Small Molecule Enhancers of Rapamycin-Induced TOR Inhibition Promote Autophagy,Reduce Toxicity in Huntington’s Disease Models and Enhance Killing of Mycobacteria by Macrophages

Upregulation of autophagy may have therapeutic benefit in a range of diseases that include neurodegenerative conditions caused by intracytosolic aggregate-prone proteins, such as Huntington’s disease, and certain infectious diseases, such as tuberculosis. The best-characterized drug that enhances autophagy is rapamycin, an inhibitor of the TOR (target of rapamycin) proteins, which are widely conserved from yeast to man. Unfortunately, the side effects of rapamycin, especially immunosuppression, preclude its use in treating certain diseases including tuberculosis, which accounts for approximately 2 million deaths worldwide each year, spurring interest in finding novel drugs that selectively enhance autophagy. We have recently reported a novel two-step screening process for the discovery of such compounds. We first identified compounds that enhance the growth-inhibitory effects of rapamycin in the budding yeast Saccharomyces cerevisiae, which we termed small molecule enhancers of rapamycin (SMERs). Next we showed that three SMERs induced autophagy independently, or downstream of mTOR, in mammalian cells, and furthermore enhanced the clearance of a mutant huntingtin fragment in cell disease models. These SMERs also protected against mutant huntingtin fragment toxicity in Drosophila. We have subsequently tested two of the SMERs in models of tuberculosis and both enhance the killing of mycobacteria by primary human macrophages.

Addendum to:

Small Molecules Enhance Autophagy and Reduce Toxicity in Huntington's Disease Models

S. Sarkar, E.O. Perlstein, S. Imarisio, S. Pineau, A. Cordenier, R.L. Maglathlin, J.A. Webster, T.A. Lewis, C.J. O'Kane, S.L. Schreiber and D.C. Rubinsztein

Nat Chem Biol 2007; 3:331-8     

Small molecules enhance autophagy and reduce toxicity in Huntington's disease models

The target of rapamycin 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-molecule modulators of mammalian autophagy. We first identified new 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 such as mutant huntingtin and A53T alpha-synuclein, which are associated with Huntington's disease and familial Parkinson's disease, respectively. These SMERs, which seem to act either independently or downstream of the target of rapamycin, attenuated mutant huntingtin-fragment toxicity in Huntington's disease cell and Drosophila melanogaster models, which suggests therapeutic potential. We also screened structural analogs of these SMERs and identified additional candidate drugs that enhanced autophagy substrate clearance. Thus, we have demonstrated proof of principle for a new approach for discovery of small-molecule modulators of mammalian autophagy.     

Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies

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 Ca(2+)-calpain-G(salpha) and cAMP-Epac-PLC-epsilon-IP(3) 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.     

Protective roles for induction of autophagy in multiple proteinopathies

Many late-onset neurodegenerative diseases, including Parkinson's disease, tauopathies, Huntington's disease and forms of spinocerebellar ataxia, are caused by aggregate-prone proteins. Previously we showed that mutant huntingtin is an autophagy substrate and that autophagy induction reduced soluble and aggregated huntingtin levels and attenuated its toxicity in cell, fly and mouse models of disease. We have recently shown in cell and fly models that autophagy induction may have general protective effects across a range of diseases caused by aggregate-prone intracellular proteins. First, we showed that this strategy reduces the levels of the primary toxin, the aggregate-prone mutant protein. Second, our recent work suggests that autophagy induction may have additional cytoprotective effects by protecting cells against a range of subsequent pro-apoptotic insults.     

