Table 1.
An overview of phenotypes observed in cells cultured from patients carrying LRRK2 mutations. Phenotypes of patient-derived cells from individuals carrying LRRK2 mutations as described in published studies are summarized here. Each line of the table corresponds to a single mutation in a single cell type from a single study and provides the test characteristics and main phenotypes observed.
| Cell Types | Nature of Patient-Derived Cells | Patient Cell Phenotypes | Reference | |
|---|---|---|---|---|
| N1437S | ||||
| 1. | Fibroblast cells | Skin biopsies obtained from four healthy, two N1437S PD patients, three G2019S patients and one R1441C patient. | No change was observed in cell adhesion between patient fibroblast and control cells in both basal and kinase inhibited state. | [57] |
| R1441C | ||||
| 2. | DA neurons | Fibroblast cells (from the Northwestern University Biorepository and NINDS human cell biorepository) were reprogrammed to iPSCs and differentiated to DA neurons. | The LRRK2 R1441C mutant depicted a reduction in lysosome specific GCase activity. The knockdown of Rab10 (bona fide substrate of LRRK2) resulted in reduced GCase activity. The R1441C mutant results in an increased level of phospho-Rab10 and over-expression of Rab10 increases the GCase activity. | [59] |
| 3. | Neural cells derived from iPSCs | The iPSC lines are derived from 2 (Twins—brother and sister) heterozygous LRRK2 R1441C (gene from father with PD) carriers. No clinical manifestation of PD in sister, however resting tremor observed in brother. | Lactate dehydrogenase (LDH) enzyme release was used in order to measure neuronal cell vulnerability, MTS assay— LDH/MTS assays, immunocytochemistry and cell counts depicted an increased vulnerability of iPSC derived neural cells from R1441C variant carriers on exposure to chemical stressors (concanamycin A, valinomycin), reduced oxygen consumption and dysfunctional mitochondrial mobility as compared to healthy controls. |
[60] |
| 4. | The iPSCs from at risk PD related R1441C variant carriers were obtained from the Coriell BioBank. | Significant mtDNA damage observed in iPSC cell lines differentiated into neural cells using two separate protocols. | [58] | |
| 5. | Fibroblast cells | Skin biopsies obtained from four healthy subjects as well as, two N1437S, three G2019S and one R1441C PD patients. | No change was observed in cell adhesion between patient fibroblast and control cells in both basal and kinase inhibited state. | [57] |
| R1441G | ||||
| 6. | DA neurons | Fibroblast cells (from the Northwestern University Biorepository and NINDS human cell biorepository) were reprogrammed to iPSCs and differentiated to DA neurons. | A reduction in lysosome specific GCase activity was reported. | [59] |
| 7. | Fibroblast from PD patients with R1441G mutation in LRRK2 (n = 2) and matched healthy subjects. Controls include Human embryonic stem cell line (H9), so as to study effects of the lentiviral reprogramming on the cells. | This study was the first to report an iPSC line with the R1441G mutation. α-synuclein levels remained unchanged in the R1441G differentiated mature DA neurons as compared to controls. |
[62] | |
| G2019S | ||||
| 8. | Neuro-progenitor cells and mature neural cells derived from iPSCs | Patient-derived fibroblasts are reprogrammed into iPSCs, three homozygous/heterozygous LRRK2 G2019S patient, three age matched healthy subjects w/o LRRK2 mutations | Significant mtDNA (mitochondrial DNA) damage observed in iPSC derived NPC and neural cells with G2019S mutation and not in the fibroblast cells or undifferentiated iPSCs. Indicating that mtDNA damage might be a neural specific phenotype. | [58] |
| 9. | Neural cells derived from iPSCs | The iPSC lines from 2 heterozygous R1441C variant carrier, 1 homozygous PD patient and healthy controls were used in this study | Lactate dehydrogenase (LDH) enzyme release was used in order to measure neuronal cell vulnerability, The G2019S mutant cells showed increased vulnerability to valinomycin, concanamycin and MPP+ (selected stressors directed to assess mitochondrial function or protein degradation) in neural cells in comparison to control fibroblast cells. |
[60] |
