P60TRP interferes with the GPCR/secretase pathway to mediate neuronal survival and synaptogenesis.
Our results suggest that p60TRP is an inhibitor of Bace1 (β-site App cleaving enzyme) and Psen.The improved cognitive functions could be attributed to increased synaptic connections and plasticity, which was confirmed by the modulation of the γ-aminobutyric acid receptor system and the elevated expression of microtubule-associated protein 2, synaptophysin and Slc17a7 (vesicle glutamate transporter, Vglut1), as well as by the inhibition of Cdh2 cleavage.In conclusion, interference with the p60TRP/ GPCR/secretase signalling pathway might be a new therapeutic target for the treatment of Alzheimer's disease (AD).
Affiliation: Department of Molecular and Cell Biology, School of Biological Sciences, College of Science, Nanyang Technological University, Singapore.
- Amyloid Precursor Protein Secretases/antagonists & inhibitors/metabolism*
- Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism*
- Receptors, G-Protein-Coupled/metabolism*
- Alzheimer Disease/metabolism/physiopathology
- Amyloid beta-Protein Precursor/metabolism
- Aspartic Acid Endopeptidases/antagonists & inhibitors
- Cell Differentiation
- Cell Line
- HEK293 Cells
- Mice, Inbred C57BL
- Mice, Transgenic
- Microtubule-Associated Proteins/biosynthesis
- Neural Stem Cells/physiology
- PC12 Cells
- RNA Interference
- Receptors, GABA-A/metabolism
- Vesicular Glutamate Transport Protein 1/biosynthesis
- tau Proteins/metabolism
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fig01: P60TRP mediates differentiation in proliferating NSCs. Western blot analyses reveal a specific p60TRP expression pattern. Left: Comparing the expression levels of p60TRP in control (C, mock/Gfp-transfected) and p60TRP-transfected NSCs (p60 = p60TRP-transfected) grown under proliferating conditions. The Western blot was performed with a N-terminal-specific p60TRP antibody which revealed a band of p60TRP at ∼120 kD (dimer), two bands at ∼60 kD (monomer and post-translationally modified monomer), 1–2 bands at ∼35 kD (cleaved products of p60TRP (CTF, C-terminal fragment), especially abundant in non-differentiated NSCs). NSCs were grown as described in supplementary ‘Materials and methods’ before analysis. Right: Comparing the expression pattern of p60TRP in non-transfected NSCs grown under proliferating and differentiating conditions. Western blot was performed with a C-terminal-specific p60TRP antibody. P60TRP undergoes a differentiation-dependent cleavage. Although the 35 kD product is clearly visible in non-differentiated NSCs, it is almost undetectable in differentiated NSCs suggesting that p60TRP and its cleavage product could be used as a potential marker for NSCs (A). P60TRP mediates neurogenesis by modulating the expression of several NSC markers, as discussed in the text. NSCs were grown as described in supplementary ‘Materials and methods’ before Western blot analyses. Of particular interest is the up-regulation of the neuronal markers Ntrk1 and Mtap2 in p60TRP-transfected cells, although Gad and Th expression were inhibited. Actin (Actb) was used as loading control (B). Quantitative analyses of the Western blots shown in (B) was performed as described in supplementary ‘Materials and methods’ (*=P < 0.05 compared with controls, n= 4) (C). P60TRP modulates the expression and phosphorylation of several signalling molecules. NSCs were grown as in (B). P60TRP-transfected NSCs demonstrated a clear increase in the phosphorylation of the anti-apoptotic proteins Bcl2 (Ser70), Akt1/2/3 and Erk1/2, as well as a clear down-regulation of pStat3, which is essential for neural differentiation [18, 32]. Tubulin (Tuba1a) was used as loading control (D). Quantitative analyses of the Western blots shown in (D) (*P < 0.05 compared with controls, n= 4) (E).
We applied the lentivirus-based transfection method to generate p60TRP overexpressing NSCs (NSC-p60) to analyse the effect of p60TRP on neuronal survival and differentiation (Fig. 1). Subsequently, we investigated the effect of p60TRP overexpression in NSCs, in both proliferating and differentiating conditions, by characterizing the expression of various signalling molecules responsible for NSC survival, self-renewal, proliferation and differentiation (Fig. 1B–E). Under proliferating conditions the common stem cell marker nestin (Nes) was down-regulated slightly in NSC-p60 whereas the higher expression of neuronal markers, such as the Ngf receptor Ntrk1 (also known as TrkA), Tubb3 and Mtap2, revealed that p60TRP induced the differentiation of NSCs even under proliferating conditions in the presence of Egf. Additionally, the glia/progenitor marker, glial fibrillary acidic protein (Gfap), was significantly up-regulated, but the oligodendrocyte markers myelin basic protein (Mbp) and 2′,3′-cyclic nucleotide 3′ phosphodiesterase were moderately down-regulated. The reduced phosphorylation of the signal transducer and activator of transcription 3 (Stat3) also supported higher levels of differentiation in NSC-p60 (Fig. 1D and E). Consistent with this observation, NSCs grown under differentiating conditions also showed that NSC-p60 possessed significantly higher numbers of Tubb3+ and Mtap2+ cells, substantiating the finding that p60TRP enhanced neurogenesis (Fig. 2A–C). Thus, our data demonstrate that p60TRP drives NSCs to differentiate particularly into neuronal progenitors.