Sovan Sarkar, Ph.D.

 

Research Associate       
Dept. of Medical Genetics,  
University of Cambridge,
Cambridge Institute for Medical Research,
Wellcome Trust/MRC Building,
Addenbrooke's Hospital, Box 139, Hills Road,
Cambridge CB2 0XY,
United Kingdom

Research

The formation of intra-neuronal mutant protein aggregates is a characteristic feature of several human neurodegenerative disorders, like Alzheimer’s disease, Parkinson’s disease, and polyglutamine disorders, including Huntington’s disease and spinocerebellar ataxias. One possible approach to treating such diseases may be to enhance degradation of the mutant proteins associated with neurodegeneration. The autophagy-lysosome and ubiquitin-proteasome pathways are the two major routes for protein clearance in eukaryotic cells. While the ubiquitin proteasome system predominantly degrades short-lived proteins and it is unclear whether it is a feasible therapeutic target, the clearance of aggregate-prone proteins (aggregate precursors) can be achieved by upregulating autophagy that can degrade long-lived proteins, protein complexes and organelles. Autophagy is an intracellular protein degradation pathway for aggregate-prone proteins causing neurodegenerative diseases, such as mutant huntingtin associated with Huntington’s disease, A53T and A30P mutants of alpha-synuclein associated with familial Parkinson's disease, ataxin 3 causing spinocerebellar ataxia type 3, and tau causing fronto-temporal dementias. Autophagic degradation of these mutant proteins correlates with reduced protein aggregation and toxicity, and therefore, enhancing autophagy may be a possible therapeutic strategy for such diseases where the mutant proteins are autophagy substrates. Thus, identification of autophagy-inducing drugs has huge therapeutic potential not only for neurodegenerative diseases, but also for other diseases where autophagy acts as a protective pathway.

Autophagy is negatively regulated by the mammalian Target Of Rapamycin (mTOR; a serine/threonine kinase), and can be induced by the mTOR inhibitor rapamycin. Since mTOR has many vital cellular functions, it is highly desirable to discover mTOR-independent, autophagy-inducing pathways/drugs as therapeutic targets. My work involves in the identification of autophagy modulators that regulate autophagy independently of mTOR.

1. Sarkar S, Floto RA, Berger Z, Imarisio S, Cordenier A, Pasco M, Cook LJ and Rubinsztein DC. Lithium induces autophagy by inhibiting inositol monophosphatase. Journal of Cell Biology 170(7):1101-1111 (2005). Access article Supplementary information

Abstract: Macroautophagy is a key pathway for the clearance of aggregate-prone cytosolic proteins. Currently, the only suitable pharmacologic strategy for up-regulating autophagy in mammalian cells is to use rapamycin, which inhibits the mammalian target of rapamycin (mTOR), a negative regulator of autophagy. Here we describe a novel mTOR-independent pathway that regulates autophagy. We show that lithium induces autophagy, and thereby, enhances the clearance of autophagy substrates, like mutant huntingtin and alpha-synucleins. This effect is not mediated by glycogen synthase kinase 3beta inhibition. The autophagy-enhancing properties of lithium were mediated by inhibition of inositol monophosphatase and led to free inositol depletion. This, in turn, decreased myo-inositol-1,4,5-triphosphate (IP3) levels. Our data suggest that the autophagy effect is mediated at the level of (or downstream of) lowered IP3, because it was abrogated by pharmacologic treatments that increased IP3. This novel pharmacologic strategy for autophagy induction is independent of mTOR, and may help treatment of neurodegenerative diseases, like Huntington's disease, where the toxic protein is an autophagy substrate.

2. Sarkar S, Davies JE, Huang Z, Tunnacliffe A and Rubinsztein DC. Trehalose, a novel mTOR-independent autophagy inducer, accelerates clearance of mutant huntingtin and alpha-synuclein. Journal of Biological Chemistry 282(8):5641-5652 (2007). Access article Supplementary information

Abstract: Trehalose, a disaccharide present in many non-mammalian species, protects cells against various environmental stresses. Whereas some of the protective effects may be explained by its chemical chaperone properties, its actions are largely unknown. Here we report a novel function of trehalose as an mTOR-independent autophagy activator. Trehalose-induced autophagy enhanced the clearance of autophagy substrates like mutant huntingtin and the A30P and A53T mutants of alpha-synuclein, associated with Huntington disease (HD) and Parkinson disease (PD), respectively. Furthermore, trehalose and mTOR inhibition by rapamycin together exerted an additive effect on the clearance of these aggregate-prone proteins because of increased autophagic activity. By inducing autophagy, we showed that trehalose also protects cells against subsequent pro-apoptotic insults via the mitochondrial pathway. The dual protective properties of trehalose (as an inducer of autophagy and chemical chaperone) and the combinatorial strategy with rapamycin may be relevant to the treatment of HD and related diseases, where the mutant proteins are autophagy substrates.