GADD34 mediates cytoprotective autophagy in mutant huntingtin expressing cells via the mTOR pathway

Increased protein aggregation and altered cell signaling accompany many neurodegenerative diseases including Huntington's disease (HD). Cell stress is counterbalanced by signals mediating cell repair but the identity of these are not fully understood. We show here that the mammalian target of rapamycin (mTOR) pathway is inhibited and cytoprotective autophagy is activated in neuronal PC6.3 cells at 24 h after expression of mutant huntingtin proteins. Tuberous sclerosis complex (TSC) 1/2 interacted with growth arrest and DNA damage protein 34 (GADD34), which caused TSC2 dephosphorylation and induction of autophagy in mutant huntingtin expressing cells. However, GADD34 and autophagy decreased at later time points, after 48 h of transfection with the concomitant increase in mTOR activity. Overexpression of GADD34 counteracted these effects and increased cytoprotective autophagy and cell survival. These results show that GADD34 plays an important role in cell protection in mutant huntingtin expressing cells. Modulation of GADD34 and the TSC pathway may prove useful in counteracting cell degeneration accompanying HD and other neurodegenerative diseases.     

Autophagy in neurodegeneration and development

Efficient protein turnover is essential for the maintenance of cellular health. Here we review how autophagy has fundamental functions in cellular homeostasis and possible uses as a therapeutic strategy for neurodegenerative diseases associated with intracytosolic aggregate formation, like Huntington's disease (HD). Drugs like rapamycin, that induce autophagy, increase the clearance of mutant huntingtin fragments and ameliorate the pathology in cell and animal models of HD and related conditions. In Drosophila, the beneficial effects of rapamycin in diseases related to HD are autophagy-dependent. We will also discuss the importance of autophagy in early stages of development and its possible contribution as a secondary disease mechanism in forms of fronto-temporal dementias, motor neuron disease, and lysosomal storage disorders.     

Lithium induces autophagy by inhibiting inositol monophosphatase

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.     

Huntington's disease: degradation of mutant huntingtin by autophagy

Autophagy is a nonspecific bulk degradation pathway for long-lived cytoplasmic proteins, protein complexes, or damaged organelles. This process is also a major degradation pathway for many aggregate-prone, disease-causing proteins associated with neurodegenerative disorders, such as mutant huntingtin in Huntington's disease. In this review, we discuss factors regulating the degradation of mutant huntingtin by autophagy. We also report the growing list of new drugs/pathways that upregulate autophagy to enhance the clearance of this mutant protein, as autophagy upregulation may be a tractable strategy for the treatment of Huntington's disease.     

Induction of autophagy with catalytic mTOR inhibitors reduces huntingtin aggregates in a neuronal cell model

Huntington's disease is a progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. This expansion produces a mutant form of the huntingtin protein, which contains an elongated polyglutamine stretch at its amino-terminus. Mutant huntingtin may adopt an aberrant, aggregation-prone conformation predicted to start the pathogenic process leading to neuronal dysfunction and cell death. Thus, strategies reducing mutant huntingtin may lead to disease-modifying therapies. We investigated the mechanisms and molecular targets regulating huntingtin degradation in a neuronal cell model. We first found that mutant and wild-type huntingtin displayed strikingly diverse turn-over kinetics and sensitivity to proteasome inhibition. Then, we show that autophagy induction led to accelerate degradation of mutant huntingtin aggregates. In our neuronal cell model, allosteric inhibition of mTORC1 by everolimus, a rapamycin analogue, did not induce autophagy or affect aggregate degradation. In contrast, this occurred in the presence of catalytic inhibitors of both mTOR complexes mTORC1 and mTORC2. Our data demonstrate the existence of an mTOR-dependent but everolimus-independent mechanism regulating autophagy and huntingtin-aggregate degradation in cells of neuronal origin.     

