In vivo rodent models in Parkinson's disease
research
Table of contents
1.
Introduction
1
2.
Pesticide induced models
2
3.
4.
5.
6.
Rotenone
2
Paraquat
2
Maneb
3
Pharmacological models
3
Reserpine
3
Haloperidol
3
Toxin induced models
4
6-OHDA
4
MPTP
4
Modeling α-syn pathology in vivo
5
PFFs
6
rAAV
6
Genetic models
7
Other genetic models
7
LRRK2
7
PINK1
8
Parkin
8
DJ-1
8
MitoPark mice
8
7.
Conclusion
9
8.
References
9
1. Introduction
Parkinson's disease (PD) is a progressive neurodegenerative disease, which is often classified as
movement disorder. PD is after Alzheimer's disease the second most common neurodegenerative
disease with a prevalence of 1% at the age of 65 and a 5% prevalence at the age of 85 1. The classical
motor symptoms of PD are resting tremor, slowness of movement (bradykinesia), muscle rigidity,
postural instability 2. However, PD is multilayered disease and there are numerous non-motor
symptoms such as such as sleeping impairment, cognitive decline and mood disorders 3. Since PD is
a progressive disease four different stages can be described: early stage, mid stage, mid-late stage
and the advanced stage. The early stage characterized by usually mild symptoms and typically occur
slowly and rarely interfere with daily activities, which lead to patients not even noticing the
Parkinson's disease in the beginning. They may have fatigue or a general sense of uneasiness just like
slight tremor, having difficulty standing, body stiffness or lack of normal movement or lack of facial
expressions. With time, the early stage transforms into the mid-stage, which is characterized by
worsening of the symptoms like tremor, muscle rigidity and movement problems which now might
interfere with daily activities. At the mid-late stage patients require help to complete daily tasks such
as walking, bathing or even just standing. The last, the fourth of four stages is the advanced stage
and by now patients require full-time nursing support and can only move around with the help of a
wheelchair. Hallucinations or delusions may occur. On average, it takes 15 years from recognition of
PD until death occurs 4.
PD is primarily characterized by the presence of cytoplasmic inclusions Lewy bodies containing
α-synuclein and ubiquitin among other components. It is believed that Lewy body pathology leads to
the degeneration of dopaminergic neurons in substantia nigra pars compacta (SNc), which is
responsible for the motor symptoms of the disease. Nevertheless, LB pathology affects other brain
regions which leads to the appearance of other symptoms as well 5. Based on current scientific
research knowledge, combination of environmental and genetic factors seem to play a crucial role in
development of PD but the complete understanding of pathomechanisms is still eluding the
researchers 6.
To understand the pathophysiology of PD, and to develop therapies, it is important to have relevant
disease models. In the following text, we will summarize the most important facts about the
available in vivo and animal models of PD and discuss their biomarker, therapeutic, AND
transnational value research. The models presented in this following writing can be categorized into
pesticide-induced, pharmacological, toxin-induced and genetic models. We will first look at the
pesticide-induced models which include rotenon, paraquat and maneb, before we continue with the
1
pharmacological models induced by reserpine and haloperidol. Afterwards we will present the
toxin-induced 6-OHDA and MPTP models. Further, we will be discussing modeling α-synuclein (α-syn)
pathology in rodents. At the end, we will also discuss a few genetic models like LRKK2, PINK1, Parkin,
DJ-1 and MitoPark mice.
Before starting with the description of different animal models, it is important to know the relevant
criteria describing the validity of the models: predictive validity, the face validity and the construct
validity. The predictive validity describes the capability of the model to identify effective drugs. Face
validity describes the similarity of the symptoms, and the construct validity describes the
pathomechanism resemblance between model and the disease. Ideally, model should fulfill all three
criteria, which is rarely a case.
