Modulation of Autophagy by Calpain Inhibition

This invention relates to the modulation of autophagy in cells, in particular through the inhibition of calpain.

Autophagy is a process that allows bulk degradation of cytoplasmic contents. It involves the formation of double membrane structures called autophagosomes , which fuse with lysosomes to form autolysosomes , in which the contents are degraded (reviewed in Klionsky and Emr, 2000) . Autophagy is involved in the clearance of the toxic, long-lived, aggregate- prone proteins which cause many neurodegenerative disorders, such as Huntington's disease (HD) and Parkinson's disease. Induction of autophagy, for example, reduces the levels of mutant huntingtin and protects against its toxicity in cells

(Ravikumar et al, 2002) and in transgenic Drosophila and mouse models of HD (Ravikumar et al, 2004) . Currently, the only suitable pharmacological strategy for up-regulating autophagy in mammalian brains is to use rapamycin, or its analogues, that inhibit the mammalian target of rapamycin (mTOR) , which is a negative regulator of autophagy. Autophagy may also be involved in the mechanisms of pathogen infections (Nakagawa et al, 2004, Gutierrez et al, 2004) .

Calpains are a family of calcium (Ca ²⁺ ) -dependent intracellular cysteine proteases, consisting of 14 members. The two ubiquitously expressed mammalian calpains, calpain 1 (μ- calpain) and calpain 2 (m-calpain) , are activated by micromolar and millimolar levels of Ca2+ respectively. Both calpain 1 and 2 are heterodimeric, consisting of a distinct 80 kDa large catalytic subunit and a common 28 kDa small regulatory subunit (reviewed in GoIl DE et al (2003) Physiol Rev 83: 731-801). The precise role of calpains in normal cellular function is poorly understood, but calpain activity is known to be preferentially directed to proteolytic modification of

cytoskeletal-membrane proteins and other proteins located at the inner surface of the plasma membrane . Although a study with an antibody recognising the active form of calpain 2 did not suggest its activation in HD (Adamec et al (2002) Acta Neuropathol 104:92-104), other calpain family members (calpain 1, 5, 7 and 10) are activated in HD tissue culture and mouse models (Gafni et al (2004) J Bio Chem 279:20211-20220). Activation of calpain has been suggested to contribute to HD pathogenesis by promoting huntingtin cleavage (Goffredo et al (2002) J Bio Chem 277:39594-39598; Kim et al (2001) Proc Natl Acad Sci USA 98:12784-12789; Gafni and Ellerby (2002) J Neurosci 22:4842-4849). This cleavage can be attenuated by- calcium chelators and calpain inhibitors (Goffredo et al (2002) J Bio Chem 277:39594-39598). Mutation of the calpain cleavage site in mutant huntingtin reduces its toxicity in HD cell models (Gafni et al (2004) J Bio Chem 279:20211-20220).

The present inventors have found that the inhibition of calpain activity induces autophagy in cells through a previously unknown mTOR independent pathway. The induction of autophagy through this novel pathway allows the treatment of diseases which are ameliorated by the induction of autophagy, including neurodegenerative disorders and pathogenic infections. Furthermore, screening methods based on this finding have led to the identification of candidate therapeutic compounds for treating these conditions.

One aspect of the invention provides a method of inducing autophagy in a cell comprising: reducing or abrogating the level or activity of calpain in said cell.

Reduction or abrogation in the level or activity of calpain induces autophagy in said cell .

Preferably, the level or activity of calpain is reduced or abrogated by contacting said cell with a calpain inhibitor.

A calpain inhibitor is a compound which reduces or inhibits the proteolytic activity of calpain. A suitable inhibitor may, for example inhibit calpain 1 (nucleic acid: AY796340, GI: 55140669, protein: P07384, GI: 115574) and/or calpain 2 (nucleic acid: AY835586, GI: 56157771, protein: P17655, GI: 60416356) .

Suitable calpain inhibitors are well known in the art and include, for example, calpastatin (Wendt et al Biol Chem. 2004 Jun;385 (6) :465-72) , ALLM, ALLN (Logie et . al . MoI Genet Metab. 2005 May; 85 (1) : 54-60) , calpeptin (Ariyoshi et al Biochem Int. 1991 Apr,-23 (S) :lθl9-33) , leupeptin, α-dicarbonyls, quinolinecarboxamides, sulfonium methyl ketones, diazomethyl ketones, Leu-Abu-CONHEt (AK275) , 27-mer calpastatin peptide and Cbz-Val-Phe-H (MDL28170) (Liu et al . Annu. Rev. Pharmacol. Toxicol. 44:349-370 (2004)).

Suitable calpain inhibitors also include, for example, calpeptin (Z-Leu-Nle-H) , α-mercaptoacrylic acids, phosphorus derivatives, epoxysuccinates, acyloxymethyl ketones, halomethylketones (Wang K. K. et al . (1997) Adv. Pharmacol.

37:117-152) and E64 (EST) (GoIl D. E. et al . (2003) Physiol.

Rev. 83 :731-801) .

In some preferred embodiments, the calpain inhibitor may be calpastatin, ALLM or calpeptin.

In other preferred embodiments, the calpain inhibitor may be an L-type Ca ²⁺ channel antagonist. Suitable L-type Ca ²⁺ channel antagonists are described in more detail below.

Methods of determining the inhibition of calpain activity are well-known in the art. For example, the effect of a test

compound on the proteolysis of one or more calpain substrates by calpain may be determined. Calpain substrates are well known in the art and include a large number of cytoskeletal proteins (e.g. α-fodrin, talin, paxillin, vinculin and tau factor) (Mehendale H.M. (2005) Trends in Pharmacol. Sci . 26,5:232-236), membrane receptors (e.g. epidermal growth factor (EGF) receptor and G proteins) , various calmodulin- binding proteins, enzymes involved in signal transduction (e.g. protein kinase C and inositol (1,4, 5) -trisphosphate kinase) and metabolic enzymes (e.g. phosphorylase kinase) .

Calpain substrates also include transcription factors (e.g. c- FOS and c-JUN) and actin-associated proteins.

An alternative approach to the inhibition of calpain employs regulation at the nucleic acid level to inhibit activity or function by down-regulating production of calpain. For instance, expression of calpain may be inhibited using anti- sense or RNAi technology. The use of these approaches to down-regulate gene expression is now well-established in the art.

Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA encoding calpain, thereby interfering with the production of calpain so that its expression is reduced or completely or substantially completely prevented. In addition to targeting coding sequence, antisense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the antisense oligonucleotides can interfere with expression control sequences. The construction of antisense sequences and their use is described for example in Peyman and Ulman (1990) Chemical Reviews 90:543-584 and Crooke (1992) Ann. Rev. Pharmacol. Toxicol. 32:329-376.

Oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo

within cells in which down-regulation is desired. Thus, double-stranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene .

The complete sequence corresponding to the coding sequence in reverse orientation need not be used. For example, fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon.

Preferably, the antisense nucleic acids are chosen among the polynucleotides of 15-200bp long that are complementary to the 5' end of a nucleic acid encoding a calpain protein. A suitable fragment may for example have about 14-23 nucleotides, e.g. about 15, 16 or 17.

Preferred antisense nucleic acids are complementary to a sequence of a calpain mRNA that contains the translational initiation codon ATG. However, the antisense nucleic acid may also be complementary to a sequence in the 3' or 5' untranslated regions or to sequences in the splice sites of the pre-mRNA precursor.

Anti-sense oligonucleotides may be deoxyribonucleotides, ribonucleotides or protein nucleic acids and may optionally comprise chemical modifications that prevent degradation by endogenous nucleases such as phosphorothioate oligonucleotides or morpholino oligonucleotides (Heasman et al (2000) Developmental Biology, 222:124-134).

An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression; Angell & Baulcombe (1997) The EMBO Journal 16 12:3675-3684; and Voinnet & Baulcombe (1997) Nature 389 553) . Double stranded RNA (dsRNA) has been found to be even more effective in gene silencing than either sense or antisense strands alone (Fire A. et al Nature 391 (1998)) . dsRNA mediated silencing is gene specific and is often termed RNA interference (RNAi) .

RNA interference is a two-step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt lengths with 5 ¹ terminal phosphate and 3' short overhangs (~2nt) . The siRNAs target the corresponding mRNA sequence specifically for destruction

(Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001)

RNAi may be also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3'- overhang ends (Zamore PD et al Cell, 101, 25-33, (2000)). Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologeous genes in a wide range of mammalian cell lines (Elbashir SM. et al . Nature, 411, 494-498, (2001)).

Another possibility is that nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific site - thus also useful in influencing gene expression. Background references for ribozymes include Kashani-Sabet and Scanlon, 1995, Cancer Gene Therapy, 2(3): 213-223, and Mercola and Cohen, 1995, Cancer Gene Therapy, 2 (1) , 47-59.

Thus, in some embodiments, a calpain inhibitor may comprise a nucleic acid molecule which consists of all or part of a

calpain coding sequence and/or the complement thereof, or example, the calpain 1 coding sequence (AY796340, GI: 55140669) or the calpain 2 coding sequence (nucleic acid: AY835586, GI: 56157771) or the complements thereof.

Such a molecule may suppress the expression of a calpain and may comprise a sense or anti-sense calpain coding sequence or may be a calpain specific ribozyme, according to the type of suppression to be employed.

