Experimental Neurology 298 (2017) 172–179

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Experimental Neurology
journal homepage: www.elsevier.com/locate/yexnr

Review Article

Experimental animal models of Parkinson's disease: A transition from MARK

assessing symptomatology to α-synuclein targeted disease modification
Wai Kin D. Koa,⁎, Erwan Bezarda,b,c
a
Motac Neuroscience Ltd, Manchester, United Kingdom
b
Univ. de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France
c
CNRS, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France

A R T I C L E I N F O A B S T R A C T

Keywords: With the understanding that α-synuclein plays a major role in the pathogenesis of Parkinson's disease (PD), novel
α-Synuclein animal models have been developed for conducting preclinical research in screening novel disease modifying
Parkinson's disease therapies. Advancements in research techniques in α-synuclein targeted disease modification have utilised
Animal models methods such as viral mediated expression of human α-synuclein, as well as the inoculation of pathogenic α-
Lewy Body
synuclein species from Lewy Bodies of PD patients, for accurately modelling progressive self-propagating neu-
rodegeneration. In applying these cutting-edge research tools with sophisticated trial designs in preclinical drug
trials, a useful platform has emerged for developing candidate agents with disease modifying actions, promising
a greater chance of success for clinical translation. In this article, we describe the transition of well-established
animal models of PD symptomatology to newly developed models of PD pathogenesis, with specific focus on
methods of viral-mediated and inoculation of pathogenic α-synuclein, that aim to aid scientific translation of
neuroprotective strategies.

1. Parkinson's disease pathogenesis
Parkinson's disease (PD) is a progressive neurodegenerative disorder
affecting approximately 1% of the population over the age of 55 years,
with the highest prevalence in ages of 85 years and over (De Rijk et al.,
1997). PD patients display a clinical syndrome of motor symptoms (in
particular bradykinesia, postural deficits and resting tremor) (Marsden,
1994), which may be preceded by a range of non-motor symptoms
(including cognitive disturbance, sleep disturbance, anosmia and con-
stipation) (Pont-Sunyer et al., 2015). These clinical manifestations of
PD appear due to the extensive loss of dopaminergic neurons of the
nigrostriatal pathway (Ehringer and Hornykiewicz, 1960), as well as
dysfunction of neurons in dopaminergic, serotonergic, adrenergic and
cholinergic neurotransmitter systems (Jellinger, 1991).
A major pathological hallmark of PD is the widespread expression of
intraneuronal proteinaceous inclusions, known as Lewy Bodies (LBs)
(Spillantini et al., 1998), found in the perikarya of neurons within the
central and peripheral nervous systems. LBs are mainly composed of α-
synuclein, a 14 kDa endogenous protein of 140 amino acid length.
Autosomal dominant forms of PD are linked to missense mutations of
the α-synuclein, specifically p.A53T, p.A30P, p.E64K, p.H50Q, p.G51D
and p.A53E (Appel-Cresswell et al., 2013; Athanassiadou et al., 1999;
Kruger et al., 1998; Lesage et al., 2013; Polymeropoulos et al., 1997;
Puschmann et al., 2009; Zarranz et al., 2004) and also the multi-
plication and triplication of the gene locus for α-synuclein (SNCA)
(Chartier-Harlin et al., 2004; Singleton et al., 2003).
In the last two decades, the α-synuclein protein has been proposed
as the main component in mediating the progressive neurodegeneration
in PD (Dehay and Fernagut, 2016; Recasens and Dehay, 2014). The
pathological structure of α-synuclein is a misfolded conformation of an
oligomeric or fibrillary nature that is of a specific phosphorylated form
(phosphor-Ser129), as seen in PD patient LB formations (Fujiwara et al.,
2002; Spillantini et al., 1997). Several studies have greatly impacted the
field of PD pathogenesis. Braak et al. first hypothesised the progression
of PD in distinct stages; beginning at the peripheral autonomic nervous
system, a region where α-synuclein pathology was identified (Braak
et al., 2006; Gelpi et al., 2014), in the anterior olfactory nucleus and
dorsal motor nucleus of the glossopharyngeal and vagal nerves, gra-
dually reaching the midbrain and cerebral cortex (Braak et al., 2003).
Pathological transmission was later suggested between neurons through
post-mortem analysis in PD patients, where LB formations were found
in embryonic mesencephalic neuron grafts in the striatum that were
implanted over a decade prior, supporting a host-to-graft transmission
process (Kordower et al., 2008; Li et al., 2010). With the transmission of

⁎
Corresponding author at: Motac Neuroscience Ltd, PO Box 363, Manchester M15 6WE, United Kingdom.
