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. 2016 Aug 2;16(5):1416-1430.
doi: 10.1016/j.celrep.2016.06.087. Epub 2016 Jul 21.

A Stem Cell Model of the Motor Circuit Uncouples Motor Neuron Death From Hyperexcitability Induced by SMN Deficiency

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A Stem Cell Model of the Motor Circuit Uncouples Motor Neuron Death From Hyperexcitability Induced by SMN Deficiency

Christian M Simon et al. Cell Rep. .
Free PMC article

Abstract

In spinal muscular atrophy, a neurodegenerative disease caused by ubiquitous deficiency in the survival motor neuron (SMN) protein, sensory-motor synaptic dysfunction and increased excitability precede motor neuron (MN) loss. Whether central synaptic dysfunction and MN hyperexcitability are cell-autonomous events or they contribute to MN death is unknown. We addressed these issues using a stem-cell-based model of the motor circuit consisting of MNs and both excitatory and inhibitory interneurons (INs) in which SMN protein levels are selectively depleted. We show that SMN deficiency induces selective MN death through cell-autonomous mechanisms, while hyperexcitability is a non-cell-autonomous response of MNs to defects in pre-motor INs, leading to loss of glutamatergic synapses and reduced excitation. Findings from our in vitro model suggest that dysfunction and loss of MNs result from differential effects of SMN deficiency in distinct neurons of the motor circuit and that hyperexcitability does not trigger MN death.

