Modulation of Heat Shock Response Proteins by ASS234, Targeted

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Cite This: Chem. Res. Toxicol. 2018, 31, 839−842

Modulation of Heat Shock Response Proteins by ASS234, Targeted for Neurodegenerative Diseases Therapy Eva Ramos,† Alejandro Romero,*,† Jose ́ Marco-Contelles,‡ Francisco Loṕ ez-Muñoz,§,⊥ and Javier del Pino*,†

Chem. Res. Toxicol. 2018.31:839-842. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 09/22/18. For personal use only.

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Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Complutense University of Madrid, 28040 Madrid, Spain ‡ Laboratory of Medicinal Chemistry (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain § School of Health, Camilo José Cela University, Villanueva de la Cañada, 28692 Madrid, Spain ⊥ Neuropsychopharmacology Unit, “Hospital 12 de Octubre” Research Institute, 28041 Madrid, Spain ABSTRACT: ASS234 is a new multitarget molecule with multiple neuroprotective actions that significantly elevate mRNA levels of NRF2 and HSF1 transcriptional factors and of HSP105, HSP90AB1, HSPA1A, HSPA1B, HSPA5, HSPA8, HSPA9, HSP60, DNAJA1, DNAJB1, DNAJB6, DNAJC3, DNAJC5, DNAJC6, HSPB1, HSPB2, HSPB5, HSPB6, HSPB8, and HSP10 heat shock proteins (HSPs) family members in SH-SY5Y cells. This NRF2 and HSF1 overexpression may explain the upregulation of both the antioxidant enzymes previously described and the members of the HSPs family observed. These findings suggest that ASS234 is a potent HSPs inductor, which might be beneficial for preventing protein misfolding aggregation and cell death in Alzheimer’s disease and other neurodegenerative diseases.

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Otherwise, heat shock proteins (HSPs) assist in protein folding, assembly, and stability, ensuring the homeostasis of the proteome (proteostasis), thus playing a key role in preventing protein misfolding aggregation, pathological inflammation processes, oxidative stress generation, and cell death.2 They are divided into several families based on their molecular weight and function; HSP110, HSP90, HSP70, HSP60, HSP40, and the small heat shock proteins (sHSPs).2 HSPs expression disruption has been described as a possible mechanism in the etiology and progression of AD.2 In this regard, our previous studies showed that ASS234 is a potent antioxidant through the overexpression of antioxidant enzymes.3 It was hypothesized that the overexpression of the evaluated antioxidant enzymes may be mediated through the upregulation of nuclear factor (erythroid-derived 2)-like 2 (NRF2), the master regulator of the NRF2 pathway.3 In this line, we use ingenuity pathways analysis (IPA) (Ingenuity H Systems, Redwood City, CA, USA) to evaluate the NRF2 pathway and find that this molecule regulates protein expression of many HSPs (Figure 2), such as HSPB8, HSP90, HSP40, which have been described to be affected in AD.2,4,5 In addition, heat shock transcription factor 1 (HSF1), the key master regulator of HSPs families gene expression, presents a cross-talk regulation of HSPs gene expression with NRF2.6 According to this information, we tested whether ASS234 induced the gene expression of NRF2 and HSF1 transcription

ain hallmarks of Alzheimer’s disease (AD), the most common neurodegenerative disorder, include intracellular deposit of hyperphosphorylated tau fibrillary aggregates and extracellular senile plaques of aggregated amyloid-beta (Aβ) peptides, which have been related to the induction of neuroinflammation, oxidative stress, and cell death.1 However, there is an absence of effective drugs to treat AD. Therefore, previously N-((5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl)methyl)-N-methylprop-2-yn-1-amine (ASS234, Figure 1), a new multitarget small molecule, has been

Figure 1. Chemical structure of ASS234.

designed for the treatment and prevention of AD. This compound is able to act simultaneously as a reversible inhibitor of human acetylcholinesterase (AChE)/butyrylcholinesterase (BuChE) and as an irreversible inhibitor of human monoamine oxidase A/B.1 In addition, it has been reported that ASS234 possesses the capacity to inhibit Αβ1−42 and Αβ1−40 selfaggregation, reducing Αβ1−42-mediated toxicity.1 However, the mechanism underlying its neuroprotective effect remains unclear. © 2018 American Chemical Society

