The chaperone GRP78 acts both as a chaperone to renature proteins but also plays a pivotal role in the abilities of cells to sense endoplasmic reticulum (ER) stress . GRP78 is located both in the ER and on the outer leaflet of the plasma membrane. In the ER, GRP78 inhibits PKR-like endoplasmic reticulum kinase (PERK), which phosphorylates and inactivates eIF2a. On the cell surface GRP78 plays roles in stabilizing plasma membrane receptors and more recently was shown to play an essential role as a co-receptor for the virus SARS-CoV-2 [9, 16]. We have previously shown that neratinib, via Rubicon-dependent LC3-associated phagocytosis (LAP), caused the internalization and subsequent macroautophagic degradation of growth factor receptors and RAS proteins [12–14]. In microglia, the uptake and degradation of APP has also been linked to LC3-associated endocytosis (LANDO) [22, 23]. Our present studies were designed to determine whether LAP / LANDO played a mechanistic role in the abilities of AR12 and neratinib to cause Tau, APP, and chaperone degradation, and whether this occurred in neurons and microglia.
In HCN2 neuronal cells and BV2 microglial cells, knock down of the essential LAP regulatory protein Rubicon suppressed autophagosome formation though did not appear to alter autophagic flux with respect to the formation of autolysosomes, i.e., vesicles that were initially GFP+ RFP+ became over time only RFP+ (Figure 1). Knock down of Beclin1 or ATG5 abolished autophagosome formation and autophagic flux (Figure 2) . HCN2 neuronal and BV2 microglial cells were transfected with plasmids to express Tau or APP, and co-transfected with siRNA molecules to knock down the expression of Rubicon, Beclin1 or ATG5. AR12 and neratinib reduced the expression of Tau and APP in HCN2 cells, that was blocked by knock down of Rubicon, Beclin1 or ATG5 (Figure 3A; Supplemental Figure 2). Knock down of Rubicon, Beclin1 or ATG5 did not significantly alter the basal expression levels of Tau or APP (data not shown). Compared to the amount of APP expressed from a transfected plasmid, endogenous APP expression in the HCN2 cells was only 5% of that value. For Tau expression, the value was only 6% of the expression compared to when Tau was expressed from a plasmid. APP was localized in the plasma membrane and in neurite-like projections whereas Tau was localized in the cytosol and appeared to have a clustered punctate appearance (Supplemental Figure 1B). AR12 and neratinib, to a significantly greater extent than observed when expressing Tau or APP from plasmids, reduced endogenous Tau and APP levels in HCN2 cells, an effect blocked by knock down of Rubicon (Figure 3B). Knock down of Rubicon, Beclin1 or ATG5 blocked the degradation of Tau and APP in BV2 microglia (Figure 3C). To confirm our Rubicon siRNA knock down data, we made use of RAW macrophages that had been genetically deleted for Rubicon. In wild type RAW macrophages, AR12 and neratinib reduced the expression of chaperones, Tau and APP, and increased eIF2a S51 phosphorylation (Figures 4A and 4B). Deletion of Rubicon in the macrophages abolished the degradation of all tested proteins and the increase in eIF2a S51 phosphorylation.
We next determined in HCN2 neuronal cells and in BV2 microglia the abilities of AR12 and neratinib to reduce the expression of mutant forms of APP and Tau [37–40]. AR12 and the drug combination reduced the expression of Tau 301L which trended to be less than the reduction of wild type Tau (Figure 5A). AR12 and the drug combination was equipotent at reducing the expression of APP, APP715 and APP 692 (Figure 5B). These findings are important for future in vivo studies as, for example, the Tau P301L mutant is used in transgenic models of Alzheimer’s Disease.
