Pluripotent Stem Cell Derived Neurons as In Vitro Models for Studying Autosomal Recessive Parkinson’s Disease (ARPD): PLA2G6 and Other Gene Loci

Abstract

Parkinson’s disease (PD) is a neurodegenerative motor disorder which is largely sporadic; however, some familial forms have been identified. Genetic PD can be inherited by autosomal, dominant or recessive mutations. While the dominant mutations mirror the prototype of PD with adult-onset and L-dopa-responsive cases, autosomal recessive PD (ARPD), exhibit atypical phenotypes with additional clinical manifestations. Young-onset of PD is also very common with mutations in recessive gene loci. The main genes associated with ARPD are Parkin, PINK1, DJ-1, ATP13A2, FBXO7 and PLA2G6. Calcium dyshomeostasis is a mainstay in all types of PD, be it genetic or sporadic. Intriguingly, calcium imbalances manifesting as altered Store Operated Calcium Entry (SOCE) is suggested in PLA2G6-linked PARK 14 PD. The common pathways underlying ARPD pathology, including mitochondrial abnormalities and autophagic dysfunction can be investigated ex vivo using the induced pluripotent stem cell (iPSC) technology and are discussed here. PD pathophysiology is not faithfully replicated by animal models, and therefore, nigral dopaminergic neurons generated from iPSC serve as improved human cellular models. With no cure till date and treatments aiming at symptomatic relief, these in vitro models derived through midbrain floor plate induction provide a platform to understand the molecular and biochemical pathways underlying PD etiology in a patient-specific manner.

1. Introduction

Parkinson disease (PD) is the second most common neurodegenerative disorder characterized by motor symptoms such as resting tremor, bradykinesia, rigidity, postural instability, stooped posture and freezing, as well as non-motor symptoms including cognitive and behavioural symptoms, sleep disorders, autonomic dysfunction, sensory symptoms and fatigue ^1–5. The pathological hallmark of PD is the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and the subsequent loss of dopamine inputs to forebrain striatal structures along with the appearance of protein inclusions called Lewy bodies (LB) composed of α-synuclein fibrils ^2,3 PD is classified into two genetic subtypes including monogenic familial forms with Mendelian inheritance and sporadic forms with no underlying genetic factors ⁶. The sporadic forms of PD are highly prevalent whereas familial PD accounts to only 5-10% of the reported cases. Monogenic familial forms of PD are rare, caused by highly penetrant disease causing mutations. Parkinsonism caused by dominant mutations including alpha-synuclein (SNCA), leucine-rich repeat kinase 2 (LRRK2), vacuolar protein sorting 35 (VPS35) and the like are largely similar to the common, late-onset sporadic PD ^7,8. Autosomal recessive PD (ARPD) results from mutations in different loci which have clinical signs typical of PD or can exhibit a wide range of other complex symptoms. The common pathways underlying ARPD are mitochondrial quality control, protein degradation processes, and oxidative stress responses among others ^9,10.

The current knowledge of PD is mostly from postmortem studies and animal models. While the former represent only the end-stage of the disease, the latter fail to reflect human disease pathology due to interspecies differences. In this context, human pluripotent stem cells (both embryonic stem cells, ESC and induced pluripotent stem cells, iPSC) are an excellent source of cells for differentiation to DA neurons in vitro. ‘Disease modelling in a dish’ by recapitulating the disease phenotypes in defined cell populations would make it possible to understand the cellular and molecular mechanisms of PD along with providing a high-throughput drug screening platform ^11–13.

2. Autosomal recessive parkinsonism

The hereditary forms of parkinsonism which are transmitted in an autosomal recessive fashion are given in Table 1. Mutations have been identified most commonly in three genes in several ethnic groups spanning different geographical locations- parkin (PRKN, PARK2), PTEN induced putative kinase 1 (PINK1, PARK6), and Parkinson protein 7 (DJ-1, PARK7). Point mutations, large genomic rearrangements, leading to deletions or multiplications presenting as homozygous or compound heterozygous variations are reported, particularly for parkin ¹⁴ Recessive mutations in several genes, including ATPase type 13A2 (ATP13A2, PARK9), phospholipase A2, group VI (PLA2G6, PARK14), F-box only protein 7 (FBXO7, PARK15), spatacsin (SPG11), and DNA polymerase gamma (POLG), cause young or juvenile onset-PD. These present with other clinical manifestations like dystonia, dementia and other disturbances ⁸. DNAJ subfamily C member 6 (DNAJC6, PARK19), synaptojanin-1 (SYNJ1, PARK 20), vacuolar protein sorting 13C (VPS13C, PARK23) are also reported to have mutations causing autosomal recessive PD ⁶.

Table 1. Genes associated with autosomal recessive Parkinson’s disease (ARPD).

Causal genes for autosomal recessive Parkinson’s disease (ARPD). Genes mapped to different PARK loci and associated with ARPD are listed together with the involved protein. Rarely, mutations in spatacsin (SPG11), and DNA polymerase gamma (POLG), cause autosomal recessive parkinsonism with juvenile onset, mostly with atypical features.