E-mail address: d.ko@motac.com (W.K.D. Ko).

http://dx.doi.org/10.1016/j.expneurol.2017.07.020
Received 17 June 2017; Accepted 28 July 2017
Available online 29 July 2017
0014-4886/ © 2017 Elsevier Inc. All rights reserved.
W.K.D. Ko, E. Bezard
α-synuclein-positive intracytoplasmic inclusions, the term ‘prionoid’
has been assigned, describing the pathogenic nature of intracellular
mechanisms, including release into extracellular space and uptake into
healthy neurons. Although mainly focused within dopaminergic neu-
rons of the SN, this self-propagating process with the pathogenic α-
synuclein acting as a template for misfolding (Brundin et al., 2008;
Dehay et al., 2016a), can lead to a spread of neurodegeneration across
interconnected brain regions causing disruption of intracellular pro-
cesses (i.e. neurotransmission, mitochondrial structure and activity,
dynamics and mitophagy, vesicular transport and protein degradation).
The pathogenic α-synuclein seeding activity is supported by detection
of monomeric, oligomeric and Ser129-phosphorylated α-synuclein
forms in human plasma and cerebrospinal fluid (Borghi et al., 2000; El-
Agnaf et al., 2003; Foulds et al., 2012; Mollenhauer et al., 2011; Tokuda
et al., 2010). Moreover, further supportive evidence comes from the use
of synthetic recombinant α-synuclein preformed fibrils (PFFs) inducing
conformation changes of endogenous soluble α-synuclein to patholo-
gical species, subsequently leading to cellular dysfunction (Hansen
et al., 2011; Luk et al., 2009; Volpicelli-Daley et al., 2011).
In recent years, the understanding of PD pathogenesis has been
greatly enhanced with the development of advanced research tools,
some taking the form of novel animal models (Angot et al., 2010; Dehay
et al., 2016b; Olanow and Brundin, 2013). In this review, we provide an
overview of the commonly used symptomatic animal models of PD and
newly developed models of PD pathogenesis, of which the latter are
expected to aid the translation of potential therapies with focus on
disease-modifying mechanisms, paving the way for the identification of
novel agents for treating neurological disorders.
2. Modelling clinical symptoms of PD
To date, neurotoxin based animal models have been well utilised in
translational research for the development of symptomatic therapies in
PD. The most commonly used neurotoxins include 6-hydroxydopamine
(6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),
which are often administered in rodents and non-human primates
(nhps), respectively, for inducing a parkinsonian state by causing severe
loss of dopaminergic neurons.
In rats, the unilateral injection of 6-OHDA into the medial forebrain
bundle causes rapid and extensive loss (> 90%) of dopaminergic
neurons that is seen within 14 days (Grealish et al., 2008). The re-
producibility and simplicity in quantifying asymmetric motor deficits,
such as rotation and forelimb bias, following administration of dopa-
minergic agents are major advantages for the use of 6-OHDA-lesioned
rats in translational research that have allowed for efficient screening of
anti-parkinsonian compounds (Cannon and Greenamyre, 2010; Lane
et al., 2006; Schwarting and Huston, 1996). In addition, the injection of
6-OHDA into the striatum permits for slower and less severe loss of
dopaminergic nigrostriatal neurons (> 60–70%), modelling more pro-
gressive neuronal degeneration (Kirik et al., 1998; Sauer and Oertel,
1994).
The gold standard model of PD motor symptoms is produced by
administration of MPTP into old world non-human primate (nhp) spe-
cies (Macaca mulatta and Macaca fascicularis). With a closer resem-
blance to humans in physiology and brain anatomy, the PD motor
features in monkeys that are induced following a regimen of MPTP
injections (typically daily doses of 0.2–2 mg/kg for 2–3 weeks) gives an
accurate model for advanced PD clinical motor signs (Bezard et al.,
2001; Bezard et al., 2003; Meissner et al., 2003). The MPTP-treated nhp
model is most commonly replicated after systemic administration of
MPTP, which readily crosses the blood brain barrier, inducing a par-
kinsonian syndrome of prolonged stillness, bradykinesia, postural def-
icit and an overall reduction in movement (Crossman et al., 1985;
Delong et al., 1985). After stabilisation of these behaviours where do-
paminergic degeneration reaches > 90% in the substantia nigra pars
compacta (SNc) (Bezard et al., 1997), subjective rating scales based on
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Experimental Neurology 298 (2017) 172–179
clinical scale criteria can be utilised to specifically assess movement
range, posture, tremor, and a calculated score for overall disability (Ko
et al., 2014). These translational tools are and have been instrumental
for evaluating the efficacy of symptomatic therapies (Bastide et al.,
2015; Johnston and Fox, 2015; Fox and Brotchie, 2010), which are
effective in providing drug efficacy in proof-of-concept tests (Ko et al.,
2014; Rylander et al., 2010). Furthermore, in recent years, sophisti-
cated technology in high definition video recording of limb trajectory
has been adapted to nhps for assessment of detailed kinematics in swing
and gait cycles during freely moving tasks (Capogrosso et al., 2016; Yin
et al., 2014). Such objective assessments of motor activity complement
subjective behavioural ratings for detecting more subtle motor deficits,
allowing for consistent endpoint assessments between MPTP-treated
nhps and PD patients, in respective preclinical and clinical trials.
