This invention relates to the induction of autophagy, and in particular to autophagy inducing compounds which may be useful in the treatment of disease .

The ubiquitin-proteasome and autophagy-lysosomal pathways are the two major routes for protein and organelle clearance in eukaryotic cells . Proteasomes predominantly degrade short-lived nuclear and cytosolic proteins . The bulk degradation of cytoplasmic proteins or organelles is largely mediated by macroautophagy, generally referred to as autophagy ⁹ . It involves the formation of double membrane structures called autophagosomes , which fuse with lysosomes to form autolysosomes . Autophagy substrates generally have long half-lives ⁹ . Autophagy can also help cells clear the toxic , long-lived, aggregate-prone proteins causing many neurodegenerative disorders , like Huntington' s disease and forms of Parkinson' s disease ³ ' ⁴ . Induction of autophagy reduces the levels of mutant huntingtin and protected against its toxicity in cells and in transgenic Drosophila and mouse models ⁶ . Currently, the only suitable pharmacological strategy for up regulating autophagy in mammals .. is to use rapamycin, or its analogues , that inhibit the mammalian target of rapamycin (mTOR) , a negative regulator of autophagy .

The present inventors have identified a novel pathway for the regulation of autophagy which involves inositol monophosphatase ( IMPase) and levels of free inositol (IP ₃ ) . Inhibition of IMPase and reductions in the level of free IP ₃ are found to induce autophagy in cells , providing a new pharmacological strategy for modulating autophagy in mammals .

One aspect of the invention provides a method of screening for an autophagy inducer comprising;

contacting an inositol monophosphatase (IMPase) polypeptide with a test compound; and, determining the interaction of the inositol monophosphatase polypeptide and the test compound .

A test compound which interacts with the polypeptide is a candidate autophagy inducer .

Interaction may include the binding of the compound to the polypeptide and/or the activity of the polypeptide in the presence of the compound .

An aspect of the invention provides a method of screening for an autophagy inducer comprising; contacting an inositol monophosphatase (IMPase) polypeptide with a test compound; and, determining the activity of the IMPase polypeptide .

A decrease in monophosphatase activity in the presence of the compound relative to its absence may indicative that the compound is an autophagy inducer .

An IMPase polypeptide (E.c. 3.1.3.25) may comprise or consist of the sequence of a eukaryotic IMPase , in particular a mammalian IMPase . A IMPase polypeptide may be a myo-inositol monophosphatase 1 (IMPAl) polypeptide comprising or consisting of the amino acid sequence of NP_005527.1 (GI : 5031789) or a myoinositol monophosphatase 2 (IMPA2) polypeptide comprising or consisting of the amino acid sequence of NP_055029.1 (GI : 7657236) or may be a fragment or variant of either of these sequences .

An IMPAl polypeptide may be encoded by a nucleotide sequence comprising or consisting of the nucleic acid sequence of NM_005536.2 (GI : 8393607) . An IMPA2 polypeptide may be encoded by

a nucleotide sequence comprising or consisting of the nucleic acid sequence of NM_014214.1 (GI : 7657235) .

A fragment or variant of a wild-type IMPase sequence as described herein may differ from the wild-type sequence by the addition, deletion, substitution and/or insertion of one or more amino acids , provided the function of hydrolysing inositol monophosphate to produce inositol is retained .

A polypeptide which is a variant of a wild-type IMPase sequence, such as human IMPAl or IMPA2 , may comprise an amino acid sequence which shares greater than about 30% sequence identity with the wild-type IMPase sequence, greater than about 40% , greater than about 45% , greater than about 55% , greater than about 65% , greater than about 70% , greater than about 80% , greater than about 90% or greater than about 95% . The sequence may share greater than about 30% similarity with the wild-type IMPase sequence, greater than about 40% similarity, greater than about 50% similarity, greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity .

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Genetics Computer Group, Madison, WI) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps . Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e . g . BLAST (which uses the method of

Altschul et al . (1990) J ^" . MoI . Biol . 215 : 405-410) , FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85 : 2444- 2448 ) , or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol . 147 : 195-197) , or the TBLASTN program, of Altschul et al . (1990) supra, generally employing default

parameters . In particular, the psi-Blast algorithm (Nucl . Acids Res . (1997) 25 3389-3402) may be used. Sequence identity and similarity may also be determined using Genomequest™ software (Gene-IT, Worcester MA USA) .

Sequence comparisons are preferably made over the full-length of the relevant sequence described herein.

Similarity allows for "conservative variation" , i . e . substitution of one hydrophobic residue such as isoleucine , valine , leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine .

Determining the activity of a polypeptide may include detecting the presence of activity, detecting the presence of activity above a threshold value and/or measuring the level of activity.

As described above , polypeptide fragments which retain all or part of the IMPase activity of the full-length protein may be generated and used in the methods described herein, whether in vitro or in vivo. Suitable ways of generating fragments include , but are not limited to, recombinant expression of a fragment from encoding DNA. For example , fragments may be generated by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers . Small fragments (e . g . up to about 20 or 30 amino acids) may also be generated using peptide synthesis methods that are well known in the art as further described below .

A fragment of a full-length sequence may consist of fewer amino acids than the full-length sequence . For example a fragment may- consist of at least 10 , at least 20 , at least 30 , at least 40 , at least 50 , at least 60 , at least 70 , at least 80 , at least 90 or at least 100 amino acids of the full length sequence but 800 or less , 700 or less , 600 or less , 500 or less , 250 or less , 200 or less , 150 or less , or 125 or less amino acids of the full length sequence .

Methods described herein may be in vivo cell-based methods , or in vitro non-cell-based methods . The precise format for performing methods of the invention may be varied by those of skill in the art using routine skill and knowledge .

The activity of IMPase (E . C. 3.1.3.25) may be determined using standard biochemical techniques . For example, IMPase activity can be measured spectrophotometrically as inorganic phosphate liberated from inositol-1-phosphate (Shaltiel G et al World J Biol Psychiatry . 2001 Apr; 2 (2 ) : 95-8. )

The level of inositol-1 , 4 , 5-triphosphate (IP ₃ ) in a cell is shown herein to correlate with the induction of autophagy and may be used in screening methods .

Another aspect of the invention provides a method of screening for an autophagy inducer comprising; contacting a cell with a test compound; and determining the level of IP ₃ in the cell .

A decrease in the level of IP ₃ in the presence of the compound is indicative that the compound is an autophagy inducer .