Locus	Gene	Protein
PARK2	Parkin	E3 ubiquitin-ligase
PARK6	Pink1	phosphatase and tensin homolog induced putative kinase1
PARK7	DJ-1	Parkinson protein 7, oncogene DJ-1
PARK9	ATP13A2	lysosomal P5-type ATPase
PARK14	PLA2G6	phospholipase A2, group VI
PARK15	FBXO7	F-box only protein 7
PARK19	DNAJC6	putative tyrosine-protein phosphatase auxilin
PARK20	SYNJ1	synaptojanin-1
PARK23	VPS13C	vacuolar protein sorting 13C
	SPG11	spatacsin
	POLG	DNA polymerase gamma

2.1. PLA2G6 (PARK14)

Phospholipase A2 group 6 (PLA2G6, iPLA2β) gene encodes a calcium-independent group 6 phospholipase A2 enzyme, which hydrolyzes the sn-2 ester bond of the membrane glycerophospholipids to produce free fatty acids and lysophospholipids ¹⁵. Various mutations in this gene have been discovered in patients with neurodegenerative disorders such as infantile neuroaxonal dystrophy (INAD), atypical neuroaxonal dystrophy (ANAD), adult-onset dystonia-parkinsonism (DP) and autosomal recessive early-onset parkinsonism (AREP), together known as PLA2G6-asscociated neurodegeneration, PLAN ¹⁶. PLAN can be classified as neurodegeneration with brain iron accumulation II (NBIA II), however a wide range of clinical variability is exhibited in these phenotypes with most PD cases devoid of brain iron deposition or cortical atrophy ¹⁷. iPLA2β protein contains an N-terminal domain, Ankyrin repeats and catalytic domains (Figure 1). iPLA2β is predominantly localized in the cytosol, but can translocate to the Golgi, ER, mitochondria, and nucleus under stimulation ^18–21. Two distinct 85 kDa (VIA-1) and 88 kDa (VIA-2) human iPLA2β isoforms have been discovered along with many N-terminal truncated forms due to proteolytic cleavage and alternate splicing ²¹. It is highly expressed in the human brain including SNpc (http://www.proteinatlas.org). The PLA2G6 gene mutations was associated with parkinsonism almost a decade ago, with R741Q and R747W being the first to be reported in adult-onset levodopa-responsive dystonia-parkinsonism ^22–24. p.R741Q has also been indicated in early-onset PD without dystonia ²⁵. Though PD-associated mutations in this gene are mostly homozygous, some of them are rare and specific to geographic areas ^26–29 while others are compound heterozygous ^30–32. The mutations that are pathogenic and causal for PD in PLA2G6 are detailed in Table 2.

Figure 1. Structure of PLA2G6 (iPLA2β) protein.

The full length protein is shown with seven ankyrin repeats (pink circles), a proline-rich motif (blue box), a glycine-rich nucleotide binding motif (magenta), a lipase motif (orange with the active site highlighted), and a proposed C-terminal calcium-dependent calmodulin binding domain (purple). Numbers indicate amino acids.

Table 2. List of PD-associated pathogenic mutations in PLA2G6 (PARK14) gene loci.

Mutations in PLA2G6 gene that cause Parkinson’s disease. Mutations in the PLA2G6 gene that are pathogenic (or likely pathogenic) and cause Parkinson’s disease are documented.

Mutation	Protein change
c.109 C >T	p.arg37-to-X (R37X)
c.216C > A	p.phe72-to-leu (F72L)
c.238 G > A	p.ala80-to-thr (A80T)
c.991G > T	p.asp331-to-tyr (D331Y)
c.1077 G > A	p.met358-llefsX6
c.1354C > T	p.gln452-to-X (Q452X)
c.1495G > A	p.ala499-to-thr (A499T)
c.1715C > T	p.thr572-to-ile (T572I)
c.1791delC	p.his597-fx69
c.1894C > T	p. arg632-to-trp (R632W)
c.1904G > A	p.arg635-to-gln (R635Q)
c.1976A > G	p.asn659-to-ser (N659S)
c.2215G > C	p.asp739-to-his (D739H)
c.2222G > A	p.argR741-to-gln (R741Q)
c.2239C > T	p.arg747-to-trp (R747W)

Widespread LB pathology is seen with PLA2G6-linked PD ^33,34. The loss of PLA2G6 in Drosophila results in impaired retromer function, ceramide accumulation, and leads to lysosomal dysfunction, leading to age-dependent loss of neuronal activity ³⁵. Dysfunction of mitochondria and increased lipid peroxidation have also been reported in PLA2G6-deficient Drosophila mimicking the human fibroblasts with R747W mutation ¹⁹. Increased sensitivity to oxidative stress, progressive neurodegeneration and a severely reduced lifespan and impaired motor co-ordination is seen in PLA2G6-knockout flies ³⁶. In yet another fly model, the loss of PLA2G6 leads to shortening of phospholipid acyl chains, resulting in ER stress and impaired neuronal activity as well as formation of α-synuclein fibrils, demonstrating that phospholipid remodeling by PLA2G6 is essential for DA neuron survival and function ³⁷. Similarly, a rodent knockin model of D331Y PLA2G6 mutation exhibited early degeneration of SNpc DA possibly via mitochondrial and ER stress, impaired autophagic mechanisms and gene expression changes ³⁸.