The MPTP-treated nhp model of PD offers further face and pre-
dictive validity (i.e. phenomenological similarities between behaviours
and measurable treatment effects as seen in the human condition, re-
spectively) with clear reversal of experimental parkinsonism following
L-3,4-dihydroxyphenylalanine (L-DOPA) treatment. The maximum L-
DOPA dose is individually titrated based on motor symptom severity
and is commonly given during the stabilisation period of model re-
plication to aid animal feeding. Daily L-DOPA treatment (in the range of
9–20 mg/kg) over 3–4 months can lead to the development of stable
and reproducible dyskinesia, with chorea and dystonia individually
scored based on severity (Fox et al., 2012). After L-DOPA priming in
MPTP-treated nhps, PD disability cannot be completely reversed
without the induction of dyskinesia, similar to that seen in PD patients.
This has been utilised to provide clinically relevant data such as ‘good
on-time’ (Ko et al., 2016; Pinna et al., 2016) allowing treatment stra-
tegies for dyskinesia to be thoroughly evaluated in dyskinetic MPTP-
treated nhps. Investigations utilising this model also provide for ad-
dressing specific intracellular mechanisms using novel biotechnological
approaches, such as gene therapy (e.g. modulation of regulators of G-
protein signalling proteins (Gold et al., 2007)) or enzymes for dopamine
synthesis (Jarraya et al., 2009), for testing potential therapies in
modifying the expression of dyskinesia.
The use of MPTP-treated nhps is also a valuable translational re-
search model for studying the non-motor symptoms of PD. For example,
chronic low dose (CLD) MPTP administration can be used to induce
cognitive deficits found in executive and attentional tasks, which are
further worsened following L-DOPA treatment, as seen in PD patients
(Gotham et al., 1988; Kulisevsky et al., 1996; Schneider et al., 2013). In
modelling both cognitive deficits and subtle motor dysfunction in
MPTP-treated nhps, the translational value allows for tests of single or
combined drug treatment strategies that produce the most desired
clinical outcome among the myriad of disease symptoms (Ko et al.,
2016). Sleep disturbances seen in PD patients such as rapid eye
movement sleep behaviour disorder (Postuma et al., 2009) can also be
evaluated in MPTP-treated monkeys using continuous electro-
encephalography, which has been used to assess the effects of different
pharmacological treatments on sleep disturbances (Barraud et al., 2009;
Hyacinthe et al., 2014). While MPTP administration causes severe loss
of dopaminergic neurons in the SNc, there is also a broad neurotoxic
effect with loss of extra-nigral dopaminergic, serotoninergic and adre-
nergic neurons (Forno et al., 1986; Mitchell et al., 1985; Perez-Otano
et al., 1991). Although this resembles some of the major neuropatho-
logical changes seen in PD patients, this animal model remains limited
for studying disease pathogenesis due to lack of true LB pathology,
albeit certain proteinaceous inclusions have been previously reported in
MPTP-treated baboons (Kowall et al., 2000).
While neurotoxin based animal models serve for the development of
symptomatic therapies, notably seen from the early success of deep
brain stimulation in PD (Benazzouz et al., 1993), these symptomatic
animal models remain limited for studying disease-modifying therapies
due to the inability to accurately replicate the nature of PD pathogen-
esis i.e. progressive self-propagating neurodegeneration with
W.K.D. Ko, E. Bezard
pathological hallmarks. In the next section, we look at the development
of recent animal models that allow for the screening of agents with
disease modifying effects.
