UV irradiation accelerates amyloid precursor protein (APP) processing and disrupts APP axonal transport

Abstract

Overexpression and/or abnormal cleavage of amyloid precursor protein (APP) are linked to Alzheimer's disease (AD) development and progression. However, the molecular mechanisms regulating cellular levels of APP or its processing, and the physiological and pathological consequences of altered processing are not well understood. Here, using mouse and human cells, we found that neuronal damage induced by UV irradiation leads to specific APP, APLP1, and APLP2 decline by accelerating their secretase-dependent processing. Pharmacological inhibition of endosomal/lysosomal activity partially protects UV-induced APP processing implying contribution of the endosomal and/or lysosomal compartments in this process. We found that a biological consequence of UV-induced γ-secretase processing of APP is impairment of APP axonal transport. To probe the functional consequences of impaired APP axonal transport, we isolated and analyzed presumptive APP-containing axonal transport vesicles from mouse cortical synaptosomes using electron microscopy, biochemical, and mass spectrometry analyses. We identified a population of morphologically heterogeneous organelles that contains APP, the secretase machinery, molecular motors, and previously proposed and new residents of APP vesicles. These possible cargoes are enriched in proteins whose dysfunction could contribute to neuronal malfunction and diseases of the nervous system including AD. Together, these results suggest that damage-induced APP processing might impair APP axonal transport, which could result in failure of synaptic maintenance and neuronal dysfunction.

Keywords: APP axonal vesicles; UV-irradiation; amyloid precursor protein; axonal transport; gamma-secretase; kinesin.

Figure 1.

UV irradiation leads to a dose-dependent and differentiation state- and cell type-independent decline in APP protein. A, Human neuroblastoma SH-SY5Y cells were exposed with 0–30 mJ/cm² UV radiation and left to recover for 0–7 h. The same amount of protein from each condition was analyzed by Western blot to determine relative amounts of APP FL, APP CTFs, TrkA, and tubulin as loading control. B, Quantification of remaining levels of APP FL, APP CTFs, and TrkA at each time point after UV exposure normalized to tubulin from three different experiments as shown in A. Two-way ANOVA followed by Bonferroni post-test was applied, and statistics are shown only when significant. For representation purposes APP FL and APP CTF data points (except mock conditions) were fitted to a nonlinear exponential decay function with R² > 0.8, and TrkA data were fitted to a straight line with R² > 0.8. C, Human neuroblastoma SH-SY5Y cells were mock-irradiated or UV-irradiated at the indicated doses. Western blot shows levels of phosphorylated-c-Jun N-terminal kinase (P-JNK) and total JNK at time 0 (−) and 1 h (+) after UV exposure. D, Quantification of detected APP FL protein by Western blot (data not shown) after 20 mJ/cm² UV radiation in human epithelial HeLa cell line, human fibroblasts (HuFB) derived from skin biopsies, and LN-CaP prostate cancer cell line, and retinoic acid (RA)-differentiated SH-SY5Y cells (100% = time 0). E, TBP-normalized APP mRNA levels analyzed by RT-qPCR from SH-SY5Y cells exposed to UV (20 mJ/cm²) and left to recover for the indicated times. Levels of mRNA from each time point are relative to corresponding times from mock-irradiated cells. Orange line represents levels of mRNA at time = 0 (n = 4, mean ± SEM).

Figure 2.

