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Holdase Activity of Secreted Hsp70 Masks Amyloid-Β42 Neurotoxicity in Drosophila

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Holdase Activity of Secreted Hsp70 Masks Amyloid-Β42 Neurotoxicity in Drosophila Holdase activity of secreted Hsp70 masks amyloid-β42 neurotoxicity in Drosophila Pedro Fernandez-Funeza,b,c,1, Jonatan Sanchez-Garciaa, Lorena de Menaa, Yan Zhanga, Yona Levitesb, Swati Kharea, Todd E. Goldea,b, and Diego E. Rincon-Limasa,b,c,1 aDepartment of Neurology, McKnight Brain Institute, University of Florida, Gainesville, FL 32611; bDepartment of Neuroscience, Center for Translational Research on Neurodegenerative Diseases, University of Florida, Gainesville, FL 32611; and cGenetics Institute, University of Florida, Gainesville, FL 32611 Edited by Nancy M. Bonini, University of Pennsylvania, Philadelphia, PA, and approved July 11, 2016 (received for review May 25, 2016) Alzheimer’s disease (AD) is the most prevalent of a large group of cell-free systems by dissociating preformed oligomers but not fi- related proteinopathies for which there is currently no cure. Here, we brils, suggesting that Hsp70 targets oligomeric intermediates (18). used Drosophila to explore a strategy to block Aβ42 neurotoxicity More recent in vitro studies show that Hsp70 and other chaperones through engineering of the Heat shock protein 70 (Hsp70), a chap- promote the aggregation of oligomers into less toxic species (19). erone that has demonstrated neuroprotective activity against several Also, Hsp70 demonstrates neuroprotection against intracellular intracellular amyloids. To target its protective activity against extra- Aβ42 in primary cultures (20), whereas down-regulation of Hsp70 cellular Aβ42, we added a signal peptide to Hsp70. This secreted form leads to increased protein aggregation in transgenic worms of Hsp70 (secHsp70) suppresses Aβ42 neurotoxicity in adult eyes, expressing intracellular Aβ42 (21). A recent study in a transgenic reduces cell death, protects the structural integrity of adult neurons, mouse model of AD overexpressing the Amyloid precursor pro- alleviates locomotor dysfunction, and extends lifespan. SecHsp70 tein with the Swedish mutation (APPswe) showed that Hsp70 binding to Aβ42 through its holdase domain is neuroprotective, overexpression improved cognition and reduced Aβ42 levels, but but its ATPase activity is not required in the extracellular space. Thus, this protective activity seemed to be indirectly mediated by ele- the holdase activity of secHsp70 masks Aβ42 neurotoxicity by pro- vated levels of insulin-degrading enzyme in glia (22). Overall, moting the accumulation of nontoxic aggregates. Combined with these observations suggest that Hsp70 exerts a robust protective other approaches, this strategy may contribute to reduce the burden activity against Aβ42 toxicity under experimental conditions that of AD and other extracellular proteinopathies. facilitate their interaction. Unfortunately, this interaction is pre- cluded under physiological conditions because Hsp70 and Aβ42 amyloid-β | Hsp70 | Drosophila | engineered chaperones | occupy different subcellular compartments. neuroprotections Drosophila has played an instrumental role in the identification of genetic and molecular mechanisms mediating the neurotoxicity lzheimer’s disease (AD) is characterized by the presence of of misfolded amyloids, with particular attention to AD (23, 24). A Aamyloid-β1–42 (Aβ42) deposition in senile plaques, tau ac- key advantage of Drosophila models is the ability to efficiently test cumulation in neurofibrillary tangles, and neuronal loss coupled candidate genes. Here, we engineered Hsp70 to force its secretion with cognitive decline and other behavioral alterations (1). Ge- (secHsp70) into the extracellular space, where it could potentially netic evidence strongly supports a causal role for Aβ42 as the interact with Aβ42. We report that secHsp70 suppresses Aβ42 triggering event in AD (2). Although the initial amyloid cascade neurotoxicity in the eye, reduces cell death in brain neurons, hypothesis posited that the accumulation of Aβ42 into insoluble protects the structural integrity of mushroom body neurons, and amyloid fibers was the key triggering event, growing evidence suppresses the progressive dysfunction of locomotor neurons. We indicates that soluble preamyloid structures are the main con- tributors to AD pathogenesis (3). These preamyloid structures Significance include both oligomers with little β-sheet structure and proto- β fibrils with a high -sheet content. Indeed, in certain experi- Heat shock protein 70 (Hsp70) is a critical protein with many β mental systems, it can be shown that soluble A 42 assemblies are protective activities inside the cell. We demonstrate that forced more potent toxins than the amyloid fibrils themselves (4, 5). secretion of Hsp70 is beneficial