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. 2016 Nov 23;36(47):12027-12043.
doi: 10.1523/JNEUROSCI.0456-16.2016.

Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons From Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization

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Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons From Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization

Nancy Stanslowsky et al. J Neurosci. .
Free PMC article

Abstract

Chorea-acanthocytosis (ChAc) is a fatal neurological disorder characterized by red blood cell acanthocytes and striatal neurodegeneration. Recently, severe cell membrane disturbances based on depolymerized cortical actin and an elevated Lyn kinase activity in erythrocytes from ChAc patients were identified. How this contributes to the mechanism of neurodegeneration is still unknown. To gain insight into the pathophysiology, we established a ChAc patient-derived induced pluripotent stem cell model and an efficient differentiation protocol providing a large population of human striatal medium spiny neurons (MSNs), the main target of neurodegeneration in ChAc. Patient-derived MSNs displayed enhanced neurite outgrowth and ramification, whereas synaptic density was similar to controls. Electrophysiological analysis revealed a pathologically elevated synaptic activity in ChAc MSNs. Treatment with the F-actin stabilizer phallacidin or the Src kinase inhibitor PP2 resulted in the significant reduction of disinhibited synaptic currents to healthy control levels, suggesting a Src kinase- and actin-dependent mechanism. This was underlined by increased G/F-actin ratios and elevated Lyn kinase activity in patient-derived MSNs. These data indicate that F-actin stabilization and Src kinase inhibition represent potential therapeutic targets in ChAc that may restore neuronal function.

Significance statement: Chorea-acanthocytosis (ChAc) is a fatal neurodegenerative disease without a known cure. To gain pathophysiological insight, we newly established a human in vitro model using skin biopsies from ChAc patients to generate disease-specific induced pluripotent stem cells (iPSCs) and developed an efficient iPSC differentiation protocol providing striatal medium spiny neurons. Using patch-clamp electrophysiology, we detected a pathologically enhanced synaptic activity in ChAc neurons. Healthy control levels of synaptic activity could be restored by treatment of ChAc neurons with the F-actin stabilizer phallacidin and the Src kinase inhibitor PP2. Because Src kinases are involved in bridging the membrane to the actin cytoskeleton by membrane protein phosphorylation, our data suggest an actin-dependent mechanism of this dysfunctional phenotype and potential treatment targets in ChAc.

Keywords: Src kinase; actin cytoskeleton; chorea-acanthocytosis; human induced pluripotent stem cells; patch-clamp electrophysiology; striatal medium spiny neurons.

