PTEN regulates AMPA receptor-mediated cell viability in iPS-derived motor neurons

Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast–iPS–motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.


Subject Category: Neuroscience
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder affecting upper and lower motor neurons (MNs) and leading to death within 2-3 years from diagnosis. Between 90 and 95% of cases are sporadic in origin, whereas the remaining 5-10% of cases are familial. Of these, B20% carry mutations in the gene encoding the superoxide dismutase 1 enzyme (SOD1). 1 Transgenic mice expressing mutant forms of human SOD1 are used as a model of familial ALS. 2 We have defined the gene expression profiles of MNs isolated from the spinal cord of G93ASOD1 transgenic mice at different stages of disease, by combining the use of laser capture microdissection (LCM) and microarray technology. This work highlighted the involvement of metabolism in the first stages of disease, along with a substantial upregulation of transcription-related transcripts. 3 Metabolic impairment suggested that astrocytes might also play a crucial role in the first stages of disease, supporting the results from other groups. [4][5][6] Hexanucleotide GGGGCC intronic expansions in the newly identified C9ORF72 (chromosome 9 open reading frame 72) gene represents the most common cause of both familial and sporadic ALS, 7,8 responsible for up to 50% of familial ALS and B10% of sporadic ALS, but to date SOD1 mutations have been the genetic subtype most widely studied and utilized to model ALS. In the presence of mutant SOD1, multiple interacting factors contribute to MN injury including protein misfolding and aggregation, defective axonal transport, excitotoxicity, mitochondrial dysfunction, dysregulated transcription and RNA processing, endoplasmic reticulum stress, apoptosis, oxidative stress as well as toxicity caused by nonneuronal cells. 9 Of these mechanisms, excitotoxicity is considered to play a key role. Routine excitatory transmission in the brain is predominantly mediated by a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In ALS, there is a body of evidence that excitotoxicity mediated by calcium-permeable AMPA receptors contributes significantly to MN injury. Riluzole, an antiexcitotoxic agent, is the only drug proven to slow the disease progression in humans. 10 AMPA receptors have been proposed to play a major role in the selective death of MNs in ALS. These characteristics are related to the way MNs handle Ca 2 þ . MNs have a low Ca 2 þ -buffering capacity and a high number of Ca 2 þ -permeable AMPA receptors. A significantly different ratio between inhibitory and excitatory synapses was present in SOD1 G93A mouse spinal cord slice cultures. SOD1 G93A MNs exhibited increased vulnerability to AMPA glutamate receptor-mediated excitotoxic stress before the onset of an overt disease phenotype. 11 The levels of the glutamate receptor 2 (GluR2) AMPA subunit, which plays an important role in the maintenance of calcium impermeability of AMPA receptors, are decreased in spinal MNs before symptom onset in conjunction with a modest increase of GluR3 expression in SOD1 G93A mice. 12 GluR2 is a subunit of the AMPA receptor, and the adenosine for the Q/R site of its pre-mRNA is converted to inosine (A-to-I conversion) by the action of adenosine deaminase acting on RNA 2 (ADAR2). Failure of A-to-I conversion at this site affects multiple AMPA receptor properties, including the Ca 2 þ permeability of the receptorcoupled ion channel. Inefficient GluR2 Q/R site editing is a disease-specific molecular dysfunction reported in the MNs of sporadic ALS patients. 13 We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents, and higher GABAmediated chloride currents, compared with spinal MNs that are vulnerable to the disease process. 14 We have also demonstrated that knockdown of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) via siRNA promotes MN survival in models of spinal muscular atrophy (SMA) 15 and ALS. 16 Inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. 17 Leptin promotes GluR1 trafficking to hippocampal synapses via PTEN inhibition. 18 Furthermore, PI3-kinase that is negatively regulated by PTEN is required for maintaining AMPA receptor clustering at the postsynaptic membranes 19,20 and AMPA receptor surface expression. 21 Thus, we speculate that manipulation of AMPA receptor expression and function by PTEN may represent a potential therapeutic strategy for clinical intervention in ALS. The breakthrough that human fibroblasts can be transformed into induced pluripotent stem (iPS) cells 22 and these could then be differentiated into MNs 23 opened new perspectives, providing a model of human origin for sporadic and familial cases of ALS. The aim of the present study is to establish a fibroblast-derived iPS-MN in vitro cell model for manipulation of AMPA receptor expression and function.