3. Modelling PD pathogenesis
With the advancements in the understanding of PD pathogenesis,
specifically the identification of misfolded α-synuclein as a pathogenic
agent, novel animal models of synucleinopathies have been developed
as a useful platform for evaluating neurodegeneration, as well as for
specifically investigating α-synuclein seeding and cell-to-cell transmis-
sion (Sato et al., 2015). In regards to the latter, viral vectors have also
been previously utilised across mice, rats and nhps for studying host-to-
graft transmission i.e. transmission models (Kordower et al., 2011;
Dehay et al., 2016b; Dehay and Fernagut, 2016). In a series of delicate
experiments, the unilateral injection of AAV2/6-huαsyn into the SN
caused an approximate 23% transmission of human α-synuclein to
implanted embryonic ventral mesencephalic neurons in striatum of the
same hemisphere (Angot et al., 2012).
The use of viral vectors has also been commonly used for inducing
neurodegeneration in mammalian species and is recognised to produce
a more acute form of PD compared to transgenic animal models, with
the avoidance of compensatory mechanisms from constitutive over-
expression, which is the case for transgenic PD models. In the first series
of demonstrations (Lo Bianco et al., 2002; Kirik et al., 2002), the re-
combinant lenti-virus was used for overexpression of human α-synu-
clein (wild-type, A30P mutant and A53T mutant) in the SN of rats,
which induced modest loss of neurons expressing tyrosine hydroxylase
(TH) (35%, 33% and 24%, respectively) (Lo Bianco et al., 2002). In
another key study, Kirik et al. (2002) was first to utilise the re-
combinant adeno-associated virus (rAAV) to overexpress wild-type and
human mutated A53T in rat nigrostriatal dopaminergic neurons. Fol-
lowing unilateral injection into rats of rAAV-α-syn in the SN, patholo-
gical features of PD were observed which included α-synuclein inclu-
sions and dystrophic neurites. In addition, the overexpression of α-
synuclein caused significant loss of nigral DA neurons (30–80%) and
striatal dopamine (~ 40%), as well as increased dopamine turnover
(~ 160%) over 6 months (Kirik et al., 2002). However in this study,
animals did not show obvious asymmetric motor deficits, possibly due
to a non-substantial loss of striatal dopamine that has been recognised
to be at a threshold level of > 50–60% (Kirik et al., 1998; Lee et al.,
1996). Later studies utilising a chimeric vector, specifically AAV1/2
carrying mutated A53T-α-synuclein cDNA, for increased neuronal
tropism and expression, demonstrated progressive neurodegeneration
in rats over 6 weeks reaching > 50% dopaminergic cell loss (Koprich
et al., 2010; Koprich et al., 2011).
The recent studies using viral mediated overexpression of α-synu-
clein in rats has denoted high translational value in modelling motor
dysfunction as well as in PD pathology. Decressac et al. (2012a, 2012b)
recently assessed behavioural deficits and neuropathological changes
caused by unilateral AAV6-α-syn injections under an optimised vector
construct (transgene driven by synapsin-1 promoter and amplified
WPRE sequence) in rats, which were compared to behavioural and
pathological deficits induced by 6-OHDA-lesions (Decressac et al.,
2012a). While loss of dopaminergic neurons in SNc of AAV6-α-syn in-
jected rats reached approximately 70%, similar to that of two-site in-
trastriatal 6-OHDA injections, the model replicated disease hallmarks of
cytoplasmic α-synuclein inclusions with significant asymmetric motor
deficits being established over 8 weeks (Decressac et al., 2012a;
Decressac et al., 2012b). Recent experiments have further characterised
the motor deficiencies in AAV9-α-syn injected rats by using high speed
motion caption of whole-body kinematics, where animals demonstrated
slow shuffling gait, reduced control of limb trajectory and medio-lateral
instability (Bourdenx et al., 2015b). The AAV-α-syn injected rat model
offers major advantages in the screening of novel neuroprotective
agents against motor deficits, as well as processes of α-synuclein
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Experimental Neurology 298 (2017) 172–179
neurotoxicity that are likely to involve processes of inflammatory re-
action (Chung et al., 2009), associated exosome release (Danzer et al.,
2012; Lee et al., 2005), ER-golgi trafficking (Cooper et al., 2006),
passive membrane diffusion (Kayed et al., 2004) and proteasome and
lysosome dysfunction (Venda et al., 2010).
