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Spinal muscular atrophy patient-derived motor neurons exhibit hyperexcitability.

Liu H, Lu J, Chen H, Du Z, Li XJ, Zhang SC - Sci Rep (2015)

Bottom Line: Using SMA induced pluripotent stem cells (iPSCs), we found that SMA MNs displayed hyperexcitability with increased membrane input resistance, hyperpolarized threshold, and larger action potential amplitude, which was mimicked by knocking down full length survival motor neuron (SMN) in non-SMA MNs.We further discovered that SMA MNs exhibit enhanced sodium channel activities with increased current amplitude and facilitated recovery, which was corrected by restoration of SMN1 in SMA MNs.Together we propose that SMN reduction results in MN hyperexcitability and impaired neurotransmission, the latter of which exacerbate each other via a feedback loop, thus contributing to severe symptoms at an early stage of SMA.

View Article: PubMed Central - PubMed

Affiliation: Waisman center, University of Wisconsin, Madison, WI, 53705, USA.

ABSTRACT
Spinal muscular atrophy (SMA) presents severe muscle weakness with limited motor neuron (MN) loss at an early stage, suggesting potential functional alterations in MNs that contribute to SMA symptom presentation. Using SMA induced pluripotent stem cells (iPSCs), we found that SMA MNs displayed hyperexcitability with increased membrane input resistance, hyperpolarized threshold, and larger action potential amplitude, which was mimicked by knocking down full length survival motor neuron (SMN) in non-SMA MNs. We further discovered that SMA MNs exhibit enhanced sodium channel activities with increased current amplitude and facilitated recovery, which was corrected by restoration of SMN1 in SMA MNs. Together we propose that SMN reduction results in MN hyperexcitability and impaired neurotransmission, the latter of which exacerbate each other via a feedback loop, thus contributing to severe symptoms at an early stage of SMA.

No MeSH data available.


Related in: MedlinePlus

AP properties of control MNs after SMN-FL knockdown.(a) Relative expression of SMN-FL mRNA measured by qPCR in MNs differentiated from control (Luc) and SMN-FL knockdown (SMNi) hESCs group (***p < 0.001; unpaired Student’s t-test. N = 3 independent experiments). (b) Relative expression of SMN-FL proteins measured by western blots in MNs differentiated from Luc and SMNi group (***p < 0.001; unpaired Student’s t-test. N = 3 independent experiments). All the groups were collected and sampled under the same conditions. The cropped blots images are shown in the figure and the full-length blots are presented in Supplementary Fig. S3b. (c) Representative traces of induced APs upon current injections (from –40 pA to 100 pA with 10 pA step for 500 ms) in MNs differentiated from Luc and SMNi ESCs. (d) The cell lysates from MNs with overexpression of Flag-tagged GFP or SMN1 in MNs from SMNi and SMA-1 groups (SMNi+GFP, SMA-1+GFP; SMNi+SMN1, SMA-1+SMN1) were analyzed by western blots using antibodies anti-Flag, -SMN1 and -NSE. Note, anti-SMN1 antibody reads two separate proteins: exogenously expressed Flag-SMN1 and endogenous residual SMN-FL produced by SMN2 gene. All the groups were collected and sampled under the same conditions. The cropped blots images are shown in the figure and the full-length blots are presented in Supplementary Fig. S3c. (e) Relative expression of total SMN-FL proteins measured by western blots in different MN groups in the 7th week after differentiation (***p < 0.001; one-way ANOVA with post hoc test. N = 3 independent experiments). (f) Representative traces of induced APs upon current injections (from –40 pA to 100 pA with 10 pA step for 500 ms) in MNs from different groups. (g) Plot of AP frequency against injected current size for each group (***p < 0.001; two-way ANOVA. N = 18 ~ 20 neurons for each condition). All data shown represent mean ± SEM.
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f4: AP properties of control MNs after SMN-FL knockdown.(a) Relative expression of SMN-FL mRNA measured by qPCR in MNs differentiated from control (Luc) and SMN-FL knockdown (SMNi) hESCs group (***p < 0.001; unpaired Student’s t-test. N = 3 independent experiments). (b) Relative expression of SMN-FL proteins measured by western blots in MNs differentiated from Luc and SMNi group (***p < 0.001; unpaired Student’s t-test. N = 3 independent experiments). All the groups were collected and sampled under the same conditions. The cropped blots images are shown in the figure and the full-length blots are presented in Supplementary Fig. S3b. (c) Representative traces of induced APs upon current injections (from –40 pA to 100 pA with 10 pA step for 500 ms) in MNs differentiated from Luc and SMNi ESCs. (d) The cell lysates from MNs with overexpression of Flag-tagged GFP or SMN1 in MNs from SMNi and SMA-1 groups (SMNi+GFP, SMA-1+GFP; SMNi+SMN1, SMA-1+SMN1) were analyzed by western blots using antibodies anti-Flag, -SMN1 and -NSE. Note, anti-SMN1 antibody reads two separate proteins: exogenously expressed Flag-SMN1 and endogenous residual SMN-FL produced by SMN2 gene. All the groups were collected and sampled under the same conditions. The cropped blots images are shown in the figure and the full-length blots are presented in Supplementary Fig. S3c. (e) Relative expression of total SMN-FL proteins measured by western blots in different MN groups in the 7th week after differentiation (***p < 0.001; one-way ANOVA with post hoc test. N = 3 independent experiments). (f) Representative traces of induced APs upon current injections (from –40 pA to 100 pA with 10 pA step for 500 ms) in MNs from different groups. (g) Plot of AP frequency against injected current size for each group (***p < 0.001; two-way ANOVA. N = 18 ~ 20 neurons for each condition). All data shown represent mean ± SEM.