Results
Generation and characterization of human iPSCs (hiPSCs) from adult human dermal fibroblasts (HDFs). Under human research subject and stem cell protocols as approved by the institutional review boards, we recruited a healthy 40-year-old Chinese female to donate a skin biopsy to be used in reprogramming studies and the production of pluripotent stem cell lines. Three other healthy controls were also used to generate induced pluripotent stem cells (iPSCs) under ethical approval in the United Kingdom. The HDFs were isolated and plated under standard fibroblast conditions (Figure 1a). After four passages, the fibroblast identity in the resulting cell lines was confirmed by TE-7 antibody staining ( Figure 1b). The cells were then used for generating iPS cells after infection with retroviruses containing human OCT3/4, SOX2, C-MYC and KLF4. As a control, we introduced green fluorescent protein (GFP) into HDF with pMXs-GFP retrovirus produced in PLAT-A packaging cells. More than 80% of HDF treated in this way expressed GFP (Figure 1c). Using this method, the dish was nearly covered with 4100 granulated colonies B25 days later. We observed distinct types of colonies that were flat and resembled hES cell-like colonies in between the granulated cells. At day 30, we picked 20 hES cell-like colonies and mechanically disaggregated them into small clumps without enzymatic digestion. The hES-like cells expanded on irradiated mouse embryonic fibroblasts (MEFs) using primate ES cell medium containing fibroblast growth factor-basic (bFGF).
To establish that reprogramming of the human fibroblasts had occurred, and that the putative hiPSCs were pluripotent, we evaluated their similarity to human embryonic stem cells (hESCs). Although we initially isolated 20 hiPSC clones, we focused on an in-depth characterization of only one of these clones. As illustrated in Figure 1d, the derived cells displayed the morphological characteristics of undifferentiated hESCs (i.e., large, compact, multicellular colonies of cells with a high nucleus-to-cytoplasm ratio) and showed high telomerase activity (Figures 1e and f). Moreover, they expressed several frequently used hESC markers (i.e., OCT3/4, SOX2, LIN28, NANOG, REX1) as shown by immunohistochemistry ( Figure 1g) and reverse transcription-PCR (RT-PCR; Figure 1h). In addition, the putative hiPSC line also maintained a normal karyotype (Figure 1i). The colonies continued to passage after at least 6 months by passaging once a week. The split ratio was 1 : 3 to 1 : 6. Bisulfite genomic sequencing analyses evaluating the methylation status of cytosine guanine dinucleotides (CpG) in the promoter region of the pluripotent-associated gene Oct4 revealed that this was highly unmethylated, whereas the CpG dinucleotides of the region were highly methylated in parental HDF (Figure 1j). These findings indicate that this promoter is active in hiPSCs.
To test pluripotency in vivo, we transplanted human iPS cells subcutaneously into the hind limb muscle of NOD/SCID mouse. At 8 weeks after injection, we observed tumor formation. Histological examination showed that the tumor contained various tissues: neural tissues (ectoderm, Figure 1k), cartilage (mesoderm, Figure 1l), muscle (mesoderm, Figure 1m) and primitive gut (endoderm, Figure 1n).
Taken together, these data demonstrate that reprogramming of wild-type fibroblasts to a pluripotent state had successfully occurred.
