Post-translational modifications on RNA-binding proteins: accelerators, brakes, or passengers in neurodegeneration?

TDP-43 pathology: From noxious assembly to therapeutic removal

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to perform a wide range of critical cellular functions by maintaining spatiotemporal regulation and organizing intracellular biochemistry. However, aberrant phase transitions are implicated in a multitude of human diseases. Here, we demonstrate that two neuronal proteins namely, tau and prion undergo complex coacervation driven by domain-specific electrostatic interactions to yield highly dynamic, mesoscopic liquid-like droplets. The acidic N-terminal segment of tau interacts electrostatically with the polybasic N-terminal intrinsically disordered segment of the prion protein (PrP). We employed a unique combination of time-resolved tools that encompass several orders of magnitude of timescales ranging from nanoseconds to seconds. These studies unveil an intriguing orchestra of molecular events associated with the formation of heterotypic condensates comprising ephemeral, domain-specific, short-range electrostatic nanoclusters. Our results reveal that these heterotypic condensates can be tuned by RNA in a stoichiometry-dependent manner resulting in reversible, multiphasic, immiscible, ternary condensates of different morphologies ranging from core-shell to nested droplets. This ternary system exhibits a typical three-regime phase behavior reminiscent of other membraneless organelles including nucleolar condensates. We also show that upon aging, tau-PrP droplets gradually convert into solid-like co-assemblies by sequestration of persistent intermolecular interactions. Our vibrational Raman spectroscopic data in conjunction with atomic force microscopy and multi-color fluorescence imaging results reveal the presence of amorphous and amyloid-like co-aggregates upon maturation. Our findings provide mechanistic underpinnings of overlapping neuropathology involving tau and PrP and highlight a broader role of complex phase transitions in physiology and disease.

Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder typically characterized by insoluble inclusions of hyperphosphorylated TDP-43. The mechanisms underlying toxic TDP-43 accumulation are not understood. Persistent activation of p38 mitogen-activated protein kinase (MAPK) is implicated in ALS. However, it is unclear how p38 MAPK affects TDP-43 proteinopathy. Here, we demonstrate that inhibition of p38α MAPK reduces pathological TDP-43 phosphorylation, aggregation, cytoplasmic mislocalization, and neurotoxicity. We establish that p38α MAPK phosphorylates TDP-43 at pathological serine 409/410 (S409/S410) and serine 292 (S292), which reduces TDP-43 liquid-liquid phase separation (LLPS) but allows pathological TDP-43 aggregation. Moreover, we show that protein arginine methyltransferase 1 methylates TDP-43 at R293. Importantly, S292 phosphorylation reduces R293 methylation, and R293 methylation reduces S409/S410 phosphorylation. R293 methylation permits TDP-43 LLPS and reduces pathological TDP-43 aggregation. Thus, strategies to reduce p38α-mediated TDP-43 phosphorylation and promote R293 methylation could have therapeutic utility for ALS and related TDP-43 proteinopathies.

/pdf/opposing-roles-of-p38a-mediated-phosphorylation-and-arginine-4cpbzbpjsc.pdf

Opposing roles of p38α-mediated phosphorylation and arginine methylation in driving TDP-43 proteinopathy

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin‐positive inclusions are associated with deposition of cytosolic inclusion bodies of TAR DNA‐binding protein 43 (TDP‐43) in brain and motor neurons. We induced phase separation of purified full‐length TDP‐43 devoid of large tags using a solution‐jump method, and monitored it with an array of biophysical techniques. The tetramethylrhodamine‐5‐maleimide‐ or Alexa488‐labeled protein formed rapidly (<1 min) apparently round, homogeneous and 0.5–1.0 μm wide assemblies, when imaged using confocal fluorescence, bright‐field, and stimulated emission depletion microscopy. The assemblies, however, had limited internal diffusion, as assessed with fluorescence recovery after photobleaching, and did not coalesce, but rather clustered into irregular bunches, unlike those formed by the C‐terminal domain. They were enriched with α‐helical structure, with minor contributions of β‐sheet/random structure, had a red‐shifted tryptophan fluorescence and did not bind thioflavin T. By monitoring with turbidimetry both the formation of the spherical species and their further clustering under different experimental conditions, we carried out a multiparametric analysis of the two phenomena. In particular, both processes were found to be promoted by high protein concentrations, salts, crowding agents, weakly by reducing agents, as the pH approached a value of 6.0 from either side (corresponding to the TDP‐43 isoionic point), and as the temperature approached a value of 31°C from either side. Important differences were found with respect to the TDP‐43 C‐terminal domain. Our multiparametric results also provide explanations to some of the solubility data obtained on full‐length TDP‐43 that were difficult to explain following the multiparametric analysis acquired on the C‐terminal domain.

https://flore.unifi.it/bitstream/2158/1290893/3/Staderini%202022%20Prot%20Sci.pdf

Biophysical characterization of full‐length TAR DNA‐binding protein (TDP‐43) phase separation

Post-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the RNA-binding protein TDP-43, is hyperphosphorylated in disease on several C-terminal serine residues, which is generally believed to promote TDP-43 aggregation. Here, we show that hyperphosphorylation by Casein kinase 1δ or C-terminal phosphomimetic mutations surprisingly reduce TDP-43 phase separation and aggregation and render TDP-43 condensates more liquid-like and dynamic. Multi-scale simulations reveal reduced homotypic interactions of TDP-43 low complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We propose that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.

https://www.biorxiv.org/content/biorxiv/early/2021/04/30/2021.04.30.442163.full.pdf

da Silva Lg

Papers

Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation