molecular mechanisms of neurodegeneration: neurodegenerative disorders webinar series part 1

Sample to Insight

Molecular Mechanisms of Neurodegeneration

Molecular Mechanisms of NeurodegenerationWei Cao, Ph.D.

[email protected]

1

Contact Technical Support:

[email protected]

Webinar-related questions: [email protected]

Sample to Insight


Welcome to our three-part webinar series on neurodegeneration

2

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

Sample to Insight


Legal disclaimer

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.

Sample to Insight

4

Signaling pathways in neurodegeneration4

Agenda

Molecular mechanisms of neurodegeneration1

Pathways that clean up proteins2

Aging – the greatest risk factor3

QIAGEN research solutions5

Sample to Insight


Neurodegeneration

5

Neurodegeneration is featured by progressive dysfunction and death of cells in selected areas of the nervous system

Alzheimer’s disease (AD) Parkinson’s disease (PD) Huntington’s disease (HD) Amyotrophic lateral sclerosis (ALS)

Common molecular mechanisms Abnormal protein assemblies (protein misfolding)

Late-life cell death in adulthood

Oxidative stress

Inflammation – induced neurotoxicity

Sample to Insight


Characteristics of neurodegenerative disorders

6

Lars Bertram and Rudolph E. Tanzi, “The genetic epidemiology of neurodegenerative disease”, 2005, J Clin Invest. 115(6): 1449

Selected areas of change: macroscopic and microscopic

Alzheimer’s disease

Parkinson’s disease

Amyotrophic lateral sclerosisHuntington’s disease

Lewy body dementia

Frontotemporal dementia

Sample to Insight


Mutant genes causing neurodegeneration

7

Lars Bertram and Rudolph E. Tanzi, “The genetic epidemiology of neurodegenerative disease”, 2005, J Clin Invest. 115(6): 1449

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

Sample to Insight

8


Agenda





Sample to Insight


Two pathways that clean up proteins

9

1. Ubiquitin – proteasome

The primary route cells use to degrade proteins

Decreased proteasome activity is observed in neurodegeneration

Mutation of a deubiquitinating enzyme (ubiquitin carboxyl-terminal hydrolase, UCHL1) has been reported in PD

Cells maintain healthy mitochondria by degrading damaged mitochondria through mitophagy; defective mitophagy is linked to PD. USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy driven by the ubiquitin ligase Parkin (Parkin ubiquitinates and tags damaged mitochondria for clearance) and protein kinase PINK1, which are encoded by two genes associated with Parkinson’s disease

2. Autophagy – lysosome

Used proteins are poor proteasome substrates

Bingol, B. et al. “The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy”, 2014 Nature, 510: 370

Sample to Insight


Autophagy in neurodegenerative disorders

10

Kesidou, E. et al. “Autophagy and neurodegenerative disorders”, 2013, Neural Regen Res. 2:2275

Essential for neuronal survival, autophagy removes damaged organelles and misfolded

proteins

Excessive or insufficient autophagic activity in neurons causes neurodegeneration

Appropriate autophagy (“self-eating”)

Autophagy – lysosomal activities serve the essential function in neurodegenerative diseases

Autophagy gene FunctionULK1,2 KinaseAtg2A,B Atg9/Atg2-Atg18 complexAtg4A,B,C,D E2-like enzymeAtg5 HydrolasesBeclin-1 E3-like activityAtg7 RegulatorMAP1LC3A,B,C E1-like enzymeGABARAP, L1,L2 Ubiquitin-like modifiers and regulatorsAtg9A,B Atg9/Atg2-Atg18 complexAtg10 E2-like enzymeAtg12 ModifierAtg13 RegulatorAtg14 RegulatorAtg16L1,2 E3-like activityRB1CC1 RegulatorWIPI-1 Atg9/Atg2-Atg18 complex

Human autophagy-related genes

Sample to Insight


Apoptosis in neurodegenerative disorders

11

Crosstalk between autophagy and apoptosis

Autophagy and apoptosis can be triggered by common upstream signals

Cells switch between the two responses in a mutually exclusive manner

Autophagy constitutes a stress adaptation that avoids cell death and suppresses apoptosis, however it may also exist as an alternative cell-death pathway