The type of suppression will also determine whether the molecule is double or single stranded and whether it is RNA or DNA. Examples of the use of siRNA to reduce or abolish calpain polypeptide expression are provided below.

The term ^v calpain' as used herein may encompass any one of calpain 1, calpain 2, calpain 3, small subunit 1, calpain 5, calpain 6, calpain 7, calpain 8a, calpain 9, calpain 10, calpian 11, calpain 12, calpain 13a or small subunit, unless context dictates otherwise. Of particular interest are calpains 1 and 2 , which are ubiquitously expressed in mammals .

In some preferred embodiments, in addition to the calpain inhibitor, the cell may be contacted with an mTOR inhibitor.

The mTOR inhibitor may be contacted with the cell sequentially or simultaneously with the calpain inhibitor.

Suitable mTOR inhibitors include rapamycin macrolides such as rapamycin or derivatives or analogues thereof, including 0- alkyl rapamycins, carboxylic acid esters, amide esters, carbamates, fluorinated esters, acetals and silyl ethers, dialdehydes, oximes, hydrazones, hydroxylamines and enols, and CCI-779 and RAD-001.

Rapamycin and its derivatives and analogues are lactam 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 produced by Streptomyces hygroscopicus, and has the structure shown below.

VC

See, e.g., 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.

One group of rapamycin analogues are 40-0 -substituted derivatives of rapamycin having the structure set out below;

wherein; X ₄ is (H, H) or O; Y ₃ is (H, OH) or O; R ₂₀ and R _2x are independently selected from H, alkyl, arylalkyl, hydroxyalkyl , dihydroxyalkyl , hydroxyalkoxycarbonylalkyl , hydroxyalkylaryalkyl , dihydroxyalkylarylalkyl , acyloxyalkyl , aminoalkyl, alkylaminoalkyl , alkoxycarbonylaminoalkyl, acylaminoalkyl , arylsulfonamidoalkyl, allyl, dihydroxyalkylallyl, dioxolanylallyl, dialkyl-dioxolanylalkyl, di (alkoxycarbonyl) -triazolyl-alkyl and hydroxyalkoxy-alkyl; wherein "alk-" or "alkyl" refers to Ci _-6 alkyl, branched or linear, preferably Ci_ ₃ alkyl,; "aryl" is phenyl or tolyl; and acyl is a radical derived from a carboxylic acid; and;

R ₂₂ is methyl or R ₂₂ and R ₂₀ together form C _2-6 alkyl; provided that R ₂₀ and R ₂₁ are not both H; and hydroxyalkoxyalkyl is other than hydroxyalkoxymethyl .

Suitable rapamycin analogues are disclosed in WO 94/09010 and WO 96/41807.

Particularly suitable rapamycin analogues 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 hydroxyesters of rapamycin, such as 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid (CCI-779) . The preparation and use of hydroxyesters of rapamycin, including CCI-779, are disclosed in U.S. Patents 5,362,718 and 6,277,983

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.

A method as described herein may further comprise determining the level of autophagy in the cell. This may be carried out, for example, by assessing clearance of known autophagy substrates, measurement of LC3-positive vesicles in cells, measurement of LC3 II band by western blot or electron microscopy.

In some embodiments, the level or activity of calpain may be reduced or abrogated in a cell in vitro. For example, a calpain inhibitor may be used to induce autophagy in a cell in vitro, as described herein.

A suitable cell may be a cell from a primary cell culture or a cultured non-neuronal or neuronal cell line. Many suitable cell lines are known, for example a Chinese hamster ovary, baby hamster kidney, COS cell (and in particular African green monkey kidney (COS-7) ) , PC12 , human neuroblastoma (e.g. SK-N- SH), or a human cervical carcinoma (e.g. HeIa).

The cell may express an aggregation-prone polypeptide and the level of aggregates of the polypeptide, and/or the formation and clearance of aggregates, may be determined. In some embodiments, the cell 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 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 system as 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) .

In other embodiments, the level or activity of calpain may be reduced or abrogated in a cell in vivo i.e. the cell may be comprised in a human or non-human mammal. Agents which reduce or abrogate the level or activity of calpain, such as calpain inhibitors, may be contacted with the cell by administering

the inhibitor to the individual. The administration of calpain inhibitors is described in more detail below.

A calpain inhibitor as described herein may be used in the manufacture of a medicament for use in the induction of autophagy in a cell. In some preferred embodiments, a calpain inhibitor and an mTOR inhibitor may be used in the manufacture of a medicament for use in the induction of autophagy in a cell . Such a medicament may be useful in the treatment of a condition which is ameliorated by increased autophagy, including for example neurodegenerative disorders and pathogenic infections .

Using the methods described herein, the inventors have found that L-type Ca ²⁺ channel antagonists induce autophagy through inhibition of calpain.

Another aspect of the invention provides a method of treatment of a neurodegenerative disorder or a method of preventing or delaying the onset of a neurodegenerative disorder comprising: administering a L-type Ca ²⁺ channel antagonist to an individual in need thereof .

Neurodegenerative disorders are described in more detail below.

A L-type Ca ²⁺ channel antagonist is a compound which reduces or inhibits the activity of a L-type Ca ²⁺ channel. Suitable L- type Ca ²⁺ channel antagonists include dihydropyridines such as nifedipine, nicardipine, nitrendipine, nimodipine, nisoldipine, felodipine, FR34235, PN200-110, isradipine, manidipine, nilvadipine, benidipine, efonidipine, amlodipine, R(+)Bay K 8644, niguldipine and lercanidipine; phenylalkylamines such as verapamil, desmethoxyverapamil, YS- 035, gallopamil, thiapamil, devapamil, loperamide, noverapamil

and bepridil; and benzodiazepines such as diltiazem and clentiazem.

Other suitable L-type Ca ²⁺ channel antagonists include include: SR 33805 oxalate, amiodarone, gabapentin, indolizinsulphones, SQ32,910, SDZ32-207-180, rhynchophylline, tetrandrine, FS-2 from dendroaspis polylepis polylepis venom, S-petasin, liensinine, osthole, calcicludine dendroaspis angusticeps, calcicludine, dendroaspis polylepis polylepis, FTX-3.3 agenelopsis aperta, taicatoxin oxyuranus, tetrahydropalmatine, fendiline, terodiline, phloretin, protopine and cinnarizine.

L-type Ca ²⁺ channel antagonists may be specific antagonists of L-type Ca ²⁺ channels, such as verapamil and nimodipine, or may also be antagonists of other types of Ca ²⁺ channel (i.e. nonspecific Ca ²⁺ channel antagonists), such as loperamide.

Other aspects of the invention provide use of an L-type Ca ²⁺ channel antagonist in the manufacture of a medicament for the treatment of a neurodegenerative disorder or a method of preventing or delaying the onset of a neurodegenerative disorder and an L-type Ca ²⁺ channel antagonist for the treatment of a neurodegenerative disorder or a method of preventing or delaying the onset of a neurodegenerative disorder.

Other aspects of the invention provide a method of treatment of a neurodegenerative disorder in an individual or a method of preventing or delaying the onset of a neurodegenerative disorder in an individual comprising: administering a calpain inhibitor and an mTOR inhibitor to an individual in need thereof; a calpain inhibitor and an mTOR inhibitor for use in the treatment of a neurodegenerative disorder or a method of preventing or delaying the onset of a neurodegenerative

disorder and the use of a calpain inhibitor and an mTOR inhibitor in the manufacture of a medicament for use in the treatment of a neurodegenerative disorder or a method of preventing or delaying the onset of a neurodegenerative disorder.

Neurodegenerative disorders may include protein aggregation disorders. Protein aggregation disorders (also known as protein conformation disorders or proteinopathies) are characterised by the formation of intracellular protein aggregates . Protein aggregation disorders include codon reiteration mutation disorders, α-synucleinopathies, prion disorders and tauopathies .

Codon reiteration mutation disorders include polyA expansion disorder and polyO expansion disorders. PolyA expansion disorders are characterised by a polyadenine (polyA) expansion mutation. PoIyQ expansion disorders are characterised by a polyglutamine (polyQ) expansion mutation and include Huntington's disease, spinocerebellar ataxias types 1, 2, 3,

6, 7 and 17, spinobulbar muscular dystrophy and dentatorubral pallidoluysian atrophy.

α-synucleinopathies are characterised by the accumulation of Lewy bodies comprising α-synuclein, and include Parkinson's Disease, LB variant Alzheimer's disease and LB dementia.

Prion disorders are characterised by the aggregation of PrP ^Sc and include familial, sporadic and new variant CJD, as well as veterinary disorders such as scrapies and BSE.

Tauopathies are characterised by the abnormal accumulation of tau protein, in particular hyperphosphorylated tau protein, and include sporadic frontotemporal dementia (FTD), Pick's disease and Alzheimer's disease.

Examples of suitable calpain inhibitors and mTOR inhibitors for use in the present methods are described above.

A individual suitable for undergoing a method of preventing or delaying the onset of a neurodegenerative disorder as described herein may have no symptoms of a neurodegenerative disorder. In some embodiments, the individual may be at risk of or susceptible to a neurodegenerative disorder, for example the individual may have one or more risk factors associated with the onset of a neurodegenerative disorder.

Other aspects of the invention provide a method of treatment of a pathogen infection in an individual comprising: administering a calpain inhibitor to an individual in need thereof; a calpain inhibitor for use in the treatment of a pathogen infection and the use of a calpain inhibitor in the manufacture of a medicament for use in the treatment of a pathogen infection.

