Inhibition of heat shock proteins increases autophagosome formation, and reduces the expression of APP, Tau, SOD1 G93A and TDP-43

Abstract

Aberrant expression and denaturation of Tau, amyloid-beta and TDP-43 can lead to cell death and is a major component of pathologies such as Alzheimer’s Disease (AD). AD neurons exhibit a reduced ability to form autophagosomes and degrade proteins via autophagy. Using genetically manipulated colon cancer cells we determined whether drugs that directly inhibit the chaperone ATPase activity or cause chaperone degradation and endoplasmic reticulum stress signaling leading to macroautophagy could reduce the levels of these proteins. The antiviral chaperone ATPase inhibitor AR12 reduced the ATPase activities and total expression of GRP78, HSP90, and HSP70, and of Tau, Tau 301L, APP, APP692, APP715, SOD1 G93A and TDP-43. In parallel, it increased the phosphorylation of ATG13 S318 and eIF2A S51 and caused eIF2A-dependent autophagosome formation and autophagic flux. Knock down of Beclin1 or ATG5 prevented chaperone, APP and Tau degradation. Neratinib, used to treat HER2+ breast cancer, reduced chaperone levels and expression of Tau and APP via macroautophagy, and neratinib interacted with AR12 to cause further reductions in protein levels. The autophagy-regulatory protein ATG16L1 is expressed as two isoforms, T300 or A300: Africans trend to express T300 and Europeans A300. We observed higher basal expression of Tau in T300 cells when compared to isogenic A300 cells. ATG16L1 isoform expression did not alter basal levels of HSP90, HSP70 or HSP27, however, basal levels of GRP78 were reduced in A300 cells. The abilities of both AR12 and neratinib to stimulate ATG13 S318 and eIF2A S51 phosphorylation and autophagic flux was also reduced in A300 cells. Our data support further evaluation of AR12 and neratinib in neuronal cells as repurposed treatments for AD.

AR12 increases BAG3 expression via ER stress signaling which is essential for Tau and APP degradation via LC3-associated phagocytosis and macroautophagy

“…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 ux 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 ux (Figure 2) [10]. 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.…”

“…Previously we had shown that AR12 and neratinib reduced Tau and APP levels via macro-autophagy and that cells expressing the autophagy protein ATG16L1 T300 were more capable of autophagosome formation and APP / Tau degradation than cells expressing the ATG16L1 A300 isoform [10]. In the present studies, knock down of a regulator of LC3-associated phagocytosis / endocytosis, Rubicon, signi cantly reduced the abilities of the drugs alone or in combination to reduce the expression of chaperone proteins, Tau and APP.…”

“…Thermo Fisher mouse: HSP70 si RNA ID: s201487 Cat #4390771; GRP78 si RNA ID: s67084 Cat #4390771; Rubicon si RNA ID: s104761 Cat #4390771; HSP90 si RNA ID: s67897 Cat #4390771. Multiple control studies have been previously presented showing on-target speci city of our siRNAs, primary antibodies, and our phospho-speci c antibodies to detect both total protein levels and phosphorylated levels of proteins and we present data in HCN2 (human) and BV2 (mouse) cells (Supplemental Figure 1A) [1][2][3][4][5][6][7][8][9][10][11][12][13][14].…”

“…AR12 (OSU-03012) is a derivative of the anti-in ammatory agent celecoxib; unlike the parent compound AR12 lacks COX2 inhibitory activity. Our group demonstrated that AR12 reduced the expression of many chaperone proteins and caused endoplasmic reticulum stress signaling with repression of protein translation, i.e., increased eIF2a S51 phosphorylation, and rapidly caused autophagosome formation, followed later by autolysosome formation, i.e., autophagic ux [1][2][3][4][5][6][7][8][9][10]. We demonstrated that the key cellular target for AR12 was the ER stress-regulatory chaperone GRP78 (aka BiP, HSPA5), followed by other ATP-dependent chaperones in the HSP90/HSP70 families.…”

“…In our recent studies in Alzheimer's Disease, using wild type and genetically modi ed HCT116 colon cancer cells as a model system expressing either ATG16L1 T300 or ATG16L1 A300, we determined whether drugs that directly inhibit the chaperone ATPase activity or cause chaperone degradation and endoplasmic reticulum stress signaling leading to macroautophagy could reduce the levels of proteins which play a pathogenic role in neurodegenerative diseases [10]. AR12 and the breast cancer drug neratinib rapidly reduced expression of Tau, amyloid precursor protein (APP), superoxide dismutase 1 (SOD1) and TAR DNA-binding protein 43 (TDP-43) [10,[12][13][14].…”