Fig. 2: Increased GNG5 promotes Aβ42 production and causes neuronal dysfunction.

A, B Establish of GNG5 overexpressed cell model SH-SY5Y-APP^OE-GNG5^OE. A Western blot detection of GNG5, APP, and PS1 levels in cells. B Enzyme-linked immunosorbent assay (ELISA) measurement of Aβ42 and Aβ40 levels in cellular supernatant. Normalized (target protein blot/reference blot) quantitative results (densitometry) calculated using Image J software are shown under each blot. C, D Knockdown of GNG5 in SH-SY5Y-APP^OE cells. C Western blot detection of GNG5, APP, PS1, and p-tau (S396) in cells. D ELISA measurement of Aβ42 and Aβ40 levels in cellular supernatant. E, F Establish of GNG5 overexpressed cell model 293T-APP^OE-GNG5^OE. E Western blot detection of GNG5, APP, PS1, and p-tau (AT8) protein levels in cells. F ELISA measurement of Aβ42 and Aβ40 levels in cellular supernatant. G, H Knockdown of GNG5 in 293T-APP^OE cells. G Western blot detection of GNG5, APP, and PS1 in cells. H ELISA measurement of Aβ42 and Aβ40 levels in cellular supernatant. I–L In vitro γ-secretase cleavage assay. I γ-secretase complex extracted from wild type 293T, 293T-NC^OE, and 293T-GNG5^OE cells. Western blot verification of PS1, PEN2, NCSTN, APH1A and GNG5 levels in the complex. J Levels of Aβ42 and Aβ40 generated in the γ-secretase cleavage assay using ELISA. Cytoplasmic proteins extracted from Neuro2a-APP^OE cells were used as substrates. K Comparation of cleavage activity of γ-secretase using recombinant C99 as substrate and γ-secretase complex extracted from WT 293T cells. Recombinant GNG5, full length PS1, and PS1 Ala₂₅₁–Ser₃₉₀ fragment were added to this system. Quantification of AICD (red arrow) was monitored using a monoclonal antibody against the C-terminal 20 amino acids (C1/6.1, BioLegend, 802801). AICD, APP Intracellular Domain, the left domain after cleavage of Aβ42 or Aβ40 from C99. L ELISA measurement of Aβ42 levels at different concentrations of recombinant GNG5 in the in vitro cleavage assay with C99 as substrate and WT γ-secretase complex. Representative confocal images depicting dendritic staining of MAP2 (M), and the statistics of total dendrite branch number and total dendrite length (N) in WT and GNG5^OE rat primary hippocampal neurons. Scale bar, 50 μm. O Representative confocal images depicting synaptic staining of presynaptic marker Synapsin I (red) and postsynaptic marker PSD-95 (green) in WT and GNG5^OE rat primary hippocampal neurons. Single synaptic number was quantified as colocalized pre- and postsynaptic puncta. The boxed areas are enlarged below the original images. Histograms depicting the relative density or size level of single synapse, PSD-95 and Synapsin I puncta in WT and GNG5^OE rat primary hippocampal neurons. n = 30 fields/group. Scale bar, 10 μm; inset, 2 μm. P Representative deconvolved images showing spine densities labeled with phalloidin in WT and GNG5^OE rat primary hippocampal neurons (left), and corresponding detailed 3D-rendered views of spines (right). Statistical results showing changes in spine density. Data are presented as the mean ± SD. p values were determined using unpaired two-tailed Student’s t-test for two groups and one-way ANOVA with Turkey post hoc test for multiple groups. *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant.