Conceived and designed the experiments: BB BC HNN RRP TDP. Performed the experiments: BB BC HNN PCL. Analyzed the data: BB BC HNN. Contributed reagents/materials/analysis tools: BS KD JWL. Wrote the paper: BB TDP RRP. Performed electrophysiological experiments: LF.
The authors have declared that no competing interests exist.
Parkinson's disease (PD) is an incurable age-related neurodegenerative disorder affecting both the central and peripheral nervous systems. Although common, the etiology of PD remains poorly understood. Genetic studies infer that the disease results from a complex interaction between genetics and environment and there is growing evidence that PD may represent a constellation of diseases with overlapping yet distinct underlying mechanisms. Novel clinical approaches will require a better understanding of the mechanisms at work within an individual as well as methods to identify the specific array of mechanisms that have contributed to the disease. Induced pluripotent stem cell (iPSC) strategies provide an opportunity to directly study the affected neuronal subtypes in a given patient. Here we report the generation of iPSC-derived midbrain dopaminergic neurons from a patient with a triplication in the α-synuclein gene (
Parkinson's Disease (PD) is a debilitating neurodegenerative disorder characterized by the loss of neurons in both the peripheral and central nervous system
Genetic studies have implicated several potential mechanisms but the etiology of Parkinson's disease remains obscure. In a large cohort of patients, Lewy bodies and Lewy neurites form in CNS and autonomic peripheral nervous system (PNS) neurons. These large intracellular proteinaceous inclusions are rich in α-synuclein and ubiquitin, an observation that suggests a role for α-synuclein and the proteasome in the molecular development of sporadic and genetic PD
Familial autosomal dominant forms of PD have been documented in families with
The subject of this study was a 48-year-old male with an
At the time of the clinical examination in summer 2008, the patient noted mild problems with recent memory. He did not experience any psychiatric symptoms such as delusions or hallucinations but reported some anxiety and slight depression. He also reported urinary urgency and occasional constipation. He indicated that he had normal sleep patterns but in speaking with his wife he did exhibit nocturnal features suggesting REM behavioral disorder. He had undergone a sleep study and apparently obstructive sleep apnea was suspected, but did not tolerate continuous positive airway pressure. The patient stated that his sense of smell was poor for at least one year and the B-SIT smell test showed abnormal sense of smell with 6/12 points. From time to time he noted periodic blurring of vision and diplopia and had obtained new corrective lenses. He was also experiencing motor fluctuations on his current medication schedule. He stated that in the “on” state he is nearly normal, but continued to experience difficulty with his handwriting. When he is in the “off” state or when the medication wears out, his speech slowed, he drooled, his handwriting worsened, and he had more difficulty with dexterity such as dressing or hygienic activities. He had not noticed any difficulty with his balance, but reported that when his medication wore off he experienced hesitation on turning. Tremor also worsened when the medication wore off. In addition, the patient noted some upper back pain. He estimated about 80% of the day was in the “on” state and had not experienced any dyskinesia. There was a mild to moderate rest tremor of his left hand and a slight tremor of his right hand in addition to an action tremor of mild degree left greater than right. He was able to rise from the chair and walk with no difficulty. His stride was normal but there was axial rigidity and loss of arm swing bilaterally. A slight hesitation on turning when asked to multitask while he walked. There was no sign of ataxia on finger nose or heel shin testing and he did not exhibit dystonia or dyskinesia. Symptoms suggested typical progression of Parkinson's disease.
