TDP-43 and FUS/TLS: Emerging Roles in RNA Processing and Neurodegeneration

1. TDP-43 and FUS/TLS: Emerging Roles in RNA Processing and Neurodegeneration By ClotildeLagier-Tourenne, MagdaliniPolymenidou, and Don W. Cleveland Powerpoint Presentation by Jonah Cader

2. Overview: Amyotrophic lateral sclerosis, commonly known as ALS or Lou Gehrig’s disease, is an adult onset, ultimately fatal, neurodegenerative disorder. Over a typical course of 1-5 years, it is characterized by progressive paralysis and the loss of both both upper and lower motor neurons. No cure and no substantial clinical treatments exist. For years, ALS research was nearly impossible (as over 90% of cases are sporadic), but the identification of toxic mutant SOD1 in ALS pathogenesis led the advent of molecular and genetic lines of inquiry. Lower Motor Neurons: Primary motor neurons of the spinal cord and brainstem- that directly innervate skeletal muscle Upper Motor Neuron: Originating in higher regionsofthe brain (ex. the motor cortex), they synapse on the lower motor neurons to convey descending commands for movement

3. Since then the field has completely shifted directions. Discoveries have been rapid as the focus has become understanding the role of RNA processing/regulation in neurodegeneration. • Most notably Cytoplasmicmislocalized aggregates of both FUS/TLS and TDP-43 have been linked to not only ALS but numerous other neurodegenerartive disorders. SOD1 Mouse Model: Breakthrough in the field; genome contains a mutation in a gene coding for superoxide dismutase 1 (an enzyme that destroys free radicals). Over 150 mutations across 3 SOD1 genes have been linked to familial ALS in humans FTLD: Frontotemporal lobar degeneration – another neurodegenerative disorder that causes various language and behavioral issues. CytoplasmicMislocalized Aggregates: Also called “inclusions,” or “pre-inclusions,” they are abnormal protein deposits within the cytoplasm

4. The Players: TDP-43 and FUS/TLS • FUS/TLS: • Name stands for fused in sarcoma/translocated in liposarcoma • Also a RNA/DNA binding protein, it is composed of 526 amino acids encoded by 15 exons • Binds single-stranded RNA and double-stranded DNA. Affinity for 3’ UTR of actin-stabilizing protein mRNA • TDP-43: • Name stands for 43 kDatransactive response (TAR) DNA-Binding protein • Protein composed of 414 amino acids, coded for by six exons and two RNA recognition motifs • C-terminal glycine-rich region is key to interaction with other proteins • Both are multifunctional proteins, mainly localized within the nucleus (though low levels accumulate in the cytoplasm of most cells). They play a series of critical roles in multiple steps of RNA processing: Transcription, splicing, transport, translation, and several other pathways

5. Proposed Physiological Roles of TDP-43 and FUS/TLS

6. In General… • Transcription: TDP-43 Binds single-stranded segments of promoter region (in TG-rich segments), blocking downstream gene transcription. FUS/TLS assumed to participate in general transcription (associated with a variety of machinery). DNA damage triggers the recruitment of FUS/TLS to promoter regions of cyclin D1 by ncRNAs, repressing CCND1 • Splicing: TDP-43 binds intronic UG segments located before alternately spliced exons thereby increasing the likelihood of their exclusion. FUS/TLS is a component of the spliceosome • miRNA Processing: Identified in a Drosha-containing complex • Nucleo-Cytoplasmic Shuttling: Both proteins move between these two regions (roles yet unknown) • Stress Granules: Form complexes with mRNA and other RNA binding proteins • RNA Transport and Local Translocation: involved in moving mRNA to dendritic spine/axonal regions where they may facilitate some local translation