Received: July 18, 2018 Published: August 22, 2018 839

DOI: 10.1021/acs.chemrestox.8b00192 Chem. Res. Toxicol. 2018, 31, 839−842

Chemical Research in Toxicology

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in gene expression were calculated using the Ct (cycle threshold) method. The expression data are presented as actual change multiples. 10 At least three replicates for each experimental condition were performed, and the presented results were representative of these replicates. Data are represented as means ± standard deviation (SD). Comparisons between experimental and control groups were performed by Student’s t-test. Statistical difference was accepted when p ≤ 0.05. Statistical analysis of data was carried out by computer using GraphPad Prism software v. 5.02. The analysis performed, after incubating ASS234 during a 24 h period, showed that this molecule was able to induce the gene expression of NRF2, HSF1, HSP105, HSP90AB1, HSPA1A, HSPA1B, HSPA5, HSPA8, HSPA9, HSP60, DNAJA1, DNAJB1, DNAJB6, DNAJC3, DNAJC5, DNAJC6, HSPB1, HSPB2, HSPB5, HSPB6, HSPB8, and HSP10 (Figure 3A,B) in a statistically significant-manner. Figure 2. NRF2-mediated oxidative stress response pathway adapted from ingenuity pathways analysis (IPA).

factors and of HSP110 (HSP105), HSP90 (HSP90AB1), HSP70 (HSPA1A, HSPA1B, HSPA5, HSPA8, HSPA9), HSP60, HSP40 (DNAJA1, DNAJB1, DNAJB6, DNAJC3, DNAJC5, DNAJC6), sHSPs (HSPB1, HSPB2, HSPB5, HSPB6, and HSPB8), and HSP10, the main members from HSPs families, in SH-SY5Y cells. Neuroblastoma SH-SY5Y cells were incubated with ASS234 (5 μM) for 24 h. We used ASS234 at a concentration of 5 μM based on previous studies of our research group where this compound was effective against several insults, oxidative stress, Tau hyperphosphorylation, or β-amyloid peptide inducedtoxicity, and at which no cytotoxic effects were evidenced in SH-SY5Y cells.3,7,8 Furthermore, previous toxicity studies of ASS234 were performed in the human cell line HepG2, where this compound exhibited less toxicity than the reference compounds, donepezil and tacrine.8,9 Total RNA was later extracted using the Trizol Reagent method (Invitrogen, catalog number 15596−026). The final RNA concentration was determined using a spectrophotometer Nanodrop 2000 (Thermo Fisher Scientific) and the quality of total RNA samples was assessed using an Experion LabChip (Bio-Rad) gel. First-strand cDNA was synthesized with 1000 ng of cRNA by using a PCR array first strand-synthesis kit (C-02; Super Array Bioscience) following the manufacturer’s instructions and including a genomic DNA elimination step and external RNA controls. After reverse transcription, we performed QPCR using prevalidated primer sets (SABiosciences) for mRNAs encoding NRF2 (QT00027384), HSF1 (PPH00164F), HSP105 (PPH01195F), HSP90AB1 (PPH01201C), HSPA1A (PPH01193B), HSPA1B (PPH01216B), HSPA5 (PPH00158E), HSPA8 (PPH01211B), HSPA9 (PPH01191A), HSP60 (QT00018970), DNAJA1 (PPH01189A), DNAJB1 (PPH01207A), DNAJB6 (PPH01212A), DNAJC3 (PPH07375A), DNAJC5 (PPH05923A), DNAJC6 (PPH05924A), HSPB1 (PPH00165F), HSPB2 (PPH01204A), HSPB5 (PPH00123B), HSPB6 (PPH17557B), HSPB8 (PPH11188B), HSP10 (PPH01190B), and ACTB (PPH00073G). We used ACTB as an internal control for normalization. Reactions were run on a CFX96 using real-time SYBR green PCR master mix PA-012 (Super Array Bioscience). The thermocycler parameters were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 72 °C for 30 s. Relative changes

Figure 3. (A) Results from real-time PCR arrays targeting select genes after ASS234 (5 μM) treatment. (B) Representative table of data in which specific gene expression was compared with controls [cells treated with DMSO (0.1%) were the negative control]. Each bar represents mean ± standard deviation (SD) of three independent experiments. ∗∗∗P < 0.001, significantly different from controls.

