Free Radical Biology and Medicine 62 (2013) 132144

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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Review Article

Parkinson disease: from pathology to molecular disease mechanisms

David T. Dexter a, Peter Jenner b,n
a
Parkinsons Disease Research Group, Centre for Neuroinammation & Neurodegeneration, Division of Brain Sciences, Faculty of Medicine, Imperial College
London, Hammersmith Hospital Campus, London, UK
b
Neurodegenerative Diseases Research Group, Institute of Pharmaceutical Science, School of Biomedical Sciences, Kings College London, London SE1 9NH, UK

a r t i c l e i n f o a b s t r a c t

Available online 4 February 2013 Parkinson disease (PD) is a complex neurodegenerative disorder with both motor and nonmotor
Keywords: symptoms owing to a spreading process of neuronal loss in the brain. At present, only symptomatic
Parkinson disease treatment exists and nothing can be done to halt the degenerative process, as its cause remains unclear.
Genes Risk factors such as aging, genetic susceptibility, and environmental factors all play a role in the onset
Oxidative stress of the pathogenic process but how these interlink to cause neuronal loss is not known. There have been
Mitochondrial dysfunction major advances in the understanding of mechanisms that contribute to nigral dopaminergic cell death,
Protein handling including mitochondrial dysfunction, oxidative stress, altered protein handling, and inammation.
Inammation However, it is not known if the same processes are responsible for neuronal loss in nondopaminergic
Neuroprotection
brain regions. Many of the known mechanisms of cell death are mirrored in toxin-based models of PD,
Free radicals
but neuronal loss is rapid and not progressive and limited to dopaminergic cells, and drugs that protect
against toxin-induced cell death have not translated into neuroprotective therapies in humans. Gene
mutations identied in rare familial forms of PD encode proteins whose functions overlap widely with
the known molecular pathways in sporadic disease and these have again expanded our knowledge of
the neurodegenerative process but again have so far failed to yield effective models of sporadic disease
when translated into animals. We seem to be missing some key parts of the jigsaw, the trigger event
starting many years earlier in the disease process, and what we are looking at now is merely part of a
downstream process that is the end stage of neuronal death.
& 2013 Elsevier Inc. All rights reserved.

Contents

Dening Parkinson disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Motor and nonmotor symptoms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Spreading pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Current approaches to treatmentno cure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Risk factors for developing PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Predisposing factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Protective factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Genetics of PDgene mutations and genome-wide association studies (GWAS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Mechanisms of neurodegeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Oxidative stress in Parkinson disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Altered mitochondrial function in PD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Altered proteolysis in PDproteasomal and lysosomal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Inammatory change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Excitotoxic mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Abbreviations: SNc, substantia nigra pars compacta; PD, Parkinson disease; NMS, nonmotor symptoms; MAO-B, monoamine oxidase-B; MPP , 1-methyl-4-phenylpyridinium;
MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 6-OHDA, 6-hydroxy dopamine; GBA, glucocerebrosidase; STN, subthalamic nucleus; DBS, deep brain stimulation; LPS,
lipopolysaccharide; UCH-L1, ubiquitin carboxyl-terminal hydrolase L1; PINK1, phosphatase and tensin homolog-inducible kinase 1; LRRK2, leucine-rich repeat kinase 2; GWAS,
genome-wide association studies; mtDNA, mitochondrial DNA; PGC-1a, peroxisome proliferator-activated receptor g coactivator-1a; UPS, ubiquitinproteasome system; LAMP2A,
lysosome-associated membrane protein type 2A; HLA, human leukocyte antigen; IL, interleukin; TNF-a, tumor necrosis factor-a
n
Corresponding author. Fax: 44 20 7848 6034.
E-mail address: peter.jenner@kcl.ac.uk (P. Jenner).

0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.freeradbiomed.2013.01.018
 D.T. Dexter, P. Jenner / Free Radical Biology and Medicine 62 (2013) 132144 133

Bringing it all together? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Translation into animal models of PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Toxin relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Gene relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
From pathogenesis to neuroprotection? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Dening Parkinson disease

Parkinson disease (PD)1 is the second most common neurode-
generative disorder after Alzheimer disease, with prevalence in
industrialized countries of approximately 0.3% of the population.
This rises with age from 1% in those over 60 years of age to 4% of
the population over 80, illustrating the effect of aging. The mean
age of onset is approximately 60 years; however, 10% of cases are
classied as young onset, occurring between 20 and 50 years of
age, which may represent a distinct disease group. PD is more
prevalent in men than in women, with reports of ratios of 1.1:1 to
almost 3:1 being quoted [135], which may be attributable to the
protective effects of estrogen in women [121]. The socioeconomic
cost of PD is high and in the United Kingdom is estimated to be
approximately 3.3 billion. In the United States, the cost per
patient per year is around $10,000, with a total economic burden
of $23 billion. The greatest proportion of cost comes in the later
stages of the illness for inpatient care and nursing homes and far
less for medication [27].
This review aims to provide a current overview of the clinical,
neuropathological, and biochemical features of PD, the genetic
components of the disease, and risk factors for its development in
relation to the pathogenic mechanisms thought to be involved in
neuronal death. Currently drug treatment provides only sympto-
matic relief and we explore how knowledge of the pathogenic
processes and the use of experimental models of PD interlink
to assist in the search for neuroprotective/neurorestorative
treatments.
Motor and nonmotor symptoms
Impaired motor function is classically used to make a clinical
diagnosis of PD. The main features are bradykinesia, rigidity,
tremor, and postural instability with an asymmetric onset spread-
ing to become bilateral with time. Other motor features include
gait and posture changes that manifest as festination (rapid
shufing steps with a forward-exed posture when walking),
speech and swallowing difculties, and a masklike facial expres-
sion and micrographia [58]. A good response to dopaminergic
medication is conrmatory of the diagnosis. Although this has
been the classical textbook description of PD, more recently it has
become recognized as a more complex illness encompassing both
motor and nonmotor symptoms (NMS), such as depression, sleep
disturbance, sensory abnormalities, autonomic dysfunction, and
cognitive decline [74]. NMS affect all patients with PD, the
frequency of which increases with disease severity, with late-
stage patients exhibiting 610 NMS. NMS create the biggest
demand on clinical resources, they are poorly diagnosed and
treated and they are the major determinant of disease outcome,
increasing disability, poor quality of life, and entry into long-term
care [13]. Whereas the causes of motor dysfunction in PD are
reasonably well understood (see later), the cause of NMS in
PD remains poorly researched and they may largely relate to
pathology outside of the basal ganglia.
From the perspective of this review, the most important
feature of NMS is that some, for example, olfactory decits,
constipation, rapid-eye-movement sleep behavior disorder, and
depression, may precede the onset of motor symptoms by many
years (although they can occur at the same time as motor
symptoms or follow the onset of motor abnormalities) [105].
NMS may in the future be used for the early diagnosis of PD,
enabling neuroprotective strategies to be introduced at an early
stage, and studies of large populations of apparently normal older
individuals are ongoing at this time to enable such early detection
to occur [6,143]. However, they provide another and perhaps vital
clue to the search for the pathogenic processes that underlie PD,
as they suggest that it is a disease of both the peripheral and the
central nervous systems and that it is a multisystem disorder that
spreads with time, affecting movement only at a relatively late
stage in its course, and may thus be a target for early diagnosis
and identication of at-risk populations. Current dopamine repla-
cement strategies for treating PD are effective against the motor
features of PD but are largely ineffective at addressing NMS.