Elevated levels of both α-synuclein and phosphorylated α-synuclein are seen in PLA2G6 knockout mice models, facilitating the formation of LB and eventually death of affected DA neurons ³⁹. In an in vitro study examining the catalytic activity of PLA2G6 proteins, recombinant proteins containing the three mutations associated with dystonia-parkinsonism (R632W, R741Q, and R747W) did not show any altered catalytic activity whereas the mutations associated with INAD led to a significant loss of enzyme activity ⁴⁰. In addition, PLA2G6-PD mutants of SHSY5Y, a neuroblastoma cell line, failed to prevent rotenone-induced death of dopaminergic cells ⁴¹. PLA2G6-PD does not present with a typical clinical scenario and continues to evolve with a wide phenotypic spectrum. It is safe to infer that the PD-relevant mutations do not significantly alter the catalytic activity of the enzyme, but induce damage through parallel mechanisms like oxidative stress, mitochondrial dysfunction or even compromised lipid remodeling.

2.1.1. PLA2G6 and Store-operated calcium entry (SOCE)

Store-operated calcium entry (SOCE) is an arm of calcium signaling activated by depletion of ER stores that triggers influx of calcium across the PM brought about by the calcium sensor STIM and the PM pore channel Orai ^42–44. Interestingly in a genetic screening of Drosophila, not only STIM1 and Orai1, but also a fly orthologue of PLA2G6 encoded by the CG6718 gene, were identified as SOCE activators ⁴⁴. Many groups have from this time identified PLA2G6 as an endogenous activator of SOCE ^45–48. The physiological relevance of neuronal SOCE is disputed ⁴⁹and its role in DA neurons, particularly, is not known. In PD, abnormal calcium homeostasis triggers a cascade of downstream events that eventually leads to cell death ⁵⁰. Interestingly, primary skin fibroblasts from idiopathic and PLA2G6-PD (R747W) patients revealed a significant deficit in endogenous SOCE and similarly, MEFs from the exon2-knockout mice exhibited deficient store-operated PLA2G6-dependent calcium signaling ⁵¹. This was also mirrored in the iPSC-derived DA neurons along with low ER calcium levels and deficient autophagic flux. The knockout mice also showed age-dependent loss of DA neurons. This study for the first time arrived at a causal relationship between PLA2G6-dependent SOCE, depleted stores, dysfunctional autophagy in DA neurons and a PD-like phenotype ⁵¹. Recently, in a patient derived (D331Y) DA neuron model, imbalance of calcium homeostasis, markedly deficient SOCE, increase of UPR proteins, mitochondrial dysfunction, increase of ROS, and apoptosis was reported. Interestingly the UPR modulator, azoramide rescued the phenotype of the mutant DA neurons, possibly via CREB signaling ⁵². These recent developments have opened new exciting areas to study the significant contributions of PLA2G6 and SOCE in PD, and may involve new undiscovered molecules providing a yet unexplored arena for PD-drug discovery.

2.2. Parkin, PINK1 and DJ-1

The E3 ubiquitin ligase parkin (PARK2) and the serine/threonine kinase PINK1 (phosphatase and tensin homolog induced putative kinase1, PARK6), act together in a mitochondrial quality control pathway and promote the selective autophagy of depolarized mitochondria (mitophagy)⁵³. PINK1 levels are low in healthy cells as it is continually cleaved inside the mitochondria in a sequential manner by proteases ⁵⁴. The import of PINK1 into mitochondria is stopped when the organelle loses its inner membrane electrochemical gradient (depolarization), which leads to the stabilization of the protein on the mitochondrial outer membrane ⁵⁵. This accumulation of PINK1 kinase on the mitochondria triggers parkin recruitment and activation resulting in ubiquitination of various outer mitochondrial membrane proteins ^56,57. The damaged mitochondria are eventually eliminated by autophagy. Pathogenic PD-associated mutations in either Parkin or PINK1 causes accumulation of impaired mitochondria, increased ROS and neuronal cell death ⁵⁸. More than 100 different Parkin mutations have been reported from PD patients, including deletions, insertions, multiplications, missense and truncating mutations and over 40 point mutations and rarely, large deletions, have been detected in PINK1 ⁵⁹. Clinically both cause young onset PD and show responsiveness to levodopa. The phenotype associated with the oncogene DJ-1 mutations has been studied in a smaller number of patients but it is overall indistinguishable from that of the patients with PINK1 or Parkin mutations ⁶⁰. DJ-1 is thought to be involved in the regulation of the integrity and calcium cross-talk between endoplasmic reticulum (ER) and mitochondria and pathogenic mutations lead to impaired ER-mitochondria association in PD ⁶¹.

