Post on 27-Nov-2018
Università degli Studi di Cagliari
DOTTORATO DI RICERCA
Tossicologia – Indirizzo Farmacologia e Farmacoterapia delle
Tossicodipendenze
Ciclo XXVIII
Influence of different neuroprotective drugs on dopamine
neurotoxicity induced by 3,4-methylenedioxymethamphetamine
and MPTP in mice
Settore scientifico disciplinare di afferenza
BIO/14
Presentata da: Dott.ssa Pier Francesca Porceddu
Coordinatore Dottorato Prof. Gaetano Di Chiara
Tutor Prof.ssa Micaela Morelli
Esame finale anno accademico 2014 – 2015
Abstract
Introduction: Parkinson‟s disease (PD) is characterized by a chronic progressive loss of
nigrostriatal dopaminergic neurons that is associated with chronic neuroinflammation. Current
treatments for PD can significantly improve symptoms but do not cure the disease or slow its
progression. An approach used in existing therapies is based on the inhibition of monoamine
oxidase (MAO), enzyme involved in the metabolic degradation of dopamine. Although, preclinical
studies showed that MAO-B inhibitors have neuroprotective activity in cellular and animal models
of PD, clinical trials did not completely confirm this result. Therefore a large number of new
molecules, with more potent MAO-B inhibitory activity and a possible neuroprotective effect, have
been proposed to replace the pre-existing MAO-B inhibitors. The profile of the recent MAO
inhibitor, SZV558, appears to be particularly interesting because of its pharmacodynamic, favorable
for disease-modifying properties and its irreversible MAO-B enzyme bind.
The enhancement of adult neurogenesis could be of great clinical interest in the management of
neurodegenerative disorders. In line with this, the metformin, a well-known antidiabetic drug, has
recently been proposed to promote neurogenesis and to have a neuroprotective effect on the
neurodegenerative processes induced by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) in a mice PD model.
Although, PD has multiple origins, one hypothesis is that amphetamine-related drugs may be part of
the wide array of factors leading to the dopaminergic neuron degeneration that causes the disease.
These hypothesis are supported by different results that showed a persistent, long-term
dopaminergic toxicity induced by 3,4-methylenedioxymethamphetamine (MDMA) in mice.
Moreover, the MDMA, altering the dopaminergic transmission, may affect neurogenesis and
synaptogenesis. On these basis, considering that the young brain is particularly sensitive to drug-
induced neurotoxicity, the consumption of MDMA during the adolescence might increase the
vulnerability of dopaminergic neurons. However, the use of amphetamine-related drugs by
adolescent and young people is often combined with caffeinated energy drinks in order to amplify
their stimulant actions. Although caffeine use is safe, the combined treatment of caffeine and
MDMA increases not only the DA release but also the microglia and astroglia activation.
Aims: During my Ph.D. I studied the influence of neuroprotective drugs, such as MAO inhibitors
and metformin, or substances, such as caffeine, on the neurodegenerative effects of two
dopaminergic toxins, MDMA and MPTP, in mice.
1. In the first phase of my study, I evaluated the neuroprotective activity of the new MAO-B
inhibitor SZV558, compared with well-known rasagiline, in a chronic mouse model of MPTP
plus probenecid (MPTPp), which induces a progressive loss of nigrostriatal dopaminergic
neurons.
2. Previous results showed that when MDMA is associated with caffeine, a more pronounced
degeneration in adolescent compared with adult mice was observed. To better clarify the
molecular mechanism at the base of the different neurotoxic effect of this drug association at
different ages, I evaluated the neuronal nitric oxide synthase (nNOS) expression, which plays a
critical role in the integration of dopaminergic and glutamatergic transmissions, in the CPu of
adolescent or adult mice treated with MDMA, alone or in combination with caffeine.
3. Finally, I investigated the neuroprotective effect of metformin against dopaminergic
neurotoxicity induced by MDMA in the CPu and SNc of adult mice.
Conclusions: These results demonstrated that the dopaminergic neurodegenerative process may be
induced or conditioned by environment stressors or substances which influence, through different
ways, the development of neurodegenerative mechanisms. In the present study I evaluated the
effects of 3 substances, known as potentially neuroprotective, in combination with two different
neurotoxins that affect the nigrostriatal dopaminergic system. The SZV558 MAO-B inhibitor and
the metformin protected the nigrostriatal pathway, usually affected in PD, by MPTP- and MDMA-
induced neurotoxicity, respectively. On the other hand, caffeine, administrated with MDMA,
showed a neurotoxic potential depending on the age of consumers, confirming the vulnerability of
adolescent brain to consumption of drug and substances that affected the dopaminergic system. In
conclusion, the study of neurodegenerative processes may be relevant to understand the human
pharmacology, the origin and development of neurodegenerative disease and to predict the
neurotoxic effect of drug abuse.
List of abbreviations:
5-HIAA 5-hydroxyl indole acetic acid
5-HT 5-hydroxytryptamine
6-OHDA 6-hydroxidopamine
ADAGIO Attenuation of disease progression with azilect given once-daily
a-MeDA a-methyldopamine
AMPK AMP-activated protein kinase
ANOVA Analysis of variance
ATP Adenosine triphosphate
BBB Blood brain barrier
BDNF Brain-derived neurotrophic factor
cAMP cyclic adenosine monophosphate
CNS Central nervous system
COMT Catechol-o-methyltransferase
CPu Caudate-putamen
CREB cAMP cresponsive element binding protein
CSF Cerebrospinal fluid
DA Dopamine
DAPI 4′,6-diamidine-2′-phenylindole dihydrochloride
DAQ DA quinone
DAT DA transporter
DOPAC 3,4-dihydroxyphenylacetic acid
DOPAL 3,4-dihydroxyphenylacetaldehyde
FDA Food and drug administration
GABA Gamma-amino butyric acid
GDNF Glial cell-derived neurotrophic factor
GFAP Glial fibrillary acidic protein
GLU Glutamate
HHA Dihydroxyamphetamine
HHMA 3, 4 –dihydroxymethamphetamine
HVA Homovanilic acid
IL Interleukin
iNOS inducible nitric oxide synthase
JPND Join programme for neurodegenerative disease
L-DOPA Levo-DOPA
LPS Lipopolysaccharide
MAO Monoaminooxidase
MAO-B Monoaminooxidase-B
MDA 3,4-methylenedioxyamphetamine
MDMA 3,4-methylenedioxymethamphetamine
MPDP+ 1-methyl-4-phenyl-2,3-dihydropyridium
MPP+ 1-methyl-4-phenylpyridinium
MPPP 1-methyl-4-phenyl-4-propionoxypiperidine
MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
MPTPp 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine plus probenecid
NA Noradrenaline
NET NA transporter
NMDA N-methyl-D-aspartic acid
N-Me-a-MeDA N-methyl-a-methyldopamine
nNOS neuronal nitric oxide synthase
NO Nitric oxide
PB Phosphate buffer
PBS Phosphate buffer solution
PD Parkinson‟s disease
PET Positron emission tomography
PKC–CREB Protein kinase C–cyclic-adenosine monophosphate (cAMP) response element-binding
protein
RNS Reactive nitrogen species
ROS Reactive oxygen species
SERT Serotonin transporter
SN Substantia nigra
SNc Substantia nigrapars compacta
SOD Superoxide dismutase
TH Tyrosine hydroxylase
TNF-α Tumour necrosis factor α
VMAT2 Vesicular monoamine transporter 2
Table of contents
INTRODUCTION ......................................................................................................................... 1
1. Neurodegeneration .................................................................................................................... 1
1.1 Definition of Neurodegeneration ....................................................................................... 1
1.2 Role of environmental factors exposure in Neurodegeneration .......................................... 1
1.3 Molecular mechanisms on the basis of Neurodegeneration................................................. 2
1.4 Dopamine vulnerability to neurodegenerative processes ........................................................ 3
2. Parkinson‟s disease .................................................................................................................... 6
2.1 PD pathophysiology ........................................................................................................... 6
2.2 PD animal models ............................................................................................................. 7
3. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ........................................................................... 8
3.1 MPTP: a doparminergic neurotoxin .................................................................................. 8
3.2 Mechanism involved in MPTP neurotoxicity ....................................................................... 9
3.2.1 Oxidative stress and neuroinflammation .................................................................... 10
3.3 Neurotoxicity in humans ........................................................................................................... 11
3.4 MPTP as PD mouse model ....................................................................................................... 12
4. 3,4-methylenedioxymethamphetamine ............................................................................................ 13
4.1 MDMA: a neurotoxic drug........................................................................................................ 13
4.2 Mechanism involved in MDMA neurotoxicity ......................................................................... 14
4.2.1 Oxidative stress ........................................................................................................ 15
4.2.2 Neuroinflammation and Hyperthermia ......................................................................... 16
4.3 Neurotoxicology in humans ..................................................................................................... 18
4.3 MDMA administration in mice: a model of dopaminergic neurotoxicity .............................. 19
4.5 MDMA neurotoxicity and adolescence .................................................................................... 20
5. PD therapy .......................................................................................................................................... 21
5.1 Current PD therapies ................................................................................................................ 21
5.2 Monoamine oxidase inhibitors therapy .................................................................................... 21
5.3 Novel (hetero)arylalkenyl propargylamine compounds.......................................................... 23
5.4 Metformin ................................................................................................................................... 24
5.5 Caffeine: Neuroprotective or Neurotoxic? ............................................................................. 25
AIMS ........................................................................................................................................... 28
MATERIALS AND METHODS ................................................................................................ 30
1. Drugs ....................................................................................................................................... 30
2. Treatments ............................................................................................................................... 30
2.1 Chronic protocol of MPTPplus probenecid MPTPp ......................................................... 30
2.2 Acute MDMA treatment in combination with caffeine ....................................................... 30
2.3 Acute MDMA treatment in combination with metformin ................................................... 31
3. Behavioral tests ......................................................................................................................... 31
3.1 Spontaneousmotor activity: Motility test ........................................................................... 31
3.2 Beam walking test............................................................................................................. 31
3.3 Inverted grid test .............................................................................................................. 32
3.4 Pellet retrieval olfactory test ............................................................................................ 32
4. Immunohistochemistry .............................................................................................................. 32
4.1 Immunohistochemistry and cresyl violet for Nissl staining ................................................ 32
4.2 Immunofluorescence for nNOS, IL-1β and TNF-α ............................................................ 33
4.3 Analisys of TH-positive cells and Nissl staining in the SNc ............................................... 33
4.4 Analisys of TH-positive fibers in CPu ............................................................................... 34
4.5 Analisys of nNOS-positive cells in CPu ............................................................................. 34
4.6 Analisys of IL-1β and TNF-α immunoreactivity in CPu ..................................................... 35
5. Statistical Analysis .................................................................................................................... 35
RESULTS .................................................................................................................................... 36
1. SZV558 administration reverts the motor impairments, olfactory dysfunction and dopaminergic
neuron degeneration induced by a chronic MPTPp treatment .................................................... 36
1.1 Changes in spontaneous motor activity: Motility test ........................................................ 36
1.2 Effect of SZV558 on motor impairment inducedby MPTPp: beam walking test ................. 36
1.3 Grasp-strength evaluation: inverted grid test.................................................................... 38
1.4 Effect of SZV558 on olfactory deficit induced by MPTPp:olfactory test ............................ 38
1.5 Effect of SZV558 on MPTPp-induced neurodegneration: TH immunohistochemistry and
Nissl staining in the SNc and CPu ................................................................................... 38
2. Effects of repeated MDMA+caffeine administration in adolescent and adult mice .................... 40
2.1 nNOS activation in the CPu of adolescent and adult mice ................................................. 40
2.2 IL-1β activation in the CPu of adolescent and adult mice ................................................. 41
2.3 TNF-α activation in the CPu of adolescent and adult mice................................................ 42
3. Neuroprotective effects of metformin administration on MDMA-induced neurodegeneration: TH
immunoreactivity and Nissl staining ......................................................................................... 44
DISCUSSION ............................................................................................................................. 46
1. The neuroprotective effects of SZV558 in a chronic MPTPp model of PD ................................ 46
2. The neurotoxic effect of caffeine on repeated MDMA administration in adult and adolescent
mice .............................................................................................................................................. 49
3. The neuroprotective effects of metformin on neurodegeneration induced by repeated MDMA
administration ........................................................................................................................... 52
CONCLUSIONS ......................................................................................................................... 54
REFERENCES............................................................................................................................ 55
1
INTRODUCTION
1. Neurodegeneration
1.1 Definition of Neurodegeneration
The term “Neurodegeneration” refers to a pathological condition characterized by dysfunction and /
or death of neurons in brain and spinal cord. Etymologically, the word is composed of the prefix
“neuro-,” which denotes relationship to the nervous system, and “degeneration,” which refers to, in
the case of tissues or organs, a process of losing structure or function (Jennekens 2014). Thus, in the
strict sense of the word, neurodegeneration corresponds to any pathological condition primarily
affecting neurons. However, this term is ever used to characterize a diverse group of neurological
disorders known as neurodegenerative disease (Przedborski et al. 2003). The European Joint
Programme for Neurodegenerative Disease (JPND) in the 2012 defined the neurodegenerative
disease as “an umbrella term for a range of conditions primarily involving neurodegeneration which
is the loss of structure or functions of neurons”. Definitions in the literature indicate that
neurodegenerative diseases are considered as to be age related, incurable, and largely untreatable
chronic progressive diseases of the central nervous system (Jennekens 2014). In general,
neurodegenerative diseases are defined as hereditary and sporadic conditions which are
characterized by progressive nervous system dysfunction. Although hundreds of neurological
disorders may fit the definition of a neurodegenerative disease, many are rare and have been found
to be caused by purely genetic factors. A small number of neurodegenerative diseases are relatively
common, such as Alzheimer‟s and Parkinson‟s disease (PD) and characterized by heterogeneous
clinical and pathological expressions affecting specific subsets of neurons in specific functional
anatomic systems (Cannon and Greenamyre 2011).
1.2 Role of environmental factors exposure in Neurodegeneration
The exact etiology at the basis of neurodegenerative processes is not well known. They involve
specific combinations of genetic predispositions and environmental stressors exposure, that trigger
oxidative and proteostasis dysfunction in vulnerable neurons, in critical ages for the development of
brain (Saxena and Caroni 2011). Instead, toxic environmental factors may be the prime suspects in
initiating neurodegenerative processes. The environmental hypothesis posits that
neurodegeneration, such as PD related degeneration, results from exposure to a neurotoxin.
Theoretically, the progressive neurodegeneration of PD could be produced by chronic neurotoxin
exposure or by limited exposure initiating a self-perpetuating cascade of deleterious events (Cannon
and Greenamyre 2011). The finding that people intoxicated with 1-methyl-4-phenyl-1,2,3,6-
2
tetrahydropyridine (MPTP) develop a syndrome nearly identical to PD (Langston et al. 1983) is a
prototypic example of how an exogenous toxin can mimic the clinical and pathological features of a
neurodegenerative disease. In fact, MPTP is able to enter and destroy dopaminergic neurons
producing a severe and irreversible parkinsonian syndrome, which is almost identical to PD
(Przedborski and Vila 2001). Numerous chemical agents may induce a behavioral phenotype known
as parkinsonism, which shares some of the behavioral features of PD, but often has different
mechanistic and pathological correlates. Consequently, the majority of the exposures may actually
bear limited relevance to the etiology of PD (Cannon and Greenamyre 2011). However, considering
the numerous environmental toxicants by which humans are exposed in the course of a life, it is
difficult to identify a single environmental factor accounting for a significant number of cases.