In some preferred embodiments, the calpain inhibitor may be used with an mTOR inhibitor. For example, an mTOR inhibitor may be administered to the individual sequentially or simultaneously with the calpain inhibitor.

A pathogen infection may be a bacterial infection, for example a mycobacterial infection such as tuberculosis, or a streptococcal infection.

In some embodiments, a calpain inhibitor and an mTOR inhibitor may be formulated into a single composition for use in the methods described herein. A pharmaceutical composition may, for example comprise a calpain inhibitor, an mTOR inhibitor and a pharmaceutically acceptable excipient, vehicle or carrier. In other embodiments, a pharmaceutical composition

may comprise an L-type Ca ²⁺ channel antagonist and a pharmaceutically acceptable excipient, vehicle or carrier.

A suitable method of producing a pharmaceutical composition may comprise admixing a calpain inhibitor, for example an L- type Ca ²⁺ channel antagonist, and an mTOR inhibitorwith a pharmaceutically acceptable excipient, vehicle or carrier.

Examples of suitable calpain inhibitors, mTOR inhibitors and L-type Ca ²⁺ channel antagonists for use in the present methods are described above .

A pharmaceutically acceptable excipient, vehicle or carrier, 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 injection, 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 injection, or injection at the site of affliction, the active ingredient will 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 Injection, Ringer's Injection, Lactated Ringer's Injection. 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.

Formulations and administration regimes which are suitable for use with rapamycin macrolides such as rapamycin, and L-type Ca ²⁺ channel antagonists, such as verapamil, loperamide and nimodipine, are well known in the art.

Administration is preferably in a "therapeutically effective amount", 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 medical practitioners .

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

Other aspects of the invention relate to the reduction or inhibition of autophagy by calpain activators . This may be useful, for example for increasing or promoting apoptosis (Levein and Yuan J. Clin Invest 2005 115 (10) 2679-88; Tsu Tsujimoto Y and Shimizu S. Cell Death Differ. 2005 12 Suppl 2:1528-34). Increased apoptosis may, for example, be useful in the treatment of cancer, type II diabetes and limb girdle muscular dystrophy 2A.

A method of decreasing autophagy in a cell may comprise inducing the level or activity of calpain in said cell .

An increase in the level or activity of calpain reduces autophagy in the cell.

Calpain activity may be induced by contacting said cell with a calpain activator. A calpain activator is a compound which induces or increases the proteolytic activity of one or more calpain polypeptides. Calpain polypeptides are described in more detail above. Calpain activators are well known in the art and include thapsigargin. Other calpain activators include L-type Ca ²⁺ channel agonists such as Bay K.

Other aspects of the invention relate to methods of identifying and/or obtaining compounds which modulate autophagy and are therefore useful in therapy.

A method of identifying and/or obtaining a compound which induces autophagy in a cell may comprise: determining the activity of a calpain polypeptide in the presence of a test compound.

A decrease in activity in the presence, relative to the absence of test compound is indicative that the test compound induces autophagy. Activity may be determined, for example, by determining the proteolytic cleavage of a calpain substrate in the presence of the test compound.

Suitable calpain polypeptides for use in the present methods include calpain 1 (P07384: swissprot) or calpain 2 (P17655: swissprot) and fragments and variants thereof.

In other embodiments, a method of identifying and/or obtaining a compound which induces autophagy in a cell may comprise:

determining the activity of a L-type Ca ²⁺ channel in the presence of a test compound.

A decrease in activity in the presence, relative to the absence of test compound is indicative that the test compound is an L-type Ca ²⁺ channel antagonist which induces autophagy. Activity may be determined, for example, by determining the Ca ²⁺ _current through the channel in the presence of the test compound.

Suitable L-type Ca ²⁺ channels for use in the present methods include Cavl .1 (L33798.1 GI:563322) Cavl .2 (L29529.1 GI:463072) Cavl .3 (voltage-gated calcium channel alphal-subunit) Human:2161aa, M76558 (M76558.1 GI :179763) , Cavl .4 (AJ224874.1 Gl: 3297874) and fragments and variants thereof.

A method may further comprise determining the ability of said compound to induce autophagy in a cell. This may be performed as described herein.

A compound which induces or promotes autophagy may be useful in the treatment of a neurodegenerative disorder.

A fragment or variant of a wild-type calpain or L-type Ca ²⁺ channel 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 activity is retained.

A polypeptide which is a variant of a wild-type sequence may comprise an amino acid sequence which shares greater than about 30% sequence identity with the wild-type 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 calpain 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 or channel 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 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 recombinant techniques and chemical synthesis techniques which are well known in the art .

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.

Screening 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 .

A compound which is found to induce autophagy may be useful in the treatment of a neurodegenerative disease or pathogen infection. A method of identifying and/or obtaining a compound useful in treatment of a neurodegenerative disease or pathogen infection may comprise: determining the activity of a calpain polypeptide or an L-type Ca ²⁺ channel in the presence of a test compound .

A decrease in activity in the presence, relative to the absence of test compound is indicative that the test compound is useful in the treatment of a neurodegenerative disease or pathogen infection.

A method of identifying and/or obtaining a compound useful in the treatment of a neurodegenerative disease or pathogen infection may comprise: contacting a cell with a calpain inhibitor compound, for example, an L-type Ca ²⁺ channel antagonist and, determining the level or amount of autophagy in said cell .

An increase in autophagy in the presence relative to the absence of inhibitor compound or antagonist may be indicative that the compound is useful in the treatment of a neurodegenerative disorder or a pathogenic infection.

A calpain inhibitor compound is a compound which inhibits the proteolytic activity of calpain. Examples of suitable calpain inhibitor compounds are described above. Further calpain inhibitor compounds may be readily identified using standard techniques. An L-type Ca ²⁺ channel antagonist is a compound which inhibits the activity of an L-type Ca ²⁺ channel. Examples of suitable L-type Ca ²⁺ channel antagonists are described above. Further L-type Ca ²⁺ channel antagonists may be readily identified using standard techniques.

A method may comprise the initial step of identifying a test compound as a calpain inhibitor compound, for example by determining the proteolytic activity of calpain in the presence of the test compound. A decrease in calpain activity in the presence of the test compound is indicative that the test compound is a calpain inhibitor compound. The proteolytic activity of calpain in the presence of the test compound may be determined by routine techniques.

A method may thus comprise: identifying a test compound as a calpain inhibitor compound, and, determining the ability of said calpain inhibitor compound to induce autophagy in a cell .

In some embodiments, a test compound may be identified as a calpain inhibitor by determining its ability to block L-type Ca ²⁺ channels. A method may comprise the initial step of identifying a test compound as a L-type Ca ²⁺ channel antagonist, for example by determining the channel activity of an L-type Ca ²⁺ channel in the presence of the test compound. A decrease in channel activity in the presence of the test compound is indicative that the test compound is a L-type Ca ²⁺ channel antagonist. The activity of an L-type Ca ²⁺ channel in the presence of the test compound may be determined by routine techniques .

A method may thus comprise: identifying a test compound as a L-type Ca ²⁺ channel antagonist , and, determining the ability of said L-type Ca ²⁺ channel antagonist to induce autophagy in a cell .

An increase in autophagy in the presence relative to the absence of the compound may be indicative that the compound is useful in the treatment of a neurodegenerative disorder or a pathogenic infection.

The calpain inhibitor may be contacted with the cell in the presence of an mTOR inhibitor. Suitable mTOR inhibitors are described in more detail above.

Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms which contain several characterised or uncharacterised components may also be used.

Combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for ability to modulate an interaction. Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances.

The amount of test compound or compound which may be added to a method of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to 1 mM or more of putative inhibitor compound may be used, for example from 0.01 nM to lOOμM, e.g. 0.1 to 50 μM, such as about 10 μM.

Suitable test compounds for screening include known calpain inhibitors, compounds such as L-type Ca ²⁺ channel antagonists which are identified herein as calpain inhibitors, and analogues and mimetics of these, for example compounds produced using rational drug design to provide candidate compounds with particular molecular shape, size and charge characteristics suitable for inducing autophagy in mammalian cells .

A method may comprise identifying the test compound as an autophagy inducer. Such a compound may for example be useful in the treatment of a neurodegenerative disorder or a pathogenic infection.

A test compound identified using one or more initial screens as having ability to increase or induce autophagy in a cell may be assessed further using one or more secondary screens. A secondary screen may, for example, involve testing for a biological function such as clearance of protein aggregates and amelioration of symptoms of neurodegenerative disorder or a pathogenic infection in animal models .

The test compound may be isolated and/or purified or alternatively, it may be synthesised using conventional techniques of recombinant expression or chemical synthesis . Furthermore, it 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 for the treatment of disease conditions such as a neurodegenerative disorder or a pathogenic infection, as described below, or for preventing or delaying the onset of such a condition. Methods of the invention may thus comprise formulating the test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier for therapeutic application, as discussed further below.

Following identification of a compound which induces autophagy, 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 desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Modification of a known active compound (for example, to produce a mimetic) may be used to

avoid randomly screening large number of molecules for a target property.

Modification of a ^λ lead' compound to optimise its pharmaceutical properties commonly comprises several steps . Firstly, the particular parts of the compound that are critical and/or important in determining the target property- are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. 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.