Viral mediated overexpression of α-synuclein has also been tested in
mice species with the potential in combining the capacity of genetic
modification with progressive disease development. However, the lack
of consensus between methods has so far shown different levels of
success for inducing neurodegeneration. For example, a number of
studies have reported limited dopaminergic neuronal degeneration
from 7 to 12 weeks post SN injection (Lauwers et al., 2003; Theodore
et al., 2008; Yasuda et al., 2009), whereas other groups, utilising AAV2/
7 with either wildtype and mutant A53T-α-synuclein have demon-
strated significant loss (82%) of nigral TH neurons at 8 weeks following
injection (Oliveras-Salva et al., 2013). In a recent study by Ip et al.
(2017), the effects of AAV1/2-induced overexpression of A53T-α-sy-
nuclein were well characterised in C57BL/6 mice (Ip et al., 2017). It
was reported that at 10 weeks post unilateral injection into the SN, A-
AV1/2-A53T- α-synuclein mice showed 33% reduction in TH neurons,
with 29% loss of dopamine active transporter binding. In addition,
HPLC analysis revealed reduction in levels of dopamine (38%) and
DOPAC (33%), respectively (Ip et al., 2017), with histological analysis
showing pathological hallmarks of LBs and Lewy-neurites (LNs) in the
nigrostriatal system. Authors also characterised the motor deficits in
AAV1/2-A53T-α-synuclein mice compared to empty vector controls,
which were seen to have forelimb bias measured using the cylinder test
at 5 and 9 weeks during the live-phase of the study. Together, the
construct validity in the proposed mouse model of PD pathogenesis was
recognised with degeneration associated with α-synuclein accumula-
tion and histological/morphological features, but this model currently
lacks predictive validation i.e. the reversal of symptomatic features
with pharmacological treatments (Ip et al., 2017).
Tests with recombinant AAV for overexpression of α-synuclein in
the SN of higher order species was originally carried out in marmosets,
successfully demonstrating PD pathogenesis (Eslamboli et al., 2007;
Kirik et al., 2003). The use of AAV2 for increased expression of wild-
type or A53T α-synuclein was found to produce 30–60% dopaminergic
neuron loss over 4 months (Kirik et al., 2003). Eslamboli et al. (2007)
further demonstrated a more reliable model of PD pathogenesis in
marmosets with use of AAV2/5-A53T α-synuclein, compared to wild-
type α-synuclein (Eslamboli et al., 2007). Recently, Koprich et al.
(2016) extended observations to macaque species, where AAV1/2-
driven overexpression of human A53T α-synuclein was used to model
PD pathogenesis, in a step towards developing of a drug screening
platform for disease modifying therapies (Koprich et al., 2016). With a
high level of transfection (86%) in nigral dopaminergic neurons after 4
separate site injections in the SN, authors demonstrated that a high titre
(20 μl of 5.1 × 1012 gp/ml) or high volume (28 μl of 1.7 × 1012 gp/ml)
of hA53T-α-synuclein caused 50% and 49% SN dopaminergic cell loss,
respectively, as well as 72% and 60% reduction of dopamine levels in
the putamen, respectively, compared to controls over 4 month ob-
servations (Koprich et al., 2016). Although further experiments are
needed for evaluating the PD behavioural phenotype in this model and
for demonstrating reproducibility, these initial studies may provide a
pathway towards validating a new model of PD pathogenesis for pre-
clinical drug development. Together, the studies utilising viral vectors
across mammalian species provides for novel models of PD pathogen-
esis with construct validity, replicating clinical hallmarks, specifically
α-synuclein related neuropathology and progressive neurodegenera-
tion.
Uncovering the mechanisms involved in PD pathogenesis has also
utilised other research methods based on host-to-graft transmission
(Angot et al., 2012; Desplats et al., 2009) which includes injection of 1.
Brain samples between α-synuclein transgenic and wild-type animals
(Luk et al., 2012b; Mougenot et al., 2012) 2. In vitro generated PFFs of
W.K.D. Ko, E. Bezard
α-synuclein (Hansen et al., 2011; Luk et al., 2012a) 3. Brain homo-
genates from patients with synucleinopathy (Bernis et al., 2015; Jones
et al., 2015) and 4. α-synuclein containing LB extracts from post-
mortem PD patient brain tissue (Recasens et al., 2014).
