Antibody-based tools for neurodegenerative disease research

64 Pages·2015·18.88 MB·English

neuroscience research

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fHoandrb onok eurodegenerative

disease research
C ontents
1

Introduction 1-1 — 1-4
2

Common themes in neurodegenerative disease 2-1 — 2-13
3

Focus on Alzheimer’s disease 3-1 — 3-21
4

Other neurodegenerative diseases 4-1— 4-18
5

Summary 5-1 — 5-5
1
Overview of neurodegenerative
diseases
Introduction
Neurodegeneration is a general term referring to
neurons that develop a loss of cellular structure
or function.1 The most common examples of
neurodegenerative diseases include Alzheimer’s
disease (AD), Parkinson’s disease (PD), Huntington’s
disease, and amyotrophic lateral sclerosis (ALS).
Neurodegenerative diseases (NDs) are debilitating, and
the global numbers of affected individuals continue to
rise sharply.2,3 Most current therapies target the specific
neurons and transmitter systems that are destroyed
during the early stages of disease.
Some examples include acetylcholinesterase inhibitors, which increase acetylcholine levels in AD, and
dopaminergic agents for PD. At best, current therapies may alleviate symptoms, but do not address
the underlying pathology and do not significantly modify or delay the disease course. Therefore,
there is a pressing need to develop new therapies that could lead to neuronal protection and
regeneration. For example, cell replacement therapies, such as those based on human embryonic
stem cells (ESCs) or induced pluripotent stem cells (iPSCs), have the potential to successfully treat
various NDs. Additionally, strategies that modulate neuroinflammatory responses may provide new
treatment options because neuroinflammation fundamentally contributes to the physiopathology of
neurodegenerative disorders.3,4
The aims of this handbook are to provide an overview of NDs and to introduce information
about new and emerging technologies and antibody-based tools that can enable and further
neuroscience research.
Contents
Overview of neurodegenerative diseases 1-2
Antibodies: powerful tools for neurodegenerative disease research 1-3
References 1-4
For Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. Find out more at thermofisher.com/antibodies
1
Overview of neurodegenerative
diseases
General statistics Risk factors
Millions of people across the globe have some form of Genetic factors are attributable to early onset, familial,
ND.5 Both AD and PD have the highest prevalence, with and sporadic forms of degenerative disorders that affect
over five million cases and up to one million cases in the the nervous system. Inherited monogenic mutations may
United States, respectively (Table 1.1). The number of be expressed in either a dominant or recessive fashion
patients with some form of a neurodegenerative condition and are predictive of disease onset for familial forms of
continues to rise. For example, the prevalence of AD in NDs, whereas other genetic mutations may predispose to
people 65 years of age or older is expected to triple by the sporadic disease development. For example, mutations
year 2050.6-16 in synuclein, alpha (non A4 component of amyloid
precursor) (SNCA), the gene that encodes alpha-syncluclein
Table 1.1. 2015 Prevalence of neurodegenerative diseases worldwide (α-synuclein) protein, are associated with PD, while various
and within the US. disruptions in the amyloid beta (A4) precursor protein
(APP) gene are linked with different forms of AD. Table 1.2
Disease Statistics
provides representative examples of genes associated with
Alzheimer’s disease 44 million people worldwide
various NDs. Multiple different risk factors, both genetic
5.2 million people in the US
and environmental, are associated with the onset of NDs
Parkinson’s disease 7–10 million people worldwide
(Figure 1.1). Additionally, advancing age is another risk factor
Up to 1 million people in the US
linked to the development of many NDs, possibly due to
Huntington’s disease 10.6–13.7 people per 100,000 in Western
the accumulation of exposure to environmental factors and
populations
30,000 people in the US a decrease in the function of cellular repair mechanisms.