Autophagy and apoptosis modulation has potential as a treatment strategy in neurodegenerative diseases Caspase-dependent and caspase-

independent routes to cell death

Ghavami, S., et al. “Autophagy and apoptosis dysfunction in neurodegenerative disorders”, 2014, Progress in Neurobiology 112:24 Maiuri, M.C., et al. “Self-eating and self-killing: crosstalk between autophagy and apoptosis”, 2007, Nature Reviews, 8:741

Cell death is one of the more common mechanisms in neurodegeneration. Programmed cell death (apoptosis) regulates the number and types of cells in brain development, making it crucial for normal neural development

Sample to Insight

12


Agenda





Sample to Insight


Aging – the greatest risk factor

13

The greatest risk factor for neurodegeneration is aging

Genes that mediate oxidative stress responses and DNA damage repair constitute the largest classes of genes upregulated in aging

Sample to Insight

Aging – oxidative stress and mitochondrial dysfunction

Title, Location, Date 14

Mitochondrial dysfunction is related to the aging process and neurodegenerative disorders through accumulation of mtDNA mutations and the generation of ROS

Mitochondria are highly abundant in brain cells and are both generators and targets of reactive species. Oxidative stress is inseparably linked to mitochondrial dysfunction

Mitochondrial turnover is dependent on autophagy, which declines with age and is

frequently dysfunctional in neurodegeneration. mTOR functions as an inhibitor of the

initiation step of autophagy

electron transport chains

Disease Major effective molecule Function

AD β-Amyloid Disrupts BER pathways, alters calcium homeostasis

PD Parkin Causes defective assembly of the OXPHOS complex

ALS SOD1 Binds to VDAC1, increasing oxidativedamage to mtDNA

HD Huntingtin Reduces respiratory chain complexfunction, generating ROS

Sample to Insight


Aging – DNA damage repair

15

Mutations in DNA surveillance and

DNA repair enzymes are associated

with neurodegeneration

The precise roles of DNA damage

response in the nervous system are

poorly understood

DNA damage repair is regulated

through autophagy

“The role of mitochondrial DNA mutation on neurodegenerative diseases”: http://www.ncbi.nlm.nih.gov/pubmed/25766619“The role of DNA repair in brain related disease pathology” : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720728/

Read review article “DNA damage repair — regulation through autophagy”https://www.qiagen.com/us/spotlight-pages/newsletters-and-magazines/articles/reviews-online-dna-damage-repair/

Sample to Insight


PI3-K / Akt / mTOR signaling pathways in aging and neurodegeneration

16

PI3-K / Akt / mTOR are therapeutic targets – may serve as essential factors for neuroprotection Cell death pathways, apoptosis and autophagy are important targets when considering

therapeutic strategies that involve PI3-K, Akt and mTOR signaling

PI3-K, Akt and mTOR pathways are tightly linked and involved in cellular senescence, aging and age-dependent diseases

mTOR is expressed at high levels in the brain

PI3-K / Akt / mTOR signaling affects a large number of cellular processes that regulate neuronal functions

Regulation of glucose metabolism Regulation of autophagy Control of mitochondrial oxidative function Modulation of lifespan – aging

Perluigi, M. et. al. “mTOR signaling in aging and neurodegeneration: At the crossroad between metabolism dysfunction and impairment of autophagy”, 2015, Neurobiology of Disease

Potential therapeutic targets

Sample to Insight

17


Agenda

Molecular mechanisms in neurodegeneration1




Sample to Insight


Download signaling pathway maps for free

18

PI3k signaling

Download over 500 signaling pathway maps at QIAGEN’s website

https://www.qiagen.com/us/products/genes and pathways/pathway central/

Akt signaling mTOR signaling Apoptosis

GeneGlobe Pathway Central

Sample to Insight


Other key signaling pathways in neurodegeneration

19

Wnt

Notch

NF-κB

CREB

Wnts are constantly released in the brain to maintain basal neural activity.