Suitable cells for use in the present methods include mammalian cells , preferably human cells . Preferably, the cell is an isolated and/or cultured cell . Any suitable cultured mammalian

cell may be used, for example Chinese hamster ovary cells , baby hamster kidney cells , COS cells, PC12 and many others .

The level of IP ₃ may be determined by using conventional biochemical techniques . For example, levels of inositol-1 , 4 , 5- trisphosphate [IP ₃ (1 , 4 , 5) ] can be measured in perchloric acid extracted samples (10 ⁷ cells/aliquot) using the [ ³ H] Biotrak Assay System (Amersham Biosciences , UK) according to manufacturer' s instruction .

A method may further comprise; contacting the test compound with a mammalian cell; and, determining the autophagy activity of said cell .

An increase in autophagy activity in the presence of the compound is indicative that the compound is a candidate agent for use in the treatment of a protein conformational disorder .

Autophagy activity may be determined by any convenient method, for example monodansylcadaverine (MDC) staining (Ravikumar et al Hu . MoI . Gen . (2003 ) 12 9 1-10) , LC3 processing (Y . Kabeya et al EMBO J . 19 5720-5728 ) , or electron microscopy visulating autophagosome numbers . Autophagy activity may be determined in the presence of proteosome inhibitors , such as epoximicin.

In some embodiments , the ability of said compound to increase the clearance of cytoplasmic protein aggregates may be determined.

A cell for use in determining autophagy activity may comprise a heterologous nucleic acid encoding an aggregation-prone polypeptide , for example A53T or A3 OP mutant forms of α- synuclein, huntingtin or GFP-tagged with expanded polyalanine repeats . An aggregation-prone polypeptide may comprise an aggregation-inducing mutation, for example a codon iteration

mutation such as a polyQ or a polyA insertion, or may have the non-mutant , wild-type sequence . Clearance of the encoded aggregation-prone polypeptide , either in an aggregated or a soluble monomeric form, may be determined. Expression of the heterologous nucleic acid may be reversible i . e . expression may be induced and repressed as required, for example by adding or removing an inducer compound. A method may comprise inducing and repressing the expression of said nucleic acid prior to contacting the mammalian cell with the test compound . Many examples of inducible and/or reversible expression systems and constructs are known in the art , including, for example the Tet- on™ expression (Clontech) , in particular in combination with the pTet-tTs™ vector (Clontech) .

An autophagy inducer identified by a method described herein may be useful in the treatment of a protein conformational disorder, in particular a neurodegenerative protein conformational disorder, or a bacterial infection, such as tuberculosis or streptococcus infection .

Protein conformational disorders which may be treated include codon reiteration mutation disorders , in particular polyQ expansion disorders such as Huntington' s disease, spinocerebellar ataxias types 1 , 2 , 3 , 6 , 7 , and 17 , Kennedy' s disease and dentatorubral-pallidoluysian atrophy. These disorders are characterised by the aggregation of mutant proteins that contain an expanded tract of repeated glutamine residues . For example , HD is characterised by an expanded polyQ stretch in exon 1 of the Huntington gene . Protein conformational disorders also include polyA expansion disorders . These disorders are characterised by the aggregation of mutant proteins which contain an expanded tract of repeated alanine residues . For example, oculapharyngeal muscular dystrophy (OPMD) is characterised by a polyadenine (polyA) expansion mutation in the polyadenine binding protein 2 gene . Protein conformational

disorders also include α-synucleiopathies such as Parkinson' s disease, LB variant Alzheimer' s disease and LB dementia . These are disorders characterised by the accumulation of cytoplasmic aggregates called Lewy bodies , which comprise α-synuclein. Protein conformational disorders also include tauopathies such as Alzheimer' s disease, which are disorders characterised by the accumulation of cytosolic aggregates of tau protein within neurons . Protein conformational disorders also include prion disorders such as Creutzfeldt-Jakob disease (CJD) , Kuru, Gerstmann-Straussler-Scheiker syndrome . CJD may include sporadic, familial , Iatrogenic and variant CJD .

Test compounds may be natural or synthetic chemical compounds used in drug screening programmes . Extracts of plants which contain several characterised or uncharacterised components may also be used .

Combinatorial library technology (Schultz, JS ( 1996) Biotechnol . Prog . 12 : 729-743 ) provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide . Suitable test compounds include analogues and derivatives of IMPase inhibitors set out below .

The amount of test substance or compound that may be added to an assay will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.1 to 100 μM concentrations of putative inhibitor compound may be used, for example from 1 to 10 μM . The test substance or compound is desirably membrane permeable in order to access intracellular targets .

The compound may be identified as an autophagy inducer using the methods described herein .

Following identification of a compound as described above, a method may further comprise modifying the compound to optimise the pharmaceutical properties thereof . The modification of a ^Λ lead' compound identified as biologically active is a known approach to the development of pharmaceuticals and may be used to avoid randomly screening large number of molecules for a target property . The optimisation commonly comprises several steps . Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined . These parts or residues constituting the active region of the compound are known as its "pharmacophore" .

Once the pharmacophore has been found, its structure is modelled to according its physical properties , e .g . stereochemistry, bonding, size and/or charge, using data from a range of sources , e . g . spectroscopic techniques , X-ray diffraction data and NMR . Computational analysis , similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process .

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted . The template molecule and the chemical groups grafted on to it can conveniently be selected so that the modified compound is easy to synthesise , is likely to be pharmacologically acceptable , and does not degrade in vivo, while retaining the autophagy inducing activity of the lead compound . The modified compounds found by this approach can then be screened to see whether they inhibit IMPase, reduce IP ₃ levels and/or stimulate autophagy, or to what extent they exhibit one or more of these activities . A method may comprise modifying a IMPase inhibitor, for example an IMPase inhibitor described below, or a IMPase substrate (such as IMP) or product (such as inositol or phosphate) to produce an

analogue or derivative , and determining the ability of said derivative to inhibit autophagy .

Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing .

The test compound may be manufactured and/or used in preparation, i . e . manufacture or formulation, of a composition such as a medicament , pharmaceutical composition or drug .

These may be administered to individuals , e . g . for any of the purposes discussed elsewhere herein. A method may comprise formulating the test compound into a pharmaceutical composition with a pharmaceutically acceptable excipient , vehicle or carrier as discussed further below.

Another aspect of the invention provides the use of an IMPase inhibitor in the manufacture of a medicament for increasing autophagy in an individual

The medicament may be useful in the treatment of a neurodegenerative protein conformational disorder, or a bacterial infection, such as tuberculosis or streptococcus infection.