Hence a greater effort needs to be made not only in under-
standing the molecular mechanisms that cause NMS but also in
how to treat them.
Spreading pathology
Neuronal loss in the substantia nigra pars compacta (SNc) and
the subsequent loss of striatal dopamine content are accepted as
being responsible for the classical motor features of PD. The
neuropathological diagnosis of PD requires the detection of
marked dopaminergic neuronal loss in the SNc and the presence
of Lewy bodies, eosinophilic inclusions consisting of a dense core
surrounded by a pale-staining halo of radiating laments. The role
of the Lewy body in pathogenesis remains unknown, but the
discovery that misfolded a-synuclein is a major component of the
radiating laments and is also present in neuronal processes as
Lewy neurites has altered views on their formation and role in
neuronal loss and has led to a major shift in thinking about the
onset and progression of the disease process from a pathological
perspective and to the classication of PD as a synucleinopathy
[21]. However, the neuropathological picture of PD has been
known to be more widespread for many decades, with many
nondopaminergic nuclei affected, including the locus coeruleus,
reticular formation of the brain stem, raphe nucleus, dorsal motor
nucleus of the vagus, basal nucleus of the Meynert, amygdala, and
hippocampus [59]. Importantly, all of these nuclei degenerate
with Lewy body pathology, suggesting a pathogenic process in
common with that occurring in the SNc. It is the Lewy body
pathology in these nondopaminergic areas that then results in
some NMS. For example, hyposmia is associated with the pre-
sence of Lewy bodies and Lewy neurites in the olfactory bulb and
brain centers such as the amygdala and perirhinal nucleus
[78,162]. But it is the presence of Lewy bodies in peripheral
tissues that takes the pathology associated with PD to another
level. Orthostatic hypotension in PD is associated with
134 D.T. Dexter, P. Jenner / Free Radical Biology and Medicine 62 (2013) 132144
sympathetic autonomic denervation of the heart with the pre-
sence of Lewy bodies [28,112]. Lewy bodies are found in the
myenteric plexus, reecting the alterations in gastrointestinal
motility occurring in PD. However, there may be a connection
between changes originating in the periphery. For example,
constipation as a common NMS is associated with neuronal loss
and the presence of Lewy bodies in the dorsal motor nucleus of
the vagus, which provides parasympathetic innervation to the
stomach and intestine [10,20,159], and changes such as these can
be initiated by injection of proteasomal inhibitors into the
stomach wall and retrograde degeneration of the dorsal motor
nucleus, at least in rats [102].
Indeed, what has become clear is that pathology in PD does not
start in the SNc, but rather Lewy body pathology and the deposi-
tion of a-synuclein are proposed to originate in the olfactory bulb
and lower brain stem, from where they spread in stages to involve
the midbrain, eventually spreading to cortical regions. A staging
of the caudorostral spread of Lewy body and a-synuclein patho-
logy in PD was rst proposed by Braak and colleagues in 2003
[11], when they proposed that a-synuclein deposition begins in
the dorsal motor nucleus of the vagus (stage 1), from where it is
thought to proceed in an upward direction via the pons (stage 2)
to the midbrain (stage 3) and from there to the basal prosenca-
phalon and mesocortex (stage 4), nally reaching the neocortex
(stages 5 and 6). It is only at stage 3 after extensive loss of
dopaminergic neurons in the SNc that motor features of PD
become apparent. Although it is recognized that not all PD cases
exactly follow this formal pattern of spread of PD pathology [68],
it is now widely accepted that spreading pathology does occur in
PD. This raises the important concept that PD pathology may be
propagated from one neuron to another [34]. Exactly how this
occurs is not known, but altered a-synuclein released by an
affected neuron that is taken up by an adjacent unaffected
neuron, or direct transfer between neurons, could act as a seed
in a prion-like mechanism to perpetuate the cycle of a-synuclein
misfolding and the spread of a-synuclein pathology [23]. Although
this hypothesis is controversial some groups have demonstrated
such a seeding mechanism in cell culture, animal models, and
fetal mesencephalic cells transplanted into the PD striatum [23].
If such a hypothesis is correct, it will have a major impact on the
molecular understanding of pathogenesis in PD.
Current approaches to treatmentno cure
The treatment of PD has not changed substantially in the past 30
years, with dopamine replacement therapy employing L-dopa and
dopamine agonists as the mainstay, supported by the use of a series
of enzyme inhibitors, namely peripheral decarboxylase inhibitors,
catechol-O-methyl transferase inhibitors, and monoamine oxidase-B
(MAO-B) inhibitors. These dopaminergic medications now come in a
variety of oral, intraduodenal, intravenous, subcutaneous, and trans-
dermal forms, providing differing degrees of drug delivery. Outside of
the dopaminergic arena only anticholinergics and the weak N-
methyl-D-aspartate receptor antagonist amantadine have had any
use. But these are all symptomatic approaches to treating the motor
decits of PD with little effect on nonmotor symptoms and no proven
effect on disease progression.
There have been several attempts to determine whether
current therapy has any effect on the disease process. The
dopamine agonists ropinirole and pramipexole show neuropro-
tective actions in preclinical models of PD of dopaminergic cell
loss, but in clinical trial (REAL-PET; the Comparison of the Agonist
Pramipexole versus Levodopa on Motor Complications of Parkin-
sons DiseaseCALM-PD) there was no slowing of progression of
motor symptoms, although imaging studies suggested a lower
rate of nigral cell degeneration compared to that seen in patients
treated with L-dopa [117,161]. However, the drugs themselves
turned out to affect the imaging markers employed [104]. Even in
mild PD, the early use of pramipexole showed no advantage on
disease progression over the later introduction of the drug
(Pramipexole on Underlying DiseasePROUD study) [131].
L-dopa has had a checkered career as a potential reason for
both acceleration of the cell death in PD and its slowing. For many
years, the ability of L-dopa to generate free radicals and to kill
dopaminergic cells in culture tainted the drug with a neurotoxic
label, but overall, based on a range of in vivo studies in animals,
postmortem studies in humans, and clinical experience, this does
not seem to be true, although the debate continues [1,108,120].
From the Earlier versus Late Levodopa (ELLDOPA) clinical trial,
among others, early use of L-dopa seems to slow disease progres-
sion based on clinical rating scales, but not when using imaging
endpoints [26].