1. Pesticide induced models
Rotenone
Rotenone
((2R,6aS,12aS)-8,9-Dimethoxy-2-(prop-1-en-2-yl)-1,2,12,12a-tetrahydro[1]benzopyrano
[3,4-b]furo[2,3-h][1]benzopyran-6(6aH)-one) is a highly lipophilic insecticide and a mitochondrial
toxin that is used for modeling PD in rodents. The advantage of this toxin is that it can easily cross the
blood-brain barrier, diffuses into neurons where it accumulates within the mitochondria and inhibits
mitochondrial complex 1 activity 7. The cause of the toxicity of rotenone ROS production, which
induces oxidative stress and causes oxidative damage in form of protein carbonyl formation in the
midbrain of treated rodents. The microglial activation in the brain caused by rotenone application is
similar with one found idiopathic PD. Another similarity with the disease is that rotenone Inhibits
proteasomal activity, which is also implicated in PD. Rotenone induces loss of dopaminergic neurons
and lees to nigro-striatal degeneration. Further α-syn and ubiquitin-positive Lewy body-like
cytoplasmic inclusions also had been found within the SNpc of the rodents accompanied with the
motor impairment 8. Unfortunately, high variability in the nature of rotenone’s effects renders this
model, still, not well characterized and mostly used for basic research.
Paraquat
Next pesticide-induced model uses herbicide paraquat ((1,1ʹ-dimethyl- 4,4ʹbipyridinium). When
paraquat is applied, it enters the brain via the neutral amino acid transporter. Once inside the cells,
paraquat leads, just like rotenone, to mitochondrial toxicity and at higher does it also inhibits
mitochondrial complex 1 activity. Paraquat induces dopaminergic system dysfunction and promotes
phosphorilated-α-syn expression (toxic α-syn form associated with aging and PD) and induces
synucleinopathy 9.
2
Maneb
The fungicide maneb (manganese ethylene-1,2-bisdithiocarbamate, polymer), which enters the brain
similarly as Paraquat, rather inhibits complex 3 of the mitochondrial respiratory chain and induces
loss of dopaminergic neurons but it does not lead to α-syn associated pathological changes. Paraquat
and maneb are commonly used in combination to enhance toxicity. Paraquat and maneb combined,
lead to motor deficits, nigral cell loss, lipid peroxidation in addition to inflammatory component of
the pathology 10. All of the three aforementioned pesticides can be applied orally, systematically,
stereotaxically or using micropumps. Unfortunately, all of them show high systemic toxicity.
2. Pharmacological models
Reserpine
The
reserpine
(methyl(3β,16β,17α,18β,20α)-11,17-dimethoxy-18-[(3,4,5-trimethoxybenzoyl)oxy]
yohimban-16-carboxylate) is a drug previously used to treat high blood pressure. It is a one of the
first compounds used to model PD in rodents 11. Reserpine is an indole alkaloid and its mode of
action is inhibition of the vesicular monoamine transporter (VMAT2). Inhibition of VMAT2 results in
loss of storage capacity of monoamines norepinephrine, 5-HT, and dopamine. It was found that two
hours after reserpine administration approximately dopamine levels are reduced 85% in the SNc and
95% in the striatum. The effect of reserpine can be prolonged by adding other chemical substances
(e.g. AMPT). Further, reserpine increases glutamate level in the nucleus subthalamicus as in PD 11.
Reserpine in rodents induces motor symptoms such as muscle rigidity and akinesia. Although the
reserpine model shows important PD-related neurochemical properties there is no SNc dopaminergic
cell degeneration. Thus, this model is mostly used targeted at symptom control research and is used
in the drug development of the following PD drugs: apomorphine, pramipexole, ropinirole, pergolide,
bromocriptine and cabergoline 12.
Haloperidol
The
another
pharmaceutic
used
to
model
PD
in
rodents
is
haloperidol
([4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl] decanoate). Haloperidol is an
antipsychotic drug that acts as antagonist of dopamine D2 and D1 receptors. Haloperidol does not
strongly mimic biochemical processes observable in PD and is more restricted to dopamine depletion
effects. Nevertheless, redictive validity of the model were confirmed with L-DOPA, bromocriptine,
pramipexole, trihexyphenidyl and amantadine drugs used as a treatment in PD 13.
3
The major flaws in the pharmacological PD models are a lack of neurodegeneration, α-syn pathology,
inflammation as well as other pathological features observable in PD. Therefore, the use of these
models is limited to effects of dopamine depletion observation.
