Quantification of the Relative Contributions of Loss-of-function and Gain-of-function Mechanisms in TAR DNA-binding Protein 43 (TDP-43) Proteinopathies

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, 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. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using both immunofluorescence and Western blotting analysis, and nuclear functional TDP-43 was measured by monitoring the sortilin 1 mRNA splicing activity. Various degrees of TDP-43 cytoplasmic accumulation and nuclear TDP-43 depletion were achieved and the resulting cellular viability was evaluated, leading to a quantitative global analysis on the relative effects of LOF and GOF on the overall cytotoxicity. These were found to be ∼55% and 45%, respectively, in both cell lines and using both readouts of cell toxicity, showing that these two mechanisms are likely to contribute apparently equally to the pathologies of ALS and FTLD-U.

Extracellular TDP‐43 aggregates target MAPK/MAK/MRK overlapping kinase (MOK) and trigger caspase‐3/IL‐18 signaling in microglia

“…Use of TDP43 (preformed TDP‐43 aggregate) preparations for in vitro studies has thus far been hampered because of the great difficulties in producing and purifying TDP‐43 protein. To study the impact of extracellular TDP‐43 aggregates on microglia, we employed previously characterized TDP‐43 aggregates produced from inclusion bodies (33, 36) that have been shown to recapitulate several features of ALS‐associated aggregates, including their size and amorphous structure, ubiquitination and phosphorylation susceptibility, and inherent neurotoxic capabilities (33). To determine the TDP‐43 content of aggregate preparations, SDS‐PAGE and protein quantification by band densitometry were used, which showed a TDP‐43 content of approximately 50% in the His‐tagged TDP43 vs. mock control aggregate preparations (Fig.…”

“…We used previously validated TDP‐43 aggregates (33, 36) and microglial cultures as a cellular ALS model, which was supported by analogous experiments with spinal cord organotypic cultures from adult‐like mice. We show that TDP‐43 aggregates are readily internalized by microglial cells and undergo a series of intracellular modifications, which are in good agreement with what has been consistently observed in neuronal cells, including the formation of cytoplasmic inclusions, ubiquitination, and the generation of cleaved TDP‐43 protein products (Fig.…”

Quantitative assessment of the degradation of aggregated TDP‐43 mediated by the ubiquitin proteasome system and macroautophagy

“…Human FL and C-term TDP-43 were overexpressed in Escherichia coli and the resulting inclusion bodies (IBs)-control IBs devoid of TDP-43, FL TDP-43 IBs, and C-term TDP-43 IBs-were purified, as previously described (6). Murine NSC34 and N2a cells were plated in 6-well plates that contained coverslips at densities of 150,000 cells/well and 30,000 cells/well, respectively (7,36). Twenty-four hours after plating, cells were washed with PBS and transfected by using the PulsIn protein delivery reagent (Polyplus, Illkirch, France), according to manufacturer instructions, with 100 ml of 20 mM Hepes buffer and 4 ml of PulsIn reagent to a final volume of 1 ml of cell culture medium without FBS that contained control IBs (4 mg/ml final concentration), FL TDP-43 IBs (5.7 mg/ml final concentration), or C-term TDP-43 IBs (5.7 mg/ml final concentration) (7).…”

“…Cells were then incubated for 60 min at 37°C with 1:500 mouse anti-TDP-43 mAbs (Novus Biologicals, Littleton, CO, USA) and for 90 min with 1:1000 diluted Alexa Fluor 488-conjugated antimouse Abs as previously reported (7). Cells were analyzed by using a Leica TCS SP5 confocal scanning microscope (Leica Microsystems, Mannheim, Germany), equipped with an argon laser source and a Leica Plan Apo 363 oil immersion objective.…”

“…It is just the concomitant accumulation of TDP-43 in the cytosol in the form of protein aggregates and its depletion from the nucleus as a soluble protein that are thought to cause the neurodegeneration in ALS and FTLD-U. Indeed, aggregates that accumulate in the cytosol are intrinsically toxic (6,7), and the depletion of the protein from the nucleus is also deleterious because of the loss of the fundamental functions played by native TDP-43 in the nucleus as a DNA-and RNA-binding protein (7)(8)(9). For this reason, it is increasingly recognized that ALS and FTLD-U originate from a combination of gain-of-function and loss-offunction mechanisms (7,(10)(11)(12).…”