Reduced Extracellular Matrix Stiffness Prompts SH-SY5Y Cell

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Reduced extracellular matrix stiffness prompts SH-SY5Y cell softening and actin turnover to selectively increase A#(1-42) endocytosis Terra M. Kruger, Kendra Bell, Thiranjeewa I. Lansakara, Alexei V Tivanski, Jonathan Alan Doorn, and Lewis L Stevens ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00366 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 2, 2018

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ACS Chemical Neuroscience

Reduced extracellular matrix stiffness prompts SH-SY5Y cell softening and actin turnover to selectively increase A(1-42) endocytosis Terra M. Kruger1, Kendra J. Bell1, Thiranjeewa I. Lansakara2, Alexei V. Tivanski2, Jonathan A. Doorn1, Lewis L. Stevens1,* 1. Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, The University of Iowa, Iowa City, IA 52242 2. Department of Chemistry, The University of Iowa, Iowa City, IA 52242 ABSTRACT Alzheimer’s disease (AD), the most common neurodegenerative disorder, is characterized by the extracellular deposition of dense amyloid beta plaques. Emerging evidence suggests that the production of these plaques is initiated by the intracellular uptake and lysosomal pre-concentration of the amyloid-beta (A) peptide. All previous endocytosis studies assess A uptake with cells plated on traditional tissue culture plastic; however, brain tissue is distinctly soft with a low-kPa stiffness. Use of an ultra-stiff plastic/glass substrate prompts a mechanosensitive response (increased cell spreading, cell stiffness and membrane tension) that potentially distorts a cell’s endocytic behavior from that observed in vivo or in a more physiologically relevant mechanical environment. Our studies demonstrate substrate stiffness significantly modifies the behavior of undifferentiated SH-SY5Y neuroblastoma, where cells plated on soft (~1kPa) substrates display a rounded morphology, decreased actin polymerization, reduced adhesion (decreased 1 integrin expression), and a reduced cell stiffness compared to cells plated on tissue culture plastic. Moreover, these neuroblastoma on softer substrates display a preferential increase in the uptake of the Aβ(1-42) compared to Aβ(140), while both isoforms display a clear stiffness-dependent increase of uptake relative to cells plated on plastic. Considering the brain is a soft tissue that continues to soften with age, this mechanosensitive endocytosis of A has significant implications for understanding age-related neurodegeneration and the mechanism behind A uptake and fibril production. Overall, identifying these physical factors that contribute to the pathology of AD may offer novel avenues of therapeutic intervention.

Keywords: amyloid, endocytosis, cell stiffness, extracellular matrix, Alzheimer’s disease, mechanosensitive * Corresponding author. Tel: (319)-335-8823; e-mail: [email protected]

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INTRODUCTION The characteristic, extracellular plaques typically associated with Alzheimer’s disease (AD) are primarily composed of the aggregated amyloid-beta (A) peptide, which most commonly consists of isoforms with either 40 or 42 residues1. A is endogenously produced from the enzymatic cleavage of the amyloid precursor protein (APP), where it is released from the cell into the extracellular space in a soluble, monomeric state2. Although the A peptide/plaques are typically found extracellularly, there is growing evidence that demonstrates that the uptake of A expedites the nucleation phase and initiates amyloid seed formation to promote the production of fibrillar aggregates. It has been demonstrated that amyloid fibril formation primarily occurs via an intracellular route in multiple cell lines3. Further, SH-SY5Y neuroblastoma endocytosis of only 100nM of Aβ leads to significant lysosomal accumulation of A with relative lysosomal concentrations reaching high M levels4. At this concentration coupled with the acidic pH of the lysosome, spontaneous fibrillation is favored, and the formation of high-molecular weight aggregates is likely to occur. Given the possibility that the A fibrillogenesis proceeds through an intracellular route, there is significant interest in detailing the specific pathway(s) of A endocytosis. Multiple routes of A endocytosis are potentially available, including: clathrin-dependent, clathrin-independent, and macropinocytosis. A endocytosis was demonstrated to be most likely receptor-mediated; while both Aβ(1-40) and Aβ(1-42) are internalized by SH-SY5Y, a scrambled version of the peptide was not4. There are a variety of reported receptors/proteins that may bind A including, NMDAr, RAGE, and Apolipoprotein E5-7. It has also been suggested that A(1-42) internalization occurs via a dynamin-dependent pathway in PC12 cells, while Aβ(1-40) is internalized passively via an energy-independent mechanism, and primarily is bound to the cell membrane, rather than accumulate in the lysosomes8. Conversely, another group demonstrated that both A isoforms are endocytosed through actin-dependent mechanisms and over 90% of the internalized peptide was trafficked to the lysosome for undifferentiated SH-SY5Y9. This study further showed a nearly two-fold preferential uptake of A(1-42) relative to A(1-40); this selectivity was suggested to be due to additional, unidentified dynamin-independent pathways specific to A(1-42). Currently, there is no consensus regarding the specific means of A uptake across different cell lines; determining this mechanism is critical for understanding the early, upstream events that precede fibril nucleation and formation. An important consideration which is absent from all previous studies of A endocytosis is the physical, mechanical influence of the extracellular matrix (ECM). The primary emphasis of this work focuses on providing a more physiologically relevant ECM environment to specifically determine the role of ECM stiffness on the uptake of either Aβ isoform. Cells function and maintain homeostasis within a narrow chemical and mechanical context10. Changes in extracellular matrix stiffness have been shown to affect cell morphology, spreading, proliferation, migration, and many other factors, including endocytosis 11-15. As reported by Wésen et al., actin-dependent mechanisms of endocytosis are activated during A internalization9. Since the actin cytoskeleton is sensitive to mechanical cues presented from the ECM, it is expected that changes in ECM stiffness could influence the efficiency, and perhaps influence the mechanism of A uptake. Multiple studies have shown this effect using both nanoparticle and peptide uptake, indicating that ECM stiffness can significantly modify endocytic efficiency(11-13). All current in vitro studies use tissue culture plastic with GPa stiffness that poorly models the physiological mechanical properties (1 – 3 kPa) of soft brain tissue. However, elastography studies confirm that brain tissue continues to soften with age, and this ECM softening is accelerated for patients diagnosed with AD16-17. This softening in vivo environment may derive significant pathophysiological consequences such as increased Aβ uptake and/or fibril 2


