1. Parkinson’s Disease
Parkinson’s disease (PD) is the second most common neurodegenerative disease in the world after Alzheimer’s disease (AD), affecting 2% of the population over the age of 60. The mean duration of the disease from the time of diagnosis to death is approximately 15 years, with a mortality ratio of 2 to 1 in the affected subjects [
1].
PD is characterized by debilitating motor deficits, such as tremor, limb rigidity and slowness of movements (bradykinesia) although non-motor features, such as hyposmia, cognitive decline, depression, and disturbed sleep are also present in later stages of the disease [
1,
2,
3]. Neuropathologically, these motor deficits are caused by the progressive preferential loss of striatal-projecting neurons of the substantia nigra pars compacta; more specifically a subtype of dopaminergic neurons (DAn) patterned for the ventral midbrain (vmDAn). Neuronal loss is typically accompanied by the presence of intra-cytoplasmic ubiquitin-positive inclusions in surviving neurons. These structures are known as Lewy bodies and Lewy neurites and they are mainly composed of the neuronal protein α-synuclein (α-syn). These protein inclusions are not only found throughout the brain but also outside of the CNS. Moreover, microglial activation and an increase in astroglia and lymphocyte infiltration also occur in PD [
4].
Approximately 90%–95% of all PD cases are sporadic with no family history. Although disease onset and age are highly correlated, PD occurs when complex mechanisms such as mitochondrial activity, autophagy or degradation via proteasome are dysregulated by environmental influence or PD-specific mutation susceptibility [
5].
Studies of rare large families showing classical Mendelian inherited PD have allowed for the identification of 11 genes out of 16 identified disease
loci. They include dominant mutations in Leucine-rich repeat kinase 2 (
LRRK2), recessive mutations in Parkin (coded by
PARK2) and PTEN-induced putative kinase (
PINK1) [
6], as well as both rare dominant mutations and multiplications in the gene encoding α-synuclein (
SNCA).
Current treatment for PD is limited to targeting only the symptoms of the disease and does not cure or delay disease progression. Therefore, the identification of new and more effective drugs to slow down, stop and even reverse PD is critical. This limited symptomatic treatment is due to the lack of clear understanding of the underlying mechanisms affected during PD. Using patient-specific iPSC-based models to recapitulate the disease from start to finish delivers a more detailed picture of the mechanisms involved in the progression of Parkinson’s disease and will aid in the discovery of disease-targeted therapies in the future.
3. Generation of PD-Specific iPSCs
In recent years, neurodegenerative disease research has quickly advanced with the help of stem cell technology reprogramming somatic cells, such as fibroblasts, into induced pluripotent stem cells (iPSC) [
15]. Human iPSC share many characteristics with human embryonic stem cells (hESC), including similarities in their morphologies, gene expression profiles, self-renewal ability, and capacity to differentiate into cell types of the three embryonic germ layers
in vitro and
in vivo [
16]. An important advantage of induced cell reprogramming is represented by the possibility of generating iPSC from patients showing sporadic or familial forms of the disease. These
in vitro models are composed of cells that carry the patients’ genetic variants, some known and others not, that are key to the contribution of disease onset and progression. Moreover, given that iPSC can be further differentiated into neurons, this technology potentially provides, for the first time, an unlimited source of native phenotypes of cells specifically involved in the process related to neuronal death in neurodegeneration
in vitro.One issue found in modeling PD with the use of iPSC is to correctly reproduce its late-onset characteristics, since aging is a crucial risk factor. Indeed, at first it was unclear whether disease-specific features of neurodegenerative disorders that usually progressively appear over several years were reproducible
in vitro over a period of only a few days to a few months. As a consequence, iPSC were initially used to model neurodevelopmental phenotypes and a variety of monogenic early-onset diseases [
17,
18,
19,
20,
21,
22,
23,
24]. However, studies using iPSC derived from patients with monogenic and sporadic forms of PD have illustrated these key features of PD pathophysiology, as a late-onset neurodegenerative disorder, after differentiating these iPSC into dopaminergic neurons. Moreover, several inducible factors that cause cell stress, such as mitochondrial toxins [
25], growth factor deficiency, or even modulated aging with induced expression of progerin (a protein causing premature aging) [
26], have also been used to accelerate and reproduce the phenotypes found during disease progression.