MN differentiation of hiPSCs. Several groups have devised protocols to differentiate hESCs/hiPSCs to functional MNs. [23][24][25] Here we chose a protocol that follows the principles of normal development and leads to a high efficiency in the production of the target MNs ( Figure 2a). We raised hiPSCs in co-culture with a MEF feeder layer, as previously described. The cultures were raised to 80-90% confluence and exhibited a uniform undifferentiated phenotype to achieve neuro-ectodermalization before inducing them to form embryoid bodies (EBs) in suspension cultures. EB aggregates are usually grown as free-floating spheres. The EBs were cultured for 7 days and then they were attached to tissue culture dishes to initiate the differentiation of neuroepithelial cells that soon emerge as neural epithelial rosettes. After 3 days, the primitive neural epithelial cells formed and expressed anterior transcription factors, such as PAX6 and OTX2 (Figure 2b). After an additional 5 days in the presence of retinoic acid (RA), definitive neuroepithelial cells tended to form neural tube-like rosettes. The neural rosettes were gently blown off and treated with both RA and sonic hedgehog (SHH), resulting in the appearance of MN progenitors. These cells then differentiated to spinal MNs in week 5 and expressed transcription factors, such as Islet1/2 ( Figure 2c). This protocol generated B50% of Islet1/2expressing MNs from the original hWT-iPSC cells. These MNs, when further cultured in the presence of neurotrophic factors, extend long axonal projections, express choline acetyltransferase (ChAT; Figure 2d) and become electrophysiologically active.
PTEN knockdown increases phosphorylation of Akt and Bad in primary cultured MNs and iPS-derived MNs. The tumor suppressor protein PTEN is a member of the protein tyrosine phosphatase family that can negatively regulate the serine/threonine kinase Akt to exert its tumor suppressor function. 26 The protein phosphatase activity of PTEN can regulate cell migration, spreading and growth. 27 PTEN is widely expressed in the mouse CNS and preferentially in neurons such as large pyramidal neurons. 28 PTEN localizes to both the nucleus and cytoplasm of neuronal and glial cells. [28][29][30] Significant progress has been made in investigating the broader role of PTEN in the brain. In addition to its normal functions such as control of neuronal migration 31,32 and neuronal size, 33,34 the PTEN protein is involved in pathological processes surrounding neuronal injury such as those associated with brain ischemia, neurological and mental disorders and drug addiction. [33][34][35][36][37][38][39] Conditional inactivation of PTEN leads to behavioral abnormalities and neuropathological changes characterized by neuronal hypertrophy. 39 Park et al. 40 reported that modulation of the  PTEN knockdown decreases AMPA receptor expression in primary cultured and iPS-derived MNs. The selective vulnerability of MNs to AMPA receptor-mediated excitotoxicity can be studied in vitro using purified MNs. These MNs are sensitive to AMPA-or kainate-induced excitotoxicity. 41 AMPA-or kainate-induced MN death can be inhibited by NBQX, an AMPA receptor antagonist. 42 In this study, we investigate the potential effects of PTEN on MN excitability in primary cultured and iPS-derived MNs. The first step was to assess whether PTEN knockdown has an effect on AMPA receptor expression. Here we showed that PTEN knockdown PTEN knockdown decreases AMPA-induced whole-cell currents in primary cultured and iPS-derived MNs. Based on the decrease of expression of AMPA receptors following PTEN silencing, we next tested the effects of PTEN knockdown on the functional output of AMPA receptors in primary cultured and iPS-derived MNs.

