Figure 5

TSP-1 rescues hippocampal neurons from Aβ42 peptide-induced reduction in synaptic density by mediating the α2δ-1 receptor. (A) α2δ-1-siRNA was transfected into hippocampal neurons overnight, after which the cells were co-cultured with hUCB-MSCs or treated with recombinant human TSP-1 under Aβ42 peptide treatment. Hippocampal neurons were separately transfected with scrambled siRNA as a control. mRNA expression of α2δ-1 was analysed using RT-PCR. (B) Representative images of hippocampal neurons stained for pre-synaptic (SYP, green) and post-synaptic (PSD-95, red) proteins (Scale bar = 25 μm). (C) Quantification of synaptic density (number of synapses per 100 μm of dendritic length, n ≥ 30 dendrites) revealed that suppression of α2δ-1 by siRNA in the hippocampal neurons did not reverse Aβ42 peptide-induced synaptic dysfunction in hippocampal neurons, despite being co-cultured with the TSP-1 secreting hUCB-MSCs or treatment with recombinant human TSP-1. (**p < 0.005 versus control-siRNA-treated hippocampal neurons). (D) In α2δ-1-knockdown hippocampal neurons, SYP and PSD-95 expression levels were determined by immunoblotting for the same conditions in (B, C). Immunoblotting also showed remarkably attenuated expression of SYP and PSD-95 in α2δ-1-siRNA-treated hippocampal neurons and significantly decreased expression of α2δ-1 in hippocampal neurons under Aβ42 peptide treatment. Right panel indicates densitometric quantification analysis. (mean ± SEM, **p < 0.005, n = 3 per group).