7. Some Specifics… • Transcription: TDP-43 associates with euchromatin (during transcription of genes). FUS/TLS interacts with RNA polymerase II possibly influencing initiation • Splicing: Splicing alterations and errors have been reported in sporadic cases. The roles of TDP-43 and FUS/TLS in these errors are still being investigated • miRNA Processing: Associate with Drosha, the protein that regulates the first step of miRNA maturation. TDP-43 may be involved in Dicer complex-mediated cleaving • RNA Regulation, Localization, Translation, and decay: Critical components of stress granules (cytoplasmic foci made of mRNA and other complexes that delay translation under stress conditions). TDP-43 may help stabilize and transport low molecular weight neurofilament mRNA to injuries sites. (NFL protein necessary for axonal repair) • Maintenance of Genome Stability: TDP-43 is necessary for early mouse embryogenesis (knockdown of TARDBP geneis embryonically lethal). FUS/TLS is very important to genomic integrity as it helps initiate DNA repair by homologous recombination according to several new studies

8. TDP-43 and FUS TLS in ALS Pathology

9. Redistribution of TDP-43 occurs early on in the progression of ALS. For some reason, mutated TDP-43 proteins clear out of the nucleus, forming ubiquitin-positive, alpha –synuclein- and tau negative, cytoplasmic aggregates • Similarly, abnormal FUS/TLS (primarily nuclear) cytoplasmic inclusions were found in neuronal and glial cells of patients presenting classic ALS phenotypes. These aggregates were were NOT immunoreceptive for TDP-43, implying that that FUS/TLS-driven neurodegenerative pathways act independently of those related to TDP-43 inclusions. • Mutated TDP-43 mouse models presented a very fast, progressive paralysis (similar to human ALS) . Moreover, overexpression of wild-type (normal) TDP-43 in primary neurons caused cell death • Studies of the link between morphologic changes and individual neuronal survival supported the idea that cytoplasmic TDP-43 is toxic to rat cortical neurons • That said, TDP-43 has been observed to undergo quick, transient cytoplasmic translocation in response to stress. The aforementioned aggregates may in fact be neuroprotectivemechansims to cope with stress that only become toxic after the recruitment of yet unidentified binding partners • According to one paper, TDP-43 interruption can cause motor problems in Drosophilia. Some of the negative effects of mutated TDP-43 metabolism may be attributable to a loss of function .

10. Neuronal TDP-43 cytoplasmic inclusions in a FTLD Patient

11. This is Your Research Lab… The three prevailing hypothesis on the roles of TDP-43 and FUST/TLS in ALS pathogenesis are: Toxic gain of function: The cytoplasmic aggregates are neurotoxic, poisoning motor neurons and causing ALS Toxic Loss of Function: The mislocalization and mutation of these two proteins means that several essential cellular processes are not proceeding and/or are severely impaired. As a result the motor neurons become diseased Natural Response to Stress: The cytoplasmic inclusions are abnormal stress granules that form in response to the pressures ALS places on the nervous system. (The aggregates are natural responses to – not symptoms of – the disease) Based on your knowledge from this presentation and the paper itself, please discuss and debate which line of inquiry you would pursue (as a group) in your fictional motor neuron research lab

12. Final Thoughts • Some combination of TDp-43 and/or FUS/TLS inclusions have been found in cases of numerous other neurodegenerative disorders including Alzheimer’s, dementia, Down syndrome, Parkinson’s, Huntington’s, and various myopathies. (See page R50 of the article). • While the exact pathogenic roles of the abnormal forms of FUS/TLS and TDP-43 have yet to be discovered, RNA/DNA binding protein research has the potential to significantly affect not only ALS but also the study of neurodegenerative diseases as a whole. (And, of course, the end goal of developing cures).

13. Sources Information comes from the journal article itself and photos were found at the following URLs: http://sandiegocaregiversblog.com/wp-content/uploads/2010/06/Lou_Gehrig.jpg http://www.nature.com/nature/journal/v445/n7125/images/445242a-i2.0.jpg http://www.springerimages.com/Images/Biomedicine/1-10.1007_s00429-010-0263-z-3