WO2015004475A2 - Compositions and methods - Google Patents

Abstract

Compositions comprising an inhibitor of β-catenin for the treatment of spinal muscular atrophy (SMA) or a related neurological condition in a subject are presented, and methods of treating SMA using the same.

Description

Compositions and Methods

The present invention relates to compositions useful for the treatment of Spinal Muscular Atrophy (SMA), and to methods for their use. In addition, the present invention relates to methods of identifying agents capable of treating SMA.

Proximal spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality, with a carrier frequency of 1 :35 in the Caucasian population and an incidence of -1 :6,000-10,000 live births (Lefebvre et al., 1995; Wirth, 2000). This autosomal recessive neuromuscular disorder is most commonly caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene, leading to reduced levels of ubiquitously-expressed full-length SMN protein (Burghes and Beattie, 2009). SMA is primarily characterised by degeneration of lower motor neurons and atrophy of skeletal muscle (Burghes and Beattie, 2009; Mutsaers et al., 2011), with functional and structural disruption of synaptic connectivity at neuromuscular junctions (NMJ) and in the spinal cord occurring during the early stages of disease pathogenesis (Murray et al., 2008; Kariya et al., 2008; Ling et al., 2010; Mentis et al., 2011 ; Imlach et al., 2012). However, recent studies have revealed that SMA, particularly in its most severe forms, is a multi-system disorder (Hamilton & Gillingwater, 2013), with other regions of the nervous system also being affected (e.g. the hippocampus; Wishart et al., 2010), as well as non-neuronal tissues and organs, including; heart (Shababi et al., 2010), vasculature

(Somers et al., 2012), liver (Hua et al., 201 1), pancreas (Bowerman et al., 2012), lung and intestines (Schreml et al., 2012). This suggests that SMN deficiency causes perturbations across a wide range of cells and tissues in SMA, albeit with motor neurons being most vulnerable.

Despite a clear understanding of the genetic causes of SMA, the mechanisms linking low levels of SMN to disease pathogenesis remain unclear. SMN protein is known to play key roles in several core canonical cellular pathways, including snRNP biogenesis and pre- mRNA splicing (Burghes and Beattie, 2009). However, attempts to link disruptions in RNA processing directly to SMA pathogenesis have proven controversial (Zhang et al., 2008; Baumer et al., 2009; Lotti et al., 2012; Praveen et al., 2012). The finding that mutations in genes encoding proteins not directly involved in RNA processing can cause related diseases (UBA1 mutations cause X-linked infantile spinal muscular atrophy; Ramser et al., 2008), alongside reports linking SMN protein to actin dynamics (Oprea et al 2008), PTEN-mediated protein synthesis pathways (Ning et al., 2010), and translational regulation (Sanchez et al., 2012), suggests that SMN may fulfil additional roles downstream from, or outside of, RNA processing pathways in the nucleus and cell body of neurons. The identification of key

l pathways downstream from SMN that are critical for the regulation of neuromuscular and system-wide pathology in SMA will be important for designing, testing and evaluating new therapeutic approaches for human SMA patients. There is a thus a significant need for new and effective treatments for SMA.

Beta-catenin (or β-catenin) is a protein that in humans is encoded by the CTNNB1 gene. It is known to have several roles in mammalian cells, but there is currently no known role associated with SMA. Beta-catenin's activity is inhibited by quercetin.

Quercetin is a plant-derived flavonoid found in fruits, vegetables, leaves and grains. For example, quercetin is found in red wine, onions, green tea, apples, berries, Ginkgo biloba, St. John's Wort, and American elder. The lUPAC name for quercetin is 2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one. Quercetin is also known by the names: Sophoretin; Meletin; Quercetine; Xanthaurine; Quercetol; Quercitin; Quertine; and Flavin meletin.

Quercetin has been shown to have potential utility as an antiviral, for the treatment of cancer, to have a possible role in modulating inflammation, and has also been indicated as having an effect on metabolic rate in rats and on the production of fat cells in vitro.

Quercetin has been sold as a nutritional supplement and is a recognised antioxidant.

Various patents relating to the use of quercetin as a nutritional supplement have been filed, e.g. US6821536 and US 2004/0101595. However, to date, quercetin has not received regulatory approval as a medicament for any specific therapeutic indication, and there the literature is somewhat conflicting as to its efficacy in treating the above conditions.

WO02/43654 discloses a hypothetical role for quercetin in modulating astrocyte activity.