2.3. ATP13A2, FBXO7, SPG11 and POLG

Mutations in ATP13A2, FBXO7, spatacsin and POLG cause juvenile-onset ARPD along with PLA2G6 ⁸. Mutations in ATP13A2 or PARK9, were first identified in 2006 in a Chilean family and are associated with a juvenile-onset, levodopa-responsive type of Parkinsonism called Kufor-Rakeb syndrome (KRS). KRS involves pyramidal degeneration, supranuclear palsy, and cognitive impairment ⁶². The ATP13A2 gene encodes a large lysosomal protein, belonging to the P5-type ATPase family of transmembrane active transporters. Its substrate specificity remains unknown. It is suggested that ATP13A2 recruits HDAC6 to lysosomes to promote autophagosome-lysosome fusion and maintain normal autophagic flux ⁶³. This, in turn is required for preventing α-synuclein aggregation in neurons. FBXO7, in turn is an adaptor protein in SCF^FBXO7 ubiquitin E3 ligase complex that mediates degradative or non-degradative ubiquitination of substrates. FBXO7 mutantions aggravate protein aggregation in mitochondria and inhibit mitophagy ⁶⁴. Parkin- and FBXO7-linked PD have overlapping pathophysiologic mechanisms and clinical features. Wild type FBXO7, but not PD-linked FBXO7 mutants has been shown to rescue DA neuron degeneration in Parkin null Drosophila ⁶⁵. Loss of activity of FBXO7 in seen in patients with PARK 15-PD and is therefore crucial for the maintenance of neurons ⁶⁶. SPG11 or spatacsin mutations present with bilateral symmetric parkinsonism at an early age with rapid deterioration and development of spastic paraplegia and thinning of the corpus callosum on MRI, typical of spastic paraplegia 11 ^67,68. An involvement of POLG, the mitochondrial DNA polymerase that is responsible for replication of the mitochondrial genome is considered in early onset-PD especially in the presence of additional symptoms, such as ophthalmoparesis, non-vascular white matter lesions and psychiatric comorbidity ⁶⁹. A role of mitochondrial DNA defects in the pathogenesis of neurodegenerative parkinsonism with POLG mutations is speculated ⁷⁰.

3. Common pathways in ARPD

There are many converging features seen at the molecular and clinical level in ARPD that are discussed in the following section. Understanding these causal molecular mechanisms is crucial to identify common targets and devise therapeutic approaches. However, some fundamental underlying processes still remain unclear. The contributions of the intracellular organelle ER in ARPD pathology via the calcium signaling pathway, SOCE is poorly understood. The mitochondrion, which is the star player in PD pathogenesis, regulates SOCE activity possibly via sub-plasmalemmal calcium buffering, the generation of mediators, local ATP modulation and regulation of STIM1⁷¹. In turn, SOCE-derived calcium significantly affects mitochondrial metabolism. Hence, the communication between SOCE and mitochondria is hypothesized to be interdependent and complex leading to fine-tuning of both SOCE and mitochondrial function⁷². Though PD-relevant mitochondrial processes are studied extensively, SOCE and its role in PD is largely unexplored. The microbiota-gut-brain axis and its imbalance by alterations in the human microbiome also represent a risk factor for PD⁷³. Such pathways that are not well-proven are omitted from this section, for the ease of understanding.

3.1. Mitochondrial pathways

The most compelling evidence for loss of mitochondrial fidelity comes from the genes PINK1 and Parkin. As mentioned earlier, PINK1 accumulates on the outer membrane of damaged mitochondria and activates Parkin’s E3 ubiquitin ligase activity. Parkin recruited to the damaged mitochondrion ubiquitinates the outer mitochondrial membrane proteins to trigger selective autophagy. In the late 1970’s when accidental exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was found to cause PD and neurodegeneration, the first causal mechanism speculated was mitochondrial dysfunction ⁷⁴. A specific defect of Complex I activity is also seen in the substantia nigra of patients with PD ⁷⁵. We now understand that the pathways included in mitochondrial quality control system are fission/fusion, mitochondrial transport, mitophagy, and mitochondrial biogenesis ⁹. The precise mechanisms by which Parkin and PINK1 regulate fission and fusion is debated but studies from Drosophila and mammalian culture systems, though contradictory, indicates unbalanced mitochondrial fission and fusion in PINK1 mutants ^9,76–79. DJ-1 ⁸⁰ and ATP13A2 ⁸¹ mutants also show fragmented mitochondria. The combined effects of Parkin and PGC-1α in the maintenance of mitochondrial homeostasis in dopaminergic neurons is demonstrated ⁸². PINK1 is also involved in mitochondrial motility along axons and dendrites of neurons. PINK1 interacts with Miro, a component of the motor/adaptor complex binding mitochondria to microtubules and allowing their movement to and from cellular processes ⁸³. Miro is phosphorylated by PINK1 and ubiquitinated by parkin, leading to its degradation and halting mitochondrial transport promoting clearance of damaged mitochondria ⁸⁴. Parkin/PINK1 is hence involved in mitochondrial trafficking ^9,85.

The clearance of damaged mitochondria or mitophagy is a pathway common to mostly all ARPD-related genes. The role of Pink1-Parkin in mitophagy is well-established ^86,87. Fbxo7 is also shown to induce mitophagy in response to mitochondrial depolarisation in a common pathway with Parkin and PINK1, and PD-associated mutations interfere in this mechanism ⁸⁸. Parkin ⁸⁹ and PINK1 ⁹⁰ is also linked to mitochondrial biogenesis therefore probably being a part of mitochondrial transcription/replication.

