Role of Membrane Tension Sensitive Endocytosis and Rho GTPases in the Uptake of the Alzheimer’s Disease Peptide Aβ(1-42)

Intraneuronal accumulation of amyloid-β (Aβ) is an early pathological signum of Alzheimer’s disease, and compartments of the endolysosomal system have been implicated in both seeding and cell–cell propagation of Aβ aggregation. We have studied how clathrin-independent mechanisms contribute to Aβ endocytosis, exploring pathways that are sensitive to changes in membrane tension and the regulation of Rho GTPases. Using live cell confocal microscopy and flow cytometry, we show the uptake of monomeric Aβ(1-42) into endocytic vesicles and vacuole-like dilations, following relaxation of osmotic pressure-induced cell membrane tension. This indicates Aβ(1-42) uptake via clathrin independent carriers (CLICs), although overexpression of the bar-domain protein GRAF1, a key regulator of CLICs, had no apparent effect. We furthermore report reduced Aβ(1-42) uptake following overexpression of constitutively active forms of the Rho GTPases Cdc42 and RhoA, whereas modulation of Rac1, which is linked to macropinosome formation, had no effect. Our results confirm that uptake of Aβ(1-42) is clathrin- and dynamin-independent and point to the involvement of a new and distinct clathrin-independent endocytic mechanism which is similar to uptake via CLICs or macropinocytosis but that also appear to involve yet uncharacterized molecular players.


Supplementary Movie 1. Cellular exposure to Aβ(1-42) and dextran 10 kDa during isotonic conditions. SH-SY5Y cells were incubated with 2 µM
and 250 µg/ml AF647-labelled dextran 10 kDa and imaged by confocal microscopy every 10 seconds during continuous incubation. The intensities have been adjusted to best represent intracellular signal. The size of the images are 113*113 µm. It can be noted that intracellular  containing vesicles are detectable towards the end of the time-lapse.

Supplementary Movie 4. Tubulation and fission of dextran 10 kDa-filled VLDs.
SH-SY5Y cells were exposed to hypotonic media (75 % MQ) for 10 min, followed by incubation with 250 µg/ml AF647-labelled dextran 10 kDa in isotonic media (recovery) for 10 min. This resulted in the formation of dextran-filled VLDs, as seen in Fig

Supplementary figures
Supplementary Figure S1. Cellular uptake of Trf and dextran 10 kDa during changes in membrane tension. SH-SY5Y cells exposed to 5 µg/ml AF488-labelled Trf and 250 µg/ml AF647-labelled dextran 10kDa for 10 minutes during either isotonic or hypotonic (75 % MQ water, 25 % cell culture medium) conditions. Recovery denotes cells in isotonic media that have been exposed to hypotonic conditions prior to incubation with Trf and dextran. The cells were imaged by confocal microscopy. The scale bar is 20 µm. Isotonic/hypotonic/recovery have been imaged with identical settings and treated in the same manner, i.e. intensities are comparable for Trf and dextran, respectively. Figure S2. CellLight Actin-GFP staining of cells exposed to changes in membrane tension. SH-SY5Y cells were stained with CellLight Actin-GFP and imaged by confocal microscopy in isotonic medium, after 10 min in hypotonic medium (75 % MQ), and after 10 min at recovery conditions in isotonic medium. The corresponding time-lapse can be seen in Supplementary Movie 5. To visualize actin, the cells were transfected with CellLight Actin-GFP BacMam 2.0 following the protocol provided by the manufacturer.

Supplementary Figure S3. Cellular uptake at increasing concentration of Aβ(1-42) and dextran 10 kDa and during changes in membrane tension. (A) Average intensity and (B) uptake relative to isotonic conditions in cells incubated with HF488-labelled Aβ(1-42) for 10 min. (C) Average intensity and (D) uptake relative to isotonic conditions in cells incubated with
AF647-labelled dextran 10 kDa for 10 min. Cells were analysed by flow cytometry (n=4) and numbers denote concentration of  and dextran, respectively. The combined data is presented in Fig. 3B of the main text. Figure S4. Uptake of Aβ(1-42) and dextran 10 kDa in SH-SY5Y cells exposed to a hypertonic shock. Quantification of cellular uptake of 2 µM  and 250 µg/ml AF647-labelled dextran 10kDa during 10 min isotonic, hypertonic (addition of 100mM NaCl to the cell culture medium) and recovery conditions, as described in the main text. Cells were analysed by flow cytometry and uptake is reported as mean cellular fluorescence intensity (N=4, n=3-4)..

Supplementary Figure S5. Uptake of Aβ(1-42) is independent on GRAF1. HeLa Flp-In T-REx cells were induced to express GFP-tagged GRAF1 after transfection with mCherry-tagged
Cdc42 Q61. The cells were incubated with 5 µM HF647-labelled Aβ(1-42) for 15 min, washed and imaged by confocal microscopy. Supplementary Movie 6 displays a time lapse of these cells, and Fig. 4B of the main text snapshots from this, but to allow for fast imaging and to be able to capture potential co-movement of Aβ(1-42) and GRAF1, these channels had so be imaged simultaneously and hence without Cdc42. This image was captured just prior to the time-lapse and shows the same field of view but also including the Cdc42 channel, with all channels imaged sequentially. The scale bar is 20 µm.