Stress Granules Protect ER-exit Sites from TDP43 Aggregation

Stress Granules Protect ER-exit Sites from TDP43 Aggregation

Hongyi Wu¹, Loo Chien Wang², Belle M. Sow¹, Damien Leow³, Jin Zhu¹, Kathryn M. Gallo^{4, 5}, Kathleen Wilsbach^{4, 5}, Roshni Gupta¹, Lyle W. Ostrow^{4, 5, *}, Crystal J. J. Yeo^{6 - 10, *}, Radoslaw M. Sobota^{2, *}, Rong Li^{1, 11, 12}

¹ Mechanobiology Institute, National University of Singapore (NUS), Singapore

² Functional Proteomics Laboratory, SingMass National Laboratory, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore

³ Department of Anatomy, Yong Loo-Lin School of Medicine, National University of Singapore, Singapore

⁴ Department of Neurology, School of Medicine, Johns Hopkins University, USA

⁵ Department of Neurology, Lewis Katz School of Medicine, Temple University, USA

⁶ National Neuroscience Institute and A*STAR, Singapore

⁷ Duke-NUS Medical School, Singapore

⁸ Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore

⁹ Department of Neurology, Feinberg School of Medicine, Northwestern University, USA

¹⁰ School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, UK

¹¹ Department of Biological Sciences, National University of Singapore, Singapore

¹² Center for Cell Dynamics and Department of Cell Biology, School of Medicine, Johns Hopkins University, USA

Background: Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that causes the loss of motor neurons (MNs). While TDP43 protein aggregation is a well-known hallmark of ALS, endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation have been consistently observed in patient MNs. As the first organelle along the secretory pathway, ER takes in secretory proteins from the cytoplasmic side, facilitates their folding, and exports them via ER-exit sites (ERES), where COPII vesicles bud to transport cargos from ER to Golgi. Although anomalies in the ER have been suggested to compromise synapse maintenance and function in ALS, the cause for ER stress and its relationship to TDP43 aggregation have been elusive.

Aims: We serendipitously discovered TDP43 aggregation in ERES in proteome-wide identification of aggregation-prone proteins and pairwise protein coaggregation analysis. We therefore investigated the formation of this type of aggregate, its relationship to TDP43 aggregation in stress granules (SGs), and physiological consequences.

Methods: We repurposed the yeast chaperone Hsp104 bearing Double Walker B (DWB) mutations as a broad-spectrum marker for protein aggregates in mammalian cells. Hsp104^DWB-mEGFP-FLAG enabled not only live imaging of stress-induced protein aggregates but also affinity-based aggregate isolation. Mass-spectrometry analysis identified over 300 proteins in aggregates purified from human cells cultured in vitro. To reveal the intracellular landscape of protein aggregation, we performed pairwise coaggregation analysis of validated aggregate proteins in human RPE-1 immortalized cell line. This led to the discovery of TDP43 coaggregation with SEC16A, the scaffold protein of ERES. We further characterized TDP43/SEC16A coaggregates using bioimaging and biochemical approaches, and identified such aggregates in ALS patient induced pluripotent stem cell (iPSC)-derived MNs and postmortem samples.

Results: Under proteotoxic stress, TDP43 aggregates in pre-formed ERES with SEC16A and COPII proteins such as SEC24D and SEC31. This occurs mostly in cells that display SG disassembly, and stimulating or suppressing SG assembly through manipulation of G3BP1 concentration respectively inhibits or exacerbates TDP43-ERES aggregation. While ordinary ERES and SGs are both “liquid-like” structures, TDP43-enriched ERES become solid aggregates. Furthermore, TDP43 aggregation in ERES impairs the latter’s function in ER-to-Golgi protein transport and frequently results in ER dilation, indicating ER stress and UPR activation. Unlike SGs, TDP43-ERES aggregates are RNA-free and enhanced by RNA-binding mutations and ALS-associated mutations that fall in the RNA-recognition motifs of TDP43. TDP43-enriched ERES are found at higher percentage in ALS patient iPSC-derived MNs and postmortem sections of the motor cortex.

Conclusion: Cytoplasmic TDP43 partitions dynamically between liquid-like SGs and solid aggregates in ERES. When the capacity of SGs to sequester TDP43 is reduced or saturated, TDP43 accumulates in ERES to compromise ER-to-Golgi vesicle transport. Our discoveries not only revealed the protective role of SGs for the secretory pathway but also links TDP43 aggregation and ER stress, both observed in ALS patient neurons.

This research was funded by grants A-0006324-03-00 and A-0007081-00-00 from Singapore Ministry of Education, and by the A*STAR core funding and Singapore National Research Foundation (under its NRF-SIS “SingMass” scheme).

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