Discussion
The aim of the present study was to determine whether PTEN knockdown has an effect on functional AMPA receptor expression and AMPA-mediated cell death in primary cultured and iPS-derived MNs. AMPA receptors are heteromeric complexes composed of various combinations of the four subunits: GLUR1 to GLUR4. The presence of the GLUR2 subunit in the assembled AMPAR determines its calcium permeability, and alternative flip or flop splicing of all subunits generates further diversity affecting the kinetic properties of AMPAR. 43,44 MNs are more susceptible to AMPA receptormediated death compared with other spinal neurons. 47,48 Deactivation and desensitization kinetics of AMPA receptors in MNs also resembled AMPA receptor kinetics in cerebellar Purkinje neurons. 47 Activation of PI3-kinase is required for AMPA receptor insertion during long-term potentiation (LTP) of mEPSCs in cultured hippocampal neurons. 44 Although the molecular control of AMPA receptor kinetics is complex and incompletely understood, subunit composition appears to be an important determinant of AMPA receptor desensitization. 52 To assess functional AMPA receptors in primary cultured and iPS-derived MNs, we measured AMPA receptormediated current in both types of cells. PTEN knockdown decreased GluR1 and GluR2 expression and AMPA-induced whole-cell currents. Downregulation of GluR1 was more significant than of GluR2. Our findings appear to imply that the ratio of GluR1 and GluR2 changes following PTEN knockdown may cause variation in the AMPA receptor properies of calcium permeability and excitatory synaptic transmission. It has been reported that the antiglutamate drug, riluzole, is able to modulate AMPA receptors by reducing the kainate-induced currents in spinal MNs in a noncompetitive and a dose-dependent manner. AMPA is considered to play an important role in the selective death of MNs in ALS. A significantly different ratio between inhibitory and excitatory synapses was present in MNs from the SOD1 G93A mouse. 11 The levels of the GluR2 AMPA subunit are decreased in spinal MNs before symptom onset in SOD1 G93A mice. 12 Inefficient GluR2 Q/R site editing appears to be a disease-specific molecular alteration reported in sporadic ALS patients. 13 At the level of the glutamatergic synapse, high concentrations of riluzole have been reported to inhibit glutamate release, attenuate excitatory amino acid receptor activation and decrease excitability of the postsynaptic cell membrane. 52 The high concentrations required, however, are unlikely to be reached in vivo using clinically approved doses. AMPA receptors are glutamate-gated cation selective channels that mediate most fast excitatory synaptic transmission in the mammalian brain. AMPA receptor desensitization protects neurons against excitotoxic effects resulting from prolonged activation, as suggested by the pharmacological blockade of AMPAR desensitization that enhances excitotoxicity in neurons, including spinal MNs. 10,[46][47][48][49]52 Here we report that PTEN inhibition decreases AMPA-induced cell death in primary cultured and iPS-derived MNs by downregulation of AMPA receptor expression and function assessed by AMPA-mediated whole-cell currents.
We have previously reported that oculomotor neurons resistant to the disease process in ALS show reduced AMPAmediated inward calcium current compared with spinal MNs that are vulnerable to the disease process. 10 These data implied that downregulation of AMPA receptor expression in spinal MNs might increase their resistance to exctitotoxic injury. We previously showed that PTEN depletion increased MN survival in models of SMA and ALS. 15,16 Here we show that PTEN inhibition decreases AMPA-mediated inward currents. Thus, PTEN depletion or inactivation may represent It has been reported that reduction of kainate-induced currents by riluzole is consistent with its neuroprotective effects, because it hyperpolarizes the membrane and attenuates spike-firing rates, leading to reduced presynaptic glutamate release. However, riluzole has a limited effect in the clinical setting because the high concentrations required to achieve these effects are unlikely to be reached in vivo using clinically approved doses. 53 PTEN silencing may achieve amelioration of AMPA receptor-mediated toxicity in vivo. Indeed, PTEN depletion has been shown to dramatically increase MN survival in vivo. 15 We have observed that the results obtained from iPS-derived and primary cultured MNs are mostly but not always consistent; for example, GluR3 decreases significantly in iPS-derived MNs (Figure 4k) but not in primary cultured MNs (Figure 4e). We used the best PTEN shRNA sequence from multiple tested sequences for the lentiviral constructs for the transduction of primary MNs. This siRNA has been tested in neuronal cell lines 38,54 and MNs in vitro 16 and in vivo. 15 The PTEN siRNA virus we used for iPS-derived MNs was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA), which has been fully characterized by the company, and its efficiency of PTEN knockdown has been tested widely by various research groups. 55,56 As both the cells and the siRNA virus are different, it would be possible to have variation for GluR3 knockdown between primary cultured and iPS-derived MNs. This difference is unlikely to represent off-target effects. We have not seen significant PTEN knockdown using scrambled siRNA sequences and other siRNA sequences. 15,16,[54][55][56] We believe therefore that the PTEN knockdown is specific in both primary cultured and iPS-derived MNs.