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production, but also supports age as the primary risk factor in AD18. Neglecting this physical contribution leaves our understanding of A endocytosis incomplete. RESULTS and DISCUSSION Development of a substrate model using PA gels PA gels are traditionally applied in 2D cell cultures to assess the influence of substrate stiffness on cell function19. These gels are well tested, easy to handle, provide reproducible stiffness for each composition, and the mechanical properties may be tuned systematically over a broad range (1 – 50 kPa) through simply adjusting the cross linking density. These factors make PA gels a suitable model to test our hypothesis that substrate stiffness may modify the endocytosis of A. Moreover, the 2D cell culture facilitates cell removal for follow-on studies including Western blot analysis and flow cytometry. The primary disadvantage is that our PA gel model remains a 2D cell culture and does not recapture apical cell-ECM and cell-cell contacts that would be present in a 3D cell culture19. This extension to 3D cell cultures is an important point and will be subject to future investigations. In order to determine an optimal polyacrylamide (PA) gel system for the purpose of this study, mechanical testing was done to characterize the substrate stiffness with changes in total polymer concentration and crosslinking density. The acrylamide monomer was varied from 810%, while the bis-acrylamide crosslinking agent was varied from 0.03-0.3%. The mechanical properties of the substrate were then characterized by measuring the real component of the shear modulus, G’, of each gel. As shown in Figure 1, by varying the total acrylamide and/or bisacrylamide, G’ was systematically varied between 1 kPa to 8 kPa. Because our hydrogels immersed in cell media (primarily water, an incompressible fluid), we assume a Poisson’s ratio of 0.5, which is consistent with the previous reports by Lee et al. and Takigawa et al20-21.The Young’s moduli calculated from these measured shear moduli provides a stiffness range of 3 – 24 kPa, this range is reasonably consistent with the typical stiffness that would be expected for brain tissue. For this manuscript, we designate the “soft” PA gel has a corresponding shear modulus of approximately 1 kPa, and the “stiff” PA gel has a corresponding shear modulus of approximately 8 kPa. Corresponding AFM nanoindentation results are consistent with Young’s modulus range (Figure S3). Mouse collagen IV was covalently bound to the surface of the gels using sulfo-SANPAH, which contains an amine-reactive N-hydroxysuccinimide ester and a photoactivatable nitrophenyl azide. When exposed to UV light at 320nm, the nitrophenyl azide forms a nitrene group that can react with the PA, while the NHS ester binds to the collagen to facilitate cell adhesion22. A BCA assay was used to quantify to the total protein present on the surface of the gels. The results of this analysis are shown in Figure 2, and there was no significant difference between the protein concentration on the soft and stiff substrates (p = 0.87; one-way ANOVA). Our selection of collagen IV was dictated by more practical reasons. When beginning this project we had tried a series of different ECM linker proteins – laminin, fibronectin and collagen IV – with expectation that each would provide suitable cell adhesion. However, in our preliminary studies, cell adhesion (and consequently cell count) was considerably improved using the collagen IV coating on our PA gels. Since cell count is important for follow-on analyses, e.g. flow cytometry, then we decided to use collagen IV. Moreover, while the brain ECM is composed of many proteins, it had been previously shown that there is a low amount of fibrous matrix proteins23. Collagen IV is not a fibril-type protein like collagen I, and thus may be considered more representative of the brain ECM. While we fully acknowledge that other ECM components (dimensionality, composition, etc.) may be play an important role in A trafficking, this paper addresses the impact of substrate stiffness and is not trying to fully recapitulate the brain ECM.

