The Central Roles of Non-coding RNAs in Neurodegenerative Disorders: Neurodegenerative Disorders Webinar Series Part 2
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This document presents a three-part webinar series on the role of non-coding RNAs (ncrnas) in neurodegenerative disorders, focusing on molecular mechanisms, the functions of miRNAs and lncRNAs, and the discovery of circulating biomarkers. It discusses the importance of ncrnas in the pathogenesis of diseases such as Alzheimer's, Parkinson's, and Huntington's, highlighting their regulatory roles in gene expression and potential as therapeutic targets. Additionally, the document outlines current approaches for studying and quantifying ncrnas to advance research in this field.
The Central Roles of Non-coding RNAs in Neurodegenerative Disorders: Neurodegenerative Disorders Webinar Series Part 2
- 1. Sample to Insight The Central Roles of Non-coding RNAs in Neurodegenerative Disorders Wei Cao, Ph.D. Wei.Cao@qiagen.com Noncoding RNAsin Neurodegeneration 1 Welcome! Contact Technical Support: BRCsupport@QIAGEN.COM 1-800-362-7737 Webinar-related questions: QIAwebinars@QIAGEN.com
- 2. Sample to Insight Welcome to our three-part webinar series on neurodegeneration Noncoding RNAsin Neurodegeneration 2 Neurodegenerative disorders: molecular mechanismsand 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 forAlzheimer’s Disease
- 3. Sample to Insight Legal disclaimer Noncoding RNAsin Neurodegeneration 3 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.
- 4. Sample to Insight The study of non-coding RNA: miRNA and lncRNA4 Agenda Molecular mechanisms of neurodegeneration1 Introduction to non-coding RNA2 Non-coding RNAin neural function and diseases3 4
- 5. Sample to Insight Molecular mechanisms of neurodegeneration Noncoding RNAsin Neurodegeneration 5 Neurodegeneration is featured by progressive dysfunction and death of cells in selected areas in the nervous system. 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
- 6. Sample to Insight 6 Non-coding RNA – the “dark matter” of the genome Over 90% of the human genome is actively transcribed, with only ~2% of the genome being protein-coding For a long time, little was known of the genome’s other functions, being referred to as “dark matter” or “junk” Non-coding RNAs: small non-coding RNAand long non-coding RNA Schw arzenbach, 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 Non-coding RNAs The proportion of non protein-coding sequences increases with complexity in eukaryotes Noncoding RNAsin Neurodegeneration
- 7. Sample to Insight 7 Short non-coding RNAs – microRNAs (miRNAs) Abundant, the most widely studied ncRNAs and roughly 21 nt in size Regulate a variety of cellular processes through post-transcriptional repression of gene expression Expression is frequently deregulated in various diseases, therefore has potential to serve as biomarkers Long non-coding RNAs (lncRNAs) Novel class of RNAs over 200 nucleotides in size Regulate protein-coding gene transcription in more complex ways than miRNAs Changes in lncRNA can be correlated with a variety of human diseases Schw arzenbach, 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 Non-coding RNAs – miRNA and lncRNA Noncoding RNAsin Neurodegeneration
- 8. Sample to Insight 8 Guil. S. and Esteller, M. (2015) “RNA–RNA interactions in gene regulation: the coding and noncoding players.” Trends in Biochemical Sciences. RNA–RNA interactions among different RNA species The central dogma and regulatory complexity One gene gives rise to one RNA to produce oneprotein. RNA has more versatile functions than its protein synthesis role RNAs regulate most cell processes, including epigenetic control, gene transcription, translation, RNAturnover, chromosomal organizationand genome defense, cellular developmental and proliferation programs RNAs always work through interactions with proteins. However, 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 more than 100 transcripts Control of splicing through direct base pairing with ncRNAs ncRNAs control translation lncRNA–miRNA interactions: lncRNAs regulate miRNA biogenesis, and miRNAs regulate lncRNAs Noncoding RNAsin Neurodegeneration
- 9. Sample to Insight miRNA biogenesis and regulatory mechanisms 9 MicroRNA (miRNA) biogenesis pathway Meza-Sosa KF, et al. “Role of microRNAs in central nervous system development and pathology”, J Neurosci Res, 2012; 90:1 Noncoding RNAsin Neurodegeneration Transcribedby RNApolymerase 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 the RNase III Drosha Pre-miRNAs are then exported to the cytosol by exportin 5 In the cytosol, the RNAse III 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
- 10. Sample to Insight miRNA biogenesis and regulatory mechanisms 10 MicroRNA (miRNA) biogenesis pathway Meza-Sosa KF, et al. “Role of microRNAs in central nervous system development and pathology”, J Neurosci Res, 2012; 90:1 miRNAs Highly expressed in the 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 Noncoding RNAsin Neurodegeneration Atx2
- 11. Sample to Insight miRNAs in neurodegenerative disorders 11 miRNAs have been implicated in neurodegenerative disorders Szafranski, K., et al. “Non-codingRNA in neuralfunction, disease, and aging”, Frontiers in genetics , March 2015 miRNAs As mediators of brain development and neuronal differentiation Playing roles in neuronal longevity and survival Regulating neurodegenerativedisease- associated pathways miRNA dysfunction downstreamof disease- linked TDP-43 alterations could represent an important pathogenic mechanism in neurodegenerativedisease Noncoding RNAsin Neurodegeneration
- 12. Sample to Insight The study of non-coding RNA: miRNA and lncRNA4 Agenda Molecular mechanisms of neurodegeneration1 Introduction to non-coding RNA2 Non-coding RNAin neural function and diseases3 12
- 13. Sample to Insight 13 lncRNAs are non-protein-codingtranscripts longer than 200 nucleotides in size Most lncRNAs are localized in the nucleus, but some are found in the cytoplasm Many lncRNAs are molecularly indistinguishable frommRNAs Although some lncRNAs (e.g., MALAT1) are highly abundant transcripts, many lncRNAs are less so. Low transcription levels do not necessarily reflect lack of functionality May contain a poly-A tail like mRNA lncRNAs are typically less conserved across species and often show low expression levels and high tissue specificity Schw arzenbach, 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 Introduction to long non-coding RNAs (lncRNAs) Noncoding RNAsin Neurodegeneration
- 14. Sample to Insight lncRNA classification and subgroup 14 Wu P. et al. “Roles of long noncoding RNAs in brain development, functional diversification and neurodegenerative diseases”, (2013). Brain Research Bulletin 97: 69 lncRNAs can exceed 100,000 nucleotides and cover a wide range of gene positions lncRNAs can be divided into three general categories: Transcribed relative to host protein-coding genes Transcribed from gene regulatory regions Transcribed from the specific chromosomal regions Intergenic Intronic Exonic Overlapping Sense Antisense Classified based on their relative position to PCG (protein-codinggenes) Noncoding RNAsin Neurodegeneration
- 15. Sample to Insight The molecularfunctions of lncRNAs 15 Martin L, Chang HY. (2012) “Uncovering the roleof genomic "darkmatter" in human disease”, J Clin Invest, 122 1589 Noncoding RNAsin Neurodegeneration
- 16. Sample to Insight The study of non-coding RNA: miRNA and lncRNA4 Agenda Molecular mechanisms of neurodegeneration1 Introduction to non-coding RNA2 Non-coding RNAin neural function and diseases3 16
- 17. Sample to Insight lncRNAs in the central nervous system (CNS) 17 The complex functions of lncRNAs coincide with the diversity and elaborate nature of the central nervous system 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 Noncoding RNAsin Neurodegeneration
