

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Solutions to miRNA and lncRNA profiling and detection

lncRNA miRNA

How can you advance your non-coding RNA research?

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miRNA expression — miScript miRNA PCR Arrays
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 (>20 arrays)  miFinder  Neurological development and disease  Pain: neuropathic & inflammatory  Apoptosis  Cell development & 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

Pre-formatted, single-use PCR arrays with wet-lab verified assays http://www.qiagen.com/products/catalog/assay-technologies/mirna/miscript-mirna-pcr-arrays Sample to Insight Noncoding RNAs in Neurodegeneration

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lncRNA databases - worldwide efforts

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The authoritative lncRNA databases

■ QIAGEN merged these two databases together for the most-up to date qPCR assay design ■ Currently cover human Gencode 19, mouse Gencode M2, RefSeq Release 65

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RT2 lncRNA qPCR system
lncRNA databases: In-house database at QIAGEN GeneGlobe provides cover > 28,000 human, 16,000 mouse lncRNA targets.


RT2 lncRNA assays: Assays laboratory-verified for optimal qPCR performance — high specificity, amplification efficiency, and sensitivity.


RT2 lncRNA qPCR Arrays: Pathway or disease relevant lncRNA assays
RT2 lncFinder PCR Array (Human and Mouse)


Custom option: Flexible custom design from the lncRNA database and qPCR database to profile mRNA and lncRNA simultanously.


lncRNA isolation: miRNeasy kits or exoRNeasy kits


Data analysis: free on-line data anlysis tool

http://www.qiagen.com/us/landing-pages/lncrna/ Sample to Insight Noncoding RNAs in Neurodegeneration

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RT2 lncRNA qPCR Array layout and controls Flexible layout and patented controls Each 96-well plate  84 lncRNA-specific assays  5 reference genes (ACTB, B2M, RPL0, RNA7SK, SNORA73A)  1 genomic DNA  3 reverse transcription controls  3 PCR controls 96-well format

Arrays are also available in 384-well plates & 100-well ring discs for the Rotor-Gene Q. Free online analysis tool

384-well format: 96x4

Species

Number of qPCR assays designed (custom designs not included)

Human

42,096

Mouse

27,425

http://www.qiagen.com/us/landing-pages/lncrna/ Sample to Insight Noncoding RNAs in Neurodegeneration

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Custom lncRNA PCR arrays – Design your own panels

Email: [email protected]

Flexibility

Cross product

Custom array (modification)

1-4 genes

mRNA & lncRNA mix ok

Custom array

Flexibility with format limitation

mRNA & lncRNA mix ok

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Experiment design: detect and profile lncRNAs 1. Isolate total RNA

Samples:  Control: healthy donor samples

2. cDNA synthesis: convert RNA to cDNA

 Patient sample

1. Total RNA isolation 3. Pre-amplification with RT2 PreAMP cDNA kit

2. cDNA synthesis: convert to total RNA to cDNA using RT2 cDNA Synthesis Kit

3. Pre-amplification: Pre-amplify the target lncRNAs with preamplification RT2 PreAMP Primer Mixes

4.

4. Run PCR: detect lncRNAs with RT-PCR

5.



Human RT2 lncRNA PathwayFinder PCR Array



Master mix: RT2 SYBR® Green qPCR Mastermix



qPCR cyclers: any instrument , such as RotorGene-Q, ABI 7900HT

5. Data analysis: GeneGlobe Data Analysis Center

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RT2 lncRNA PCR Arrays Compatible with a wide range of qPCR instruments

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We provide service – send samples to us & receive results .

Whole Genome  

.

Pathway / Focused Panel      

.

Mutation Detection Methylation qPCR NGS

Sample Preparation – DNA, RNA extraction & purification   

.

Mutation Profiling Methylation PCR Array lncRNA PCR Array miRNA PCR Array NGS

Individual Gene / Locus    

.

Illumina Gene Expression Profiling Illumina Genotyping

Cells, Tissue or Biofluids Fixed Tissue Small Sample

http://www.qiagen.com/products/catalog/services/

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Solutions at QIAGEN Non-coding RNA Expression 

miScript miRNA PCR Arrays & Assays



RT2 lncRNA PCR Arrays & Assays



Custom PCR Arrays

Protein 

EpiTect ChIP Arrays



ELISArray kits

Functional Studies 

Cignal Reporter Assays



shRNA & siRNA

Service Solutions

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Thank you for attending

Questions?

Thank you for attending today’s webinar! Contact QIAGEN Call: 1-800-426-8157 Email: [email protected]

Wei Cao, Ph.D. [email protected]

Sample to Insight Noncoding RNAs in Neurodegeneration

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Welcome to our 3-part webinar series on neurodegeneration Neurodegenerative disorders: molecular mechanisms and circulating biomarker discovery – a 3-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

Sample to Insight Noncoding RNAs in Neurodegeneration

36