In conclusion, we have shown for the first time that PTEN knockdown decreases the expression and activity of AMPA receptors in MNs. Modulation of AMPA receptors may underlie some of the neuroprotective effects of PTEN inhibition or reduced expression of the PTEN protein. In future work, we will use ALS patient fibroblasts to generate iPS-derived MNs and investigate the effect of PTEN knockdown on MN survival in patient iPS-derived MNs from specific subtypes of ALS patients. This may represent a useful strategy for drug screening to identify compounds that may modulate disorders involving neuronal hyperexcitability, including ALS.

Materials and Methods
Human fibroblast and Plat-A culture. Under human research subject and stem cell protocols as approved by the institutional review boards, we recruited a healthy 40-year-old Chinese female to donate a skin biopsy to be used in reprogramming studies and the production of pluripotent stem cell lines. Three other healthy controls were also used to generate iPSCs under ethical approval in the United Kingdom. The dermal fibroblasts were isolated by 0.25% trypsin (Gibco, Shanghai, China, 25200). HDFs and Plat-A cells were maintained in medium containing Dulbecco's modified Eagle's medium (DMEM; Gibco, 11965-092), 10% fetal bovine serum (FBS, Thermo, Shanghai, China), 1% nonessential amino acids (NEAA, Gibco, 11140-050) and 1% GlutaMAX (Gibco, 35050).
At 48 h after transfection, the medium was collected as the first virus-containing supernatant and replaced with new medium that was collected 24 h later as the second virus-containing supernatant. The virus-containing supernatants were filtered through a 0.45 mm pore-size filter and supplemented with 10 mg/ml polybrene (Sigma, St. Louis, MO, USA, H9268). Equal amounts of virus-containing supernatants containing each of the four retroviruses were mixed before transduction, transferred to the fibroblast dish and incubated overnight. Approximately 50 000 fibroblasts per well of a six-well plate were transduced twice over 48 h. Then, the transduced cells were passaged on plates containing irradiated MEFs. The medium was replaced with iPSCs medium, containing DMEM/F12 (Gibco, 11330), 20% knockout serum replacement (KSR, Gibco, 10828-028), 1% nonessential amino acids (NEAA, Gibco, 11140-050), 1% GlutaMAX (Gibco, 35050), 0.1 mM b-mercaptoethanol (Sigma) and 4 ng/ml bFGF (R&D, St. Louis, MO, USA). The first hiPSC colonies appeared B2 weeks later and they could be picked after 1-2 additional weeks of culture. Individual colonies were picked and either transferred into a single well of 12-well plates containing iPSC medium and irradiated MEFs. For passaging, hiPSCs were incubated with DMEM/F12 containing collagenase IV (1 mg/ml) at 371C for 10-15 min. When colonies at the edge of the dish were dissociating from the bottom, the enzyme was removed and washed by iPSC medium without bFGF. Cells were collected by gently pipetting.
RNA isolation and PCR analysis. Total RNA was isolated using the RNAsimple Total RNA Kit (Tiangen, Shanghai, China). Total RNA at 1 mg was used for the reverse transcription reaction with the RevertAid First Strand cDNA synthesis kit (Fermentas, Beijing, China). The cDNA from MEFs was used as a negative control, whereas that from hESCs was used as a positive control. RT-PCR was performed using specific sequences: 57 hOCT3/4- Alkaline phosphatase staining and immunocytochemistry. Alkaline phosphatase staining was performed using the Alkaline Phosphatase Detection kit (Si Dan Sai, Shanghai, China, 1102). For immunocytochemistry, cells were fixed with phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 10 min at room temperature. After washing with PBS, the cells were treated with PBS containing 4% normal donkey serum (Jackson Immuno-Research, West Grove, PA, USA, 017-000-121), 1% bovine serum albumin (BSA, Sigma) and 0.1% Triton X-100 for 45 min at room temperature. Primary antibodies included SSEA3 (1 : 100 Teratoma formation. One million hWT-iPSCs cells were injected subcutaneously into the hind limb muscle of the NOD/SCID mouse (Jackson Laboratory, Bar Harbor, ME, USA). Paraffin sections of formalin-fixed teratoma specimens were prepared 6-8 weeks after injection, and analysis of H&E-stained tissue sections was performed for each specimen. All animal experiments were performed in accordance with the institutional guidelines.
