Aggregation-prone TDP-43 sequesters and drives pathological transitions of free nuclear TDP-43

Cellular and Molecular Life Sciences (2023) 80:95 https://doi.org/10.1007/s00018-023-04739-2 Cellular and Molecular Life Sciences ORIGINAL ARTICLE Aggregation‑prone TDP‑43 sequesters and drives pathological transitions of free nuclear TDP‑43 Sean S. Keating1 · Adekunle T. Bademosi1 · Rebecca San Gil1 · Adam K. Walker1 Received: 1 November 2022 / Revised: 22 February 2023 / Accepted: 24 February 2023 © The Author(s) 2023 Abstract Aggregation of the RNA-binding protein, TDP-43, is the unifying hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43-related neurodegeneration involves multiple changes to normal physiological TDP-43, which undergoes nuclear depletion, cytoplasmic mislocalisation, post-translational modification, and aberrant liquid–liquid phase separation, preceding inclusion formation. Along with toxic cytoplasmic aggregation, concurrent depletion and dysfunction of normal nuclear TDP-43 in cells with TDP-43 pathology is likely a key potentiator of neurodegeneration, but is not well understood. To define processes driving TDP-43 dysfunction, we used CRISPR/Cas9-mediated fluorescent tagging to investigate how disease-associated stressors and pathological TDP-43 alter abundance, localisation, self-assembly, aggregation, solubility, and mobility dynamics of normal nuclear TDP-43 over time in live cells. Oxidative stress stimulated liquid–liquid phase separation of endogenous TDP-43 into droplet-like puncta, or spherical shell-like anisosomes. Further, nuclear RNAbinding-ablated or acetylation-mimicking TDP-43 readily sequestered and depleted free normal nuclear TDP-43 into dynamic anisosomes, in which recruited endogenous TDP-43 proteins remained soluble and highly mobile. Large, phosphorylated inclusions formed by nuclear or cytoplasmic aggregation-prone TDP-43 mutants also caused sequestration, but rendered endogenous TDP-43 immobile and insoluble, indicating pathological transition. These findings suggest that RNA-binding deficiency and post-translational modifications including acetylation exacerbate TDP-43 aggregation and dysfunction by driving sequestration, mislocalisation, and depletion of normal nuclear TDP-43 in neurodegenerative diseases. Keywords Acetylated TDP-43 · Endogenous gene tagging · LLPS · TDP-43 loss of function · Motor neuron disease Introduction Pathological aggregation of TAR DNA-binding protein 43 (TDP-43) in neurons is a key hallmark of almost all amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal dementia (FTD) cases [1, 2]. TDP-43 is an essential DNA-/RNA-binding protein that predominantly resides in the nucleus and physiologically regulates stability, transport, splicing, and translation of many thousands of mRNA transcripts [3–6]. In disease, TDP-43 undergoes * Rebecca San Gil * Adam K. Walker 1 Neurodegeneration Pathobiology Laboratory, Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, St. Lucia, QLD 4072, Australia progressive changes including nuclear depletion, cytoplasmic mislocalisation, misfolding, post-translational modification, aberrant liquid–liquid phase separation, and assembly into distinct pathological species before forming inclusions [7]. Cytoplasmic accumulation of TDP-43 has been regarded as a primary driver of neuronal toxicity and dysfunction [8, 9], however, loss of normal nuclear TDP-43 has also been reported even in the absence of cytoplasmic TDP-43 inclusions in post-mortem FTD [10, 11] and ALS brains [12]. Indeed, TDP-43 nuclear depletion can independently mediate neurodegeneration [13], via dysregulation of TDP-43-dependent transcription [14] and mRNA splicing functions [3, 15, 16]. As nuclear loss and cytoplasmic accumulation of TDP-43 occur concurrently in many disease models [13, 17], the driving mechanisms and relative contributions of these processes to neuronal dysfunction or toxicity remain elusive. Post-translational modifications (PTMs) are a major feature of TDP-43 pathology in ALS and FTD, and likely play diverse roles in TDP-43 dysfunction, aggregation propensity, 13 Vol.:(0123456789) 95 Page 2 of 23 and neurotoxicity. For example, phosphorylation is a widely recognised hallmark of pathology progression [18–20]. Less is understood regarding the pathogenic role of aberrant acetylation of TDP-43, which marks cytoplasmic inclusions in ALS spinal cord and is thought to affect RNA binding functions [21–23]. TDP-43 acetylation sites are found within the RNA-recognition motifs (RRMs), namely lysine residues 145 and 192 [21, 22, 24]. Mechanisms and consequences for TDP-43 acetylation have been investigated with acetylation-mimicking TDP-43 ‘2KQ’ variants, in which lysine-to-glutamine amino acid substitution at these RNA-binding residues replicates changes in electrostatic charge thought to be mediated by acetylation [21, 24, 25]. Indeed, acetylation of TDP-43 RRMs leads to substantially decreased RNA affinity and loss of splicing activity [21, 22]. Acetylation-mimicking TDP-43-2KQ mutants recapitulate native interactions of acetylated wild-type TDP-43 proteins, and further promote putative acetylation while forming disease-reminiscent insoluble and phosphorylated cytoplasmic inclusions [21, 22]. RNA deficiency promotes TDP-43 instability [26, 27], and aberrant liquid–liquid phase separation (LLPS), forming assemblies which may seed pathological aggregation [25, 28–34]. TDP-43 LLPS occurs physiologically, but can be enhanced in response to cellular stress or RNA dyshomeostasis, giving rise to diverse biomolecular condensates, characterised by ‘liquid droplet-like’ properties of subcellular mobility, fusion, fission, and dynamic exchange of comprising proteins with surrounding intracellular TDP-43 [25, 28, 30, 32, 33, 35–38]. The effects of RNA binding on TDP-43 assembly and aggregation, independent of other pathological modifications, have been studied using TDP-43 ‘FL’ mutants in which 4 or 5 essential RNA-binding phenylalanine residues in the RRMs are mutated to leucine, preventing RNA interactions [21, 25, 31, 39, 40]. RNA-binding-ablated FL mutants demonstrate increased aggregation propensity and toxicity [21, 25, 32, 39], reminiscent of truncated TDP-43 species lacking RRMs, or TDP-43 containing ALS-associated RRM mutations [29, 41–43]. Importantly, mutant RNAbinding-ablated TDP-43-5FL and acetylation-mimicking TDP-43-2KQ proteins over-expressed in the nucleus have recently been shown to undergo a distinct type of LLPS in association with molecular chaperones, readily assembling into dynamic spherical shell-like structures, termed ‘anisosomes’ [25]. TDP-43 anisosomes exclude RNA, and exhibit a ‘liquid-like’ core void of TDP-43 occupied by phase-separated heat-shock protein 70 (Hsp70), which maintains the structure and solubility of these assemblies [25]. While direct links to ALS and FTD pathology remain to be established, it is thought that a loss of chaperone ATPase activity (...truncated)