3.2. Autophagy-lysosomal pathways

In addition to impaired mitophagy, protein degradation pathways, especially the autophagy-lysosomal pathway are affected in PD. ATP13A2 is suggested as a regulator of the autophagy-lysosome pathway. ATP13A2 acts in concert with another PD-protein SYT11 and its loss of function results in dysfunctional autophagy-lysosomal pathway as seen in PD ⁹¹. α-synuclein-independent neurotoxicity due to endolysosomal dysfunction has also been demonstrated in ATP13A2 null mice ⁹² Parkin knockout neurons too show perturbed lysosomal morphology and mitochondrial stress ⁹³. DJ-1 is associated to chaperone-mediated autophagy (CMA) and its deficiency aggravates α-synuclein aggregation by inhibiting CMA activation ⁹⁴ Loss of DJ-1 could also lead to impaired autophagy and accumulation of dysfunctional mitochondria ⁹⁵. Autophagic defects are a mainstay in PLA2G6-PD. Genetic or molecular impairment of PLA2G6-dependent calcium signalling is a trigger for autophagic dysfunction, progressive loss of DA neurons and age-dependent L-DOPA-sensitive motor dysfunction in a mouse knockout model ⁵¹.

3.3. Cell death and oxidative stress

Oxidative stress and apoptosis are frequently involved in ARPD pathogenesis. ROS accumulation plays a key role in the initiation and acceleration of cell death compromising neuronal function and structural integrity ⁹⁶. The protein products of Parkin, PINK1 and DJ-1 are associated with disrupted oxidoreductive homeostasis in DA neurons. Impaired cell survival in part due to defective oxidative stress response is implicated in PARK2 knockout neurons ⁹⁷. Further transgenic overexpression of the parkin substrate, aminoacyl-tRNA synthetase complex interacting multifunctional protein-2 (AIMP2) leads to a selective, age-dependent progressive loss of dopaminergic neurons via activation of poly(ADP-ribose) polymerase-1 (PARP1) ⁹⁸. Similarly PINK1 is also shown to exert a neuroprotective effect by inhibiting ROS formation and maintaining normal mitochondrial membrane potential and morphology in cultured SN dopaminergic neurons ⁹⁹. The profiles of oxidative damage in the whole brain and neurochemical metabolites in the striatum of PINK1 knockout rats at different ages and genders was studied and oxidative damage revealed as a crucial factor for PD ¹⁰⁰. Loss of PINK1 inhibits the mitochondrial Na(+)/Ca(2+) exchanger (NCLX), resulting in impaired mitochondrial calcium extrusion which was, however, fully rescued by activation of the protein kinase A (PKA) pathway ¹⁰¹. DJ-1 also has a role in cell death and combating oxidative stress. It suppresses PTEN activity, thereby promoting cell growth and promoting cellular defense against ROS through PI3K/Akt signaling ¹⁰². Reduced anti-oxidative stress mechanisms have been reported in PD patients with mutant DJ-1 protein ¹⁰³. It is also described that Daxx, the death-associated protein, translocated to the cytosol selectively in SNpc neurons due to MPTP mediated downregulation of DJ-1 after treatment with the neurotoxin in mouse models ¹⁰⁴. DJ-1 is also hypothesized to regulate the expression of UCP4 by oxidation and partially via NF-κB pathway in its protective response to oxidative stress ¹⁰⁵. DJ-1, particularly in its oxidized form is documented as a biomarker for many diseases including PD. DJ-1 may also work by increasing microRNA-221expression through the MAPK/ERK pathway, subsequently leading to repression of apoptotic molecules ¹⁰⁶. Additionally cell permeable Tat-DJ-1 protein exerts neuroprotective effects in cell lines and mouse models of PD ¹⁰⁷. Data from the field indicate that DJ-1 may become activated in the presence of ROS or oxidative stress, but also as part of physiological receptor-mediated signal transduction and acts as a transcriptional regulator of antioxidative gene batteries ¹⁰⁸. ATP13A2, on the contrary is thought to protect against hypoxia-induced oxidative stress ¹⁰⁹. A recent study revealed a conserved neuroprotective mechanism that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export ¹¹⁰. PLA2G6 protein is also indicated in oxidative stress-related pathways ^19,52