Netherless, recent several clinical reports founding that patients diagnosed with neurodegenerative
diseases, such as PD, had a higher rate of exposure to amphetamine-related drugs at a young age,
compared with the general population (Parrott et al. 2004; Callaghan et al. 2010; Christine et al.
2010; Curtin et al. 2015). These data suggest a possible correlation between amphetamine-related
drugs and the PD etiology.
1.3 Molecular mechanism on the basis of Neurodegeneration
Although the neurodegenerative diseases show different pathophysiology, they have in common
specific molecular mechanisms, such as mitochondrial dysfunctions and oxidative stress,
aggregated protein deposits, neuroinflammation.
The presence in tissue of proteinaceous deposits is a hallmark of all neurodegenerative disease.
Although the composition and localization (intra- or extracellular) of protein aggregates differ from
disease to disease, this common feature suggests that protein deposition per se, or some related
event, is toxic to neurons. Aggregated or soluble misfolded protein could be neurotoxic through a
variety of mechanisms. Protein aggregates could directly cause damage, perhaps by deforming the
cell or indirectly interfering with the proteosomal functions (Dauer and Prezedborki 2003).
Another important factor which has been associated with many chronic neurodegerative conditions
is the inflammatory response in the central nervous system (CNS) (Stoll and Jander 1999). In fact
recent studies have demonstrated that glial activation participates in the events that induce neuronal
damage (Barcia et al. 2011). Investigating the specific correlation between neuroinflammation and
neurodegeneration, several studies have showed the presence of a glial response both concurrently
and after the dopaminergic neurodegeneration (Araki et al. 2001; Wu et al. 2002; Barcia et al. 2004;
Novikova et al. 2006; Yazdani et al. 2006). Some researchers speculated that increased pro-
inflammatory response could result in a delayed and progressive loss in dopaminergic neurons in
3
the substantia nigra (SN), similar to that seen in PD (Qin et al. 2007). The chronic
neuroinflammation might be involved in neurodegenerative processes through the chronic release of
toxic mediators, such as proiflammatory cytokines, which would attack surrounding neurons
eventually contributing to their death through apoptotic mechanisms, potentiating
neurodegenerative processes (Wu et al. 2002; Hirsch et al. 2003; Hald and Lotharius 2005; Tansey
et al. 2007). On this basis, it is plausible to speculate that drugs which prevent or counteract the
detrimental consequences of stress on inflammatory pathways may offer novel treatments for a
variety of neurodegenerative pathologies (Hurley and Tizabi, 2013).
Finally, abnormalities in mitochondrial functions detected in a range of neurodegenerative disease,
and in evidences from disease models suggest that mitochondrial dysfunctions may play a role in
disease pathogenesis (Dauer and Przedborski 2003). In particular, these pathological dysfunctions
trigger the production of oxidative stress factors. The oxidative stress is the result of the imbalance
between reactive oxygen species (ROS), such as peroxidase and free radicals, and the ability of the
biological system to detoxify them. ROS causes lipid peroxidation, cytoskeleton disorganization
and DNA phenomena that convey in cell death (Luo et al. 1998).
1.4 Dopamine vulnerability to neurodegenerative processes
It has been speculated that dopaminergic neurons present an elevated vulnerability to
neurodegenerative processes. In vitro studies demonstrated that the application of dopamine (DA)
induces death of striatal cells (Cheng et al. 1996). At physiological concentrations DA do not
exhibits toxicity, but malfunctions on DA release and/or her metabolism could lead
neurodegeneration. Although, the mechanisms is still unclear, several evidences showed that the
DA neurotoxic effects are associated with the production of ROS caused by DA metabolites such
as, DA-quinone (DAQ) (Cadet et al. 1997; Blum et al. 2001; Wersinger et al. 2004). Dopaminergic
neurons may be a particularly fertile environment for the generation of ROS, due to the metabolism
of DA producing hydrogen peroxide and superoxide radicals, and the auto-oxidation of DA
producing DAQ (Graham 1978), a molecule that damages proteins by reacting with cysteine
residues and inhibits ROS scavenger enzymes (Hauser et al. 2013).
The oxidative stress seems initiating with the interaction of DA with mitochondrial oxidative
phosphorylation system causing inhibition of complex 1 and decreasing adenosine triphosphate
(ATP) (Ben-Shachar et al. 2004). In particular, inhibition of complex 1 increases the production of
the superoxide, which may form toxic hydroxyl radicals or react with nitric oxide (NO) to form
peroxynitrite. These molecules may cause cellular damage by reacting with nucleic acids, proteins,
and lipids. However, one target of these reactive species may be the electron transport chain itself
4
(Cohen 2000), leading to mitochondrial damage and further production of reactive oxygen species
(ROS). This mitochondria-related energy failure may disrupt vesicular storage of DA, causing the
free cytosolic concentration of DA to rise and allowing harmful DA-mediated reactions to damage
cellular macromolecules (Fig. 1).
Finally, DA activates apoptotic signaling through mechanisms of oxidation (Luo et al. 1998) and
necrotic cell death (Di Filippo et al. 2006).
Fig. 1. Factors that trigger oxidative stress in neurodegenerative processes.
Dopaminergic neurons of the substantia nigra pars compacta (SNc), the area most affected in PD,
appear to be particularly vulnerable to oxidative stress induced by mitochondrial dysfunction
(Biskup and Moore, 2006). Studies addressing the excitability properties of these neurons have
provided that the vulnerability is induced by particular channels (Cav1.3 low voltage-dependent L-
type Ca-channels) that open at relatively hyperpolarized membrane potentials, leading to high Ca
flux loads in DA SNc neurons (Chan et al. 2007), producing oxidative stress (Guzman et al. 2010).
However, this neuronal SNc property if associated with mitochondrial dysfunction that
compromises the ability of mitochondria to accumulate Ca++
, may induce a neurodegenerative
5
process (Autere et al. 2004). The susceptibility of dopaminergic neurons to mitochondrial
dysfunction, could even explain the ability of several toxins, such as MPTP and 3,4-
methylenedioxymethamphetamine (MDMA), to induce cell death in DA neuronal populations
(Biskup and Moore 2006).
6
2. Parkinson‟s Disease
2.1 PD pathophysiology
PD is the second most common neurodegenerative disease, affecting 1% of the population over 55
years of age (Lees et al. 2009). The main features of PD are tremor, muscle rigidity, bradykinesia,
and postural instability, whose intensity increases as the neurodegenerative process progresses;
however, these motor manifestations can be accompanied by non-motor symptoms such as
olfactory deficits, sleep impairments, and neuropsychiatric disorders (Forno 1996; Chaudhuri et al.
2006). The evident disease is characterized by the loss of over 70% of the dopaminergic neurons in
the SNc, a profound decrease of DA in the striatum, and the presence of intracytoplasmic inclusions
called Lewy bodies, which are composed mainly of α-synuclein and ubiquitin (Lees et al. 2009).
Although a variety of possible pathogenetic mechanisms have been proposed over the years,
including excessive release of oxygen free radicals, dysfunction of protein degradation, activation
of glia and impairment of mitochondrial function, the pathogenesis of PD is still largely uncertain
(Pringsheim et al. 2014; Block and Hong 2005).
As mentioned before, the SNc is particularly vulnerable to neurodegeneration induced by
mitochondrial dysfunction (Biskup and Moore 2006).
The hypothesis that mitochondrial dysfunctions play a role in the pathogenesis of PD was fueled by
the discovery that MPTP block the mitochondrial electron transport chain by inhibiting complex 1
(Nicklas et al. 1987). Subsequently, several studies identified abnormalities in complex 1 activity in
PD (Greenamyre et al. 2001). In vitro studies indicate that complex 1 defect may subject cells to
oxidative stress and energy failure. However, several biological markers of oxidative damage,
consistent with increased ROS, are elevated in the SNc of PD brains (Sian et al. 1994).
Moreover, other neurodegenerative conditions, such as the neuroinflammation, play vital roles in
the degeneration of dopaminergic neurons (Dauer and Przedborski 2003). However, emerging
evidence indicates that sustained inflammatory responses, T cell infiltration and glial cell activation
are common features of both human PD patients and animal models of PD (Hirsch et al. 2012; Lv et
al. 2015). In particular the involvement of neuroinflammation in PD pathogenesis has been
suggested by positron emission tomography (PET) studies that showed in the SNc of PD patients, a
pronounced activation of microglia, one of the major cell types which are involved in the
inflammatory responses in the CNS (Bartels et al. 2010; Gerhard et al. 2006). Further biochemical
analysis reveals higher levels of proinflammatory mediators, released by reactive microglia,
including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), in the midbrain of PD patients
(Boca et al 1994; Mogi et al. 1994; Brodacki et al. 2008). These data confirm the involvement of
7
immune components in PD pathology. A great body of studies shows that even astrocytes play a
role in the neuroinflammatory processes in PD. Like microglia, astrocytes respond to the
inflammatory stimulations such as Lipopolysaccharide (LPS), producing pro-inflammatory
cytokines both in vitro and in vivo (Saijo et al. 2009; Tanaka et al. 2013). Finally, reactive
astrogliosis characterized by the increased expression levels of glial fibrillary acidic protein (GFAP)
and hypertrophy of cell body and cell extensions have been reported in various PD animal models.
2.2 PD animal models
The most direct method to understand etiology, pathology, and molecular mechanisms of PD is the
use of various animal models. For the past several decades, animal models of PD have come in a
variety of forms. Typically, they can be divided into those using environmental or synthetic
neurotoxins or those utilizing the in vivo expression of PD-related mutations. Although the
identification of different genetic mutations (α-synuclein, parkin, LRKK2, PINK1, DJ-1) has led to
the development of genetic models of PD (Dawson et al. 2010), it is important to remember that,
only ∼10% of PD cases are due to genetic mutations (Dauer and Przedborski 2003), while the vast
majority of PD cases are sporadic and from unknown origins.
In the neurotoxic models have been used compounds that produce both reversible (reserpine) and
irreversible (MPTP, 6-hydroxydopamine (6-OHDA), paraquat, rotenone) effects. Recent studies
have focused more on irreversible toxins to produce PD-related pathology and symptomatology.
Neurotoxin-based models produced by 6-OHDA and MPTP administration are the most widely
used toxic models, while paraquat and rotenone are more recent additions to the stable of toxic
agents used to PD model (Dauer and Przedborski 2003; Betarbet et al. 2002). This strategy quite
popular among PD researchers is based on the premise that dopaminergic neurons have a
stereotyped death cascade that can be activated by a range of insults, including neurotoxins. A
common feature of all toxin-induced models is their ability to cause an oxidative stress and cell
death in DA neuronal populations. As mentioned above, oxidative stress results from increased
production of extremely reactive free radicals, which may be formed during a number of cellular
processes, including mitochondrial oxidative respiration and DA metabolism.
In addition to oxidative stress and mitochondrial deficits, many pathogenic mechanisms such as
chronic neuroinflammation, autophagy, and proteasomal dysfunction are believed to sustain and
amplify the neurodegenerative process, in PD animal models (Barcia et al. 2003; Hirsch et al. 2003;
Olanow 2007; Zhou et al. 2008).
8
3. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
3.1 MPTP: a dopaminergic toxin
As mentioned before, over the years, a variety of toxins of uncertain relevance inducing PD have
been used as agents to destroy dopaminergic neurons. However, none of the validated toxic models
of PD is a homolog of the disease, even though these models replicate many, but never all, of the
features of PD. Having stated this limitation, it is fair to say that among the various toxic models of
PD, the MPTP model has become one of the most commonly used. In fact, administration of MPTP
to humans and experimental animals causes the degeneration of dopaminergic neurons in SNc and
in striatum, causing a clinical picture in both humans and monkeys, indistinguishable from PD
(Langston et al. 1983).
Figure 2. Chemical structure of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
MPTP is a by-product of the chemical synthesis of a meperidine analog with potent heroin-like
effects (Fig. 2). It was made by Barry Kidstone, a 23 years old graduate student who set up a home
laboratory to synthesize 1-methyl-4-phenyl-4-propionoxypiperidine (MPPP), but after four
injections of what he thought to be MPPP, he started to show severe bradykinesia (Langston and
Ballard 1983). However, similar to patients with idiopathic PD, he responded to treatment with
Levo-DOPA (L-DOPA) and developed the same complications associated with L-DOPA therapy
(Langston and Ballard 1983). Successive investigations confirmed that he had unconsciously
synthesized a new dopaminergic neurotoxin capable to produce a reliable and reproducible lesion of
the nigrostriatal dopaminergic pathway after its systemic administration. Successive
neuropathologic studies of MPTP-exposed addicts have revealed a loss of dopaminergic neurons
restricted to SNc, similar to PD (Langston et al. 1999). The responses, as well as the complications,
to traditional antiparkinsonian therapies are virtually identical to those seen in PD. However,
previous data suggest that, following the main phase of neuronal death in PD, MPTP-induced
neurodegeneration may continue to progress “silently” over several decades, at least in humans
intoxicated with MPTP (Vingerhoets et al. 1994; Langston et al. 1999). The severity of MPTP-
9
induced lesion depends on the regimen and route of administration, and on the species considered.
Since its discovery, MPTP has been widely used to create animal models of PD in a variety of
species (Jakowec and Petzinger 2004; Kopin 1987; Kurosaki et al. 2004), though the most used
species are currently the non human primates and the mice. Non human primates are the species
most sensitive and rats the lowest sensitive to MPTP neurotoxicity, whereas mouse strains widely
vary in their sensitivity to the toxin, with the C57BL/6J being the most susceptible (Hamre et al.
1999; Sedelis et al. 2000).
3.2 Mechanism of action and neurotoxicity
The mechanism of MPTP toxicity is quite similar among humans, non-human primates and mice.
MPTP is a highly lipophilic protoxin which rapidly crosses the blood-brain barrier (BBB) after
systemic administration. Once in the brain, MPTP enters astrocytes and is bioactived to the unstable
intermediate 1-methyl-4-phenyl-2,3-dihydropyridium (MPDP+) by monoamine oxidase-B (MAO-
B) (Ekblom et al. 1993). Subsequently, MPDP+ spontaneously oxidizes to 1-methyl-4-
phenylpyridinium (MPP+) at least in vitro (Chiba et al. 1985; Fritz et al. 1985), whereas it is not
clear if this reaction may occur in vivo. Another mechanism for MPDP+ oxidation to MPP+
involves HO• radicals (Castagnoli et al. 1985), which appears in line with the evidence showing that
transgenic mice expressing high levels of superoxide dismutase are resistant to MPTP (Przedborski
et al. 1992). Recent findings show that once released from the astrocytes into the extracellular space
via the Organic Cation Transporter 3 (Cui et al. 2009), MPP+ is taken up into the neuron by the DA
transporter (DAT) (Chiba et al. 1985; Heikkila et al.1985). Consequently, mice lacking the DAT are
protected from MPTP toxicity (Bezard et al. 1999; Gainetdinov et al. 1997). Once inside the
neuron, MPP+ can follow three routes. It can remain in the cytosol to interact with cytosolic
enzymes, especially those carrying negative charges (Klaidman et al. 1993). In the second way, the
MPP+ can be stored in vesicles via uptake by the vesicular monoamine transporter (VMAT2) (Del
Zompo et al. 1993; Wimalasena et al. 2008), inducing the efflux of DA out into the intercellular
space. Here DA can be metabolized into a number of compounds, including metabolites and
products which lead the formation of ROS (Fig. 3) (Burke et al. 2008; Panneton et al. 2010).
