Introduction
Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons
of the substantia nigra pars compacta (SNpc). Proteostasis impairment at the level
of the endoplasmic reticulum (ER) is emerging as a driving factor of dopaminergic
neuron loss in PD. ER stress engages the activation of an adaptive reaction known
as the unfolded protein response (UPR) to recover proteostasis or trigger apoptosis
of damaged cells. The therapeutic potential of the UPR as a target has been recently
validated using pharmacological and gene therapy approaches. A complex view is emerging
where ER stress may have a dual role in PD, both in maintaining cell survival during
initial stages of the diseases and trigger neuronal degeneration when the stress levels
are sustained. Here we overview recent advances in determining the impact of ER stress
to PD.
PD is a progressive neurodegenerative disease that affects movement control, characterized
by the loss of dopaminergic neurons in the SNpc. In most PD cases the presence of
intracellular inclusions, termed Lewy bodies (LBs) is observed, where fibrillar aggregates
of αSynuclein constitute a major component. Many cellular processes are altered in
PD, including redox control, mitochondrial function, autophagy/lysosomal function,
protein quality control mechanisms, and vesicle trafficking, among other processes.
Accumulating evidence supports disruption in the secretory pathway as a triggering
factor of proteostasis dysfunction in PD, mediating in part the selective degeneration
of dopaminergic neurons (Chua and Tang, 2013; Mercado et al., 2013). Importantly,
in addition to PD, ER stress is emerging as a relevant driver of most common neurodegenerative
diseases (Hetz and Mollereau, 2014).
ER stress activates the UPR, a complex signaling transduction pathway that mediates
cellular adaptation to restore ER function (reviewed in Ron and Walter, 2007; Hetz,
2012). In this article we discuss recent insights on the significance of ER stress
as a driver of dopaminergic neuron loss in PD and the potential of targeting UPR components
to augment the homeostatic capacity of the ER and reduce pro-apoptotic signals.
ER stress signaling
The UPR is a signaling network mediated by the activation of three stress sensors
located at the ER membrane, including inositol requiring kinase 1α (IRE1α), activating
transcription factor 6 (ATF6), and protein kinase RNA-like ER kinase (PERK) (Figure
1A). These UPR transducers control the expression of a variety of genes involved in
almost every aspect of the secretory pathway, resulting in a reduction in the load
of misfolded proteins at the ER. Activation of the UPR improves the efficiency of
protein folding and quality control mechanisms, in addition to enhance ER and Golgi
biogenesis, protein secretion and the clearance of abnormally folded proteins through
the autophagy and ER-associated degradation (ERAD) pathways. However, under chronic
ER stress UPR sensors shifts their signaling toward induction of cell death by apoptosis
(Urra et al., 2013).
Figure 1
Involvement of ER stress in PD. (A) Schematic representation of the three branches
of the UPR. (B) Knockout (KO) animals for XBP1, CHOP or ATF6 have been tested to manipulate
the UPR in PD models. (C) Images modified from Valdes et al. (2014): Wild-type mice
were injected into the with (i) AAV expressing an shRNA against XBP1 (shXBP1/GFP),
(ii) EGFP alone, or (iii) a vector to overexpress XBP1s. One month after injection,
experimental PD was induced using the 6-OHDA model to monitor dopaminergic neuron
loss at the SNpc. Green: AAV transduced cells expressing GFP. Red: dopaminergic neurons
stained with anti-tyrosine hydroxylase (TH). Scale bar: 200 μm.
IRE1α is an endoribonuclease that processes the mRNA encoding the transcription factor
X-Box binding protein-1 (XBP1) which results in the expression of a more stable and
active transcription factor, termed XBP1s (Ron and Walter, 2007). Upon activation,
ATF6 traffics to the Golgi and undergoes subsequent proteolytic processing to release
ATF6f, an active transcriptional factor (Ron and Walter, 2007). PERK is an ER-located
kinase that upon activation phosphorylates the eukaryotic initiation factor 2α (eIF2α),
attenuating general protein translation. In turn, eIF2α phosphorylation leads to the
specific translation of activating transcription factor 4 (ATF4), which up-regulates
many important genes functioning in redox control, amino acid metabolism and protein
folding (Harding et al., 2003). Under chronic stress, ATF4 regulates the expression
of pro-apoptotic genes such as CHOP.