4. Induced pluripotent stem cells (iPSC) in Parkinson’s disease research

Yamanaka’s discovery in 2007 where key transcriptional factors (Oct4, Sox2, Klf4 and c-Myc) were used to reprogram adult cells to a de-differentiated, poised cell type called Induced pluripotent stem cells (iPSCs) revolutionized the field of human disease modeling ¹¹¹. Reprogrammed iPSCs are similar to embryonic stem cells or ESCs, are pluripotent and can differentiate to multiple lineages. iPSCs when derived from a PD patient has the patient’s complete genetic background and provides a valuable platform to study the impact of genetic mutations. Within a year of the discovery of iPSCs, PD patient-derived iPSCs ¹¹² and DA neuron differentiation from iPSCs were reported ¹¹³. iPSC models have successively been established from various sporadic and familial PD patients. To date, iPSCs are the most robust cellular system to understand PD and generate disease-relevant cell types for PD ¹¹⁴

iPSC studies typically involve few participants and random selection of cases and controls which results in heterogeneous models in vitro. Consequently, a large sample size is required to increase statistical power and sample sizes of 10-30 individuals per iPSC study may be required to achieve a statistical power of 80% ^115,116. These are, in turn, labor-intensive and expensive; hence it is unlikely that these requirements are met. A smaller number may be used if clinically and genotypically homogeneous subjects are used to reduce the variance in the cellular phenotypes. Hence a preponderance of familial PD is seen in these studies. A recent report elegantly summarizes a meta-analysis of 385 iPSC-derived neuronal lines modeling mutations/deletions/triplications in LRRK2, PRKN, PINK1, GBA and SNCA ¹¹⁶. The authors discuss the importance of using the right controls in such studies. When healthy subjects are used as controls, differences in genetic background may give rise to variance in neuronal phenotypes studies that are not caused by disease mutations. Gene editing techniques (TALEN, ZFN, CRISPR/Cas9) aids in the generation of isogenic lines that differ in only one single mutant gene and this circumvents the issue of variance due to genetic background. CRISPR/Cas9 system, an RNA-based endonuclease, is the most common and effective tool used in the iPSC model for introducing the genetic changes seen in PD, including but not limited to knockout, knockin and gene correction ^117–120. Disease-causing mutations, therefore can be inserted in healthy ESC or iPSC lines or gene-correction of a single mutation can be performed in PD-lines to include comparative isogenic control lines ¹¹⁶.

Differentiation protocols for DA neurons mimic embryologic development in utero. Unlike cortical neurons, midbrain DA neurons are derived from the ventral floorplate of the neural tube ¹²¹. The molecular mechanisms that regulate the development of midbrain DA neurons in vivo, and how taking cues from this, one can generate in vitro human midbrain DA neurons from iPSCs is systematically reviewed previously ¹²². Dual-SMAD inhibition along with modulation of sonic hedgehog (SHH) and WNT signaling by CHIR99021 (GSK3β inhibitor), and addition of FGF8 is routinely used to generate midbrain floor-plate precursors ^123–125. BDNF, GDNF, TGFβ3, dbcAMP, ascorbic acid, DAPT and ActivinA are used to enhance the purity and maturity of DA neurons which express the key marker TH (tyrosine hydroxylase) ^126–128. A schematic and generalized diagram outlining the midbrain DA differentiation protocol from iPSCs is shown in Figure 2 (the starting population can also be ESCs). It is important to note that, irrespective of the protocols used a heterogeneous cell population is attained, with neurons, glia and NSCs. To attain a high percentage of TH⁺ DA neuron population, several strategies have been employed. Sorting of DA progenitors which are CD184^high/CD44^{- 129} or CORIN^{+ 130} is shown to increase the TH⁺ DA neuron yield. CRISPR/ Cas9-based knockin of a fluorescent reporter to visually identify and purify TH⁺ DA neurons has also been attempted ¹³¹. A monolayer-based neural differentiation protocol was described recently that reproducibly generates ~70-80% midbrain DA neurons ^132,133. A higher concentration of 300 ng/ml SHH (100-200ng/ml is used normally) in combination with a lower concentration of 0.6μM CHIR99021 (0.8 μM-3 μM is used normally) and passaging and replating in the early differentiation and patterning stages maximized the yield of midbrain DA neurons as early as day 30. This monolayer platform is amenable to imaging and functional assessments of autophagy/ mitophagy ¹³². In an interesting study, autophagic dysfunction and premature aging was shown by PD-patient derived NSCs ¹³⁴. One of these patients had early onset-PD with a novel mutation in PLA2G6 gene. The authors hypothesize that developmental defects, and the subsequent depletion of NSC pool size could lead to lower DA neuron number and this impacts the onset and severity of the disease progression ¹³⁴. Hence not only iPSC-derived DA neurons, but also the developmentally upstream NSCs could be a disease-relevant phenotype for prediction analyses and design of intervention therapies.

Figure 2. A generalized schematic protocol for differentiation of human induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (DA) neurons.

Human iPSCs are treated with small molecules, such as GSK3β inhibitors (GSK3i) and SMAD inhibitors along with Shh/ FGF8b to induce midbrain floor plate formation and subsequent midbrain DA specification. This is done either by means of direct differentiation from iPSCs or through embryoid bodies (EBs). The DA progenitors can be sorted with a midbrain cell surface marker like CORIN to achieve higher purity of DA neurons via elimination of unwanted contaminant cells. Mature midbrain DA neurons are generated from these progenitors by addition of mentioned factors at the end of 40-70 DIV (days in vitro) in total. * indicates factors that may be used in the final differentiation step, but not compulsory. ActA, activin A; AA, ascorbic acid; BDNF, brain derived neurotrophic factor; DAPT, γ-secretase inhibitor; dbcAMP, dibutyryl cyclic adenosine monophosphate; FGF8, fibroblast growth factor 8; GDNF, glial cell-derived neurotrophic factor; Shh, sonic hedgehog; TGFβ3, transforming growth factor beta-3.

