Azadiradione ameliorates polyglutamine expansion disease inDrosophilaby potentiating DNA binding activity of heat shock factor 1

Vinod K Nelson,Manas Jana,Subhash C Lakhotia,Gopal Chakrabarti,Vibha Dwivedi,Anup K Misra,Asif Ali,Naibedya Dutta,Soumyadip Paul,Arnab Ganguli,Andrei Komarov,Nihar R Jana,Mahadeb Pal,Suvranil Ghosh,Subhrangsu Chatterjee,Subhash C Mandal

doi:10.18632/oncotarget.12930

Abstract

Aggregation of proteins with the expansion of polyglutamine tracts in the brain underlies progressive genetic neurodegenerative diseases (NDs) like Huntington's disease and spinocerebellar ataxias (SCA). An insensitive cellular proteotoxic stress response to non-native protein oligomers is common in such conditions. Indeed, upregulation of heat shock factor 1 (HSF1) function and its target protein chaperone expression has shown promising results in animal models of NDs. Using an HSF1 sensitive cell based reporter screening, we have isolated azadiradione (AZD) from the methanolic extract of seeds of Azadirachta indica, a plant known for its multifarious medicinal properties. We show that AZD ameliorates toxicity due to protein aggregation in cell and fly models of polyglutamine expansion diseases to a great extent. All these effects are correlated with activation of HSF1 function and expression of its target protein chaperone genes. Notably, HSF1 activation by AZD is independent of cellular HSP90 or proteasome function. Furthermore, we show that AZD directly interacts with purified human HSF1 with high specificity, and facilitates binding of HSF1 to its recognition sequence with higher affinity. These unique findings qualify AZD as an ideal lead molecule for consideration for drug development against NDs that affect millions worldwide.

Highlights

Neurodegenerative diseases (NDs) like polyglutamine based diseases which include spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), Machado-Joseph disease MJD/SCA3), several spinocerebellar ataxias (SCA), and Huntington’s, Parkinson’s- and Alzheimer’s diseases affect millions of people worldwide [1, 2]
Screening for activator of heat shock factor 1 (HSF1) was carried out using cell-based reporter system harboring renilla luciferase (Rluc) and GFP independently under the control of six tandem copies of heat shock element (HSE) (6xHSE)
The purification was verified by an increase in the specific activity by Rluc assay at each purification step (Figure 1A)

Summary

Introduction

Joseph disease MJD/SCA3), several spinocerebellar ataxias (SCA), and Huntington’s, Parkinson’s- and Alzheimer’s diseases affect millions of people worldwide [1, 2]. People affected with these diseases survive in a debilitated condition, which imposes a heavy financial and psychological burden on the society. Accumulation of non-native protein aggregates, defective cellular heat shock response (HSR) and compromised protein quality control pathways are common hallmarks of various NDs [5,6,7,8]. HSF1 as a homotrimer binds to the recognition element HSE (repetitive 5’-nGAAn-3’) on its target chaperone genes to activate their expression which helps in refolding of mis- or unfolded proteins or removal of the non-native protein aggregates [13]. Chemical inhibition of HSP90 and proteasome results in HSF1 activation [12, 16]

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Results

Conclusion