It has been the MAO inhibitors selegiline and rasagiline,
however, that have attracted most attention as potential disease
modiers. It was the ability of these drugs to prevent 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity to nigral
dopaminergic neurons through inhibition of 1-methyl-4-phenyl-
pyridinium (MPP ) by blocking MAO-B activity [60]. However,
both drugs also are associated with a range of actions that
encompass antiapoptotic actions, antioxidant effects, antigluta-
matergic effects, and neurotrophic actions, among others, that
underlie a belief in their ability to alter the rate of loss of
dopaminergic neurons [62,84]. Both selegiline and rasagiline have
been examined in detail in clinical trials to assess whether disease
modication occurs in humans. The Deprenyl and Tocopherol
Antioxidative Therapy of Parkinsonism (DATATOP) study initially
suggested that selegiline treatment could delay the need for the
introduction of L-dopa in early PD but this now seems largely due
to a symptomatic effect of the drug, although open label exten-
sion studies in other clinical trials still suggest that disease
progression is slower in those patients with PD that receive early
selegiline treatment [115,119]. Similarly, for rasagiline, the
results of the TEMPO (TVP-1012 in Early Monotherapy for
Parkinsons Disease Outpatients) and ADAGIO (the Attenuation
of Disease Progression with Azilect Given Once Daily) studies
suggested that early use had a positive effect on clinical scores
compared to later introduction of the drug, most notably in those
patients with the highest degree of motor disability [109,118].
However, so far none of the studies undertaken have convinced
the regulatory authorities to label either selegiline or rasagiline as
disease modifying.
One other approach to current therapy deserves mention,
namely, suppression of glutamatergic activity in PD. As will be
explained later, excitotoxicity may contribute to the pathogenic
process and as a consequence inhibition of glutamatergic function
may inuence disease progression. This has stimulated new
interest in the actions of amantadine, which is currently used
largely to suppress dyskinesia in PD. However, there is anecdotal
evidence that treatment with amantadine from the beginning of
therapy may lead to a better outcome than would otherwise be
expected, and new clinical studies are being started with the drug
to provide evidence-based medicine for its effects.
Risk factors for developing PD
Predisposing factors
Age represents the biggest predisposing factor for PD (young-
onset cases may form a different patient population) for the
majority of individuals developing the illness. However, it
 D.T. Dexter, P. Jenner / Free Radical Biology and Medicine 62 (2013) 132144 135

remains unknown whether it is chronological age or the aging
process that is responsible [69]. Loss of estrogen production in
women with age may also remove a protective effect and there is
some evidence that early menopause, hysterectomy, or removal
of the ovaries increases risk in women to that seen in men
[121,123]. Genetic causes are becoming more common as gene
hunting continues (see later) and may account for up to 40% of
those at risk in the population from autosomal dominant, reces-
sive, and susceptibility genes [41]. However, there are also
environmental factors that contribute to the risk of developing
PD [148].
The discovery of the toxicity of MPTP to nigral dopaminergic
cells generated interest in environmental causes of the disease,
although no other MPTP-like toxins have been discovered [53].
The closest has been the ability of the structurally related
paraquat, a commercial weed killer, to destroy dopaminergic cells
in rodents and this links nicely to the increased risk of developing
PD that repeatedly emerges from epidemiological studies,
although there is no association with any specic compound
[90,149,150]. The same might apply to the complex I inhibitor
rotenone as a constituent of derris, which is commonly used by
gardeners as a pesticide, but no cases have specically been
reported [7]. In contrast, exposure to annonacin derived from
fruits in Guadeloupe was shown to produce parkinsonism [75].
Other environmental causes include solvent exposure (n-hexane,
methanol), carbon monoxide poisoning, hydrogen sulde intox-
ication, and perhaps manganese, although the resulting syndrome
involves striatal cell degeneration and dystonic features and
there now seems to be no increased risk in welders, who can be
exposed to manganese vapor [70].
One last factor worthy of note is head trauma, as it has
repeatedly emerged as borderline signicant for increased risk
of PD in population studies. A recent study in ex-National Football
League players in the United States suggests that head trauma
does increase the risk of developing PD [81] and so this may be a
more relevant factor than previously thought, although contro-
versial [128].
Protective factors
Factors that protect against the development of PD may reveal
a lot about underlying pathogenic mechanisms. Perhaps the
protective effect of cigarette smoking is the best documented
and most reproducible of observations [72]. Nicotine is the
immediate candidate for a neuroprotective action and there is
currently interest in developing nicotinic agonists, for example, a-
7 nicotinic agonists, for both symptomatic treatment and neuro-
protection [122]. However, cigarette smoke contains more than
4000 components, so other agents may contribute to the protec-
tive effects, for example, smoking decreases MAO-B activity in
brain [165]. Additionally, it may be that a genetic disposition
against reward-seeking habits, such as cigarette smoking, may
prevail in those who go on to have a clinical diagnosis of PD.
Caffeine intake also appears protective against the development
by negative reports on these compounds [5,146,148,169]. Perhaps
surprisingly, but of enormous potential interest, is a role for
exercise and metabolic factors associated with the risk of developing
PD [169].
Genetics of PDgene mutations and genome-wide association
studies (GWAS)
Investigation of familial PD has so far revealed at least 17
autosomal dominant and autosomal recessive gene mutations
responsible for variants of the disease [47]. These include
a-synuclein mutations and triplication, parkin, ubiquitin carboxyl-
terminal hydrolase L1 (UCH-L1), DJ-1, phosphatase and tensin
homolog-inducible kinase 1 (PINK1), leucine-rich repeat kinase 2
(LRRK2), and glucocerebrosidase (GBA) (see Table 1). Of these
parkin and LRRK2 are probably the most common genetic link to
young-onset and late-onset PD, respectively, whereas GBA muta-
tions may be the most common risk factor. Autosomal dominant
mutations are typied by mutations in a-synuclein and LRRK2.
Although a-synuclein mutations are only rarely encountered,
their discovery led directly to a-synuclein being identied as a
major component of Lewy bodies and Lewy neurites and the
labeling of PD as a synucleinopathy, underpinning much of the
consensus on the nal stages of neuronal loss in PD being related
to altered protein aggregation [33,35]. Mutations in LRRK2 seem
to alter kinase/GTPase of this mixed lineage-like kinase found in
cytoplasm and outer membrane of mitochondria [17,39]. LRRK2
interacts with another PD-related protein, parkin, and mutant
LRRK2 induces apoptotic cell death in cultured neurons. Auto-
somal loss of function mutations include those in the ubiquitin
E3 ligase parkin, which in combination with the ubiquitin-
conjugating enzyme causes the attachment of ubiquitin as a
marker on proteins destined for destruction by the proteasome.
Additionally mutations in the mitochondrial PINK1 protect cells
from mitochondrial stress/dysfunction, and the rarest mutation,
in the redox-sensitive chaperone DJ-1, protects cells against
oxidative stress [17,39]. Familial forms of PD and the associated
gene mutations currently account for approximately 10% of cases
and they have distinct clinical and pathological phenotypes.
However, there is sufcient overlap with sporadic PD that
investigations into such gene mutations have revealed important
clues as to the molecular mechanisms that underlie the disease
process in PD (see Fig. 1). Indeed, many of these familial PD
neurodegeneration mechanisms overlap with the pathogenic
mechanisms discovered in sporadic PD, such as mitochondrial
dysfunction, oxidative stress, and altered protein handling (see
below and Fig. 1) [47]. Furthermore a combination of advances
in genetic analysis techniques, the ability to genotype cost
Table 1
Common gene mutations causing familial Parkinson disease.
