Activation of AMPK-induced autophagy ameliorates Huntington disease pathology in vitro

Highlights

• AMPK activation by A769662 can induce autophagy in an mTOR independent manner.

• Autophagy induction by A769662 or overexpression of a constitutively active form of AMPKα improves HD phenotype.

• Validation of AMPK as a promising therapeutic target to treat HD.

• Identification of A769662 as a potential therapeutic compound to facilitate the clearance of mutant huntingtin.

Abstract

The expansion of a polyglutamine repeat in huntingtin (HTT) causes Huntington disease (HD). Although the exact pathogenesis is not entirely understood, mutant huntingtin (mHTT) causes disruption of various cellular functions, formation of aggregates and ultimately cell death. The process of autophagy is the main degradation pathway for mHTT, and various studies have demonstrated that the induction of autophagy leads to an amelioration of aggregate formation and an increase in cell viability. Commonly, this is achieved by inhibition of the mammalian target of rapamycin (mTOR), a prominent regulator of cell metabolism. Alternatively, non-canonical AMPK or mTOR-independent autophagy regulation has been recognized. Given mTOR's involvement in major cellular pathways besides autophagy, its inhibition may come with potentially detrimental effects. Here, we investigated if AMPK activation may provide a target for the induction of autophagy in an mTOR-independent manner. We demonstrate that activation of AMPK by A769662 and overexpression of a constitutively active form of AMPKα in STHdh cells and mouse embryonic fibroblasts (MEFs), leads to increased expression of the autophagosomal markers LC3 and p62, suggesting efficient autophagy induction. The induction of autophagy was independent of mTOR, and accompanied by a decrease of mHTT-containing aggregates as well as improved cell viability.

Therefore, we validated AMPK as a promising therapeutic target to treat HD, and identified A769662 as a potential therapeutic compound to facilitate the clearance of mHTT.

Introduction

Macroautophagy, herein referred to as autophagy, is the most important degradation pathway in cells, besides the proteasomal degradation system (Ciechanover, 2005). Cellular components, which have to be degraded and may consist of long-lived proteins, protein complexes, damaged organelles and even pathogens, are engulfed by a double membrane structure, the so-called autophagosome, are degraded by the lysosomal degradation pathway (Dunn, 1994). Via macroautophagy, cell constituents can be recycled, viruses or bacteria can be rendered harmless, and the cells can get rid of disturbing and ultimately toxic substances (Yuk et al., 2012).

Thus, the autophagic system is of major importance for maintaining proper cell function especially in non-regenerating cells such as neurons (Mehrpour et al., 2010). Defective or ineffective autophagic degradation has been shown to be associated with numerous diseases such as cancer, viral and bacterial infections, and, as discovered more recently, with neurodegenerative diseases (Qu et al., 2003, Liang et al., 1998, Levine, 2005, Rubinsztein, 2006). Especially in neurodegenerative diseases characterised by the formation of protein aggregates, such as tauopathies or polyglutamine (polyQ) diseases including Huntington Disease (HD), autophagy holds an important role regarding the depletion of toxic aggregation products (Ravikumar et al., 2002).

HD is a progressive neurodegenerative disease which is associated with motor dysfunction, psychiatric disturbances and cognitive decline. The disease is caused by an expanded CAG repeat in the huntingtin gene (The Huntington's Disease Collaborative Research Group, 1993), which translates into an elongated polyQ stretch in the huntingtin protein (HTT), and which results in functional changes of the protein, as well as misfolding and aggregation in nucleus and neuronal processes (Vonsattel et al., 1998). Due to the large protein size and the presence of the polyQ stretch in mutant huntingtin (mHTT), both soluble and aggregated forms are mainly degraded by the autophagosomal degradation pathway (Ravikumar et al., 2002). Several studies have shown that the induction of autophagy leads to a decrease in soluble and aggregated mHTT, as well as a reduction of mHTT-mediated cytotoxicity in HD cell and animal models (Ravikumar et al., 2004, Sarkar et al., 2005).

In these studies, the induction of autophagy was most frequently evoked by inhibition of the mammalian target of rapamycin (mTOR), the major negative regulator of autophagy, via rapamycin (Ravikumar et al., 2004). However, several studies have reported a dysregulation of the mTOR signalling pathway in different HD cell and animal models that has been interpreted as a survival mechanism of the affected cells (Xifró et al., 2011, Gines et al., 2003). Therefore, mTOR might not provide the best target for autophagy induction, and a non-canonical mTOR-independent (Codogno et al., 2011) strategy should be considered for therapeutic approaches.

In this regard, AMP-activated kinase (AMPK), a major energy sensor (Steinberg and Kemp, 2009), might provide a potential alternative as a target for autophagy induction. AMPK is composed of a heterotrimeric structure, in which the α-subunit exhibits catalytic, and the β– and γ-subunits possess regulatory function (Xiao et al., 2011). AMPK can be activated by different upstream kinases and by phosphorylation of its catalytic α-subunit at its Thr172 residue (Carling et al., 2008).

Besides its key role in metabolism, AMPK has been shown to regulate autophagy. The activation of AMPK induces autophagy in two different ways, on the one hand by inhibition of mTORC1 (Inoki et al., 2003, Dubbelhuis and Meijer, 2002) and on the other hand by phosphorylating the mammalian homologue of ATG1, the Unc-51-like autophagy activating kinase 1 (ULK1) (Egan et al., 2011, Meijer and Codogno, 2011). Activation of AMPK by the compounds metformin and resveratrol has been shown to have neuroprotective effects, and metformin even prolonged the life span and improved the motor phenotype in an HD mouse model (Ma et al., 2007, Kumar et al., 2006). Additionally, the two substances neferine and onjisaponin B has been shown to induce autophagy by activating AMPK in an mTOR dependent manner and to improve HD phenotypes in PC12-cells (Wong et al., 2015, Wu et al., 2013).

Using two different HD cell models and automated high-throughput autophagy assessments (Thost et al., 2015) we analysed the relationship between AMPK activation, either by using the small molecule A769662 or by overexpressing a constitutively active form of AMPKα, and autophagy in an mTOR independent manner. We demonstrate that AMPK activation leads to autophagy induction in the absence of altered mTOR activity, a decrease in mHTT aggregation, and a subsequent increase in cell viability. Thus, targeting AMPK may provide a promising therapeutic strategy to mediate mHTT clearance for HD without interfering with mTOR signalling.