Figure 1.
Generation and use of iPSC modelling in PD. Somatic cells from a diseased patient are isolated and then reprogrammed to a pluripotent state (iPSCs). iPSCs can be maintained in culture or induced to differentiate along tissue- and cell-type specific pathways. Differentiated cells can be used to elucidate disease mechanism pathways, as well as for the development of novel therapies.
Figure 1.
Generation and use of iPSC modelling in PD. Somatic cells from a diseased patient are isolated and then reprogrammed to a pluripotent state (iPSCs). iPSCs can be maintained in culture or induced to differentiate along tissue- and cell-type specific pathways. Differentiated cells can be used to elucidate disease mechanism pathways, as well as for the development of novel therapies.
In this review, the recent work on iPSC-based PD modeling for both sporadic and familial cases will be discussed, as well as how iPSC-based studies are helping in the advancement of novel drug discoveries. These studies give insight for the fundamental understanding of PD pathogenesis, which is critical for the development of new treatments.
4. Modeling Sporadic and Familial PD Using iPSC
Over the last few years, several studies have reported the generation of iPSC from patients suffering from sporadic and genetic forms of PD (
Table 1). The first group generated PD-specific iPSC from a sporadic PD patient in 2008 [
27]. Over the following year, the Jaenisch’s group was able to demonstrate that iPSC derived from PD patients were able to differentiate towards DAn, however, no characteristic signs of progressive neurodegeneration or disease-related phenotypes were observed in those cells [
28]. The Jaenisch group generated gene-free iPSC lines from skin fibroblasts of five idiopathic PD patients. Using
in vivo experiments, they showed that PD-specific iPSC-derived DAn were able to survive and engraft in the rodent striatum for at least 12 weeks. A small number of these cells co-expressed tyrosine hydroxylase (TH) and G-protein-gated inwardly rectifying K+ channel subunit (GIRK2), which are the hallmark characteristics of vmDAn. Remarkably, injection of these iPSC-derived DAn into the brains of 6-OHDA-lesioned rats resulted in motor symptoms improvement [
29].
Table 1.
Summary of the described PD iPSC modeling publications in this review.
Table 1.
Summary of the described PD iPSC modeling publications in this review.
Gene | Publication | Mutation | Number of patients | Isogenic Controls | Cell Type Differentiation | Findings |
---|
SNCA | Devine et al., 2011 [30] | Triplication | 1 | NO | Floor-plate DAn differentiation (21–30 days): 28%–37% TH+/TUJ1+ | mRNA doubled expression of SNCA |
Byers et al., 2011 [31] | Triplication | 1 | NO | DAn differentiation (50 days): 6%–11% TH+ | Double expression of SNCA, increased susceptibility to OS |
Chung et al., 2013 [32] | A53T | 2 | YES | Neuronal differentiation (56–84 days): DAn yield not specified. | Increased nitrosative stress, and ER stress, reversed by adding NAB2. |
Ryan et al., 2013 [25] | A53T | 1 | YES | Kriks’s Floor-plate DAn differentiation: ~80% A9 DAn of total neurons. | Diminished spare respiration mitochondrial capacity; increased ROS/RNS and attenuation of MEF2/PGC1α neuroprotective pathway |
GBA1 | Mazzulli et al., 2011 [33] | N370S/84GG insertion | 1 | NO | DAn diff. (30 days): 80% TUJ1+, 10% TH+/TUJ1+ | Formation of soluble α-syn oligomers, correlated with a decline of lysosomal proteolysis. |
Schöndorf et al., 2014 [34] | GBA1 (RecNcil/wt) GD (N370S; L444P) | 4 GBA1 4 GD | YES | Kriks’s Floor-plate DAn differentiation: 15%–20% TH+/GIRK2+/FOXA2+/VMAT2+ There is also further purification of DAn by FACS | Causal relation of GBA1 mutations with increased a-syn and LB inclusions, correlated with autophagic/lysosomal system impairment |
PARK2 | Jiang et al., 2012 [35] | Exon 3/5 deletion | 2 | NO | DAn differentiation (70 days): yield not specified | Loss of Parkin function; decreased DA uptake and incorrectly folded DAT protein, with increased OS susceptibility. Transduction of WT PARK2 reversed OS sensitiveness. |
Imaizumi et al., 2012 [36] | Exons 2–4 and 6,7 homozygous deletion | 2 | NO | DAn differentiation (10 days): yield not specified | Abnormal mitochondrial morphology and impaired mitochondrial homeostasis. |