Gene Locus/disease Mode of inheritance Age of onset (years)

of PDmore so in men than in women and more so in those not SNCA PARK1/4 Autosomal dominant 2085
using hormone replacement therapy [114]. Caffeine has been LRRK2 PARK8 Autosomal dominant 3279
demonstrated to exert neuroprotective effects in a variety of GRN FTDP-17 Autosomal dominant 4583
experimental models of PD, possibly via the involvement of the MAPT FTDP-17 Autosomal dominant 2576
DCTN1 Perry syndrome Autosomal dominant 3561
adenosine A2a receptor [14]. This has highlighted the potential
PRKN PARK2 Autosomal recessive 1672
neuroprotective effect of A2a antagonists, such as istradephylline PINK1 PARK6 Autosomal recessive 2040
and preladenant, which are currently under development as DJ1 PARK7 Autosomal recessive 2040
symptomatic treatments for PD. FBXO2 PARK15/PPS Autosomal recessive 1019
Other potentially protective factors are not so well dened NR4A2/NURR1 Unknown 4567
POLG Unknown 2026
but there have been suggestions of an effect of antihypertensives
(notably calcium antagonists), nonsteroidal anti-inammatory FTDP-17, frontotemporal dementia with parkinsonism linked to chromosome 17;
drugs, and antilipidemics, among others, although all are balanced PPS, palidopyramidal syndrome.
136 D.T. Dexter, P. Jenner / Free Radical Biology and Medicine 62 (2013) 132144
Parkin
-synuclein
Ubiquitin
Misfolded
protein
Dopaminergic neuronal
environment
Iron
Oxidative
stress
Cytokines
ROS
Lack of
growth
Activated factor
Microglia support
Innate
Inflammation
Fig. 1. Key molecular mechanisms that are widely accepted to contribute to the neurodegenerative process in dopaminergic neurons in the substantia nigra in Parkinson
disease. Blue double-headed arrows indicate molecular mechanisms that may not only be toxic in their own right but importantly may also inuence other molecular
mechanisms known to be features in Parkinson disease. Double helix structures identify some of the common gene mutations found in familial PD and brown arrows
indicate where the altered protein may interfere with cell function and overlap with known mechanisms of cell death in sporadic Parkinson disease. ROS, reactive oxygen
species. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)
effectively, and the formation of large patient sample consortia,
for example, the International PD Genomics Consortium, has
facilitated highly powered GWAS in idiopathic PD, which have
revealed some 14 risk gene loci for PD, including a-synuclein,
LRRK2, human leukocyte antigen (HLA), and tau [55,106] (see
Table 2). Although the presence of such risk gene loci is not a
predictor for the development of PD, they highlight the fact that
PD is a complex disease involving both genetic risk and environ-
mental factors in its etiology.
Mechanisms of neurodegeneration
Oxidative stress in Parkinson disease
Oxidative stress remains a cornerstone of the concepts under-
lying the loss of dopaminergic neurons in PD. Since the 1980s
there has been an exponential growth in publications that
implicate the formation of reactive oxygen species as a nal step
in neuronal death of whatever origin. Starting from the idea of
free radical production resulting from increased chemical and
enzymatic oxidation of dopamine, through to the mechanism of
action of toxins such 6-hydroxydopamine (6-OHDA) and para-
quat, to evidence from postmortem and clinical investigations,
oxidative stress and the resulting oxidative damage have emerged
largely unscathed [36,61,63,64,141]. Whereas direct evidence for
increased free radical generation is almost impossible to detect,
the potential inducers of oxidative stress and markers of its effects
make a substantial case for its occurrence. Enhanced lipid peroxida-
tion (malondialdehyde, lipid hydroperoxides, 4-hydroxynonenal, and
advanced glycation end products), protein oxidation (protein carbo-
nyls), and DNA oxidation (8-hydroxyguanosine) in the SNc in PD and,
perhaps, more generally throughout the body, all point in the same
direction. More recently, the nding of a negative correlation between
UCH-L1
Glutamate
ExcitotoxicityParkin
Proteasome
Proteasome Ca2+
Dysfunction
PINK1
Aggregated
Protein
Mitochondria DJ-1
Energy
Apoptosis
depletion
Neurodegeneration
plasma urate levels and disease progression in PD has raised the
spectrum of altered antioxidant activity that reects the decrease
previously reported in reduced glutathione levels (along with changes
in catalase and glutathione peroxidase) [3,4,15,36,138].
The source of the oxidative stress remains hotly contested, with
both neuronal and glial sources being implicated, but there seems
little doubt that the most likely contributor is increased radical
formation originating from mitochondria (see later) and, perhaps,
endoplasmic reticulum. Altered accumulation of iron in SNc, changes
in calcium channel activity, altered proteolysis (proteasomal and
lysosomal), changes in a-synuclein aggregation, and the presence of
mutant proteins (for example DJ-1) are all examples of how oxidative
stress might be induced in PD [126,133,146,152]. This emphasizes the
diverse nature of the causes of cell death in this illness but also the
common nal pathways through which neuronal loss occurs. There is
nothing specic about a role for oxidative stress in PD, as it also
contributes to cell degeneration in a variety of other disease states.
However, it may offer the opportunity to inuence disease progres-
sion although attempts to date have been unsuccessful in clinical
trials despite a multitude of encouraging reports from both in vitro
and in vivo models of PD. However, ongoing studies with centrally
active iron-chelating drugs and the elevation of urate levels through
administration of uric acid or inosine administration offer new hope.
Altered mitochondrial function in PD
Mitochondrial involvement in cell death in PD has returned to
center stage and provides part of a unifying concept of how
neuronal loss occurs in both sporadic and inherited disease.
The discovery of the neurotoxicity of MPTP through its metabolite
MPP identied a role for the inhibition of complex I in pathogenesis
that is shared by other substances toxic to dopaminergic neurons,
including rotenone and annonacin [32,76,124]. Very quickly, an
inhibition of complex I was shown to be present in the SNc that
 D.T. Dexter, P. Jenner / Free Radical Biology and Medicine 62 (2013) 132144
Table 2
GWAS risk genes for Parkinson disease.
Locus SNP
MAPT rs2942168
SNCA rs356221
BST1 rs4698412
LRRK2 rs1491942
GAK rs11248051
HLA-DRB5 rs3129882
ACMSD rs10928513
STK39 rs2102808
HIP1R rs10847864
MCCC1/LAMP3 rs11711441
SYT11 rs34372695
GPNMB rs156429
STX1B rs4889603
FGF20 rs591323
was tissue and disease specic, although also detectable in platelets
and muscle in PD [85,129,130,132]. A key Krebs cycle enzyme, a-
ketoglutarate dehydrogenase, was also shown to be impaired [103].
The construction of cybrids using mitochondrial DNA (mtDNA) from
patients with PD has clearly demonstrated the encoding of the
complex I defect [38], but no consistent alterations in mtDNA have
been demonstrated, although deletions associated with complex IV
and oxidative damage occur in PD. Perhaps importantly, only about
30% of patients with PD have a clear complex I defect, suggesting,
perhaps, that they form an important subgroup within the disease
that offers a specic target for interference with the disease process.
