Common molecular mechanisms and pathways leading to neurodegeneration, such as Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease or Multiple Sclerosis, are presented in this slideshow. Learn more about research and therapeutic strategies as well as how these discoveries and tools can be used to facilitate your neurodegeneration research.

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Molecular Mechanisms of Neurodegeneration
Wei Cao, Ph.D.
Wei.Cao@qiagen.com
Molecular Mechanisms of Neurodegeneration 1
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Welcome to our three-part webinar series on neurodegeneration
Molecular Mechanisms of 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

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Legal disclaimer
Molecular Mechanisms of 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
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Signaling pathways in neurodegeneration4
Agenda
Molecular mechanisms of neurodegeneration1
Pathways that clean up proteins2
Aging – the greatest risk factor3
4
QIAGEN research solutions5

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Neurodegeneration
Molecular Mechanisms of 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

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Characteristics of neurodegenerative disorders
Molecular Mechanisms of Neurodegeneration 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

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Mutant genes causing neurodegeneration
Molecular Mechanisms of 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

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Signaling pathways in neurodegeneration4
Agenda
Molecular mechanisms of neurodegeneration1
Pathways that clean up proteins2
Aging – the greatest risk factor3
8
QIAGEN research solutions5

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Two pathways that clean up proteins
Molecular Mechanisms of Neurodegeneration 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

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Autophagy in neurodegenerative disorders
Molecular Mechanisms of Neurodegeneration 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 Function
ULK1,2 Kinase
Atg2A,B Atg9/Atg2-Atg18 complex
Atg4A,B,C,D E2-like enzyme
Atg5 Hydrolases
Beclin-1 E3-like activity
Atg7 Regulator
MAP1LC3A,B,C E1-like enzyme
GABARAP, L1,L2 Ubiquitin-like modifiers and regulators
Atg9A,B Atg9/Atg2-Atg18 complex
Atg10 E2-like enzyme
Atg12 Modifier
Atg13 Regulator
Atg14 Regulator
Atg16L1,2 E3-like activity
RB1CC1 Regulator
WIPI-1 Atg9/Atg2-Atg18 complex
Human autophagy-related genes

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Apoptosis in neurodegenerative disorders
Molecular Mechanisms of Neurodegeneration 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

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Signaling pathways in neurodegeneration4
Agenda
Molecular mechanisms of neurodegeneration1
Pathways that clean up proteins2
Aging – the greatest risk factor3
12
QIAGEN research solutions5

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Aging – the greatest risk factor
Molecular Mechanisms of Neurodegeneration 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

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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 oxidative
damage to mtDNA
HD Huntingtin
Reduces respiratory chain complex
function, generating ROS

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Aging – DNA damage repair
Molecular Mechanisms of Neurodegeneration 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/

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

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Signaling pathways in neurodegeneration4
Agenda
Molecular mechanisms in neurodegeneration1
Pathways that clean up proteins2
Aging – the greatest risk factor3
17
QIAGEN research solutions5

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Download signaling pathway maps for free
Molecular Mechanisms of Neurodegeneration 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

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Other key signaling pathways in neurodegeneration
Molecular Mechanisms of 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)

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Neurodegeneration is a novel class of development disorders
Molecular Mechanisms of Neurodegeneration 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

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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
Molecular Mechanisms of Neurodegeneration

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microRNAs in neurodegenerative disorders
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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 disorders
Disorders 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
Molecular Mechanisms of Neurodegeneration

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Signaling pathways in neurodegeneration4
Agenda
Molecular mechanisms in neurodegeneration1
Pathways that clean up proteins2
Aging – the greatest risk factor3
23
QIAGEN research solutions5

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Research solutions at QIAGEN
24
Visit QIAGEN’s Neurodegenerative Disease Research Portal
https://www.qiagen.com/us/products/life-science-research/neurodegenerative-disease/
Molecular Mechanisms of Neurodegeneration

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Research solutions at QIAGEN
Title, Location, Date 25
 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

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Gene expression at mRNA level – pathway-focused solutions
Title, Location, Date 26
 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

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RT2 Profiler PCR Array introduction
Molecular Mechanisms of Neurodegeneration 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

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PCR arrays are available for 14 species!
Molecular Mechanisms of Neurodegeneration 28
 Human
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 Chinese hamster ovary (CHO)
 C. elegans
Browse by species: https://www.qiagen.com/us/products/genes and pathways/species-portal/

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Custom PCR arrays
Molecular Mechanisms of Neurodegeneration 29
 RNA-seq verification
 Biomarker discovery
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 Bioprocessing optimization
 Biological response investigation
 Interspecies study (14 species available)
Email: BRC.Custom@qiagen.com

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miRNA expression — miScript miRNA PCR Arrays
Molecular Mechanisms of Neurodegeneration 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

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DNA methylation screening – methylation PCR arrays
Molecular Mechanisms of Neurodegeneration 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

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ChIP PCR Arrays – protein and DNA interactions
Molecular Mechanisms of Neurodegeneration 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

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qBiomarker Copy Number PCR Arrays
Molecular Mechanisms of Neurodegeneration 33
qBiomarker Copy Number PCR Arrays
Focused CNV analysis for any real-time instrument
 Alzheimer’s disease
 Intellectual disability
 Glioma
 Birth defects
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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

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Mutation detection – qBiomarker Somatic Mutation PCR Arrays
Molecular Mechanisms of Neurodegeneration 34
 PI3K-Akt pathway
 Receptor tyrosine kinases (RTKs)
 Brain cancer
http://www.qiagen.com/search/qbiomarker-somatic-mutation-pcr-arrays
Workflow

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ELISArrays – Multi-Analyte ELISArray Kits
Molecular Mechanisms of Neurodegeneration 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

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We provide service – send samples to us and receive results
Molecular Mechanisms of Neurodegeneration 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
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. Sample preparation – DNA / RNA extraction and
purification
 Cells, tissue or biofluids
 Fixed tissue
 Small sample
http://www.qiagen.com/products/catalog/services/

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Solutions at QIAGEN
Title, Location, Date 37
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
 qBiomarker Copy Number PCR Arrays
Protein
 EpiTect ChIP Arrays
 ELISArray kits
Functional studies
 Cignal Reporter Assays
 shRNA and siRNA
Service Solutions

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Email: BRCsupport@QIAGEN.com
Wei Cao, Ph.D.
Wei.Cao@QIAGEN.com
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Molecular Mechanisms of Neurodegeneration
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