Figures

Figure 1.
Figure 1.
Characterization of hiPSC lines. A, All hiPSC lines used in this study expressed the pluripotency markers NANOG, OCT4, TRA1–81, and SSEA4 as seen by immunocytochemical stainings. B, To show pluripotency, iPSC lines were differentiated in vitro into cells expressing markers of all three germ layers: ectoderm (TUBB3), endoderm (AFP), and mesoderm (SMA). For A and B, WT1–1 and Mut1 are shown as representative lines. Nuclei were counterstained with Hoechst. Scale bars, 100 μm. C, qRT-PCR revealed that all iPSC lines expressed a comparable level of pluripotency markers as the hESC line HUES6. D, Retroviral transgenes used for reprogramming were efficiently silenced in established iPSC lines. Expression was normalized to a freshly infected human fibroblast sample 4 d postinfection (dpi). Data are presented as means ± SEM using GAPDH and BACT as housekeeping genes.
Figure 2.
Figure 2.
Sequential induction of hiPSCs toward MSNs. A, Schematic summary of the differentiation procedure. BD, Phase-contrast images during in vitro differentiation. hiPSC colonies were detached from the feeder layer and cultured in suspension as EBs in the presence of small molecules as indicated. On days 12–14, EBs were plated on Matrigel-coated cell culture dishes. Neuronal cells started to spread out and matured through the addition of various growth factors. Scale bars, 100 μm.
Figure 3.
Figure 3.
Differentiation of hiPSCs into MSNs. A, The WNT inhibitor IWP2 was added either at the beginning of differentiation or at day 5 and telencephalic identity was monitored by analyzing FOXG1 mRNA expression by qRT-PCR on day 5, 10, and 13. Only when IWP2 was added from day 0 the expression of the forebrain marker FOXG1 was strongly induced. Data were exemplarily generated with the hiPSC line WT1–2. B, When EBs were plated at day 8 of differentiation and fixed 2 d later, cells stained positive for the neural progenitor cell marker NESTIN and FOXG1. C, At day 21 of the differentiation process, neurons marked by the expression of TUBB3 mostly expressed FOXG1. D, At day 30, different hiPSC lines of ChAc patients (Mut 1 and 2) and controls (WT 1–1 and 1–2) showed comparable expressions of the genes FOXG1, GAD67, and MAP2 as analyzed by qRT-PCR and normalized to the average expression level in undifferentiated hiPSCs. MSNs were replated as single cells at ∼day 30. Two days later, most of the TUBB3+ neurons (E) stained also positive for FOXG1 and GABA and ∼40% of patient and control MSNs expressed DARPP32 (F). The stainings were performed in triplicate (FOXG1 and DARPP32) or duplicate (GABA). Data are shown as means ± SEM. For DF, the lines WT1–1, WT1–2, Mut1, and Mut2 were used. G, Mature MSNs at ∼day 30 or older expressed TUBB3, DARPP32, MAP2, GAD67, and GABA, as shown by representative immunocytochemical staining. Scale bars, 100 μm.
Figure 4.
Figure 4.
Neuronal morphology and GABAergic synaptic density of control and ChAc patient-derived cells. Control (A, C) and patient-derived (B, D) iPSCs were stained for neuronal marker βIII-tubulin (red) and for GABAergic synapses (green). βIII-tubulin staining was combined with the ImageJ-based software NeurphologyJ to detect neuronal somata, neurites, attachment points of neurites at the soma, and end points of neurites automatically. E, Total neurite outgrowth was quantified normalizing neurites by somata. Therefore, we divided the area of the neurites by the area of the somata or the number of neurites (# neurites) by the number of somata (# somata). These parameters showed a tendency to be increased in patient-derived cells. In addition, total neurite length was normalized by the number of somata, which was significantly enhanced in ChAc neurons (p < 0.05, Student's t test). F, The number of end points of the neurites within an image was normalized by the number of attachment points at the soma giving a quantitative expression for an increased total ramification of patient-derived cells. Moreover, we used the ImageJ-based plugin SynapCountJ to quantify GABAergic synapses as stained in C and D. There was no significant difference in the synaptic density of ChAc neurons and controls. The bar graph gives the synaptic density as synapses per 100 μm neurite (all bars show means ± SEM, n = 3, where n represents a culture seeded at different dates).
Figure 5.
Figure 5.
Altered Na+ currents in neurons from ChAc patients. A, Voltage-gated ion currents of neurons differentiated from iPSCs for 40 d in vitro recorded in whole-cell voltage-clamp by depolarizing steps in increments of 10 mV from a holding potential of −70 to 40 mV. Inward currents were completely blocked by TTX, identifying them as being Na+ driven. Outward currents showed TEA sensitivity, indicating a potassium dependency. B, Although the maximal INa and IK normalized to individual cell capacitances did not differ significantly in either group, sodium channels in ChAc neurons activated at lower voltages and the sodium current amplitudes at −30 and −20 mV were significantly larger in ChAc cells. Data are presented as means ± SEM (p < 0.05, ANOVA). C, Expression of several sodium channel subunits tended to be upregulated in ChAc neurons as seen by qRT-PCR, which could explain the altered sodium current kinetics.
Figure 6.
Figure 6.
Elevated AP amplitudes in ChAc neurons. A, Differentiated cells from ChAc patients and healthy controls fired single APs in response to depolarizing current pulses in current-clamp mode that could be blocked by 1 μm TTX. B, Although the percentage of cells with APs did not differ in either group, the AP amplitudes were significantly higher in ChAc neurons compared with controls. C, Spontaneous AP firing was measured at the neuronal membrane potential over a period of 11 s. D, Spontaneous APs were observed only in a minor cell population without significant differences in percentage or frequency, but higher amplitudes in ChAc neurons compared with controls. Data represent means ± SEM (p < 0.05, Mann–Whitney test).
