Figure 7. Enhanced synaptic vesicle release in T21 neurons mediated by elevated APP and DYRK1A levels.

a) Synaptic vesicle (SV) release was assessed in T21, T21-rev, fAD and fADcorr neurons using live cell imaging of SypHy. SypHy is a fusion protein consisting of synaptophysin and ecliptic pHluorin, a pH-sensitive GFP variant targeted to the vesicular lumen. pHluorin is quenched by the resting acidic pH (5.5) within vesicles and fluoresces upon externalization to the extracellular solution (pH 7.4) via neuronal stimulation with KCl leading to vesicle fusion. Neurons were transduced on DIV17 with two lentiviruses expressing SV2-tdTomato (to identify synapse rich areas) and SypHy. On d21, neurons were imaged continuously in the presence of media before and during stimulation with KCl. At the end of the assay, NH₄Cl was applied, which unquenches SypHy fluorescence by uniformly raising intravesicular pH, was used to determine responsive puncta (i.e. at least 2-fold increase in fluorescence). Regions of interest (ROIs) were selected based upon SV2-tdtomato fluorescence and multiple metrics were quantified for each ROI.

b,c) Quantification of the percent NH₄Cl responsive puncta, the percent synaptic vesicles responsive to KCl, and the percent of the total pool released by KCl across lines. Data in (b) are from person #1 and data in (c) are from person #2.

d-f) The average (+/−SEM) of SypHy signal of all NH₄Cl responsive puncta across the experimental time course is quantified across lines derived from person #1 (d), person #2 (e), and fAD isogenic pair (f). Shaded areas represent time points with KCl stimulation.

g) Quantification of the percent NH₄Cl responsive puncta, the percent synaptic vesicles responsive to KCl, and the percent of the total pool released by KCl for fAD and fADcorr neurons.

h-j) The same SV release assay was performed using “wild type” iNs derived from a not cognitively impaired individual with low AD pathology in their brain and normal karyotype at death (BR93,(59)). These neurons were transduced with lentivirus expressing wild type APP or APP harboring fAD mutations (“NLF”). ELISA was performed to confirm elevation of Aβ levels present in media with APP overexpression, and an elevation of Aβ42:40 ratio with fAD mutation (h). Quantification of the percent NH₄Cl responsive puncta, the percent synaptic vesicles responsive to KCl (k), and the percent of the total pool released by KCl across conditions (i-j).

Total numbers of coverslips (“N”) and regions of interest (“n”) per genotype: T21 person #1 N=9, n=940; T21-rev person #1 N=3, n=314; T21–2xAPP N=6, n=521 ROIs; T21–2xDYRK1A N=9, n=894; T21 person #2 N=3, n=275; T21-rev person #2 N=3, n=258; fAD N=5, n=394; fADcorr N=4, n=358, BR93 APP-NLF N=3, n=383. One-way ANOVA with Dunnett’s multiple comparison tests were performed in b,h,j, and Mann-Whitney tests performed in c,g. p-values as shown.