In a variant of this approach, the three-dimensional structure of the compound which induces autophagy is modelled. This can be especially useful where the compound changes conformation, allowing the model to take account of this in the optimisation of the lead compound.

A template molecule is then selected, onto which chemical groups that 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 biological

activity of the lead compound. The modified compounds found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Modified compounds include mimetics of the lead compound.

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

As described above, a compound identified and/or obtained using the present methods may be formulated into a pharmaceutical composition.

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.

Database accession numbers recited herein refer to the sequence entry which was current under this accession number at the filing date of the present application.

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 below.

Figure 1 shows that L-type Ca ²⁺ channel antagonists enhance the clearance of mutant aggregate-prone proteins in stable inducible PC12 cells which express the A53T α-synuclein mutant. A53T α-synuclein was detected by densitometry relative to actin.

Figure 2 shows that L-type Ca ²⁺ channel antagonists enhance the clearance of mutant aggregate-prone proteins in stable inducible PC12 cells which express the EGFP-HDQ74 mutant. ξGFP-HDQ74 was detected by densitometry relative to actin.

Figure 3 shows SK-N-SH cells transfected with EGFP-HDQ74 construct for 4h and treated with or without lμM verapamil, lμM loperamide, iμM nimodipine for 48h post-transfection. DMSO was control . The proportions of EGFP-positive cells with aggregates or cell death were expressed as odds ratios and the control was taken as 1.

Figure 4 shows SK-N-SH cells transfected with EGFP-HDQ74 construct for 4h were treated with or without lμM bay K for 48h post-transfection. DMSO was control. The proportions of EGFP-positive cells with aggregates or cell death were expressed as odds ratios and the control was taken as 1.

Figure 5 shows the effect of different concentrations of calpain inhibitors on calpain activity. The activation or inhibition of calpain in COS-7 cells was analysed by densitometry of the ratio of activated calpain small subunit (Cp active) to actin.

Figure 6 shows the percentage of pEGFP-HDQ74 positive COS-7 cells with aggregates and apoptotic morphology (cell death) , treated with or without 10 μM calpastatin, 50 μM ALLM, 50 μM calpeptin or DMSO for 48 h, expressed as odds ratios and the controls (untreated or DMSO-treated) were taken as 1.

Figure 7 shows the percentage of pEGFP-HDQ74 positive SK-N-SH cells with aggregates and apoptotic morphology (cell death) treated with or without 10 μM calpastatin, 50 μM ALLM, 50 μM

calpeptin or DMSO for 48 h, expressed as odds ratios and the controls (untreated or DMSO-treated) were taken as 1.

Figure 8 shows clearance of mutant huntingtin fragment in stable inducible PC12 cells expressing EGFP-HDQ74, after inducing expression with doxycycline for 8 h, then switching it off for 96 h, with (+) or without (-) lOμM calpastatin.

Figure 9 shows clearance of mutant huntingtin fragment in stable inducible PC12 cells expressing EGFP-HDQ74 after inducing expression with doxycycline for 8 h, then switching expression off for 96 h, with (+) or without (-)50 μM ALLM or 50 μM calpeptin.

Figure 10 shows A53T α-synuclein clearance in mitotic stable inducible PC12 cell lines expressing A53T α-synuclein mutant, treated with or without 10 μM calpastatin for 24 h..

Figure 11 shows A53T α-synuclein clearance in non-mitotic neuronally differentiated (with 100 ng/ml NGF for 5 days) stable inducible PC12 cell lines expressing A53T α-synuclein mutant, treated with or without 10 μM calpastatin for 24 h..

Figure 12 shows that calpain inhibitors facilitate clearance of mutant proteins by induction of autophagy, wherein the percentage of EGFP-HDQ74-positive cells with aggregates in transfected COS-7 cells, treated for 48 h with (+) or without (-) 10 mM 3 -MA or 10 μM lactacystin were expressed as odds ratio.

Figure 13 shows the percentage of EGFP-HDQ74 -positive cells with aggregates in transfected COS-7 cells, treated for 48 h with 10 mM 3-MA with (+) or without (-) 10 μM calpastatin, expressed as odds ratio.

Figure 14 shows the percentage of EGFP-HDQ74-positive cells with aggregates in transfected COS-7 cells, treated for 48 h with 10 μM lactacystin with or without 10 μM calpastatin in presence or absence of 10 rciM 3 -MA, expressed as odds ratio.

Figure 15 shows clearance of A53T α-synuclein in stable inducible PC12 cells expressing A53T α-synuclein mutant, after inducing expression with doxycycline for 48 h, then switching expression off for 24 h, treated for 24 h with with (+) or without (-) 10 μM lactacystin, 10 μM calpastatin or both, in presence or absence of 10 πiM 3 -MA.

Figure 16 shows the proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in SK-N-SH cells, treated for 48h with DMSO or 1OmM 3 -MA with or without iμM verapamil.

Figure 17 shows the proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in SK-N-SH cells, treated for 48h with DMSO or 1OmM 3 -MA with or without lOμM lactacystin and IμM verapamil in presence or absence of 1OmM 3 -MA

Figure 18 shows the results of treating COS-7 cells with DMSO (control) or 50μM calpeptin for 24h, fixed and assessed by electron microscopy for the number of autophagosome-like structures (A) and mitochondria (B) per 50 cell profiles. The data was analysed using Student's t-test and the error bar denotes standard error of the mean. **, p < 0.01.

Fiqure 19 shows the proportions of EGFP-positive cells with EGFP-HDQ74 aggregates or cell death in COS-7 cells treated with DMSO (control) or 2.5μM thapsigargin (thap) for last 24h of 48h post-transfection period.

Figure 20 shows the clearance of A53T α-synuclein in stable PC12 cells as in Figure IA, treated with DMSO (control) or 2.5μM thapsigargin for 24h.

Figure 21 shows the proportions of EGFP-positive cells with

EGFP-HDQ74 aggregates or cell death in COS-7 cells transfected with EGFP-HDQ74 construct along with empty vector (pCDNA3.1) or constitutive active S50E human m-calpain at 1:3 ratio. The proportions of EGFP-positive cells with aggregates or cell death were assessed after 48h.

Figure 22 shows the percentage of EGFP-HDQ74-positive cells with aggregates and cell death in transfected COS-7 cells, treated with 2.5 μM thapsigargin in presence or absence of 10 μM calpastatin or 50 μM ALLM for the last 24 h of the 48 h post-transfection period, expressed as odds ratio. Cells were pretreated with calpain inhibitors for 15 min prior to treatment with thapsigargin.

Figure 23 shows inhibition of clearance of A53T α-synuclein in stable PC12 cells expressing A53T α-synuclein mutant, treated with DMSO (control) or 2.5 μM thapsigargin in presence or absence of 10 μM calpastatin for 24 h.

Figure 24 shows the percentage of EGFP-HDQ74-positive cells with aggregates and cell death in COS-7 cells transfected with EGFP-HDQ74 and constitutive active S50E human m-calpain constructs (1:3 ratio) for 4h treated with or without lOμM calpastatin, or 50μM ALLM for 48h, and the control represents untreated S50E m-calpain-transfected cells.

Figure 25 shows the percentage of EGFP positive cells with GFP-LC3 vesicles in COS-7 cells transfected with pEGFP-LC3, treated with 2.5 μM thapsigargin with or without 10 μM calpastatin or 50 μM ALLM for 2h, expressed as odds ratios.

Figure 26 shows A53T α-synuclein clearance in stable PC12, treated with DMSO (control) or 2.5 μM thapsigargin in presence or absence of 50 μM ALLM for 24 h. Cells were pretreated with ALLM for 15 min before adding thapsigargin .

Figure 27 shows the clearance of A53T α-synuclein in stable PC12 cell line, treated with DMSO (control) or 2.5μM thapsigargin in presence or absence of 20μM caspase inhibitor I (Z-VAD-FMK) for 24h, was analysed by densitometry analysis relative to actin. Cells were pretreated with Z-VAD-FMK for 15min prior to thapsigargin treatment.

Figure 28 shows the percentage of EGFP-positive cells with EGFP-LC3 vesicles in COS-7 cells, treated with 2.5μM thapsigargin with or without 20μM Z-VAD-FMK for 2h, was expressed as odds ratio. Cells were pretreated with Z-VAD-FMK for 15min before thapsigargin addition. *, p < 0.05; NS, Non- significant.

Figure 29 shows stored intracellular calcium in Fura2-loaded COS-7 cells, treated with DMSO (dashed) , thapsigargin (black) , thapsigargin and calpastatin (dark grey) or thapsigargin and ALLM-treated (light grey) .

Figure 30 shows peak ionomycin-releasable calcium (mean ± s.d., n = 3) , under the same conditions as in Figure 26.

Figure 31 shows the proportions of EGFP-positive cells with

EGFP-HDQ74 aggregates or cell death in SK-N-SH cells, treated with DMSO (control) or lμM bay K in presence or absence of lμM verapamil for 48h post-transfection.

Figure 32 shows the proportions of EGFP-positive cells with EGFP-HDQ74 aggregates or cell death in SK-N-SH cells as in Figure IG, treated with DMSO (control) or lμM bay K in presence or absence of lOμM calpastatin for 48h post- transfection.

Figure 33 shows A53T α-synuclein clearance in COS7 cells treated with DMSO (control) or lμM Bay K in presence or absence of lμM verapamil for 24h. Cells were pretreated with verapamil for 15min before Bay K treatment.

