Supplemental Discussion
1. Relevance of intrastriatal 6-hydroxydopamine lesions as a model of Parkinson´s
Disease
Intrastriatal injections of 6-hydroxydopamine provide a model of retrograde
nigrostriatal degeneration (Berger et al., 1991, Bjorklund et al., 1997, Ichitani et al.,
1991, Kirik et al., 1998). The degeneration of dopaminergic neurons starts in the
striatum, where the toxin produces a severe lesion of dopamine axon terminals around
the injection site. At the same time, the toxin is retrogradely transported to the cell
body region, causing a partial loss of dopamine cells in the substantia nigra pars
compacta (Berger et al., 1991, Ichitani et al., 1991, Sauer and Oertel, 1994). Time
course studies combining a fluorescent retrograde axonal tracer (fluorogold) with TH
immunohistochemistry have showed that intrastriatal 6-hydroxydopamine causes a
biphasic neurodegenerative process. A first phase of rapid dopamine cell loss occurs
within one week post-lesion, and this is followed by a mild additional loss of nigral
dopamine neurons proceeding over several weeks (Bjorklund et al., 1997, Kirik et al.,
1998, Sauer and Oertel, 1994). The use of a retrograde labeling method has allowed
investigators to establish that the disappearance of TH-immunoreactive cells in the
nigra reflects actual neuronal death rather than downregulation of the TH enzyme
(Bjorklund et al., 1997, Sauer and Oertel, 1994). Indeed, a parallel and equivalent
decline in the numbers of TH-positive neurons and fluorogold-labeled cells was found
to occur in the substantia nigra pars compacta during the first 5 weeks post lesion
(Sauer and Oertel, 1994). However, downregulation of TH occurred in the surviving
dopamine neurons, which become atrophic in the phase immediately preceding their
death (Sauer and Oertel, 1994). This combination of cell death and atrophic changes
in the surviving neurons, protracted over the time, not only confers some progressive
feature to the model but also provides a substrate for testing potential neuroprotective
or neurorestorative interventions (Bjorklund et al., 1997).
The biphasic time course of the intrastriatal 6-hydroxydopamine model is
suitable to reproduce the evolution of nigrostriatal dopamine degeneration in human
Parkinson´s Disease. A recent pathological study assessing the extent of nigrostriatal
degeneration at different intervals after Parkinson diagnosis (Kordower et al., 2013)
has revealed a rapid decline of dopaminergic markers in the first years of clinical
disease, reaching a plateau-like phase by 4-5 years (Kordower et al., 2013).
Importantly, the loss of dopaminergic markers in the dorsal putamen appeared
complete at post diagnostic intervals of 4-5 years, while spared dopamine fibers could
still be detected in ventral and medial striatal regions (Kordower et al., 2013). At the
same intervals, the loss of dopamine cell bodies in the substantia nigra (assessed by
counting melanine-containing neurons) varied between 33% and 80% (Kordower et
al., 2013).
In addition to the biphasic time course of nigrostriatal degeneration, the
regionally heterogeneous pattern of striatal dopamine denervation and the partial loss
of nigral dopamine neurons are reproduced by the intrastriatal 6-hydroxydopamine
lesion model used in the present study. Indeed, while virtually complete (~ 99%)
dopamine denervation occurred in the dorsolateral striatum (which included the sites
of toxin injection), partial (~55%) dopamine denervation occurred in the dorsomedial
region (cf. Fig 1). The percentage loss of TH-immunoreactive cells in the ipsilateral
substantia nigra pars compacta was approximately 60% (cf. Fig 3). This pattern of
nigrostriatal
degeneration
is
quite
different
from
that
produced
by
6-
hydroxydopamine injections in the medial forebrain bundle, causing a very fast and
nearly complete loss of both striatal dopaminergic fibers and nigral cell bodies,
without any regional specificity (Francardo et al., 2011).
2. Significance of the delayed start-treatment experiment
While chronic treatment with PRE-084 promoted functional recovery when started on
the day of the lesion, it failed to achieve significant effects when started one week
post-lesion. In the main body of the manuscript, we have discussed that the first week
post-surgery represents a critical window of opportunities for treatments that boost
endogenous plasticity mechanisms in 6-hydroxydopamine-lesioned rodents.