γ-Secretase activity mediates UV-induced APP CTF decay. A, Left, Representative Western blot of SH-SY5Y cells treated with the γ-secretase inhibitor Compound E (1 μm) or the proteasome inhibitor MG132 (20 μm) for the indicated times and probed for APP CTFs and tubulin as loading control. Expected sizes for β-CTFs and α-CTFs are marked on the right. Middle and right, Quantification of APP CTF remaining levels at each time point after each indicated drug treatment to tubulin from three different experiments as shown in A. B, SH-SY5Y cells were exposed to UV (20 mJ/cm²) in the presence of vehicle (first column), MG132 (20 μm; second column), Compound E (1 μm; third column), or Compound E and cycloheximide (50 μg/ml) simultaneously (fourth column), and left to recover for the indicated times in the presence of indicated treatment. Equivalent amounts of protein were loaded and probed by Western blot for APP FL, APP CTFs, and tubulin as loading control. Expected sizes for β-CTFs and α-CTFs are indicated on the right. C, Protein biosynthesis in SH-SY5Y cells was inhibited using cycloheximide (50 μg/ml) for the indicated times and analyzed by Western blot for APP FL, APP CTFs, and tubulin as a loading control. D, MEFs derived from Psen1/2 dKO (Psen1/2 dKO cells; top three rows) or Psen1/2 dKO cells rescued with human PSEN1 and PSEN2 (+hPSEN1/2; bottom three rows) were mock-irradiated or UV-irradiated (20 mJ/cm²) and analyzed by Western blots to probe for APP FL, APP CTFs, and actin as loading control.

Figure 3.

UV irradiation induces degradation of APLP1 and APLP2, but not other γ-secretase substrates. A, Equivalent protein amounts from SH-SY5Y cells mock-irradiated or UV-irradiated (20 mJ/cm²) were analyzed by Western blots and probed for BACE1, PSEN1 CTF, and PSEN2 CTF, Nicastrin, Pen2, and tubulin as loading control. Graphs depict quantification of relative levels of indicated protein before UV exposure (gray bars) and seven hours after irradiation (red bars) from three different experiments as shown on left panels. B, We probed Western blot analysis of SH-SY5Y untreated or exposed to UV radiation (20 mJ/cm²), allowed to recover for indicated times, for APLP1, APLP2, ErbB4, N-cad FL, N-cad CTF, Notch, and Tubulin as loading control. Graphs depict quantification of relative levels of indicated protein before UV exposure (gray bars) and seven hours after irradiation (red bars) from three different experiments as shown on left panels. C, TBP-normalized APLP1 and APLP2 mRNA levels analyzed by RT-qPCR from SH-SY5Y cells exposed to UV (20 mJ/cm²) and relative to untreated cells (orange line represents levels of corresponding mRNA at time = 0) and left to recover for indicated times (n = 3, mean ± SEM). D, Protein biosynthesis in SH-SY5Y cells was inhibited using cycloheximide (50 μg/ml) for indicated times and samples were probed for APLP1 and APLP2 and tubulin as a loading control. E, SH-SY5Y cells treated for 1 h with Compound E (1 μm) alone or Compound E and cycloheximide were exposed to UV radiation and left to recover for the indicated times in the presence of indicated drugs. Levels of APP FL, APP CTFs, and tubulin as loading control were determined by Western blots.

Figure 4.

Endosomal/lysosomal pathways contribute to UV-induced APP processing. A, SH-SY5Y cells untreated (control), treated with NH₄Cl (50 mm) for 1 h or the endocytosis inhibitor Dynasore (100 μm) for 4 h were mock or UV irradiated (20 mJ/cm²) and left to recover for the indicated times. Corresponding drug was maintained in conditioned medium at all time points. Levels of APP FL and CTFs were analyzed by Western blot using tubulin as a loading control. B, Graphs show the ratio of remaining APP FL and APP CTFs, each normalized to tubulin and to time = 0, between samples exposed to UV and mock treated at each time point analyzed. Each treatment condition was similarly analyzed (n = 3, mean ± SEM). C, Same as B but for Dynasore-treated cells. Experiments were performed four times *p < 0.05, **p < 0.01, ***p < 0.001 by t test at each time point comparing conditions.

Figure 5.