- 18. Sample to Insight Dysregulated lncRNAs in neurodegenerative diseases 18 lncRNAs Associated disease Biological function BACE1-AS AD Increase BACE1 mRNA stability through a post-transcriptional feed-forward mechanism NAT-Rad18 AD Downregulate DNA repair protein Rad18, giving the neuron more sensitivity to apoptosis 17A AD Impair GABAB signalingpathway by decreasing GABAB R2 transcription GDNFOS AD Modulate the expression of endogenous GDNF in human brain Sox2OT AD, PD Regulate co-transcribed Sox2 gene expression to suppress neurogenesis 1810014B01Rik AD, PD Unknown BC200 AD, PD Modulate local proteins in postsynaptic dendritic microdomains to maintain long-term synaptic plasticity naPINK1 PD Stabilize svPINK1 resulting in disturbed mitochondrial respiratory chain, increase sensitivity to apoptosis HAR1F HD Aberrant nuclear-cytoplasmic REST / NRSF trafficking causedby 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 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 Noncoding RNAsin Neurodegeneration
- 19. Sample to Insight lncRNAs are essential for brain development 19 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/ Noncoding RNAsin Neurodegeneration
- 20. Sample to Insight lncRNAs in Alzheimer’s disease (AD) 20 Faghihi MA, et al (2008) Expression of a noncoding RNA is elevated in Alzheimer's disease and drives rapid feed-forward regulation of beta- secretase. 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 AD is the most common neurodegenerativedisorder 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 expressedin AD patients and directly implicated in the increasedabundance of Aβ42 in AD BACE1-AS increases BACE1 mRNA stability and generates additional Aβ42 through a post-transcriptional feed-forwardmechanism Noncoding RNAsin Neurodegeneration
- 21. Sample to Insight lncRNAs in Parkinson’s disease (PD) 21Noncoding RNAsin Neurodegeneration PD is the most frequent motor disorder, and deep-brain- stimulation (DBS) treatment alleviates symptoms RNAseq experiment: PD patients’leukocytes pre- and post-DBS treatment were compared to healthy controls 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
- 22. Sample to Insight lncRNAs alteration in Huntington's disease (HD) brains 22 Rory Johnson. “Long non-coding RNAs in Huntington's disease neurodegeneration”, 2012, Neurobiology of Disease. 46:245 Possible mechanism of lncRNAs in HD HD is caused by an expansion of a CAG triplet repeat stretch within the Huntington gene REST (RE1 Silencing Transcription Factor) is a target of the mutated Huntington gene Many lncRNAs are direct targets of and regulated by REST Noncoding RNAsin Neurodegeneration
- 23. Sample to Insight The study of non-coding RNA: miRNA and lncRNA4 Agenda Molecular mechanisms of neurodegeneration1 Introduction to non-coding RNA2 Non-coding RNAin neural function and diseases3 23
- 24. Sample to Insight Current lncRNA quantification approaches 24 Sample isolation Amplification qPCR Data analysis and interpretation miRNeasy exoRNeasy Serum / Plasma Kit miScript miRNA PreAMP PCR Kit RT² lncRNA PreAMP PCR Kit miScript miRNA PCR Array RT2 lncRNA PCR System Free data analysis tool RNAseq (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 Test and verify your hypothesis with miScript miRNAPCR Array and RT2 lncRNAqPCR Assays or Custom PCR Arrays Noncoding RNAsin Neurodegeneration
- 25. Sample to Insight Real-time PCR quantification of non-coding RNAs 25 The gold standard for gene quantification The method of choice to confirm next-generation sequencing and microarray results Simple and easy to carry out High sensitivity and specificity High throughput compatible, automatable Very low template amounts necessary Noncoding RNAsin Neurodegeneration
- 26. Sample to Insight Solutions to miRNA and lncRNA profiling and detection 26 How can you advance your non-coding RNA research? Noncoding RNAsin Neurodegeneration
- 27. Sample to Insight miRNA expression — miScript miRNA PCR Arrays 27 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 Noncoding RNAsin Neurodegeneration
- 28. Sample to Insight 28 lncRNA databases – world wide efforts Noncoding RNAsin Neurodegeneration
- 29. Sample to Insight The authoritative lncRNA databases 29 QIAGEN has merged these two databases together for the most up- to-date qPCR assaydesign Currently covers human GENCODE 19, mouse GENCODE M2 and RefSeqRelease 65 Noncoding RNAsin Neurodegeneration