IMPase inhibitors are well known in the art and include L- 690330 , L-690488 (Atack JR et al J Pharmacol Exp Ther . 1994 JuI ; 270 (1) : 70 -6) , lithium, valproate and carbemazapine . Other examples of suitable IMPase inhibitors are described in Fauroux CM, Freeman S . J Enzyme Inhib . 1999 _/ 14 (2) : 97-108 , Miller DJ et al . Org Biomol Chem. 2004 Mar 7 ,- 2 (5) : 671-88. and Atack JR et al J Pharmacol Exp Ther . 1994 Jul ; 270 (1) : 70-6.

For example, IMPase inhibitors may include inositol-1- monophosphate analogues or variants , for example with one or

more cyclohexane hydroxyl groups deleted or substituted (Fauroux CM, Freeman S . J Enzyme Inhib . 1999 _/ 14 (2 ) : 97-108)

IMPase inhibitors may also include phosphonates such as ( - ) - (Ii?, 222, 422, 6i?) -2 , 4 , 6-trihydroxycyclohexyl cyclohexylammonium 1- methylphosphonate; ( - ) - (IR, 2R ₁ AR, 6R) -6-Propyloxy-2 , 4- dihydroxycyclohexyl cyclohexylammonium 1-methylphosphonate ; ( - ) - (Ii?, 2R ₁ AR, 6R) -6-Propyloxy-2 , 4 -dihydroxycyclohexyl cyclohexylammonium 1-ethylphosphonate ; ( - ) - (1S, 2R, AS, 6R) -6- Methylamino-2 , 4-dihydroxycyclohexyl cyclohexylammonium 1- methylphosphonate; ( -) - (IS, 2R, AS, 6R) -6-Hexylamino-2 , 4 - dihydroxycyclohexyl 1-methylphosphonate , ( - ) - (15, 223, 4S, 6.R) -6- (2 - Phenylethyl) amino-2 , 4-dihydroxycyclohexyl 1-methylphosphonate and analogues , derivatives and salts thereof (Miller DJ et al Org Biomol Chem. 2004 Mar 7 ; 2 (5) : 671-88 ) .

IMPase inhibitors may also include inositol analogues such as ( ^■ ) - (IS, 2R, AS, 6R) -6- (2-phenylethyl) aminocyclohexane-1, 2 , 4-triol ; ( - ) - ( 1S, 2R, AS, 6R) -β-Hexylaminocyclohexane-l ^ , 4-triol ; ( - ) - ( IS, 2R ₁ AR ₁ 6R) -6-Propyloxycyc1ohexane-1 , 2 , 4-triol ; ( - ) - (IS, 2R, AS, 6R) -6-Hexyloxycyclohexane-l , 2 , 4-triol ; ( - ) - (IS, 2R ₁ AS, 6R) ~6- [4- (2 -Hydroxyphenyloxy) butyloxy] cyclohexane- 1, 2 , 4-triol ; ( -) - (1S, 2R, AS, 6R) -6- (2-Aminoethyloxy) cyclohexane- 1 , 2 , 4-triol ; ( - ) - (IS, 2R ₁ AS, 6R) -6- (3 -Aminopropyloxy) cyclohexane- 1 , 2 , 4-triol ; ( - ) - (IS, 2R ₁ AS, 6R) -6-Aminocyclohexane-l, 2 , 4-triol; ( ^■ ) - (1.3, 2.22, 4-?, 6R) -6-Ethylaminocyclohexane-l , 2 , 4-triol ; ( - ) - (IS, 222, 4.3, 6i2) -δ-Butylaminocyclohexane-l , 2 , 4-triol ; ( - ) - (1S, 222, 4S, 6i2) -δ-Octylaminocyclohexane-l, 2 , 4-triol ; ( - ) - (IS, 2R, AS, 6R) -6- (4-Phenylbuty1) aminocyclohexane-1 , 2 , 4-triol , ( - ) - (IS, 222, 4S, 622) -6- [N- (4-phenylbutyl) -N- (2-aminoethyl) - amino] cyclohexane-1 , 2 , 4-triol and analogues , derivatives and salts thereof .

IMPase inhibitors may also include bisphosphonates , such as 1- hydroxyethylidene-1, 1 bisphosphonic acid and hydroxymethylene

bisphosphonic acid, terpenoids such as sesquiterpene L-671776 from Memnoniella echinata and puberulonic acid from Penicillum spp and tropolones , in particular hydroxyl substituted tropolones such as 7-hydroxytropolone and 3 , 7- dihydroxytropolone and analogues , derivatives and salts thereof .

Other IMPase inhibitors may be analogues or variants of L- 690330 , L-690488 , lithium, valproate and carbemazapine .

In some embodiments , IMPase inhibitors other than lithium may be employed .

Inhibition of both mTOR and IMPase is shown herein to produce a cumulative effect on the stimulation or induction of autophagy .

Other aspects of the invention provide a combination of an mTOR inhibitor and an IMPase inhibitor for use in increasing autophagy in an individual , a pharmaceutical composition comprising an mTOR inhibitor, an IMPase inhibitor and a pharmaceutically acceptable excipient and the use of an mTOR inhibitor and an IMPase inhibitor in the manufacture of a medicament for increasing autophagy in an individual .

An mTor inhibitor may include a rapamycin macrolide such as rapamycin or a salt , analogue or derivatives of rapamycin. Suitable rapamycin macrolides are described in more detail below. An IMPase inhibitor may include a compound described above .

The combination of an mTOR inhibitor and an IMPase inhibitor may be useful in the treatment of a neurodegenerative disorder, or a bacterial infection, such as tuberculosis , or streptococcus infection, as described herein.

Pharmaceutical compositions are described in more detail below.

Lithium and other IMPase inhibitors are commonly used in the treatment of mood disorders . The data presented herein indicates that other compounds which modulate autophagy may also be useful in the treatment of mood disorders .

Another aspect of the invention provides a method of screening for a compound useful in the treatment of a mood disorder comprising; contacting the test compound with a mammalian cell ; and, determining the autophagy activity of said cell .

A change in autophagy activity in the presence of the compound, for example an increase or decrease , is indicative that the compound is a candidate agent for use in the treatment of a mood disorder .

Autophagy activity may be determined by measuring the ability of said compound to increase the clearance of cytoplasmic protein aggregates . Aggregate clearance may be determined for example , in the presence of proteosome inhibitors such as epoximicin. The determination of autophagy activity and the clearance of cytoplasmic protein aggregates are described in more detail above .