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SH-SY5Y response to ECM stiffness Multiple studies have demonstrated that cells adapt to mechanical changes in their environment11-12, 24. Generally this mechanoadaptive response physically manifests as changes in cell morphology, phenotypic differentiation, mobility, adhesion, and cytoskeletal reorganization. Cell spreading is a dynamic process; however, this is particularly evident at the early stages of cell adhesion and spreading. For late-stage cell spreading, an equilibrium is established and rounded (for soft substrates) or spread (for stiff substrates) cell morphologies are consistently observed25. All cells were equilibrated for three days prior to fixation and imaging. Undifferentiated SH-SY5Y cells display a consistent response mechanism, e.g. confocal imaging of cells stained for F-actin with phalloidin demonstrate a clear morphological change in response to substrate stiffness. As displayed in Figure 3, on the softest substrate, SH-SY5Y cells exhibit a rounded morphology, while those cells on the stiff substrate and on tissue culture plastic display much higher cell spreading and cell branching. Also shown in Figure 3, this qualitative observation was confirmed quantitatively with area analysis done in FIJI and is consistent with previous reports for multiple different cell lines 11-12. With an increase in cell spreading, our confocal images displayed in Figure 3 demonstrate a stiffness dependent cytoskeletal response from SH-SY5Y cells. For increasing substrate stiffness, there is a clear increase in F-actin polymerization, as shown by the increasing green fluorescence intensity and the presence of stress fibers distributed throughout the cell interior. With regard to fixation, a few publications are available that indicate cell fixation may induce cell blebbing or shrinkage depending upon the temperature, time and osmolarity of the fixative. However, this fixativeinduced cell blebbing was reported to arise from fixation at room temperature, while we used an ice-cold fixative26. If fixation alone were to cause cell rounding, then all cell cultures (plastic, stiff PA gel and soft PA gel) should have given a similar morphology and spread area; however, our results indicate this clearly is not the case. Cytoskeletal reorganization is a common mechanoadaptive response to changes in substrate stiffness27. To better quantify how the physical properties of the cell change as a result of cytoskeletal reorganization, we measured SH-SY5Y live-cell stiffness as a function of substrate stiffness using AFM indentation. Complementary to our observed increase in cell spreading and F-actin polymerization, our AFM results demonstrate that the SH-SY5Y cells also display a significant increase in stiffness with increasing substrate stiffness (Figure 4). All representative force-displacement curves and cell images are provided as Supporting Information. Integrins are heterodimeric, transmembrane proteins that are the mechanoreceptors of the cell28. These proteins negotiate mechanical stimuli presented outside the cell into an intracellular signaling cascade. One primary mechanism performed by integrins is regulation of cell adhesion. Typically cell adhesion is reduced when cells are plated on soft substrates. For SH-SY5Y cells, adhesion is reduced in a stiffness-dependent manner with cells on the softest substrates displaying a significant reduction in 1 integrin expression, as shown in Figure 5. A uptake dependence on substrate stiffness To determine whether the uptake of A is influenced by substrate stiffness, SH-SY5Y cells were plated on our soft and stiff substrates, as well as tissue culture plastic. The cells were dosed with TAMRA-labelled A (0.5 M) for 24 hours, and were then analyzed quantitatively with flow cytometry and qualitatively using confocal microscopy. Our flow-cytometry results displayed that uptake of both A isoforms significantly increased as the substrate stiffness decreased, with maximum uptake observed on our soft substrate (Figure 6). Moreover, while the internalization of A(1-40) and A(1-42) were similar for cells on tissue culture plastic, after 4


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a 24 hr exposure the uptake of A(1-42) on the softest substrate demonstrated a significantly higher uptake relative to A(1-40). If intracellular A accumulation precedes fibrillogenesis, the higher uptake of A(1-42) on soft substrates that physiologically mimic the mechanical environment of the brain, suggests immediate consequences for the potential production of highly toxic A(1-42) oligomeric and high molecular weight species. Figure 7 displays phase contrast images that suggest that both A isoforms were distributed intracellularly, and were not merely adhered to the cell membrane, and is consistent with the previous studies of A endocytosis in SH-SY5Y cells4, 9. Cold culture (at 4°C) is an approach to identify energy-dependent uptake mechanisms by stopping or significantly reducing a cell’s metabolic activity. The endocytosis of both A isoforms was determined to be energy dependent, as shown in Figure 8, by a significant decrease in uptake at 4°C. To aid identifying a specific mechanism of uptake, SH-SY5Y cells were dosed with a series of established pharmacological inhibitors that act to block specific modes of endocytosis. After a pre-treatment period of one hour, all inhibitors demonstrated varying effects on limiting the uptake of either isoform. Our preliminary, inhibition studies were performed on cells plated on tissue culture plastic, and the results of these studies are provided as Supporting Information. Of all inhibitors, methyl-- cyclodextrin (MCD), cytochalasin D (cytD), and nocodazole, demonstrated some inhibition of A endocytosis and thus these three inhibitors were chosen to evaluate whether the mechanism of uptake is sensitive to substrate stiffness. The results of these studies are provided in Figure 8. While the inhibitory effect of cytD and nocodazole were largely insensitive to substrate stiffness, it appears that MCD was less effective on the soft substrates compared to the stiff substrate or the tissue culture plastic. Membrane cholesterol increases with substrate stiffness MCD acts to inhibit endocytosis through the extraction of cholesterol from the plasma membrane29. Because MCD was less effective on the softer substrates, we were interested in whether membrane cholesterol was also dependent upon substrate stiffness. The rationale for this expectation is that commensurate with actin remodeling, changes in cell adhesion and morphology, the plasma membrane tension is mechanosensitive and is a contributing regulator of cell shape and actin turnover. Membrane tension can be modified by its composition, and cholesterol content may vary in response to changes in membrane tension. The membrane cholesterol content was analyzed for the cells plated on each substrate using an Amplex Red cholesterol assay. The cells that were plated on either of the soft or stiff substrates showed a significantly decreased membrane cholesterol than those cells plated on the tissue culture plastic, as shown in Figure 9. The cells plated on the soft and stiff substrates displayed a cholesterol content of 388 and 375 pmol/µg, respectively, while those cells plated on the tissue culture plastic had nearly 75% more cholesterol at 642 pmol/µg. PA substrates for systematically introducing a mechanosensitive cellular response The results from this study are significant evidence that undifferentiated SH-SY5Y cells are sensitive to their mechanical environment. Since the BCA quantitation of collagen deposition on the surface of each PA substrate displayed equivalent protein concentrations, any cell behavior changes observed on the varying substrates is not due to an inconsistent ECM-linker protein distribution but rather due to the mechanical properties of the underlying substrate. Rheological characterization of our substrates demonstrated a range of shear moduli from nominally 1 – 8 kPa are consistent with the results from recent magnetic resonance elastography studies that determine the Young’s moduli of brain tissue to be in the low kPa regime30-32. Reasonably matching this unique mechanical environment, which is considerably softer than other tissues (e.g. muscle, bone, vascular, etc.) is vital for providing a more physiologically relevant environment to better model A endocytosis in vitro. The physical forces 5