Amyotrophic lateral 6 people per 100,000 total population Medical conditions, such as cardiovascular disease and
sclerosis >20,000 people in the US injuries resulting in head trauma, may also increase the risk
Multiple sclerosis 2.5 million people worldwide for developing AD, while smoking—and certain infectious
400,000 people in the US agents—may increase the risk for MS.17-20
Risk factors
Inflammation
Genetic Environment
Aging Oxidative stress
risk factors risk factors
Growth factor loss Mood
disturbance
Protein aggregation Motor
DNA damage disturbance
Monogenic mutations Mitochondrial dysfunction Neurodegeneration
Apoptosis
Excitotoxicity Cognitive
impairment
Sensory
disturbance
Figure 1.1. Many neurodegenerative diseases share common risk factors and mechanisms that predispose patients to disease development.
1-2 Antibody-based tools for neurodegenerative disease research | Introduction
1
Antibodies:
powerful tools for neurodegenerative
disease research
Antibodies are used for multiple applications, including
immunofluorescence imaging, immunohistochemistry, western
blotting, ELISA, flow cytometry, and other applications. Our
antibodies are validated by many citations worldwide, helping
you achieve superior experimental results. To access our
antibody search tool and find the right antibody for your
research, go to, thermofisher.com/neuroantibodies
Table 1.2. Representative genes and gene products associated with NDs.
Gene
Disease Target Antibody Cat. No. Representative publications referencing our antibodies
symbol
AD APP Amyloid Amyloid Precursor Protein Brodney MA, Barreiro G, Ogilvie K, et al. (2012) Spirocyclic sulfamides as
Precursor Monoclonal Antibody (mAβP2-1) β-secretase 1 (BACE-1) inhibitors for the treatment of Alzheimer’s disease:
Protein (OMA103132) utilization of structure based drug design, WaterMap, and CNS penetration
studies to identify centrally efficacious inhibitors. J Med Chem 55(21):9224–
9239.
PS1 Presinilin 1 Presenilin 1 Monoclonal Antibody Shimada IS, LeComte MD, Granger JC, et al. (2012) Self-renewal and
(APS 18) differentiation of reactive astrocyte-derived neural stem/progenitor
(MA1-752) cells isolated from the cortical peri-infarct area after stroke. J Neurosci
32(23):7926–7940.
PS2 Presinilin 2 Presenilin 2 Monoclonal Antibody Diehlmann A, Ida N, Weggen S, et al. (1999) Analysis of presenilin 1 and
(APS 26) presenilin 2 expression and processing by newly developed monoclonal
(MA1-754) antibodies J Neurosci Res 56(4):405–419.
PD SNCA Alpha- Alpha Synuclein Antibody (14H2L1), Coelho-Cerqueira E, Carmo-Gonçalves P, Pinheiro AS, et al. (2013)
synuclein ABfinity™ Rabbit Monoclonal α-Synuclein as an intrinsically disordered monomer--fact or artefact? FEBS J
(701085) 280(19):4915–4927.
SNCA Monoclonal Antibody Sánchez-Danés A, Richaud-Patin Y, Carballo-Carbajal I, et al. (2012) Disease-
(2B2D1, 2B2A11) specific phenotypes in dopamine neurons from human iPS-based models of
(MA5-15290) genetic and sporadic Parkinson’s disease. EMBO Mol Med 4(5):380–395.
ALS SOD1 Superoxide Superoxide Dismutase 1 Polyclonal Chen X, Choi IY, Chang TS, et al. (2009) Pretreatment with interferon-gamma
dismutase 1 Antibody protects microglia from oxidative stress via up-regulation of Mn-SOD. Free
(LF-PA0013) Radic Biol Med 46(8):1204–1210.
HD HD Huntingtin Huntingtin/Htt Neoepitope 552 Leyva MJ, Degiacomo F, Kaltenbach LS, et al. (2010) Identification and
Polyclonal Antibody evaluation of small molecule pan-caspase inhibitors in Huntington’s disease
(PA1-003) models. Chem Biol 17(11):1189–1200.