3. Toxin induced models
6-OHDA
6-OHDA (6-hydroxydopamine) is a selective catecholaminergic neurotoxin. Unlike MPTP
(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), 6-OHDA cannot cross the blood-brain barrier.
Therefore, 6-OHDA needs to be delivered stereotaxically. Since 6-OHDA can enter in neurons using
monoamine transporters it has neurotoxic effects on both dopaminergic and noradrenergic neurons.
For that reason, 6-OHDA protocols commonly include administration of noradrenergic reuptake
inhibitors 14. When applied to SNc 6-OHDA causes progressive retrograde neuronal degeneration of
the dopaminergic neurons causing motor function impairment. 6-OHDA mechanism of action
includes oxidative stress and an inhibition of mitochondrial complexes I and VI 15. Although toxin
induced models mimic several crucial aspects of the disease, such as neurodegeneration, dopamine
loss, inflammation, mitochondrial dysfunction, 6-OHDA models target relatively small regions of
brain (usually SNc) and dopamine neurons, the effects of α-syn associated pathology as well as the
pathological processes in different brain regions cannot be observed.
MPTP
MPTP is a neurotoxin that causes the destruction of dopaminergic cells and causes the damage to
the nigrostriatal DA pathway. While rats are resistant to this toxin other animals, including mice are
commonly used 16. Unlike 6-OHDA, MPTP is able to cross the blood-brain barrier of organisms quickly,
which means that no surgical intervention is required, however surgical procedure can increase the
neurodegeneration selectivity. In the brain, MPTP is metabolized by glial cells to MPP+ in which is
then transported by vesicular monoamine transporter (VMAT+) to dopamine neurons. MPTP is then
accumulated by mitochondria where it inhibits the electron transport in the respiratory chain in
mitochondrial complex I. This leads to a cascade reaction where ATP production is reduced and ROS
and superoxide radicals are produced, which ultimately leads to neuronal cell death.
Shortcomings of the toxin induced models include lack of Lewy body pathology and relatively fast
process of neurodegeneration which does not appear in PD 17. Nevertheless, these models have a
strong construct validity and they were extensively used in both basic, translational and drug
development studies.
4
4. Modeling α-syn pathology in vivo
The α-syn protein is encoded by the SNCA gene and is highly expressed neuronal protein that
regulates vesicle transport and is also involved in neurotransmitter release 18. The α-syn is also the
main component of Lewy-bodies as well as Lewy-neurites, pathological hallmark of the disease 19.
Mutations of α-syn are directly responsible for the different familiar forms PD. Missense mutations in
SNCA A30P, A53T as well as E46K were described as a causes of a few familial forms of PD. Aside
point mutations that cause familiar forms of PD, doubling or tripling of SNACA gene are already
sufficient to cause the autosomal dominant form of PD 20. Accordingly, the α-syn level is an important
determinant in the progression of PD. Under normal conditions, α-syn is in an equilibrium of
unfolded monomers as and a-helical tetramers. An increase in the unfolded state and thus shift in
equilibrium favor the accumulation of α-syn. Thus, misfolded α-syn can trigger the activation of
microglia, resulting in a positive feedback loop because since microglia can promote the aggregation
and propagation of α-syn pathology
. Potential triggers for misfolding of α-syn are diverse. For
21
example, misfolding may be the result of neuroinflammation, mitochondrial dysfunction, oxidative
stress, lysosomal dysfunction, and ubiquitin-proteasome system
. The α-syn is subject to
22
post-translational modification, such as phosphorylation as well. For instance, phosphorylated of
serine 129 is one of the most important markers for PD, as the disease is associated with increase in
phosphorylation of α-syn. In the diseased brain 90% of α-syn is phosphorylated, whereas only 4% is
phosphorylated in the healthy brain
23
. However, it remains to be determined whether
phosphorylation leads to accumulation or is it instead a mechanism for labeling and removal of
excessive α-syn.