introduced through the mechanical ECM are potent regulators of cell function and homeostasis33. Not accommodating for this contribution can lead to a distorted cellular response where pronounced changes in cytoskeletal structure, membrane tension, and protein expression may significantly alter the biochemical endpoints of a specific study. Accounting for the mechanical contribution from the ECM our in vitro A endocytosis studies may more suitably represent neuronal endocytosis in vivo. Previous studies have demonstrated substrate stiffness can affect numerous cellular behaviors, and our work supports similar results11-12, 15. Qualitative observations for SH-SY5Y display a morphological stiffness dependence with cells on softer substrates displaying a rounded morphology distinct from the spreading (and increasing cell area) observed for cells on the stiffest substrates. Consistent with these morphological changes is a restructuring of the actin cytoskeleton. The actin cytoskeleton, in combination with microtubules and intermediate filaments, provides mechanical integrity to the cell27, and thus mechanosensitive changes to cytoskeleton are expected to be similarly displayed in the cell’s mechanical properties. This interdependence between cell area, F-actin and cell stiffness had previously been demonstrated by Solon et. al. for NIH 3T3 fibroblasts24. Using a similar PA substrate model, fibroblasts adopted a spread morphology on stiff substrates with increased cell area. Plotting fibroblast cell stiffness against cell area displayed a positive correlation and also displayed an increase in Factin polymerization. Our AFM nanoindentation results shown in Figure 4 are consistent with these previous studies and confirm that with increasing F-actin polymerization, SH-SY5Y cell stiffness (E, Young’s modulus) more than doubles from cells on soft substrates compared to cells plated on glass. A further consideration is cell adhesion mediated by integrins, a family of heterodimeric, transmembrane proteins that act as the mechanoreceptors of the cell34. Changes in the mechanical microenvironment are communicated through the integrins into a biochemical response that is regulated by the specific recruitment of proteins to integrin’s cytosolic tail or nascent focal adhesion35-36. Increasing cell adhesion physically manifests as an increase in cell spreading/area37, and as previously mentioned, SH-SY5Y adopt a spread morphology on stiff substrates suggesting higher cell adhesion. Our ECM linker protein is mouse collagen IV, and our specific interest focuses on the 1-integrin. Our Western blot results display a clear stiffness dependence for 1 integrin protein expression, cells on soft substrates displaying the lowest 1 expression. This reduced 1 expression limits SH-SY5Y adhesion to the soft substrate and consequently results in a rounded cell morphology with a reduced cell stiffness. Lastly, membrane tension is a further physical cell property that adjusts in response to substrate stiffness. This has clearly been demonstrated by Huang et al. whereby lifetime fluorescence measurements were used to support the conclusion that bovine aortic endothelial cells (BAECs) plated on soft substrates resulted in a significant decrease in membrane tension11. This decrease in membrane tension was consistent with a decrease in both cell area and F-actin polymerization. Overall, our data collectively illustrate that SH-SY5Y cells sensitively respond to changes in substrate stiffness; our next interest is understanding how these physical cell adjustments may act to modulate A endocytosis. Mechanoadaptation and A endocytosis Provided the multiple mechanisms through which substrate stiffness may adjust cell function, our expectation is that endocytosis, an actin-dependent process of trafficking cargo across the cell membrane, may equally be affected38. Only a few previous studies have explicitly tested this expectation; however, all of them consistently demonstrated that substrate stiffness can significantly modify the uptake of both particles and peptides. For the BAEC cells studied by Huang et al., the overall uptake of 100 nm carboxylated polystyrene nanoparticles was determined by the net contribution of cell area and membrane tension. Based on their 6