| 10. | Neurons differentiated from iPSCs | Fibroblast from two patients harboring the LRRK2 G2019S mutations reprogrammed into iPSCs, further, differentiated into neurons. Nonisogenic healthy controls iPSCs were generated from healthy women. | The authors reported a prominent dysregulation by means of alterations in gene expression of CPNE8 (role in calcium mediated intracellular processes), MAP7 (involved in microtubule dynamics), UHRF2 (involved in cell-cycle regulation), ANXA1 (vital role in phospholipid binding), CADPS2 (involved in exocytosis of vesicles with neurotransmitters/neuropeptides), in differentiated neurons out of which 4 genes are known contributors of DA neurodegeneration. The mutation also results in an increase in extracellular signal regulated kinase 1/2 (ERK) phosphorylation | [97] |
| 11. | PD patient LRRK2 heterozygous G2019S iPSCs derived neurons and tested against control. | The study concludes that the LRRK2 plays a pivotal role in the Endoplasmic reticulum (ER) Ca2+ homeostasis, the upregulated kinase activity of the G2019S mutant. Alteration of calcium homeostasis along with ER stressors potentially result in the neurite collapse. | [142] | |
| 12. | Midbrain DA neurons derived from iPSCs | Fibroblast cells (from the Northwestern University Biorepository and NINDS human cell biorepository) were reprogrammed to iPSCs and differentiated to DA neurons. | Significant a reduction in the GCase activity of LRRK2 G2019S iPSC neuronal cells in comparison to the healthy controls, which was reversed on correction of the mutation using CRISPR-Cas9. | [59] |
| 13. | Fibroblast from 2 PD patients with G2019S mutation in LRRK2 and matched healthy subjects. Controls include Human embryonic stem cell like cells (H9) | α-synuclein levels were two-fold higher in the G2019S differentiated mature DA neurons than controls. Marked increase in autophagic mediator p62 (responsible for clearance of α-synuclein). The G2019S mutant results in an impaired NF-κB signaling and the gene transcription of NF-κB was altered in LRRK2 knockdown experiments. | [62] | |
| 14. | G2019S iPSCs generated from dermal fibroblast cells. Further differentiated into midbrain DA neurons. | No difference was observed between differentiated and undifferentiated iPSC cell from the G2019S and WT iPSCs and hESCs in terms of pluripotency markers, general morphology, differentiation potential, epigenetics and gene expression profiles. G2019S affected neurons displayed higher than normal expression of stress response genes altered α-synuclein proteins accumulations and increased vulnerability of neurons to neurotoxins (compared to unaffected controls). | [99] | |
| 15. | iPSCs- derived DA, glutaminergic and sensory neurons | Human iPSCs were obtained from two homozygous LRRK2 G2019S patient cells, one heterozygous G2019S carrier and three healthy controls from publicly available samples from NINDS Coriell Institute. | The study indicates that the G2019S results in an impaired mitochondrial respiration pattern in the iPSC derived DA and glutaminergic neurons, however, sensory neurons showed no such trends. The G2019S iPSC derived DA neurons displayed alternate mitochondrial distribution and trafficking patterns as well as cell specific bio-energetic modifications caused by reduced NAD+ and sirturin deacetylatse which is quite indicative of a disease specific phenotype. | [100] |
| 16. | Neuro-epithelial cells (NESCs) | The study looked at thirteen human iPSC-derived NESC lines obtained from three patients carrying the LRRK2-G2019S mutation with four healthy individuals (age/gender matched). Four isogenic NESC lines were generated, two of which had a mutation introduced and the other two in which the mutation was corrected. | Patient NESC have an increased number of fragmented mitochondria, reduced membrane potential and reduced mitophagic clearance via lysosomes in comparison to isogenic control lines. | [102] |
| 17. | Astrocytes | A Co-culture of iPSC derived astrocytes and ventral midbrain DA neurons (vmDAns) from familial G2019S PD patients | Chemical enhancement of Chaperone Mediated autophagy (CMA) protected PD astrocytes and vmDAns via clearance of α-synuclein accumulations. Non-cell autonomous contribution of astrocytes during PD pathogenesis. Possibility of exploring a therapeutic strategy of blocking pathogenic cross talk between neurons and glial cells. Dysfunctional CMA (chaperone mediated autophagy), impaired macroautophagy, progressive α-synuclein accumulation. | [107] |
| 18. | Fibroblast cells | 3 skin fibroblast groups - 1) Control (Patients who did not develop PD) 2) Idiopathic PD—IPD (Patients without G2019S PD) 3) G2019S PD patients The overall goal of the study is to examine variation in the levels of histone acetyltransferase (HAT) and histone deacetylase (HDAC), described as the key players of autophagy. |