More recently, interest in the role of mitochondria has stemmed
from genetic investigations in familial PD. Notably mutations in a-
synuclein, parkin, PINK1, and DJ-1 and perhaps LRRK2 have been
associated with altered mitochondrial function [129,130,133] (see
Fig. 1). These mutations can lead to altered protein localization in
mitochondria in PD, abnormalities in mitochondrial structure and
function, and a decrease in complex I assembly and activity. Loss of
function of, notably, DJ-1, but also parkin and PINK1, decreases
mitochondrial protection against oxidative stress, which in turn
increases mitochondrial dysfunction. Another important role for
parkin and PINK1 is in the turnover of mitochondria by autophagy,
specically mitophagy; they act in tandem to regulate this
process. This may be critically important in PD, in which autophagy
seems impaired, reducing the cells ability to remove damaged
mitochondria.
GWAS, laser-captured human dopaminergic neuron studies, and
SNc transcriptome studies have added to the evidence pointing to the
key role of decits in the mitochondrial electron transport chain in PD
[25]. Genes controlling cellular bioenergetics that are expressed in
response to peroxisome proliferator-activated receptor g coactivator-
1a (PGC-1a) are underexpressed in PD, whereas PGC-1a expression
is upregulated in broblasts from PD patients [113,137,168]. This has
important functional consequences, as the loss of dopaminergic
neurons in primary culture induced by mutant a-synuclein or
rotenone was prevented by activation of PGC-1a and associated
with increased expression of nuclear-encoded subunits of the mito-
chondrial respiratory chain.
Altered proteolysis in PDproteasomal and lysosomal
The presence of multiple proteins in Lewy bodies, most notably
a-synuclein, led to the idea that the catabolism of unwanted,
damaged, or mutated proteins might be disrupted in PD, leading
to cellular aggregation and neuronal death [31,136,140]. This led
to investigation of the roles of the ubiquitinproteasome system
(UPS) and lysosomes in pathogenesis in PD. UPS involvement was
137
Protein
Microtubule-associated protein tau
a-Synuclein
Bone marrow stromal cell antigen 1
Leucine-rich repeat kinase-2
Cyclin G-associated kinase
Major histocompatibility complex, class II, DR b5
Aminocarboxymuconate semialdehyde decarboxylase
Serine threonine kinase 39
Huntingtin-interacting protein 1-related
Lysosomal-associated membrane protein 3
Synaptotagmin-11
Glycoprotein (transmembrane) NMB
Syntaxin 1B
Fibroblast growth factor 20
supported by the discovery of mutations in parkin and UCH-L1, which
function as a ubiquitinprotein ligase and in ubiquitin recycling.
Examination of the 26S proteasome in PD revealed selective changes
in its catalytic activity and composition in the SNc that were
associated, perhaps wrongly, with impaired degradation of a-synu-
clein [97,100,151]. In cell culture and after direct intracerebral
injection, proteasomal inhibitors, such as lactacystin and proteasome
inhibitor 1 (PSI), were found to selectively destroy dopaminergic
neurons [88,94,98,163]. In cell culture, the initiation of cell death
using proteasomal inhibitors led to a cascade of events involving
increases in oxidative and nitrative stress and damage and alterations
to mitochondrial function [50]. Indeed, there is a close association
between mitochondrial electron transport activity and the regulatory
caps of the 26S proteasome, which are ATPases. The potential role of
the UPS has been conrmed many times over and supported by
microarray and transcriptional studies on laser-captured microdis-
sected nigral dopaminergic neurons from PD [37]. However, the
concept lost credibility when a slow, progressive degeneration of
neurons in the brain that resembled that occurring in PD was claimed
after systemic administration of proteasomal inhibitors [99], but this
was never substantively reproduced (compare [73,86] with [12,167]).
As a consequence attention turned to lysosomes and to
autophagy (macro-autophagy, micro-autophagy, and chaperone-
mediated autophagy) (see [133]). Because autophagy-related
proteins and autophagosomes are present in Lewy bodies, this
seemed a viable target for disruption related to dopaminergic cell
death. Indeed, the expression of the chaperone-mediated autop-
hagy proteins lysosome-associated membrane protein type 2A
(LAMP2A) and heat shock protein 70 is reduced in SNc in PD [2].
In addition, silencing of LAMP2A activity in a dopaminergic cell
line reduced chaperone-mediated autophagy and increased the
half-life of a-synuclein. Altered handling of a-synuclein can also
be induced by disruption/mutation of the lysosomal membrane
protein ATPase (ATP13A2), which is affected in one form of
familial PD [154]. The potential involvement of altered lysosomal
function in PD was supported by studies in MPTP-treated mice
[18,19]. Toxin treatment caused a depletion of lysosomes in
dopaminergic cells secondary to the onset of oxidative stress that
led to the accumulation of autophagosomes and cellular degen-
eration. The toxicity of MPTP was attenuated by pharmacological
and genetic treatments that enhanced lysosomal formation.
Alterations in autophagy and mitophagy also occur in response
to nigral neuronal degeneration induced by 6-OHDA [87,139].
Another strand supporting lysosomal involvement in neuronal
loss in PD comes from Gaucher disease, a lysosomal storage
disorder due to mutations in the GBA gene. This leads to a
deciency of the lysosomal enzyme GCase, which catalyzes the
138 D.T. Dexter, P. Jenner / Free Radical Biology and Medicine 62 (2013) 132144
metabolism of the sphingolipid glucosylceramide to ceramide.
Gaucher mutations lead to a 20- to 30-fold increase in the risk of
developing PD and 510% of patients with PD have a GBA
mutation (see for example [156]). Recently, a GCase deciency
was reported in the substantia nigra of patients with PD and GBA
mutations but, importantly, also in those with sporadic PD [30].
Because not all individuals with GBA mutations develop PD, the
probability is that these lead to increased susceptibility to other
factors involved in the pathogenic process, such as a-synuclein
accumulation and oxidative stress occurring as a result of
altered mitochondrial function. Indeed, GCase deciency leads to
a-synuclein accumulation both in vitro and in vivo [89,164].
Inammatory change
The concept of inammatory change in the brain in PD started
with the description of activated HLA-positive microglia in SNc [92].
Subsequently, alterations in the cytokines interleukin-1a (IL-1a),
IL-1b, and tumor necrosis factor-a (TNF-a) were found in the brain
and cerebrospinal uid, and postmortem studies showed inducible
nitric oxide (NO) synthase to be present in activated microglia [45].
Inducible NO synthase is a source of NO, which in turn can react with
superoxide from glial or neuronal sources to form the highly reactive
peroxynitrite. This can nitrate proteins and other biomolecules, and 3-
nitrotyrosine adducts are found in the SNc in PD. In addition, there is
evidence for the presence of nitrated a-synuclein in Lewy bodies [31].
Microglia activation and inammatory change were thought to be a
consequence of neuronal destruction but there is evidence for a more
general systemic inammatory reaction in PD, suggesting it to be a
primary cause of neuronal loss in some cases. In addition, peripheral
inammation may enhance the adverse effects of inammatory
change occurring in the substantia nigra [43]. This point was brought
into focus by recent GWAS that showed HLA to be a risk factor for the
occurrence of PD [40].