Finally, the MPP+ can be concentrated within the mitochondria by a mechanism that relies on the
mitochondrial transmembrane potential (Ramsay and Singer 1986). In the mitochondria, MPP+ is
able to inhibit complex 1, resulting in the release of ROS as well as reduced ATP production
(Mizuno et al. 1987; Richardson et al. 2007). The ability to interfere with mitochondrial respiration
at the level of complex 1 is a key mechanism in the toxic effects of MPP+ (Nicklas et al. 1987;
Suzuki et al. 1990). Importantly, the cytotoxic effects of MPP+ are marked in cells that are
10
particularly sensitive to a deficiency in aerobic energy metabolism, a condition that applies to
dopaminergic neurons (Marey-Semper et al. 1993 and 1995) (Fig. 3)
Figure 3. Neurotoxic mechanism of the MPTP metabolite, MPP+, on dopaminergic terminals.
3.2.1 Oxidative stress and neuroinflammation
Through his metabolites, MPDP+ and MPP
+, the MPTP induces the formation of ROS and reactive
nitrogen species (RNS) (Drechsel and Patel 2008; Smith and Bennett 1997), which results in
oxidative stress (Adams et al. 1993; Meredith and Kang 2006). In this regards, it is worth
mentioning that mice transgenic for superoxide dismutase-1 (SOD-1), a key ROS scavenging
enzyme, are resistant to MPTP-induced dopaminergic neurodegeneration (Przedborski et al. 1992).
An excitotoxic mechanism for MPTP neurotoxicity has also been proposed, based on the
observation that intrastriatal administration of MPP+ to rats can induce a marked increase in
extracellular glutamate (GLU) (Carboni et al. 1990). Overstimulation of N-methyl-D-aspartic acid
(NMDA) receptors can lead to increase the intracellular Ca++
levels, an effect that causes the
activation of variety of proteases and kinases and results in the breakdown of cytoskeletal proteins
and the formation of ROS (Sattler and Tymiansky 2000 and 2001). However, the NMDA induced
the activation of nitric oxide synthase (nNOS) and, consequently, the production of NO.
Concurrently, depending on the level of NO production and the oxidative conditions, NO may
interact with H2O2 to produce peroxynitrite, or with Fe++
and Cu++
to generate ROS and to increase
the oxidative stress (Fig. 1) (Itzhak and Ali 2006).
Another mechanism by which MPTP is able to increase the oxidative stress in neurons is the
chronic neuroinflammation. In chronic neuroinflammatory response, elevated levels of pro-
inflammatory cytokines trigger oxidative and nitrosative stress that can aggravate the
11
neurodegenerative process (Hemmerle et al. 2012). Several evidences indicate that an intense
inflammatory reaction has been detected in the SNc of post mortem brains from MPTP-lesioned
addicts and monkeys after MPTP exposure (Barcia et al. 2003; McGeer et al. 2003). In this regard,
Liberatore and colleagues (1999) have shown that microglial cells not only increase in number after
MPTP injection, but also can flood dopaminergic neurons with large amounts of RNS, supporting a
role of activated microglia in MPTP-induced neurotoxicity in mice (Fig. 1) (Liberatore et al. 1999).
However, Hirsch and Hunot (2000) suggested that MPTP acts directly on the induction of cytokines
that activate inducible nitric oxide synthase (iNOS) (Hirsch and Hunot 2000), triggering the toxic
NO effects. Finally, blockade of neuroinflammation has been associated to a neuroprotective effect
in several models of dopaminergic degeneration, such as the chronic MPTP model, confirming that
glia may participate in MPTP-induced neurotoxicity (Wu et al. 2002; Schintu et al. 2009).
3.3 Neurotoxicity in humans
In the first cases of MPTP intoxication, patients showed immobility, marked generalized increase in
tone, inability to speak intelligibly, a fixed stare, marked diminution of blinking and other motor
impairment such as short steppes, slow shuffling gait and generalized bradykinesia, briefly the
classical PD symptoms (Langston and Ballard 1984). As in experimental animals, MPTP
administration in humans causes the degeneration of dopaminergic neurons in SNc and the
depletion of DA in striatum (Javitch et al. 1984). All cases of MPTP intoxication in humans were
caused by one, or few repeated administrations of the toxin that could lead to active
neurodegeneration, years after the initial exposure (Langston 1987). The loss of dopaminergic
neurons restricted to the SNc is the most important similarity with PD and has been revealed for the
first time in human through a neuropathologic study of brains of three MPTP-exposed addicts
(Langston et al. 1999). Moreover, PET studies using [18F]-DOPA have revealed that MPTP-
intoxicated individuals display a severely reduced DA uptake similar to that of late-stage idiopathic
PD (Calne et al. 1985; Snow et al. 2000; Vingerhoets et al. 1994). Finally, the depletion of nigral
dopaminergic neurons was found to be consistently present together with gliosis and clustering of
microglia around nerve cells (Langston et al. 1999).
One typical neuropathologic feature of PD has, until now, been lacking in the MPTP model: the
eosinophilic intraneuronal inclusions, called Lewy bodies have not been convincingly observed in
MPTP-induced parkinsonism (Forno et al. 1993). Netherless, the absence of Lewy bodies may be
due to the young age at the onset of MPTP-induced parkinsonism, since age may be an important
factor for development of these aggregates (Gibb and Lees 1988).
12
3.4 MPTP as PD mouse model
In general MPTP is administered to mice either in an acute or in subchronic regimen (Heikkila et al.
1984; Sonsalla and Heikkila 1986). In these models, MPTP can produce death of dopaminergic
neurons in SNc by at least the 40% in C57BL/6J mice and significant depletions in striatal level of
DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)
(Ricaurte et al. 1986). MPTP administration in mice induces a neuroinflammatory effect in the SNc,
striatum (Członkowska et al. 1996; Kohutnicka et al. 1998; Kurkowska-Jastrzebska et al. 1999), and
hippocampus (Luellen et al. 2003; Costa et al. 2014). Bradykinesia, akinesia, altered balance and
other motor features can be observed in MPTP-treated mice through various behavioral analyses
(Fleming et al. 2013; Sedelis et al. 2001; Tillerson et al. 2002). Whole-body tremor and postural
abnormalities also have been reported, but chiefly in the first day after lesioning (Sedelis et al.
2001). Despite the evidence of DA reductions, mice that receive MPTP acutely do not always
exhibit motor dysfunctions or motor abnormalities (Heikkila et al. 1989; Meredith and Rademacher
2011). Acute MPTP treatment induces a rapid and transient neurodegeneration, which does not
allow the development of chronic pathogenic mechanisms and/or motor disabilities. In contrast, the
chronic administration induces a gradual and persistent degeneration of nigrostriatal neurons
associated with motor deficit (Bezard et al. 2000, Fornai et al. 2005). Accordingly, chronic
exposure to low doses of MPTP over several weeks, in combination with the clearance inhibitor
probenecid, has been shown to reproduce several aspects of the human disease and to be a most
suitable model for studying drugs with neuroprotective potential (Carta et al. 2013, Petroske et al.
2001, Schintu et al. 2009). In addition, typical PD pathological features, such as chronic
inflammatory response in the SNc, alpha-synuclein positive inclusions, Lewy-bodies like deposits,
altered glutamate function, apoptotic neuronal demise, have been described in the chronic MPTP
model, suggesting that this model might extensively reproduced the neuropathology of PD
(Meredith at al. 2002; Dervan et al. 2004; Novikova et al. 2006).
Moreover, when MPTP is co-administered with probenecid (MPTPp), which retards the renal and
CNS clearance of the toxic metabolites of MPTP, the degeneration of dopaminergic neurons takes
place over a period of 5-8 weeks. This chronic regimen induces apoptosis, no mortality and mice
survive in a healthy state to the treatment. Moreover, the chronic treatment induces olfactory deficit,
one of the symptoms that characterizes the early as well as late stages of the disease. Progression in
a model with MPTP is a very important requirement since it allows studying the efficacy of
neuroprotective drugs during the progressive DA neurodegeneration, reproducing more closely the
human situation (Schintu et al. 2009).
13
4. 3,4-methylenedioxymethamphetamine
4.1 MDMA: a neurotoxic drug
Although PD has multiple origins, one hypothesis is that amphetamine-related drugs may be part of
the wide array of factors leading to the dopaminergic neuron degeneration that causes the disease
(Obeso et al. 2010). However, recent studies have showed that MDMA (ecstasy) has a neurotoxic
effect that is selective for the nigrostriatal pathway (Granado et al. 2008a). On these basis, it is
conceivable that prolonged exposure to MDMA, similar to that proposed for other amphetamine-
related drugs (Garwood et al. 2006), may damage the dopaminergic neurons in the human SNc
(Moratalla et al. 2015). Therefore, these damaged neurons could die earlier, depleting the reserve of
neural cells necessary for normal neurological functions and ending up in the manifestation of PD
(Garwood et al. 2006; Todd et al. 2013). On these bases, the study of MDMA neurotoxicity appears
particularly useful to understand the molecular mechanisms that induce the neuronal degeneration
which cause the disease.
Figure 4. Chemical structure of 3,4-methylenedioxymethamphetamine (MDMA).
Although it has been synthesized in 1912 (Fig. 4), MDMA got popular as a recreational drug since
the mid 1980s, because of its effects on mood and social relations (Hall and Henry 2006). Similar to
other amphetamine-related drugs, MDMA induces a state of “high”, mainly characterized by
disinhibition in social relations, openness of spirit, increased empathy towards other people,
increased self-esteem and self-confidence, euphoria, increased vigilance, improvement of mood,
and decrease of fatigue (Downing 1986; Greer and Tolbert 1986; Kirkpatrick et al. 2014). For these
positive properties of inducing feeling of well being and increasing communication (Watson and
Beck 1991), in the beginning of 1976, it was introduced in clinical psychotherapeutic practice
(Shulgin 1990). Although MDMA generally elicits “positive” effects, the 25% of MDMA users
report having had at least one adverse reaction to the substance (Davison and Parrott 1997; Green et
al. 2003; Morton 2005). Acute toxicity elicited by MDMA in humans and experimental animals
includes effects on the neuroendocrine and thermoregulatory systems, in particular induction of
hyperthermia, and on the cardiovascular system (Gordon et al. 1991; Vollenweider et al. 1998; de la
14
Torre et al. 2000). Following several cases-reported toxicity and death induced by exposure to large
dose of MDMA, the UK classified the MDMA as a Schedule 1 drug, thus prohibiting to posses, sell
or give away the substance (Dowling et al. 1987). Finally, on 1 July 1985 the Food and Drug
Administration (FDA) placed the compound on schedule I control substance. Netherless, actually
the MDMA is the most common psychostimulant consumed by adolescent and young people in
dance-clubs.
4.2 Mechanisms involved in the MDMA neurotoxicity
MDMA is usually consumed in pills and numerous studies have indicated that MDMA is well
absorbed by the oral route, and reaches its maximum concentration after 1.5-3 hours from its intake,
with a plasma half-life around 6-7 hours (de la Torre et al. 2004). MDMA is able to easily cross the
BBB and enter the brain without significant delay, by means of his highly lipid solubility.
A number of reports have demonstrated that, in the CNS, MDMA binds to all three presynaptic
monoamine transporters, whit an interspecies different affinity (Moratalla et al. 2015). As substrate
for the monoamine transporters the MDMA is translocated to the cytoplasm. Here, MDMA causes
the dissipation of the proton gradient between the vesicles and the cytosol that is necessary for the
proper functioning of the VMAT2. The VMAT2 dysfunction inhibits the influx and proper storage
of serotonin (5-HT), DA, and noradrenalin (NA) (Rudnick and Wall 1992; Cozzi et al. 1999).
Because its ability to cause functional reversal of both VMAT2 and monoamine transporters,
MDMA is able to increase the extracellular levels of 5-HT, DA, and NA in multiple brain regions
(Gudelsky and Yamamoto 2008). However, the partial inhibition of the MAO-B, located in the
outer mitochondrial membrane, boots the stay of monoamine in the neuronal terminal (Leonardi and
Azmitia, 1994). In this regard, it has been confirmed in the mouse striatum a MDMA dose-
dependently increase of both DA and 5HT release (Górska and Gołembiowska 2015) (Fig. 5).
Several studies have reported that the microinjection of MDMA in different brain areas does not
induce neurotoxicity, unless it was administrated at doses much higher than those having neurotoxic
effects when administered peripherally (Paris and Cunningham 1992; Esteban et al. 2001; Escobedo
et al. 2005). These latter findings indicate that MDMA has to be systemically metabolized to
produce its neurotoxic effects, and suggest that MDMA metabolites are responsible for these
effects.
As stated earlier, MDMA is N-demethylated to 3,4-methylenedioxyamphetamine (MDA). MDMA
and MDA are O-demethylenated respectively to N-methyl-a-methyldopamine (N-Me-a-MeDA),
also called 3,4-dihydroxymethamphetamine (HHMA) and a-methyldopamine (a-MeDA), also
called 3,4-dihydroxyamphetamine (HHA) (Lim and Foltz 1988; Kumagai et al. 1994). These
15
catechols can undergo oxidation to o-quinones that are highly redox-active molecules and produce
free radicals, ROS or RNS (Green et al. 2003; de la Torre et al. 2004; Farré et al. 2004). N-Me-a-
MeDA, a-MeDA and theo-quinones may be conjugated with glutathione to form a glutathionyl
adduct (Hiramatsu et al. 1990; Bai et al. 2001). This conjugate remains redox-active, being readily
oxidized to the quinone thioether and many of the metabolites pharmacologically active (Easton et
al. 2003; Easton and Marsden 2006; de la Torre et al. 2004; Capela et al. 2006).
Figure 5. Neurotoxic mechanisms of MDMA on dopaminergic terminals.
4.2.1 Oxidative stress and excitotoxicity
In addition to its toxic metabolites, MDMA is able to lead in the production of ROS through three
different known pathways. Firstly, the elevated synaptical DA concentration induced by MDMA,
may undergo over auto-enzymatic oxidation, resulting in the production of ROS and toxic
metabolites (Marchitti et al. 2007). The second pathway that lead in ROS production involves
glutamatergic system that through increasing of the intracellular Ca++
levels, triggers the formation
of ROS (Sattler and Tymiansky 2000 and 2001) and the production of NO (Itzhak and Ali 2006).
The mechanism through with NO mediate the MDMA toxicity is not completely understood,
although neuronal nitric oxide synthase (nNOS) inhibitors prevent the MDMA induced
dopaminergic neurotoxicity in mice (Colado et al. 2001).
More recent evidence suggests an important role of the mitochondrial electron transport chain
defect in mediating the MDMA toxic effects. In particular, Puerta and coworkers (2010) recently
reported that inhibition of complex 1 of the mitochondrial electron transport chain is one of the
16
earlier events that take place in MDMA-induced neurotoxicity in mice (Puerta et al. 2010).
Similarly to PD, the inhibition of complex 1 induced by MDMA increases the production of ROS
and others dangerous free-radicals. However, as mentioned above, the mitochondria-related energy
failure in dopaminergic cells, may disrupt vescicular storage of DA, increasing the DA release and
the formation of DA toxic metabolites (Fig. 5). In this regard, the inhibition of MAO-B induced by
MDMA, increases the cytosolic DA levels (Leonardi and Azmitia 1994), promoting the
autoxidation of DA in DAQ.