Inhibition of endoplasmic reticulum stress counteracts neuronal cell death and protein aggregation caused by N-terminal mutant huntingtin proteins

Accumulation of abnormal proteins occurs in many neurodegenerative diseases including Huntington's disease (HD). However, the precise role of protein aggregation in neuronal cell death remains unclear. We show here that the expression of N-terminal huntingtin proteins with expanded polyglutamine (polyQ) repeats causes cell death in neuronal PC6.3 cell that involves endoplasmic reticulum (ER) stress. These mutant huntingtin fragment proteins elevated Bip, an ER chaperone, and increased Chop and the phosphorylation of c-Jun-N-terminal kinase (JNK) that are involved in cell death regulation. Caspase-12, residing in the ER, was cleaved in mutant huntingtin expressing cells, as was caspase-3 mediating cell death. In contrast, cytochrome-c or apoptosis inducing factor (AIF) was not released from mitochondria after the expression of these proteins. Treatment with salubrinal that inhibits ER stress counteracted cell death and reduced protein aggregations in the PC6.3 cells caused by the mutant huntingtin fragment proteins. Salubrinal upregulated Bip, reduced cleavage of caspase-12 and increased the phosphorylation of eukaryotic translation initiation factor-2 subunit-alpha (eIF2alpha) that are neuroprotective. These results show that N-terminal mutant huntingtin proteins activate cellular pathways linked to ER stress, and that inhibition of ER stress by salubrinal increases cell survival. The data suggests that compounds targeting ER stress may be considered in designing novel approaches for treatment of HD and possibly other polyQ diseases.     

Protective Roles for Induction of Autophagy in Multiple Proteinopathies

Many late-onset neurodegenerative diseases, including Parkinson’s disease, tauopathies, Huntington’s disease and forms of spinocerebellar ataxia, are caused by aggregate-prone proteins. Previously we showed that mutant huntingtin is an autophagy substrate and that autophagy induction reduced soluble and aggregated huntingtin and attenuated its toxicity in cell, fly and mouse models of disease. We have recently shown in cell and fly models that autophagy induction may have general protective effects across a range of diseases caused by aggregate-prone intracellular proteins. First, we showed that this strategy reduces the levels of the primary toxin, the aggregate-prone mutant protein. Second, our recent work suggests that autophagy induction may have additional cytoprotective effects by protecting cells against a range of subsequent pro-apoptotic insults.     

Role of autophagy in the clearance of mutant huntingtin: a step towards therapy?

Macroautophagy (henceforth referred to simply as autophagy) is a bulk degradation process involved in the clearance of long-lived proteins, protein complexes and organelles. A portion of the cytosol, with its contents to be degraded, is enclosed by double-membrane structures called autophagosomes/autophagic vacuoles, which ultimately fuse with lysosomes where their contents are degraded. In this review, we will describe how induction of autophagy is protective against toxic intracytosolic aggregate-prone proteins that cause a range of neurodegenerative diseases. Autophagy is a key clearance pathway involved in the removal of such proteins, including mutant huntingtin (that causes Huntington’s disease), mutant ataxin-3 (that causes spinocerebellar ataxia type 3), forms of tau that cause tauopathies, and forms of alpha-synuclein that cause familial Parkinson’s disease. Induction of autophagy enhances the clearance of both soluble and aggregated forms of such proteins, and protects against toxicity of a range of these mutations in cell and animal models. Interestingly, the aggregates formed by mutant huntingtin sequester and inactivate the mammalian target of rapamycin (mTOR), a key negative regulator of autophagy. This results in induction of autophagy in cells with these aggregates.     

Inositol and IP3 levels regulate autophagy: biology and therapeutic speculations

We recently showed that lithium induces autophagy via inositol monophosphatase (IMPase) inhibition, leading to free inositol depletion and reduced myo-inositol-1,4, 5-triphosphate (IP3) levels. This represents a novel way of regulating mammalian autophagy, independent of the mammalian target of rapamycin (mTOR). Induction of autophagy by lithium led to enhanced clearance of autophagy substrates, like mutant huntingtin fragments and mutant alpha-synucleins, associated with Huntington's disease (HD) and some autosomal dominant forms of Parkinson's disease (PD), respectively. Similar effects were observed with a specific IMPase inhibitor and mood-stabilizing drugs that decrease inositol levels. This may represent a new therapeutic strategy for upregulating autophagy in the treatment of neurodegenerative disorders, where the mutant protein is an autophagy substrate. In this Addendum, we review these findings, and some of the speculative possibilities they raise.     