According to Kyoto Encyclopedia of Genes and Genomes database (KEGG),11 HSPs overexpression could lead cells under pathological conditions to repair and refold misfolded proteins or to degradation of irreparable proteins avoiding neurodegeneration (Figure 4). In this sense, the complex of HSP90 and HSP70/HSP40 (DNAJ) is capable of suppressing Aβ formation and aggregation and has been reported to degrade phosphorylated tau proteins, preventing cell death.1,5 DNAJB6 inhibits the aggregation of Αβ1−42 and the formation of fibrils from polyglutamine peptides, which are involved in neurodegenerative disorders such as AD and Huntington disease, respectively.11 Conversely, DNAJB1 presents an insignificant effect by itself on the polyglutamine and Aβ-42 peptides aggregation, although it acts to enhance the aggregation inhibition effect of HSP70.12 HSP105 upregulates HSP70 gene expression and collaborates with DNAJA1, DNAJB1, and HSP70 (HSPA1A) to disaggregate misfolded and aggregated polypeptides.13 HSP60/HSP10 system is involved in mitochon840



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resistance against H2O2 and superoxide anions, although the mechanism is unknown.27 Overexpression of HSP27 or treatment with rHSP27 has been shown to protect cells from oxidative damage and cell death.28 Thus, the use of therapeutic tools that could induce the regulation of HSPs pathways that could be relevant for neurodegenerative diseases treatment. These data support our previous studies showing an ASS234mediated increase of antioxidant enzyme expression,3 which could be induced through NRF2 upregulation. NRF2 and HSF1 overexpression may explain the upregulation of the HSPs families described, which could protect against AD. In conclusion, according with the reports shown, the increase on the expression of these genes could counteract oxidative stress, accumulation of misfolded proteins, and neuroinflammation processes. Therefore, these effects may well contribute to the ASS234 neuroprotective properties, revealing it as a potential tool for neurodegenerative diseases’ treatment.

Figure 4. Modified with permission from Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway map of protein processing in human endoplasmic reticulum (Bip = HSPA5, GRP94 = HSP90AB1, NEF = HSP105). The shaded genes were determined to be significant from the statistical analysis after ASS234 (5 μM) treatment. Green represents down-regulation, while red depicts up-regulation. White represents no tested. A solid red line represents the most probable route regulation of protein processing in human endoplasmic reticulum.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +34-913943836. *E-mail: [email protected]. Phone: +34-913943970. ORCID

Eva Ramos: 0000-0001-5791-0687 Alejandro Romero: 0000-0001-5483-4973 Javier del Pino: 0000-0001-6937-7701

drial refolding of misfolded proteins and play a decisive role in many neurodegenerative disorders like Alzheimer’s and Parkinson’s disease.14 sHSPs are neuroprotective molecules that act against neurodegenerative disorders diminishing mitochondrial dysfunctions, attenuating accumulation of misfolded proteins, and reducing neuronal apoptosis.15 sHSPs could inhibit amyloid formation and reduce the amount of Aβ oligomers and tau peptides that had been preformed, decreasing their subsequent cytotoxicity.16 HSPs and HSF1 overexpression has been described to reduce protein aggregation of Tau and Aβ, cell death, and cognitive dysfunctions induced by stress factors.17−20 Finally, HSPs overexpression has been reported to be able to modulate inflammation, block reactive oxygen species (ROS) formation, and apoptosis induction.21 HSPs overexpression upregulates antiapoptotic proteins such as BCL-2 and affects caspase processing, protecting against cell death.21 HSP70 and HSP60 have been described to be members of the autoantigen complex, which elicits inmunoregulatory cascades, blocking the immune response.21 HSP70 reduces the release of proinflammatory factors such as nuclear factor kB (NF-kB), matrix metalloproteinases (MMPs), and ROS.22 Overexpression of HSP70 was associated with down-regulation on expression of several representative NF-kB dependent pro-inflammatory genes such as TNF-α and IL-1β.22 Besides, administration of HSP70 has been describe to prevent or block inflammatory damage and induce production of anti-inflammatory cytokines in some chronic inflammatory disorders.23 Otherwise, NRF2 overexpression has been reported to upregulate antioxidant enzymes,24 which supports our previous studies and could explain the ASS234 antioxidant effect observed. In addition, aggregation of misfolded proteins such as amyloid aggregates has been reported to induce oxidative stress,25 and the HSPs overexpression may attenuate this effect through preventing misfolded protein aggregation, which could also explain the ASS234 antioxidant effect. Moreover, HSPs have been reported to attenuate ROS formation. In this regard, HSPB4 binds to transition metal ions and prevents ROS formation.26 HSP60 overexpression has been reported to produce a greater cellular

Funding

This work was supported by MINECO (Grant Nos. SAF2012− 33304 and SAF2015−65586-R) and by HISCHEMAO (Project 2015−21). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.M.-C. is indebted to MINECO for its support. The authors thank Camilo José Cela University for its continued support. REFERENCES