Figure 34 shows A53T α-synuclein clearance in COS7 cells, treated with DMSO (control) or lμM Bay K in presence or absence of lOμM calpastatin for 24h. Cells were pretreated with calpastatin for 15min before Bay K treatment .

Figure 35 shows the proportion of GFP-positive cells with aggregates or cell death in COS-7 cells transfected with pEGFP-HDQ74 along with empty vector (pCDNA3.1) or pRheb, expressed as odds ratios.

Figure 36 shows the proportion of GFP-positive cells with aggregates or cell death in COS-7 cells transfected with pEGFP-HDQ74 along pRheb, expressed as odds ratio. The rheb- transfected cells were treated with or without 10 μM calpastatin, 50 μM ALLM or 50 μM calpeptin, and the control represents untreated rheb-transfected cells.

Figure 37 shows the percentage of EGFP-HDQ74-positive cells with aggregates and cell death in transfected COS-7 cells, treated with 2.5 μM thapsigargin with or without 0.2 μM rapamycin for the last 24 h of the 48 h post-transfection period, expressed as odds ratio.

Figure 38 shows clearance of A53T α-synuclein in stable inducible PC12 cells, treated for 24 h with 2.5 μM thapsigargin with (+) or without (-) 0.2 μM rapamycin.

Figure 39 shows the percentage of EGFP-positive cells with GFP-LC3 vesicles in COS-7 cells transfected with pEGFP-LC3, treated with 2.5 μM thapsigargin with or without 0.2 μM rapamycin for the 2 h, was expressed as odds ratio.

Figure 40 shows the percentage of EGFP-HDQ74-positive cells with aggregates in transfected COS-7 cells, treated with (+) or without (-) 10 μM calpastatin, 0.2 μM rapamycin or both for 48 h, expressed as odds ratios.

Figure 41 shows the percentage of EGFP-HDQ74-positive cells with cell death in transfected COS-7 cells, treated with (+) or without (-) 10 μM calpastatin, 0.2 μM rapamycin or both for 48 h, expressed as odds ratios.

Figure 42 shows clearance of soluble EGFP-HDQ74 in stable PC12 cells expressing EGFP-HDQ74, inducing expression with doxycycline for 8 h, then switching it off for 96 h, treated with (+) or without (-) 10 μM calpastatin, 0.2 μM rapamycin or both for 48 h.

Figure 43 shows clearance of A53T α-synuclein in stable PC12 cells expressing A53T α-synuclein mutant, after inducing expression with doxycycline for 48 h, then switching expression off for 24 h, treated with (+) or without (-) 10 μM calpastatin, 0.2 μM rapamycin or both for 8 h.

Figure 44 shows the percentage of levels of calpain 1 relative to calpain 2 in HeLa cells either transfected with siGLO(control) or siRNAs for calpain 1 for 96 h.

Figure 45 shows the percentage of levels of calpain 2 relative to calpain 1 in HeLa cells either transfected with siGLO(control) or siRNAs for calpain 2 for 96 h.

Figure 46 shows the percentage of EGFP- and siGLO-positive cells with GFP-LC3 vesicles in HeLa cells transfected with siGLO with or without calpain 1 siRNA or calpain 2 siRNA for 96 h, followed by transfection with pEGFP-LC3 for 4, expressed as odds ratios .

Figure 47 shows the percentage of EGFP- and siGLO-positive cells with EGFP-HDQ74 aggregates and cell death in HeLa cells transfected with pEGFP-HDQ74 along with siGLO in presence or absence of calpain 1 siRNA or calpain 2 siRNA for 96h, expressed as odds ratios .

Figure 48 shows that flies treated with verapamil hydrochloride show a shift in the distribution of the number of rhabdomeres compared to flies treated with DMSO (control) alone. Rhabdomere counts from all 3 independent experiments are included. rz=945 ommatidia (DMSO) and n=930 ommatidia (verapamil hydrochloride treatment) . Verapamil was also found to be protected at other concentrations (Figures 46-48) . Mann Whitney p value <0.0001; Student's t-test p value = 0.038. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, Nonsignificant .

Figure 49 shows the distribution of the number of rhabdomeres in flies treated with 1.25M verapamil hydrochloride.

Figure 50 shows the distribution of the number of rhabdomeres in flies treated with 1.87M verapamil hydrochloride

Figure 51 shows the distribution of the number of rhabdomeres in flies treated with 2.5M verapamil hydrochloride.

Figure 52 shows the effect of L-type Ca2+ channel antagonists and agonists in PC12 cells. Intracellular Ca2+ measurements of Fura2-loaded PC12 cells were determined during activation of voltage-gated Ca2+ channels by membrane depolarisation achieved by addition of extracellular KCl (60 mM) and subsequent addition (arrow) of loperamide (1 μM; dark grey) , Bay K (1 μM; light grey) or vehicle alone (black) . Traces shown are representative of at least 3 separate experiments . Similar reductions in intracellular Ca2+ were observed following addition of verapamil and nimodipine

Examples

Material and Methods

Compounds Cells were treated with various compounds including lμM verapamil hydrochloride, lμM loperamide hydrochloride, iμM nimodipine, IμM nitrendipine, lμM amiodarone hydrochloride, lμM Bay K 8644 (Sigma), lOμM calpastatin, 50μM ALLM, 50μM calpeptin, 20μM caspase inhibitor I (all from Calbiochem) , 2.5μM thapsigargin, 0.2μM rapamycin, 40OnM bafilomycin Al, 1OmM 3 -methyladenine (3-MA) and lOμM lactacystin (all from Sigma) for various time-points as stated under different experimental conditions.

Plasmids

Huntington' s Disease (HD) gene exon 1 fragment with 74 polyglutamine repeats (Q74) in pEGFP-Cl (Clontech) was described and characterised previously (Narain Y. et al . J. Med. Genet. 36: 739-746 (1999)).

Mammalian cell culture and transfection

African green monkey kidney cells (COS-7) , human neuroblastoma cells (SK-N-SH) and human cervical carcinoma cells (HeLa) were maintained in Dulbecco's Modified Eagle Medium (DMEM, Sigma) supplemented with 10% Fetal Bovine Serum (FBS, Sigma), 100 U/ml Penicillin/Streptomycin and 2 mM L-Glutamine (Sigma) at 37 ⁰ C,

5% Carbon dioxide (C02) . Cells were plated in six-well dishes at a density of 1x105 cells per well for 24 h and transfected with pEGFP-HDQ74 (1.5μg/well of 6-wells plate) using LipofectAMINE reagent for COS-7 cells and LipofectAMINE PLUS reagent for SK-N-SH cells using manufacturer's protocol

(Invitrogen) . The transfection mixture was replaced after 4 h incubation at 37 ⁰ C by various compounds, including 10 μM calpastatin, 50 μM ALLM, 50 μM calpeptin, 0.2 μM rapamycin, 10 mM 3-methyladenine (3-MA), 10 μM lactacystin, and 2.5 μM thapsigargin. Transfected cells were fixed with 4% paraformaldehyde (Sigma) after 48 h 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 3x105 cells per well and treated for 24 h. HeLa cells stably expressing UbG76V-GFP reporter (Dantuma N. P. et al. Nat. Biotechnol. 18: 538-543 (2000)) were grown in the same media used for COS-7 cells supplemented with 0.5 mg/ml G418.

Inducible PC12 stable cell lines expressing EGFP-tagged exon 1 of HD gene (EGFP-HDQ74) (Wyttenbach A. et al . Hum. MoI. Genet. 10: 1829-1845 (2001) and HA-tagged A53T α-synuclein mutant (Webb J. L. et al . J. Biol. Chem. 278: 25009-25013 (2003)), previously characterised, were maintained at 75 μg/ml hygromycin B (Calbiochem) in standard DMEM with 10% horse serum (Sigma) , 5% FBS, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, and 100 μg/ml G418 (GIBCO) at 37 ⁰ C, 10% C02.

Quantification of aggregate formation and cell death

Approximately 200 EGFP-positive cells were counted for the proportion of cells with aggregates, as described previously (Narain Y. et al . J. Med. Genet. 36: 739-746 (1999)). 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 (Wyttenbach A. et al. Hum. MoI. Genet. 11: 1137-1151 (2002)). Analysis was performed using Nikon Eclipse E600 fluorescence microscope (plan-apo 60x/l.4 oil immersion lens at room temperature) 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. Images were acquired with 4Ox plan-fluor 40x/0.75 lens at room temperature .

Clearance of mutant huntingtin and A53T α-synuclein Stable inducible PC12 cell lines expressing EGFP-HDQ74 or A53T α-synuclein mutant were plated at 3x105 per well in 6-well dishes and induced with 1 μg/ml doxycycline (Sigma) for 8 h and 48 h respectively. Expression of the transgene was switched off by removing doxycycline from the medium (Ravikumar B. et al . Hum. MoI. Genet. 11: 1107-1117 (2002); Webb J. L. et al . J. Biol. Chem. 278: 25009-25013 (2003)) and cells were treated with or without calpain inhibitors for 96 h for EGFP-HDQ74 clearance and 24 h for A53T α-synuclein clearance. DMSO was used as a control. To study the additive effect of calpastatin with rapamycin, treatment was done for 48 h for EGFPHDQ74 clearance and 8 h for A53T α-synuclein clearance. Compounds were replenished every 24 h for EGFP- HDQ74 clearance studies. Clearance of mutant proteins was measured by immunoblotting with EGFP for soluble EGFP-HDQ74 clearance or HA for A53T α-synuclein clearance.