"Wnts play a central role in the formation and function of neuronal circuits." (J. Mol. Cell Biol. 2014; 6: 75)

"Deregulation in Wnt signaling might have a fundamental role in the origin of neurological diseases, by altering the synaptic function at stages where the phenotype is not yet established but when the cognitive decline starts" (Front. Cell. Neurosci. 2013; 7: 1)

Sample to Insight


Neurodegeneration is a novel class of development disorders

20

Neurodegenerative diseases may represent fundamental disorders of neurogenesis and neural development

Adult neurogenesis (the emergence of new neurons in the mature brain) is altered in neurodegeneration

Signaling pathways regulate neurogenesis Wnt Notch Hedgehog Growth and neurotrophic factors BMP (bone morphogenetic proteins) Neurotransmitters Transcription factors Epigenetic modulators

Faigle R., et al. “Signaling mechanisms regulating adult neural stem cells and neurogenesis”, 2013, Biochim Biophys Acta.1830:2435

Sample to Insight

Inflammation in neurodegeneration – neuroinflammation

21

Inflammation is linked to many neurodegenerative diseases Alzheimer's disease Parkinson's disease ALS (amyotrophic lateral sclerosis) Huntington’s disease (HD)

Factors of neuroinflammation Inducers Sensors Transducers Effectors

Signaling pathways and Cytokines: TNF-α, Interleukin-1, 6 and 10, TGF-β MAPK, NF-κB, PPAR-γ

A remarkable convergence exists between the mechanisms responsible for the sensing, transduction and amplification of inflammatory responses that result in the production of neurotoxic mediators Glass, C.K., et al. “Mechanisms Underlying Inflammation in Neurodegeneration”. Cell 2010;140: 918


Sample to Insight

microRNAs in neurodegenerative disorders

22

MicroRNA (miRNA) biogenesis pathway miRNAs Highly expressed in the CNS, including the brain

and spinal cord Key modulators of both CNS development and

plasticity Have been implicated in neurodegenerative

disorders Represent a novel class of therapeutic targets for

neurodegenerative disordersDisorders Implicated miRNAs Potential Targets

AD miR-29a, miR-29b-1↓, miR-107↓, miR-298, miR-328↓ BACE1

AD miR-146a↑ complement factor H

AD miR-20a, miR-17-5p and miR-106b, miR-101 APP

AD miR-15a, miR-195 and miR-497 (only miR-15a decreased in AD) ERK1

PD miR-133b↓ PITX3

PD miR-433 FGF20

PD miR-7↓ αSYN

HD miR-9, miR-9*↓ REST, CoREST (RCOR1)

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


Sample to Insight

23


Agenda

Molecular mechanisms in neurodegeneration1




Sample to Insight

Research solutions at QIAGEN

24

Visit QIAGEN’s Neurodegenerative Disease Research Portal

https://www.qiagen.com/us/products/life-science-research/neurodegenerative-disease/


Sample to Insight

Research solutions at QIAGEN


Gene expression – RT2 Profiler PCR Arrays miRNA regulation – miScript miRNA PCR Arrays DNA methylation – EpiTect DNA Methyl qPCR

Arrays DNA mutation detection – EpiTect ChIP qPCR

Arrays Copy number alteration – qBiomarker Copy

Number PCR Arrays Somatic mutation – qBiomarker Somatic Mutation

PCR Arrays ELISArray Cell-based reporter assays siRNA and shRNA mutation

Sample to Insight

Gene expression at mRNA level – pathway-focused solutions


Oxidative stress Unfolded protein response (ER stress) DNA damage Aging Autophagy Apoptosis Synaptic plasticity Alzheimer’s disease Parkinson’s disease Huntington’s disease Multiple sclerosis Mitochondria Neurogenesis Neuronal ion channels Neurotransmitters and receptors Neurotrophins Dopamine and serotonin pathways GABA and glutamate Pain – neuropathic and inflammatory Immunotoxicity Phagocytosis

http://www.qiagen.com/RT2-Profiler

Key signaling pathways

Cellular senescence p53 signaling PI3K-AKT signaling mTOR signaling TGFβ / BMP signaling Hedgehog signaling Notch signaling WNT signaling

RT2 Profiler PCR Array System

Sample to Insight


RT2 Profiler PCR Array introduction

27

Pathway-specific genes of

interest (84)

Housekeeping genes (5)

Genomic DNA contamination

control (GDC)

Reverse transcription

controls (RTC) (3)

Positive PCR controls (PPC)

(3)

B2M, HPRT, RPL13A, GAPDH, HGDC

Total RNA extraction cDNA mix with master-mix / load the plate run qPCR data analysis

Sample to Insight


PCR arrays are available for 14 species!