- 30. Sample to Insight RT2 lncRNA qPCR system 30 lncRNAdatabases: in-house database at QIAGEN GeneGlobe covers over 28,000 human and 16,000 mouse lncRNAtargets RT2 lncRNAassays: laboratory-verifiedfor optimal qPCR performance – high specificity, amplification efficiency and sensitivity RT2 lncRNAqPCR Arrays: Pathway- or disease-relevant lncRNA assays RT2 lncFinder PCR Array (human and mouse) Custom option: flexible custom design from the lncRNAdatabase and qPCR database to profile mRNA and lncRNAsimultanously lncRNAisolation: miRNeasy kits or exoRNeasy kits Data analysis: free online data anlysis tool http://www.qiagen.com/us/landing-pages/lncrna/ Noncoding RNAsin Neurodegeneration
- 31. Sample to Insight RT2 lncRNA qPCR Array layout and controls 31 Flexible layout and patented controls Each 96-well plate contains: lncRNA-specific assays (84) Reference genes (5) (ACTB, B2M, RPL0, RNA7SK, SNORA73A) Genomic DNA control (1) Reverse transcription controls (3) PCR controls (3) Arrays are also available in 384-well plates and 100-well ring discs for the Rotor-Gene Q Free online analysis tool http://www.qiagen.com/us/landing-pages/lncrna/ Species Number of qPCR assays designed (custom designs not included) Human 42,096 Mouse 27,425 384-well format: 96x4 96-well format Noncoding RNAsin Neurodegeneration
- 32. Sample to Insight Custom lncRNA PCR arrays – design your own panels 32 Email: BRC.Custom@qiagen.com Flexibility Cross product Custom array (modification) One to four genes mRNA and lncRNA mix ok Custom array Flexibility with format limitation mRNA and lncRNA mix ok Noncoding RNAsin Neurodegeneration
- 33. Sample to Insight Experiment design: detect and profile lncRNAs 33 2. cDNAsynthesis: convert RNAto cDNA 3. Pre-amplification with RT2 PreAMP cDNAkit 1. Isolate total RNA 4. 5. Samples: Control: healthy donor samples Patient sample 1. Total RNA isolation 2. cDNA synthesis: convert to total RNA to cDNA using RT2 cDNA Synthesis Kit 3. Pre-amplification: pre-amplify the target lncRNAs with pre- amplification RT2 PreAMP Primer Mixes 4. Run PCR: detect lncRNAs with qPCR Human RT2 lncRNA PathwayFinder PCR Array Master mix: RT2 SYBR® Green qPCR Mastermix qPCR cyclers: any instrument, such as RotorGene-Q or ABI 7900HT 5. Data analysis: GeneGlobe Data Analysis Center Noncoding RNAsin Neurodegeneration
- 34. Sample to Insight RT2 lncRNA PCR Arrays 34 Compatible with a wide range of qPCR instruments Noncoding RNAsin Neurodegeneration
- 35. Sample to Insight We provide service – send samples to us and receive results 35 . Whole genome Illumina Gene Expression Profiling Illumina Genotyping . Pathway-focused panel Mutation profiling Methylation PCR array lncRNA PCR array miRNA PCR array NGS . Individual gene / locus Mutation detection Methylation qPCR NGS . Sample preparation – DNA/ RNAextraction and purification Cells, tissue or biofluids Fixed tissue Small sample http://www.qiagen.com/products/catalog/services/ Noncoding RNAsin Neurodegeneration
- 36. Sample to Insight Solutions at QIAGEN 36 Non-coding RNAexpression miScript miRNA PCR Arrays and Assays RT2 lncRNAPCR Arrays and Assays Custom PCR Arrays Protein EpiTect ChIP Arrays ELISArray kits Functional studies Cignal Reporter Assays shRNAand siRNA Service Solutions Noncoding RNAsin Neurodegeneration
- 37. Sample to Insight 37 Thank you for attending today’s webinar! Contact QIAGEN Call: 1-800-426-8157 Email: BRCsupport@QIAGEN.com Wei Cao, Ph.D. Wei.Cao@QIAGEN.com Questions? Thank you for attending Noncoding RNAsin Neurodegeneration 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.
Editor's Notes
- #12: Taken together, these studies identify miRNAs as crit- ical players in fundamental brain development processes such as neuronal differentiation. Thus, disruption of miRNA biogen- esis can compromise neurodevelopmental and neural differen- tiation processes that are known to occur at various stages of life. in the setting of neurodegenerative disease, loss of key homeostatic functions downstream of miRNA biogen- esis can induce non-cell autonomous neuronal degeneration miRNA dysfunction may trigger overt neuronal degeneration as well as more subtle deficits in the setting of neurodegenerative disease.
- #23: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146038/