Taken together, these mechanisms leading to the formation of reactive intermediates, ROS, and/or
toxic oxidation products may represent the triggering factors responsible for the toxicity exerted by
this amphetamine. Finally, these elevated levels of free radicals and the reduced levels or
inactivation of antioxidant enzymes, such as catalase and superoxide dismutase (SOD), induced by
MDMA results in oxidative stress (Cadet et al. 2001; Sanchez et al. 2003). In line with this, MDMA
produces a less marked oxidative stress and striatal depletion of both DA and 5-HT in transgenic
mice overexpressing SOD than in wild-type mice (Jayanthi et al. 1999). Furthermore, treatment
with antioxidant agents has been found to afford neuroprotection in MDMA-treated rats (Gudelsky
1996; Aguirre et al. 1999; Shankaran et al. 2001).
4.2.2 Neuroinflammation and Hyperthermia
A relevant issue related to MDMA-induced neurotoxicity is that MDMA can trigger inflammatory
processes in those brain areas that exhibit dopaminergic and/or serotonergic terminal degeneration,
but not in brain areas where no modifications in either DA or 5-HT levels occur (Yamamoto et al.
2010). Several preclinical have demonstrated that MDMA elicits astroglial and microglial activation
in the striatum (Granado et al. 2008a; Costa et al. 2013; Frau et al. 2016), as well as in the cortex
(Herndon et al. 2014; Costa et al. 2014), and hippocampus (Costa et al. 2014; Lopez-Rodriguez et
al. 2014). However, elevated levels of glia-generated reactive species, such as NO, superoxide and
cytokines, have been shown to correlate with neurodegeneration induced by amphetamine-related
drugs (Castelli et al. 2014; Thomas et al. 2004). In support of the hypothesis that the
neuroinflammation is one of the factors involved in MDMA toxicity, Zhang and coworkers showed
that minocycline, an anti-inflammatory drug, has a neuroprotective effect against MDMA induced
neurotoxicity (Zhang et al. 2006).
Another mechanism that may be implicated in MDMA neurotoxicity is the hyperthermic response
that is influenced by dose, ambient temperature and other housing conditions (Moratalla et al.
2015). In this regard, it is worth mentioning that the typical environmental conditions featuring
dance clubs, where music is deafening and room temperatures are high due to crowding, together
17
with the fact that club-goers usually consume little water and considerable amounts of ethanol, are
crucial to amplifying MDMA-induced hyperthermia (Green et al. 2003; Moratalla et al. 2015).
The mechanism of MDMA-induced hyperthermia is complex, and involves not only serotonergic
and dopaminergic systems, but also adrenergic transmission (Sprague et al. 1998). The increased
release of these monoamines induced by MDMA administration may stimulate receptors involved
in thermoregulation (Shankaran and Gudelsky 1999). Although previous studies associated the
hyperthermia with the increased 5-HT release (Shankaran and Gudelsky 1999), recent evidences
have indicated that the primary mechanism involved in the hyperthermic response is the dopamine
release. In fact, several studies reported that the activation of D1 receptors induces hyperthermia in
mice (Zarrindast and Tabatabai 1992), while the inactivation of D1 receptors attenuated MDMA-
induced hyperthermia and prevented the striatal loss of DA (Granado et al. 2014).
However, a role of GLU has been suggested by a significant decrease of MDMA-induced
hyperthermia in rats treated with NMDA receptor antagonists (Nisijima et al. 2012).
Studies in experimental animals have indicated that hyperthermia induced by MDMA, being
harmful per se by leading to dehydration and altered hydrosaline homeostasis (Green et al. 2004;
Baylen and Rosenberg 2006), may be one of the factors that promote glial activation and
neurotoxicity caused by this amphetamine-related drug (Miller and O‟Callaghan 1995; Colado et al.
1998; Mechan et al. 2001; Touriño et al. 2010). With regard to the possible relationship among
MDMA-induced hyperthermia and glial activation it is interesting to mention a recent study by our
group, which demonstrated the existence of a positive correlation between the elevation in body
temperature and the degree of glial activation in mice treated with MDMA (Frau et al. 2013).
However, previous studies in rats have demonstrated that hyperthermia induced by MDMA is
associated with enhanced production of free radicals and proinflammatory cytokines, such as IL-1β,
IL 6, and TNF-α, (Green et al. 2004; Orio et al. 2004). At the moment, is not clear if the increased
concentrations of proinflammatory cytokines could be a consequence, or the cause of MDMA-
induced hyperthermia. Further support to a possible relationship among hyperthermia and
neurotoxicity comes from evidence showing that in experimental animals high body temperature
enhances the formation of toxic metabolites of MDMA, which are known to increase oxidative
stress (Cadet et al. 2007). On this basis, it has been speculated that hyperthermia may have an effect
on the other neurotoxic mechanisms of MDMA, such as ROS and reactive RNS production,
eventually causing nerve terminal damage (Goni-Allo et al. 2008; Malberg et al. 1998), and glial
activation (O'Callaghan and Miller 1994; Thomas et al. 2004).
18
4.3 Neurotoxicity in humans
To elucidate the toxic mechanisms which lead to higher incidence of PD in MDMA abusers
(Callaghan et al. 2012; Curtin et al. 2015), several researchers investigated the MDMA-induced
neurotoxic effect in both humans and experimental animals. The features of neurotoxic damage
induced by MDMA seem to vary, depending on the gender and strain of animals, which may
influence the response to different dosing regimens and administration routes of MDMA (Ricaurte
et al. 1988; Colado et al. 1995; Itzhak et al. 2003).
In humans, MDMA displays higher affinity for noradrenalin transporter (NET), and lower, but
similar, affinities for serotonin transporter (SERT) and DAT (Verrico et al. 2007). However, the
ability to release intracellular monoamines is higher in SERT-expressing cells than in either DAT-
or NET-expressing cells, and this may justify the reduction of SERT density observed in ecstasy
users compared to controls (Haddad et al. 2002; Reneman et al. 2001a,b; Semple et al. 1999).
Moreover, the neurotoxic potential of MDMA in humans has been evaluated indirectly by
measuring the concentration of 5-HIAA (5-hydroxyl indole acetic acid) in cerebrospinal fluid (CSF)
in recreational ecstasy users. Several studies reported significantly lower levels of 5-HIAA in
recreational ecstasy users compared to polydrug users who had never used ecstasy (McCann et al.
1999 and 1994; Ricaurte et al. 1990). In this regard, Kish and colleagues (2000) demonstrated
severe depletion (50-80%) of striatal 5-HT and 5-HIAA in the brain of a 26-year-old male who had
taken MDMA regularly for 9 years (Kish et al. 2000). More direct evidence supporting that MDMA
produces long-term neurotoxic effects on brain 5-HT systems emerged from neuroimaging studies:
increase in 5-HT2A receptor density detected by SPECT, in heavy MDMA abusers compared to
controls, showed a long-term compensatory response to 5-HT depletion (Reneman et al. 2000).
While several studies have suggested that MDMA may harm the serotonergic system in the human
brain, it is less clear whether MDMA may be toxic for human dopaminergic neurons.
In this regard, it is noteworthy that MDMA has significant affinity for DAT (Verrico et al. 2007)
and promotes the release of DA in multiple brain regions. This consideration with the reported
higher incidence of PD in heavy MDMA-abusers, suggest that MDMA may be toxic for the human
dopaminergic system (Moratalla et al. 2015).
The evaluation of the long-term effects of MDMA, including neurotoxicity, in humans is complex,
since MDMA users frequently consume it in the context of poly-drug abuse, together with other
psychoactive substances, such as ethanol, cannabis, and cocaine (Schifano et al. 1998). However,
there are many variables that could not be controlled, e.g. dose and purity of MDMA, number of
times MDMA was used, set and setting, and the time between interview and the last use of MDMA.
19
Thus, animal models afford the unique opportunity to evaluate the effects of MDMA without many
complicating factors.
4.4 MDMA administration in mice: a model of dopaminergic neurotoxicity
To evaluate the MDMA toxicity on dopaminergic system, the murine model is the most validated.
In fact, although in rats MDMA has the highest affinity for the SERT, and lower affinities for NET
and DAT (Steele et al. 1987; Rudnick and Wall 1992), in mice MDMA seems to act as a
dopaminergic neurotoxin (Kindlundh-Högberg et al. 2007). Therefore, the study of MDMA actions
on DA in mouse appears particularly interesting, since DA mediates several behaviors that occur
after MDMA administration, such as hyperactivity, alterations in mental state, hyperthermia
(Fantegrossi et al. 2003; Green et al. 2003; Miller and O‟Callaghan 1995) and the reported higher
incidence of PD in MDMA abusers (Callaghan et al. 2012; Curtin et al. 2015). When administered
to mice, MDMA produced damage of DA terminals and decreases the concentrations of DA,
DOPAC, and HVA in several brain regions (Moratalla et al. 2015). Moreover, and most
importantly, MDMA produces long-term degeneration of dopaminergic nerve terminals (Brodkin et
al. 1993; Colado et al. 2001; Izco et al. 2010; Costa et al. 2013) and a decrease in tyrosine
hydroxylase (TH), the rate-limiting enzyme for DA synthesis, in the striatum (Green et al. 2003;
Costa et al. 2013). Moreover, a study by Granado and coworkers demonstrated that MDMA
administration induces a significant decrease in TH-positive neurons in the SNc but not reduce the
synthesis of TH, supporting the toxic effect of MDMA on the nigrostriatal system. Interestingly, the
same group observed that MDMA induces a loss of TH and DAT fibers in the striatum, but not in
the nucleus accumbens, indicating that the dopaminergic neurotoxicity of MDMA targets the
nigrostriatal system, while sparing the mesolimbic pathway (Granado et al. 2008a). These results
are in line with earlier evidence describing dopaminergic terminal loss in the mouse striatum
following MDMA administration (Fornai et al. 2004). Moreover, it damages striatal dopaminergic
terminals, with an effect that appears more pronounced in the striosomal compartment than in the
matrix (Granado et al. 2008b). Furthermore, and most notably, a similar striosomal damage has
been observed following the MPTP administration (Iravani et al. 2005); this feature is one of the
similarities between the effects of MDMA and those of a dopaminergic neurotoxin (Moratalla et al.
2015). In fact, MDMA, as MPTP, is able to induce mitochondrial dysfunction that, as explained
above, trigger oxidative stress in the vulnerable dopaminergic neurons of SNc (fig. 3 and 5).
20
4.5 MDMA Neurotoxicity and adolescence
It must be considered that an increased risk of developing drug abuse and drug-related problems is
often associated with the age of consumers (Hawkins et al. 1992). In fact, MDMA is largely
consumed by adolescents, during raves and club parties. In both humans and experimental animals,
adolescence is a critical period for the development of the brain, which undergoes major
developmental changes (Casey et al. 2000; Piper and Meyer 2004). In fact, the adolescent brain
appears to be particularly vulnerable to the long-term noxious effects of exogenous substances,
including drugs of abuse (Cadoni et al. 2015; Daza-Losada et al. 2009; Sisk and Zehr 2005; Spear
2000). In this contest it is important to highlight that although the adolescent brain is particularly
vulnerable to neurotoxicity; a lower sensitivity to MDMA effects in adolescent compared with adult
mice, was described by previous studies (Reveron et al. 2005; Teixeira-Gomes et al. 2015; Frau et
al. 2016). In particular, repeated MDMA administration induced a loss of dopaminergic neurons in
SNc of adult and adolescent mice, whereas TH-positive fibers in striatum were decreased in adult
mice only (Reveron et al. 2005; Frau et al. 2016). These results are confirmed by microdialysis
experiments that showed lower DA extracellular concentrations in adult compared whit adolescent
mice, 7 days after repeated MDMA administrations (Reveron et al. 2005). Although the lower
striatal neurotoxic effect, the abuse of MDMA during adolescence, such as other amphetamine-
related drugs, may lead to long-lasting effects, which could eventually render the brain more
sensitive to toxic insults later in life. Considering the MDMA property to affect neurogenesis and
synaptogenesis, leading to the disruption of DA homeostasis, the consumption of MDMA during
the adolescence could increase the vulnerability of dopaminergic neurons (Rice and Barone 2000).
In particular, MDMA may interfere with dopamine synapse development in a phase of life crucial
to the development of the dopaminergic system (Fasano et al. 2008; Goffin et al. 2010), rendering
this system more vulnerable at a later time (Costa et al. 2014; Frau et al. 2016). The alteration in
dopaminergic transmission and function could be a negative factor in the development of
neurodegenerative diseases, such as PD, which principally involves the dopaminergic system. In
line with this, adult mice treated with MDMA during adolescence showed more marked
neuroinflammatory and neurotoxic effects MPTP-induced in motor (SNc and striatum), and non-
motor (hippocampus and medial prefrontal cortex) brain areas, compared with mice non-pretreated
with MDMA (Costa et al. 2013 and 2014).
21
5. Parkinson disease therapy
5.1 Current PD therapies
Current treatments for Parkinson disease, based fundamentally on the replacement of DA, can
significantly improve symptoms but, unfortunately, do not cure the disease or slow down its
progression. Although the number of drugs has increased and our sophistication in using them has
improved, L-DOPA and dopaminergic agonists, remain the principal current treatment of PD. A
necessary prerequisite for these approaches, however, is the presence of functioning dopaminergic
nerve terminals in the striatum, meaning that with its progression the disease becomes increasingly
refractory to pharmacological treatment. Moreover, these approaches have many side effects, such
as dyskinesia or motor fluctuations, which can not easily be tolerated during life-long treatment and
could be a starting point for further complications (dopamine dysregulation syndrome or impulse
control disorder) (Vitale et al. 2013).
Several non dopaminergic therapies have been explored in the treatment of PD and the adenosine
A2A receptors antagonists seem promising. Furthermore, epidemiological studies have shown that
the incidence of PD is lower in consumers of high doses of caffeine, an antagonist of adenosine A1
and A2A receptors (Ross et al. 2000). A2A receptor antagonists have also been shown to have a
neuroprotective role in several experimental rodent models of PD (Carta et al. 2009; Ikeda et al.
2002; Pierri et al. 2005). Antagonism of adenosine A2A receptors facilitates GABA release in
striatum, reducing striatopallidal neuronal overactivity. This reduction helps to increase indirect
inhibitory output from the striatum to the globus pallidus, restoring balance between the basal
ganglia output pathways (Ferré et al. 1997; Mori and Shindou 2003). Moreover, several data have
shown that A2A receptor antagonism counteracts neuroinflammatory processes (Carta et al. 2009;
Huang et al. 2006) by inhibiting astroglial and microglial activation (Ikeda et al. 2002; Pierri et al.
2005). These data have been confirmed by Carta and coworkers that showed a reduced astrogliosis
and microgliosis after subchronic MPTP administration in A2A receptor knockout mice (Carta et al.
2009). Finally, preclinical studies and clinical trials suggest that these compounds may increase the
therapeutic efficacy of L-DOPA without exacerbating L-DOPA-associated dyskinetic effects (Bara-
Jimenez et al. 2003; Grondin et al. 1999; Hauser et al. 2008; Kanda et al. 2000; LeWitt et al. 2008;
Morelli 2003; Schwarzschild et al. 2006; Stacy et al. 2008).