Figure 7.
Figure 7.
Enhanced spontaneous synaptic transmission in neurons derived from ChAc patient iPSCs. A, Higher percentages of ChAc neurons showed spontaneous mPSCs, which exhibited an almost 2-fold increase in frequency and a significantly higher mean amplitude in ChAc cells compared with controls, indicating a higher synaptic activity. B, C, Application of the AMPA receptor blocker NBQX reduced mPSC frequency just slightly, whereas the GABAA receptor blocker bicucullin decreased current frequency by 66% and 90% in ChAc and control neurons, respectively, indicating a predominantly GABAergic synaptic input, which is consistent with the high number of GABAergic cells in our cultures. Data are presented as means ± SEM (p < 0.05, Mann–Whitney test).
Figure 8.
Figure 8.
ChAc neurons and controls showed similar spontaneous and neurotransmitter-induced Ca2+ signaling. A, Representative traces of transient cytosolic Ca2+ changes induced by bath application of GABA (100 μm), acetylcholine (ACh, 100 μm), glutamate (Glut, 50 μm), glycine (Gly, 100 μm), and KCl (50 mm) in fura-2-loaded neurons from control iPSCs. Intracellular Ca2+ changes are presented as ratios of the fluorescence signals obtained at 340 and 380 nm (F340/F380). B, Summary of cytosolic Ca2+ response amplitudes normalized to the basal Ca2+ level of the investigated cells (n) showed no significant differences between ChAc neurons and healthy controls. C, A similar fraction of ChAc and control cells responded to neurotransmitter application obtained from experiments as shown in A. Note: GABA and glycine induced depolarizing Ca2+ signals in ∼20% of the cells. Therefore, the GABAergic and glycinergic synaptic transmission partly have an excitatory function in ChAc neurons and controls. D, Analyzing spontaneous Ca2+ transients revealed no significant difference in the percentage of spontaneously active cells (∼30%), Ca2+ current frequency, or amplitudes in ChAc neurons compared with controls. E, Genomic expression of voltage-gated calcium channel subunits analyzed by qRT-PCR did not differ significantly between ChAc neurons and healthy controls. Data represent means ± SEM (p < 0.05, Mann–Whitney test).
Figure 9.
Figure 9.
Treatment with the F-actin stabilizer phallacidin and the Src kinase inhibitor PP2 rescued the functional phenotype seen in ChAc neurons (n = 33–98). Treatment of differentiating cells with 100 nm phallacidin (AC) or 10 μm PP2 (DF) 72 h before analysis decreased the frequency and amplitudes of mPSCs to the level of control cells (n = 8–101). C, F, AP amplitudes were reduced significantly by phallacidin and PP2 as well. All data are given as means ± SEM of individual cell lines and as averaged values (p < 0.05, Mann–Whitney test).
Figure 10.
Figure 10.
Actin regulation and Lyn kinase activity in differentiated neurons. A, After at least 3 months of MSN differentiation from iPSCs, cell-culture dishes of ChAc neurons (n = 5 clones, red) contained significantly fewer cells (data are given as Whiskers minimum to maximum, box 25% to 75%, median, p < 0.05, Student's t test) than healthy controls (n = 4 clones, blue) when investigated with a Hoechst-based quantification assay. B, G-/F-actin ratio (soluble globular G-actin over filamentous F-actin) of MSNs (right bars) cultures differentiated for 6 weeks is increased significantly (p < 0.05, Student's t test) in ChAc cells (n = 9 clones, red) compared with healthy controls (n = 17 clones, blue), whereas cultivation into mDANs (9 ChAc and 16 control clones, left bars) did not lead to marked differences. Data represent means ± SD. C, Western blots of patient and control MSNs were analyzed with antibodies against the Rho-kinase downstream targets cofilin (left) and profilin (right). Results show different phosphorylation patterns between patient-derived and control cells. D, Phosphorylated and nonphosphorylated levels of cofilin (black) and profilin (gray) were quantitatively measured and ratios calculated. Although expression levels and phosphorylation state of controls did not differ, one ChAc cell line (Mut 2) showed a strong hyperphosphorylation of cofilin and cells from another patient (Mut 1) displayed hypophosphorylation of profilin. E, Western blots with specific antibodies against active Lyn kinase (phospho-Lyn 396) and total Lyn in MSNs. The representative experiment shown is one of three separate similar experiments, each with MSNs derived from healthy controls (Ctrl) or individual ChAc patients and each with similar results. GAPDH was used as a loading control (left). Shown is a densitometric analysis of the immunoblots; data are shown as either a single experiment (empty circle) or as means ± SD (black circle) (n = 3; *p < 0.05, Student's t test). F, Western blot with specific antibodies against active Lyn (phospho-Lyn 396) and total Lyn in mDANs. The representative experiment shown is one of three separate similar experiments, each with mDANs derived from healthy controls (Ctrl) or individual ChAc subjects and each with similar results. Actin was used as a loading control (left). Shown is a densitometric analysis of the immunoblots; data are shown as either a single experiment (empty box) or as means ± SD (black circle) (n = 3; *p < 0.05, Student's t test).

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