ER stress in PD
The mechanisms leading to ER stress in PD and the actual impact of the UPR on the
degeneration cascade are just starting to be uncovered. A genetic screening in yeast
revealed that one of the major physical targets of αSynuclein is Rab1, an essential
component of the ER-to-Golgi trafficking machinery (Cooper et al., 2006; Gitler et
al., 2008). Over-expression of Rab1 in animal models of PD reduced stress levels and
protected dopaminergic neurons against degeneration (Coune et al., 2011). Importantly,
the generation of neuronal cultures from induced pluripotent stem cells (iPSC)-derived
from PD patients revealed major proteostasis alterations (Chung et al., 2013). The
authors provided evidence indicating that ER stress is a salient molecular signature
of human PD neurons. There are many other studies linking other PD genes with alteration
of the secretory pathway, including LRRK2, Parkin, Pael-R, DJ-1, ATP13A2 (reviewed
in Mercado et al., 2013), and VPS35 (Zimprich et al., 2011). These reports suggest
that secretory pathway dysfunction is a common hallmark of PD, which may result in
pathological levels of ER stress contributing to the etiology of the disease.
The UPR and cell fate in PD
Genetic manipulation of essential UPR components in the context of PD had been performed
only in a few studies (Figure 1B). For example, ATF6α knockout animals showed increased
accumulation of ubiquitin-positive inclusions and enhanced loss of dopaminergic neurons
induced by a PD-triggering neurotoxin (Egawa et al., 2011). Although ATF6 is not essential
for development and survival of dopaminergic neurons in mice, this stress sensor controls
the levels of the chaperone BiP and ERAD components under resting conditions in these
neurons (Egawa et al., 2011). A recent study determined that ATF6 is a direct target
of αSynuclein. Expression of αSynuclein was shown to inhibit the processing of ATF6
through a physical association, leading to an impaired up-regulation of ERAD genes,
which sensitized cells to apoptosis (Credle et al., 2015).
We recently reported a set of in vivo studies uncovering the significance of the UPR
transcription factor XBP1 in controlling the survival of dopaminergic neurons (Valdes
et al., 2014). We found that the developmental ablation of Xbp1 in the nervous system
preconditioned dopaminergic neurons and rendered them resistant to the PD-triggering
neurotoxin 6-hydroxydopamine (6-OHDA) (Figure 1B). This neuroprotective effect was
accompanied by the up-regulation of several UPR effectors in the SNpc of animals in
the absence of pro-apoptotic markers such as Chop. This phenotype correlated with
the presence of poly-ubiquitinated proteins and large inclusion bodies in dopaminergic
neurons of XBP1 deficient animals, resembling the classical alterations observed in
PD. Remarkably, dopaminergic neurons were prompt to undergo proteostasis alterations
in the absence of XBP1, a phenomenon not observed in other brain areas including cortex,
striatum, or spinal cord (Hetz et al., 2009; Valenzuela et al., 2012; Vidal et al.,
2012; Valdes et al., 2014). We proposed that developmental targeting of XBP1 provides
neuroprotection through an “ER-hormesis” mechanism where the occurrence of mild non-lethal
ER stress engages an adaptive response that sustains neuronal function in the absence
of XBP1, which also renders dopaminergic neurons more resistance to a PD-inducing
stimulus. In agreement with this concept, establishment of an ER-hormesis condition
(Matus et al., 2012) by the administration of low doses of the ER stress agent tunicamicyn
on a rodent and fly model of PD selectively engaged adaptive UPR signaling events
involving the expression of XBP1s (Fouillet et al., 2012).
