TDP-43 enhances translation of specific mRNAs linked to neurodegenerative disease

Published online 24 October 2018 Nucleic Acids Research, 2019, Vol. 47, No. 1 341–361 doi: 10.1093/nar/gky972 TDP-43 enhances translation of specific mRNAs linked to neurodegenerative disease Nagammal Neelagandan1 , Giorgio Gonnella2 , Stefan Dang2 , Philipp C. Janiesch1 , Katharine K. Miller1 , Katrin Küchler1 , Rita F. Marques1 , Daniela Indenbirken3 , Malik Alawi3,4 , Adam Grundhoff3 , Stefan Kurtz2 and Kent E. Duncan1,* 1 Received September 05, 2018; Revised October 03, 2018; Editorial Decision October 04, 2018; Accepted October 08, 2018 ABSTRACT INTRODUCTION The RNA-binding protein TDP-43 is heavily implicated in neurodegenerative disease. Numerous patient mutations in TARDBP, the gene encoding TDP43, combined with data from animal and cell-based models, imply that altered RNA regulation by TDP43 causes Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. However, underlying mechanisms remain unresolved. Increased cytoplasmic TDP-43 levels in diseased neurons suggest a possible role in this cellular compartment. Here, we examined the impact on translation of overexpressing human TDP-43 and the TDP-43A315T patient mutant protein in motor neuron-like cells and primary cultures of cortical neurons. In motor-neuron like cells, TDP-43 associates with ribosomes without significantly affecting global translation. However, ribosome profiling and additional assays revealed enhanced translation and direct binding of Camta1, Mig12, and Dennd4a mRNAs. Overexpressing either wild-type TDP-43 or TDP-43A315T stimulated translation of Camta1 and Mig12 mRNAs via their 5 UTRs and increased CAMTA1 and MIG12 protein levels. In contrast, translational enhancement of Dennd4a mRNA required a specific 3 UTR region and was specifically observed with the TDP-43A315T patient mutant allele. Our data reveal that TDP-43 can function as an mRNA-specific translational enhancer. Moreover, since CAMTA1 and DENND4A are linked to neurodegeneration, they suggest that this function could contribute to disease. TDP-43 is an RNA-binding protein and a major component of ubiquitinated aggregates in motor neurons that are pathological hallmarks of two related neurodegenerative diseases: Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) (1–3). In support of a causal link between altered TDP-43 function and disease, numerous patient mutations have been identified in the TARDBP gene, which codes for TDP-43 (4,5). Nevertheless, most patients do not have TDP-43 mutations, suggesting that altered function of wild-type (WT) TDP-43 may be important in these patients. In healthy cells, TDP-43 is primarily localized in the nucleus, whereas in disease it is significantly increased in the cytoplasm, sometimes concomitant with depletion from the nucleus. Altered TDP-43 localization has also been observed in other neurodegenerative diseases, including Alzheimer’s (6), as well as in traumatic brain injury (7). Disease models based on altered TDP-43 expression in animals and cultured cells have revealed common features of TDP-43 pathophysiology (8). For example, TDP43 s RNA-binding activity is essential for toxicity (9) and disease-like symptoms do not depend on formation of aggregates per se (10). While other cells are clearly involved in ALS pathology (11), expression of mutant TDP-43 in motor neurons alone can lead to symptoms (12). Moreover, simply overexpressing WT hTDP-43 at a high enough level can lead to disease symptoms (13) and mutant alleles may lead to higher TDP-43 protein levels (14). Collectively, these studies support a model in which altered regulation of one or more cellular RNAs bound by TDP-43 causes disease (15). Experiments carried out to study the function of TDP43 have revealed its direct physical RNA targets in specific * To whom correspondence should be addressed. Tel: +49 40 7410 56274; Fax: +49 40 7410 53436; Email: Present address: Stefan Dang, AKRA GmbH, 20095 Hamburg, Germany.  C The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Neuronal Translational Control Research Group, Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), Hamburg 20251, Germany, 2 Universität Hamburg, MIN-Fakultät, ZBH––Center for Bioinformatics, Hamburg 20146, Germany, 3 Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg 20251, Germany and 4 Bioinformatics Core, University Medical Center Hamburg-Eppendorf (UKE), Hamburg 20251, Germany 342 Nucleic Acids Research, 2019, Vol. 47, No. 1 mic TDP-43 might directly regulate translation of specific mRNAs comes from CLIP-Seq experiments demonstrating that TDP-43 binds to many mRNAs in their 3 UTR, a major region for translational control by RNA-binding proteins (16,17). In Drosophila, TDP-43 negatively regulates translation of futsch (26) and hsc 70-4 (27). Two studies in cultured mammalian neurons also provide additional evidence for translational repression by TDP-43 of Rac1, Map1b and GluR1 (GluA1) mRNAs (28,29). These directed studies with individual mRNAs indicate that TDP-43 represses translation of specific mRNAs under some circumstances. They also motivate a broader investigation of TDP43 s impact on translation and how it might contribute to neurodegenerative disease. Here, we apply genome-wide ribosome profiling to two cell culture models of the cell types affected in ALS and FTD patients: spinal motor neurons and cortical neurons. In combination with several downstream assays, this enabled us to identify three new translational target mRNAs of the human TDP-43A315T mutant: Camta1, Mig12 and Dennd4a. Interestingly, CAMTA1 and DENND4A both have strong links to neurodegenerative disease. We show that TDP-43 directly binds to these mRNAs via crosslinkIP, supporting direct regulation. Moreover, overexpressing either WT hTDP-43 or the TDP-43A315T patient mutant protein could stimulate translation of Camta1 and Mig12 5 UTR reporter mRNAs and led to increased levels of endogenous proteins. In contrast, translational enhancement of Dennd4a mRNA was only observed with the patient mutant allele via the 3 UTR. Collectively, our data demonstrate a novel function for TDP-43 as an mRNAspecific translational enhancer and further suggest that this function could be an important component of the diseasecausing mechanism. MATERIALS AND METHODS Animal welfare and approvals All animal care and experimental procedures were performed according to UKE Animal Research Facility institutional guidelines and conformed to the requirements of the German Animal Welfare Act. Relevant approvals: ORG 765 and G14/003 Zucht Neuro. Cell line and primary neuronal culture (...truncated)