A skin biopsy was taken and iPSCs were generated from primary human dermal fibroblasts (Trpl-HDF) and from HDFs isolated from his unaffected sister (Ctrl-HDF) with a normal
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Other characteristics of the iPSCs also matched those of hESCs. The
To generate neurons, independently derived iPSC clones from the
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Immunofluorescent (IF) staining was used to evaluate differentiated cultures for the presence of nestin, a type VI intermediate filament protein indicative of neural progenitor cells; doublecortin (DCX), a microtubule-associated protein found in immature neurons; and tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, indicative of dopaminergic neurons. After 50 days of differentiation, the cultures contained nestin-positive neural progenitors, DCX-positive immature neurons and TH-positive mature DA neurons (
Neurons were often localized in islands of differentiating nestin-positive progenitor cells. These dense aggregates produced extensive axonal outgrowth (
Scoring of the relative abundance of TH-positive neurons indicated that there were no significant differences between iPSC or hESC lines (
To further characterize the progression of events that accompanies differentiation, gene expression profiles were evaluated at different stages during the differentiation process using microfluidic, high-density quantitative PCR (qPCR). Samples of 500–1000 cells were manually collected from replicate cultures of undifferentiated pluripotent cells, cultures highly enriched for neural rosettes, and cultures enriched for mature neurons (days 0, 28 and 50, respectively). Cells were lysed and processed for expression analysis of a 96-gene panel diagnostic of pluripotent stem cells, endoderm, mesoderm, neural crest and CNS cells, including neurons and glia (
We used cluster analysis to examine gene expression and time points across the sample groups (
(
To determine whether protein aggregation was accompanied by an increase in cell stress, the transcription level of 14 oxidative stress and protein aggregation-related genes (
(
To determine whether cell death resulted from this increase in cell stress, we stained differentiated neurons for cleaved caspase-3 protein, a key regulator in cell apoptosis. DA neurons showed no significant differences in the abundance of caspase-3 positive cells under baseline conditions. However, when the antioxidant ascorbic acid was withdrawn from the culture media and cells were challenged with 200 µM hydrogen peroxide for 24-hours, significantly higher numbers of Caspase-3 positive TH neurons were observed in Trpl17 and Trpl43 compared to controls (
Parkinson's disease results from a complex interplay of genetics and environment. Not all patients exhibit identical features and future clinical approaches may benefit from diagnostic methods that predict disease mechanism and classify patients by virtue of neuron-intrinsic disease features. Here, we present data that indicates that differentiation of pluripotent stem cells may provide a cell-based model to detect cell autonomous disease processes based on protein deregulation/accumulation. The cell lines that we generated were derived from a PD patient with a naturally-occurring genomic triplication of the
Although the data presented here indicates that elevated wild-type α-synuclein is sufficient for cell-intrinsic vulnerability, the mechanism of pathogenic action is still unknown. The elevation of oxidative stress and aggregation of interacting proteins in the diseased line are consistent with free radical accumulation, perhaps as a result of protein aggregation-induced alterations in ubiquitin-proteasome function. Such perturbations in protein dynamics may influence the efficient removal or neutralization of oxidative molecules from the mitochondria's electron transport chain or tyrosine hydrolysis in dopaminergic neurons leading to increased neuron vulnerability. Although loss of midbrain dopaminergic neurons produces the prominent clinical features of PD, the degenerative process extends well beyond the midbrain dopaminergic neurons to affect autonomic function, cognition and mood. We confirm here that both TH-positive and TH-negative neurons display ubiquitinated intracellular inclusions. This is consistent with the dominant disease inheritance of the wild-type SNCA triplication with expression of SNCA in all neurons, and indicates that patient-derived iPSC can be used to study disease mechanisms and selective vulnerably across the many neuron subtypes involved in PD.
SNCA is not the only gene linked to hereditary PD. Mutations in LRRK2, UCHL1, PARKIN and others can lead to autosomal dominant inherited forms of PD, suggesting that the proteins encoded by these genes play an important role in neural cell homeostasis. In parallel work, we have shown that vulnerability and PD related phenotypes can be detected in iPSC-derived DA neurons, however, the apoptotic vulnerability was lower in the SNCA triplication lines than observed in DA neurons derived from a LRRK2-G2019S homozygous iPSC line
Protein accumulation, oxidative stress marker increases and caspase activity in SNCA triplication iPSC-derived DA neurons demonstrates that PD linked mutations can produce detectable features of disease in young neurons, and we anticipate that the derivation of iPSC from both genetic and idiopathic patients will ultimately define whether a given mutation or more complex polygenic background confers a cell-autonomous vulnerability or whether the disease process in a given class of patients requires additional environmental or epigenetic contribution to confer selective vulnerability in dopaminergic neurons. The ability to observe disease-related phenotypes in PD iPSC-derived neurons across patient groups provides a useful platform for studying patient-specific molecular mechanisms of disease, environmental impacts and potential therapeutic applications.