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observations, they constructed a thermodynamic model to describe the uptake process and conclude that while uptake may increase linearly with cell area, it also decreases exponentially with increasing membrane tension11. Similar studies were reported by Wang et al. for the substrate-dependent internalization of 22 nm sized micelles into two breast cancer cell lines (MCF-7 and MDA-MB-231) with distinctly separate motilities12. Lastly, for the caveolaemediated uptake a small peptide YARA, Brugano demonstrated that human pleural mesothelial cells plated on soft substrates endocytosed this peptide with significantly higher efficiency than cells plated on stiff substrates13. It has been suggested that A uptake is primarily trafficked through an actin-dependent mechanism9, thus mechano-adaptive cytoskeletal restructuring on the softer substrates may rationally influence peptide uptake. Using a cold culture, the uptake of both A isoforms into SHSY5Y cells was determined to follow an energy dependent process, and this supports the previous results reported by Wésen et. al. To identify whether a specific mechanism of endocytosis was preferentially activated, a series of pharmacological, endocytic inhibitors were used: genistein, nocodazole, nystatin, MCD, cytochalasin D, amiloride and chlorpromazine. Our results for all inhibitors for both A(1-40) and A(1-42) are provided as Supporting Information. For SH-SY5Y cells plated on tissue culture plastic, the three most effective inhibitors of the endocytosis of both A isoforms were nocodazole, cytD, and MCD. Pre-treatment with each of these inhibitors reduced A uptake by approximately 20% relative to controls. Both nocodazole and cytD affect the cytoskeleton of the cell through distinctly separate mechanisms39-40. However, MCD binds to the cholesterol on the cell membrane, and therefore disrupts caveolae formation to prevent caveolae-dependent endocytosis. Chlorpromazine inhibits clathrin-mediated endocytosis by translocating clathrin from the cell surface to the intracellular endosomes39. Although chlorpromazine was less effective than the three preceding inhibitors, this was the only inhibitor that displayed a significant, selective inhibition between the two A variants, indicating that A(1-42) is preferentially endocytosed through a clathrinmediated process relative to A(1-40). These results are in line with those previously reported by Wésen et al.; however, their follow-on studies with SH-SY5Y cells transfected to express a C-terminal variant of the clathrin-binding domain of AP180 led to the conclusion that Aβ endocytosis was clathrin-independent, and the inhibitory effect of chlorpromazine was attributed to off-target affects. While we do utilize a series of pharmacological inhibitors to identify a potentially predominant mechanism of endocytosis for each A isoform, it is clear that multiple mechanisms of endocytosis are operable in A uptake. Pharmacological inhibitors are routinely criticized for their lack of specificity, i.e. while a certain targeted pathway may be arrested, offtarget effects can influence other endocytic pathways as well41. Moreover, since multiple modes of endocytosis are involved in the uptake of A, a compensatory mechanism may be initiated whereby the blockade of one mode of endocytosis simply simulates the use of another. Recent endocytic inhibitors, dynasore and Pitstop 2, may have been included; however, they too suffer from a lack of specificity. For dyansore the primary mode of action is blocking dynamin-mediated fission of the clathrin-coated endosome; however, as reported Preta et al., dynasore can destabilize cholesterol homeostasis in the plasma membrane and subsequently affect macropinocytosis and caveolae-mediated uptake42. As further reported by Rodal et al., disruption of cholesterol can affect both clathrin- and caveolae-dependent mechanisms of uptake43. Similarly, Pitstop 2 is not a completely targeted agent, and can affect both clathrindependent and clathrin-independent modes of endocytosis39. Alternatively, siRNA may be used to inhibit endocytosis, however, the knockdown of proteins central to a specific mode of endocytosis can lead to off-target, indirect effects that modify cell function39. Overall, further studies are needed to precisely define the mechanism(s) of A endocytosis. 7