Suitable cells and test compounds are described in more detail above .

Mood disorders include depression, bipolar disorder (manic- depression) , cyclothymia and dysthymia .

mTOR inhibitors , such as rapamycin are known to stimulate autophagy and may be particularly useful in the treatment of disorders previously treated with IMPase inhibitors , such as mood disorders .

Another aspect of the invention provides the use of an mTOR inhibitor in the manufacture of a medicament for use in the treatment of a mood disorder .

mTOR inhibitors include rapamycin and other rapamycin macrolides . A macrolide is a macrocyclic lactone, for example a compound having a 12-membered or larger lactone ring . Lactam macrolides are macrocyclic compounds which have a lactam (amide) bond in the macrocycle in addition to a lactone (ester) bond. Rapamycin is a lactam macrolide produced by Streptomyces hygroscopicus (McAlpine J . B . et al . J.Antibiotics ( 1991) 44 : 688 ; Schreiber, S . L . et al . J . Am. Chem. Soc . ( 1991) 113 : 7433 ; US3 , 929 , 992 ) . A rapamycin macrolide as described herein may include rapamycin or a salt , analogue or derivative of rapamycin .

Suitable rapamycin analogues well known in the art (see for example WO 94/09010 and WO 96/41807) and include 40-O- (2 - hydroxy) ethyl-rapamycin, 32 -deoxo-rapamycin, 16-O-pent-2 -ynyl- 32 -deoxo-rapamycin, 16-0-pent-2 -ynyl-32 -deoxo-40-0- (2- hydroxyethyl) -rapamycin, 16-O~pent-2-ynyl-32- (S) -dihydro- rapamycin and 16-O-pent-2-ynyl-32- (S) -dihydro-40-O- (2 - hydroxyethyl) -rapamycin . Other rapamycin analogues include carboxylic acid esters as set out in WO 92/05179 , amide esters as set out in US5 , 118 , 677 , carbamates as set out in US5 , 118 , 678 , fluorinated esters as set out in US5 , 100 , 883 , acetals as set out in US5 , 151 , 413 , silyl ethers as set out in US5 , 120 , 842 and arylsulfonates and sulfamates as set out in US5 , 177 , 203. Other rapamycin analogues which may be used in accordance with the invention may have the methoxy group at the position 16 replaced with alkynyloxy as set out in WO 95/16691. Rapamycin analogues are also disclosed in WO 93/11130 , WO 94/02136 , WO 94/02385 and WO 95/14023.

Administration of a compound for the treatment of a disorder, as described herein, is preferably in a "prophylactically effective amount " or a "therapeutically effective amount" (as the case may be , although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual . The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment , e . g . decisions on dosage etc , is within the responsibility of general practitioners and other medical doctors .

A composition may be administered alone or in combination with other treatments , either simultaneously or sequentially dependent upon the condition to be treated.

Pharmaceutical compositions may include , in addition to active ingredient , a pharmaceutically acceptable excipient , carrier, buffer, stabiliser or other materials well known to those skilled in the art . Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient .

The precise nature of the carrier or other material will depend on the route of administration, which may be oral , or by inj ection, e . g . cutaneous , subcutaneous or intravenous .

Pharmaceutical compositions for oral administration may be in tablet , capsule , powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant . Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils , mineral oil or synthetic oil . Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol , propylene glycol or polyethylene glycol may be included.

For intravenous , cutaneous or subcutaneous inj ection, or inj ection at the site of affliction, the active ingredient will

IS

be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example , isotonic vehicles such as Sodium Chloride Inj ection, Ringer ' s Inj ection, or Lactated Ringer ' s Inj ection. Preservatives , stabilisers , buffers , antioxidants and/or other additives may be included, as required .

Examples of techniques and protocols mentioned above can be found in Remington' s Pharmaceutical Sciences , 16th edition, Osol , A. (ed) , 1980.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure . All documents mentioned in this specification are incorporated herein by reference in their entirety .

The invention encompasses each and every combination and sub- combination of the features that are described above .

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above and tables described below .

Figure 1 shows the activity of lithium in reducing mutant huntingtin aggregates in COS-7 cells transfected with pEGFP- HDQ74 and treated with or without 1OmM LiCl or 1OmM NaCl for 48h . The effects of treatment on the percentage of EGFP-HDQ74- positive cells with aggregates or apoptotic morphology (cell death) are expressed as odds ratios ¹⁰ .

Figure 2 shows the activity of lithium in reducing mutant huntingtin aggregates in SK-N-SH cells transfected with pEGPP-

HDQ74 and treated with or without 1OmM LiCl or 1OmM NaCl for 48h. The effects of treatment on the percentage of EGFP-HDQ74- positive cells with aggregates or apoptotic morphology (cell death) are expressed as odds ratios ¹⁰ .

Figure 3 shows mutant huntingtin fragment (EGFP-HDQ74) clearance by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 12Oh in the presence (+) or absence ( - ) of 1OmM LiCl .

Figure 4 shows the percentage of GFP-positive cells with aggregates in stable PC12 cells expressing EGFP-HDQ74 after switching on EGFP74 expression then switching off for 12Oh in the presence (+) or absence ( - ) of 1OmM LiCl . Data are expressed as odds ratio compared to control condition (120h off) .

Figure 5 shows A53T α-synuclein mutant clearance in stable inducible PC12 cell lines by showing the amount of α-synuclein mutant remaining after switching on expression then switching off for 24h in the presence (+) or absence ( - ) of 1OmM LiCl or 1OmM NaCl .

Figure _, 6 shows A3 OP α-synuclein mutant clearance in stable inducible PC12 cell lines by showing the amount of α-synuclein mutant remaining after switching on expression then switching off for 24h in the presence (+) or absence ( - ) of 1OmM LiCl or 1OmM NaCl .

Figure 7 shows the percentage of EGFP-HDQ74-positive cells with aggregates and cell death in COS-7 cells as in (Ia) , treated with or without lOμM SB216763 or lOOμM L-690330 for 48h, expressed as odds ratios .

Figure 8 shows the percentage of EGFP-HDQ74-positive cells with aggregates and cell death in SK-N-SH cells as in (figure 1) ,

treated with or without lOμM SB216763 or lOOμM L-690330 for 48h, expressed as odds ratios .

Figure 9 shows mutant huntingtin fragment (EGFP-HDQ74) clearance by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 17Oh in the presence (+) or absence ( - ) of lOμM SB216763.