Potential patients with specific genetic disease and healthy volunteers were informed of the project through poster advertising and referrals through patient advocacy and disease focus organizations (Parkinson's Institute). Written informed consent was obtained form all patients involved in the study. A Stanford institutional review board approved the primary cell line derivation (approval IRB-15028) and Stanford's stem cell research oversight committee (approval SCRO-212) approved all subsequent tissue use and experimentation. Potential donors were screened for men and women between the ages of 18 and 75 that had the genetically linked Parkinson's disease. After informed consent was obtained, primary human dermal fibroblasts (HDF) from the medial arm dermis of a 42-year-old male with SNCA triplication and his 46-year-old unaffected sister were obtained by first cleaning the region was cleaned with an alcohol swab and injecting 2–3 ml of 1% lidocaine with 1∶100,000 diluted epinephrine. Then, a 4 mm dermal specimen was removed with a core punch biopsy instrument and placed in sterile PBS, while the skin defect was closed with a 4-0 nylon suture and covered with a double antibiotic ointment bandage. Sutures were removed after 2 weeks. The skin tissue biopsy was washed in Ca2+ and Mg2+ free Dulbecco PBS (Invitrogen, Carlsbad, CA) and minced into small pieces before being seeded onto gelatin coated 6-well cell culture flasks (Corning, Acton, MA) containing DMEM/F12 supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin (Invitrogen), 10% FBS (DMEM/FBS culture media) and cultured at 37°C37C in 5% CO2. A minimal amount of culture media was used to promote tissue attachment to the gelatin-coated surface (1 ml of culture media per well). The media was brought up to 4 ml per well once the skin fragments attached and the media was changed every 2 days. Once fibroblasts began to migrate out, the attached biopsy fragments and any connected epithelial cells were removed and the fibroblasts were cultured to 80–90% confluence. This primary culture was passaged by brief exposure to 0.15% trypsin-EDTA (Invitrogen, Grand Island, NY) and seeded into four gelatin-coated 175 cm cell culture flasks with fresh DMEM-F12/FBS culture media. These somatic cells were cultured until they reached 90% confluence and subsequently frozen in DMEM/FBS culture media supplemented with 10% dimethyyl sulphoxide (DMSO, Sigma, St. Louis, MO) in aliquots of one million cells per cryovial. These somatic cells were thawed as required for reprogramming studies.
Mouse embryonic fibroblasts (MEFs) were prepared by sacrificing pregnant CF-1 mice (Charles River Laboratories), transferring fetuses to fresh PBS and rinsing until blood is absent. Animal work was approved under Stanford guidelines for animal safety and received approval from Stanford's administrative panel on laboratory animal care (approval APLAC-16146) and SCRO (approval SCRO-212) boards. Fetal heads and visceral organs were mechanically removed and the remaining carcass was rinsed in PBS, finishing in a rinse containing 5 ml Trypsin. Carcass tissue was cut into small pieces using scalpels, transferred from individual fetuses to a 15 ml centrifuge tube and incubated in 5% CO2 at 37°C37C for 20 min. 10 ml feeder medium was added to neutralize the Trypsin and the solution was pipetted up and down with a 25 ml stripper pipette. The sample was centrifuged at 1000 RPM for 5 min, the supernatant was aspirated and the sample was resuspended in 10 ml fresh feeder medium – repeated until solution was devoid of blood. Cells were plated at one fetus per T175 gelatin coated flasks with 30 ml feeder medium and incubated in 5% CO2 at 37°C.37C. Cells were subsequently passaged every 2–6 days (when flasks neared 90% confluency) for 5–7 passages, irradiated with 3000 rad gamma waves and then frozen down in DMSO.
293FT cells were cultured in T175 flasks to ∼90% confluence on the day of transfection. For each 293FT T175 flask, two premixes were prepared; (1) 10 ug VSVG, 15 ug delta8.9, 10 ug retroviral vectors carrying Oct3/4, Sox2, Klf4 and c-Myc in 10 ml Opti-MEM; and (2) 120 ul Lipofectamine in 5 ml Opti-MEM. The premix solutions were incubated for 5 min at room temperature and then mixed gently by hand inversion. The resulting mix was then allowed to sit for 20 min at room temperature. The 293FT cells were treated with the resulting 15 ml mix and incubated in 5% CO2 at 37°C37C for 6 hours, after which the transfection mixture as replaced with 18 ml of 10% FBS in DMEM+Glutamax and incubated in 5% CO2 at 37°C37C for 72 hours. Harvest the supernatant in 50 ml conical tubes and spin down at 2000 rpm for 5 minutes. Filter the supernatant through Millex-HV 0.45 filter unit and store briefly for concentrating. To concentrate the virus, 30 ml fresh viral supernatant was concentrated 100× by centrifugation at 17,100 rpm for 2:20 hours at 20°C20C in a Beckman Coulter Optima L-80XP Ultracentrifuge and re-suspended in 300 ul of 10% FBS/DMEM. 100× viral stock stored at −80°C80C. Target fibroblasts were prepared at 1×105 cells per well of a 6-well plate. The four prepared viral supernatants were mixed to the appropriate concentrations with fresh MEF medium and supplemented with 8 ng/mL polyprene and cultured with the cells overnight. The next day, cells were washed once with medium and incubated overnight with MEF medium to allow recovery from the infection. The retroviral infection was repeated for a total of two rounds.