4.1. iPSC-derived two dimensional (2D) and three dimensional (3D) culture models of ARPD

Mutations in the PARK2 gene, encoding the protein parkin, have been identified as the most common cause of ARPD. Unsurprisingly the limited in vitro iPSC-derived ARPD models primarily examine the cellular pathologies of this gene. Human iPSC-derived neurons with PARK2 knockout is known to demonstrate severe mitochondrial dysfunction even in the absence of external stressors. PARK2 patient iPSC-derived neurons showed increased oxidative stress, higher α-synuclein accumulation and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway ¹³⁵. Interestingly iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis in their study. In a similar study, the loss of parkin significantly increased the spontaneous DA release independent of extracellular calcium and showed decreased dopamine uptake by reducing the total amount of correctly folded DAT along with DA-dependent oxidative stress. All these phenotypes could be rescued by overexpression of parkin, but not its PD-linked T240R mutant or GFP ¹³⁶. Mitochondrial dysfunction, elevated α-synuclein, synaptic dysfunction, DA accumulation, and increased oxidative stress and ROS has been reported in PARK2 and PINK1 patient-derived neurons in a floor-plate-based but not a neural-rosette-based directed differentiation strategy ¹³⁷. Impairment of mitophagy via formation of S-nitrosylated PINK1 (SNO-PINKI) has also been shown in iPSC-derived parkin mutant neurons ¹³⁸. In a recent study, PARK2 knockout neurons from isogenic lines exhibited lysosomal impairments and autophagic perturbations, suggesting an impairment of the autophagy-lysosomal pathway in parkin-deficient cells ⁹³. The same group had earlier shown disturbances in oxidative stress defense, mitochondrial respiration and morphology, cell cycle control, and cell viability of parkin deficient neurons ⁹⁷.

Midbrain-specific 3D cultures are at present a powerful tool for modeling PD in vitro. The use of microwells by Tieng et al was the very first attempt in this direction to generate embryoid bodies, which were then placed on an orbital shaker before being seeded and grown at air-liquid interface ¹³⁹. DA progenitor cells expressed FOXA2 and LMX1A as well as TH within a short span of 3 weeks. Subsequently a number of reports have been published for midbrain organoids with neuromelanin expression seen in long-term cultures ^{127,140–143}. However PD modeling with midbrain organoids is largely focused on dominant mutations like LRRK2 ^128,140, SNCA ¹⁴⁴and also an only report on sporadic PD ¹⁴⁵. A very recent study used CRISPR-Cas9 genome editing to develop isogenic loss-of-function 3D models of early-onset autosomal recessive PD (PARKIN^-/-, DJ-1, and ATP13A2) to identify common pathways ¹⁴⁶. The DA neuronal population was markedly reduced in RRKR organoids but no significant differences were observed in the other two cell lines. The death of newly differentiated TH⁺ neurons and higher expression of VTA marker CALB1 in the PRKN organoids were indicative of A9-like neurons being more severely affected than others. A dysregulation of the autophagy-lysosomal pathway and upregulated ROS in all cell lines and an upregulation of pathways associated with oxidative phosphorylation, mitochondrial dysfunction, and Sirtuin signaling, as well as a significant depletion of mitochondrial proteins was seen in the PRKN-/- DA neurons ¹⁴⁶. Astrocytic pathologies in human PRKN-mutated iPSC-derived midbrain organoids were revealed for the first time, suggesting a non-autonomous cell death mechanism for dopaminergic neurons in brains of PRKN-mutated patients ¹⁴⁷. Mutations in PINK1 have also been reported to generate reduced TH⁺ counts in midbrain organoids ¹⁴⁸. Human midbrain organoid/spheroid cultures are a scalable and reproducible system to obtain DA neurons expressing markers of terminal differentiation along with neuromelanin production in a 3D environment that replicates the neuronal and glial cytoarchitecture of the human midbrain ¹⁴⁹. They can hence provide a crucial platform to explore the molecular basis of ARPD, and also to delineate the associated cellular pathologies.

Cell replacement therapy with iPSC-based DA derivatives- The various challenges pertaining to the safety and efficacy of stem cell-based cell transplantations in PD has been elegantly reviewed and described ¹⁵⁰. The right neural cell type for transplantation is of utmost importance. FGF8b inclusion in the differentiation protocols helps in acquisition of a caudal midbrain fate and promotes high dopaminergic graft volume, density and yield as evidenced by deep sequencing of more than 30 human ESC-derived midbrain tissues ¹⁵¹. Dopaminergic precursors beyond the floorplate progenitor stage but before formation of TH⁺ dopaminergic neurons are found to be most efficient for graft survival, integration and function in animal models ^130,152 Grafting of these precursors into the putamen area, where SNpc dopaminergic neurons innervate, is an approach most likely to succeed ¹⁵⁰. The number of cells to be transplanted is still debated. Takahashi’s group reported a minimum of 16,000 TH⁺ cells in a primate model to see improvements in PD score and motor function ¹³⁰. The generation and implantation of iPSC-derived autologous dopaminergic progenitor cells in a patient with idiopathic PD is reported with clinical and imaging results suggesting possible benefit over a period 24 months ¹⁵³. A global consortium, GForce-PD (http://www.gforce-pd.com), was set up in 2014, with major academic networks in Europe, the US, and Japan working on developing stem cell-derived neural cell therapies for PD ¹⁵⁴. The clinical trials using human ESCs are ongoing in Australia (NCT02452723) and China (NCT03119636), with their pre-clinical data published ^155,156. A clinical trial (JMA-IIA00384, UMIN000033564) in Japan to treat PD patients by using iPSC-derived DA progenitors (DAPs) was started in 2018 by Takahashi and colleagues ^157,158. The results of these trials are eagerly anticipated.