We hypothesized that expression of Tau or APP may alter the behavior of signaling pathways when treated with AR12 and neratinib. HCN2 cells were treated with AR12 and neratinib for 6h, after which alterations in protein expression and protein phosphorylation were determined. As we have observed in other cell types, neratinib reduced both the expression and the phosphorylation of the plasma membrane receptors ERBB1/2/3 (Figure 6). Neither expression of Tau nor expression of APP altered the levels of drug-induced protein degradation or protein phosphorylation when compared to empty vector transfected cells. Degradation of KRAS and NRAS was observed. Alterations in the phosphorylation of intracellular signaling pathways also were near identical comparing empty vector control plasmid transfected compared to cells expressing Tau or APP. Notably, compared to other cell types we have previously treated with neratinib as a single agent, we observed a profound increase in the phosphorylation of AMPKa T172 and ULK1 S317 and profound reductions in the phosphorylation of ULK1 S757, mTORC1 S2448, mTORC2 S2481 and p70 S6K T389. Increased S317 phosphorylation concomitant with lower S757 phosphorylation results in a very high level of ULK1 catalytic activity which drives autophagosome formation. Knock down of AMPKa prevented the alterations in protein phosphorylation observed in mTOR and ULK1 (Figure 7A). The most surprising data was that AR12 and neratinib combined to not only reduce p70 S6K T389 phosphorylation but also to reduce p70 S6K protein levels. Knock down of the macro-autophagy regulatory proteins Beclin1 or ATG5 prevented p70 S6K degradation (Figure 7B). As p70 S6K signaling has been linked to enhanced Tau phosphorylation, we hypothesize that the portions of p70 S6K complexed with Tau were being degraded by macroautophagy in our cells.
In HCN2 cells combined exposure of transfected cells to AR12 and neratinib reduced APP expression by 30% and Tau expression by 31%. Transfecting cells to express an activated mutant form of mTOR reduced the ability of the drugs to reduce APP levels, from a 30% reduction to an 11% reduction, and to reduce Tau levels, from a 31% reduction to a 9% reduction. Thus, the ability of AR12 and neratinib to inactivate mTOR, which required the AMPK, plays a key role in the ability of the drug combination to cause the degradation of Tau and APP.
In HCN2 neuronal and BV2 microglial cells, AR12 reduced the expression of GRP78 (cell surface and total), HSP70 and HSP90 (Figures 8 and Supplemental Figure 3). AR12 and neratinib interacted to further reduce the expression of HSP90 and to inactivate eIF2a. Chaperones in cells are complexed with other proteins including BAG3 (associated with HSP70) and AHA1 and CDC37 (associated with HSP90). BAG3, AHA1 and CDC37 have all been linked to AD pathology [24–30]. BAG3 has been shown to enhance Tau degradation by autophagy [22–24]. HSP90 and AHA1 promote Tau pathogenesis . And CDC37 with HSP90 also acts to maintain Tau stability [28–30]. AR12 alone as well as the drug combination increased BAG3 expression (Figure 8 and Supplemental Figure 3). The drug combination did not alter the expression of CDC37 but did reduce the levels of AHA1. The histone deacetylase HDAC6 regulates HSP90 activity; increased HSP90 acetylation reduces chaperone function . AR12 and the drug combination reduced HDAC6 expression, which will increase HSP90 acetylation concomitant with a further reduction in overall HSP90 chaperoning activity (Figure 8 and Supplemental Figure 3).
In neuronal cells and microglia, knock down of the LAP / LANDO regulatory protein Rubicon or the macroautophagy regulatory proteins Beclin1 or ATG5 prevented the drugs alone or in combination from reducing the expression of Tau and APP (Figures 9A and 9B). Knock down of Beclin1 or ATG5 prevented AR12 alone or in combination from enhancing eIF2a S51 phosphorylation in the microglia whereas knock down of Rubicon did not (Figure 9B) [32–35]. In both the HCN2 and BV2 cells, knock down of Rubicon prevented AR12 as a single agent from reducing total GRP78 expression but did not block the reduction when combined with neratinib. Knock down of Rubicon, Beclin1 or ATG5 abolished the abilities of the drugs alone or in combination from reducing cell surface GRP78 levels. Similar data was also obtained when examining the expression of HSP70 and HSP90. Collectively, this data demonstrates a subtle differential regulation of GRP78 expression / degradation by our drugs in neuronal cells and microglia based on its sub-cellular location.