Regardless of the specific mechanism of endocytosis, both A isoforms displayed a significant, endocytic increase for SH-SY5Y cells plated on our soft (1 kPa) substrate. Moreover, substrate softening led to the selective uptake of A(1-42) which significantly outpaced A(1-40) after a 24 hour exposure period. Confocal fluorescent images qualitatively confirm an intracellular distribution of both peptides. From our studies and previous work, the internalization of A is an actin-dependent process, and pre-treatment with cytD results in a significant decrease in Aβ. However, the actin depolymerization associated with cell softening on the softer gels yielded an increase in overall Aβ uptake. CytD is reported to bind with high affinity to the growing, barbed, end of F-actin filaments and prevents further polymerization with G-actin39. Polymerization of actin is an important element of macropinocytosis and drives membrane ruffling and lamellipodia formation and extension44. This pharmacological disruption to the actin cytoskeleton is likely distinct from that initiated by substrate stiffness. Hence the combination of substrate stiffness and cytD pre-treatment does not synergistically deactivate actin-dependent endocytosis, as demonstrated in Figure 8. The mechanosensitive increase in A uptake could be caused by a decrease in the membrane tension for those cells on the soft substrates. Moreover, due the depolymerization of actin, which may frustrate certain actin-dependent modes of endocytosis, a compensatory affect may arise that shifts favor toward alternative modes of endocytosis. Although the substratedependent cell membrane tension was not measured, all other correlative indicators (decreased cell area, decreased F-actin, lower 1 expression and reduced cell stiffness) measured in this study would strongly suggest that SH-SY5Y membrane tension decreases with decreasing substrate stiffness. Particularly for budding-type modes of endocytosis, like caveolae-mediated or clathrin-dependent mechanisms, where the invagination develops inward and is eventually closed off, decreased membrane tension can exert a significant alleviate the energy expended for payload wrapping. Caveolae/raft-mediated endocytosis is further sensitive to cholesterol content45; however, our studies indicate membrane cholesterol decreases as the substrate softens, as shown in Figure 9. This is further supported by the reduced efficacy of MCD, which sequesters cholesterol, observed for cells plated on the softest substrate. Thus, it is more likely that Aβ uptake through clathrin-mediated endocytosis is directly influenced by the decreased membrane tension, and thus lead to the observed increase in A(1-40) and A(1-42) uptake for SH-SY5Y cells on the soft substrates. This is directly consistent with theoretical treatment of receptor-mediated endocytosis reported by Gao et al.46. Moreover, it has been suggested that an increase in membrane tension could lead to an “assault” on the production of clathrin coated pits47. Membrane tension determines the actin dependence of clathrin-coat assembly, where the mature pits are formed in the absence of actin polymerization. This would mean, for the cells that are plated on the stiff substrates and the tissue culture plastic, uptake substantially decreases because of the increased actin polymerization and the increased membrane tension for those cells on stiff substrates. This may further explain the conclusion drawn by Wésen et al. that suggests for SH-SY5Y cells on tissue culture plastic A uptake is clathrin-independent. However, upon decreasing membrane tension, the actin-dependent endocytosis modes become less favorable, and the predominant mechanism shifts to a clathrin-dependent mode to compensate and exploit the energetic benefit of a reduced membrane tension. It is important to recognize the both mechanical and biochemical factors combine to regulate cell function. Physical factors presented from the ECM are often ignored, but a growing literature base is collectively demonstrating the critical role of mechanics is cell biology and disease. Our studies clearly identify that Aβ endocytosis is sensitive to substrate stiffness and the preferential uptake of A(1-42) into softened neuroblastoma has immediate implications toward the potential aggregation of this A variant and the production to neurotoxic, highmolecular weight aggregates. Overall, delineating the mechanical cues that underscore A processing and clearance will provide a fresh perspective to the pathophysiology of AD, and 8


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potentially open research avenues into new sites of therapeutic intervention and novel therapeutic strategies. METHODS Peptide purchase and storage conditions Both TAMRA-A(1-40) and TAMRA-A(1-42) were purchased from Anaspec (Fremont, CA) and received in small, hermetically sealed vials. After opening a vial, the lyophilized peptide was solubilized in dimethylsulfoxide and aliquoted at 200µM. Solutions were stored at 80°C and confirmed stable (no spontaneous fibrillation) by absence of a strong Thioflavin T fluorescent response prior to dosage. We observed that solutions of either isoform were stable under these concentration and storage conditions for approximately three months, though our samples were used within two weeks. Substrate preparation method Polyacrylamide (PA) substrates with varying stiffness were prepared as done previously48. Briefly, 18mm round glass coverslips were coated with a uniform film of sodium hydroxide by evaporating a 500L solution of 0.1M NaOH on a hot plate set to 80°C. The coverslips were then silanated in a 1% aminopropyltriethoxysilane (APTES) solution for 10 minutes. After extensive washing with water, the coverslips were exposed to 0.5% glutaraldehyde for 30 minutes, then were dried with a Kimwipe. Glass slides were treated with 200 L of dichlorodimethylsilane (DCDMS), then washed extensively with water. The polyacrylamide gel solutions were made using varying solutions of Dulbeccos’s phosphate buffered saline, 2% N,N’ methylene bisacrylamide, and 40% acrylamide solutions. The final concentrations and our relative designations for each substrate were: “Soft” (8% acrylamide, 0.03% BIS), and “Stiff” (10% acrylamide, 0.30% BIS). 40L of the stock solutions were sandwiched between the silanated glass slide and the treated glass coverslip and were allowed to polymerize for 30 minutes. Sulfo-SANPAH was covalently cross-linked to the surface of the substrate using a 0.2mg/mL solution under a UV light at 395nm. They were then washed in 50mM HEPES buffer, and incubated with 10µg/mL mouse collagen (IV) overnight at 4°C. The coverslips were sterilized under a UV light in a biosafety cabinet and transferred to a 12-well plate for cell seeding. Substrate characterization 1. Rheology The mechanical properties of each substrate was characterized using a RheoStress One rheometer with a 35mm standard stainless steel plate geometry. The PA substrates were prepared as described, and 1mL of solution was allowed to polymerize on the base of the steel plate. After the substrate was fully polymerized, the upper plate was lowered, leaving a 1mm gap. Frequency and stress sweeps were performed to find the linear viscoelastic range of each gel. Using an oscillatory stress of 250 Pa, frequency was varied from 0.1-10Hz, measuring 8 points per decade. Then, using a frequency of 0.25 Hz, the oscillatory stress was varied between 100 and 1000 Pa. Each frequency and stress sweep were performed at 37°C, and each data point is averaged between three independently prepared samples. We report the average of these measurements with the error represented by the standard deviation. 2. BCA for protein characterization To ensure all of the PA substrates were equivalent in the extracellular matrix sensed by the cells, the ECM protein was quantified using a BCA assay following the manufacturer’s instructions for the enhanced test-tube protocol. The PA substrates were prepared as described, and transferred to sterile 12-well plates. The substrates were incubated with 2mL of 9