Huntingtin/Htt Neoepitope 513 Hermel E, Gafni J, Propp SS, et al. (2004) Specific caspase interactions and
Polyclonal Antibody amplification are involved in selective neuronal vulnerability in Huntington’s
(PA1-002) disease. Cell Death Differ 11(4):424–438.
Multiple different mutations in the Synuclein, alpha (non A4 component of amyloid
precursor) or SNCA gene encode proteins that oligomerize, agregate, and form inclusions
in the brain that are characteristically associated with PD. In addition to their role in PD,
α-Synuclein oligomers are also linked to NDs such as dementia with Lewy bodies.21
Alpha-synuclein (14H2L1), Rabbit Monoclonal ABfinityTM Antibody.
Immunohistochemistry analysis of paraffin-embedded human brain tissue with anti–alpha
synuclein antibody (Cat. No. 701085) showing staining in the cytoplasm (right) compared to
a negative control without primary antibody (left).
To discover our full lline of antibodies, ELISAs, and LuminexTM assay kits visit
thermofisher.com/antibodies
Antibody-based tools for neurodegenerative disease research | Introduction 1-3
1
References
1. Przedborski S, Vila M, Jackson-Lewis V (2003) Neurodegeneration: what is it and
where are we? J Clin Invest 111(1):3–10.
2. Davis M, Stroud C, eds (2013) Neurodegeneration: Exploring Commonalities Across
Diseases: Workshop Summary USA: National Academy of Sciences. p4-15. http://
www.nap.edu/catalog.php?record_id=18341. Accessed September 2015.
3. Aoun S, McConigley R, Abernethy A Currow DC et al. (2010) Caregivers of people
with neurodegenerative diseases: profile and unmet needs from a population-based
survey in South Australia. J Palliat Med 13(6):653–661.
4. Amor S, Puentes F, Baker D et al. (2010) Inflammation in neurodegenerative
diseases. Immunology 129(2):154–169.
5. Neurodegenerative Diseases. National Institute of Environmental Health Sciences.
https://www.niehs.nih.gov/research/supported/diseases/neurodegenerative/index.
cfm. Accessed July 2015.
6. Alzheimers.net 2015 Alzheimer’s Statistics. http://www.alzheimers.net/resources/
alzheimers-statistics/. Accessed July 2015.
7. Statistics on Parkinson’s. Parkinson’s Diseae Foundation http://www.pdf.org/en/
parkinson_statistics Accessed July 2015.
8. Bates GP, Dorsey R, Gusella J, et al. (2015) Huntington Disease. Nat Rev Dis
Primers 1:1–21.
9. Kinsley L, Siddique (2001) T Amyotrophic Lateral Sclerosis Overview. [Updated
2015 Feb 12]. In: GeneReviews™ [Internet]. Pagon RA, Adam MP, Ardinger HH et
al., editors. Seattle (WA): University of Washington, Seattle; 1993–2015.
10. Epidemiology of ALS and Suspected Clusters. ALS Association. http://www.alsa.
org/als-care/resources/publications-videos/factsheets/epidemiology.html. Accessed
July 2015.
11. Epidemiology of MS. MS International Federation. http://www.msif.org/research/
epidemiology-of-ms/. Accessed July 2015.
12. Tullman MJ (2013) Overview of the epidemiology, diagnosis, and disease
progression associated with multiple sclerosis. Am J Manag Care 2013;19(2
Suppl):S15–S20.
13. Facts You Should Know. ALS Association. http://www.alsa.org/about-als/facts-you-
should-know.html. Accessed July 2015.
14. What is ALS/MND? International Alliance of ALS/MND Associations. http://www.
alsmndalliance.org/what-is-alsmnd/. Accessed July 2015.
15. Huntington’s Disease. Remedy’s Health Communities.com. http://www.
healthcommunities.com/huntingtons-disease/overview-of-huntingtons.shtml.
Accessed July 2015.