RNA was purified using QIAGEN Quick Prep-Mini kit or cell sorting directly into the pre-amplificaiton reaction mix. Samples were then reverse transcribed and pre-amplified with 5 ul CellsDirect 2× Reaction Mix, 10 ul Superscript III TR/Platinum Taq Mix (Invitrogen, CellsDirect One-Step qRT-PCR kit), 2.5 ul of 0.5× pooled primers and probes, 1.5 ul TE Buffer (QIAGEN) and 0.1 ul SUPERaseIn (Applied Biosystems). Rt-PCR was performed in 20 ul volumes with 10 ul ABI 2× Reaction Mix (Applied Biosystems), 1 ul FAM probe, 1 ul VIC probe, 0.5–2 ul of pre-amplified sample, fill remaining volume with water. Pre-amp thermocycle: 95°C95C for 10 min, 18 cycles of 95C for 15 seconds, 60C for 4 min, and then hold at 4°C.4C. Expression was normalized to the geometric mean of four housekeeping genes: GAPDH, CTNNB1, EEF1A1, CENTB3 (Centrin). Genes were then grouped into catagories representing developmental stages and cell types, with a fold change induction, normalized to the undifferentiated state, computed over the sample time points.
Alkaline phosphatase (AP) staining was performed with Vector® Red Alkaline Phosphatase Substrate Kit I (Vector Laboratories, CA), according to the manufacturer's protocol. For immunocytochemistry, cells were fixed in 4% paraformaldehyde/PBS for 10 minutes, washed twice with PBS, and blocked with 1–3% donkey, goat or chicken serum in PBS for 1 hour—all procedures were done at room temperature. For nuclear or intracellular staining, after fixation, the cells were permeabilized with 0.3% Triton-X100 for 30 minutes at room temperature. Subsequently, the primary antibodies were added to PBS and incubated for 1 hour at room temperature or overnight at 4°C. The cells were then washed with PBS before fluorescent-conjugated secondary antibodies were added and incubated for an hour at room temperature. Finally, the cells were rinsed with PBS three times and counterstained with DAPI. Stained samples were imaged directly on a LEICA inverted microscope or, if the samples were on coverslips, were mounted in PVA-DAVCO overnight and then imaged on an inverted Zeiss Confocal. Primary antibodies and their dilutions were as follows: Oct4 (diluted at 1∶100, Santa Cruz), Sox2 (1∶200, Millipore), SSEA1 (1∶200, Millipore), SSEA4 (1∶200, Millipore), TRA1-60 (1∶200, Millipore), TRA1-81 (1∶200, Millipore), Nanog (1∶100, Abcam), α-Fetoprotein (1∶200, Abcam), βIII-Tubulin (1∶200, Abcam), α-Smooth muscle actin (1∶200, Abcam), Vasa (1∶200, Abcam) Nestin (1∶200, Santa Cruz), Doublecortin (1∶200, Santa Cruz), Tyrosine hydroxylase (1∶500, Pel-Freez Biologicals), SNCA (1∶500 or 1∶1600, NeoMarkers), SNCA (1∶200, Thermo Scientific), SNCA (1∶200, Labvision), Ubiquitin (1∶250, Millipore), FoxA2 (1∶50, Santa Cruz). Secondary antibodies were raised in either donkey, goat or rabbit with conjugates: Alexa 488-conjugated anti-rabbit IgG (1∶500, Invitrogen), Alexa 594-conjugated anti-rabbit (1∶500, Invitrogen), Alexa 647-conjugated anti-rabbit (1∶500, Invitrogen), Alexa 488-conjugated anti-mouse IgM (1∶500, Invitrogen) and Alexa 488 anti-mouse IgG (1∶500, Invitrogen), FITC anti-Mouse conjugated, Cy3 anti-Rabbit, and Cy5 anti-Goat (1∶500 Jackson Immuno). In micrographs DAPI, FITC, Cy3, Cy5, are visualized as Blue, Green, Red, and Yellow (or Blue, when DAPI is removed).