Western Blot Analysis Cell pellets were lysed on ice in Laemmli buffer (62.5 πiM 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 subjected to SDS- polyacrylamide gel electrophoresis and proteins were transferred to nitrocellulose membranes (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) , all from Cell Signaling Technology, anti-calpain small subunit (μ- or mcalpain) (MAB3083, Chemicon) , anti-calpain 1 (MAB3104, Chemicon) , anti- calpain 2 domain III (208755, Calbiochem) , anti-LC3 (gift from T. Yoshimori) and anti-actin (A2066, Sigma) . Blots were probed with anti-mouse or anti-rabbit IgG-HRP (Amersham) and visualised using ECL or ECL Plus detection kit (Amersham) .

Immunocytochemistry

COS-7 cells were fixed with 4% paraformaldehyde. Primary antibodies included anti- LC3 , anti-Phospho-S6 Ribosomal Protein (Ser235/236) and anti-Phospho-4E-BPl (Thr37/46) . Standard fluorescence methods were used for detection and secondary antibodies used were goat anti-rabbit Alexa 488 Green and Alexa 594 Red (Cambridge Biosciences) . Images were acquired on a Zeiss LSM510 META confocal microscope (63x 1.4NA plan-apochromat oil immersion) at room temperature using Zeiss LSM510 v3.2 software.

Measurement of stored intracellular calcium Following 24 h treatment with 2.5 μM thapsigargin alone or in combination with 10 μM calpastatin or 50 μM ALLM (as previously described) , COS-7 cells were preincubated with 1 μM Fura2-AM and an equal volume of Pluronic F127 (both Molecular Probes) for 45 min at room temperature in HEPES-buffered saline (HBS) containing 2 mM CaC12 and stored on ice. Aliquots of cells (~5 x 105/run) were resuspended in nominally Ca2+- free HBS. Stored intracellular Ca2+ was released into the cytoplasm by addition of 10 μM ionomycin and quantified by monitoring Fura-2 fluorescence using a Cairn Spectrophotometer

as previously described (Melendez A. et al . J. Biol. Chem. 273: 9393-9402 (1998) ) .

RNA Interference

SMARTpool siRNA (pool of four different siRNA duplexes) against calpain 1 and calpain 2 (Dharmacon) were used for knockdown of the respective calpains . siGLO RISC-free siRNA (Dharmacon) , which is fluorescent and does not target any human or mouse genes, was used as a control. HeLa cells were transfected with siRNAs (200 nM per well) for 96h using Oligofectamine (Invitrogen) according to manufacturer's protocol. For experiment with pEGFP-LC3, HeLa cells were transfected with siGLO alone or in combination with calpain 1 siRNA or calpain 2 siRNA (total 320 nM per well in 1:3 ratio) for 96h followed by transfection with pEGFP-LC3 (lμg per well) using LipofectAMINE PLUS (Invitrogen) for 4h. Cells were fixed after 2h and analysed by fluorescence microscopy. For experiment with pEGFP-HDQ74, HeLa cells were transfected with pEGFP-HDQ74 (1.5μg per well) along with siGLO in presence or absence of calpain 1 siRNA or calpain 2 siRNA (1:3 ratio as above) for 96h using LipofectAMINE 2000 (Invitrogen) according to manufacturer's protocol. Cells were fixed after transfection and analysed by fluorescence microscopy. The total amount of siRNA in the above experiments (all in 12- wells plate) is identical in control (siGLO) and calpain knockdown cells.

Statistical analysis for aggregate formation, cell death and EGFP-LC3 vesicles

Approximately 200 EGFP-positive cells per sample were counted for the proportion of cells with green fluorescent EGFP-HDQ74 aggregates, as described previously (Narain et al . , 1999) . If an EGFP-positive cell has one or many aggregates, the aggregate score is 'one' . If an EGFP-positive cell does not

have any aggregate, the aggregate score is ^λ zero' . For example, the statement ^v calpastatin significantly reduced EGFP-HDQ74 aggregates' means that calpastatin significantly reduced the proportion of ξGFP-positive cells with EGFP-HDQ74 aggregates. Only EGFP-positive cells were counted so that we count only the transfected cells.

For EGFP-LC3 vesicles, we considered an EGFP-positive cell as having a score of 'zero' if there were 5 or fewer vesicles (as cells have basal levels of autophagy) and cells scored One' if they had >5 LC3-positive vesicles (Sarkar et al . , 2005) .

Fly culture Fly culture and crosses were carried out at 25°C and at 70% humidity, using Instant Fly Food (Philip Harris, Ashby de Ia Zouch, UK) unless otherwise stated. Flies were raised with a 12h light: 12h dark cycle. Aliquots of 0.5M verapamil hydrochloride (Sigma, Poole, UK) in DMSO, or DMSO alone were added to the water that was used to prepare the instant fly food.

Virgin female flies of the genotype y w; gmr-httNterm(l- 17DQ120 igmrQ120) (Jackson et al . , 1998) were allowed to mate with male flies from an isogenised w ¹¹¹⁸ stock (Ryder et al . , 2004) in food vials for 48h. Flies were then transferred to vials containing instant fly food containing either verapamil hydrochloride in DMSO or DMSO alone. Progeny were collected 0- 4h after eclosion, kept on food of the same composition as they had been reared on, and scored for photoreceptor degeneration using the pseudopupil technique (Franceschini, 1972) two days after eclosion.

We analysed toluidine blue-stained plastic sections of gmr- httQ120 Drosophila eyes and characterized the rhabdomere loss

in detail. Consistent with a previous report (Jackson et al . , 1998) , we observed loss of rhabdomeres followed by degeneration of the eyes, which manifested as structural disorganization but only subtle and low levels of photoreceptor loss (Berger et al . , 2006) . No loss of rhabdomeres is seen in either wild-type flies, or transgenic flies expressing otherwise identical huntingtin transgenes with 23 glutamines . Since we have observed some variability in the eye disorganization in Q120 flies and since it is difficult to quantify structural changes, we have used the pseudopupil technique as a quantifiable read-out. The loss of visible rhabdomeres in this model preceded photoreceptor death/loss assessed by toludine-blue staining of plastic sections and is a progressive degenerative phenotype seen only in flies expressing the mutant transgene (not wild-type) and was not present at eclosion (Jackson et al . , 1998) .

Pseudopupil analysis

For pseudopupil analysis, heads were removed from adult male flies and mounted on microscope slides using nail polish. The ommatidia were analyzed using a 10Ox objective and bright field optics with bright illumination, with the observer blinded to the identity of the flies (Franceschini, 1972) . Rhabdomere counts were carried out by analyzing 15 ommatidia per fly with around 20 flies per experiment for each treatment . The experiment was repeated on three independent occasions. Raw data from individual ommatidia were analyzed nonparametrically using a Mann-Whitney U test to determine significance levels, with the STATVIEW software, version 4.53 (Abacus Concepts) . A paired Student's t-test was used to compare the three experiment-average scores for each treatment .

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 (Wyttenbach A. et al . Hum. MoI. Genet. 10: 1829- 1845 (2001), Wyttenbach A. et al . Hum. MoI. Genet. 11: 1137- 1151 (2002)) . 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 on the immunoblots was done by Scion Image Beta 4.02 software (Scion Corporation) . ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, Non-significant . Densitometry analysis was shown relative to actin.

Results

Identification of Autophagy Promoting Compounds

250 drugs, selected on the basis of being either currently in use in man for other indications, or in clinical trials were screened for autophagy enhancers. Such well-characterised drugs do not require primary safety and pharmacokinetic testing.

The drugs were tested for enhancement of the clearance of A53T α-synuclein, a known autophagy substrate that is also cleared by proteasomes (Webb et al . , 2003) but not by chaperone- mediated autophagy (a pathway distinct from macroautophagy) (Cuervo et al . , 2004) . We used a stable doxycycline-inducible PC12 (neuronal precursor) cell line expressing A53T α- synuclein, where transgene expression is first induced by adding doxycycline and then switched off by removing doxycycline from the medium. If A53T α-synuclein levels are followed at various times after switching off expression, after an initial induction period, the effect of different

drugs on its clearance can be assessed (Webb et al . , 2003) . Using this paradigm, a number of compounds were identified that appeared to modulate A53T α-synuclein clearance, including known autophagy inducers like rapamycin and valproate (Sarkar et al . , 2005; Webb et al . , 2003).

5 L-type Ca ²⁺ channel antagonists were found to enhance A53T α- synuclein clearance, and Bay K [an L-type Ca ²⁺ channel agonist (Greenberg et al . , 1984)] had the reverse effect, we prioritised our validation studies on L-type Ca ²⁺ channel antagonists, focusing on 3 drugs that act at different sites on these receptors - verapamil, loperamide and nimodipine (Church et al . , 1994; Hockerman et al . , 1997; Sharp et al . , 1987; Striessnig et al . , 1987). Each of these drugs enhanced A53T α-synuclein clearance in multiple experiments whereas Bay K had opposite effects (Figure 1) .