Additionally, there seems to be some cross-talk between the
central nervous system innate inammatory response and the
peripheral immune system. Microarray gene expression studies
have identied modied neuroimmune signaling in peripheral blood
mononucleated cells from PD patients, and changes in neuroin-
ammatory markers in PD have more recently been detected by
studies demonstrating increased concentrations of IL-2 [145], TNF-a,
IL-6 [22], osteopontin, and RANTES/chemokine (CC motif) ligand 5
[125] in serum in PD. In particular, serum levels of RANTES, a
chemokine produced by activated microglia, were not only higher in
PD compared to normal subjects but signicantly correlated with
Unied Parkinsons Disease Rating Scale scores [125]. Such ndings
have supported the concept that a selective blood biomarker could
be developed and used to rene the clinical diagnosis of PD.
Multiple experimental studies have been utilized to investi-
gate the role of inammatory change in PD in greater detail.
Initially toxins that directly destroy dopaminergic neurons, such
as 6-OHDA, MPTP, and rotenone, were shown to activate micro-
glia and induce inammatory change. But it has been the use of
lipopolysaccharide (LPS) in both in vitro cellular models and
in vivo studies that focused on glial activation [44,56,57,95,96].
These studies have shown that dopaminergic neuronal loss can
occur as a direct consequence of microglial activation that is
accompanied by increased cytokine formation, increased produc-
tion of reactive oxygen and nitrogen species, and decreased
secretion of trophic factors responsible for the normal mainte-
nance of neuronal viability. A potentially important nding is that
damage after toxin application to SNc is accompanied by the
presence of monocytes, suggesting bloodbrain barrier damage
and permeability. Indeed, altered gene and protein expression for
the adhesion molecule intercellular adhesion molecule-1 occurs
in PD and in experimental models of the illness [101]. The current
consensus seems to be that a mild to moderate peripheral
inammation can exacerbate the loss of dopaminergic neurons
initiated by another toxic insult.
All of these data suggest that one way of attempting to reduce
cell loss in PD may be by reducing inammatory change, and
certainly this seems to work in experimental systems (but see
later). This may be relevant to both the initiation and the
progression of neuronal destruction irrespective of whether this
starts at the level of neurons or glia. The importance of the glial
activation in PD might lie in its long duration. In both humans and
nonhuman primates, activated microglia can be detected years
after exposure to MPTP, suggesting a propagating role in ongoing
pathogenic change [93]. Conversely, this long-term activation
may prevent microglia from performing their normal support
role for neuronal viability, for example, through the release of
trophic factors. Although the emphasis on the role of glia has
centered on activated microglia, it should be borne in mind that
astrocytosis also occurs in PD and it too may play a signicant
role in the sequence of events that lead to cell death.
Excitotoxic mechanisms
Excitotoxicity is always included in the list of pathogenic mechan-
isms that are thought to contribute to cell death in PD. However, the
degree of direct evidence for this is small. The major contributor is the
overactivity of the subthalamic nucleus (STN), which releases gluta-
mate and which innervates the substantia nigra pars compacta and
the internal segment of the globus pallidus [8,142]. In addition, there
is evidence of altered glutamatergic input from the corticostriatal
pathway. Excitotoxicity might also arise from the dysfunction of
mitochondria that occurs in PD. However, experimental studies show
that excitotoxins can cause destruction of nigral dopaminergic
neurons and that this process can be blocked by various classes of
glutamate antagonists [48,67,142,153,157,158]. Similarly, cell loss
induced by toxins acting through other mechanisms, such as 6-OHDA,
is diminished by drugs that either modulate glutamate release or
diminish its action on target cells through actions on either inotropic
or metabotropic glutamate receptors. The scenario would be that as
PD develops with the onset of neuronal loss in the SNc, the increased
activity of the glutamatergic input from the STN acts to amplify cell
death. Indeed, lesions of the STN have been shown to reduce the
loss of dopaminergic neurons normally induced by the injection of
6-OHDA into the SNc [12,99,135]. Overactivity of the glutamatergic
output pathways from the STN to the globus pallidus also contributes
to the evolution of some of the clinical symptoms of PD. Indeed, deep
brain stimulation (DBS) can correct the overactivity of the STN,
producing a clinical benet in PD. Perhaps importantly, recent
investigations utilizing DBS in PD have associated it with a lack of
progression of motor symptoms [147]. Assuming that DBS would also
decrease the overactivity of the STN pathways innervating the SNc,
this does add some support to the concept that excitotoxicity plays a
role in cell death in PD.
Bringing it all together?
In the past 20 years, there have been signicant advances in the
understanding from a mechanistic perspective of the events that lead
to the death of nigral dopaminergic neurons in PD. It is worth
remembering, however, that the pathology of PD is widespread and
that the same rigor has not been applied to looking at the underlying
causes of cell death in nondopaminergic nuclei, such as the locus
coeruleus and raphe nuclei. Indeed, there has been a presumption
that because these neuronal groups also degenerate with the appear-
ance of Lewy bodies, the same cell death sequences are operative,
but in reality, this is not known. Cartoons of the sequence of events
 D.T. Dexter, P. Jenner / Free Radical Biology and Medicine 62 (2013) 132144
that underlie nigral cell loss have been published regularly (and to
which we have added in this review) but these have not changed
much over the years in terms of the overall concepts espoused, as
there has been a trend to make new information on pathogenic
processes in PD t into the existing framework of conceptual belief.
This may or may not be correct.
Certainly the events that are commonly related to pathogen-
esis in PD t nicely togetherfor example, mitochondrial dys-
function with oxidative stress and altered proteolysis. The
evidence from gene mutations identied in inherited PD has
added to this accepted interaction and has again highlighted
mitochondrial abnormalities and altered protein handling as key
events. The general acceptance of PD as a synucleinopathy has
added to the weight of the arguments for a common thread
underlying nigral cell loss originating in both sporadic and
inherited disease [33,35]. There is also a degree of comfort
associated with containing novel information within an accepted
arena. But is this the right thing to do?
A skeptical view would be that it is not surprising that all the
major organelles of a cell are somehow involved in the demise of
the neuron. This is emphasized by a range of studies in which cell
death is initiated at a point in the accepted cycle of pathogenic
events in PD. These illustrate very nicely that it is difcult to
distinguish what is horse and what is cart. For example, if cell
death is initiated by inhibiting complex I, then this entrains
changes in oxidative and nitrative stress and proteasomal activity.
Inhibiting proteasomal activity leads to altered mitochondrial
function and increased oxidative and nitrative stress and so on,
with the same applying to the introduction of mutant parkin or
a-synuclein into cell lines [5052,79,80].
A more difcult conclusionand one that would decrease an
authors chances of publicationwould be that many of the key
events that are identied as being involved in cell death in PD and
that result from gene mutations represent separate pathogenic
pathways that dene different forms of what is currently deemed
to be PD. Indeed, the current view is that PD is not a single disease
but rather a syndrome of multiple causes and manifestations.
A simple example is the general absence of Lewy bodies in the
SNc in cases with parkin mutations [42]. Another example from
sporadic PD is that only 30% of individuals have a complex I defect
that is outside of the normal range. Our own limited investiga-
tions failed to show any correlation in postmortem nigral tissue
between iron accumulation, the extent of oxidative stress, and
mitochondrial dysfunction [65].