To determine methylation regions, bisulfite sequencing was performed on genomic DNA isolated from hESCs and iPSCs, grown on feeder-free media, with Methyl Easy Xceed Rapid DNA Bisulfite Modification Kit (Human Genetic Signatures, Sydney, New South Wales, Australia) per manufacturers directions. The promoter regions of Oct3/4 and Nanog were amplified by PCR, as previously described
To prepare metaphase spreads, 10 µl/mL colcemid was added to the cell culture and incubated for up to 2 hours. The growth medium was removed and collected and the cells were rinsed with HBSS. Cells were treated with 2 mL trypsin and incubated at 37°C for 5–7 min. The collected colcemid medium from the earlier step was re-applied to the cells to stop neutralize the trypsin and resuspend the cells. Resulting solution was centrifuged at 1000 RPM for 6 min and the supernatant was partially aspirated and resuspended in the native solution by flicking the tube. 5 drops of pre-warmed hypotonic solution were slowly added against the side, while flicking with a finger, until 1 ml had been added. Volume was then brought to 2 mL with hypotonic solution. The sample was incubated at 37°C for 7 min and then centrifuged at 1000 RPM for 6 min. Medium was added to resuspend the cells. To fix the cells, 5 drops of fixative were slowly added against the side of the tube and the volume was brought to 2 mL with fixative. Cells were gently mixed and fixed for 30 min at room temperature. After fixing, the sample was centrifuged, aspirated, and resuspended as above. Any clumps were removed by vacuum from the side of tube, and 2 ml of fixative were added to the tube. Sample was then mixed, incubated for 20 min at room temperature, centrifuged, aspirated, and resuspended in 2 ml of fixative. Samples were transferred onto pre-cleaned slides in ∼100 ul drops, left to dry overnight, and then analyzed on a SKY microscope (Spectral-Imaging, Vista, CA).
SNCA gene copy number analysis: Genomic DNA was isolated from frozen cell pellets using a DNeasy Blood & Tissue Kit (Qiagen 69504). A FAM-MGB Taqman Copy Number Assay against the 3rd intron of SNCA (Hs04791950_cn) and a VIC-TAMRA Taqman Copy Reference Assay against RNase P (#4403326) were used with Taqman Genotyping MasterMix (#4371353; Applied Biosystems) to co-amplify 20 ng of genomic DNA in quintuplicate from each sample on a Stratagene Mx3000P RT-PCR System. The absolute copy number of SNCA for each sample is reported by calculating the relative copy number (SNCA/RNase P) for each reaction (2−ΔCT) and then adjusting by a factor of 2 which reflects the normal allelic copy number of RNase P in the genome. 2 alleles of SNCA are normally present, a duplication of SNCA yields 3 alleles, and a triplication of SNCA yields 4 alleles.
Primer sequences used for determining exogenous and endogenous expression of the OCT4, SOX2, KLF4 and c-MYC. Exogenous: pMXs-AS3200:
To investigate
As sated previously, animal work was approved under Stanford guidelines for animal safety and received approval from Stanford's administrative panel on laboratory animal care (approval APLAC-16146) and SCRO (approval SCRO-212). To determine iPSC potential to form all three germ layers
To direct differentiation of pluripotent cell colonies towards a midbrain dopaminergic (DA) neuron cell fate, a modified version of the protocol outlined by Perrier et al. was utilized25. Two or three hESC or iPSC colonies were mechanically harvested and gently dissected into 4–6 pieces per colony. Cells were then plated onto a 6 cm co-culture dish with irradiated MS-5 (xMS-5) stromal cells (MS-5 cells were expanded in MS-5 stromal cell culture medium: 455 ml a-MEM, 2 mM L-glutamine, 50 ml heat inactivated FBS, Pen-strep) and cultured for 16 days in Serum Replacement Media (15% KOSR in KO-DMEM, GIBCO), with media changes every two days. After 16 days, media was changed to N2+/+ (progesterone 20 nM, putrescine 100 uM, sodium selenite 30 nM, insulin 5 ug/ml, transferrin 0.1 mg/ml in DMEM-F12, GIBCO). At day 28, neural rosettes in the colonies were passaged through mechanical micro-dissection and transferred onto Poly/Laminin coated (15 ug/ml polyornithine, 1 ug/ml laminin, SIGMA) 6-well plates and cultured for one week in 200 ng/ml Sonic Hedgehog (SHH-C24II (a more active form than the unmodified protein), R&D Systems), 100 ng/ml Fibroblast Growth Factor 8 (FGF-8, R&D Systems), 20 ng/ml Brain Derived Neurotrophic Factor (BDNF, R&D Systems), and 0.2 mM Ascorbic Acid (AA, SIGMA) in N2+/+ media. On day 35, when cultures were approximately 80% confluent, colonies were passaged through digestion in Ca2+/Mg2+ free HBSS (GIBCO) at room temperature for 1 hour and subsequent mechanical dissociation. Removed cell aggregates were centrifuged at 200 g for 5 minutes, resuspended and plated on Poly/Laminin coated dishes at 50–100×103 cells/cm2 in N2+/+ media with the previously noted, day 28, growth factors. On day 42, differentiation was induced for 8 days through growth factor withdrawal by changing media to N2+/+ with 20 ng/ml BDNF, 20 ng/ml Glial Derived Neurotrophic Factor (GDNF, R&D Systems), 1 ng/ml Transforming Growth Factor B3 (TGF-B3, R&D Systems), 1 mM dibutyryl Cyclic Adenosine MonoPhosphate (cAMP, SIGMA) and 0.2 mM AA in N2+/+ media. On day 50, cell cultures were harvested for analysis.