Studies in rodents have shown host-to-graft transmission of α-sy-
nuclein between neural cells. An example of this is the use of transgenic
mice overexpressing α-synuclein (line 61 and line F28) that lead to α-
synuclein immunoreactivity in grafted cells from 4 to 6 weeks post-
transplantation (Desplats et al., 2009; Hansen et al., 2011). In vivo
studies have also utilised the injection of brain extracts from α-synu-
clein transgenic mice into healthy transgenic animals to demonstrate
pathogenic transmission (Mougenot et al., 2012). In a study by Luk
et al., brain homogenates from old transgenic mice (M83) were injected
into cortex and striatum of young asymptomatic transgenic mice, which
caused the spread of α-synuclein pathology in central nervous system
(CNS) over 90 days (Luk et al., 2012b). Although this demonstrates that
the pathogenic protein is within the brain homogenate sample for in-
itiating disease pathogenesis, the seed pathogen remains to be eluci-
dated within the vast content encompassed in the inoculated material.
Pathological and behavioural abnormalities that resemble PD, such as
loss of dopaminergic neurons in the SNc and motor deficits, has also
been demonstrated in wild-type mice from a striatal injection of re-
combinant α-synuclein PFFs (at 5 μg), a process dependent on host
expression of endogenous α-synuclein, which supports a seeding me-
chanism induced by pathogenic species (Luk et al., 2012a). However,
while providing useful insights into the disease developmental process,
it remains noted that large amounts of PFFs (within the microgram
range) are required to initiate pathology, raising questions of the direct
association with disease pathogenesis and calling for further studies in
identifying the pathogenic subspecies of oligomers and fibrils.
In the past few years, much progress has been made in modelling PD
pathogenesis in nhps using human α-synuclein species derived from LBs
of post-mortem PD patients, where it was hypothesised that patient-
derived aggregated α-synuclein holds the pathogenic candidate. In the
study performed by Recasens et al. (2014), nigral LB samples from 3 PD
patient brain samples, purified by sucrose gradient fractionation and
filter retardation, were injected into the i. motor striatum (100 μl) at
two rostrocaudal levels or ii. substantia nigra (10 μl) in rhesus macaque
monkeys (Recasens et al., 2014). The PD-derived LB-extracts caused
progressive nigrostriatal neurodegeneration, where it was found that
exogenous human α-synuclein was internalised by neurons initiating
conversion of host α-synuclein, which spread across anatomically in-
terconnected brain areas. During the live phase of the study, dopami-
nergic neuron loss (~ 34%) was shown at 9 months post striatal LB
injection by positron emission scans using radioligand marker 11C-
DTBZ. Post-termination analysis conducted at 14 months post injection,
showed that nhps injected with LBs into the striatum or SNc, had sig-
nificant dopamine cell loss in SNc (~ 40% and ~15%, respectively).
With more extensive loss at the striatal dopaminergic axon terminals
than SNc cell bodies, authors alluded to a putative mechanism of LB
extract induced retrograde nigral neuronal degeneration or possible
initiation of pathological cascades that induced distal neurodegenera-
tion (Recasens et al., 2014). Moreover, the detailed cell analysis showed
most pathological (Ser129 phosphorylated/proteinase K resistant) α-
synuclein diffusely accumulated in the cytosol and in the presynaptic
terminals. In mice, it is found that the potent reactivity species of
purified LB samples causing neurodegeneration is within a picogram
dose range (Recasens et al., 2014), in comparison to a microgram dose
range that is needed for recombinant PFFs to induce cell loss, sug-
gesting a higher pathogenic potential of infectivity of the former. To
support these in vivo investigations, an in vitro research platform has
been developed specifically for compound assessment against α-synu-
clein cell toxicity and cell transmission (neuron/glia ↔ glia/neuron).
Using 96-well plate high-throughput screening in primary cortical
neuronal cultures, drug efficacy against α-synuclein toxicity can be
monitored through blockade of changes to cell morphology, such as
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Experimental Neurology 298 (2017) 172–179
neurite length and branching. Using high content time-lapse imaging,
cell culture experiments have so far demonstrated the dose-dependent
effect of α-synuclein-induced toxicity on neurite outgrowth of cortical
neurons over a 7 day period (Cavaliere et al., 2015). These in vitro
methods in addition to the LB-seeding animal models in mice and nhps
provide for a powerful platform for studying disease modifying agents.
For example, the ability to monitor gradual neuronal degeneration in-
duced by purified LB patient samples as described by Recasens et al.
(2014) provides for screening of neuroprotective compounds that can
halt or slow the disease process. Further ex vivo analysis of post-
mortem brain tissue of animal models can be used to identify whether
potential disease modifying agents are effective against major disease
processes such as α-synuclein aggregation and cell-to-cell transmission.