Figures

Figure 1
Figure 1. Smn deficiency induces MN death through cell autonomous mechanisms
(A) Schematic representation of the lentiviral vectors used to generate the ES(Hb9:GFP)-SmnRNAi cell line. (B) RT-qPCR analysis of Smn mRNA levels in WT and RNAi ES cells cultured with and without Dox for 4 days. RNA levels in Dox-treated cells are expressed relative to those in untreated cells. Data are represented as mean and SEM from independent experiments (n=3). (C) Western blot analysis of Smn protein levels in WT and RNAi ES cells cultured with and without Dox for 4 days. A two-fold serial dilution of WT ES cell extract is analyzed on the left. (D) RT-qPCR analysis of Smn mRNA levels in FACS-purified MNs differentiated from WT and RNAi ES cells cultured with and without Dox for 5 days. RNA levels in Dox-treated cells are expressed relative to those in untreated cells. Data are represented as mean and SEM from independent experiments (n=3). (E) Western blot analysis of Smn protein levels in mixed cultures of ES-MNs cultured with and without Dox for 5 days after differentiation from WT and RNAi ES cells. A two-fold serial dilution of the extract from WT ES-MNs is analyzed on the left. (F) Representative 96-well image of GFP+ MNs differentiated from WT ES cells at 5DIV. Scale bar=800µm. (G) MetaMorph software analysis of GFP+ MNs from the image in F. Scale bar=800µm. (H) Survival analysis of GFP+ MNs in mixed cultures following differentiation from WT, RNAi and RNAi+SMN ES cells cultured with and without Dox. Normalized data of Dox-treated relative to untreated cells are shown as mean and SEM from independent experiments (n≥6). (I) Survival analysis of FACS purified GFP+ MNs following differentiation from WT, RNAi and RNAi+SMN ES cells cultured with and without Dox. Normalized data of Dox-treated relative to untreated cells are shown as mean and SEM from independent experiments (n=3). See also Figure S1.
Figure 2
Figure 2. Smn deficiency induces MN hyperexcitability non-cell autonomously
(A) Representative images of intracellularly filled control (−Dox) and Smn-deficient (+Dox) MNs differentiated from RNAi ES cells and cultured under mixed conditions for 5 days. Upper panel: Alexa-555 fill in red; lower panel: merged with GFP in green. Scale bar=20µm. (B) Superimposed membrane responses (upper traces) following current injection (lower traces) in control and Smn-deficient MNs as in A. Scale bars=20mV, 40pA, 40ms. (C) Current/voltage relationships for the MNs shown in B (Input resistance: −Dox= 534MΩ, +Dox= 782MΩ) and averages for input resistance (Rin) and time constant (τ) of control (n=15) and Smn-deficient (n=14) MNs at 5DIV. (D) Representative images of intracellularly filled control (−Dox) and Smn-deficient (+Dox) MNs differentiated from RNAi ES cells and cultured for 5 days after FACS purification. Upper panel: Alexa-555 fill in red; lower panel: merged with GFP in green. Scale bar= 20µm. (E) Superimposed membrane responses (upper traces) following current injection (lower traces) of control and Smn-deficient FACS purified MNs as in D. Scale bars= 20mV, 40pA, 40ms. (F) Current/voltage relationships for the two FACS purified MNs shown in E (Input resistance: −Dox=582MΩ, +Dox=598MΩ) and average input resistance and time constant of FACS purified control (n=15) and Smn-deficient (n=15) MNs at 5DIV. See also Figure S2.
Figure 3
Figure 3. Smn deficiency does not alter the somato-dendritic area of MNs
(A) Single-plane confocal images of Alexa-555 intracellularly-filled control (−Dox) and Smn-deficient (+Dox) MNs differentiated from RNAi ES cells in mixed cultures at 5DIV (upper panels) and Neurolucida reconstructions of their dendritic trees (bottom panels). (B) Quantification of dendritic tree, soma area and capacitance of control (n=13) and Smn-deficient (n=12) MNs in mixed cultures at 5DIV. (C) Single-plane confocal images of Alexa-555 intracellularly-filled control (−Dox) and Smn-deficient (+Dox) FACS purified MNs differentiated from RNAi ES cells at 5DIV (upper panels) and Neurolucida reconstruction of their dendritic trees (bottom panels). (D) Quantification of dendritic tree, soma area and capacitance of control (n=9) and Smn-deficient (n=7) MNs in FACS purified cultures at 5DIV. Arrows indicate putative axons. Scale bars=40µm. See also Figure S3.
Figure 4
Figure 4. Development of ES cell lines for the purification of INs with inducible Smn knockdown
(A) Schematic representation of the lentiviral vector used to generate WT and RNAi ES(Hb9::GFP;Syn::mCherry) cell lines. (B) Representative images of ES(Hb9::GFP;Syn::mCherry)-derived neuronal cultures expressing mCherry (red) and stained with Tuj1 (green) and DAPI (blue) at 5DIV. (C) Quantification of mCherry+/Tuj1+ INs in neuronal cultures differentiated from WT and RNAi ES(Hb9::GFP;Syn::mCherry) cells as in B (n≥3). Scale bar=100µm. (D) RT-qPCR analysis of Smn mRNA levels in WT and RNAi ES(Hb9::GFP;Syn::mCherry) cells cultured with and without Dox for 4 days. RNA levels in Dox-treated cells are expressed relative to those in untreated cells. Data are represented as mean and SEM from independent experiments (n=3). (E) Western blot analysis of Smn protein levels in WT and RNAi ES cells cultured as in D. A two-fold serial dilution of WT ES cell extract is analyzed on the left. (F) RT-qPCR analysis of Smn mRNA levels in FACS-purified INs differentiated from WT and RNAi ES cells cultured with and without Dox for 5 days. RNA levels in Dox-treated cells are expressed relative to those in untreated cells. Data are represented as mean and SEM from independent experiments (n=3). (G) Western blot analysis of Smn protein levels in mixed cultures of ES-INs cultured with and without Dox for 5 days after differentiation from WT and RNAi ES cells. A two-fold serial dilution of the extract from WT ES-INs is analyzed on the left. See also Figure S4.
Figure 5
Figure 5. Smn deficiency in pre-motor INs induces MN hyperexcitability
(A) Schematic representation of the design for co-culture experiments with FACS purified MNs and INs. (B) Representative confocal images of an intracellularly filled (blue), GFP+ (green) MN co-cultured with mCherry+ (red) INs. Scale bar=50µm. (C) Superimposed membrane responses (upper traces) following current injection (lower traces) and current/voltage relationships for MNs recorded in all four combinations. Scale bars=20mV, 40pA, 40ms. For ease of comparison, in each graph the black dotted line represents the current/voltage relationship under wild-type conditions. (D) Mean input resistance (Rin) and time constant (τ) of MNs recorded in the four different combinations. MN(WT)+IN(WT), n=12; MN(RNAi)+IN(RNAi), n=13; MN(RNAi)+IN(WT), n=18; MN(WT)+IN(RNAi), n=17. See also Figure S5.
Figure 6
Figure 6. Smn deficiency in INs leads to loss of excitatory synapses onto MNs
(A–B) Superimposed images of VGluT2 (green) and Syp (red) immunoreactivity on the soma and the dendrites of a Cascade blue intracellularly-filled MN (blue) in co-cultures of MNs and INs differentiated from either WT or RNAi ES cells as indicated. Arrows indicate VGluT2 synapses. (C) Number of VGluT2 synapses relative to all Syp+ synapses onto the soma and dendrites of MNs from all four co-culture combinations. MN(WT)+IN(WT), n=11; MN(RNAi)+IN(RNAi), n=12; MN(RNAi)+IN(WT), n=10; MN(WT)+IN(RNAi, n=10. (D–E) Superimposed images of VGAT (green) and Syp (red) immunoreactivity on the soma and the dendrites of MNs as in A. Arrows indicate VGAT synapses. (F) Number of VGAT synapses relative to all Syp+ synapses onto the soma and dendrites of MNs from two co-culture combinations. MN(WT)+IN(WT), n=8; MN(RNAi)+IN(RNAi), n=7. Scale bar=10µm (soma) and 5µm (dendrites). See also Figure S6.
Figure 7
Figure 7. Block of excitatory neurotransmission induces hyperexcitability but not death of MNs
(A) Superimposed membrane responses (upper traces) following current injection (lower traces) of WT MNs in mixed cultures under different pharmacological conditions. Graphs show current/voltage relationships for the corresponding MNs in each treatment group. The current/voltage relationship of the control MN (blue dotted line) is included in each graph for ease of comparison. Scale bars=20mV, 40pA, 40ms. (B) Mean input resistance (Rin) and time constant (τ) for all experimental groups. Number of recorded MNs: control, n=24; TTX, n=15; Ach blockers, n=12, Gly+GABA blockers, n=13, Glu blockers, n=11. (C) Survival curves of MNs for all experimental groups normalized to day 0. Data represent mean and SEM from independent experiments (n=3). See also Figure S7.

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