MN culture. Cultures of embryonic spinal MNs were prepared as previously described. 15 Briefly, the ventrolateral part of the E13 spinal cord was dissected and incubated at 371C for 15 min in 0.1% trypsin in Hanks' balanced salt solution. After trituration, cells were plated on dishes precoated with anti-p75 nerve growth factor (NGF) receptor antibody (Abcam, ME20.4, ab8877) in Neurobasal (Gibco) for 30 min. The cells were washed three times with Neurobasal, and the attached cells were isolated from the plate with depolarizing solution (0.8% NaCl, 35 mM KCl) and collected in full media (Neurobasal supplemented with 2% horse serum, 2% B27 (Gibco), 0.1 mM b-mercaptoethanol (Sigma) and 1 Â Glutamax (Gibco)). For staining, 2000 cells were plated on poly-DL-ornithine (Sigma) and mouse laminin (Invitrogen)-coated coverslips in four-well dishes (Greiner, Frickenhausen, Germany). For western blots, 100 000 cells were plated on each well of 12-well plate also coated with poly-ornithine and mouse laminin. MNs were transduced at 24 h after plating with multiplicity of infection (MOI) of 10. For all assays, brainderived neurotropic factor was used at concentrations of 5 ng/ml and MNs were cultured at 371C with 5% CO 2 . Medium was replaced after 24 h and then every 2 days. AMPA treatment for survival (TUNEL assay) and electrophysiology were carried out from day 7 onwards. Cells were treated with 50, 100, 200, 300, 400, 500 and 600 mM (S)-AMPA to determine dose-response effects of AMPA treatments. Of the neurons, 50% were TUNEL positive at 24 h following exposure to 300 mM AMPA for 1 h. For AMPA inhibition, 50 mM CNQX (Sigma) was added to culture medium 1 h before MNs were exposed to 300 mM (S)-AMPA (Sigma) for 1 h and then fresh medium was applied for 24 h before performing a TUNEL survival assay. 58 The iPS-derived MN differentiation. The iPS cells were maintained on gelatinized tissue-culture plastic in iPS media containing DMEM/F12 (Gibco, 11330), 20% knockout serum replacement (KSR, Gibco, 10828-028), 1% NEAA (Gibco, 11140-050), 1% GlutaMAX (Gibco, 35050), 0.1 mM b-mercaptoethanol (Sigma) and 4 ng/ml bFGF (R&D) at 371C, 5% CO 2 . Medium was changed every 24 h. To generate MNs, [23][24][25] iPS cells were passaged using dispase (1 mg/ml) and triturated into small cell clumps and placed into ultralow adherent culture dishes (Corning, Corning, NY, USA). For the first 3 days, cells were kept in suspension in iPS medium, to enhance single cell survival, and 20 ng/ml bFGF (Invitrogen) was added to enhance growth. At day 4, EBs were switched to neural induction medium (DMEM/F12 with L-glutamine; NEAA; penicillin/streptomycin; heparin, 2 mg/ml; N2 supplement; Invitrogen). At day 6, all-trans retinoic acid (RA; 1 mM; Sigma) was added. The same medium was changed with fresh RA daily.