The present inventors have discovered that β-catenin plays a significant role in SMA pathogenesis and that the β-catenin inhibitor, quercetin, has a significant effect in reducing SMA pathology in vivo in animal models of SMA. Furthermore, they have elucidated details of the molecular pathways of SMA which explain this observed efficacy. Furthermore, based on this new understanding of the molecular mechanisms which lead to SMA, it is now possible to screen for active agents with potential utility in treating SMA.

Statements of the Invention According to a first aspect of the invention there is provided a composition comprising an inhibitor of β-catenin for the treatment of spinal muscular atrophy (SMA) or a related neurological condition in a subject. Particularly, but not exclusively, the present invention relates to the treatment of autosomal recessive proximal spinal muscular atrophy (known as 'standard' SMA). 'Standard' SMA is a disorder associated with a genetic mutation on the SMN1 gene on chromosome 5q (locus 5q13), affecting people of any age, but in its most severe form being the most common genetic cause of infant death.

Other SMA sub-types include: X-linked spinal muscular atrophy (XLSMA); Distal spinal muscular atrophy (DMSA) - also known as spinal muscular atrophy with respiratory distress type 1 (SMARD1) and hereditary motor neuropathy type 6 (HMN6); Autosomal dominant distal spinal muscular atrophy (ADSMA); Scapuloperoneal spinal muscular atrophy

(SPSMA); Juvenile asymmetric segmental spinal muscular atrophy (JASSMA); Spinal muscular atrophy with lower extremity predominance (SMA-LED); Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME); Spinal muscular atrophy with congenital bone fractures (SMA-CBF); and Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH).

The general term 'spinal muscular atrophy' or 'SMA' is intended to include all of the abovementioned SMA sub-types. Specific sub-types will be referred to where appropriate. As mentioned above, in a particularly significant embodiment of the present invention, the SMA condition to be treated is 'standard' SMA, which is by far the most common type of SMA. There are other forms of spinal muscular atrophies which typically result from mutations in genes other than SMN1 - known as localised spinal muscular atrophies.

However, these are very rare in comparison, with 'standard' SMA being responsible for 90- 95% of all cases of spinal muscular atrophies. The present invention is based upon the discovery that β-catenin is a central factor though which altered SMN activity exerts pathogenesis in SMA, especially in respect of 'standard' SMA. Thus the present invention is founded upon the understanding that β-catenin inhibitors are able to ameliorate SMA-associated pathogenesis. In addition to SMA, the present invention proposes that inhibitors of β-catenin can be used to treat neurological conditions related to SMA, for example: - Hereditary motor and sensory neuropathies (HMSN) - also known as Charcot-Marie- Tooth disease (CMT) and peroneal muscular atrophy (PMA);

- Spinal and bulbar muscular atrophy (SBMA);

- Peripheral neuropathies (e.g. diabetic neuropathy / HIV neuropathy); and

- Motor neuron disease (ALS etc.).

Known inhibitors if β-catenin include:

- Quercetin

- The compounds disclosed as inhibitors of β-catenin in "Inhibition of β-Catenin/Tcf

Signaling by Flavonoids", Seyeon Park and Jaemin Choi, Journal of Cellular

Biochemistry 1 10:1376-1385 (2010) (the contents of which are incorporated by reference), which include:

- Genistein, kaempferol, isorhamnentin, and baicalein.

- The compounds disclosed as inhibitors of β-catenin in "Wnt/beta-Catenin Signaling and Small Molecule Inhibitors" Current Pharmaceutical Design, 2013, 19, 634-664, (the contents of which are incorporated by reference), which include:

- PKF115-584, PNU-74654, PKF118-744, CGP049090, PKF118-310, ZTM000990, and BC21. It is preferred that inhibitors of the present invention are inhibitors of transcriptional activity of β-catenin, e.g. though interaction of β-catenin with the Tcf complex.

Preferably the β-catenin inhibitor is a flavonoid, or a derivative or variant thereof. More preferably the β-catenin inhibitor is a flavonol, or a derivative or variant thereof.

Flavonols are a class of flavonoids that have the 3-hydroxyflavone backbone (lUPAC name: 3-hydroxy-2-phenylchromen-4-one), shown below as Formula 1.

Formula 1

Diversity among the flavonols stems from the different positions and numbers of phenolic OH groups.