4. Animal model considerations
The implementation of novel methods in modelling PD pathogenesis
through technological breakthroughs provides a potentially effective
avenue for the rapid development of disease-modifying agents for
clinical use. These newly developed research approaches, gives high
translational value in modelling progressive neurodegeneration in the
closest species related to humans, mimicking effects on behaviour and
pathophysiology.
With the use of the novel research tools, screening for candidate
agents against neurodegeneration can be carried out relatively rapidly,
where it remains important to refine and strictly orient experimental
measures and designs for clinical translation (Camus et al., 2015). As
successful translation of research to the clinical domain has suffered
from poor experimental designs and methodological approaches in
animal studies (Hackam, 2007; Martic-Kehl et al., 2012; Van Der Worp
et al., 2010), which has led to exaggerated findings (Button et al., 2013;
Hackam, 2007; Ioannidis, 2008; Van Der Worp and Macleod, 2011), the
use of animal models in PD can benefit by strictly adhering to rando-
misation, treatment/assessment blinding and sample size calculated
designs (Bebarta et al., 2003; Button et al., 2013; Perel et al., 2007; Van
Der Worp et al., 2005), to avoid poor data reproduction and ineffective
translational progress (Hackam, 2007; Hackam and Redelmeier, 2006;
Sena et al., 2007; Van Der Worp et al., 2010). Along these lines, tech-
nical approaches in the range of animal models can value from stan-
dardising certain research methods. In the new models for example,
consistency in experimental factors, such as rodent strain, serotype,
promotor, vector (lentivirus vs. adenovirus), aggregates of wild-type or
forms of mutated/phosphorylated of α-synuclein and titre for sample
preparations, are likely to enhance experimental data reproducibility.
Indeed, differences in research techniques have so far shown incon-
sistencies in neuropathology in rodent models, where dopaminergic
neuron loss has been reported to range from extensive (~ 70%)
(Decressac et al., 2012a; Decressac et al., 2012b) to moderate (~ 30%)
(Mcfarland et al., 2009; Recchia et al., 2008).
Moreover, experiments using the most relevant animal model are to
be ideally designed with similar or exact endpoints/outcomes to those
used in patients. These include the use of biomarkers for measuring
neurodegeneration (Delatour et al., 2006; Jack et al., 2009) i.e. in vivo
SPECT imaging measurement of [99mTc]-TRODAT-1 binding (Fernagut
et al., 2010; Meissner et al., 2003; Prunier et al., 2003). As such, proof
of concept animal trials, especially in the range of new animal models
of PD may mitigate the risk of taking a preclinical compound to clinical
trials.
5. Developing animal models of disease pathogenesis
With the new methods for developing drug candidates, the tech-
nological approaches employed also provide for greater ways for un-
covering neurological disease pathogenesis. The nature of disease de-
velopment in new PD pathogenesis models e.g. > 9 months in LB-
injected nhps or > 4 months in AAV1/2-huA53T injected nhps,
W.K.D. Ko, E. Bezard
presents a significant time frame for deciphering neurodegeneration
across brain regions that may relate to non-motor impairments during
the pre-motor symptomatic phase (Jellinger, 2011), simultaneously
giving translational value for development of such therapies. Recent
studies have been extended to investigating peripheral transmission
into the CNS, which may help elucidate unknown processes in prion-
like pathogenesis like site-specific initiation in anatomical regions i.e.
olfactory bulb and internal plexuses. In wild-type mice, Rey et al.
(2013) showed the injection of molecular species of recombinant
human α-synuclein (monomeric, oligomeric and fibrillary forms) into
the olfactory bulb, rapidly spread to interconnected brain regions in-
cluding anterior olfactory bulb, frontal cortex, striatum and amygdala
(Rey et al., 2013). In rat studies, the injection of rAAV serotype ex-
pressing human wild type α-synuclein into the left vagus nerve in rats
caused expression of human α-synuclein in the medulla oblongata and
caused a caudo-rostral spread to interconnected brain regions (Ulusoy
et al., 2013). In addition to this, pathogenic species in the form of
human α-synuclein from SN of PD patients and distinct recombinant α-
synuclein forms (including monomers, oligomers and fibrils) can be
seen to pass into the CNS through the vagal nerve following injection
into the intestinal walls of rats (Holmqvist et al., 2014). The gastro-
intestinal route of entry for pathogenic species has also been previously
investigated using the environmental toxin rotenone. Following the
Braak hypothesis, peripheral transmission was determined through in-
tragastric administration of retonone in mice, which caused α-synuclein
pathology in both the enteric nervous system and CNS (Pan-Montojo
et al., 2010). These new insights into the pathogenic basis of PD present
greater opportunities for developing disease modifying strategies,
whereby specific treatments may be designed and directed to key
anatomical regions for effectively preventing α-synuclein pathology
and subsequent disease progression.