the working reagent at 60°C for 30 minutes. After cooling to room temperature, the supernatant in each well was transferred to a 96-well plate and the absorbance was measured at 562nm using a SpectraMax M5 spectrophotometer. Cell culture SH-SY5Y neuroblastoma were obtained from ATCC. Cells were maintained at 37°C and 5% CO2, and grown in Opti-MEM supplemented with 10% fetal bovine serum, 1mM sodium pyruvate, MEM nonessential amino acids (Gibco), and 1% penicillin/streptomycin. All uptake experiments used Opti-MEM without phenol in the media. Cell and Gel Mechanical Property Evaluation Using Atomic Force Microscopy (AFM) Nanoindentation All force versus indentation depth data were collected using a Molecular force probe 3D (Asylum Research) AFM in a liquid cell. SH-SY5Y cells plated on either glass coverslips or PA substrates were submerged in DPBS for the AFM measurements. Nanoindentation measurements were collected within an hour after submersion in the buffer solution. Two different cantilevers (Novascan) were used that had a polystyrene bead with radius of 2.25 µm attached at the end of the cantilever for force measurements. A nominal spring constant was 0.06 N/m and the actual spring constants were determined using a built-in thermal noise method.49 SH-SY5Y cells were located using the AFM top view video camera prior to force measurements. Force measurements were performed at the approximate center of the cells under the cantilever. Force versus indentation depth curves were collected in the contact mode with maximum force range of 1 – 4 nN using a 0.7 µm/s tip approach velocity. Force measurements were collected on three different samples, (I) SH-SY5Y cells grown on bare glass coverslips, (II) SH-SY5Y cells grown on a soft substrates and (III) SH-SY5Y cells grown on stiff substrates. In addition to the measurements over cells, force spectroscopy was also performed in an area clear from cells on top of polyacrylamide gels to obtain the stiffness of the gel. To determine the Young’s moduli for live SH-SY5Y cells and polyacrylamide gels, the approach data of the force-indentation curves collected in buffer solution were fit to the Hertzian elastic contact model.50-52 For the Young’s moduli calculations, a Poisson’s ratio of 0.5 was used for both SH-SY5Y cells and polyacrylamide gels.53-54 Due to the high water content in both cells and gels, we model these materials as isotropic and incompressible, and therefore a Poisson’s ratio of 0.5 is appropriate. Typically, approximately 20 % of the force curves collected were excluded from the analysis due to probing on the edge of the cell.55 Higher stiffness yielding force curves are excluded from the analysis due to contribution of substrate effect which are a result of probing cells on the edge. Averaged Young’s moduli values were obtained by recording 10 consecutive measurements on each individual cell with a minimum of 12 different cells for each different substrate on which they were grown. Assessment of A uptake efficiency and mechanism 1. Flow cytometry Cells were plated on each substrates at 1 x105 cells per well. Because of the poor attachment of the cells to the soft substrate, cells were plated at 1.5 x 105 cells per well, and grown for 24 hours in complete medium. Cells were then dosed with 0.5 M of either isoform (A(1-40) or A(1-42)) of TAMRA-labelled A in phenol-free media. The media was removed, cells were washed with DPBS, and replaced with phenol-free media containing either TAMRAlabelled A isoform (0.5 M) for 24 hours. The cells were washed 2x with ice cold DPBS, then coverslips were removed from their well and inverted in a clean well to detach the cells from the 10


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substrate using trypsinization. Cell viability was ensured by both a separate MTS assay which indicated no toxicity to either A isoform (see Supporting Information) and by using a live/dead violet stain (ThermoFisher). Flow cytometry was run on a Becton Dickinson LSR II. All uptake experiments were done with n = 3 in duplicate with each analysis consisting of 10,000 cells. The results are presented as the ratio of the geometric means of fluorescence * 100 (to give a ‘percentage’ of uptake compared to the untreated control). A detailed description our gating protocol and flow-cytometry data analysis is provided as Supporting Information. 2. Endocytic inhibitors SH-SY5Y cells were dosed with methyl- cyclodextrin (20mM), nocodazole, nystatin, cytochalasin D, or amiloride for 1 hour. To determine whether A uptake was an energydependent process, an uptake study was also done at 4°C, where the cells were placed at 4°C 1 hour prior to an A dose for 2 hours. Flow cytometry analysis was consistent with our protocol for the uptake studies. 3. Confocal microscopy for A uptake and cell spreading After growing on the substrates for 3 days, the cells were fixed for 10 minutes in 4% paraformaldehyde. The cells were then permeabilized with a 0.1% solution of Triton-X for 5 minutes. The cells were incubated with 0.165 M AlexaFluor488-labelled phalloidin to stain for F-actin. The substrates were then mounted on glass slides with VectaSheild mounting media containing DAPI. The fluorescent images were captured using a Zeiss 710 confocal microscope operating at a 63X magnification. Based on these images, relative cell spreading area was calculated using Fiji with a minimum of n = 50 cells per group. Cholesterol Assay Assay was completed using an Amplex Red Cholesterol Assay (ThermoFisher) and used according to the manufacturer’s instructions. This assay is based on the reaction of cholesterol with cholesterol oxidase to yield H2O2, which is then detectable using the Amplex Red reagent. SH-SY5Y cells were plated onto soft, stiff, and tissue culture plastic substrates in 12-well plates at 1x106 cells per well, and grown for 3 days in complete medium. The media was removed and the cells were washed with DPBS. The cells on the PA substrates were lysed by transferring the gels to new 12-well plates and inverting the coverslip onto 100µL lysis buffer [50mM TRIS HCl (pH = 8.5), 100mM NaCl, 5mM MgCl2, 1mM EDTA, 0.5% Nonidet P-40, and protease inhibitor mix (Roche Diagnostics)] and were lysed for 30 min on a shaker table at approximately 4°C. The cholesterol was normalized to the total protein content in the sample that had been previously analyzed using a BCA assay. Western blot – 1 integrin expression SH-SY5Y were plated onto PA gel substrates, and lysed as described earlier. The total protein concentration of each lysate was determined using a BCA according to the manufacturer’s instructions. 10ug of protein were loaded onto a 10% SDS-PAGE gel, transferred electrophoretically onto a nitrocellulose membrane, and probed with 4B7R monoclonal antibody. Supporting Information: - Description of the AFM nanoindentation data analysis for SH-SY5Y cells and PA gels - Representative force-displacement curves and fitting analysis for PA gels - Representative force-displacement curves and fitting analysis for SH-SY5Y cells - Mechanical data for SH-SY5Y cells and PA gels - Complete data set for all inhibited endocytosis studies 11