28

Human Mouse Rat Rhesus macaque Drosophila Dog Pig

Cow Chicken Horse Zebrafish Rabbit Chinese hamster ovary (CHO) C. elegans

Browse by species: https://www.qiagen.com/us/products/genes and pathways/species-portal/

Sample to Insight


Custom PCR arrays

29

RNA-seq verification Biomarker discovery CRISPR / RNAi knockdown verification Bioprocessing optimization Biological response investigation Interspecies study (14 species available)

Email: [email protected]

Sample to Insight


miRNA expression — miScript miRNA PCR Arrays

30

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

Sample to Insight


DNA methylation screening – methylation PCR arrays

31

EpiTect Methyl qPCR Array workflow

Apoptosis

Cell cycle

DNA repair

Wnt signaling

Notch signaling

Mental disorders

EpiTect DNA Methyl PCR Arrays

Analyze 22 or 94 genes per sample without bisulfite conversion

http://www.qiagen.com/products/catalog/assay-technologies/epigenetics/epitect-methyl-ii-pcr-arrays

Sample to Insight


ChIP PCR Arrays – protein and DNA interactions

32

Apoptosis

Inflammatory responses

DNA damage signaling pathway

TGFb signaling pathway

Wnt signaling pathway

Notch signaling pathway

http://www.qiagen.com/search/epitect-chip-qpcr-arrays

EpiTect ChIP qPCR Arrays

Simultaneous analysis of 84 pathway-focused genes

Requires as few as one million cells per array

Streamlined procedure, ready to use on most cyclers

Pathway-focused PCR array

Sample to Insight


qBiomarker Copy Number PCR Arrays

33


Focused CNV analysis for any real-time instrument

Alzheimer’s disease

Intellectual disability

Glioma

Birth defects

Autism spectrum disorders

Schizophrenia

http://www.qiagen.com/products/catalog/assay-technologies/real-time-pcr-and-rt-pcr-reagents/qbiomarker-copy-number-pcr-arrays

qBiomarker Copy Number PCR Array layout

Sample to Insight


Mutation detection – qBiomarker Somatic Mutation PCR Arrays

34

PI3K-Akt pathway

Receptor tyrosine kinases (RTKs)

Brain cancer

http://www.qiagen.com/search/qbiomarker-somatic-mutation-pcr-arrays

Workflow

Sample to Insight


ELISArrays – Multi-Analyte ELISArray Kits

35

Analysis of multiple cytokines or chemokines

You can measure 12 cytokines or chemokines for six samples at same time!

http://www.qiagen.com/products/catalog/assay-technologies/protein-and-cell-assays/multi-analyte-elisarray-kits

Sample to Insight


We provide service – send samples to us and receive results

36

. Whole genome Illumina Gene Expression Profiling Illumina Genotyping

. Pathway-focused panel Mutation profiling Methylation PCR Array miRNA PCR array NGS

. Individual gene / locus Mutation detection Methylation qPCR NGS

. Sample preparation – DNA / RNA extraction and purification Cells, tissue or biofluids Fixed tissue Small sample

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

Sample to Insight

Solutions at QIAGEN


Gene expression RT2 Profiler PCR Arrays and Assays

Custom PCR Arrays

Epigenetics miScript miRNA PCR Arrays and Assays

EpiTect Methyl qPCR Arrays and Assays

qBiomarker Somatic Mutation PCR Arrays


Protein EpiTect ChIP Arrays

ELISArray kits

Functional studies Cignal Reporter Assays

shRNA and siRNA

Service Solutions

Sample to Insight

38

Thank you for attending today’s webinar!

Contact QIAGENCall: 1-800-426-8157

Email: [email protected]

Wei Cao, [email protected]

Questions?

Thank you for attending


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.

molecular mechanisms of neurodegeneration: neurodegenerative disorders webinar series part 1

Healthcare