S. Longhi’s team guest: Fabrizio Chiti, University of Florence, Italy – “Self-assembly of TDP-43: from phase separation to inclusion formation and implications in disease”

Abstract

Cytoplasmic deposition of the nuclear TAR DNA-binding protein 43 (TDP-43) in brain and motor neurons is associated with, and thought to be the primary cause of, a number of diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-TDP) and limbic-predominant age-related TDP-43 encephalopathy (LATE). One complexity of this process is the ability of TDP-43 to form liquid-phase membraneless organelles in cells. We induced phase separation of purified full-length TDP-43, of a construct containing the three folded domains without the unstructured low-complexity domain (LCD-less TDP-43) and the previously characterized LCD, using a solution-jump method and monitoring phase-separation with an array of biophysical techniques. Both complementary LCD and LCD-less fragments formed bona-fide round droplets able to coalesce and with FRAP-positive internal molecular diffusion. By contrast, full-length TDP-43 formed apparently round assemblies with limited internal diffusion and without coalescence, rather clustering into irregular bunches. The pH- and salt-dependence of the processes for the three constructs were distinct, providing a rationale of the complex electrostatic forces governing phase separation of full-length TDP-43 and its fragments. Expression of full-length TDP-43 in cultured NSC-34 cells showed the partial colocalization of the protein with stress-granule biomarkers, indicating its embedding into these liquid species, and suggesting that RNA and protein factors are necessary for this process to occur in biology for TDP-43 and associated diseases.

On another issue, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both, given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation. To address this issue we have  transfected murine NSC-34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs) so that depletion of nuclear TDP-43 and accumulation of cytoplasmic TDP-43 aggregates occur separately and independently of each other. The resulting cellular viability were attributed to ∼55% LOF and 45% GOF, respectively, in both cell lines and using two distinct readouts of cell toxicity. A similar conclusion was achieved by overexpressing human TDP-43 in NSC-34 cells and monitoring in real-time nuclear depletion, cytoplasmic accumulation and cell toxicity as oxidative stress and mitochondrial impairment, using the MitoSOX and MTT reduction assays, respectively. Bot sets of results, therefore, show that these two mechanisms are likely to contribute apparently equally to the TDP-43 proteinopathies. Finally, by taking advantage of stimulated emission depletion (STED) microscopy and using a quantitative biology approach based on mathematical fitting of multiple kinetic traces, we attributed neuronal dysfunction associated with cytoplasmic deposition component to the formation of the largest inclusions, independently of stress granules.