against the extracellular protein However, the likely reality is that both soluble preamyloid and aggregates typical of Alzheimer’s disease (AD). Engineering amyloid assemblies contribute to disease but perhaps in different Hsp70 enables its interaction with the amyloid-β42 peptide, the β ways. Thus, neutralizing the toxicity of different A 42 assemblies main pathogenic agent in AD. This interaction suppresses am- poses extraordinary challenges. yloid-β toxicity in the eye, reduces cell death in brain neurons, In addition to AD, self-association of misfolded proteins into and protects neuronal architecture and function. Interestingly, toxic assemblies is the main pathological event in a large number of secreted Hsp70 exerts this protective activity without utilizing neurodegenerative diseases collectively known as protein misfolding its refolding activity and without decreasing the levels and disorders or proteinopathies (6). These intracellular protein aggre- aggregation of amyloid-β42. These results suggest a protective gates led several teams to propose molecular chaperones as cellular mechanism mediated by direct binding to amyloid-β42, which machines with key roles regulating pathology. Early in vitro studies blocks amyloid-β42 neurotoxicity. We discuss here the poten- confirmed the regulatory role for chaperones in protein misfolding tial therapeutic benefits of secreted Hsp70. and aggregation (7–9). Then, N. Bonini’s group demonstrated that the Heat shock protein 70 (Hsp70), the main cytosolic chaperone in Author contributions: P.F.-F., J.S.-G., and D.E.R.-L. designed research; P.F.-F., J.S.-G., L.d.M., eukaryotes, suppressed the toxicity of Ataxin-3–78Q (Atx3) and Y.Z., Y.L., S.K., and D.E.R.-L. performed research; P.F.-F. and D.E.R.-L. contributed new α Drosophila reagents/analytic tools; P.F.-F., J.S.-G., L.d.M., Y.Z., Y.L., and D.E.R.-L. analyzed data; and mutant -Synuclein in (10, 11). Hsp70 proved later its P.F.-F., T.E.G., and D.E.R.-L. wrote the paper. neuroprotective activity in transgenic mice expressing mutant Atx1, The authors declare no conflict of interest. – Androgen receptor, and tau (12 14). These results suggest the This article is a PNAS Direct Submission. therapeutic potential of Hsp70 activation (15, 16). Hsp70 is found in 1To whom correspondence may be addressed. Email: [email protected] or diego. a variety of pathological protein aggregates within cells and dem- [email protected]. onstrates a notable ability to regulate proteotoxic assemblies (17). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Remarkably, Hsp70 inhibits the early stages of Aβ42 misfolding in 1073/pnas.1608045113/-/DCSupplemental. E5212–E5221 | PNAS | Published online August 16, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1608045113 Downloaded by guest on October 2, 2021 also find that secHsp70 interacts with Aβ42 in brain neurons but cytosolic/nuclear amyloid (27). Compared with control flies ex- PNAS PLUS does not alter Aβ42 steady-state levels or aggregation. In- pressing LacZ alone (Fig. 2 A and F), flies coexpressing Atx3-78Q terestingly, secHsp70 promotes the formation of Aβ42 aggregates with LacZ exhibited glassy and depigmented eyes with abnormal in a cell-based assay that is reversed by addition of ATP, sug- lenses (Fig. 2 B and G). As described previously, Hsp70 completely gesting that the secHsp70 binding is critical to mask neurotoxic suppressed the Atx3-78Q eye phenotypes (Fig. 2 C and H)(10), Aβ42 epitopes. Based on these results, we demonstrate that the demonstrating its potent antiamyloid activity. However, neither ATPase activity of secHsp70 that mediates its foldase activity is ER-bound BiP nor secHsp70 modified the glassy and depig- not required for its protective activity. We also confirmed through mented eye induced by Atx3-78Q (Fig. 2 D, E, I,andJ). Flies mutations that disrupt the substrate-binding domain (SBD) of coexpressing Atx3-78Q and a secreted GFP reporter (secGFP), a secHsp70 that the holdase activity of secHsp70 is sufficient to control for the artificial secretion of Hso70, exhibited the same eye mask Aβ42 neurotoxic epitopes. These studies report a potent phenotype as flies coexpressing LacZ (Fig. S1 A and B). Thus, BiP mechanism for blocking Aβ42 neurotoxicity. and secHsp70 are confined to the secretory compartment and do not leak into the cytosol based on the lack of suppression of the Results Atx3-78Q phenotype. Generation of SecHsp70. To force Hsp70 secretion, we analyzed Next, we determined the ability of the different Hsp70 con- virtual fused human Hsp70 (HSPA1L) (10) with the signal structs to suppress the toxicity of the human Aβ42 peptide in the peptide of the human Ig heavy chain V-III, which is compatible eye. Coexpression of Aβ42 and LacZ dramatically reduced the size with Drosophila and mammals and displayed a perfect match of the eye, disrupted
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