5.2 Monoamine oxidase inhibitors therapy
A different approach currently used as an add-on therapy, is based on the inhibition of enzymes
involved in the metabolic degradation of dopamine (monoamine oxidase, (MAO), catechol-O-
methyltransferase, (COMT)) (Youdim and Riederer 2004). Selective MAO-B inhibitors block the
22
enzyme responsible for the intracellular degradation of DA, thereby increasing DA availability in
the nigrostriatal pathway and prolonging the effectiveness of L-DOPA replacement therapy.
a b
Figure 6. Chemical structures of (R)-N-methyl-N-(1-phenylpropan-2-yl) prop-2-yn-1-amine (selegiline) (a)
and N-propargyl-1(R)-aminoindan (rasagiline) (b).
During the last decade, the well-known MAO-B inhibitors, selegiline [deprenyl, (R)-N-methyl-N-
(1-phenylpropan-2-yl) prop-2-yn-1-amine] and rasagiline [Azilect, N-propargyl-1(R)-aminoindan]
(Fig. 6a and b), have been shown to be neuroprotective against dopaminergic cell death. In line with
these results, several reports demonstrated the neuroprotective effects of the MAO-B inhibitors in
parkinsonian animal models (Mandel et al. 2007). Their neuroprotection has been attributed to both
antioxidant and antiapoptotic properties of the parent compounds or their metabolites (Reznichenko
et al. 2010; Wu et al. 1996). Inhibition of MAO-B may afford neuroprotection through a reduced
production of ROS and aldehydes, protecting against oxidative stress (Youdim et al. 2006;
Hauptmann et al. 1996; Youdim and Lavie 1994; Mallajosyula et al. 2008). In particular, Youdim
and coworkers showed that rasagiline, as other propargylamine compounds, prevents the fall in the
mitochondrial potential induced by oxidative stress and increases the activity of antiapoptotic
factors and antioxidant enzymes (Youdim et al. 2001 and 2006). Although the primary effect of
rasagiline in PD is presumably done by MAO-B inhibition, resulting in a slower metabolism of
endogenous and exogenous DA and thus providing symptomatic benefits (Finberg et al. 1996 and
1998), it also possesses neuroprotective activity unrelated to its MAO-B inhibition. In particular
rasagiline has been shown to increase the levels of brain-derived- and glial cell line-derived
neurotrophic factors (BDNF, GDNF) (Maruyama et al. 2004; Weinreb et al. 2004) and it is able to
increase cell survival in cell culture and animal models of PD (Bar-Am et al. 2007; Zheng et al.
2005). The neurorestorative activity of this MAO-B inhibitor was demonstrated in a progressive
apoptotic model of neuronal death induced by long-term serum deprivation (Bar-Am et al. 2005 and
2007), and also in a post-MPTP-administration model in mice (Mandel et al. 2007; Sagi et al.
2007).
23
Nevertheless, in the recent ADAGIO (Attenuation of Disease Progression with Azilect Given Once-
Daily) trials, a delayed start study design was applied to separate symptomatic benefit from disease-
modifying effect, and the results failed to define a clear, dose-dependent disease-modifying action
of rasagiline (Ahlskog et al. 2010).
5.3 Novel (hetero)arylalkenyl propargylamine compounds
On the basis of the controversial neuroprotective activity of the more useful irreversible MAO-B
inhibitors (Olanow and Rascol 2010; Weinreb et al. 2010), a large number of new molecules, that
contain a propargyl ring and showed more potent MAO-B inhibitory activity and neuroprotective
effects, have been proposed to replace the pre-existing MAO-B inhibitors. In particular, a novel
series of (hetero)arylalkenylpropargylamine compounds that demonstrate highly potent MAO-B
inhibitory activity with remarkable selectivity over other types of amine oxidase enzymes, were
synthesized (Huleatt et al. 2015). The design of these novel propargylamines is based on the
knowledge that the propargylamino group has been demonstrated to be a potent anti-apoptotic agent
(Maruyama et al. 2002), while MAO-B selectivity and potency as well as overall neuroprotective
profile were tuned through judicious choice of the skeleton and its substituents (Huleatt et al. 2015).
Figure 7. Chemical structure of N-Methyl-2-phenyl-N-(prop-2-yn-1-yl)prop-2-en-1-amine Hydrochloride
(SZV558).
A number of compounds among this series displayed cytoprotective properties against 6-OHDA-
and rotenone-induced cell death in PC12 cells (Huleatt et al. 2015). In particular, to elicit cell death
was used in the first series of experiments 6-OHDA, whereas in the second series, rotenone. The
compounds effects were compared to rasagiline treated-cells. In line with literature data (Zheng et
al. 2005; Milusheva et al. 2010; Bar-Am et al. 2007), rasagiline exerted significant 48% protective
effect. Of the compounds tested, the SZV558 [N-Methyl-2-phenyl-N-(prop-2-yn-1-yl)prop-2-en-1-
amine Hydrochloride] (Fig. 7) showed >40% protection against 6-OHDA-induced cell death
(Huleatt et al. 2015). Specifically, the profile of SZV558 appears to be particularly interesting
because of its pharmacodynamic, which is favorable for disease-modifying properties, since it
24
irreversibly binds the MAO-B enzyme, and has a long half-life. In particular the peak of the effect
of SZV558 was obtained 18 hours after the administration, and a tendency of protection was present
up to 42 hours (Huleatt et al. 2015). This compound appears to be superior to rasagiline with respect
to both selectivity (human MAOA/B selectivity ratio: 58) and potency, having an IC50 of 60 nM
(Huleatt et al. 2015).
However, culture-based cell survival assays represent oversimplified experimental systems, and
they might not reflect the complexity of in vivo systems. On these bases, in a recent study, the
protective activity of SZV558 was evaluated in various in vivo models of PD, using different
neurotoxins (rotenone and MPTP). The results displayed that SZV558 inhibited selectively the
oxidative stress induced by pathological DA release in the rotenone-treated rat striatum slices. In
this case, the new compound appeared to provide higher protective efficacy than rasagiline (Baranyi
et al. 2016). Moreover, the effect of SZV558 was dose-dependent and, at low doses, its efficacy was
higher than rasagiline against the depletion of DA, even after the acute administration of MPTP.
These data were confirmed by behavioral tests, such as the open-field test and rotarod test, used to
evaluate respectively locomotor activity and motor coordination (Hu et al. 1991; Shiotsuki et al.
2010; Meredith and Kang 2006). Therefore, SZV558 fulfills the criterion of a multi-target anti-PD
compound.
5.4 Metformin
Several therapies have been explored in the treatment of PD, such as the identifying well-tolerated,
medications that enhance adult neurogenesis that could be of great clinical interest in the
management of neurodegenerative disorders. In line with this, the metformin, a well-known
antidiabetic drug, has recently been proposed to promote neurogenesis and to have a
neuroprotective effect on the neurodegenerative processes induced by MPTP in a mice PD model
(Patil et al. 2014).
Figure 8. Chemical structure of metformin (1,1-dimetilbiguanida).
Metformin is an antidiabetic medication, member of biguanide class (Fig. 8). It is the first-line drug
of choice for the treatment of type 2 diabetes, that effectively lowers plasma glucose levels
25
primarily by decreasing hepatic glucose production and by improving lipid metabolism in both liver
and muscle tissues (Perriello et al. 1994; Stumvoll et al. 1995). At cellular level, metformin
activates AMP-activated protein kinase (AMPK), that is the key mechanism by which this drug acts
on liver and muscle but also on the intestine (Pieri et al. 2010; Sakar et al. 2010).
Moreover, study conducted by Labuzek et al. (2010 a, b) demonstrated that orally administered
metformin rapidly crosses the BBB and is carried in the neurons by organic cation transporter 1
(Łabuzek et al. 2010a, b), usually involved in the carriage of endogenous compounds, such as DA
(Becker et al. 2011). On this base, in addition to its antidiabetic potential, the therapeutical effects
of metformin on various CNS disorders, such as PD or Huntington disease, has been studied. (Ma et
al. 2007; Ng et al. 2012).
Clinical trials conducted in a Taiwanese population cohort showed that incident PD risk in type 2
diabetes increases 2.2-fold. While, the sulfonylureas treatment further increases risk by 57%, the
combination with metformin avoids completely the risk (Wahlqvist et al. 2012). In line with this,
Wang and colleagues demonstrated that the metformin activates in adult neural stem cells (Wang et
al. 2012), the protein kinase C–cyclic-adenosine monophosphate (cAMP) response element-binding
protein (PKC–CREB) pathway, which responds to a wide range of neurological disorders, such as
PD and Alzheimer‟s disease (Emsley et al. 2005; Potts and Lim 2012).
In the CNS, metformin is able to stimulate AMPK, increasing glucose uptake by neurons (Amato
and Man 2011) and affecting neural metabolism (Potts and Lim 2012). Recent studies suggest that
AMPK activation might prevent neuronal cell death and play a pivotal role as a survival factor in
PD. In fact, inhibition of AMPK increases MPP+-induced cell death while overexpression of
AMPK raises cell viability after exposure to MPP+ in SH-SY5Y cells (Choi et al. 2010). In line
with this, metformin was found to alleviate dopaminergic dysfunction and mitochondrial
abnormalities through the AMPK activation in drosophila models of PD (Ng et al. 2012). Moreover,
previous studies suggest that metformin prevents the oxidative stress-related cellular death in non-
neuronal cell lines (Łabuzek et al. 2010a, b), conserving the activities of antioxidant enzymes
(Pavlovic et al. 2000; Chakraborty et al. 2011; Esteghamati et al. 2013). Finally, metformin was
found to be neuroprotective by inhibiting apoptosis in neuronal cortical cells (El-Mir et al. 2008)
and more recently by promoting neurogenesis in the olfactory bulb and in hippocampus of adult
mice by PKC-CBP pathway (Wang et al. 2012).
5.5 Caffeine: neuroprotective or neurotoxic?
As mentioned above, several epidemiological evidences showed that caffeine as well as coffee
consumption is linked to a reduced risk of developing PD (Ross et al. 2000; Ascherio et al. 2001;
26
Schwarzschild et al. 2003). In this regard, considerable research data have suggested that the
caffeine may protect against the PD neurodegeneration by A2A receptor blockade (Morelli et al.
2010). The primary action of caffeine is the blockade of A1 and A2A receptors which leads to
secondary effects on many classes of neurotransmitters (Fredholm et al. 1999). The A2A receptor
are implicated as mediators or modulators of dopaminergic neurodegeneration (Morelli et al. 2010),
consequently the blockade of this receptor by caffeine might reduce the neurodegenerative
progression. Moreover, caffeine is able to attenuate the loss of striatal DA induced by acute MPTP
administration in mice, confirming the neuroprotective potential of A2A receptor blockade in PD
(Chen et al. 2001).
Although caffeine use is safe (Ascherio et al. 2001; Fredholm et al. 1999; Schwarzschild et al.
2003), the consumption of caffeinated energy drinks might influence the adverse effects of other
substances which are taken concomitantly (Mohamed et al. 2011; Morelli and Simola 2011; Simola
et al. 2006). The use of amphetamine-related drugs is often combined with beverages containing a
high quantity of caffeine in order to amplify their stimulant actions (Mohamed et al. 2011; Reissig
et al. 2009). Caffeine has been reported to influence the toxicity of amphetamine-related drugs, as
shown by potentiation of seizures, hyperthermia, tachycardia, and mortality (Camarasa et al. 2006;
Derlet et al. 1992; McNamara et al. 2006, 2007; Vanattou-Saïfoudine et al. 2012). When co-
administered with MDMA, caffeine potentiated the MDMA effect not only on the DA but also on
5-HT release, as measured by an in vivo microdialysis in mice (Gorska and Gołembiowska 2015).
Gorska and co-workers, using selective antagonists of A1 and A2A receptors, showed that both
adenosine receptors markedly enhanced the DA and 5-HT release induced by MDMA in the mouse
striatum (Gorska and Gołembiowska 2015). These data suggest that DA release induced by
combined treatment of MDMA and caffeine is mediated by blockade of both adenosine receptors.
Moreover, hyperthermia and neuroinflammation after acute but not chronic administration of
caffeine and MDMA have been reported to cause neurotoxic effects in rodents (Khairnar et al.
2010; McNamara et al. 2006; Ruiz-Medina et al. 2013; Vanattou-Saïfoudine et al. 2011). On the
other hand, exacerbation of MDMA-induced hyperthermia by caffeine is proposed to result from
the inhibition of A2A receptors (Vanattou-Saïfoudine et al. 2010).
However, caffeine has been reported to potentiate the glia-activation in the striatum of adult and
adolescent mice, when it has given together with MDMA (Khairnar et al. 2010; Frau et al. 2016).
Probably, by blocking both A1 and A2A receptors, caffeine may facilitate neuroinflammatory
processes induced by MDMA. However, the promotion of temperature elevation induced by
caffeine (McNamara et al. 2006) might contribute to the increase in astroglia reactivity observed
after MDMA plus caffeine administration, suggesting another possible mechanism potentiating
27
MDMA effects (Moratalla et al. 2015). Interestingly, adolescent mice showed a higher sensitivity to
dopaminergic neurodegeneration induced by the association caffeine - MDMA both in striatum and
SNc, compared with adults, suggesting that caffeine may worsen the toxicity elicited by MDMA in
adolescents (Frau et al. 2016).
Finally, the caffeine ability to affect the absorption of MDMA in intestinal epithelial cells and
increase the area under the plasma concentration curve of MDMA (Kuwayama et al. 2007), as well
as the increased DA release, excessive oxidative stress or formation of toxic metabolites may have a
role in caffeine‟s potentiation of MDMA effects (Gołembiowska et al. 2009; Gołembiowska and
Dziubina 2012).
Contrary to the acute treatment, the chronic administration of caffeine before an acute MDMA
treatment exerts a protective role against MDMA induced neuroinflammation (Ruiz-Medina et al.
2013). One explanation of this paradox is that mice pretreated with caffeine did not experience
temperature increase after MDMA administration; in fact after 21 days of chronic daily caffeine
administration, tolerance to elevated body temperature induced by caffeine, may be developed.
These data demonstrated that depending on the dose and/or administration pattern (acute versus
chronic), caffeine could contribute or attenuate brain damage (Ruiz-Medina et al. 2013).
28
AIMS
The therapy of PD is based on the treatment of symptoms while neuroprotective treatments, the
only able to stop the disease progression, are not present yet. Starting from the assumption that both
MPTP and MDMA in mice are able to affect the nigrostriatal pathway inducing a specific
dopaminergic neurodegeneration, and considering that one of the possible multiple PD origins is the
chronic exposure to dopaminergic neurotoxins, the nigrostriatal neurodogeneration induced by
either MDMA or MPTP administration were used as models to test the effects of substances
potentially neuroprotective.
On this basis, the present study evaluated the influence of drugs, such as MAO inhibitors and
metformin, or substances, such as caffeine, on the neurodegenerative effects of MDMA and MPTP
in mice.
1. In the first phase of my study, I evaluated the neuroprotective activity of the new MAO-B
inhibitor compound, SZV558, compared with well-known rasagiline, in a chronic mouse model
of MPTP plus probenecid administration, characterized by the progressive development of
parkinsonian symptoms, neurodegeneration, and neuroinflammation (Schintu et al. 2009).
Consequently, the first aim of my study was to investigate whether the SZV558 administration
could revert the motor impairments, the olfactory dysfunction and the loss of nigrostriatal
neurons, induced by MPTPp chronic treatment in adult mice.
To pursue this first aim, the following experiments were carried out:
- Assessment of the olfactory deficit, usually exhibited after a chronic MPTP treatment or in
the early PD stages and evaluated by pellet retrieval olfactory test.
- Assessment of the spontaneous motor activity, the motor performance and coordination,
and finally the skilled forepaw use, evaluated by, respectively, motility test, beam walking
test and inverted grid test after the last MPTP administration.