After 60 days of iPSC patterning and differentiation into dopamine neurons, neurons were depolarized to evoke dopamine release. Media was removed and 1 ml of N2 media supplemented with 56 mM KCl was added per well (6 well dish) and incubated at 37°C for 15 minutes. The medium was collected and immediately frozen in liquid nitrogen and stored at −80°C until assay. Protein lysates were used to estimate relative cell number in each well. Lysates were made in ice cold 25 mM Tris supplemented with a Complete Mini protease inhibitor cocktail tablet (Roche). Lysates were sonicated and cleared by maximum speed centrifugation in a tabletop microcentrifuge. Soluble protein concentrations were measured by a standard Bradford assay. To measure dopamine, media samples were thawed, stabilized at a final concentration of 0.4 N perchloric acid, and centrifuged at 15,000 rpm at 4°C for 12 minutes to clear debries. The supernatant was then collected and dopamine was assayed by HPLC with electrochemical detection (Coularray detector, ESA, Chelmsford, MA) using a reverse phase C18 column (Perkin Elmer Instruments, Shelton, CT). The mobile phase consisted of a mixture of 90 mM sodium acetate, 35 mM citric acid, 130 uM ethylenediaminetetraaceticacid (EDTA), 230 uM 1-octanesulfonic acid and 10% (v/v) methanol, with a flow rate of 1 mL/min. DA concentration was quantified by comparison of AUC to known standard dilutions. Dopamine release was normalized to protein determinations of cleared protein lysates.
IPSC-derived cells cultured on glass coverslips were accessed via whole-cell patch clamp and assayed for their ability to generate action potentials in current clamp (CC). Electrophysiological recordings were obtained in Tyrode media ([mM] 150 NaCl, 4 KCl, 2 MgCl2, 2 MgCl2, 10 D-glucose, 10 HEPES, pH 7.35 with NaOH) using a standard internal solution ([mM] 130 KGluconate, 10 KCl, 10 HEPES, 10 EGTA, 2 MgCl2, pH 7.3 with KOH) in 3–5 MΩ glass pulled pipettes. Square pulse current steps of varying sizes and frequencies were injected to patched cells held at resting potential. Cells were chosen for patching based on either 1) their proximity or apparent extension of neuronal processes or 2) morphological similarity to typical neurons.
Western blotting was performed to determine α-synuclein in iPSC-derived DA neuron cultures possessing an SNCA triplication. 20 ug of protein lysates harvested from Trpl17, Ctrl2 and H9 cell derived neuron cultures (60 DIV, 2 independent wells per sample) were subjected to SDS-PAGE and stained for α-synuclein (Thermo Scientific), stripped and re-stained for GAPDH (SIGMA). Monomeric α-synuclein was observed at approximately 17 kDa. Band intensities were measured using Image J, and ratio of monomeric α-synuclein/GAPDH was calculated for each sample. Results were reported as the means of two samples with error bars representing SEM. A one-way ANOVA with Duncan's post-hoc analysis found that Trpl17 neuronal cultures (possessing 4 genomic copies of
Student's t-test was used for comparing two samples with adequate sample numbers. For determining statistical significance between more than two groups, a one-way ANOVA was used with either Duncan's or Bonferroni's post-hoc analysis. Data was analyzed either in InStat Version 3.0a, Microsoft Excel v12.20 or MATLAB 2008.
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