Mentions: MNs from SMA transgenic mice1415 and patients (this study) display hyperexcitability. To establish the relationship between the reduction of SMN-FL and the hyperexcitability, we performed two sets of experiments. First, we examined the AP activities on MNs differentiated from our established hESC lines with RNAi knockdown of SMN-FL (SMNi) or luciferase (Luc, as a control)25. qPCR and Western blot revealed a reduction of nearly 80% SMN-FL mRNA (Fig. 4a) and protein (Fig. 4b and Supplementary Fig. S3b). Using a similar recording regimen, we found that MNs from SMNi ESCs displayed an increased AP frequency and smaller rheobase as compared to the Luc group (Fig. 4c,g). This result suggests that SMN reduction results in MN hyperexcitability.


Spinal muscular atrophy patient-derived motor neurons exhibit hyperexcitability.

Liu H, Lu J, Chen H, Du Z, Li XJ, Zhang SC - Sci Rep (2015)

AP properties of control MNs after SMN-FL knockdown.(a) Relative expression of SMN-FL mRNA measured by qPCR in MNs differentiated from control (Luc) and SMN-FL knockdown (SMNi) hESCs group (***p < 0.001; unpaired Student’s t-test. N = 3 independent experiments). (b) Relative expression of SMN-FL proteins measured by western blots in MNs differentiated from Luc and SMNi group (***p < 0.001; unpaired Student’s t-test. N = 3 independent experiments). All the groups were collected and sampled under the same conditions. The cropped blots images are shown in the figure and the full-length blots are presented in Supplementary Fig. S3b. (c) Representative traces of induced APs upon current injections (from –40 pA to 100 pA with 10 pA step for 500 ms) in MNs differentiated from Luc and SMNi ESCs. (d) The cell lysates from MNs with overexpression of Flag-tagged GFP or SMN1 in MNs from SMNi and SMA-1 groups (SMNi+GFP, SMA-1+GFP; SMNi+SMN1, SMA-1+SMN1) were analyzed by western blots using antibodies anti-Flag, -SMN1 and -NSE. Note, anti-SMN1 antibody reads two separate proteins: exogenously expressed Flag-SMN1 and endogenous residual SMN-FL produced by SMN2 gene. All the groups were collected and sampled under the same conditions. The cropped blots images are shown in the figure and the full-length blots are presented in Supplementary Fig. S3c. (e) Relative expression of total SMN-FL proteins measured by western blots in different MN groups in the 7th week after differentiation (***p < 0.001; one-way ANOVA with post hoc test. N = 3 independent experiments). (f) Representative traces of induced APs upon current injections (from –40 pA to 100 pA with 10 pA step for 500 ms) in MNs from different groups. (g) Plot of AP frequency against injected current size for each group (***p < 0.001; two-way ANOVA. N = 18 ~ 20 neurons for each condition). All data shown represent mean ± SEM.
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Related In: Results  -  Collection