- Assessment of the dopaminergic neuronal damage in SNc and caudate-putamen (CPu)
studied by the TH immunohistochemistry and the neuronal death in SNc by Nissl staining.
2. In a second phase of my studies, on the basis of previous results that showed a more
pronounced degeneration in adolescent compared with adult mice when MDMA is associated
with caffeine, I studied the neurotoxic proprieties of MDMA and caffeine administrated in
association in adolescent or adult mice.
Consequently, the second aim was to clarify the molecular mechanism at the base of the
different neurotoxic effect of this drug association at different ages. To pursue this second aim,
I measured by immunofluorescent evaluation, the nNOS expression, which plays a critical role
29
in the integration of dopaminergic and glutamatergic transmissions, and the levels of
proinflammatory cytokines IL-1β and TNF-α in the CPu of adolescent or adult mice treated
with MDMA, alone or in combination with caffeine.
3. In the third part of my PhD, starting from the assumptions that the antidiabetic drug metformin
is able to decrease the loss of dopaminergic neurons induced by MPTP, I investigated whether
the neuroprotective effect of metformin is able to counteract the dopaminergic neurotoxicity
induced by repeated MDMA administration in adult mice. The third aim of my thesis was to
evaluate the effects of metformin administration on nigrostriatal dopaminergic loss of neurons
induced by acute MDMA treatment.
To pursue this third aim, I assess the dopaminergic neuronal damage in SNc and CPu by TH
immunohistochemistry and the neuronal death in SNc by Nissl staining.
30
MATERIALS AND METHODS
1. Drugs
The following drugs were used:
- (hetero)arylalkenylpropargylamine derivative (SZV558) and (R)-N-(prop-2-ynyl)-2,3-
dihydro-1H-inden-1-amine (rasagiline),which were synthesized according to the method
used by Huleatt and coworkers (Huleatt et al. 2015).
- Probenecid, MPTP-HCl, metformin and caffeine were obtained from Sigma (Sigma-
Aldrich, Milan, Italy).
- MDMA–HCl was synthesized at the Department of Life and Environmental Sciences,
University of Cagliari (Frau et al. 2013).
SZV558, rasagiline, MPTP and metformin were dissolved in distillated water. While probenecid
was dissolved in 5% NaHCO3 and MDMA was dissolved in saline. All solutions were freshly
prepared on the day of use.
2. Treatments
Experimental procedures were approved by the Ethics Committee of the University of Cagliari in
compliance with the European Communities Council Directives (2010/63/EU; L.276; 22/09/2010).
Efforts were made to minimize the number of animals used and to maximize humane treatment.
2.1 Chronic protocol of MPTP plus probenecid (MPTPp)
Experiments were performed on adult (12-week-old) male C57Bl/6J mice (Charles River, Italy)
treated with vehicle (saline solution) plus probenecid, or MPTP (25 mg/kg i.p.) plus probenecid
(100 mg/kg i.p.), administered 30 min before each MPTP administration (MPTPp), twice a week for
5 weeks, alone, or in the presence of the SZV558 (1 mg/kg i.p.) or rasagiline (1 mg/kg i.p.)
administered 18 hours before each MPTPp administration. At the end of treatment, mice were tested
behaviorally (motility test, beam-walking test, inverted grid test and olfactory test) to evaluate the
motor and olfactory performance impairments. Animals were sacrificed 3 days after the last
administration of MPTPp.
2.2 Acute MDMA treatment in combination with caffeine
Adult and adolescent (28-day-old) male C57BL/6J mice, were treated with vehicle, caffeine (2×10
mg/kg, 4-hour interval, i.p.), MDMA (4×20 mg/kg, 2-hour intervals, i.p.) or MDMA (4×20 mg/kg,
2-hour intervals) plus caffeine (2×10 mg/kg, before the first and third MDMA administration). On
31
the second day, mice received vehicle or caffeine (2×10 mg/kg, 12-hour interval), and on the third
day, mice received vehicle or caffeine (1×10 mg/kg). Animals were sacrificed 48 hours after the last
MDMA administration.
2.3 Acute MDMA treatment in combination with metformin
Following the previous MDMA treatment, adult male mice, were treated with metformin (2×200
mg/kg, 10-hour interval, o.s.) or MDMA plus metformin (2×200 mg/kg, 1h before the first MDMA
administration and 4h after the last). On the next two days, mice received one daily administration
of vehicle or metformin (200 mg/kg). 48 hours after the last administration of MDMA, mice were
sacrificed and transcardially perfused.
3. Behavioral tests
3.1 Spontaneous motor activity: motility test
Spontaneous motility was assessed 2 days before the MPTPp treatment and 1 day after the last
MPTPp administration, in a quiet isolated room. Mice were placed individually in plexiglass cages
(length 47 cm, height 19 cm, width 27 cm), with a metal grid over the floor, and equipped with
infrared photocell emitters-detectors situated along the long axis of each cage (Opto-Varimex Mini;
Columbus Instruments). The interruption of a photocell beam was detected by a counter that
recorded the total number of photocell beam interruptions. The counter recorded two different types
of motor activity: locomotor activity due to the locomotion of the mouse along the axes of the cage
and total motor activity due to locomotion plus non-finalized movements (stereotyped behaviors,
such as grooming, rearing, and sniffing). The counter recognized the stereotyped movements
because of the continuous interruption of the same photocell beam, whereas locomotion along the
cage produced interruptions of different photocell beams. Motility was detected as soon as the
mouse entered into the cage and was evaluated for 60 min (Baiguera et al. 2012).
3.2 Beam-walking test
The motor performance and coordination of mice were evaluated with the beam-walking test
(Fleming et al. 2004). In this test, mice were trained to traverse the length of a plexiglass beam that
was divided into four sections of 25 cm each (1 m total length). Each section of the beam had a
different width: 4, 3, 2, and 1 cm; the beam was placed on a table and ended in the animal‟s home
cage. Mice received 2 days of training before testing. On the first day, mice received two assisted
trials, involving the placement of the mouse on one extremity of the beam with the home cage in
close proximity to the animal. This encourages forward movement along the beam. After two
32
assisted trials, mice were able to traverse the entire length of the beam unassisted. Days 1 and 2 of
training ended when all animals had completed five unassisted runs across the entire length of the
beam. To increase the difficulty further, on the day of the test, a mesh grid (1 cm squares) of
corresponding width was placed over the beam surface. The test was performed 2 days after the last
MPTPp administration and mice were videotaped for a total of five trials. An error was counted
when, during a forward movement, a limb slipped through the grid. By scoring each limb slip
individually, the severity of the error could be measured. The number of steps and the number of
errors were calculated across all five trials and averaged for each group (Fleming et al. 2004).
3.3 Inverted grid test
The inverted grid test was used to assess skilled forepaw use, especially related to the distal
musculature and digit manipulations. Mice were placed in the center of a horizontal square grid (15
cm2) consisting of a wire mesh (mesh 0.5 cm
2) surrounded by wooden walls. The grid was placed
20 cm above a tabletop and was rotated upside down allowing mice to move freely. The time the
mice took before falling down was recorded. If a mouse fell from the mesh grid within 10 s,
additional trials were allowed (maximum: three trials) within an interval of 5 min; in this case,
latencies before falling were measured. The mean ± SEM of three trials was calculated. Moreover,
for each trial, the number of steps and forelimb fault per step was rated and compared with controls.
No training was performed, but a pre-test was carried out (Meredith and Kang 2006). The time the
mice took before falling down was measured 2 days after the last MPTPp administration.
3.4 Pellet retrieval olfactory test
Mice were food-deprived for 20 hours before the olfactory test. The test was conducted in a clean
plastic cage (length 42 cm, height 15 cm, width 24 cm). A smelling pellet was buried under the
bedding (1 cm) in a cage corner. The mouse was positioned in the center of the cage and the time to
retrieve the pellet and bite it was measured (Schintu et al. 2009). The retrieval time of the buried
pellet was measured 3 days after the last MPTPp administration.
4. Immunohistochemistry
4.1 Immunohistochemistry and cresyl violet for Nissl staining
Three days after the last dose of MPTPp or 48 hours after the last MDMA administration, the mice
were anesthetized with chloral hydrate (400 mg/kg i.p.), transcardially perfused with 4%
paraformaldehyde in phosphate buffered (PB 0.1 M, pH 7.4), and their brains removed and used for
33
immunohistochemistry. Coronal sections (40 μm thick) were cut on a vibratome. Free-floating
sections were incubated overnight with TH antibody (polyclonal rabbit anti-TH, 1:1000, Millipore,
USA). The primary antibody was prepared in Phosphate buffer solution (PBS) plus Triton solution
containing normal goat serum. After careful washing, the sections were incubated in proper
biotinylated secondary antibody (Vector, UK). For visualization, avidin-peroxidase protocol (ABC,
Vector, UK) was applied, using 3,3′-diaminobenzidine (Sigma, Italy) as chromogen. After washing,
the sections were mounted on gelatin-coated slides, air-dried, dehydrated in ascending
concentrations of ethanol, and cleared with xylene (Frau et al 2011).
Adjacent SNc sections were stained with cresyl violet for the Nissl staining to evaluate cell death in
this area. For TH and cresyl-violet-stained cells immunohistochemistry in the SNc, three sections
were sampled (anterior–posterior: -2.92 to -3.28 mm from bregma) according to the atlas of Paxinos
and Franklin 2001. For each mouse, three sections from the CPu (anterior-posterior: 1.10 mm to
0.62 mm from bregma) were analyzed for TH.
4.2 Immunofluorescence for nNOS, IL-1β and TNF-α
Sections from the CPu (50 μm thick) were cut coronally on a vibratome and incubated with the
primary antibodies for IL-1β, TNF-α and nNOS, (polyclonal goat anti-IL-1β and polyclonal goat
anti-TNF-α, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA; polyclonal rabbit anti-nNOS,
1:3000, Millipore, Temecula, CA, USA) for 48 h at 4°C in PBS containing Triton X-100, BSA, and
NGS. For fluorescent immunostaining of nNOS, AlexaFluor® 488-labeled goat anti-rabbit IgG
(1:400, Jackson ImmunoResearch Europe, Suffolk, UK) was used as a secondary antibody, while a
three-step detection was used to increase the signal of IL-1β and TNF-α antibodies by combining
biotin-conjugated IgG (1:200, rabbit anti-goat, Vector, Peterborough, UK) and AlexaFluor® 488-
labeled streptavidin–fluorescein (1:200, Jackson ImmunoResearch Europe, Suffolk, UK). To allow
visualization of cell nuclei, sections were finally incubated for 10 min with 4′,6-diamidine-2′-
phenylindole dihydrochloride (DAPI, 1:10,000, Sigma-Aldrich, Milan, Italy). Finally, all sections
were rinsed and mounted on slides using VectaShield anti-fade mounting media (Vector Inc.).
Standard control experiments were performed by omission of either the primary or secondary
antibody, and yielded no cellular labeling.
4.3 Analysis of TH-positive cells and Nissl staining in the SNc
Three sections of SNc were captured at 10x magnification; in each section, the whole left and right
SNc area were analyzed. Images were digitized (PL-A686 video camera, Pixelink, Canada) under
constant-light conditions. The stained cells were counted manually by a blind experimenter. The
34
number of TH-positive cells and Nissl stained neurons was obtained separately for each SNc level.
Thereafter, in order to obtain an average value from all levels analyzed, the number of cells/level
from each mouse was normalized with respect to the vehicle. Values from the three levels were than
averaged to generate a mean.
4.4 Analysis of TH-positive fibers in CPu
Images were digitized in gray scale with a video camera (Pixelink PL-A686) and TH
immunoreactivity analysis was performed using the Scion Image analysis program (Scion Corp.
USA). The average gray values from white matter were subtracted from each section to correct for
background immunoreactivity. For each level of CPu, the obtained value was first normalized with
respect to vehicle, and values from different levels were averaged thereafter.
4.5 Analysis of nNOS-positive cells in CPu
Analysis of nNOS immunoreactivity for each animal was performed on one tissue section out of
every 3 successive sections for a total of 6 sections containing the CPu. We have chosen the total
size of the examined area in which the nNOS-positive neurons were counted in order to include
almost all of the CPu, according to the extension of the region under analysis. The selected coronal
levels of these sections corresponded to the levels of plates 11–29 for the CPu (AP: +1.1 to +0.02).
The number of nNOS-positive neurons was counted bilaterally in 6 sections per animal. In these
sections, 12 non-overlapping randomly selected ROIs of 0.15 mm2, were examined with a 20×
objective of Olympus IX 61 microscope by two trained observers blind to drug treatment. In order
to ensure that the ROIs did not overlap, their limits were defined based on structural details within
the tissue sections. We did not count the nNOS-positive neurons touching the inferior or the right
sides of the ROIs. The number of nNOS-positive neurons was expressed as mean/mm2 ± SEM.
4.6 Analysis of IL-1β and TNF-α immunoreactivity in CPu
Images of single wavelengths were obtained with an epifluorescence microscope (Axio Scope. A1,
Zeiss, Oberkochen, Germany) connected to a digital camera (1.4 MPixels, Infinity 3-1, Lumenera,
Nepean, Canada). In each brain section, two portions from the CPu (dorsolateral and ventromedial),
left and right, were acquired using a 40× objective. The images of both IL-1β and TNF-α were
digitally merged with the images of DAPI, using the Image J software (U.S. National Institutes of
Health, USA). In the merged image, the levels of IL-1β and TNF-α were quantified. For each
animal, three sections from the CPu (A: 1.10 mm; 0.74 mm; 0.38 mm from bregma) were analyzed.
35
For each section of the CPu, the obtained value was first normalized with respect to vehicle, and
then values from different sections were averaged.
5. Statistical analysis
Behavioral results were statistically compared with a one-way analysis of variance (ANOVA),
followed by Newman-Keuls post hoc test, for comparison between experimental groups. For
immunohistochemistry experiments, results were statistically analyzed by one-way ANOVA
followed by Tukey‟s post hoc test (for unequal N). Results were considered significant at P< 0.05.
36
RESULTS
1. SZV558 administration reverts the motor impairments, olfactory dysfunction and dopaminergic
neuron degeneration induced by a chronic MPTPp treatment
1.1 Changes in spontaneous motor activity: motility test
To evaluate the modification of the spontaneous motor activity induced by the chronic MPTPp
treatment, the total activity and the ambulatory activity were evaluated through the motility test
before the treatment and 1 day after the last MPTPp administration. We observed no significant
changes for total activity and ambulatory activity (Table 1) after MPTPp administration. Moreover,
in animals treated with MPTPp compared with vehicle and MPTPp plus SZV558 or rasagiline
groups, the one-way ANOVA did not indicate a significant effect of treatment in the ambulatory
activity.
Table 1. Motility test pre- and post-chronic MPTPp treatment.
MOTILITY TEST
TOTAL ACTIVITY LOCOMOTOR ACTIVITY
Pre-treatment Post-treatment Pre-treatment Post-treatment
Mean SEM Mean SEM Mean SEM Mean SEM
Vehicle 11503.7 983.5 8737.5 1651.8 8727 983.5 3690.2 1651.8
MPTPp 5803.8 1162.2 6587 771.6 4004.6 916.6 2773.4 343.8
MPTPp+SZV558 9063.1 1055.9 7904.8 982.4 6929.8 791.6 3415.9 463.7
MPTPp+Rasagiline 9941.6 1055.6 8867.8 1214.2 7497 928.8 3938 639.8
Motor activity was measured 2 days before the first MPTPp administration and 1 day after the last MPTPp
administration. Values are expressed as mean ± SEM.
