Blocking p62-dependent SMN degradation ameliorates spinal muscular atrophy disease phenotypes
Natalia Rodriguez-Muela, … , Rajat Singh, Lee L. Rubin
Natalia Rodriguez-Muela, … , Rajat Singh, Lee L. Rubin
Published April 19, 2018
Citation Information: J Clin Invest. 2018;128(7):3008-3023. https://doi.org/10.1172/JCI95231.
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Research Article Neuroscience

Blocking p62-dependent SMN degradation ameliorates spinal muscular atrophy disease phenotypes

Abstract

Spinal muscular atrophy (SMA), a degenerative motor neuron (MN) disease, caused by loss of functional survival of motor neuron (SMN) protein due to SMN1 gene mutations, is a leading cause of infant mortality. Increasing SMN levels ameliorates the disease phenotype and is unanimously accepted as a therapeutic approach for patients with SMA. The ubiquitin/proteasome system is known to regulate SMN protein levels; however, whether autophagy controls SMN levels remains poorly explored. Here, we show that SMN protein is degraded by autophagy. Pharmacological and genetic inhibition of autophagy increases SMN levels, while induction of autophagy decreases these levels. SMN degradation occurs via its interaction with the autophagy adapter p62 (also known as SQSTM1). We also show that SMA neurons display reduced autophagosome clearance, increased p62 and ubiquitinated proteins levels, and hyperactivated mTORC1 signaling. Importantly, reducing p62 levels markedly increases SMN and its binding partner gemin2, promotes MN survival, and extends lifespan in fly and mouse SMA models, revealing p62 as a potential new therapeutic target for the treatment of SMA.

Authors

Natalia Rodriguez-Muela, Andrey Parkhitko, Tobias Grass, Rebecca M. Gibbs, Erika M. Norabuena, Norbert Perrimon, Rajat Singh, Lee L. Rubin

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Figure 1

Autophagy regulates SMN protein levels.

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Autophagy regulates SMN protein levels. (A) Representative immunoblot an...
(A) Representative immunoblot and quantification of protein lysates from human control fibroblasts after treatment with lysosomal inhibitors (NL), serum starvation (S–), or a combination of both (S–/NL) for 24 hours (n = 5 independent experiments). (B) Representative image of immunostaining against SMN (green) on human control fibroblasts after the indicated treatments (nuclei are labeled with DAPI, blue). Scale bar: 50 μm. (C) Representative immunoblot and quantification of protein lysates from WT mouse ESC–derived MNs infected with lentivirus carrying shRNA against Atg7 (Atg7i) or empty control (NS) for 7 days and treated for the last 24 hours of the culture with rapamycin or control media (results are expressed relative to NS control cells; n = 3 independent experiments). (D) Representative IP from HEK293T lysates transfected with HA-SMN plasmid and treated with lysosomal inhibitors (NL and HCQ), rapamycin, or the proteasome inhibitor MG132 (MG) for 24 hours and immunoblotted against ubiquitin, HA, LC3, and actin. (E) Representative immunoblot from HEK293T lysates transfected with empty vector (–) or the HA-tagged versions of SMN-FL or SMNΔ7 (Δ7) and cultured in control or amino acid–free media (EBSS), rapamycin, or both for 24 hours. (F) Representative immunoblot and quantification from human control fibroblasts after a 24-hour treatment with serum-free media, NL, or both, showing the levels of the SMN-binding partner gemin2 (member of the SMN complex) (n = 3 independent experiments). *P < 0.05 and **P < 0.01, by 2-tailed t test. All results are shown as the mean ± SEM. Ct, control; Rapa, rapamycin; Ub, ubiquitin.