5. Limitations to iPSC-based disease modeling of PD

Although iPSCs and their derivatives are currently in the forefront as PD models, there are many challenges which remain unaddressed. The most significant drawback of in vitro models is the absence of LB formation in PD iPSC-derived DA neurons. Increased levels of phosphorylated pS129 α-synuclein however has been observed ¹⁵⁹. Additionally, the efficiency of generating DA neurons varies significantly between different methods and approximately 20–30% of the final cells are identified as DA neurons even with the most robust method such as the floor-plate induction protocol ¹⁶⁰. Sorting of DA progenitors with markers such as CORIN seems to aid in a better yield of mature and functional DA neurons ^158,161. Knocking in a reporter gene in the endogenous TH locus has been attempted to quantify the final yield of DA neurons ¹⁶⁰ to understand the efficiency of different published protocols. However, no significant progress has been made to analyze if the DA neurons derived in vitro are similar to the SNpc neurons impacted in a PD patient. A TH⁺ DA neuron is necessarily not a representation of the A9 or SNpc nuclei of the brain, though GIRK2/TH positivity and low Calbindin is considered as an A9 signature ^162–164 A reliable strategy would be multiplexing markers for reliable subtype identification¹⁶⁵. Another difficulty in modelling PD with iPSCs is the induction of ‘aging’ in a culture dish. Pharmacological inhibition of telomerase by the inhibitor BIBR1532 demonstrates moderate disease-relevant phenotypes in PINK1 and PARKIN DA neurons ¹⁶⁶. Progerin (the protein associated with premature aging) overexpression as a strategy to induce aging is also reported ¹⁶⁷ but interpretation is complex as disease-relevant phenotypes and progerin-phenotypes are indistinguishable ¹⁶⁸. Moreover, contrary to what is seen in PD pathology, an exogenous stressor is always necessary to observe disease phenotypes in an iPSC-DA system. In sa PD patient derived-iPSC model, DA neurons exhibit apoptosis only after exposure to stressors including hydrogen peroxidase, MG132 and 6-OHDA ¹⁶⁹ Lastly, 2D culture systems that are normally used to differentiate DA neurons do not mimic the complex in vivo environment. 3D organoids fill this gap by representing a more physiologically relevant model system. However, the tremendous progress seen in the field is largely limited to cortical or cerebral organoids. A few midbrain spheroid or organoid culture systems are nonetheless reported ^{128,140–142}. Results from these studies indicate that 3D midbrain cultures are definitely an improvement over 2D cultures to model PD. A recent study describes the robust generation of midbrain organoids with homogeneous distribution of midbrain DA neurons along with other neuronal subtypes as well as functional glial cells, including astrocytes and oligodendrocytes ¹⁷⁰. Nevertheless, an overall low efficiency of generation and heterogeneity within the midbrain organoids along with its ethical considerations raises contentious questions towards a bench-to-bedside approach.

6. Conclusions

Human pluripotent stem cells, iPSCs in particular, are an invaluable tool to help us better understand PD pathology by generating functional, DA neurons with A9-like identity and also reproducing the midbrain cell composition. The improvement in differentiation protocols and 3D culturing techniques combined with genome editing technologies aids in better PD modeling studies. Additionally, these cultures exhibit key features of PD, such as α-syn accumulation, autophagic defects, oxidative stress and impairment of mitochondrial function. However, it may be advantageous to include other cell types like microglia in PD studies rather than focusing on midbrain-specific organoids to understand the disease pathology in a relevant way. The bloodbrain barrier (BBB) and its dysfunction in PD should also be emphasized. Further, the future direction in investigating PD should make use of the organ-on-chip or organoids-on-chip model with a multi-organ configuration to study different cell types and involvement of various organs in PD progression and pathology. Lastly, ARPD genes other than PRKN and PINK1, though rare may provide insights into the common molecular pathways of the monogenic disease forms and should be included in such detailed studies.

Abbreviations

ARPD

autosomal recessive Parkinson’s disease

DA

dopaminergic

ER

endoplasmic reticulum

ESC

embryonic stem cell iPSC - induced pluripotent stem cell LB - Lewy bodies

NSC

neural stem cell

PD

Parkinson’s disease

PM

plasma membrane

ROS

reactive oxygen species

SNpc

substantia nigra pars compacta

SOCE

store operated calcium entry

TH

tyrosine hydroxylase