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

Abstract

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are neurodegenerative disorders that share the cytosolic deposition of TDP-43 (TAR DNA-binding protein 43) in the CNS. TDP-43 is well known as being actively degraded by both the proteasome and macroautophagy. The well-documented decrease in the efficiency of these clearance systems in aging and neurodegeneration, as well as the genetic evidence that many of the familial forms of TDP-43 proteinopathies involve genes that are associated with them, suggest that a failure of these protein degradation systems is a major factor that contributes to the onset of TDP-43-associated disorders. Here, we inserted preformed human TDP-43 aggregates in the cytosol of murine NSC34 and N2a cells in diffuse form and observed their degradation under conditions in which exogenous TDP-43 is not expressed and endogenous nuclear TDP-43 is not recruited, thereby allowing a time zero to be established in TDP-43 degradation and to observe its disposal kinetically and analytically. TDP-43 degradation was observed in the absence and presence of selective inhibitors and small interfering RNAs against the proteasome and autophagy. We found that cytosolic diffuse aggregates of TDP-43 can be distinguished in 3 different classes on the basis of their vulnerability to degradation, which contributed to the definition-with previous reports-of a total of 6 distinct classes of misfolded TDP-43 species that range from soluble monomer to undegradable macroaggregates. We also found that the proteasome and macroautophagy-degradable pools of TDP-43 are fully distinguishable, rather than in equilibrium between them on the time scale required for degradation, and that a significant crosstalk exists between the 2 degradation processes.-Cascella, R., Fani, G., Capitini, C., Rusmini, P., Poletti, A., Cecchi, C., Chiti, F. Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy.

TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis

“…Although there is controversy regarding whether TDP-43 is overexpressed in ALS patients [13], the presence of CTFs could represent a consequence of loss in protein homeostasis that leads to protein accumulation. This is supported by the finding that TDP-43 accumulation in the cytoplasm deregulates autophagy and proteasome functions [14][15][16]. Further, the presence of CTFs could represent a cellular way to cope with the increase in protein accumulation, by cleaving it in smaller pieces that could more easily enter the ubiquitin-proteasome system (UPS) and autophagy systems [17,18].…”

Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration

“…We next wanted to extend our evaluation of TFEB involvement in aggregate clearance of the protein TARDBP/TDP-43 (TAR DNA binding protein) typically characterizing sporadic forms of ALS and of fronto-lateral temporal dementia, as well as familial forms of ALS (fALS) linked to several human SOD1 (superoxide dismutase 1) mutations. In patients and cellular models of these diseases, TARDBP is cleaved releasing a neurotoxic fragment of 25 kDa (TARDBP-25/TDP-25), and we recently reported that this cleaved fragment is highly prone to aggregate into sodium dodecyl sulfate-insoluble species resistant to autophagy clearance and capable of impairing autophagy [63,64]. While no significant variations were found for the soluble fraction of the GFP-tagged TARDBP-25 fragment evaluated in WB (Figure 3(e)), similar to the AR model, we found that trehalose induced the clearance of the aggregated form of this fragment retained on FRA (Figure 3(f), quantification in Figure 3(g)).…”

Tdp-25 Routing to Autophagy and Proteasome Ameliorates its Aggregation in Amyotrophic Lateral Sclerosis Target Cells

“…In mammalian cells, the clearance of aberrantly folded or misfolded proteins is mediated by the intracellular protein quality control (PQC) system 11 . The PQC system is composed of chaperone/co-chaperone proteins, which recognize, bind to and target aberrant proteins to degradation, and the degradative systems, like the ubiquitin proteasome system and the autophagy 12 – 14 . Chaperones and co-chaperones work in complex; one example is represented by BAG3, a co-chaperone of HSP70 that also binds to the chaperone HSPB8, and the E3 ligase CHIP.…”