The fibroblasts from G2019S cells display an increased clearance of defective mitochondria where as those of IPD show a reduction of mitophagy and increased ROS. The acetylation proteins between the two groups vary, however, HDAC activity is reduced in IPD cells (HAT activity unchanged), the imbalance of autophagy enzymes leads to cell death. The inhibition of the HATs in IPD cell lines could be considered as cyto-protective in this case. | [103] |
| 19. | Skin biopsies collected from 5 individuals of confirmed G2019S PD diagnosis, results compared to 5 healthy age matched controls. | 1. Mitochondrial membrane potential (decrease by 45%) and ATP levels are reduced in LRRK2 mutant fibroblast cells 2. Increased elongation and interconnectivity of mitochondria in LRRK2 mutant patient cells. |
[105] | |
| 20. | Skin biopsies donated by four G2019S PD patients and four healthy controls. | Lysosomes were enlarged/swollen, through microscopy they could be characterized by large translucent areas and clustered around the nucleus in G2019S patient-derived fibroblasts. These defects were reversed by inhibition of kinase activity using LRRK2In1, silencing of TPC2 (Two pore channels—acidic vesicles that comprise the endolysosomal system) and pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns (3,5)P2] crucial to autophagy. | [109] | |
| 21. | Fibroblasts cultured from punch skin biopsies obtained from nine presymptomatic PD G2019S mutation carriers and age matched healthy controls | A reduction in the secretion of Progranulin (PGRN) was observed in supernatants of cultured human fibroblasts isolated from presymptomatic LRRK2 (G2019S) mutation carriers, and mitochondrial function remained unaffected. | [110] | |
| 22. | Fibroblast cells were obtained via skin biopsies from LRRK2 G2019S mutation carriers both with and without PD as well as healthy controls. | Cells from the G2019S mutant carriers w/o symptoms show an enhanced mitochondrial performance as well as upregulated autophagic flux in comparison to healthy controls and G2019S patients. Therefore, the hyperactivity of mitochondrial complex as well as autophagy could be the driving force for carriers to develop symptoms. | [106] | |
| 23. | Two separate (1 male, 1 female) LRRK2 G2019S PD patient fibroblast cell lines were obtained from the Coriell biorepository | Amplified mitophagy is observed in patient fibroblasts as compared to healthy controls, this is further supported by the evidence indicating a significant loss of mitochondrial membrane potential, reduction in mitochondrial mass and reduced citrate synthase activity accompanied by increased autophagic flux. These results indicate that G2019S mutant accelerates autophagy in cells. | [104] | |
| 24. | Skin biopsies were obtained from four healthy, two N1437S PD patients, three G2019S patients and one R1441C patient. | The G2019S mutant did not result in any change in cell adhesion patterns in fibroblast cells as compared to healthy control fibroblasts, neither in basal conditions nor in conditions of inhibition of LRRK2 kinase activity. | [57] | |
| 25. | Skin biopsies from three PD G2019S patients and four healthy individuals were obtained | An increase in kinase activity and autophagic flux in the mutant fibroblast cells was observed. However, MEK/ERK (MAPK signaling pathway) inhibition reduced the autophagic flux and sensitivity of the G2019S mutants in comparison to healthy controls. | [143] | |
| 26. | Skin biopsies obtained from G2019S mutation carriers (not directly related to each other) without clinical symptoms of PD. Control fibroblasts obtained from age/gender matched controls (n = 5) | Increased sensitivity of LRRK2 mutant fibroblasts to LatA (drug known to disrupt microfilament organization, cell shape [144]). They also observe a significant increase in F-Actin bundles with a decrease in filopodial length. This depicts a pattern of LRRK2 mutant dependent alteration of F-Actin dynamics. | [111] | |
| 27. | Lymphoblastoid cells (LCLs) | LCLs samples from six patients with heterozygous G2019S mutation, thirteen sporadic PD patients and thirteen unrelated gender/age matched controls. For the study additionally three gender/age matched LCL controls, six gender/age matched LCLs from heterozygous G2019S LRRK2 patients were obtained from the NINDS Coriell Cell Repository. | The authors concluded that the G2019S LCLs and a subset of LCLs derived from sporadic PD patients displayed a centrosomal cohesion deficit, reverted by LRRK2 kinase inhibition. | [114] |