Recent breakthroughs in techniques for site–specific gene editing
and transfection is also paving a new way forward for studying disease
progression in nhps (Yang et al., 2008). It has been demonstrated that
intravenous injection of adeno-associated virus serotype 9 variant
(scAAV9) in newly born macaques (post-natal day 1) mediates effica-
cious neuronal gene transduction in brain areas such as the cortex,
midbrain, hippocampus and cerebellum (Dehay et al., 2012). Further
efforts are being made for homogenous gene expression in the cortex
and deep brain structures using in utero delivery of viral vectors to
monkey embryos, which have so far demonstrated widespread neuronal
transduction with scAAV2/2-GFP (Bourdenx et al., 2015a), offering the
potential for mutant gene expression for the study of neurological dis-
orders. In addition, specific gene editing techniques in one cell embryos
for modification of the monkey genome has been shown with the
CRISPR/Cas9 system allowing for two gene modifications (Niu et al.,
2014). Transcription activator-like effector nuclease (TALEN)-mediated
gene modification has also been employed in macaque species for
mutation of methyl-CpG binding protein 2 (MECP2) gene in modelling
Rett syndrome (Liu et al., 2014). Recent data has shown that the mi-
croinjection of MECP-2-targeting TALEN plasmids to nhp zygotes can
produce monkeys that exhibit autistic-like behaviours (repeated cir-
cling, increased stress response and reduced social interactions), as well
as germline transmission of transgene to F1 progeny, modelling general
phenotypes seen in patients (Liu et al., 2016). These studies to date
demonstrate the efficient use of genetic engineering techniques in nhps
for investigating disease pathogenesis and their potential use for de-
velopment of new therapies.
6. Preclinical tests of potential disease modifying agents
Several studies to date have utilised novel PD pathogenesis animal
models for testing candidate agents in preventing α-synuclein pa-
thology. In a series of experiments by Tran et al. (2014), α-synuclein
monoclonal antibodies for misfolded α-synuclein (Syn211 and Syn303)
were shown to reduce LBs and LNs pathology, blocking α-synuclein PFF
176
Experimental Neurology 298 (2017) 172–179
entry and cell-to-cell transfer in primary hippocampal cell cultures
(Tran et al., 2014). It was also demonstrated that repeated systemic
injection of Syn303 at 10 mg/kg in PFF-inoculated wild-type mice at-
tenuated SN dopaminergic degeneration and accumulation syn506-
positive aggregates compared to control IgG-treated mice, where be-
havioural deficits in grip strength and motor coordination were also
improved in the group treated with α-synuclein monoclonal antibodies
(Tran et al., 2014). Another novel candidate agent currently being
tested is the specific oligomer modulator named Anle138b, a compound
discovered through high-throughput screening. So far, in vivo tests
have demonstrated that Anle138b has blood brain barrier permeability
targeting the pathological aggregation process of pathological α-synu-
clein oligomers, binding specifically to aggregated α-synuclein (Wagner
et al., 2013). Following treatment in the LB-seeding mouse model of PD,
a reduction of oligomer accumulation and slower disease progression
has been reported. This compound is now being tested in LB-injected
nhps and has so far demonstrated a promising pharmacokinetic profile.
Further therapies in stages of development are vaccinations with short
peptide affitopes® for preventing α-synuclein aggregation by targeting
the C-terminus region of α-synuclein, which has shown efficacy in
transgenic mouse models for decreasing α-synuclein oligomers in axons
and synapses and reduced loss of dopaminergic neurons (Mandler et al.,
2014; Masliah et al., 2011). The above mentioned studies together
demonstrate the active shift in development of disease modifying
therapies using the range of novel animal models of PD pathogenesis, in
order to fulfil the major unmet clinical need in the treatment of neu-
rological disorders.
7. Conclusions
The recognition of the α-synuclein as the major component in the
pathogenesis of PD has given way to newly developed animal models
allowing for cutting-edge research studies in screening novel agents
with disease modifying actions. These novel disease models of patho-
genesis include the use of two established methods that are viral vector
induced α-synuclein expression and LB seeding, which replicate the
progressive substantial loss of neurodegeneration and the self-propa-
gating spread of pathogenic α-synuclein. Although these disease models
require further predictive validity for disease modifying drug devel-
opment, it remains a great leap towards developing a translational
platform for achieving successful clinical outcomes.
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