Data were statistically compared with one-way ANOVA, followed by the Newman–Keuls post hoc test.
Number of independent experiments: 4–7/group
1.2 Effect of SZV558 on motor impairment induced by MPTPp: beam-walking test
The number of steps and errors made by mice were recorded for each animal 2 days after the 10th
MPTPp administration, in order to evaluate, respectively, the motor performance and coordinat ion.
The analysis of the time to traverse the beam did not show any difference between the four groups
(data not shown). In contrast, the analysis of the number of steps showed an increase in the MPTPp
group, indicating that MPTPp-treated mice, as parkinsonian patient performed smaller steps.
37
Repeated treatment with SZV558 or rasagiline during MPTPp administration prevented the
impairment induced by MPTPp, as showed by the post hoc test (Newman-Keuls) (Fig. 9a).
Analysis of the total errors showed that MPTPp administration induced a significant increase in
total errors compared with vehicle, demonstrating motor function impairment (Fig. 9b). Motor
function improvement, demonstrated by the decrease in total errors, was observed in the groups
treated with SZV558 or rasagiline compared with the MPTPp group, as indicated in the post hoc
test.
Figure 9. The effect of SZV558 on the behavior in the chronic MPTPp induced PD in mice.
a-b Beam-walking test. To evaluate the number of steps (a) and the total errors (b), the mice were
videotaped for a total of five trials, 2 days after the 10th MPTPp administration. The number of steps and the
number of errors were calculated across all five trials and averaged for each group. *P< 0.05 compared with
vehicle, # P< 0.05 compared with MPTPp. (c) Inverted grid test. To assess skilled forepaw use, the latency
time of falling down was measured 2 days after the 10th MPTPp administration. (d) Olfactory test. To
evaluate the olfactory deficit, the retrieval time of a buried smelling pellet was measured 3 days after the 10th
MPTPp administration. *P< 0.05 compared with MPTPp. Values are expressed as mean ± SEM. Data were
statistically compared with one-way ANOVA, followed by the Newman-Keuls post hoc test.
38
1.3 Grasp-strength evaluation: inverted grid test
The inverted grid test was used to assess forepaw use, especially related to distal musculature and
digit manipulations. This test was done 2 days after the end of treatment. No significant changes
with either MPTPp or rasagiline were observed, indicating that the mice did not have any distal
musculature deficit (Fig. 9c).
1.4 Effect of SZV558 on olfactory deficit induced by MPTPp: Pellet retrieval olfactory test
To reveal any effect of the compounds on olfactory deficit, elicited by MPTPp, mice were evaluated
for olfactory function 3 days after the end of treatment, through the retrieval time of a buried
smelling pellet.
The results of olfactory test showed an increase of retrieval time of a buried smelling pellet,
demonstrating an olfactory deficit, in the MPTPp group. In contrast in mice treated with MPTPp
plus SZV558 or rasagiline, a decrease in retrieval time was observed compared with the MPTPp
group (Fig. 9d).
1.5 Effect of SZV558 on MPTPp-induced neurodegeneration: TH immunohistochemistry and Nissl
Staining in the SNc and CPu
To investigate the effect of SZV558 on dopaminergic cell death, TH immunohistochemistry and
Nissl Staining were performed in the SNc and CPu. MPTPp induced a significant loss of TH-
positive cells in the SNc, as measured by TH immunoreactivity (Fig. 10a). Analysis of Nissl-
positive nigral neurons showed a decrease in number of neurons demonstrating a neuronal death of
MPTPp treated mice compared to vehicle, confirming the dopaminergic neuron degeneration
induced by MPTPp. The combined treatment with MPTPp plus SZV558 or rasagiline prevented the
loss of dopaminergic neurons in the SNc, observed in the mice treated with MPTPp alone, as
demonstrated by the post hoc analysis (Tukey‟s test) of TH immunoreactivity (Fig. 10b). The cresyl
violet staining confirmed the 30% of reduction in number of neurons in the SNc in MPTPp treated
mice. However, this loss of dopaminergic neurons were not observed in Nissl stained SNc of mice
pretreated with SZV558 and rasagiline (Fig. 10c). The loss of TH-positive fibers in the CPu of mice
treated with MPTPp confirmed dopaminergic neuron degeneration (Fig. 10d and e). Both SZV558
and rasagiline prevented neurodegeneration in the CPu compared with the MPTPp group, as shown
by the post hoc analysis (Tukey‟s test) of TH-positive fibers in the CPu.
39
Figure 10. Immunoreactivity for TH in the SNc and CPu in chronic MPTPp treatment.
Representative coronal sections of SNc immunostained for TH show the TH positive neuronal reduction
induced by MPTP treatment (a). The graphs show the number of TH immunoreactive cells (b) and of Nissl-
stained cells (c) in the SNc, expressed as a percentage with respect to vehicle-treated mice. SZV558 and
rasagiline prevented the loss of TH-positive neurons in the SNc, as compared with the animals treated with
MPTPp. Representative coronal sections of striatum immunostained for TH at 5× magnification (d). The
graph shows the mean density of gray value of TH expressed as a percentage with respect to vehicle-treated
mice (e). The TH density reduction confirms the neurodegeneration induced by MPTP. The two compounds
prevent the loss of TH positive fiber induced by MPTPp. Values are expressed as mean ± SEM. *P< 0.05
compared with vehicle. # P< 0.05 compared with MPTPp. Data were statistically compared with one-way
ANOVA, followed by Tukey‟s post hoc test.
D
40
2. Effects of repeated MDMA+caffeine administration in the CPu of adolescent and adult mice
As MPTP, MDMA was shown to produce DA neuron degeneration offer acute-repeated or chronic
treatments. Moreover, caffeine was shown to potentiate DA neurons degeneration. To obtain a
better understanding of the possible molecular mechanism at the basis of the neurodegeneration
observed in CPu of adult and adolescent mice treated with MDMA+caffeine, I evaluated the nNOS,
IL-1β and TNF-α expression (Fig.11a, 12a and 13a).
2.1 nNOS activation in the CPu of adolescent and adult mice
Our results showed a significant increase in nNOS-positive neurons in the CPu of adult mice treated
with MDMA alone (Granado et al. 2008a), caffeine alone, and MDMA plus caffeine, compared
with vehicle (Fig. 11c). In contrast, in adolescent mice, MDMA administration did not increase the
number of nNOS-positive neurons, while caffeine alone or in combination with MDMA
significantly increased the number of nNOS-positive neurons (Fig. 11b).
A
B C
41
Figure 11. Effect of combined 3,4-methylenedioxymethamphetamine (MDMA) plus caffeine neuronal nitric
oxide synthase (nNOS) levels in the CPu.
Representative coronal sections of striatum immunostained for Nnos. Scale bar: 100μm (a). Histograms of
nNOS levels in the CPu of adolescent (b) and adult male (c). Values are expressed as mean
SEM.*P<0.05;**P<0.002 compared with vehicle-treated mice; by Tukey‟s post hoc test.
2.2 IL-1β activation in the CPu of adolescent and adult mice
Very low levels of IL-1β were observed in the CPu of adult and adolescent mice treated with
vehicle or caffeine alone. Conversely, an increase in the levels of IL-1β was found in adult mice
treated with MDMA alone, compared with mice treated with vehicle or caffeine alone (Fig. 12c),
whereas the same treatment induced no modification in the levels of IL-1β in adolescent mice (Fig.
12b). Combined treatment with MDMA plus caffeine did not further raise the levels of IL-1β
induced by MDMA alone in adults (Fig. 12c), whereas in adolescent mice, an increase in the levels
of IL-1β was observed, compared with vehicle-treated mice (Fig. 12b).
A
B C
42
Figure 12. Effect of combined 3,4-methylenedioxymethamphetamine (MDMA) plus caffeine IL-1β levels in
the CPu.
Representative coronal sections of striatum immunostained for IL-1β. Scale bar: 100μm (a). Histograms of
IL-1β levels in the CPu of adolescent (b) and adult male (c). Values are expressed as mean
SEM.*P<0.05;**P<0.002 compared with vehicle-treated mice; ^^P<0.002 compared with caffeine-treated
mice by Tukey‟s post hoc test.
2.3 TNF-α activation in the CPu of adolescent and adult mice
Accordingly with IL-1β results, vehicle or caffeine treated groups showed very low levels of TNF-α
in the CPu of adult and adolescent mice. Increased levels of TNF-α was observed in adult mice
treated with MDMA alone, compared with vehicle-treated mice (Fig. 13c), but adolescent MDMA-
treated mice did not show modification in the levels of TNF-α (Fig. 13b). Combined treatment with
MDMA plus caffeine elevated the levels of TNF-α induced by MDMA alone in both adult and
adolescent mice (Figs. 13b and c).
A
B C
43
Figure 13. Effect of combined 3,4-methylenedioxymethamphetamine (MDMA) plus caffeine neuronal TNF-α
levels in the CPu.
Representative coronal sections of striatum immunostained for TNF-α. Scale bar: 100μm (a). Histograms of
TNF-α levels in the CPu of adolescent (b) and adult male (c). Values are expressed as mean SEM.
**p<0.002 compared with vehicle-treated mice; ^^p<0.002 compared with caffeine-treated mice; ##
p<0.001
compared with MDMA-treated mice by Tukey‟s post hoc test.
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3. Neuroprotective effects of metformin administration on MDMA-induced neurodegeneration:
TH immunoreactivity and Nissl staining
Since the antidiabetic drug metformin has been shown to have neuroprotective activity, in order to
confirm the neuroprotective property of metformin on neurodegenerative processes, induced by an
acute MDMA treatment, I studied its effect on TH immunoreactivity.
In line with previous results, acute repeated MDMA administration induced a significant loss of
TH-positive neurons in the SNc (Fig 14a and b), as measured by TH immunoreactivity. Cresyl
violet staining confirmed a reduction in the number of neurons in the SNc of MDMA-treated mice
by about 30% compared with the vehicle group (data not shown). Moreover, a decrease in TH-
positive fibers in the CPu was observed in MDMA-treated mice compared with the vehicle group
(Fig 14c and d), confirming the MDMA-induced nigrostriatal neurodegeneration.
Metformin treatment during MDMA administration prevented the MDMA-induced decrease in TH-
positive neurons and fibers in the SNc (Fig 14b) and CPu (Fig 14d), respectively. Finally, analysis
of Nissl-positive neurons demonstrated a recovery in the number of neurons in the SNc of mice
treated with MDMA plus metformin compared with the MDMA group.
45
Figure 14. Immunoreactivity for TH in the SNc and in CPu in mice treated with MDMA and metformin
coadministration.
Representative coronal sections of SNc immunostained for TH (a). Mice were treated with vehicle,
metformin (2×200 mg/kg, i.p.), MDMA (4×20 mg/kg, i.p.), and MDMA (4×20 mg/kg, i.p.) plus caffeine
(2×200 mg/kg, i.p.) and sacrificed 48 hours after the last MDMA administration. The graph shows the
number of TH positive neurons in the SNc (b).
Values are expressed as mean ± SEM.*P< 0.05 compared with vehicle; #P< 0.05 compared with MDMA.
Data were statistically compared with one-way ANOVA, followed by Tukey‟s post hoc test.
46
DISCUSSION
The study of neurodegenerative effects induced by neurotoxins, allows understanding the influence
of internal or external factors, such as respectively the age and the simultaneous consumption of
other substances, on the development of neurodegenerative processes.
On these bases, the research work of my thesis was based on the study of new compounds that
many display neuroprotective effects in PD animal models. To this end, firstly I evaluated the
neuroprotective effect of a new MAO-B inhibitor, SZV558, on a chronic MPTPp mouse PD model;
then I studied the influence of the two known neuroprotective drugs, caffeine and metformin, on the
neurodegenerative effect of repeated MDMA administration in mice.
1. The neuroprotective effect of SZV558 in a chronic MPTPp model of PD
The results of the present study, consistent with previous studies, show that chronic administration
of MPTPp in mice induced dopaminergic neurodegeneration as evidenced by a decrease in TH
immunoreactivity in the CPu and SNc, associated with motor and olfactory deficits. Administration
of the new (hetero)arylalkenyl propargylamine compound SZV558, similarly to the well-known
MAO-B inhibitor rasagiline, counteracts these deficits and restores both motor function and normal
DA neuron innervation.
The current PD therapies are largely unsatisfactory since DA-replacement therapy only counteracts
the symptoms of the disease without affecting the course of neurodegeneration. Therefore, the
search for therapies that may delay or stop the disease progression is very active. To pursue this
scope, it is necessary to utilize PD models that closely reproduce the disease neurodegeneration that
is slow and starts several years before the motor symptoms appear.
The model chosen to test SZV558, differently from acute MPTP treatments that induce a rapid
degeneration of dopaminergic nigrostriatal neurons, induces a more progressive degeneration, with
neurons continuing to die after completion of toxin administration (Meredith and Ramedacer 2011).
Moreover, when MPTP is co-administered with probenecid, the degeneration of dopaminergic
neurons takes place over a period of 5–8 weeks, inducing apoptosis. The chronic model utilized in
this study has been well characterized and was validated for utilization in a protocol of drug-
induced neuroprotection (Carta et al. 2013).
In the last decade, the MAO-B inhibitors have been proposed as supplementation of the DA
replacement therapy, but the clinical studies fallen to confirm their protective effects (Ahlskog et al.
2010). Moreover, recent studies showed several adverse effects, such as control disorder (Vitale et
al. 2013). Consequently, researchers focalized the attention on new drugs contain the anti-apoptotic
47
propargyl ring and a high MAO-B selectivity. The SZV558 had showed its neuroprotective activity
in in vitro experiments, and it has confirmed this property in the chronic MPTPp treatment.
In the present study the rescue of neurodegeneration was indicated by the immunohistochemical
results that showed that dopaminergic neurodegeneration, evaluated through TH
immunohistochemistry, was rescued by SZV558. The drug, similarly to rasagiline, completely
counteracted the decrease in TH-positive neurons and terminals observed after chronic MPTPp in
the SNc and CPu, respectively.
However, the beam-walking test showed that MPTPp increases the number of steps and errors,
indicating an impairment of gait. It is interesting to note that the beam-walking test strictly reflects
the deficits that characterize PD, such as slowness of movements, indicated by the increased
number of steps to traverse the length of the beam, together with an unstable gait, indicated by the
number of errors (Fleming et al. 2013). SZV558, similarly to rasagiline, counteracts the slowness of
movement, together with the unstable gait induced by chronic MPTPp. In contrast, no impairment
after chronic MPTPp was observed in the grid test, indicating that limb strength, was not impaired
by the chronic regimen of MPTPp administration and no modifications were produced by either
SZV558 or rasagiline. Not surprisingly, no differences were observed in locomotor activity and
total motility after chronic MPTPp, as adaptive changes in motility are consistently observed when
the DA neuron lesion does not exceed 70–80% (Chia et al. 1996; Meredith and Ramedacer 2011).
Confirming previous studies, MPTPp treatment has a significant impact on olfactory ability.
Olfactory deficits and abnormalities in olfaction-related structures, one of early symptoms of PD,
have been showed years before diagnosis of the disorder and appearance of motor symptoms. The
impairment usually affects all areas of olfaction, including identification and discrimination of
odors and reduced sensitivity (Doty 2012 a, b). SZV558 counteracted the olfactory deficit induced
by chronic MPTPp as measured by the decreasing pellet retrieval time.