Fig 10 shows mutant huntingtin fragment clearance by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 17Oh in the presence (+) or absence ( - ) of lOOμM L-690330.

Figure 11 shows levels of IPi _-2 measured in COS-7 cells treated with 1OmM LiCl or lOOμM L-690 , 330 for 24h .

Figure 12 shows levels of IP ₃ measured in COS-7 cells treated with 2μM Bradykinin, 1OmM LiCl or lOOμM L-690 , 330 for 5min.

Figure 13 shows the percentage of EGFP-HDQ74-positive cells with aggregates and cell death in COS-7 cells treated with or without 50μM CBZ or ImM VPA for 48h, expressed as odds ratios .

Figure 14 shows IP ₃ levels measured in COS-7 cells treated for 5min with or without 2μM bradykinin, 1OmM LiCl or 1OmM LiCl pre- treated for 5min with ImM myo-inositol (Ins) or 24μM prolyl endopeptidase inhibitor (PEI) .

Figure 15 shows the percentage of EGFP-HDQ74-positive cells with aggregates in COS-7 cells , either left untreated or treated with 1OmM LiCl with (+) or without (-) ImM myo-inositol or 24μM PEI for 48h, were expressed as odds ratios .

Figure 16 shows the percentage of EGFP-HDQ74-positive cell death in COS-7 cells , either left untreated or treated with 1OmM LiCl

with (+) or without (-) ImM myo-inositol or 24μM PEI for 48h, expressed as odds ratios .

Figure 17 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 12Oh in the presence (+) or absence (- ) of 1OmM LiCl and ImM myoinositol or 24μM PEI .

Figure 18 shows IP ₃ levels measured in COS-7 cells treated with or without ImM myo-inositol , 24μM PEI or 0.2μM rapamycin (Rap) for 5min.

Figure 19 shows the percentage of EGFP-HDQ74 -positive cells with aggregates and cell death in COS-7 cells treated with or without ImM myo-inositol or 24μM PEI for 48h, expressed as odds ratios .

Figure 20 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 120h in the presence (+) or absence ( - ) of ImM myo-inositol or 24μM PEI .

Figure 21 shows COS-7 cells transfected with pEGFP-HDQ74 along with empty vector (pCDNA3.1) or pRheb at 1 : 3 ratio . The proportion of GFP-positive cells with aggregates or cell death were assessed after 48h and expressed as odds ratios .

Figure 22 shows COS-7 cells transfected with pEGFP-HDQ74 and pRheb and treated with or without 1OmM LiCl or IOOM L-690 , 330. The control represents untreated rheb-transfected cells . The proportion of GFP-positive cells with aggregates or cell death were assessed after 48h and expressed as odds ratios .

Figure 23 shows the percentage of EGFP-HDQ74-positive cells with aggregates in COS-7 cells as in (Ia) , either left untreated or treated with 0.2μM rapamycin with (+) or without (-) ImM myoinositol (Ins) or 24μM prolyl endopeptidase inhibitor (PEI) for 48h, expressed as odds ratios .

Figure 24 shows the percentage of EGFP-HDQ74-positive cells with cell death in COS-7 cells either left untreated or treated with 0.2μM rapamycin with (+) or without (-) ImM myo-inositol (Ins) or 24μM prolyl endopeptidase inhibitor (PEI) for 48h, expressed as odds ratios .

Figure 25 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 12Oh in the presence (+) or absence ( - ) of 0.2μM rapamycin and ImM myo-inositol or 24μM PEI .

Figure 26 shows the percentage of EGFP-HDQ74-positive cells with aggregates (upper) and cell death (lower) in COS-7 cells , treated with (+) or without (-) 1OmM LiCl , 0.2μM rapamycin or both for 48h, expressed as odds ratios .

Figure 27 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 72h in the presence (+) or absence ( - ) of 1OmM LiCl , 0.2μM rapamycin or both.

Figure 28 shows the percentage of EGFP-HDQ74-positive cells with aggregates (upper) and cell death (lower) in COS-7 cells treated with (+) or without (-) lOOμM L-690 , 330 , 0.2μM rapamycin or both for 48h, expressed as odds ratios .

Figure 29 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 72h in the presence (+) or absence ( -) of lOOμM L-690 , 330 , 0.2μM rapamycin or both .

Figure 30 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 12Oh and 17Oh in the presence (+) or absence ( - ) of 1OmM NaCl .

Figure 31 shows the percentage of GFP-positive PC12 cells expressing EGFP-HDQ74 with aggregates after treatment for 17Oh with or without 1OmM LiCl or 1OmM NaCl , expressed as odds ratio, compared to the control untreated cells (17Oh off) .

Figure 32 shows the percentage of EGFP-HDQ74 -positive cells with aggregates in COS-7 cells treated with (+) or without (-) 1OmM 3 -MA, 1OmM LiCl or both for 48h, expressed as odds ratio .

Figure 33 shows a schematic diagram of some molecular targets of lithium and the relevant pathways . Lithium affects different cellular processes , the two main pathways being the Wingless (Wnt) pathway and the inositol cycle ² ' ¹⁷ . Lithium inhibits glycogen synthase kinase-3β (GSK-3β . of the Wnt pathway and activates β-catenin, which mediates Tcf-mediated transcription. Lithium also inhibits inositol monophosphatase (IMPase) to reduce free inositol that decreases intracellular inositol- 1 , 4 , 5-triphosphate (IP ₃ ) levels and a rundown of the inositol cycle ⁵ .

Figure 34 shows the percentage of EGFP-HDQ74-positive cells with aggregates in COS-7 cells treated with (+) or without (-) 1OmM 3 -MA, lOOμM L-690 , 330 or both for 48h, expressed as odds ratio . *** , p < 0.001 ; ** , p < 0.01 ; * , p < 0.05 ; NS, Non-significant .

Figure 35 shows the percentage of EGFP-HDQ74-positive cells with aggregates in COS-7 cells treated with (+) or without (-) lOμM lactacystin (Lact) , lOOμM L-690 , 330 or both for 48h, expressed as odds ratio . *** , p < 0.001 ; ** , p < 0.01 ; * , p < 0.05 ; NS, Non-significant .

Figure 36 shows a schematic diagram of the inositol cycle and its modulation to reverse the effects of lithium. Lithium inhibits inositol monophosphatase (IMPase) to reduce free inositol that decreases intracellular inositol-1, 4 , 5 - triphosphate (IP ₃ ) levels ² ' ⁵ , leading to enhanced clearance of mutant proteins through autophagy by a rundown of the inositol cycle .