Hedgehog signaling was initiated on day 15 by application of SHH (100 ng/ml, R&D) and purmorphamine (PUR, 1 mM). The same media was changed with freshly daily. At day 28, basal medium was changed to Neurobasal (Invitrogen), containing all previous factors, with the addition of 10 ng/ml of insulin-like growth factor 1 (IGF-1) and 1 mM cAMP (Sigma). At day 35, EBs were dissociated with 0.05% trypsin (Invitrogen) and plated onto poly-lysine/laminin-coated dishes or coverslips. Plated neurons were cultured in the same medium as above with addition of ascorbic acid (200 ng, Sigma). Cells were transduced with human shRNA PTEN lentiviral vectors with MOI of 10 for 7 days before assessment. Cells were treated with 50, 100, 200, 300, 400, 500 and 600 mM (S)-AMPA to determine dose-response effects of AMPA treatment. Approximately 50% of the cells were TUNEL positive 24 h after exposure to 300 mM AMPA for 1 h. Exposure to AMPA with assays for survival (TUNEL assay) and electrophysiology were carried out from day 45 onwards. For AMPA inhibition, 50 mM CNQX (Sigma) was added to culture medium 1 h before MNs were exposed to 300 mM (S)-AMPA (Sigma) for 1 h and then fresh medium was applied for 24 h before performing a TUNEL survival assay. 58 Electrophysiology. Whole-cell electrophysiological experiments were recorded as previously described. 14 Voltage clamp recordings were performed using an Axon Multi-Clamp 700B amplifier (Axon Instruments, Sunnyvale, CA, USA) using unpolished borosilicate pipettes placed at the cell soma. Pipettes had a resistance of 2-4 MO when filled with intracellular solution. Intracellular solution for AMPA-or kainate-induced currents consisted of 120 mM CsF, 3 mM MgCl 2 , 5 mM EGTA and 10 mM HEPES (pH adjusted to 7.25 with 12 mM CsOH). Cs þ in the pipette solution was included to block K þ -dependent membrane conductance. Cells were accepted for study if a stable seal formed with a whole-cell resistance of at least 120 M O and a series resistance of o10 MO. Receptors were activated by focal perfusion of agonists from a micropipette with its tip located 30-50 mm from the cell. Three cells were used for dose-response recordings for AMPAinduced whole-cell currents. Currents were recorded in 20 mM extracellular Na þ at À 60 mV, in response to AMPA concentrations ranging from 5 mM to 5 mM. The extracellular perfusion buffer consisted of 15.3 mM NaCl, 4.7 mM NaOH, 2 mM CaCl 2 , 10 mM HEPES, 10 mM glucose and 228 mM sucrose, pH 7.40. For the measurement of Ca 2 þ permeability, 100 mM AMPA, close to the EC 50 from the dose-response recordings, was used. All extracellular solutions were supplemented with MK-801 (10 mM), tetrodotoxin (1.0 mM) and Cd 2 þ (100 mM) to block NMDA receptors, voltage-gated Na þ channels and Ca 2 þ channels, respectively. Cells were held at a membrane potential of À 60 mV. All recordings were performed at room temperature of 21-231C. Current recordings were sampled onto an IBM-PC compatible computer using pClamp10 software (Axon). Data were filtered at 3 kHz and sampled at 20-40 kHz.
TUNEL staining. Primary cultured or iPS-induced neurons were stained by the TUNEL technique (ApopTag@Red In Situ Apoptosis Detection Kit) according to the manufacturer's instructions (Millipore). MNs were fixed in 4% PFA in PBS at room temperature for 15 min. MNs were incubated in equilibration buffer for 1 h at room temperature. The buffer was then removed and samples were incubated in reaction buffer and TdT enzyme mix at 371C for 90 min. The mix was then replaced with the stop buffer. Samples were kept at 371C for 3 h. They were then washed in PBT and incubated with gentle shaking in blocking solution and anti-DIG-rhodamine mix overnight at 41C. After that, rabbit anti-tubulin primary antibody (1 : 500; Cell Signaling) followed by CF488A-conjugated donkey antirabbit IgG (1 : 800, Biotium) was stained in green as described in the immunocytochemistry methods. Hoechst staining was used to stain nuclei in blue. Mounted MNs were viewed using a Leica TCS SP5 confocal microscope.