Therefore, the results showed a strict association between behavioral and biochemical markers of
dopaminergic neurodegeneration.
Previous results by Baranyi (2016) showed that SZV558 decreased the formation of DAQ, the toxic
DA metabolite well known as an effector molecule of dopaminergic neuro-specific cytoxicity
(Asanuma et al. 2003). The DAQ production has been showed in neurodegenerative disease such as
PD, and in exposure to dopaminergic toxins, such as MPTP and MDMA. In fact, it usually
produced only in case of precedent mitochondrial dysfunction (Baranyi et al. 2016). In particular,
DAQ reduces mitochondrial function and permeability (Berman et al. 1999; Bisaglia et al. 2010; Li
et al. 1997) and inhibits ROS scavenger enzymes (Hauser et al. 2013; Belluzzi et al. 2012), leading
oxidative stress. However, DAQ also inhibits proteasomal activity (Zafar et al. 2006) and
48
inactivates functional proteins of dopaminergic neurons, such as TH (Kuhn et al. 1999; Xu et al.
1998), DAT (Whitehead et al. 2001) and alpha-synuclein (Conway et al. 2001).
All together, these mechanisms may contribute to the protective effects exerted by SZV558 on
MPTP toxicity. In order to verify the real death of dopaminergic neurons in the SNc, Nissl stain was
evaluated. The results of this analysis confirmed that chronic MPTPp causes a degeneration of
dopaminergic cells and that coadministration of SZV558 produces neuroprotection.
The effect of SZV558 lasts for up to 42 hours, consistent with the irreversible inhibition of the
MAO-B by the compounds (Baranyi et al. 2016). Moreover, previous results showed that SZV558
is protective against the depletion of DA even when administered after subchronic MPTP, when
MPTP was already converted to the toxic metabolite MPP+
by MAO-B (Jackson-Lewis and
Przedborski 2007), suggesting that SZV558 protects from progressive dopaminergic degeneration,
not only through irreversible MAO-B inhibition, but through a mechanism independent of the
inhibition of MAO-B enzyme (Baranyi et al. 2016). Finally, to confirm that SZV558 protection is
independent from MAO-B inhibition, even if it was administrated 18 hours before MPTP treatment,
our collaborator Baranyi (2016) detected the MPTP/MPP+ ratio 72 hours after an MPTP acute
treatment. The results did not show any significant difference between the MPTP and MPP+ levels
detected in mice pretreated with SZV558 and mice pretreated with vehicle, confirming that SZV558
has a neuroprotection completely independent by MAO-B inhibition (Baranyi et al. 2016). On these
bases, even in the chronic MPTPp treatment, where the compound was administrated 18 hours
before each MPTPp administration, the SZV558 could have a protective action that is independent
of the inhibition of MAO-B enzyme.
In conclusion, the SZV558 showed a neuropretective efficacy in the MPTPp protocol, a model that
closely reproduces neurodegeneration in PD in which dopaminergic neurodegeneration occurs over
time. PD is a multifactorial disease and several risk factors contribute to the vulnerability of DA
neurons (Sulzer and Surmeirer 2013), therefore, SZV558 by affecting multiple targets, may,
through these differentiated mechanisms have a more favorable action than other MAO-B inhibitors
as neuroprotective agent in the therapy of PD.
The results of this study have been published as part of the article:
“Novel (Hetero)arylalkenyl propargylamine compounds are protective in toxin-induced models of
Parkinson's disease”. Baranyi M, Porceddu PF, Gölöncsér F, Kulcsár S, Otrokocsi L, Kittel Á,
Pinna A, Frau L, Huleatt PB, Khoo ML, Chai CL, Dunkel P, Mátyus P, Morelli M, Sperlágh B.
Mol Neurodegener. 2016 Jan 13;11(1):6.
49
2. The neurotoxic effect of caffeine on repeated MDMA administration in adult and adolescent
mice
The use of MDMA in association with caffeinated energy drinks is very common among
adolescents and young adults. Earlier evidences showed the existence of noxious interactions
between MDMA and caffeine, since as reported by Khairnar and coworkers (2010), caffeine is able
to increase the MDMA-induced neuroinflammation in mice (Khairnar et al. 2010). Moreover recent
results suggest that caffeine is able to potentiate the MDMA neurotoxicity (Frau et al. 2016). As
mentioned above, the effect of caffeine on neurodegenerative processes is controversial and
depends on the dose and/or administration pattern (Ruiz-Medina et al. 2013). Netherless several
studies demonstrated that the acute administration of caffeine during repeated administration of
MDMA, potentiated the neurotoxic MDMA effects. In this regard, previous results showed that
combined treatment of MDMA plus caffeine induced an increased loss of striatal DA fibers
compared with MDMA, only in adolescence, but not in adult mice (Fig. 15) (Frau et al. 2016),
suggesting that the abuse of MDMA during the adolescence could render the neurons more
sensitive to the combined effects of other substances.
To examine in depth the caffeine effects and to understand the age-linked differences in MDMA
neurotoxicity, in collaboration with the group of Dr. Castelli I evaluated, the nNOS expression and,
in collaboration with Dr. Costa I measured the levels of the cytokines IL-1β and TNF-α in CPu of
adult and adolescent mice treated with a combined MDMA plus caffeine.
The nNOS, the enzyme synthesis of NO, is involved in many pathological processes, such as the
neurodegenerative effects of amphetamine-related drugs (Dawson and Dawson 1998; Granado et al.
2008a; Castelli et al. 2014). nNOS has an important role in the integration of dopaminergic and
glutamatergic transmission, since activation of NMDA receptors, following the increase in GLU
release, activates nNOS that produces NO. NO increases DA and GLU release, and, depending on
the level of NO production and the oxidative levels, may interact with H2O2 to produce
peroxynitrite (Itzhak et al. 2006). Moreover, oxidative stress increased by peroxynitrite, promotes
the oxidation of DA in the toxic metabolite DAQ (LaVoie and Hastings 1999a, b) inducing DA
neurotoxicity. The involvement of nNOS expression in the MDMA neurotoxicity was previously
demonstrated by the protective effect of nNOS inhibitor on loss of striatal DA fibers induced by
MDMA (Colado et al. 2001 and 2004) and by the resistance of nNOS knockout mice to MDMA
neurotoxicity (Itzhak et al. 2004). On these bases, the elevation of nNOS by MDMA observed in
adult but not in adolescent mice, confirmed the lower sensitivity of adolescents to MDMA
neurotoxic effect. Accordingly with these data, an increased expression of IL-1β and TNF-α by
50
MDMA in adults has been observed, while in adolescent MDMA-treated mice, cytokines levels
were similar to vehicle group. These results are in line with previous studies showing
proinflammatory cytokines as a marker correlated with dopaminergic neuron degeneration. In fact
elevated levels of cytokines trigger oxidative and nitrosative stress that can induce the
neurodegenerative process (Hemmerle et al. 2012).
The age of consumers is an important factor that may alter the MDMA neurotoxic effects (Hawkins
et al. 1992). In particular, the neurons during the adolescence appear to be particularly vulnerable to
specific drugs abuse (Cadoni et al. 2015; Daza-Losada et al. 2009; Sisk and Zehr 2005; Spear
2000). Netherless, the CPu of adolescent mice showed a lower sensitivity to MDMA
neurodegenerative effect, compared with adults (Reveron et al. 2005; Frau et al. 2016). Moreover,
repeated MDMA administration induces a decrease of striatal TH density in adult, but not in
adolescent mice (Frau et al. 2016). On these bases, I can speculate that low nNOS and cytokines
levels, as observed in adolescent MDMA-treated mice, are not able to lead to process related to
oxidative stress and the resulting neurodegeneration.
Figure 15. Reported current results of striatal neurodegeneration and neuroinflammation in adult and
adolescent mice treated with caffeine alone (2x10mg/kg), MDMA alone (4x20mg/kg) or in combination
(MDMA 4x20mg/kg plus caffeine 2x10mg/kg).
51
On the other hand, caffeine induces an elevation of nNOS in adolescent but not in adult mice treated
with repeated MDMA administration, showing the potentiation of MDMA effects induced by
caffeine in adolescents (Fig. 15). As shown by Gorska and Gołembiowska (2015), this potentiation
could be explained by an increase of MDMA-stimulated DA release by concomitant administration
of caffeine. In particular, Gorska and Gołembiowska (2015) demonstrated that caffeine potentiates
the DA release induced by MDMA through the antagonism of both A2A and A1 receptors (Gorska
and Gołembiowska 2015). On this basis, the blockade by caffeine of A2A receptors, localized in
striatal glutamatergic terminals and involved in the modulation of GLU release (Ciruela et al. 2006),
could play a role in the nNOS expression. Moreover, the A1 blockade by caffeine might increase
the release of proinflammatory cytokines. In fact, elevated levels of proinflammatory cytokines,
such as IL-1β, are observed in A1 knockout mice (Tsutsui et al. 2004). Another hypothesis that can
explain the elevated cytokines release by combined administration of MDMA and caffeine, is the
ability of caffeine to potentiate the hyperthermia induced by MDMA (McNamara et al. 2006). The
MDMA-induced hyperthermic response is mediated by elevated levels of IL-1β (Green et al. 2004),
consequently it is conceivable that caffeine is able to increase the cytokines release through the
strengthening of hyperthermic mechanisms.
Caffeine alone does not induce any decrease in striatal TH-positive fibers (Fig. 15) (Frau et al.
2016), consequently the only increase in nNOS does not appear to be correlated with any
neurodegenerative process, since the NO may induce oxidative stress only if it interacts with H2O2.
These data allow inferring that the nNOS may lead neurodegeneration only if there is an oxidative
process underway. Therefore, increased nNOS levels together with a neuroinflammatory processes
(demonstrated by elevated levels of IL-1β and TNF-α) which increases oxidative stress, as seen
after MDMA alone and MDMA plus caffeine (Fig. 15), might create a neurotoxic environment
causing dopaminergic neuron degeneration.
In conclusion, the use of caffeine in association with MDMA during adolescence may worsen the
neurotoxicity and neuroinflammation elicited by MDMA, confirming that the adolescence is a
critical phase of life not only for the development of addiction, but also for the possibility of
causing neurotoxicity to dopaminergic neurons.
These results have been published as part of the article: “Influence of caffeine on 3,4-
methylenedioxymethamphetamine-induced dopaminergic neuron degeneration and
neuroinflammation is age-dependent”. Frau L, Costa G, Porceddu PF, Khairnar A, Castelli MP,
Ennas MG, Madeddu C, Wardas J, Morelli M.
J Neurochem. 2016 Jan;136(1):148-62.
52
3. The neuroprotective effect of metformin on neurodegeneration induced by repeated MDMA
administration
The neurotoxicity to dopaminergic neurons induced by MDMA in adult mice has been
demonstrated by several studies, using different drug doses and different administration protocols
(Granado et al. 2008a and 2011; Costa et al. 2013).
In line with previous studies, acute repeated MDMA administration induces a significant loss of
TH-positive neurons and fibers, respectively, in the SNc, and CPu of adult mice (Fig. 15).
The reduction in the number of neurons in the SNc of MDMA-treated mice by about 30% compared
with the vehicle group, was confirmed by cresyl violet staining, showing that MDMA doesn‟t
reduce TH expression, but induces dopaminergic neurodegeneration (Granado et al. 2008a).
Metformin treatment prevented the MDMA-induced decrease in TH-positive neurons and fibers in
the SNc and CPu, respectively, confirmed by the analysis of Nissl-positive neurons that
demonstrated a recovery in the number of neurons in the SNc of mice treated with MDMA plus
metformin by about 30% compared with the MDMA group.
The neuroprotective effect of metformin observed in this study could be particularly useful,
considering the higher incidence of PD reported in amphetamine-related drugs abusers (Brust 2010;
Christine et al. 2010; Callaghan et al. 2012; Curtin et al. 2015) and is in line with the
neuroprotective proprieties of metformin demonstrated in epidemiological and animal models of PD
(Wahlqvist et al. 2012; Patil et al. 2014). In this regard, metformin, attenuating the generation of
ROS and RNS (Chakraborty et al. 2011), and increasing antioxidant enzymes activity (Patil et al.
2014), may counteract the oxidative stress, one of the principal mechanisms involved in
neurodegenerative disease and in MDMA or MPTP neurotoxicity (Fig. 3 and 5) (Meredith and
Kang 2006; Puerta et al. 2010; Górska et al. 2014 and 2015).
The mechanism proposed for the neuroprotection of metformin in the CNS is the activation of
AMPK which is a key regulator of cellular energy metabolism. On the other hand, in drosophila
models of PD, AMPK activation induced by metformin, alleviated dopaminergic dysfunction and
mitochondrial abnormalities (Ng et al. 2012). However, the activation of AMPK exerts a protective
effect against the neurotoxic effects of MPP+ and it has been proposed as a survival factor for
dopaminergic neurons in PD (Choi et al 2010). AMPK has also a potential anti-inflammatory
activity, in fact it is a marker of M2 macrophages which are generally anti-inflammatory (Sag et al.
2008). In this regard, a recent study showed a decreased production of the proinflammatory
cytokine IL-1β and increase in antinflammatory IL-10, induced by metformin in LPS-activated
macrophages, suggesting that this antidiabetic drug may modulate the inflammatory response in
53
microglia (Kelly et al. 2015). Considering the strong correlation between neurodegeneration and
neuroinflammation, the antinflammatory property of metformin has been to take in consideration as
possible neuroprotective mechanism.
Finally, metformin has been reported as promoter of neurogenesis by activating an atypical PKC–
CREB pathway. Moreover, CREB a transcription factor played a major role in neurodevelopment,
synaptic plasticity, and neuroprotection (Sakamoto et al. 2011), promotes the transcription of BDNF
and TH (Piech-Dumas and Tank 1999).
As shown by studies performed in vivo and in vitro, MDMA, altering dopaminergic transmission,
may affect neurogenesis and synaptogenesis (Rice and Barone 2000; Fasano et al. 2008; Goffin et
al. 2010). On this basis, the restorative number of TH-positive neurons in mice treated with MDMA
plus metformin suggests that another mechanism for metformin protection may be the neurogenesis,
which is affected in MDMA-treated animals.
The findings of this study showed that metformin treatment prevents the neurodegenerative effects
of repeated MDMA administration in adult mice, confirming the therapeutic potential of this
antidiabetic medication in the treatment of neurodegenerative processes.
These results and findings have been submitted for publication to the journal Neurotoxicity
Research: “Metformin protects against dopaminergic neurotoxicity induced by 3,4-
methylenedioxymethamphetamine administration”. Porceddu PF, Ishola OI, Morelli M.
54
CONCLUSIONS
These results demonstrated that the dopaminergic neurodegenerative process may be induced or
conditioned by environment stressors or substances which influence, through different ways, the
development of neurodegenerative mechanisms.
In the present study I evaluated the effects of three substances, known as potentially
neuroprotective, in combination with two different neurotoxins that affect the nigrostriatal
dopaminergic system. The SZV558 MAO-B inhibitor and the metformin protected the nigrostriatal
pathway, affected by MPTP- and MDMA- induced neurotoxicity. On the other hand, caffeine,
administrated with MDMA, showed a neurotoxic potential depending on the age of consumers,
confirming the vulnerability of adolescent brain to consumption of drugs and substances that
affected the dopaminergic system.
In conclusion, the study of neurodegenerative processes may be relevant to understand the human
pharmacology, the origin and development of neurodegenerative disease and to predict the
neurotoxic effect of drug abuse.
55
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