Figure 37 shows inositol-1 , 4 , 5-triphosphate (IP ₃ ) levels measured in COS-7 cells treated with 1OmM LiCl for 0 min, 1 min, 5 min, 1 h and 24 h. *** , p < 0.001 ; ** , p < 0.01 ; * , p < 0.05 ; NS , Non-significant .

Figure 38 shows a schematic diagram of the regulation of autophagy in mammalian cells , which is governed by the activity of mammalian target of rapamycin (mTOR) .

Examples

Materials & Methods

Plasmids

Huntington' s Disease (HD) gene exon 1 fragment with 74 polyglutamine repeats (Q74) in pEGFP-Cl (Clontech) was described and characterised previously ¹² .

Mammalian cell culture and transfection African green monkey kidney cells (COS-7) and human neuroblastoma cells (SK-N-SH) were maintained in Dulbecco ' s Modified Eagle Medium (DMEM, Sigma) supplemented with 10% Fetal

Bovine Serum (FBS , Sigma) , lOOU/ml Penicillin/Streptomycin and 2mM L-Glutamine (Sigma) at 37°C, 5% Carbon dioxide (CO ₂ ) . Cells were plated in six-well dishes at a density of IxIO ⁵ cells per well for 24h and transfeσted with pEGFP-HDQ74 using LipofectAMINE reagent for COS-7 cells and LipofectAMINE PLUS reagent for SK-N-SH using manufacturer' s protocol (Invitrogen) . Transfection mixture was replaced after 4 hours incubation at 37 ⁰ C by various compounds like 1OmM lithium chloride (LiCl) , 0.2μM rapamycin, 1OmM 3 -methyladenine (3 -MA) , lOμM lactacystin, 50μM carbamazepine (CBZ) , ImM myo-inositol (all from Sigma) , 1OmM NaCl (BDH) , 10μM SB216763 (Tocris) , lOOμM L-690 , 330 (Tocris) or 24μM prolyl endopeptidase inhibitor 2 (Z-PP-CHO, Calbiochem) . Transfected cells were fixed with 4% paraformaldehyde (Sigma) after 48h and mounted in 4' , 6- diamidino-2 -phenylindole (DAPI , 3 mg/ml , Sigma) over coverslips on glass slides and analysed for aggregation and cell death . For immunoblotting, COS-7 cells were plated at a density of 3xlO ^s cells per well and treated for 24h . Inducible PC12 stable cell lines expressing EGFP-tagged exon 1 of HD gene (EGFP-HDQ74) ²⁴ and HA-tagged A53T and A30P α-synuclein mutants ⁴ , previously characterised, were maintained at 75 μg/ml hygromycin B (Calbiochem) in standard DMEM with 10% horse serum (Sigma) , 5% FBS , 100U/ml penicillin/streptomycin, 2mM L-glutamine, and lOOμg/ml G418 (GIBCO) at 37°C, 10% CO ₂ .

Quantification of aggregate formation and cell death . Approximately 200 EGFP-positive cells were counted for proportion of cells with aggregates , as described previously ¹² . Nuclei were stained with DAPI and those showing apoptotic morphology (fragmentation or pyknosis) were considered abnormal . These criteria are specific for cell death, which highly correlate with propidium iodide staining in live cells ²⁵ . Analysis was performed using fluorescence microscope with observer blinded to identity of slides . Slides were coded and

the code was broken after completion of experiment . All experiments were done in triplicate at least twice .

Clearance of mutant huntingtin and α-synucleins . Stable inducible PC12 cell lines expressing EGFP-HDQ74 , or A53T or A30P α-synuclein mutants , were plated at 3xlO ⁵ per well in S- well dishes and induced with lμg/ml doxycycline (Sigma) for 8h and 48h respectively . Expression of transgenes was switched off by removing doxycycline from the medium ³ ' ⁴ and cells were treated with or without various compounds for 12Oh for EGFP-HDQ74 clearance and 24h for mutant α-synuclein clearance . For additive effect of LiCl or L-690 , 330 with rapamycin, treatment was done for 72h for EGFP-HDQ74 clearance and 8h for mutant α-synuclein clearance . The medium with various compounds was changed every 24h. Cells were either fixed and mounted in DAPI , or processed for immunoblotting analysis with EGFP for soluble EGFP-HDQ74 clearance or HA for mutant α-synuclein clearance .

Western Blot Analysis . Cell pellets were lysed on ice in Laemmli buffer (62.5mM Tris- HCl pH 6.8 , 5% β-mercaptoethanol , 10% glycerol , and 0.01% bromophenol blue) for 30 min in the presence of protease inhibitors (Roche Diagnostics) . Samples were subj ected to SDS- polyacrylamide gel electrophoresis and proteins were transferred to nitrocellulose membrane (Amersham Pharmacia Biotech) . Primary antibodies used include anti-EGFP (8362 -1 , Clontech) , anti-HA (12CA5 , Covance) , anti-mTOR (2972) , anti-Phospho-mTOR (Ser2448) (2971) , anti-p70 S6 Kinase (9202 ) , anti Phospho-p70 S6 Kinase (Thr389) ( 9206) , anti-4E-BPl (9452 ) , anti-Phospho-4E-BPl (Thr37/46) (9459) , anti-S6 Ribosomal Protein (2212) and anti-

Phospho-S6 Ribosomal Protein (Ser235/236 ) (2211) , all from Cell Signaling technology, anti-LC3 (gift from T . Yoshimori , National Institute of Genetics , Japan) , anti-actin (A2066, Sigma) and anti-tubulin (Clone DM IA, Sigma) . Blots were probed with anti-

mouse or anti-rabbit IgG-HRP (Amersham) and visualised using ECL or ECL Plus detection kit (Amersham) .

Iimunocytochemistry.

Cos-7 cells were fixed with 4% paraformaldehyde . Primary antibodies included anti-LC3 (gift from T . Yoshimori , National Institute of Genetics , Japan) and anti-Phospho-S6 Ribosomal Protein (Ser235/236) (2211 , Cell Signalling technology) . Standard fluorescence method was used for detection and secondary antibodies used were goat anti-rabbit Alexa 488 Green and Alexa 594 Red (Cambridge Biosciences) .

Labelling autophagic vacuoles with monodansylcadaverine. Autophagic vacuoles were labelled with monodansylcadaverine (MDC, Sigma) by incubating Cos-7 cells on coverslips with 0.05mM MDC for 1 hour at 37°C ^1S . Cells were then rinsed thrice with PBS _r and analysed immediately using fluorescent microscope .