(1) Marco-Contelles, J., Unzeta, M., Bolea, I., Esteban, G., Ramsay, R. R., Romero, A., Martínez-Murillo, R., Carreiras, M. C., and Ismaili, L. (2016) ASS234, As a New Multi-Target Directed Propargylamine for Alzheimer’s Disease Therapy. Front. Neurosci. 10, 294. (2) Gorenberg, E. L., and Chandra, S. S. (2017) The Role of Cochaperones in Synaptic Proteostasis and Neurodegenerative Disease. Front. Neurosci. 11, 248. (3) Ramos, E., Romero, A., Marco-Contelles, J., and Del Pino, J. (2016) Upregulation of Antioxidant Enzymes by ASS234, a Multitarget Directed Propargylamine for Alzheimer’s Disease Therapy. CNS Neurosci. Ther. 22 (9), 799−802. (4) Zhu, Z., and Reiser, G. (2018) The small heat shock proteins, especially HspB4 and HspB5 are promising protectants in neurodegenerative diseases. Neurochem. Int. 115, 69e79. (5) Ou, J. R., Tan, M. S., Xie, A. M., Yu, J. T., and Tan, L. (2014) Heat shock protein 90 in Alzheimer’s disease. BioMed Res. Int. 2014, 796869. (6) Dinkova-Kostova, A. T. (2012) The Role of Sulfhydryl Reactivity of Small Molecules for the Activation of the KEAP1/NRF2 Pathway and the Heat Shock Response. Scientifica 2012, 606104. (7) Del Pino, J., Ramos, E., Aguilera, O. M., Marco-Contelles, J., and Romero, A. (2014) Wnt signaling pathway, a potential target for Alzheimer’s disease treatment, is activated by a novel multitarget compound ASS234. CNS Neurosci. Ther. 20 (6), 568−570. (8) Bolea, I., Gella, A., Monjas, L., Pérez, C., Rodríguez-Franco, M. I., Marco-Contelles, J., Samadi, A., and Unzeta, M. (2013) Multipotent, permeable drug ASS234 inhibits Aβ aggregation, possesses antioxidant

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properties and protects from Aβ-induced apoptosis in vitro. Curr. Alzheimer Res. 10 (8), 797−808. (9) Serrano, M. P., Herrero-Labrador, R., Futch, H. S., Serrano, J., Romero, A., Fernandez, A. P., Samadi, A., Unzeta, M., Marco-Contelles, J., and Martínez-Murillo, R. (2017) The proof-of-concept of ASS234: Peripherally administered ASS234 enters the central nervous system and reduces pathology in a male mouse model of Alzheimer disease. J. Psychiatry. Neurosci. 42 (1), 59−69. (10) Livak, K. J., and Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta DeltaC(T)) Method. Methods 25, 402−408. (11) Kanehisa, M., and Goto, S. (2000) KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27−30. (12) Månsson, C., Arosio, P., Hussein, R., Kampinga, H. H., Hashem, R. M., Boelens, W. C., Dobson, C. M., Knowles, T. P., Linse, S., and Emanuelsson, C. (2014) Interaction of the molecular chaperone DNAJB6 with growing amyloid-beta 42 (Aβ42) aggregates leads to substoichiometric inhibition of amyloid formation. J. Biol. Chem. 289 (45), 31066−31076. (13) Mattoo, R. U., Sharma, S. K., Priya, S., Finka, A., and Goloubinoff, P. (2013) Hsp110 is a bona fide chaperone using ATP to unfold stable misfolded polypeptides and reciprocally collaborate with Hsp70 to solubilize protein aggregates. J. Biol. Chem. 288 (29), 21399−21411. (14) Bie, A. S., Fernandez-Guerra, P., Birkler, R. I., Nisemblat, S., Pelnena, D., Lu, X., Deignan, J. L., Lee, H., Dorrani, N., Corydon, T. J., Palmfeldt, J., Bivina, L., Azem, A., Herman, K., and Bross, P. (2016) Effects of a Mutation in the HSPE1 Gene Encoding the Mitochondrial Co-chaperonin HSP10 and Its Potential Association with a Neurological and Developmental Disorder. Front. Mol. Biosci. 3, 65. (15) Treweek, T. M., Meehan, S., Ecroyd, H., and Carver, J. A. (2015) Small heat-shock proteins: important players in regulating cellular proteostasis. Cell. Mol. Life Sci. 72, 429−451. (16) Wilhelmus, M. M., Otte-Höller, I., Wesseling, P., De Waal, R. M., Boelens, W. C., and Verbeek, M. M. (2006) Specific association of small heat shock proteins with the pathological hallmarks of Alzheimer’s disease brains. Neuropathol. Appl. Neurobiol. 32 (2), 119−130. (17) Abisambra, J. F., Jinwal, U. K., Jones, J. R., Blair, L. J., Koren, J., and Dickey, C. A. (2011) Exploiting the diversity of the heat-shock protein family for primary and secondary tauopathy therapeutics. Curr. Neuropharmacol. 9 (4), 623−31. (18) Kasza, Á ., Hunya, Á ., Frank, Z., Fülöp, F., Török, Z., Balogh, G., Sántha, M., Bálind, Á ., Bernáth, S., Blundell, K. L., Prodromou, C., Horváth, I., Zeiler, H. J., Hooper, P. L., Vigh, L., and Penke, B. (2016) Dihydropyridine Derivatives Modulate Heat Shock Responses and have a Neuroprotective Effect in a Transgenic Mouse Model of Alzheimer’s Disease. J. Alzheimer's Dis. 53 (2), 557−571. (19) Pierce, A., Podlutskaya, N., Halloran, J. J., Hussong, S. A., Lin, P. Y., Burbank, R., Hart, M. J., and Galvan, V. (2013) Over-expression of heat shock factor 1 phenocopies the effect of chronic inhibition of TOR by rapamycin and is sufficient to ameliorate Alzheimer’s-like deficits in mice modeling the disease. J. Neurochem. 124 (6), 880−893. (20) Sun, Y., Zhang, J. R., and Chen, S. (2017) Suppression of Alzheimer’s disease-related phenotypes by the heat shock protein 70 inducer, geranylgeranylacetone, in APP/PS1 transgenic mice via the ERK/p38 MAPK signaling pathway. Exp. Ther. Med. 14 (6), 5267− 5274. (21) Ikwegbue, P. C., Masamba, P., Oyinloye, B. E., and Kappo, A. P. (2018) Roles of Heat Shock Proteins in Apoptosis, Oxidative Stress, Human Inflammatory Diseases, and Cancer. Pharmaceuticals 11 (1), 2. (22) Kim, J. Y., and Yenari, M. A. (2013) The immune modulating properties of the heat shock proteins after brain injury. Anat Cell Biol. 46 (1), 1−7. (23) Giffard, R. G., Han, R. Q., Emery, J. F., Duan, M., and Pittet, J. F. (2008) Regulation of apoptotic and inflammatory cell signaling in cerebral ischemia - the complex roles of Heat Shock Protein 70. Anesthesiology 109 (2), 339−348. (24) Kumar, H., Kim, I. S., More, S. V., Kim, B. W., and Choi, D. K. (2014) Natural product-derived pharmacological modulators of Nrf2/ ARE pathway for chronic diseases. Nat. Prod. Rep. 31, 109−139.

(25) Sharma, S., Verma, S., Kapoor, M., Saini, A., and Nehru, B. (2016) Alzheimer’s disease like pathology induced six weeks after aggregated amyloid-beta injection in rats: increased oxidative stress and impaired long-term memory with anxiety-like behavior. Neurol. Res. 38 (9), 838−850. (26) Nagaraj, R. H., Nahomi, R. B., Mueller, N. H., Raghavan, C. T., Ammar, D. A., and Petrash, J. M. (2016) Therapeutic Potential of αCrystallin. Biochim. Biophys. Acta, Gen. Subj. 1860 (1), 252−257. (27) Cabiscol, E., Bellí, G., Tamarit, J., Echave, P., Herrero, E., and Ros, J. (2002) Mitochondrial Hsp60, resistance to oxidative stress, and the labile iron pool are closely connected in Saccharomyces cerevisiae. J. Biol. Chem. 277, 44531−44538. (28) Alvarez-Olmedo, D. G., Biaggio, V. S., Koumbadinga, G. A., Gómez, N. N., Shi, C., Ciocca, D. R., Batulan, Z., Fanelli, M. A., and O’Brien, E. R. (2017) Recombinant heat shock protein 27 (HSP27/ HSPB1) protects against cadmium-induced oxidative stress and toxicity in human cervical cancer cells. Cell Stress Chaperones 22 (3), 357−369.

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Modulation of Heat Shock Response Proteins by ASS234, Targeted

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