PARK2 PINK1 | Miller et al., 2013 [26] | PINK1 (Q456X) Parkin (V324a) | 1 1 | NO | Kriks’s Floor-plate DAn differentiation yield not specified | Loss of dendrite lenght and decreased neuronal survival, as seen by decreased p-ATK values, when exposing mDA neurons to progerin. |
PINK1 | Seibler et al., 2013 [37] | C1366T, C509G | 3 | NO | Floor-plate DAn differentiation: 11%–16% TH+/TUJ1+ | Endogenous mutant PINK1 diminished Parkin recruitment to the mitochondrial membrane under the presence of valynomycin. WT PINK1 rescued Parkin recruitment. |
(PINK1) | Cooper et al., 2012 [38] | Q456X | 2 | NO | DAn differentiation (22 days): 35% TUJ1+; 10% TH+ | Increased vulnerability of neural cells to chemical stressors, with common defects to protect against OS. |
LRRK2 | Nguyen et al., 2011 [39] | G2019S, R1441C | 2 | NO | Floor-plate DAn differentiation (30–35 days): 3.6%–5% TH+ | α-syn accumulation, increased OS genes, and increased susceptibility to hydrogen peroxide. |
Sánchez-Danes et al., 2012 [40] | G2019S | 7 Sporadic 4 LRRK2 (G2019S) | NO | DAn diff (Lentiviral-mediated forced expression LMX1A in neural precursors) (75 days): 55% TH+/TUJ1+ (Majority TH+GIRK2+) | Reduced neurite lenght and number. Accumulation of α-syn in LRRK2 DAn. Reduction of autophagic flux and accumulation of early autophagosomes. |
Orenstein et al., 2013 [41] | G2019S | 4 LRRK2 (G2019S) | NO | As described in [40] | Blockage of the CMA degradation pathway due to accumulated α-syn with correlated increased expression of LAMP-2A. |
Reinhardt et al., 2013 [42] | G2019S | 2 | YES | Floor-plate DAn differentiation (30–35 days): 20% TH/TUJ1/DAPI | Decreased neurite lenght levels. Increased ERK activation levels, and discover of novel genes dysregulated in LRRK2 DAn. |
Many laboratories have now successfully recapitulated in vitro some of the characteristics of PD, using iPSC as a model compared to the aforementioned studies in which no signs of Parkinson’s disease were observed. However, given that PD is a progressive aging disease that affects several cellular mechanisms involving different cell types, each iPSC model highlights only some PD-associated characteristics. Nevertheless, each one of these models has helped to understand some of the fundamental underlying mechanisms as a proof-of-concept. In the last few years, iPSC-model reliability has rapidly improved and has paved the way for the discovery of new complex biomolecular interactions in the pathogenesis of PD. Thus, iPSC modeling has shown to be promising as a tool for drug-screening platforms in the future.
Recently, iPSC-derived DA neurons carrying a triplication of
SNCA, the coding gene for α-syn protein, have been generated [
30,
31]. These cells showed enhanced α-syn mRNA and protein levels [
30] and increased cell death vulnerability when exposed to oxidative-stress inducers [
31]. Using an iPSC model based on the rare missense A53T
SNCA mutation, Chung
et al. observed early pathogenic phenotype in patient-derived neurons, compared to isogenic gene-corrected controls. In particular, they observed a connection between nitrosative and ER stress in the context of α-syn toxicity. Interestingly, the levels of CHOP (CCAAT enhancer binding protein homologous protein), a component of ER stress-induced apoptosis, did not change, indicating that in this model cellular pathology was still at an early stage [
32]. iPSC-derived DAn, carrying the A53T
SNCA mutation, also showed α-syn aggregation, altered mitochondrial machinery, thus enhancing basal ROS/RNS production [
25]. The increase of RNS production leads to
S-nitrosylation of the
pro-survival transcription factor MEF2 and its consequent inhibition, reducing the expression of the mitochondrial master regulator PGC1α and genes that are important for the development and survival of A9 DAn [
43]. Interestingly, Ryan
et al., postulated that the MEF2-PGC1α pathway contributes to the appearance of late-onset phenotypes in PD due to the complex interaction between environmental factors and gene expression. Indeed, when PD-associated pesticides were added below EPA-accepted levels, this was enough to exacerbate oxidative/nitrosative stress, inhibiting MEF2-PGC1α and inducing apoptosis, a late-onset phenotype [
25].