3. Sarkar S*, Perlstein EO*, Imarisio S, Pineau S, Cordenier A, Maglathlin RL, Webster JA, Lewis TA, O’Kane CJ, Schreiber SL and Rubinsztein DC. Small molecules enhance autophagy and reduce toxicity in Huntington’s disease models. Nature Chemical Biology 3(6):331-338 (2007). Access article Supplementary information Chemical compounds Cover page  *Joint first authors

Abstract: The target of rapamycin (TOR) proteins regulate various cellular processes including autophagy, which may play a protective role in certain neurodegenerative and infectious diseases. Here we show that a primary small-molecule screen in yeast yields novel small-molecules modulators of mammalian autophagy. We first identified novel small-molecule enhancers (SMER) and inhibitors (SMIR) of the cytostatic effects of rapamycin in Saccharomyces cerevisiae. Three SMERs induced autophagy independently of rapamycin in mammalian cells, enhancing the clearance of autophagy substrates like mutant huntingtin and A53T alpha-synuclein, associated with Huntington's disease (HD) and familial Parkinson's disease, respectively. These SMERs, which appear to act either independently, or downstream, of TOR, attenuated mutant huntingtin-fragment toxicity in HD cell and Drosophila models, suggesting therapeutic potential. We also screened structural analogs of these SMERs and identified additional candidate drugs enhancing autophagy. Thus, we have demonstrated proof-of-principle for a novel approach for discovery of small-molecule modulators of mammalian autophagy.

4. Sarkar S, Krishna G, Imarisio S, Saiki S, O’Kane CJ and Rubinsztein DC. A rational mechanism for combination treatment of Huntington's disease using lithium and rapamycin. Human Molecular Genetics 17(2):170-178 (2008). Access article Supplementary information

Abstract: Huntington's disease (HD) is caused by a polyglutamine expansion mutation in the huntingtin protein that confers a toxic gain-of-function and causes the protein to become aggregate-prone. Aggregate-prone proteins are cleared by macroautophagy, and upregulating this process by rapamycin, which inhibits the mammalian target of rapamycin (mTOR), attenuates their toxicity in various HD models. Recently we demonstrated that lithium induces mTOR-independent autophagy by inhibiting inositol monophosphatase (IMPase) and reducing inositol and IP(3) levels. Here we show that glycogen synthase kinase-3beta (GSK-3beta), another enzyme inhibited by lithium, has opposite effects. In contrast to IMPase inhibition that enhances autophagy, GSK-3beta inhibition attenuates autophagy and mutant huntingtin clearance by activating mTOR. In order to counteract the autophagy inhibitory effects of mTOR activation resulting from lithium treatment, we have used the mTOR inhibitor rapamycin in combination with lithium. This combination enhances macroautophagy by mTOR-independent (IMPase inhibition by lithium) and mTOR-dependent (mTOR inhibition by rapamycin) pathways. We provide proof-of-principle for this rational combination treatment approach in vivo by showing greater protection against neurodegeneration in an HD fly model with TOR inhibition and lithium, or in HD flies treated with rapamycin and lithium, compared to either pathway alone.

5. Williams A*, Sarkar S*, Cuddon P*, Ttofi EK, Saiki S, Siddiqi FH, Jahreiss L, Fleming A, Pask D, Goldsmith P, O’Kane CJ, Floto RA and Rubinsztein DC. Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nature Chemical Biology 4(5):295-305 (2008). Access article Supplementary information Chemical compounds  *Joint first authors

Abstract: Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca²⁺ channel antagonists, the K⁺_ATP channel opener minoxidil, and the G_i signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP₃ levels, influencing calpain activity, which completes the cycle by cleaving and activating G_sa, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca²⁺ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.

6. Sarkar S, Ravikumar B, Floto RA and Rubinsztein DC. Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies. Cell Death and Differentiation 16(1):46-56 (2009). Access article

Abstract: The formation of intra-neuronal mutant protein aggregates is a characteristic of several human neurodegenerative disorders, like Alzheimer's disease, Parkinson's disease (PD) and polyglutamine disorders, including Huntington's disease (HD). Autophagy is a major clearance pathway for the removal of mutant huntingtin associated with HD, and many other disease-causing, cytoplasmic, aggregate-prone proteins. Autophagy is negatively regulated by the mammalian target of rapamycin (mTOR) and can be induced in all mammalian cell types by the mTOR inhibitor rapamycin. It can also be induced by a recently described cyclical mTOR-independent pathway, which has multiple drug targets, involving links between Ca²⁺–calpain–G_sa and cAMP–Epac–PLC-e–IP₃ signalling. Both pathways enhance the clearance of mutant huntingtin fragments and attenuate polyglutamine toxicity in cell and animal models. The protective effects of rapamycin in vivo are autophagy-dependent. In Drosophila models of various diseases, the benefits of rapamycin are lost when the expression of different autophagy genes is reduced, implicating that its effects are not mediated by autophagy-independent processes (like mild translation suppression). Also, the mTOR-independent autophagy enhancers have no effects on mutant protein clearance in autophagy-deficient cells. In this review, we describe various drugs and pathways inducing autophagy, which may be potential therapeutic approaches for HD and related conditions.