A stress sensitive ER membrane-association domain in Huntingtin protein defines a potential role for Huntingtin in the regulation of autophagy

We have recently published the precise definition of an aminoterminal membrane association domain in huntingtin, capable of targeting to the endoplasmic reticulum and late endosomes as well as autophagic vesicles. In response to ER stress induced by several pathways, huntingtin releases from membranes and rapidly translocates into the nucleus. Huntingtin is then capable of nuclear export and re-association with the ER in the absence of stress. This release is inhibited when huntingtin contains the polyglutamine expansion seen in Huntington's disease. As a result, mutant huntingtin expressing cells have a perturbed ER and an increase in autophagic vesicles. Here, we discuss the potential function of the huntingtin protein as an ER sentinel, potentially regulating autophagy in response to ER stress. We compare these recent findings to the well characterized mammalian target of rapamycin, mTor, a protein described over a decade ago as related to huntingtin structurally by leucine-rich, repetitive HEAT sequences. Since then, the described functional similarities between Huntingtin and mTor are striking, and this new information about huntingtin's direct association with autophagic vesicles indicates that this structural similarity may extend to functional similarities having an impact upon ER functionality and autophagy.     

Novel targets for Huntington's disease in an mTOR-independent autophagy pathway

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(s)alpha, 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.     

Insulin htts on autophagy

Macroautophagy has been shown to participate in the degradation and clearance of polyglutamine (polyQ) tract-containing proteins generated by trinucleotide repeat expansion mutations. Large expansions are the genetic cause of diseases such as Huntington's Disease that lead to neuronal dysfunction due to polyQ protein aggregates. Recently, a functional screen performed by Yamamoto et al to investigate proteins that regulate such autophagic processes revealed a novel role for insulin signaling in the promotion of autophagy of mutant protein aggregates. This suggests that insulin/insulin-like growth factor signaling pathways not only prevent the induction of autophagy, but paradoxically may promote autophagy of deleterious proteins in certain circumstances.     

Small molecule enhancers of autophagy for neurodegenerative diseases

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders, including Huntington's disease and various spinocerebellar ataxias, are associated with the formation of protein aggregates. These aggregates and/or their precursors are thought to be toxic disease-causing species. Autophagy is a major degradation pathway for intracytosolic aggregate-prone proteins, including those associated with neurodegeneration. It is a constitutive self-degradative process involved both in the basal turnover of cellular components and in response to nutrient starvation in eukaryotes. Enhancing autophagy may be a possible therapeutic strategy for neurodegenerative disorders where the mutant proteins are autophagy substrates. In cell and animal models, chemical induction of autophagy protects against the toxic insults of these mutant aggregate-prone proteins by enhancing their clearance. We will discuss various autophagy-inducing small molecules that have emerged in the past few years that may be leads towards the treatment of such devastating diseases.     

Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein

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.     

Isolation of a 40-kDa Huntingtin-associated protein

Huntington's disease is caused by an expanded CAG trinucleotide repeat coding for a polyglutamine stretch within the huntingtin protein. Currently, the function of normal huntingtin and the mechanism by which expanded huntingtin causes selective neurotoxicity remain unknown. Clues may come from the identification of huntingtin-associated proteins (HAPs). Here, we show that huntingtin copurifies with a single novel 40-kDa protein termed HAP40. HAP40 is encoded by the open reading frame factor VIII-associated gene A (F8A) located within intron 22 of the factor VIII gene. In transfected cell extracts, HAP40 coimmunoprecipitates with full-length huntingtin but not with an N-terminal huntingtin fragment. Recombinant HAP40 is cytoplasmic in the presence of huntingtin but is actively targeted to the nucleus in the absence of huntingtin. These data indicate that HAP40 is likely to contribute to the function of normal huntingtin and is a candidate for involvement in the aberrant nuclear localization of mutant huntingtin found in degenerating neurons in Huntington's disease.