Braak et al.24 hypothesized that PD does not start in the SNc but there are two starting points in the
disease progression of PD, namely the olfactory bulb and intestinal nerves. From there, the disease
progression spreads to other brain regions via the olfactory tract or vagus nerve. Further, he
described six stages of PD pathology progression, with each stage describing in the formation of
α-syn aggregations in the different brain areas. These hypotheses lead to proposing that prion like
mechanism is responsible for α-syn propagation 25.
In the PD research in the context of α-syn propagation and associated pathology three approaches
are most commonly used: injection of preformed α-syn fibrils (PFFs), the injection of recombinant
adono-associated viral vectors (rAAV) which mediate overexpression of α-syn and transgenic animals
that overexpress different types of α-syn.
PFFs
Injection of α-syn PFFs in rodents triggers formation of LB inclusions in the targeted and neighboring
networked brain areas. Furthermore, presence and propagation of PPFs triggers neuroinflammation,
5
which in turn contributes to the spread of PD pathology
. The α-syn PFFs models are most
26
commonly used for studies investing mechanisms of α-syn propagation. The models of α-syn PFFs are
useful for development of therapies that prevent the formation of α-syn aggregations at early stages
of disease progression.
However, these models also carry some disadvantages. Correct injection of PFFs is immensely
important, as the fibrils can hardly spread through the tissue. Therefore, in most of the experiments,
injection is performed in the striatum, since this provides a relatively large area that is easy to reach,
among other reasons. Rodents injected with different pathogen strains of PFFs show different
disease courses as well as differences in behavior. Furthermore, different α-syn pathogen strains also
have different efficacies, some can cause loss of dopaminergic neurons when injected to SNc 26.
Another shortcoming is a very fast propagation of the pathology which does not correspond to what
is observed in PD patients.
rAAV
rAAV has several advantages over the standard lentivirus viral vector. For example, the particles are
smaller, allowing more rAAV particles to be injected per titer, and because of their size, they migrate
more easily through tissue. Also, it is rare for mutagenesis to occur as a result of integration into the
host genome. However, rAAV mediated α-syn do not have a propagation mechanism as seen in α-syn
PFFs. Additionally, in rAAV α-syn, transgene efficiency and expression depend on titer, injection site,
promoter, and serotypes. For example, previously used serotype 2 induced mild immune responses
in human hepatocytes and required a high dose for efficient transduction of neurons. The new
rAAV2, have increased efficiency without eliciting immune responses 27.
Both rats and mice are commonly used for the rAAV injected method of α-syn overexpression. Rats
are more convenient because a viral vector requires stereotactic injection and since rats have a
bigger brain it is easier to target regions of interest. Mice on the other hand offer more transgenic
tools that can be useful in experimental design. Parameters such as species, strain, and age of the
animals all play a central role in the pathology of PD in these models. For example, older animals are
more affected by cell loss from dopaminergic neurons than younger ones.
Models that make use of viral vectors have a clearly visible dopaminergic neurodegeneration, in
contrast to all transgenic α-syn mice. Injecting human WT α-syn or mutant A53T α-syn into rat SNc
yields an age-dependent progressive course of PD including loss of DA neurons, motor deficits, and
even aggregations of α-syn similar to LB. The loss of cells was accompanied by degenerative changes
in striatal axons, and α-syn aggregations were also found in axons and dendrites 28. However, it is
worth mentioning that α-syn levels after rAAV injection is higher than levels in idiopathic PD and PD
6
with SNCA multiplications. Further, rAAV models usually target small brain regions restricting PD
associated pathology to a which is not the case in PD. Further, in these models, α-syn pathology is
restricted exclusively to neurons transduced by rAAV that express α-syn 27.
Genetic models
There a several transgenic mouse models use an α-syn overexpression to mimic the PD features in
mice. Commonly human wild type or mutant (A53T, A30P) is expressed under different, usually
pan-neuronal promotors, such as Thy1, platelet-derived growth factor (PDGF), prion promotor (PRP)
etc., to ensure neuron specific overexpression of α-syn
29,30
. These mice usually show motor
impairments, some extent of α-syn associated pathology (usually no Lewy body-like inclusions) some
impairment in dopamine system function and pathological features such as inflammation and
mitochondrial dysfunction. However, dopamine neuronal loss is not present 31. The validity of these
models depends on factors such as type of the promotor, type of the transgene as well as the age of
the animal. Nevertheless, these models provide the opportunity to observe the effects of PD
pathology in the whole brain together with non-motor symptoms. Further these models are often
used for the studies involving other synucleinopathies such as multiple system atrophy and dementia
with Lewy bodies.