Interestingly, α-syn is one of the main pathological readouts for many of the sporadic and familial PD cases that are not related with mutations in
SNCA [
44]. For example, the clinical link between the lysosomal storage disorder Gaucher disease (GD) and PD appears to be based on the fact that mutations in acid
GBA1 gene, which causes GD, contributes to the pathogenesis of synucleinopathies [
33,
34].
GBA1 encodes the lysosomal enzyme β-Glucocerebrocidase (GCase), which cleaves the β-glucosyl linkage of GlcCer. Functional loss of GCase activity in iPSC-derived neurons has been associated with compromised lysosomal protein degradation, which in turn induces α-syn accumulation, resulting in neurotoxicity through aggregation-dependent mechanisms [
33]. In addition, iPSC-derived neurons carrying the heterozygous mutation in
GBA1 also have shown increased levels of GlcCer, changes in the autophagic/lysosomal system and calcium homeostasis, which may cause a selective threat to DA neurons in PD [
34].
Similarly to mutations in
GBA1, mutations in
PINK1 and
PARK2 are also associated with early onset recessive forms of familial PD [
45]. Both proteins, PINK1 and Parkin, are involved in the clearance of mitochondrial damage. Therefore their mutations cause a PD characterized by mitochondrial stress as main feature [
46,
47,
48]. Under physiological conditions, Parkin, which is localized in the cytoplasm, is translocated to damaged mitochondria in a PINK-dependent manner triggering mitophagy [
49]. This has been confirmed in iPSC-derived DA neurons carrying a mutation in
PINK1. In these cells, Parkin recruitment to mitochondria was impaired and only over-expression of WT
PINK1 was able to rescue the function [
37]. On the other hand, iPSC models for mutation in
PARK2 revealed an increase of oxidative stress. Jiang and colleagues showed that iPSC from patients carrying mutations in
PARK2 enhanced the transcription of monoamine oxidase, the spontaneous release of dopamine and significantly decreased dopamine uptake, increasing susceptibility to reactive oxygen species [
35]. Although the incremented oxidative stress has been confirmed in a parallel study, in this study no difference in monoamine oxidase was observed [
36]. On the contrary, the oxidative stress was accompanied by a compensation mechanism that involved the activation of the reducing Nrf2A pathway [
36].
Mutations in
LRRK2 have been one of the most studied mutations in PD, not only because they are the most common cause of familial PD, but also because clinical symptoms of
LRRK2-PD are similar to those of idiopathic PD [
50]. The most common mutation is the G2019S, which results in hyper-activity of the LRRK2 kinase domain. Although penetrance of this gene has shown to be variable between individuals’ age, iPSC model of a G2019S
LRRK2-PD has recapitulated characteristic features of PD, such as accumulation of α-syn, increase in genes responsible for oxidative stress and enhanced susceptibility to hydrogen peroxide, which is displayed through caspase-3 activation [
39]. Furthermore, the expression of key oxidative stress-response genes and α-syn were found to be increased in neurons from
LRRK2-iPSC, when compared to those differentiated from control iPSC or hESC.
Our group has generated iPSC lines from seven patients with idiopathic PD and four patients carrying G2019S mutation in the
LRRK2 gene [
40]. We observed morphological alterations in PD-derived iPSC vmDAn (fewer and shorter neurites) as well as an increase in the number of apoptotic neurons over a long-time culture (2.5 months). Moreover, we found an accumulation of α-syn in
LRRK2-iPSC derived DAn after a 30 days culture.
Sporadic forms of PD are not as well defined, given that they may be caused by several genetic variants, as well as a strong environmental effect. However, our study revealed that DAn, which were derived from idiopathic PD patients, also showed an increased susceptibility to degeneration
in vitro after long-term culture [
40].