Here we would like to discuss the implications of our delayed-start experiment
with respect to a potential application of Sigma-1 receptor agonists as a diseasemodifying treatment for Parkinson´s disease. If the first week post-lesion in our mice
recapitulates the extent of nigrostriatal degeneration occurring in the first 4-5 years of
clinical disease (see above), our data indicate that treatment with Sigma-1 receptor
agonists is unlikely to be effective in Parkinson´s patients if started at post-diagnostic
intervals longer than 3-4 years. We acknowledge, however, that the extent to which
results from laboratory animals sustaining acute lesions can be extrapolated to human
Parkinson´s disease remains always uncertain. Indeed, the clinically manifest phase of
Parkinson´s disease is preceded by a phase of non-dopaminergic neurodegeneration
by many years, possibly impacting on the brain´s neuroregenerative potential.
3. Significance of trophic factor upregulation and signaling pathway activation by
PRE-084 treatment.
In our study, we found higher protein levels of GDNF, BDNF, pAkt and pERK1/2 in
the striatum and the substantia nigra of mice chronically treated with PRE-084 (0.3
mg/kg/day) compared to saline-treated animals (cf. Figs 4 and 5). These molecular
effects support the interpretation that, at a regimen producing behavioural restoration,
treatment with PRE-084 had promoted endogenous plasticity and defense
mechanisms (see main Discussion).
Here we would like to review some of the evidence linking the above
mentioned molecular changes to the pro-survival and pro-plasticity effects produced
by several treatments tested in animal models of Parkinson´s disease. Several studies
have shown that the Akt pathway is activated by compounds exerting protective
effects on the dopaminergic system (Lim et al., 2008, Sagi et al., 2007, Weinreb et al.,
2007, Nair and Olanow, 2008, Yu et al., 2008). This pathway has been shown to
mediate not only anti-apoptotic but also neurorestorative events in a murine model of
PD (Ries et al., 2006). Moreover, a recent study has shown that activation of the Akt
signaling cascade (following rho kinase inhibition) was associated with perikaryal and
axonal protection in the MPTP mouse model of Parkinson´s Disease (Tonges et al.,
2012). Activation of ERK1/2 signaling in nigral dopamine neurons appears to mediate
GDNF-induced neuroprotection against toxic damage both in vitro and in vivo
(Lindgren et al., 2008, Onyango et al., 2005, Ugarte et al., 2003, Lindgren et al.,
2012).
In addition to being beneficial to the nigrostriatal dopamine pathway, trophic
factor upregulation and ERK1/2 and Akt pathway activation have the potential to
boost plasticity and repair mechanisms in several neural systems. Accordingly,
treatment with Sigma-1 receptor agonists has been reported to promote functional
recovery in several disease models (reviewed in the main manuscript). In our study, in
addition to a pro-dopaminergic effect, treatment with PRE-084 increased striatal
serotonin levels (cf. Table 1). Elevated levels of BDNF have been shown to increase
the expression of tryptophan hydroxylase 2 in serotonin neurons, while also exerting
potent neurotrophic effects on serotonin axon terminals (Mattson et al., 2004). Along
with other studies (Ago et al., 2011), these considerations suggest that
pharmacological
stimulation
of
Sigma-1
receptors
potentiates
serotonergic
neurotransmission via upregulation of BDNF levels. Importantly, serotonin and
BDNF have been described to co-regulate one another, acting as a “dynamic duo” to
regulate neuronal survival and synaptic plasticity (Mattson et al., 2004).
Since Parkinson´s disease is not only a disorder of dopaminergic neurons, but
involves all monoaminergic systems in the brain (Mann and Yates, 1983, Scatton et
al., 1983, Halliday et al., 1990), a treatment able to promote neurochemical and
synaptic restoration in several monoaminergic pathways (and in other systems as
well) may potentially improve both motor and non-motor features in Parkinson´s
patients.
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