Perhaps in what is termed sporadic PD, we are missing key
pieces of the jigsaw and what we are observing are secondary
changes that are the result of one or more primary events that
remain unknown. It becomes increasingly possible that patho-
genic events in SNc do not originate in the brain but are the result
of some peripheral event that is then transmitted into brain,
initiating cell death that propagates itself in a stoichiometric
fashion through synucleinopathy to eventually reach the SNc and
that explains the long prodromal period and the occurrence of
nonmotor symptoms that are now an accepted part of what used
to be considered a movement disorder (see earlier).
Translation into animal models of PD
Toxin relevance
Numerous toxins are in use in experimental models of PD
largely based on their ability to destroy dopaminergic neurons
through a variety of relevant pathogenic mechanisms. In cell
culture and in primary neuronal cultures, the concentration of
toxin to which cells are exposed and the duration of that exposure
139
can be controlled, although the time course of such studies is
seldom more than a few days. In this manner, it is possible to
study the role of specic pathogenic pathways in the induction
and progression of cell death, although the environment is
entirely articial and the cells may not be representative of the
susceptibility of dopaminergic neurons in the aging brain in
humans that are affected in PD. For example, cell lines may not
have a full dopaminergic phenotype, they may not be derived
from a neuronal source, and they will not be accompanied by glial
cells. Primary cultures are derived from fetal sources, they are
immature and may be from species in which susceptibility to
toxin action is different from that in humans. Even the cell culture
medium may not be reective of the normal environment for cells
in that it may be high in iron and low in antioxidants and so
provide a highly pro-oxidant environment without the natural
antioxidant defense mechanisms being operative [16]. Never-
theless, as a primary means of assessing cell death processes,
these are highly useful techniques and capable of being manipu-
lated, for example, by gene transfection, to study both sporadic
and familial PD.
The use of toxins in in vivo models of PD is also a highly useful
way of investigating dopaminergic cell death and drug action, but
has limitations that have proved difcult to overcome (see
[9,24,91] for recent reviews; see Table 3). Most toxins require
direct intracerebral injection into the SNc, medial forebrain
bundle, or striatum. Most need to be injected in concentrations
that rapidly kill the majority of dopaminergic neurons to allow
motor decits to be observed. It is much more difcult to control
the toxin concentration, the degree of neuronal exposure, and the
time course of toxin effect than when using in vitro culture
systems. Certainly, specic pathogenic mechanisms, for example,
oxidative stress and complex I inhibition and inammatory
change, can be modeled using 6-OHDA, MPP , and LPS, respec-
tively, but toxin exposure is usually acute, in young animals, and
in a species that differs in susceptibility to humans. The biggest
drawback is that most approaches are one off and do not in any
way reect the progressive nature of cell loss in PD. One or two
exceptions are the injection of 6-OHDA in to the striatum, which
causes retrograde degeneration of dopaminergic neurons, and the
use of infusion pumps to provide longer periods of toxin expo-
sure. But even here, we are talking about days rather than weeks
or months or years. In addition, most pathogenesis-based toxin
studies are focused entirely on the SNc and there have been only
limited attempts at destroying other neuronal systems affected in
PD, which do not mimic one of the key features of PD, that being
altered protein deposition.
There are, however, exceptions to most of the above criticisms.
The peripheral administration of 6-OHDA to neonatal rats
destroys dopaminergic and noradrenergic neurons as it passes
the incomplete bloodbrain barrier. MPTP can be used systemi-
cally in both mice and monkeys to destroy dopaminergic neurons
in the SNc although, again, the time course of these studies is
short and the effects specic to dopaminergic systems. Peripheral
administration of LPS has been reported to induce nigral
Table 3
Toxins used in animal models of PD that reect the pathogenic process.
Pathology modeled Toxin
Oxidative stress 6-OHDA and paraquat
Nitrative stress LPS
Mitochondrial dysfunction Rotenone, MPTP, and MPP
Excitotoxicity Quinolinic acid, ibotinic acid
Proteasomal dysfunction PSI, epoximycin, lactacystin
Glial cell activation LPS
140 D.T. Dexter, P. Jenner / Free Radical Biology and Medicine 62 (2013) 132144
dopaminergic cell loss but not consistently, although it may
increase susceptibility to the central actions of other toxins.
Systemic administration of rotenone provides an interesting
possibility despite its inherent toxicity [7]. There seems to be
agreement that with careful administration, rotenone kills nigral
dopaminergic cells as well as affecting neurons in other nuclei
and perhaps with sequential pathology [54,116]. The biggest
question is whether these are relevant to PD or more reective
of the widespread pathology occurring in multiple-system atro-
phy, another Parkinson-like disorder [46]. Most intriguing is the
use of proteasomal inhibitors such as epoximycin and PSI. There is
no doubt that these are toxic to dopaminergic neurons in vitro
and on stereotaxic injection into the SNc (see above). But reports
of the slow progressive onset of neuronal loss in a pattern that
resembles that occurring in PD have proved difcult to reproduce
in some laboratories but not others. This would be a marked
advance in toxin-based models of PD if viable, as cell death
progresses through the brain, as suggested by Braak and is
accompanied by a-synuclein aggregate formation. In fact, recent
data suggest that PSI does not enter the brain but rather such
proteasomal inhibitors can induce Lewy body-like pathology in
the gut and other organs (unpublished observations) and this
then invades the brain through the dorsal motor nucleus of the
vagus [102]. If this is a reality, it would explain much that has
consumed the debate over the progression of PD in recent years.
Gene relevance
Understanding the function of wild-type and mutated proteins
associated with familial PD has been possible through their
transfection into cell lines and this has led to advances in
discerning their role in the pathogenic process through altera-
tions in oxidative stress, mitochondrial function, and altered
proteasomal and lysosomal activity. But it has been the transla-
tion into in vivo experimental models that has proved more
daunting. Despite promising ndings in Drosophila and Caenor-
habditis elegans, there are no transgenic, overexpression, knock-
out, or knock-in models of wild-type or mutant a-synuclein that
have fully recapitulated the pathological picture of PD (see
[9,83,166] for recent reviews). Viral vector-induced expression
of wild-type and mutant a-synuclein in rats and primates has
been associated with nigral dopaminergic cell degeneration but
this has not been a consistent nding. The picture has been
similar when the same approaches have been attempted using
parkin, DJ-1, PINK1, and LRRK2. Changes in dopaminergic func-
tion in striatum, altered morphology of dopaminergic neurons,
changes in other monoaminergic systems, degeneration of motor
neurons, gliosis, and inammatory changes occur, among others,
along with mild motor abnormalities, but not the expression of
PD that was hoped for. However, there is still value in this
approach, as the generation of these animals allows an in vivo
approach to explaining the roles of mutated proteins in the
pathogenic process dened using other techniques. It also pro-
vides a test bed in which to examine potential neuroprotective
therapies, for example, kinase/GTPase inhibitors in LRRK2 trans-
genic animals [77,82] and the role of passive vaccination in
a-synuclein transgenic mice [155]. There also seems to be a
signicant role for studying nonmotor symptoms of PD in genetic
models of PD, with reports of everything from cognitive impair-
ment to altered gastrointestinal motility and constipation
[91,160]. It could be questioned whether mice provide the right
background to test the pathogenic potential of mutated proteins
in PD, as total knockout of single wild-type proteins does
not seem to result in alterations in dopaminergic function in
the brain of mice that are null for parkin, DJ-1, and PINK1;
nothing untoward seems to occur as they age [71]. Increased
susceptibility to subsequent toxic insult may be enhanced and it
may be that combinations of genetic models and toxin-based
approaches will yield important information on pathogenesis
related to nigral cell death. However, one recent study suggests
that models more closely related to PD can be generated under
appropriate conditions. A subset of DJ-1 null mice backcrossed
into a normal background was reported to show early onset of
unilateral nigral dopaminergic cell loss that became bilateral with
time and later involved the locus coeruleus and the onset of
motor decits [127].