UV irradiation impairs APP axonal transport in primary mouse hippocampal cultures. A, To test whether transfected APP-YFP responds to UV, SH-SY5Y cells were transiently transfected with APP tagged with YFP at its C-terminal end (APP-YFP) and treated with vehicle or the γ-secretase inhibitor Compound E (1 μm) 1 h before UV (20 mJ/cm²) exposure and left to recover for the indicated times in the presence of indicated drug treatment. Equivalent amounts of protein from each sample were analyzed by Western blots using GFP antibodies, which detect the YFP moiety. Full-length APP-YFP, α-CTF-YFP, and AICD-YFP bands are indicated on the right. Tubulin was used as a loading control. B, Scheme summarizing imaging experiment layout for hippocampal mouse neurons. Primary mouse hippocampal cells incubated 10 d in vitro (div) were transiently transfected with APP-YFP. Fifteen hours later the cells were treated with vehicle or γ-secretase inhibitor Compound E (CE; 1 μm) for 1 h before mock or UV radiation (20 mJ/cm²). Following a 4 h recovery period, 15 s of APP-YFP movement was recorded per axon and the data transformed into kymographs displaying the trajectories of stationary, anterograde, and retrograde YFP-containing puncta. Kymograph displayed was obtained from APP-YFP transfected in control cells. Immediately after imaging, cells were processed for Western blot analysis to detect endogenous APP CTFs, and tubulin as loading control. C, Graph depicting the number of anterograde, retrograde, and total APP-YFP puncta/micrometer in control, UV irradiated (20 mJ/cm²), CE treated, and UV irradiated (20 mJ/cm²) after CE-treated hippocampal cultures. Horizontal lines within the column represent the median. The vertical lines (whiskers) mark the minimum and the maximum of all data points in each condition. The bottom and the top of the box mark the 25% percentile and 75% of all data, respectively (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001 by Mann–Whitney U test.

Figure 6.

APP FL and APP CTF cofractionate with motor proteins and secretase components to LVPs in mouse synaptosomes. A, Mouse brain cortices were fractionated to obtain the synaptosome-enriched fraction LP2. The same amount of protein from each fraction was loaded and probed using antibodies to the proteins indicated on the left. B, LP2 fraction was sedimented on a continuous sucrose velocity gradient (10–38%). Twelve fractions were collected and equivalent volumes from each fraction were analyzed by Western blots for the proteins indicated on the left. Two distinct vesicle pools were discernable by size and protein content, the SVP corresponding to fractions 3 and 4 was defined by its high enrichment in bona fide synaptic vesicle component VAMP (left box). The LVP comprising fractions 7–10 was defined by enrichment of larger Rab-5-containing endosomal organelles (right box). C, EMs of vesicle populations present in pooled SVP fractions 3 and 4 (left), and pooled LVP fractions 7–9 (right). Insets are magnified images from representative EM pictures from each vesicle pool. Arrowheads point to ∼50 nm vesicles present in SVP fraction, and arrow points to an example of larger organelles present in LVP fraction. Scale bar, 100 nm.

Figure 7.

APP immunoisolates contain morphologically heterogenous organelles containing motor proteins and the secretase machinery. A, EM of LVP fraction incubated with magnetic beads coated with APP antibodies and GFP as negative control. Representative EMs show no material on GFP-coated magnetic bead surface. Representative EMs show a heterogeneous population of vesicles (arrowheads) decorating the surface of APP beads. B, Electron micrograph of SVP fraction incubated with magnetic beads coated with APP, synaptotagmin (SYT), or GFP antibodies. Representative EMs show no material on GFP-coated magnetic bead surface. Representative EMs show a homogenous population of ∼50 nm vesicles (arrowheads) decorating the surface of APP- and SYT-coated beads. C, APP immunoisolates from LVP from either wild-type or APP KO synaptosomes were analyzed by Western blots with indicated antibodies. D, Immunoisolates from LVP fractionated from wild-type mice using APP, BACE1, KLC1, and GFP antibodies were analyzed by Western blot and probed for indicated antibodies on the left. E, Venn diagram of predicted proteins obtained from APP and syd immunoisolates (Abe et al., 2009) obtained from mouse synaptosomal LVP fractions.