Since genetic manipulations during development can lead to compensatory mechanisms
that mask the direct biological effects of a certain gene, we then targeted XBP1 in
adult animals locally at the SNpc (Valdes et al., 2014). Knocking down XBP1 resulted
in chronic ER stress involving the up-regulation of Chop, causing spontaneous neurodegeneration
of dopaminergic neurons (Figure 1C). These results highlight the importance of XBP1
in sustaining dopaminergic neuron function and viability, reinforcing the concept
that ER stress is a factor underling their differential neuronal vulnerability. Therapeutic
strategy to artificially engage a UPR adaptive program has been developed to pre-adapt
dopaminergic neurons to a PD-inducing event. Using a gene therapy approach, we delivered
active XBP1s into the SNpc of adult mice using adeno-asociated viral (AAVs) vectors
(Valdes et al., 2014). This strategy conferred a dramatic protection against 6-OHDA
(Figure 1C), in addition to reduce striatal denervation. Similarly, a previous report
also indicated that XBP1s gene transfer also protects dopaminergic neurons against
the PD-inducing neurotoxin MPTP (Sado et al., 2009).
XBP1 has a conserved role in sustaining dopaminergic neuron survival. Recently, the
over-expression of XBP1 was shown to protect against αSynuclein-induced dopaminergic
neuron degeneration in C. elegans, whereas neuron-specific RNAi knockdown of xbp1
exacerbates the neurodegeneration process (Ray et al., 2014). The unconventional splicing
of XBP1 mRNA, in addition to require the endoribonuclease IRE1α, it involves the RNA
ligase RTCB-1. This ligase also confers protection to dopaminergic neurons against
αSynuclein overexpression in C. elegans, uncovering for the first time a functional
relationship between XBP1 and its ligase in the regulation of proteostasis in neurons
(Ray et al., 2014). XBP1 expression has been shown to be neuroprotective also when
it is delivered into neural stem cells that are then transfer into the brain. This
strategy, increased the survival of the graft and improved the motor performance in
a rotenone-induced rat model of PD (Lihui et al., 2012). Finally, an AAV-based gene
therapy strategy to enhance the folding capacity of the ER was also evaluated on a
genetic model of PD (Gorbatyuk et al., 2012). Thus, increasing evidence indicates
that the local modulation of the UPR in the nigrostriatal circuit may have important
therapeutic potential in PD.
The UPR is a double-edged sword, cytoprotective when activated to a moderate extent,
but degenerative when it is sustained over time. Markers of PERK/eIF2α activation
have been found in PD post-mortem brain tissue, where nigral dopaminergic neurons
displaying αSynuclein inclusion are also positive for phosphorylated PERK and eIF2α
(Hoozemans et al., 2007). Deletion of the pro-apoptotic factor CHOP protects dopaminergic
neurons against 6-OHDA and MPTP (Silva et al., 2005) (Figure 1B). Several strategies
are now available to modulate PERK signaling in different disease contexts, including
inhibitors of PERK activity, eIF2α phosphatases, and ATF4 expression (reviewed in
Hetz et al., 2013). Salubrinal, a small compound that enhances eIF2α (Boyce et al.,
2005), was shown to delay disease onset and attenuate motor deficits induced by αSynuclein
over-expression (Colla et al., 2012). Unexpectedly, although salubrinal treatment
attenuated disease symptoms, its administration did not protect dopaminergic neurons
from degeneration (Colla et al., 2012). In the last 2 years, new exciting findings
implicate the PERK/ATF4 signaling branch of the UPR as an interesting target to treat
neurodegenerative diseases (Halliday et al., 2014). In this scenario, additional tools
are available to systematically test the consequences of inhibiting the PERK pathway
in PD models at the level of PERK, eIF2α, or ATF4, respectively.
Perspective
Many important questions remain to be solved in this growing field. Since distinct
UPR signaling branches could have specific and even opposite consequences on neuronal
survival depending on the disease input (Hetz and Mollereau, 2014), a systematic approach
is needed to determine what are the optimal components of the UPR pathway as possible
targets to develop future therapeutic interventions. Gene therapy strategies are currently
been developed in PD patients and the first results of phase I and II clinical trials
are available showing excellent safety profiles (Coune et al., 2012). In this context,
the possible therapeutic potential and side effects of delivering active UPR components
into the SNpc in the long term remains to be determined in non-human primates since
most of the available studies only used rapid-evolving PD rodent models. Another interesting
aspect to explore in the future is the cell-non-autonomous control of the UPR in PD,
which may propagate protective responses to other brain areas and tissues (Mardones
et al., 2015). Overall all these novel insights have placed ER proteostasis in the
center of the etiology of PD, which may translate in the near future into the development
of prototypic strategies to alleviate dopaminergic neuron loss.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.