5. Other genetic models
LRRK2
Mutations of leucine-rich repeat kinase 2 (LRRK2) provide a late-onset autosomal dominant familiar
form of PD. The most common mutations in LRKK2 include G2019S, which is a point mutation in the
kinase domain, and R1441C, which is a mutation in the guanosine triphosphate domain. These
mutations as well as LRRK2 knockout are used to generate mouse transgenic models of PD.
Overexpression of G2019S leads to a mild, progressive course of DA neurodegeneration in SNc, but
without motor deficits or changes in DA levels 32. LRRK2 BAC transgenic mice showed age-related and
progressive motoric deficits. In addition, slight reductions in striatal DA release were present but no
neurodegeneration
33
. Other LRRK2 transgenic models show no functional abnormalities in the
nigrostriatal DA neurons of SNc, however accumulation and aggregations of α-syn and ubiquitin do
occurs 32.
PINK1
PTEN-induced kinase 1 (PINK1) is mitochondrial serine/threonine-protein kinase encoded by the
PINK1 gene. Mutations in PINK1 are the second most frequently occurring cause of autosomal
recessively inherited early-onset PD. PINK1-deficient mice are a model of the PARK6 variant of PD. PD
PINK1 KO mouse models exhibit some typical signs of PD such as decreased levels of DA in the
7
striatum and as well as decreased locomotor activity, accompanied by mitochondrial disfunction.
However, this models show no changes in DA neurons, no LBs, and likewise no degeneration of
neurons 34,35.
Parkin
Parkin is a 465-residue E3 ubiquitin ligase and mutations in Parkin are associated with autosomal
recessive PD with an early onset. More than 100 mutations in the Parkin gene have been found up to
date. In mouse models of Parkin KO, several exons in the PRKN gene are deleted. All of these models
have in common that none of them show any abnormality in behavior or loss of SNc neurons. Only
the Parkin-Q331X-DAT-BAC mouse model have a late-onset of a progressive motor deficits and
age-dependent loss of DA neurons in SNc 36.
DJ-1
Protein deglycase (DJ-11) mutation is autosomal recessive associated to induce early-onset PD37. DJ-1
is redox sensitive chaperone that inhibits the aggregation of α-syn. KO mouse models in which exon 2
of DJ-1 gene has been deleted show deficits in locomotion, but without further relevant pathological
features of PD. However, DJ-1 KO C57 mice backcrossed to C57/BL6 show age-dependent
degeneration of the nigrostriatal system,neurodegeneration in locus coeruleus and mild behavioral
disturbances 38.
MitoPark mice
The importance of mitochondrial function impairment in PD is well documented. MitoPark mice
were created with a purpose to enable researchers to more closely examine how impaired
mitochondrial function contributes to PD pathology. MitoPark mice have a conditional knockout of
mitochondrial transcription factor A in dopaminergic neurons. The exhibit many of the important
core symptoms and pathologies observable in PD. For instance, MitoPark mice show
neurodegeneration, a progressive behavioral change, intraneuronal inclusions reminiscent of LB, cell
death of dopaminergic neurons in the SNc, and they respond to levodopa (L-dopa), which is used in
treatment of PD 39.
6. Conclusion
Modeling complex disease such as PD is not easy. Many different factors need to be taken into
account. Usually, model provides opportunity to observe certain features of the disease pathology,
but not all of them. For example MPTP and 6-OHDA provide opportunity to observe dopaminergic
neuronal loss and motor symptoms but do not mimic α-syn associated pathological features of the
disease. On the other hands transgenic models of α-syn overexpression provide insight into
pathological features caused by α-syn, but rarely show Lewy body pathology and dopaminergic
8
neuronal loss. Therefore, the need of the study dictates the selection of the model and sometimes
several different models need to be used for researchers to gain the answer to the asked questions.
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