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f4: AP properties of control MNs after SMN-FL knockdown.(a) Relative expression of SMN-FL mRNA measured by qPCR in MNs differentiated from control (Luc) and SMN-FL knockdown (SMNi) hESCs group (***p < 0.001; unpaired Student’s t-test. N = 3 independent experiments). (b) Relative expression of SMN-FL proteins measured by western blots in MNs differentiated from Luc and SMNi group (***p < 0.001; unpaired Student’s t-test. N = 3 independent experiments). All the groups were collected and sampled under the same conditions. The cropped blots images are shown in the figure and the full-length blots are presented in Supplementary Fig. S3b. (c) Representative traces of induced APs upon current injections (from –40 pA to 100 pA with 10 pA step for 500 ms) in MNs differentiated from Luc and SMNi ESCs. (d) The cell lysates from MNs with overexpression of Flag-tagged GFP or SMN1 in MNs from SMNi and SMA-1 groups (SMNi+GFP, SMA-1+GFP; SMNi+SMN1, SMA-1+SMN1) were analyzed by western blots using antibodies anti-Flag, -SMN1 and -NSE. Note, anti-SMN1 antibody reads two separate proteins: exogenously expressed Flag-SMN1 and endogenous residual SMN-FL produced by SMN2 gene. All the groups were collected and sampled under the same conditions. The cropped blots images are shown in the figure and the full-length blots are presented in Supplementary Fig. S3c. (e) Relative expression of total SMN-FL proteins measured by western blots in different MN groups in the 7th week after differentiation (***p < 0.001; one-way ANOVA with post hoc test. N = 3 independent experiments). (f) Representative traces of induced APs upon current injections (from –40 pA to 100 pA with 10 pA step for 500 ms) in MNs from different groups. (g) Plot of AP frequency against injected current size for each group (***p < 0.001; two-way ANOVA. N = 18 ~ 20 neurons for each condition). All data shown represent mean ± SEM.
Mentions: MNs from SMA transgenic mice1415 and patients (this study) display hyperexcitability. To establish the relationship between the reduction of SMN-FL and the hyperexcitability, we performed two sets of experiments. First, we examined the AP activities on MNs differentiated from our established hESC lines with RNAi knockdown of SMN-FL (SMNi) or luciferase (Luc, as a control)25. qPCR and Western blot revealed a reduction of nearly 80% SMN-FL mRNA (Fig. 4a) and protein (Fig. 4b and Supplementary Fig. S3b). Using a similar recording regimen, we found that MNs from SMNi ESCs displayed an increased AP frequency and smaller rheobase as compared to the Luc group (Fig. 4c,g). This result suggests that SMN reduction results in MN hyperexcitability.

Bottom Line: Using SMA induced pluripotent stem cells (iPSCs), we found that SMA MNs displayed hyperexcitability with increased membrane input resistance, hyperpolarized threshold, and larger action potential amplitude, which was mimicked by knocking down full length survival motor neuron (SMN) in non-SMA MNs.We further discovered that SMA MNs exhibit enhanced sodium channel activities with increased current amplitude and facilitated recovery, which was corrected by restoration of SMN1 in SMA MNs.Together we propose that SMN reduction results in MN hyperexcitability and impaired neurotransmission, the latter of which exacerbate each other via a feedback loop, thus contributing to severe symptoms at an early stage of SMA.

View Article: PubMed Central - PubMed

Affiliation: Waisman center, University of Wisconsin, Madison, WI, 53705, USA.

ABSTRACT
Spinal muscular atrophy (SMA) presents severe muscle weakness with limited motor neuron (MN) loss at an early stage, suggesting potential functional alterations in MNs that contribute to SMA symptom presentation. Using SMA induced pluripotent stem cells (iPSCs), we found that SMA MNs displayed hyperexcitability with increased membrane input resistance, hyperpolarized threshold, and larger action potential amplitude, which was mimicked by knocking down full length survival motor neuron (SMN) in non-SMA MNs. We further discovered that SMA MNs exhibit enhanced sodium channel activities with increased current amplitude and facilitated recovery, which was corrected by restoration of SMN1 in SMA MNs. Together we propose that SMN reduction results in MN hyperexcitability and impaired neurotransmission, the latter of which exacerbate each other via a feedback loop, thus contributing to severe symptoms at an early stage of SMA.

No MeSH data available.


Related in: MedlinePlus