16. 2015 Alzheimer’s Disease Facts and Figures. Alzheimer’s Association. https://www.
alz.org/facts/downloads/facts_figures_2015.pdf. Accessed July 2015.
17. Hindle JV (2010) Ageing, neurodegeneration and Parkinson’s disease. Age Ageing
39(2):156–161.
18. Risk Factors. Alz.org http://www.alz.org/alzheimers_disease_causes_risk_factors.
asp. Accessed July 2015.
19. Simon KC, Schmidt H, Loud S (2015) Risk factors for multiple sclerosis,
neuromyelitis optica and transverse myelitis. Mult Scler 21(6):703–709.
20. Van Deerlin VM (2012) The genetics and neuropathology of neurodegenerative
disorders: perspectives and implications for research and clinical practice. Acta
Neuropathol 124(3):297–303.
21. Klein C, Westenberger (2010) A Genetics of Parkinson’s disease. Cold Spring Harb
Perspect Med 2(1):a008888.
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1-4 Antibody-based tools for neurodegenerative disease research | Introduction
2
Common themes in
neurodegenerative disease
Introduction
Traditionally, neurodegenerative diseases (NDs) have been
researched as separate entities with differing etiologies
and pathologies. Recently, however, it has become clear
that certain molecular, cellular, and genetic mechanisms
likely span a variety of NDs. These findings suggest that
investigation into commonalities may not only provide
insight into basic processes, but also into shared
diagnostic biomarkers and therapeutic targets.1
In addition to similar features of neuronal dysfunction
across various brain regions, NDs may share specific
pathophysiological processes. For example, many of
these conditions involve the production, accumulation, and
decreased removal of misfolded proteins that culminate in
the appearance of insoluble plaques. Furthermore, common mechanisms of neuronal malfunction
and destruction occur in multiple diseases and manifest through inflammation, oxidative stress,
mitochondrial dysfunction, and enhanced excitotoxicity. (Table 2.1).2-12
Many decades of research have yielded current therapies that, in many cases, target the specific
neurons and transmitter systems that are destroyed during the course of these diseases. These
therapies include acetylcholinesterase inhibitors to increase levels of acetylcholine in Alzheimer’s
disease (AD), and dopaminergic agents for Parkinson’s disease (PD). At best, current therapies
alleviate or delay symptoms, but do not address the underlying pathology and do not modify or delay
the disease course. Moreover, current therapies do not preserve or restore neuronal function. In
contrast, emerging therapies in development are designed to target specific pathological processes
involved in neuronal destruction, and provide neuronal protection and regeneration. For example, stem
cell–based therapies, such as those using mesenchymal stem cells or cells derived from induced
pluripotent stem cells (iPSCs), and immunotherapeutic strategies may lead the next generation of
modalities for treating a variety of NDs.
Contents
Overview of neurodegenerative diseases 2-2 Oxidative stress and neurodegeneration 2-9
Common themes in neurodegeneration 2-3 Emerging areas of neurodegenerative disease research 2-11
Protein misfolding and neurodegeneration 2-4 References 2-13
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For Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc.
2
Overview of neurodegenerative
diseases
Overview of neurodegenerative disease
Various defining features are associated with different NDs. For
example distinct regions of the brain may be involved and unique
cell types may play a role in the pathogenesis of different NDs.
For example, AD affects cholinergic neurons, PD is associated
by the degradation of dopaminergic neurons, and MS impacts
oligodendrocytes that form the myelin sheath surrounding neurons of
the CNS. Despite these disease-specific characteristics, NDs share
many common pathologies, such as protein misfolding, chronic
inflammation, and high levels of oxidative stress that induces cell
death. Table 2.1 provides an overview of features associated with
several neurodegenerative disorders.13–20
PSD95 Mouse Monoclonal Antibody (6G6-1C9).
Immunofluorescent analysis of U251 glioma cells with
anti-PSD95 antibody (Cat. No. MA1-045). Nuclei were
stained with DAPI (blue).
