Identification and therapeutic rescue of autophagosome and glutamate receptor defects in C9ORF72 and sporadic ALS neurons
Yingxiao Shi, … , Berislav V. Zlokovic, Justin K. Ichida
Yingxiao Shi, … , Berislav V. Zlokovic, Justin K. Ichida
Published August 8, 2019; First published July 16, 2019
Citation Information: JCI Insight. 2019;4(15):e127736. https://doi.org/10.1172/jci.insight.127736.
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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 4

C9ORF72 and sporadic ALS iMNs have elevated glutamate receptor levels that are normalized by 3K3A-APC.

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C9ORF72 and sporadic ALS iMNs have elevated glutamate receptor levels t...
(A) Immunofluorescence images showing NR1+ punctae on neurites of iMNs treated with 10 nM inactive 3K3A-APC or 3K3A-APC for 6 days. Scale bar: 2 μm. This experiment was repeated 3 times with similar results. (B and C) NR1+ punctae per unit area in control, C9-ALS (B), or sporadic ALS (C) iMNs. Each gray circle represents the number of NR1+ punctae per area unit on a single neurite (1 neurite quantified per iMN). n = 33 (controls and C9-ALS) or 13 (sporadic) iMNs quantified per line per condition from 2 biologically independent iMN conversions of 2 CTRL, 2 C9-ALS, or 6 sporadic ALS lines. Median ± interquartile range. Kruskal-Wallis testing. (D) Number of calcium transients per 30 seconds in control or C9-ALS iMNs treated with 10 nM inactive 3K3A-APC or 3K3A-APC. n = 21 iMNs per line per condition from 3 biologically independent iMN conversions of 3 CTRL and 3 C9-ALS lines. For the C9-ALS plus 3K3A-APC condition, n = 19 iMNs per line. Median ± interquartile range. Kruskal-Wallis testing. (E) Number of calcium transients per 30 seconds in control or sporadic ALS iMNs treated with inactive 3K3A-APC or 3K3A-APC. n = 20 iMNs per line per condition from 3 biologically independent iMN conversions of 3 CTRL and 1 sporadic line. Median ± interquartile range. Kruskal-Wallis testing. (F) Immunoblotting of surface NR1 after surface protein biotinylation in C9-ALS iMNs generated with NGN2, ISL1, and LHX3 and treated with 10 nM inactive 3K3A-APC or 3K3A-APC for 6 days. (G) Quantification of NR1 immunoblotting from F. n = 4 biologically independent iMN conversions. Each gray circle represents an individual sample. The ratio of surface to total transferrin receptor was used to normalize for the membrane protein extraction efficiency and TUJ1 was used to normalize for neuron number. (H) Immunoblotting of surface NR1 after surface protein biotinylation in sporadic ALS iMNs (1 patient) generated with NGN2, ISL1, and LHX3 and treated with 10 nM inactive 3K3A-APC or 3K3A-APC for 6 days. The full blot for total TUJ1 is shown. (I) Quantification of NR1 immunoblotting from H. n = 4 biologically independent iMN conversions. Each gray circle represents an individual sample. The ratio of surface to total transferrin receptor was used to normalize for the membrane protein extraction efficiency and TUJ1 was used to normalize for neuron number. The day of differentiation stated on each panel indicates the day of differentiation on which the experimental treatment or time course was initiated. TF, transferrin.