Statistical Analysis .

Pooled estimates for the changes in aggregate formation or cell death, resulting from perturbations assessed in multiple experiments , were calculated as odds ratios with 95% confidence intervals . We have used this method frequently in the past to allow analysis of data from multiple independent experiments ¹⁰ ' ²⁴ ' ²⁵ . Odds ratios and p values were determined by unconditional logistical regression analysis , using the general log-linear analysis option of SPSS 9 software (SPSS , Chicago) . Densitometry analysis was done by Scion Image Beta 4.02 software (Scion Corporation) on immunoblots from three independent experiments . Significance for clearance of mutant proteins was determined by factorial ANOVA test using STATVIEW software, version 4.53 (Abacus Concepts) . *** , p < 0.001; ** , p < 0.01 ; * , p < 0.05 ; NS , Non-significant .

Inositol phosphate measurements .

IPi, ₂ were assayed as previously described ²⁶ . Briefly, COS-7 cells labelled with myo- [ ³ H] inositol (lμCi/10 ⁶ cells) for 24h, stimulated and then subj ected to chloroform/methanol (1 : 2) extraction followed by Bligh-Dyer phase separation. Levels of IPi, ₂ were determined by liquid scintillation counting of fractions eluted following Dowex (formate form) ion exchange chromatography of aliquots of the aqueous phase . Results were calculated as a percentage of total incorporated radioactivity. Levels of inositol-1 , 4 , 5-trisphosphate [IP ₃ ( 1 , 4 , 5 ) ] were measured in perchloric acid extracted samples (10 ⁷ cells/aliquot) using the [ ³ H] Biotrak Assay System (Amersham Biosciences , UK) according to manufacturer' s instruction. Data presented as mean ± SD of triplicate measurements and representative of at least three separate experiments .

Results

Lithium was confirmed to significantly reduced aggregation and cell death in COS-7 (non-neuronal) and SK-N-SH (neural precursor) cells (Figs . 1 and 2 ) , caused by truncated forms of mutant huntingtin, the aggregate-prone protein that causes

Huntington' s disease (HD) ¹¹ . Expression levels of EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats (EGFP-HDQ74) correlate with aggregate formation (the proportion of transfected cells with aggregates) in such cell models ³ ' ¹² .

To investigate if the reduced aggregation of the huntingtin construct was due to enhanced clearance,

Clearance of an aggregation prone huntingtin construct was determined using a stable doxycycline-inducible PC12 cell line expressing EGFP-HDQ7. The transgene expression was first induced by adding doxycycline and then switched off by removing doxycycline from the medium. The clearance of the construct at various times after switching off expression after an initial induction period can be used to assess whether specific agents

alter the clearance of the transgene product , as its expression decays when synthesis is stopped ³ .

Lithium was observed to significantly enhance clearance of soluble EGFP-HDQ74 (Fig . 3 ) and reduce aggregates at 12Oh and 17Oh (Figs 4 & 31) , whereas sodium chloride had no effect (Figs 30 & 31) . Furthermore, clearance of A53T and A3OP α-synuclein mutants , which cause autosomal dominant forms of Parkinson' s disease ¹³ ' ¹⁴ was assessed. After an induction of stable inducible PC12 cell lines and a subsequent removal of doxycycline , lithium treatment for 24h enhanced the clearance of A53T (Fig . 5) and A30P (Fig . 6) α-synuclein mutants whereas no effects were observed with sodium chloride .

We assessed if lithium induced autophagy, as the clearance of EGFP-HDQ74 and the A53T and A30P α-synuclein mutants are strongly dependent on this pathway ³ ' ⁴ ' ⁶ . In COS-7 cells , lithium increased the number of monodansylcadaverine (MDC) stained vesicles that have been shown to correlate with autophagic activity ¹⁵ . Rapamycin was used as a positive control in these experiments . The increased autophagic activity induced by lithium was confirmed with an independent readout , which is reported to be specific for autophagosome number . The microtubule-associated protein 1 light chain 3 (LC3 ) , a homologue of Apg8p essential for autophagy in yeast , is processed post-translationally into LC3 -I , which is cytosolic , and LC3 -II , which associates with autophagosome membranes ¹⁶ .

Lithium increased the number of LC3 -positive autophagic vesicles in COS-7 cells , like rapamycin, and increased the levels of LC3 - II . The modest increase in LC3 -II is similar to what has been observed previously when autophagy is induced ¹⁶ .

If lithium facilitates clearance of EGFP-HDQ74 and mutant α- synucleins through autophagy, then it should be blocked by the autophagy inhibitor, 3 -methyladenine (3 -MA) . 3 -MA itself increased EGFP-HDQ74 aggregate formation in COS-7 cells ³ (Fig .

32) and delayed clearance of A53T α-synuclein ⁴ . Furthermore , 3 - MA abolished the effect of lithium in reducing EGFP-HDQ74 aggregation (Fig . 32) , or promoting A53T α-synuclein clearance . Thus, lithium induces autophagy and enhances the clearance of known autophagy substrates like EGFP-HDQ74 and mutant α- synucleins .

Lithium inhibits a number of enzymes , including glycogen synthase kinase-3β (GSK-3β) and inositol monophosphatase (IMPase) ² ' ¹⁷ . In order to test if either of these enzymes were involved in autophagy regulation, we used specific inhibitors of GSK-3β (SB216763 ) ¹⁸ and IMPase (L-690 , 330 ) " (Fig 33 ) . L-690 , 330 reduced aggregation and cell death caused by EGFP-HDQ74 in both COS-7 and SK-N-SH cells , while SB216763 increased EGFP-HDQ74 aggregates but reduced cell death (Fig . 7) . L-690 , 330 facilitated clearance of EGFP-HDQ74 (Fig . 10) and the mutant α- synucleins , while SB216763 had no effect on clearance of soluble EGFP-HDQ74 (Fig . 9) and appeared to slow clearance of mutant α- synucleins in stable PC12 cell lines . This supports the role of IMPase in autophagy regulation.