- MTS viability results for SH-SY5Y cells exposed to monomeric A(1-40) and A(1-42) - Optical images of SH-SY5Y cells prior to AFM measurement - Description of flow-cytometry data analysis and representative scatter plots with gating applied - Representative flow-cytometry histograms for A endocytosis studies Author Contributions: T.M.K. carried out cell culture, substrate preparation and rheological characterization, flow-cytometry endocytosis studies, inhibitor studies, and the membrane cholesterol assay. T.M.K. also lead in writing the manuscript. K.J.B. assisted with cell culture, gel preparation and cell stiffness measurements. T.I.L. and A.V.T. carried out the AFM indentation experiments, data analysis and interpretation of the cell stiffness and PA gel stiffness measurements. J.A.D. provided the SH-SY5Y cells and equipment necessary for Western blotting. T.M.K. and L.L.S. conceived and planned the experiments. All authors contributed to the collective interpretation of the results and each have carefully reviewed the manuscript.

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FIGURES Figure 1. Shear modulus of polyacrylamide gels with varying polymer and cross-linker concentrations.

10000 8000 6000 4000 2000 0 8% -0.03% 8% -0.06% 8% -0.15% 8% -0.30% 10% -0.30%

Acrylamide% - bis-acrylamide%

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Figure 2. Protein concentration (mouse collagen IV) remains consistent across the soft and stiff polyacrylamide gels. (n = 3, p = 0.87)

Collagen concentration ( g/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 3. SH-SY5Y show an increased cell spreading area (n = 50, p < 0.05) and increased Factin polymerization (as shown by the green fluorescent phalloidin stained confocal images) as the substrate stiffness increases. Cell spreading was calculated using groups on n > 50.

SH-SY5Y cell spreading

*

150

* 100

50

0 Soft

Stiff

Glass

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Figure 4. AFM indentation measurements of cell stiffness for SH-SY5Y cells plated on soft and stiff PA gels, as well as on glass. (n = 12, p < 0.05)

0.5

Cell Stiffness, kPa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.4 0.3 0.2

*

*

0.1 0.0 Soft

Stiff

Glass

Figure 5. SDS-PAGE displays an increase in 1 integrin expression for SH-SY5Y plated on tissue culture plastic compared to those plated on softer substrates. (One-way ANOVA with Tukey’s post hoc test n = 3, p < 0.05)

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Figure 6. Uptake of A(1-40) and A(1-42) shows a significant decrease in uptake as the ECM environment becomes stiffer. A(1-42) uptake is even more enhanced than the uptake of A(140).

Percent Control uptake (Plastic)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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Figure 7. Phase contrast images suggest an increased uptake for SH-SY5Y plated on soft substrates. SH-SY5Y were given a dose of 500nM TAMRA-labelled Aβ(1-42) or Aβ(1-40) for 24 hours.

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Figure 8. The effect of chemical inhibitors on the overall uptake of A(1-40) and A(1-42). Cytochalasin D shows a similar effect on uptake, regardless of substrate stiffness or A isoform. MβCD was not effective at all on the softer substrates for either isoform. Nocodazole was ineffective at inhibiting the uptake of Aβ(1-40) on the soft substrates, but did significantly inhibit uptake for A(1-42).

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Figure 9. Cholesterol content for SH-SY5Y cells is decreased on the softer substrates compared to SH-SY5Y plated on tissue culture plastic

Cholesterol content (ng/ g protein)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Reduced extracellular matrix stiffness prompts SH-SY5Y cell softening and actin turnover to selectively increase A(1-42) endocytosis Terra M. Kruger1, Kendra J. Bell1, Thiranjeewa I. Lansakara2, Alexei V. Tivanski2, Jonathan A. Doorn1, Lewis L. Stevens1,*

Figure for Table of Contents


Reduced Extracellular Matrix Stiffness Prompts SH-SY5Y Cell

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