| 28. | Endogenous LRRK2 activity was studied in EBV-transformed LCLs derived from a PD patient with homozygous G2019S mutation and one healthy control (no LRRK2 mutations) | LRRK2 levels were similar in both cell lines, however, a three-fold increase in kinase activity was observed in the G2019S mutant. The S910, S935 sites in both the mutant and healthy LCLs were equally phosphorylated. However, on treatment with kinase inhibitors (H-1152, Sunitib), the phosphorylation of S910, S935 was more potently inhibited in the G2019S mutant than the healthy control. | [112] | |
| 29. | Six iPSC line derived LCLs and healthy age matched controls were used for the study (Parkinson institute or NINDS Coriell biorepository). Further, skin biopsies from three G2019S PD patient and three healthy individuals were obtained to study fibroblast cells. | An increase in the mtDNA damage was observed in LCLs of G2019S mutation carriers (with PD) as compared to LCLs from age matched healthy controls. However, no change was observed in mtDNA damage in fibroblast cells obtained from G2019S mutation carriers in comparison to healthy subjects. | [113] | |
| G2019S Organoids | ||||
| 30. | Midbrain floor plate neural progenitor cells (mfNPCs) | mfNPC are differentiated into 2D midbrain DA neurons (mDANs) and 3D human midbrain-specific organoids (hMOs) from PD patients carrying the LRRK2-G2019S mutation. | The expression of DA neuron markers TH, AADC, VMAT2, and DAT are markedly decreased in LRRK2-G2019S organoids as compared to the controls at day 60. Mature neuronal genes such as NURR1, PITX3, EN1, TH, and MAPT are also reduced in the LRRK2-G2019S mutant organoids but not in 2D cultures (where their expression is very low regardless of genotype). The DA neurons in LRRK2-G2019S organoids exhibited a decrease in neurite length. | [115] |
| I2020T | ||||
| 31. | Neurons | I2020T PD patient fibroblast derived iPSCs were reprogrammed to neurons (Japanese Sagamihara kindred) | The I2020T neurons displayed reduced levels of phospho-AKT in comparison to control neurons, with an observable increase in apoptosis. An increase in glycogen synthase kinase-3β (GSK-3β)—a central intracellular regulator of vital functions—proliferation, migration, glucose metabolism) which is implicated in a number of diseases and high tau phosphorylation. Post-mortem histopathological studies reveal deposits of neurofibrillary tangles, increased tau phosphorylation of neurons. | [145] |
| G2294R | ||||
| 32. | Peripheral blood mononuclear cells (PBMC) | Monocytes were isolated from PBMCs of G2294R PD patient and gender matched controls. Further monocytes were differentiated to macrophages | LRRK2 protein levels are increased in LRRK2 G2294R monocytes and reduced in macrophages. Further Rab8 and Rab10 levels were also decreased in macrophages. | [133] |
| G2385R | ||||
| 33. | PBMC | PBMCs from G2385R patients were reprogrammed into iPSC cells and used to generate stable cell lines. | No characterization was reported. | [146] |
Legend—An overview of phenotypes observed in cells cultured from patients carrying LRRK2 mutations. The studies have been grouped in order of the occurrence of the variant in the LRRK2 protein and the cell type. About 31 studies have been looked at to summarize patient-derived cell phenotypes. iPSCs—induced pluripotent stem cells, DA—Dopaminergic neurons, PD—Parkinson’s disease, GCase—glucocerebrosidase, LDH—Lactate dehydrogenase, MPP+, WT—Wild Type, w/o—Without, mtDNA—mitochondrial DNA, NPC—Neural Progenitor Cells, ERK—Extracellular regulated kinase 1/2, ATP—Adenosine Triphosphate, BAC mice—Bacterial Artificial Chromosome generated transgenic mice, CMA—Chaperone mediated autophagy, EOPD—Early onset Parkinson’s disease, ER—Endoplasmic Reticulum, GCase—Glucocerebrosidase, GSK3β—Glycogen synthase kinase 3 β, H9 cells—Human embryonic stem cell line, HAT—Histone acetylase, HDAC—Histone deactetylase, hESCs—Human Embryonic Stem Cells, LCLs—Lymphoblastoid cells, LOPD—Late onset Parkinson’s disease, LRRK2—Leucine rich repeat kinase 2, MPP+—1-methyl-4-phenylpyridinium (neurotoxin), NAD+—Nicotinamide adenine dinucleotide, NESCs—Neuro-epithelial cells, NF-κB—nuclear factor kappa-light-chain-enhancer of activated B cells, PBMCs—Peripheral Blood Mononuclear Cells.