Inhibitors of autophagy (3 -MA) and the proteasome (lactacystin were used to test whether the enhanced clearance of these proteins by L-690 , 330 was by autophagy or the proteasomal route . Both these inhibitors reduced clearance of A53T α-synuclein in the PC12 stable line ⁴ and increased EGFP-HDQ74 aggregates in COS-7 cells ³ (Figs 34 & 35) . When autophagy was inhibited by 3 - MA, L-690 , 330 could not facilitate clearance of A53T α- synuclein or reduce EGFP-HDQ74 aggregates (Fig 34) . However, L- 690 , 330 enhanced clearance of A53T α-synuclein and significantly reduced EGFP-HDQ74 aggregates in cells treated with the proteasome inhibitor lactacystin (Fig 35) . Thus L-

690 , 330 enhanced clearance of these proteins through autophagic route, which was confirmed by more LC3 -stained autophagic vesicles in COS-7 cells upon L-690 , 330 treatment . Thus , the

IMPase activity of lithium can account for its induction of autophagy.

Lithium and L-690 , 330 were confirmed as inhibiting IMPase in our experimental conditions , as they increased levels of inositol mono- and bis-phosphate (IP _1-2 ) (Fig . 11) and decreased myo- inositol-l , 4 , 5-triphosphate (IP ₃ ) levels (Fig . 12 ) , a consequence of reduced free inositol ^s . Lithium significantly reduced IP ₃ levels even at time-points from 1 min to 24h (Fig . 37) . Inositol depletion is a common mechanism for mood stabilizing drugs like lithium, carbamazepine (CBZ) and valproic acid (VPA) ⁷ . Consistent with a role for inositol depletion in autophagy regulation, CBZ and VPA significantly reduced EGFP- HDQ74 aggregates and attenuated polyglutamine toxicity in COS-7 cells (Fig . 13 ) , and also enhanced clearance of A3OP α- synuclein . Thus these drugs may be of therapeutic value in HD and related neurodegenerative diseases .

In order to test the importance of inositol levels in autophagy regulation, we tested whether the effect of lithium on autophagy could be overcome by addition of extracellular inositol in the form of myo-inositol ⁷ . We also used an inhibitor of prolyl oligopeptidase activity called prolyl endopeptidase inhibitor 2 (PEI) , which elevates intracellular IP ₃ and abolishes some other effects of lithium ²⁰ (Fig 36) . Lithium may deplete inositol not only by inhibiting IMPase but also by decreasing the transport of myo-inositol into cells ²¹ . Accordingly, cells were pretreated with myo-inositol and PEI before adding lithium. As predicted, both myo-inositol and PEI pretreatment raised IP ₃ levels in COS- 7 cells treated with lithium, compared to lithium treatment alone (Fig . 14) . Myo-inositol and PEI significantly reversed the protective effect of lithium on EGFP-HDQ74-induced aggregation and cell death in COS-7 cells (Figs . 15 and 16) . These compounds also inhibited the effect of lithium on the clearance of soluble EGFP-HDQ74 (Fig 17) and A53T α-synuclein in stable PC12 cell lines . Single treatments of myo-inositol or PEI , which itself

increased intracellular IP ₃ (Fig . 18 ) , increased EGFP-HDQ74 aggregates and cell death in COS-7 cells (Fig . 19) and inhibited the clearance of soluble EGFP-HDQ74 (Fig. 20) . These data provide indication that lithium induces autophagy through its IMPase activity, acting at the level of (or downstream of) lowered IP ₃ .

The activity of mTOR, a protein kinase , can be inferred by the levels of phosphorylation of its substrates , S6K1 (p70S6K) and 4E-BP1 at Thr389 and Thr37/46 , respectively (Fig 38 ) , and the phosphorylation of the S6 ribosomal protein (S6P) , a substrate of S6K1. While rapamycin reduced phosphorylation of S6K1 , S6P and 4E-BP1 in COS-7 cells as expected, lithium or L-690 , 330 did not have any effect on their phosphorylation, which provides indication that their effects are independent of mTOR inhibition . Overexpression of small G-protein rheb, which greatly enhances mTOR signalling ²³ , markedly increased EGFP- HDQ74 aggregates and cell death in COS-7 cells ⁵ (Fig . 21) . However, lithium or L-690 , 330 reduced EGFP-HDQ74 aggregates and cell death in rheb-transfected cells (Fig . 22 ) , suggesting that induction of autophagy by IMPase inhibition occurs even when mTOR is activated. The effect of IP ₃ on autophagy is unlikely to be a downstream consequence of mTOR inhibition, as rapamycin had no effect on IP ₃ levels (Fig . 18) . Furthermore , neither myoinositol nor PEI abolished the protective effect of rapamycin on polyglutamine toxicity in COS-7 cells (Fig . 23 and 24) and the increased clearance of soluble EGFP-HDQ74 and A53T α-synuclein in stable PC12 cell lines (Fig . 25) , in contrast to what we observed in the context of lithium. Therefore, rapamycin and intracellular inositol appear to independently regulate autophagy. Since the effect of rapamycin is not reduced by myoinositol or PEI , it appears that the inhibition of mTOR is a dominant mechanism. Lithium and rapamycin were observed to have additive effects in reducing EGFP-HDQ74 aggregates and cell death in COS-7 cells (Fig . 26) , compared to the single

treatments of lithium or rapamycin. Furthermore, lithium and rapamycin together facilitated greater clearance of soluble EGFP-HDQ74 at 72h (Fig . 27) and A53T α-synuclein at 8h, compared to single either compound alone . In order to clearly demonstrate this effect , early time-points were chosen at which obvious reductions of the levels of these proteins are not yet observed when the cells are treated with either of the compounds alone . The rapamycin concentration used in these experiments was close to saturating, as a further increase in the rapamycin dose did not lead to more clearance of soluble EGFP-HDQ74. Consistent with the above observations , the combination of L-690 , 330 and rapamycin had an enhanced protective effect on EGFP-HDQ74 mediated toxicity in COS-7 cells (Fig . 28) and also facilitated greater clearance of soluble EGFP-HDQ74 and A53T α-synuclein at early time-points in stable PC12 cell lines , compared to the individual treatments (Fig . 29) .

Lithium induces autophagy by inhibiting inositol monophosphatase , which in turn decreases free inositol leading to enhanced clearance of mutant proteins . These effects are reversed by free inositol (myo-inositol) , which itself inhibits clearance of autophagy substrates , providing further support for the role of IMPase in autophagy regulation. The abrogation of the effect of lithium on autophagy by PEI provides indication that these effects are mediated via IP ₃ .

These findings reveal a novel pathway for autophagy regulation independent of the mTOR pathway, which is currently the only known autophagy pathway that can be manipulated pharmacologically in man. This may be of potential value to neurodegenerative diseases caused by aggregate-prone proteins , such as HD ⁶ .

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