These 3 drugs were tested in stable inducible PC12 cell models expressing a mutant fragment of huntingtin with 74 glutamines tagged to enhanced green fluorescent protein (EGFP-HDQ74) , which we have previously validated as an autophagy substrate (Ravikumar et al . , 2002) . Using a switch on/switch off paradigm similar to that described for A53T α-synuclein, we observed that these 3 different L-type Ca ²⁺ channel antagonists enhanced soluble EGFP-HDQ74 clearance and that Bay K had opposite effects (Figure 2) .

To test whether the L-type Ca ²⁺ channel antagonists were protective against polyQ toxicity caused by the mutant huntingtin fragment, we transfected SK-N-SH (neuronal precursor) cells with an EGFP HDQ74 construct. Expression levels of EGFP-HDQ74 correlates with aggregate formation (the proportion of transfected cells with aggregates) in such cell models (Narain et al . , 1999; Ravikumar et al . , 2002). Verapamil, loperamide and nimodipine significantly reduced

EGFP-HDQ74 aggregates and toxicity (Figure 3) whereas Bay K showed the opposite effects (Figure 4) .

Calpain inhibitors reduce mutant huntingtin fragment toxicity and facilitate its clearance

Calpains were activated with the endoplasmic reticulum Ca2+/Mg2+ ATPase inhibitor, thapsigargin, which increases intracellular Ca2+ levels (Thastrup 0. et al . Proc . Natl. Acad. Sci. USA 87: 2466- 2470 (1990)). The small calpain regulatory subunit is converted from a 28 kDa protein (Cp inactive) to a 21 kDa polypeptide (Cp active) upon activation with increased Ca2+ levels (reviewed in GoIl D. E. et al . Physiol. Rev. 83: 731-801 (2003)). The cells were pre-treated with calpain inhibitors or vehicle alone before adding thapsigargin.

Inhibition of calpain 1 and calpain 2 was inferred from the levels of the activated calpain small (regulatory) subunit. On this basis we used 10μM calpastatin, 50μM ALLM and 50μM calpeptin in subsequent experiments. Figure 5 shows the effect of different concentrations of calpain inhibitors on the activity of calpain. COS-7 cells were treated with DMSO (control) or 2.5 μM thapsigargin in presence or absence of 10 μM, 25 μM or 50 μM of calpastatin, ALLM or calpeptin for 30 min, and the activation or inhibition of calpain was analysed. The activation or inhibition of calpain was analysed by densitometry of the ratio of activated calpain small subunit to actin.

An EGFP-tagged huntingtin exon 1 fragment with 74 polyglutamine repeats (EGFP-HDQ74) in COS-7 (non-neuronal) and SK-N-SH (neural precursor) cells was used to test whether calpain inhibitors were protective against polyglutamine toxicity caused by a mutant huntingtin fragment, . Expression levels of EGFP-HDQ74 correlate with aggregate formation (the

proportion of transfected cells with aggregates) in such cell models (Narain Y. et al . J. Med. Genet. 36: 739-746 (1999); Ravikumar B. et al . Hum. MoI. Genet. 11: 1107-1117 (2002)). The calpain inhibitors, calpastatin, ALLM and calpeptin, significantly reduced aggregation and cell death in COS-7 (Figure 6) and SK-N-SH cells (Figure 7) caused by EGFP-HDQ74.

To investigate if the reduced aggregation of the huntingtin construct was due to enhanced clearance, we used a stable doxycycline-inducible PC12 cell line expressing EGFP-HDQ74, where transgene expression is first induced by adding doxycycline and then switched off by removing doxycycline from the medium. If the clearance of this construct is followed at various times after switching off expression after an initial induction period, the effect of specific agents on the clearance of the transgene product can be assessed, as its expression decays when synthesis is stopped (Ravikumar B. et al. Hum. MoI. Genet. 11: 1107-1117 (2002)). Calpain inhibitors were observed to enhance clearance of soluble EGFP- HDQ74 (Figures 8 and 9) . Furthermore, the calpain inhibitors enhanced the clearance of the insoluble mutant huntingtin that gets retarded in the stacking gel. Thus, calpain inhibitors were protective against toxicity causes by mutant huntingtin fragment by enhancing its clearance from the cells.

Calpain inhibition enhances clearance of aggregate-prone proteins by autophagy

Inhibitors of autophagy (3-MA) and the proteasome (lactacystin) were used to test whether the enhanced clearance of EGFP-HDQ74 mediated by the calpain inhibitors was through autophagy or the proteasomal route. We also used stable doxycycline-inducible PC12 cell line expressing the A53T mutant of α-synuclein (which causes autosomal dominant Parkinson's disease (Polymeropoulos M.H. et al . Science 276: 2045-2047 (1997) ) . A53T α-synuclein is a substrate for both

macroautophagy and the proteasome (Webb J. L. et al . J. Biol. Chem. 278: 25009-25013 (2003)), but is not cleared by a different pathway called chaperone-mediated autophagy (Cuervo, A.M. et al. Science 305: 1292-1295 (2004)). Both 3-MA and lactacystin increased EGFP-HDQ74 aggregates in COS-7 cells (Ravikumar B. et al . Hum. MoI. Genet. 11: 1107-1117 (2002)) (Figure 10) and impaired clearance of A53T α-synuclein in the PC12 stable line (Webb J. L. et al . J. Biol. Chem. 278: 25009- 25013 (2003) ) . Calpastatin (the most specific of the calpain inhibitors) enhanced A53T α-synuclein clearance (Figures 10 and 11) . When autophagy was constitutively inhibited by 3-MA, calpastatin could neither reduce EGFP-HDQ74 aggregates (Figures 12 to 14) , nor facilitate clearance of A53T α- synuclein (Figure 15) . However, cells treated with both calpastatin and lactacystin had significantly reduced EGFP-

HDQ74 aggregates (Figure 14) and enhanced clearance of A53T α- synuclein (Figure 15) , compared to cells treated with lactacystin alone, providing indication that calpain inhibition can enhance the clearance of these proteins even when the proteasome is inhibited. When 3-MA was used together with lactacystin, these beneficial effects of calpastatin were lost (Figures 14 and 15) . These data provide indication that calpastatin enhanced clearance of the aggregate-prone proteins like EGFP-HDQ74 and A53T α-synuclein through the autophagic route. Similar effects were seen with verapamil, where the reduction of EGFP-HDQ74 aggregates was also dependent on autophagy - 3-MA abolished the verapamil effect (Figure 16) , whereas in cells treated with both verapamil and lactacystin fewer aggregates were observed, compared to lactacystin alone (Figure 17) . Likewise, the effects of verapamil on A53T α- synuclein clearance were blocked by another autophagy inhibitor (bafilomycin Al) , while enhanced A53T α-synuclein clearance was seen with both verapamil and lactacystin, compared to lactacystin alone.

Autophagosome numbers were assessed using the microtubule- associated protein 1 light chain 3 (LC3) fused to EGFP (EGFP- LC3) (Mizushima, 2004) . LC3 is processed post-translationally into LC3-I, then converted to LC3-II, which associates with autophagosome membranes (Kabeya et al . , 2000) . Quantification of the number of cells with LC3-positive vesicles or LC3-II levels (versus actin) allows for a specific and sensitive assessment of autophagosome number in large numbers of cells (Mizushima, 2004) . As ξGFP-LC3 overexpression does not affect autophagic activity, numbers of EGFP-LC3 vesicles have frequently been used to assess autophagosome number (Mizushima et al . , 2004) . Calpastatin, ALLM and calpeptin increased the number of LC3-positive autophagic vesicles in COS-7 cells. Likewise, calpeptin increased autophagosome numbers in stable EGFP-LC3 HeLa cells (Bampton et al . , 2005). Similar trends were observed with ALLM and calpastatin. Calpeptin also increased the number of autophagosome-like structures and decreased the numbers of mitochondria [which are endogenous autophagy substrates (Klionsky and Emr, 2000)] in COS-7 cells as evaluated by electron microscopy (EM) (Figure 18A and 18B) .

LC3-II accumulates with increased autophagosome formation but also if there is impaired autophagosome-lysosome fusion. We assayed LC3-II in the presence of bafilomycin Al, which blocks autophagosome-lysosome fusion (Yamamoto et al . , 1998) . As expected, bafilomycin Al treatment increased the levels of LC3 II. To test if the calpain inhibitors induce autophagy, we pretreated EGFP-LC3 HeLa cells (Bampton et al . , 2005) with the inhibitors for 24h and then added bafilomycin Al to the cells for a further 4h. Calpastatin, ALLM and calpeptin resulted in increased LC3-II in the presence of bafilomycin Al, compared to bafilomycin Al alone, providing indication of the induction of autophagy. This result was confirmed in non-mitotic neuronally differentiated PC12 cells with calpeptin by analyzing the levels of endogenous LC3-II.

LC3-II levels were also increased by verapamil and loperamide in the presence of bafilomycin Al, compared to bafilomycin Al alone. However, Bay K did not affect LC3-II levels, providing indication that it probably inhibited autophagy at an early stage .

Activation of calpain inhibits clearance of aggregate-prone proteins Calpain activation with thapsigargin was observed to significantly increase EGFP-HDQ74 aggregates and cell death in COS-7 cells (Figure 19) and delayed A53T α-synuclein clearance (Figure 20) . Calpain activation did not inhibit proteasome activity, assessed with HeLa cells stably expressing the UbG76V-GFP reporter (Dantuma N. P. et al . Nat. Biotechnol. 18: 538-543 (2000) ) , a fluorescent specific proteasome substrate, in contrast to lactacystin. Consistent with these data, a constitutively active S50E human m-calpain (Glading et al . , 2004) increased EGFP-HDQ74 aggregation and toxicity in COS-7 cells (Figure 21) .