It should also be remembered that genetic changes not directly
linked to PD have yielded important animal models in which
nigral dopaminergic cell death does occur and which again add to
the knowledge of pathogenic mechanisms. These include the
aphakia mouse lacking the homeobox transcription factor Pitx3,
the mitopark mouse lacking mitochondrial transcription factor A,
and Nurr-1 decient mice [29,49,66].
From pathogenesis to neuroprotection?
Based on the extent of knowledge of pathogenic mechanisms
leading to dopaminergic cell death in PD, it would seem likely
that treatment strategies aimed at stopping or slowing the
progression of the disease process would be available but this is
not the case. Neuroprotection has proved a more difcult nut to
crack than most had envisaged and the current situation is
starting to look like that in stroke. Based on experimental studies,
a range of compounds have been advanced into clinical develop-
ment, but so far, none have proved to have a convincing effect
on the worsening of motor symptoms [107,110,111,134,144].
This has involved antioxidants, dopamine agonists, monoamine
oxidase inhibitors, agents affecting mitochondrial function, cal-
cium antagonists, antiapoptotic agents, and neurotrophic factors,
among others. So far, only the monoamine oxidase B inhibitor
rasagiline has been shown to have some effect on motor scores,
but this was not sufcient for it to be labeled as disease-
modifying [109]. Although the pace has lessened, there are a
number of ongoing studies looking to see whether centrally active
iron chelators, precursors of urate, or stimulators of glial cell-
derived neurotrophic factor production in brain are able to do
what has eluded previous attempts. But perhaps the worrying
issue is whether the current concepts of the pathogenic processes
in PD and the nature of PD itself are correct.
We base the animal models of PD on the processes that we
believe to be operative in cell death in humans. Classically, there
is great reliance on using MPTP-treated mice and the 6-OHDA-
lesioned rats to model oxidative stress and mitochondrial dys-
function, respectively. But these may have deceived us as so far
there has been no translation into clinical effect. The surprising
thing is that we continue to use these models to nd further
potential neuroprotective strategies despite their history, perhaps
because there is nowhere else to turn in terms of toxin-related
effect. Interestingly, there has been relatively little work in
rotenone- or PSI-treated rodents, which may have more to offer.
What is particularly worrying is the large numbers of compounds
reported as positive in MPTP-treated miceif it were that easy,
we would have neuroprotection in PD by now. There is clearly a
need to reassess the models employed and to ensure that we are
working from a rm knowledge base from postmortem studies
and the living PD patient with respect to the events that occur
during neuronal loss and their time course. The last may be very
important as it may be too late to expect a neuroprotective
treatment to have much effect by the time a diagnosis of PD is
made on classical grounds, as neuronal loss will be too advanced.
Perhaps ongoing studies to identify premotor signs of PD will
 D.T. Dexter, P. Jenner / Free Radical Biology and Medicine 62 (2013) 132144
identify an earlier patient population in which to study neuro-
protection and maybe the animal models will turn out to be
correct in their prediction of effect.
The gene defects associated with familial PD may lead to new
potential targets for neuroprotection and, at present, mitochon-
drial dysfunction and lysosomal defects seem attractive. The
problem has been, and to a large extent remains, that most
aberrant proteins identied do not offer an obvious target for
therapeutic intervention and, in many cases, their physiological
function remains unknown. This applies to a-synuclein, parkin,
DJ-1, and PINK1, among others. LRRK2 mutations are linked to
altered kinase/GTPase activity and this could be an attractive
target, but to nd a centrally active kinase inhibitor that focuses
on the areas of the brain affected in PD, that does not impair the
physiological function of the protein, and that does not affect
other kinases, is challenging. Indeed, the pharmaceutical industry
may nd it difcult to identify viable targets at the current time
on which to unleash the medicinal chemists and preclinical
teams. The other danger is that the defects in familial disease
have nothing to do with the processes that underlie neuronal loss
in the majority of PD patients with sporadic disease. The means of
assessing molecules that might potentially rectify the effects of
gene defects also needs assessment. For example, it is perhaps not
too surprising that kinase inhibitors prevent the neuronal loss in
mice induced by the introduction of a LRRK2 mutation that
increases kinase activity. The acceptance that PD is a synucleino-
pathy suggests that removal of the protein could slow disease
progression whatever the initial cause. But what type of
a-synuclein should be removed and how much of it? Whether this
should be aggregates, brils, or protobrils or inhibition of the
formation of the protein, all of it, some of it, or none of it remains
to be determined. Certainly approaches such as passive vaccina-
tion can remove a-synuclein and prevent the consequences of its
aggregation in a-synuclein overexpressing mice, but will this
work in PD? The worry is that several similar approaches using
human monoclonal antibodies against amyloid have recently
failed in Alzheimer disease despite encouraging preclinical data.
Finally, it may be the nature of PD that has so far prevented the
translation of pathogenic mechanisms into a neuroprotective
treatment. Clinical trials are still undertaken in the classical
manner of comparing the effect of an agent between large groups
of patients in a placebo arm and an active treatment arm, looking
for a statistically relevant outcome. But this presumes that PD is a
single illness resulting from the same pathogenic processes and
this is increasingly being challenged. Rather there should be
attempts to look at drug effect in individuals with common
features, for example, a mitochondrial complex I defect. At
present signicant effects of a specic drug targeted at one
component of the cell death pathway may be missed in a small
proportion of patients in a clinical trial because of dilution of the
outcome by the majority who have PD associated with other
pathogenic mechanisms and who did not respond. Indeed, it may
be that we have to think about neuroprotection for small portions
of the PD population who have the disease for specic reasons
and, notably, this may be those with inherited disease, for
example, a LRRK2 mutation, as a druggable target. The downside
is that such an approach may not be economically viable for the
pharmaceutical industry. The alternative is to adopt the oncology
approach and to use cocktails of drugs that exert multiple effects
on the pathogenic cascade.
Concluding remarks
Clearly we have made great strides in improving our under-
standing of the nature and causes of PD. However, there are
141
several fundamental gaps in our knowledge, which hamper our
ability to develop effective neuroprotective strategies for PD.
Importantly, we still do not understand the molecular mechan-
isms that account for the spreading pathology of the disease.
Additionally, the failure to generate an accurate model of sporadic
Parkinson disease and the clinical failure of neuroprotective
strategies developed from such models highlight that we are
missing a key section of the cell death mechanism jigsaw. Indeed,
the mechanisms currently used to explain pathogenesis in PD
may just be the downstream consequence of a so far unknown
trigger. Certainly, it is clear that PD is not a single disease with a
single cause but a syndrome in which the variants have in
common nigral dopaminergic cell death and motor dysfunction,
but around which multiple other neuronal systems degenerate
and which have multiple primary causes involving a range of
pathogenic processes.
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