Importantly, the appearance of the neurodegenerative phenotypes in differentiated DAn from either idiopathic or
LRRK2-associated PD was shown to be the consequence, at least in part, of impaired autophagy. Blockade of autophagy by lysosomal inhibition showed a specific reduction in autophagic flux by LC3-II immunoblotting, suggesting that the clearance of autophagosomes was compromised [
40]. Proteins may also enter the autophagic process directly at the lysosome level, via chaperone-mediated autophagy (CMA). Increased co-localization of α-syn with LAMP2A puncta in iPSC-derived
LRRK2 DAn, revealed a compromised degradation of α-syn by CMA [
41]. Although both wild-type and mutant LRRK2 inhibit CMA, G2019S LRRK2 protein was more resistant to the CMA-mediated degradation, resulting in α-syn accumulation [
41]. Furthermore, the same phenotype was induced by over-expression of wild-type or G2019S
LRRK2 in control iPSC-derived cultures [
40] and rescued by LRRK2 inhibition [
42]. Indeed, iPSC-derived DAn cultures from isogenic G2019S
LRRK2 lines (mutation being the sole experimental variable) exhibited an increased mutant-specific apoptosis and decreased neurite outgrowth, as well as alterations in the expression of several pERK (phosphorylated ERK) controlled genes, all of which could be rescued by the inhibition of LRRK2 [
42]. Moreover, the genetic correction of LRRK2 mutation resulted in the phenotypic rescue of differentiated neurons with improved neurite length to levels comparable to those of controls.
5. Patient-Derived Stem Cells Could Improve Drug Research for PD
An important goal of humanized stem cell-based PD model systems is the screening of potential new drugs that could affect the neurodegenerative process at several levels during its development in specifically affected human cells. Moreover, the availability of such patient-specific stem cell-based model systems could help identifying new pharmacological strategies for the design of personalized therapies. Recently, iPSC-derived forebrain neurons have been used as a platform to screen disease-modifying drugs, highlighting the possibilities of iPSC technology as an
in vitro cell-based assay system for AD research [
51]. A recent study has also taken a significant leap towards personalized medicine for PD patients, by investigating signs of the disease in patient-specific iPSC-derived neurons and testing how the cells respond to drug treatments [
38]. The study showed that neurons derived from PD patients carrying mutations in the
PINK1 or
LRRK2 genes display common signs of distress and vulnerability such as abnormalities in mitochondria and increased vulnerability to oxidative stress. However, they found that oxygen consumption rates were lower in cells with mutations in
LRRK2 and higher in cells with the mutations in
PINK1. Notably, they were able to rescue the phenotype caused by toxins to which the cells were exposed to with various drug treatments, including the antioxidant coenzyme Q10 and rapamycin. Most importantly, the response of iPSC-derived neurons was different depending on the type of familial PD, since drugs that prevented damage to neurons with mutations in
LRRK2, did not protect neurons with mutations in
PINK1 [
38].
In addition, Ryan and colleagues performed a high-throughput screening (HTS) to identify molecules that are capable of protecting DAn from the toxic effect of PD-associated pesticides. They observed that the MEF2-PGC1α pathway contributes to the late-onset PD phenotypes due to the interaction between environmental factors and gene expression [
25]. They performed HTS for small molecules capable of targeting the MEF2-PGC1α pathway and they identify isoxazole as new potential therapeutic drug. Isoxazole, not only drove the expression of both MEF2 and PGC1α, but also protected A53T DAn from pesticide-induced apoptosis [
25].
Chung and colleagues investigated yeast and iPSC PD models in parallel to discover and reverse phenotypic responses to α-syn. In conjunction to what was previously reported, they showed a connection between α-syn toxicity, accumulation of NO and ER stress [
32]. With these results, they took a step further by screening for possible α-syn toxicity suppressors in their iPSC model, to compare with their previous yeast screenings [
52,
53,
54]. In particular they showed that the ubiquitin ligase Nedd4 and its chemical activator NAB2 [
53] are able to rescue the α-syn toxicity in patient-derived neurons [
32], opening a door to a new potential drug treatment.
These results encourage the use of iPSC technology as a tool to discover potential therapeutic drugs. However, concluding for what recent studies have unveiled up until now focusing only on genetic forms of PD, it remains to be determined whether this advanced technology can be used also in sporadic patients with uncertain genetic cause of the disease.