Table 2.1. Features associated with several neurodegenerative disorders.
Neurodegenerative Affected brain Cell types Protein components/
Defect Pathology
disease regions involved biomarkers
Alzheimer’s disease Progressive Hippocampus; cerebral Cholinergic Senile plaques, Aβ amyloid, tau, vimentin,
dementia cortex neurons; neurofibrillary actin, α-synuclein, TDP-43,
astrocytes; tangles, Lewy ubiquitin, and MAP2
microglial cells bodies, neuronal
inclusions
Parkinson’s disease Movement Substantia nigra; Dopamine Lewy bodies Cytoplasmic aggregates of
disorder hypothalamus producing neurons α-synuclein; may include other
proteins: Parkin (PARK2), DJ-1
gene product (PARK7), LRRK2
(PARK8; Dardarin)
Huntington’s disease Dementia, Striatum; cerebral GABAergic Neural inclusions Nuclear aggregation of
motor and cortex; thalamus neurons huntingtin (HTT); huntingtin-
psychiatric interacting protein (HIP)
problems
Amyotrophic lateral Movement Motor neurons of the Neurons; glial cells Neural inclusions Ubiquitin-positive, Tardbp-
sclerosis disorder cortex, brainstem, positive, intra-cytoplasmic
spinal cord; non- inclusions; FUS/TLS, SOD1
motor neurons may be
affected in later disease
Multiple sclerosis Sensory, Sensorimotor neurons Neurons; Axonal MBP, PLP, MOG, lesions/
motor, of the central nervous microglial cells, demyelinating proinflammatory cytokines
and visual system oligodendrocytes; lesions/plaques (i.e., IL-17)
disturbances lymphocytes; of immune cells
antigen-presenting
cells
2-2 Antibody-based tools for neurodegenerative disease research | Common themes in neurodegenerative disease
2
Common themes in
neurodegeneration
Select methods for evaluating biomarkers Obtaining human samples from the CNS to directly
and proteins visualize pathology presents major limitations; however,
Biomarker discovery is essential for developing strategies postmortem tissue assessment using histological and
that may lead to earlier detection of NDs and to novel immunohistological analysis of proteins associated with
therapies for treating these debilitating conditions. In AD and other NDs is commonly used to aid diagnoses,
addition, determining the localization and distribution and in vitro cellular experiments with primary neuronal
patterns of proteins within cells provides insight about cells and cell lines also lend well to experiments that
mechanisms that promote pathogenesis. A number of implement antibody-based tools.22 These tools are often
approaches are employed for evaluating biomarkers used for examination of surrogate compartments such as
that correlate with specific NDs and for elucidating the the CNS and for neural tissue analysis frequently included
cellular localization of proteins. Physical biomarkers may as experimental endpoints for characterizing models of
be categorized under brain imaging methodologies. neurodegeneration developed with a wide-range of different
Examples of techniques for identifying and quantitating species, including non-human primates and rodents. One
proteins present in blood and cerebrospinal fluid (CSF) major challenge with preclinical models is that they do not
include ELISA and various proteomics platforms such as fully recapitulate the complexities associated with human
Luminex.21 An example of a method that is increasingly used NDs; however use of experimental models has aided efforts
for identifying neuronal populations is immuno-electron to identify biomarkers, test agents, and cell-based therapies
microscopy (immuno-EM), which enables detection of designed to treat neurodegenerative conditions, and have
amino acids, peptides, and proteins. This technique involves provided insight into the cellular and molecular mechanism
a specialized tissue preparation protocol and coupling involved in ND-pathogenesis and progression.23
antibodies to electron-dense markers such as colloidal gold
particles (Table 2.2 and Figure 2.1).
Table 2.2. Select methods for evaluating neurodegenerative
proteinopathies.