AR12 and neratinib interacted to increase autophagosome formation in neuronal cells and microglia that was followed by autolysosome formation; autophagic flux (Figure 10, upper graphs). Knock down of eIF2a significantly reduced autophagosome formation and abolished the interaction between AR12 and neratinib. AR12 and the drug combination reduced expression and of p62 and LAMP2 (Figure 10, lower Tables). In HCN2 and BV2 cells transfected to express Tau or APP, knock down of eIF2a prevented their degradation. Knock down of eIF2a also prevented the drugs from reducing the levels of HDAC6, p62 and LAMP2. (Figure 11). Thus, we hypothesize that the initial inactivation of GRP78 catalytic function by AR12 facilitates an initial increase in eIF2a phosphorylation which in turn is essential for greater levels of eIF2a phosphorylation, greater levels of macroautophagy and eventually leading to significant amounts of APP/Tau/chaperone protein degradation.
Based on our data showing reduced chaperone expression following drug exposure, we next defined the chaperones which played the most important roles in regulating APP and Tau stability; in HCN2 cells (Figure 12); in BV2 cells (Figure 13); in RAW macrophages (Supplemental Figure 4). Over-expression of GRP78, HSP70 or HSP90, or knock down of GRP78, HSP70 or HSP90 surprisingly did not significantly alter the basal expression levels of APP and Tau (data not shown). This data demonstrates that over-expression of either GRP78, HSP70 or HSP90 prevented the drug-induced degradation of APP. Over-expression of GRP78, but not of HSP70 or HSP90, prevented the drug combination from reducing Tau expression. Knock down of GRP78, but not of HSP70 or HSP90, enhanced the ability of AR12 alone and the drug combination to reduce APP expression. Knock down of GRP78 also further enhanced the ability of AR12 as a single agent to reduce Tau levels. Our GRP78 data is congruent with our earlier findings when knocking down eIF2a expression. Collectively these findings strongly argue that the chaperone GRP78 and translation regulator eIF2a play key roles in regulating the ability of AR12 and neratinib to reduce Tau and APP protein levels.
The co-chaperone BAG3 has been shown to facilitate the degradation of Tau and APP [24–26]. Over-expression of GRP78, HSP70 or HSP90, or knock down of GRP78, HSP70 or HSP90 surprisingly did not significantly alter the basal expression level of BAG3 (data not shown). Knock down of GRP78, HSP70 or HSP90 significantly enhanced the ability of AR12, alone or in combination with neratinib, to enhance BAG3 expression (Figure 14A). Equally, over-expression of GRP78, HSP70 or HSP90 significantly reduced the ability of AR12, alone or in combination with neratinib, from enhancing BAG3 levels (Figure 14B). Thus, reduced chaperone levels facilitate more drug-induced BAG3 expression.
Knock down of BAG3, by 74%, reduced drug-induced autophagosome formation and autophagic flux (Figure 14C). AR12 and neratinib, as previously observed, profoundly reduced the expression of Tau and APP, and knock down of BAG3 almost abolished the abilities of AR12 and neratinib to cause degradation of Tau (~92%) and APP (~91%) (Figure 14D). Knock down of BAG3 also significantly reduced the abilities of AR12 and neratinib to reduce the expression of GRP78 (total and cell surface) and of HDAC6 (Figure 15A). Notably and in contrast to Tau and APP, a trend of GRP78 degradation was observed even in drug-treated BAG3 knock down cells, with a reduction in degradation of only ~48%. This data suggests that the regulation of APP and Tau expression after AR12 / neratinib exposure, compared to GRP78, is exquisitely dependent upon BAG3 expression.
Knock down of BAG3 prevented the degradation of HSP90, p62 and LAMP2 which is congruent with our prior autophagy data (Figure 15B). AR12 and neratinib activated PERK and significantly increased eIF2a S51 phosphorylation (Figure 15C). Knock down of BAG3 reduced the ability of the drugs to increase PERK and eIF2a phosphorylation below significance, although a trend of increased phosphorylation was noted. This data again suggests that the initial catalytic inhibition of GRP78 by AR12 that facilitates ER stress signaling, which in turn leads to autophagy, and degradation of GRP78, acts in a feed-forward fashion and is required to fully activated ER stress signaling. Finally, we attempted to link cause-and-effect for the actions of AR12 upon the expression of BAG3 and the role of autophagy and ER stress signaling in that process. Knock down of Beclin1, ATG5, ULK1, eIF2a or PERK significantly reduced AR12-induced BAG3 expression (Figure 15D). This data further supports the concept that the drugs cause a feed-forward signaling loop to degrade Tau and APP.