Identification and therapeutic rescue of autophagosome and glutamate receptor defects in C9ORF72 and sporadic ALS neurons
Yingxiao Shi, Shu-Ting Hung, Gabriel Rocha, Shaoyu Lin, Gabriel R. Linares, Kim A. Staats, Carina Seah, Yaoming Wang, Michael Chickering, Jesse Lai, Tohru Sugawara, Abhay P. Sagare, Berislav V. Zlokovic, Justin K. Ichida
Yingxiao Shi, Shu-Ting Hung, Gabriel Rocha, Shaoyu Lin, Gabriel R. Linares, Kim A. Staats, Carina Seah, Yaoming Wang, Michael Chickering, Jesse Lai, Tohru Sugawara, Abhay P. Sagare, Berislav V. Zlokovic, Justin K. Ichida
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Research Article Neuroscience Stem cells

Identification and therapeutic rescue of autophagosome and glutamate receptor defects in C9ORF72 and sporadic ALS neurons

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease with diverse etiologies. Therefore, the identification of common disease mechanisms and therapeutics targeting these mechanisms could dramatically improve clinical outcomes. To this end, we developed induced motor neuron (iMN) models from C9ORF72 and sporadic ALS patients to identify targets that are effective against these types of cases, which together comprise approximately 90% of patients. We find that iMNs from C9ORF72 and several sporadic ALS patients share 2 common defects — impaired autophagosome formation and the aberrant accumulation of glutamate receptors. Moreover, we show that an anticoagulation-deficient form of activated protein C, 3K3A-APC, rescues these defects in both C9ORF72 and sporadic ALS iMNs. As a result, 3K3A-APC treatment lowers C9ORF72 dipeptide-repeat protein (DPR) levels, restores nuclear TDP-43 localization, and rescues the survival of both C9ORF72 and sporadic ALS iMNs. Importantly, 3K3A-APC also lowers glutamate receptor levels and rescues proteostasis in vivo in C9ORF72 gain- and loss-of-function mouse models. Thus, motor neurons from C9ORF72 and at least a subset of sporadic ALS patients share early defects in autophagosome formation and glutamate receptor homeostasis and a single therapeutic approach may be efficacious against these disease processes.

Authors

Yingxiao Shi, Shu-Ting Hung, Gabriel Rocha, Shaoyu Lin, Gabriel R. Linares, Kim A. Staats, Carina Seah, Yaoming Wang, Michael Chickering, Jesse Lai, Tohru Sugawara, Abhay P. Sagare, Berislav V. Zlokovic, Justin K. Ichida

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Figure 3

Rescue of autophagosome formation by 3K3A-APC improves proteostasis.

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Rescue of autophagosome formation by 3K3A-APC improves proteostasis. (A–...
(A–C) Immunostaining (A) and quantification (B and C) to determine endogenous poly(GR)+ punctae in control or C9-ALS iMNs with 10 nM inactive 3K3A-APC or 3K3A-APC treatment for 6 days. Quantified values represent the average number of nuclear poly(GR)+ punctae in n = 30 iMNs (controls) or 40 to 44 iMNs (C9-ALS) per line per condition from 2 control or 2 C9-ALS patient lines. For each line, iMNs were quantified from 2 independent iMN conversions per line per condition. Median ± interquartile range. Each gray circle represents the number of poly(GR)+ punctae/unit area in a single iMN. Mann-Whitney testing. Solid and dotted lines in A outline the cell body and nucleus, respectively. Scale bars: 5 μm. (D and E) Dot blot (D) and quantification (E) of poly(GR)+ levels in iMNs from 2 C9-ALS patient lines with 10 nM inactive 3K3A-APC or 3K3A-APC treatment for 6 days. Each gray circle represents 1 dot blot sample. Mean ± SD. n = 3 independent iMN conversions per line per condition. One-way ANOVA with Tukey’s correction across all comparisons. (F) Survival of control iMNs without excess glutamate with overexpression of eGFP or GR(50)-eGFP and 10 nM inactive or active 3K3A-APC. n = 90 iMNs per condition, iMNs quantified from 3 biologically independent iMN conversions. Two-sided log-rank test, corrected for multiple comparisons, statistical significance was calculated using the entire survival time course. n = 90 iMNs per condition. (G–J) Immunofluorescence analysis of total TDP-43 (G) and quantification of the ratio of nuclear to cytoplasmic TDP-43 in control, C9-ALS (H), or sporadic ALS iMNs (I and J). Ratio of nuclear to cytoplasmic TDP-43 in individual C9-ALS iMNs treated with 10 nM inactive 3K3A-APC or 3K3A-APC for 6 days. iMNs from 2 controls, 2 C9-ALS, and 4 sporadic ALS patients were quantified. n = 30 iMNs per line (control and C9-ALS) per condition or n = 26 iMNs (I), 30 iMNs (J) (inactive 3K3A-APC), or n = 35 iMNs (J) (3K3A-APC) (sporadic ALS) per condition per line from 2 biologically independent iMN conversions were quantified. Each gray circle represents a single iMN. For H, median ± interquartile range. Kruskal-Wallis testing. For I and J, mean ± SEM. Unpaired t test with Welch’s correction. Scale bars: 5 μm. Dotted lines outline the nucleus and cell body. The day of differentiation stated on each panel indicates the day of differentiation on which the experimental treatment or time course was initiated.