The cellular expression and proteolytic processing of the amyloid precursor protein is independent of TDP-43

Abstract

Alzheimer's disease (AD) is a neurodegenerative condition, of which one of the cardinal pathological hallmarks is the extracellular accumulation of amyloid β (Aβ) peptides. These peptides are generated via proteolysis of the amyloid precursor protein (APP), in a manner dependent on the β-secretase, BACE1 and the multicomponent γ-secretase complex. Recent data also suggest a contributory role in AD of transactive response DNA binding protein 43 (TDP-43). There is little insight into a possible mechanism linking TDP-43 and APP processing. To this end, we used cultured human neuronal cells to investigate the ability of TDP-43 to interact with APP and modulate its proteolytic processing. Immunocytochemistry showed TDP-43 to be spatially segregated from both the extranuclear APP holoprotein and its nuclear C-terminal fragment. The latter (APP intracellular domain) was shown to predominantly localise to nucleoli, from which TDP-43 was excluded. Furthermore, neither overexpression of each of the APP isoforms nor siRNA-mediated knockdown of APP had any effect on TDP-43 expression. Doxycycline-stimulated overexpression of TDP-43 was explored in an inducible cell line. Overexpression of TDP-43 had no effect on expression of the APP holoprotein, nor any of the key proteins involved in its proteolysis. Furthermore, increased TDP-43 expression had no effect on BACE1 enzymatic activity or immunoreactivity of Aβ1-40, Aβ1-42 or the Aβ1-40:Aβ1-42 ratio. Also, siRNA-mediated knockdown of TDP-43 had no effect on BACE1 immunoreactivity. Taken together, these data indicate that TDP-43 function and/or dysfunction in AD is likely independent from dysregulation of APP expression and proteolytic processing and Aβ generation.

Keywords: Alzheimers disease; Amyloid precursor protein; BACE1; Gene regulation; TDP-43; proteolysis.

Figure 1. TDP-43 does not co-localise with either the APP holoprotein or its intracellular domain

SH-SY5Y or OX1-19 iPSCs were cultured and differentiated as described, followed by fixation and immunocytochemistry using primary antibodies against TDP-43 and (A) APP holoprotein (antibody 22C11) and (B) AICD. iPSN denotes iPSC-derived neurons.

Figure 2. AICD is present in nucleoli

SH-SY5Y cells were cultured and differentiated as described, followed by fixation and immunocytochemistry using primary antibodies against (A) FE65, AICD and Tip60 (B) AICD and fibrillarin. (C) Cells were treated with actinomycin D (0.02 µg/ml, 3 h) and immunocytochemistry performed to assess the effect on AICD localisation.

Figure 3. APP overexpression or knockdown does not affect TDP-43 expression

SH-SY5Y cells were cultured as described and (A) stably transfected with each of the three APP isoforms. Expression was confirmed by immunoblot, compared with TDP-43 expression and (B) changes in protein expression quantified. (C) Cells were nucleofected with two independent siRNA targeting APP and protein expression of APP and TDP-43 assessed by immunoblot and (D) quantified; n=4. *P<0.05 and ***P<0.001.

Figure 4. Inducible TDP-43 overexpression does not affect APP processing

A stable cell line with doxycycline-inducible TDP-43 expression (pT-REx-DEST30-TDP-43-FLAG) was generated and (A) TDP-43 overexpression confirmed by immunoblot. APP and its proteolytic enzymes were also probed by immunoblot and (B) changes quantified by densitometry. (C) Cells were nucleofected with two independent siRNA targeting TDP-43 and protein expression of TDP-43 and BACE1 assessed by immunoblot and (D) quantified. **P<0.01 and ***P<0.001.

Figure 5. TDP-43 overexpression does not modulate BACE1 mRNA, activity or the generation of Aβ peptides

TDP-43 expression was induced in SH-SY5Y (pT-REx-DEST30-TDP-43-FLAG) cells and cells were analysed for (A) BACE1 mRNA levels, (B) β-secretase activity and culture medium assessed for (C) Aβ1-40 and (D) Aβ1-42 followed by (E) calculation of the Aβ1-40: Aβ1-42 ratio (F) sAPPα immunoreactivity (using antibody 22C11). Total protein was assessed using Amido Black stain to ensure equal loading and (G) protein changes were quantified by densitometry.