Journal: Human Molecular Genetics

Article Title: The pro-domains of neurotrophins, including BDNF, are linked to Alzheimer's disease through a toxic synergy with Aβ

doi: 10.1093/hmg/ddv130

Figure Lengend Snippet: The pro-domain of BDNF and Aβ 1–42 exhibits synergistic toxicity in SH-SY5Y human neuroblastoma cell cultures. ( A ) Addition of 25, 100 and 1000 n m oligomeric Aβ 1–42 to the culture medium of SH-SY5Y human neuroblastoma cells for 48–60 h resulted in significant toxicity when compared with vehicle. Each bar represents mean ± SEM ( n = 3). Statistical comparisons were made by one-way analysis variance test. * P < 0.05. ( B ) Cells were treated with 200 ng/ml recombinant pro-domain of BDNF for 48–60 h. Met66 BDNF pro-domain alone did not have an effect on cell viability in control cells. ( C ) Cells were treated with Met66 pro-domain of BDNF (200 ng/ml) in combination with 25 n m Aβ 1–42 for 48–60 h. In the presence of Aβ 1–42 cell death was increased upon addition of Met66 BDNF pro-domain ( n = 9) performed. Each bar represents mean ± SEM ( n = 9). Statistical comparisons were made by paired t test. ** P < 0.01. ( D ) Twenty-six paired experiments indicated that cell death was higher when a particular preparation of Aβ 1–42 (25 n m , n = 9 preparations of Aβ 1–42 ) was supplemented with Met66 BDNF pro-domain ( y -axis) than for that preparation of Aβ 1–42 alone ( x -axis, dashed line indicates the null hypothesis of no effect of the Met66 BDNF pro-domain). ( E ) Similar results were seen when non-differentiated and ( F ) differentiated cells were treated with the Met66 BDNF pro-domain (200 ng/ml) in combination with 1 μ m Aβ 1–42 for 48 h. ( G ) Cells were treated with the Met66 BDNF pro-domain (200 ng/ml) in combination with 25 n m Aβ 1–42 oligomers for 48–60 h before SDS–PAGE and western blotting with antibodies against p75 NTR and sortilin. β-Actin expression was used as a loading control. Western blots indicated that levels of p75 NTR but not sortilin were higher following Aβ 1–42 oligomer treatment in cultures of human neuroblastoma cells. Representative blots are shown ( n = 3). ( H ) The p75 NTR and ( I ) sortilin intensities from the western blots were normalized against the level of β-actin. Each bar represents mean ± SEM ( n = 3). Statistical comparisons were made by one-way analysis variance test. * P < 0.05.

Article Snippet: Twenty four hours after plating, cells were washed and maintained in 0.5% (v/v) FBS-containing medium for 18 h. After that cells were replaced with 0.2% (v/v) FBS-containing medium and then incubated further 48–72 h in the presence of Aβ 1–42 oligomers with or without recombinant human wild type (Val66) or variant (Met66) BDNF pro-domain (Alomone labs).

Techniques: Recombinant, SDS Page, Western Blot, Expressing