Detection methods
Magnetic resonance imaging (MRI)
Positron emission tomography (PET) scan
Western blot
Histology
Immunohistochemistry
Immunofluorescence imaging
Immuno–electron microscopy (immuno-EM)
Enzyme-linked immunosorbent assay (ELISA) Figure 2.1. Immuno-EM may be employed to identify
Multiplex protein assays antigen-specific localization and distribution of antigens
in tissue. Post-embedding immuno-gold detection of vesicular
Mass spectrometry
glutamate transporter 1 (VGLUT1) in rat spinal cord section.
Note synaptic bouton with gold particles over synaptic vesicles,
presence of mitochondria, apposition with dendrite, and synaptic
active zone.
Antibody-based tools for neurodegenerative disease research | Common themes in neurodegenerative disease 2-3
2
Protein misfolding and
neurodegeneration
Mechanisms of protein misfolding Transmembrane stress sensors and downstream
Protein misfolding is a hallmark feature of many transcription factors comprise the UPR.26 Examples
neurodegenerative diseases, including AD, PD, HD, and of ER membrane proteins acting as associated stress
amyotrophic lateral sclerosis (ALS). Misfolded proteins sensors include: inositol-requiring transmembrane
generate signals to initiate a set of endoplasmic reticulum kinase/endoribonuclease 1 (IRE1), protein kinase–like
(ER) stress responses, collectively termed the unfolded eukaryotic initiation factor 2α kinase (PERK), and activating
protein response (UPR), which act to protect cells from the transcription factor-6 (ATF6).25 Once activated, these
detrimental buildup of proteins.12 Although many wild type proteins regulate multiple processes, including the rate of
proteins are capable of misfolding or forming aggregates, protein production, the expression of proteins that aid in
genetic mutation can result in the generation of a protein protein folding, the prevention of protein aggregation, and
with an altered amino acid sequence that enhances the the promotion of retrotranslocation and the degradation of
tendency of the protein to fold and aggregate. proteins produced in the ER (Table 2.3).12
For example, in AD, multiple different APP mutations
Table 2.3. Representative ER membrane stress sensors.
drive production of defective Aβ peptides that oligomerize
and contribute to fibril formation (Figure 2.2). Enhanced Antibody
Target Function
Cat. No.
aggregation may also result from overproduction of a
protein product, or increased metabolic, oxidative, and IRE1 Transcription of X-box-binding IRE1 alpha
inflammatory stress events. protein; promoter for genes Polyclonal
regulating ER retrotranslocation Antibody
and degradation of misfolded (PA1-16928)
An elegant mechanism of quality control for protein
proteins
processing, folding, and trafficking exists within the
PERK Phosphorylates eIF2α to prevent PERK Polyclonal
endoplasmic reticulum (ER) to prevent the buildup of
ER influx of pre-modified proteins Antibody
misfolded proteins. Foldases and molecular chaperones
(PA5-15305)
play an intimate role in this process. Misfolded proteins
ATF6 Transcription factor mediates ATF6 Polyclonal
may be retained within the ER, or may be broken down by upregulation of chaperone Antibody
autophagy or the proteasome-dependent ER-associated proteins (PA5-20215)
protein pathway.24,25 XPB1 Transcription factor for ER XPP-1 Monoclonal
stress-related proteins regulates Antibody (9B7E5)
ER retrotranslocation and (MA5-15768)
degradation of misfolded proteins
Alzheimerʼs Amyloid-β
disease
Native monomer
Parkinsonʼs α-synuclein
disease
Misfolding
Abnormal protein
Huntingtonʼs disease Huntingtin
misfolding and
(HD SCAs) ataxins
aggregation
β-sheet
Amyotrophic lateral TDP-43 oligomers
sclerosis SOD1
Neurodegeneration
Frontotemporal Amyloid fibrillar
Tau
lobar degeneration aggregates
Figure 2.2. Protein misfolding and neurodegeneration.
2-4 Antibody-based tools for neurodegenerative disease research | Common themes in neurodegenerative disease