Cells were pre-treated with calpastatin or ALLM prior to thapsigargin treatment. Addition of calpain inhibitors prior to thapsigargin treatment in COS-7 cells alone, drastically reduced calpain activation in the presence of thapsigargin

(Figure 22) . Pre-treatment of thapsigargin-treated cells with calpastatin or ALLM reduced the proportion of EGFP-HDQ74- transfected COS-7 cells with aggregates and cell death (Figure 22) and enhanced the clearance of A53T α-synuclein in a stable inducible PC12 cell line (Figure 23) , compared to thapsigargin alone. Likewise, calpastatin and ALLM reduced aggregation and cell death in COS-7 cells co-transfected with EGFP-HDQ74 and constitutively active m-calpain constructs (Figure 24) . To verify that this effect of calpain inhibitors was due to increased autophagic activity, we transfected COS-7 cells with

LC3 fused to green fluorescent protein (GFP-LC3) . GFP-LC3 localizes only to autophagic membranes but not to other membrane structures (Kabeya Y. et al . EMBO J. 19: 5720-5728 (2000) ) . As it does not affect autophagic activity, numbers of GFP-LC3 vesicles have frequently been used to assess autophagic activity (Mizushima N. et al . MoI. Biol. Cell 15: 1101-1111 (2004)). COS-7 cells transfected with pEGFP-LC3 for 4 h were treated with 2.5 μM thapsigargin with or without 10 μM calpastatin or 50 μM ALLM for 2h, and analysed by fluorescence microscopy. Cells were pre-treated with calpain inhibitors for 15 min before thapsigargin addition. The effects of treatment on the percentage of EGFP positive cells with GFP-LC3 vesicles were expressed as odds ratios. Thapsigargin-treated cells pre-treated with calpastatin or ALLM showed significantly more GFP-LC3 vesicles than thapsigargin-treated cells only (Figures 25 and 26) . These effects were not due to caspase activation by thapsigargin, as the broad caspase inhibitor Z-VAD-FMK did not increase A53T α- synuclein clearance or the proportion of cells with EGFP-LC3 vesicles (Figures 27 and 28) . This is consistent with the decreased EGFP-HDQ74 aggregation and increased A53T a- synuclein clearance mediated by calpain inhibitors in thapsigargin-treated cells being largely due to enhanced autophagy.

Since the autophagy inhibitory effect of thapsigargin was rescued by calpain inhibitors, we assessed levels of stored intracellular Ca2+. COS-7 cells were treated for 24h with either DMSO or thapsigargin, in presence or absence of calpastatin or ALLM, and loaded with cytoplasmic Ca2+ indicator Fura2. Addition of ionomycin in the absence of extracellular Ca2+ permitted quantification of stored intracellular Ca2+. Thapsigargin treatment was found to reduce stored intracellular Ca2+, in comparison to DMSO-treated (control) cells (Figures 29 and 30) . Moreover, there were no

significant differences between the thapsigargin-treated cells with or without calpain inhibitors (Figure 30) , providing indication that the enhancement of autophagy by calpain inhibitors under these conditions is not mediated by effects on the levels of stored intracellular Ca ²⁺ .

L-type Ca ²⁺ channel antagonists are Calpain inhibitors We next addressed how the L-type Ca ²⁺ channel modulators influenced aggregate-prone protein clearance.

L-type Ca ²⁺ channel antagonists were found to reduce intra- cytosolic Ca ²⁺ levels, while Bay K increased Ca ²⁺ (figure 52) .

Bay K, which increased intracellular Ca ²⁺ levels, was found to activate calpains . Pre-treatment of cells with L-type Ca ²⁺ channel antagonists (verapamil or loperamide) or calpain inhibitors (ALLM) prevented calpain activation in the presence of Bay K. Moreover, treatment of Bay K-treated cells with verapamil or calpastatin significantly reduced the proportions of SK-N-SH cells with EGFP-HDQ74 aggregates and cell death

(Figures 31 and 32) , and significantly enhanced the clearance of A53T α-synuclein clearance (Figures 33 and 34) , compared to Bay K alone .

Induction of autophagy by calpain inhibition is mTOR- independent

The activity of mTOR, a protein kinase, can be inferred by the levels of phosphorylation of its substrates, ribosomal S6 protein kinase (S6K1, also known as p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1) at Thr389 and Thr37/46 respectively (Schmelzle and Hall, 2000) . While rapamycin (a specific mTOR inhibitor) reduced phosphorylation of S6K1 and 4E-BP1 in COS-7 cells as expected, calpastatin,

ALLM and calpeptin 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 (Manning B.D. et al . Trends Biochem. Sci. 28: 573-576 (2003)), markedly increased EGFP-HDQ74 aggregates and cell death in COS-7 cells (Ravikumar B. et al . Nat. Genet. 36: 585-595 (2004)) (confirmed in Figure 35). 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 48 h. However, all the calpain inhibitors reduced EGFP- HDQ74 aggregates and cell death in rheb-transfected cells (Figure 36) , providing indication that induction of autophagy by calpain inhibition occurs even when mTOR is activated.

Rapamycin significantly reduced EGFP-HDQ74 aggregation and cell death in COS-7 cells in presence of thapsigargin (Figure 37) , and also enhanced clearance of A53T α-synuclein in a PC12 stable cell line (Figure 38), compared to thapsigargin alone. This effect can be accounted for by increased autophagic activity due to mTOR inhibition, as rapamycin significantly increased GFP-LC3 vesicles in thapsigargin-treated COS-7 cells (Figure 39) . Rapamycin treatment was started 15 min prior to thapsigargin addition. These data also provide further indication that mTOR and calpains regulate autophagy via different apparently independent pathways .

Inhibition of calpain and mTOR has additive effects on the clearance of aggregate-prone proteins

Calpastatin and rapamycin were observed to have additive effects in reducing EGFP-HDQ74 aggregates and cell death in COS-7 cells, compared to the single treatments of calpastatin or rapamycin (Figures 40 and 41) . Furthermore, calpastatin and

rapamycin together significantly facilitated greater clearance of soluble EGFPHDQ74 at 48h (Figure 42) and A53T α-synuclein at 8h (Figure 43), compared to single either compound alone. In these experiments, we have chosen early time-points at which we do not yet observe obvious reductions of the levels of these proteins when the cells are treated with either of the compounds alone. It is important to note that 0.2μM rapamycin is as efficient at clearing EGFP-HDQ74 as 0.4μM rapamycin, providing indication that this dose is saturating. However, co-treatment with calpastatin and verapamil (both acting through the same pathway) did not facilitate any further clearance of the aggregate-prone proteins at this early time point .

Calpain knockdown induces autophagy and reduces mutant huntingtin toxicity siRNAs against human calpain 1 and calpain 2 were used to knockdown these respective calpains to study their effect on autophagy. HeLa cells were transfected with SMARTpool siRNA (pool of four different siRNA duplexes) against calpain 1 and calpain 2 respectively. We also used siGLO RISC-free siRNA as a control in this experiment, which does not target any human or mouse genes .

HeLa cells were transfected with siGLO with or without calpain 1 siRNA or calpain 2 siRNA (1:3 ratio) for 96 h, followed by transfection with pEGFP-LC3 for 4 h. Cells were fixed after a further 2 h and analysed by fluorescence microscopy. The percentage of EGFP- and siGLO-positive cells with GFP-LC3 vesicles were expressed as odds ratios.

Maximal knockdown of both the calpains was observed at 96h post-transfection. The level of knockdown of calpain 1 or calpain 2 was measured as a percentage relative to each other (Figures 44 and 45) . Calpain 1 showed >60% reduction in

protein levels while calpain 2 showed a reduction of >80%. To verify if calpain knockdown induces autophagy, we transfected HeLa cells with siGLO with or without siRNAs to calpain 1 or calpain 2 for 96h, followed by transfection with GFP-LC3 construct . siGLO-positive HeLa cells with either calpain 1 or calpain 2 knockdowns showed more GFP-LC3 vesicles, compared to siGLO-treated control cells without calpain siRNAs (Figure 46) . Thus, inhibition of either calpain 1 or capain 2 increased autophagic activity in mammalian cells.

Increased autophagic activity is due to calpain knockdown has a protective effect on mutant huntingtin fragment toxicity. HeLa cells were cotransfected with EGFP-HDQ74 and siGLO with or without siRNAs to calpain 1 or calpain 2 for 96h. Knockdown of either calpain 1 or calpain 2 reduced mutant huntingtin aggregates and cell death (Figures 47) .

Verapamil attenuated neurodegeneration in an Insect Model

The therapeutic potential of L-type Ca ²⁺ channel antagonists in vivo was tested using a Drosophila HD model expressing the first 171 residues of mutant huntingtin with 120 glutamines in photoreceptors, using the pseudopupil technique (justified in supplementary methods) . This method assesses the number of visible rhabdomeres by light microscopy and has been widely used to quantify the toxicity of proteins with long polyQs in the fly eye (Jackson et al . , 1998; Marsh and Thompson, 2004; Ravikumar et al . , 2004; Steffan et al . , 2001). Verapamil attenuated neurodegeneration in Drosophila expressing mutant huntingtin, compared to flies treated with the vehicle (DMSO) (Figures 48 to 51) .

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