In a preferred embodiment of the present invention the β-catenin inhibitor is quercetin, or a derivative or variant thereof. Quercetin is a flavonol having the structure shown in Formula 2 below:

Formula 2

The term 'derivative' includes compounds which comprise the relevant flavonoid linked to another moiety. A derivative can be a glycoside. Quercetin, for example, is known in various forms linked to sugar moieties (glycones) to form glycosides, and such glycosides can be considered a type of 'quercetin derivative'. For example, the glycosides rutin and quercitrin are formed when quercetin is linked to rutinose and rhamnose, respectively. Other known glycosides of quercetin include spiraeoside, troxerutin, isoquercitin and hyperoside. Other derivatives included in the present application include various physiologically acceptable salts. In addition, quercetin is known to be sulphated to form quercetin 3-0- sulphate in human plasma. For example, quercetin 3-O-sulphate would appear to be the predominant, and possibly sole, quercetin metabolite in humans. Alkali metal salts of flavonols (including quercetin) are described in US2012/0183587, which show improved solubility and bioavailability compared to the native flavonol.

The term 'variant' includes entities which are derived or obtained from the flavanoid, which have a different chemical structure, but which retain similar, or have improved, biological activity or pharmacological properties. In particular, the relevant biological activities are the inhibition of β-catenin activity and resultant ability to ameliorate SMA pathogenesis in a subject. Relevant pharmacological activities include solubility and bioavailability. The invention thus extends to any quercetin derivative or variant which is able to exhibit the desired therapeutic effect in vivo, i.e. amelioration of SMA-associated pathogenesis through inhibition of β-catenin activity. Whether any such quercetin derivate exhibits the desired biological activity can be readily determined by the skilled person, using his normal skills, in combination with the various experimental protocols and animal models described below.

However, in a preferred embodiment the aglycone form of quercetin is used in the various aspects of the present invention. Myrestin and kaempferol are close structural relatives to quercetin, and therefore are likely to exhibit similar effects to quercetin in vivo, and thus provide alternative inhibitors for use in the present invention.

Suitably the treatment of SMA is the treatment of neuromuscular aspects of SMA

pathogenesis. Administration of a β-catenin inhibitor, quercetin, has been shown to have a particularly pronounced ability to ameliorate neuromuscular pathology in SMA. However, recent findings suggest that SMA is a complex condition, and pathogenesis in other tissues or organs is not significantly dictated by β-catenin levels or activity. Accordingly, in certain embodiments of the invention the β-catenin inhibitor can be administered together with another active to treat other aspects of SMA pathogenesis.

Preferably the subject is a human. However, the subject could be any mammal which is affected by SMA and would therefore benefit from treatment. The invention provides various actives formulated for pharmaceutical use, and optionally further comprising a pharmaceutically acceptable diluent, excipient and/or carrier.

The invention therefore includes pharmaceutical formulations which may include, in addition to the active ingredient, a pharmaceutically acceptable diluent, excipient and/or carrier. Such formulations may be used in the methods of the disclosure. Additionally or

alternatively, pharmaceutical formulations may include a buffer, stabiliser and/or other material 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 any suitable route, for example by oral administration, or via a parenteral route and particularly by infusion or injection (with or without a needle). The route of administration may be subcutaneous injection. The route of administration may be intravenous injection or infusion. Other routes of administration which may be used include administration by inhalation or intranasal administration. Compositions are provided that include one or more of the actives that are disclosed herein in a carrier. The compositions can be prepared in unit dosage form for administration to a subject. The amount and timing of administration are at the discretion of the treating physician to achieve the desired purposes. The active may be formulated for systemic or local administration. In one example, the active formulated for parenteral administration, such as subcutaneous or intravenous administration.

The composition of the present invention may be administered orally. An active may be administered orally in a liquid dosage form or a solid dosage form. Examples of solid dosage forms are tablets, capsules, granules, powders, beads and microcapsules. An active agent of the disclosure, with or without at least one additional therapeutic agents, that is administered in a solid dosage may be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. A solid oral dosage form may be designed to release the active portion of the formulation at the point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. At least one additional agent may be included to facilitate absorption of an active of the disclosure and/or any additional therapeutic agents. In such solid dosage forms, the active compound is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as lactose, sodium citrate or dicalcium phosphate and/or one or more: a) fillers or extenders for example starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders for example carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants for example glycerol; d) disintegrating agents for example agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents for example paraffin; f) absorption accelerators for example quaternary ammonium compounds; g) wetting agents for example cetyl alcohol and glycerol monostearate; h) absorbents for example kaolin and bentonite clay and i) lubricants for example talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or high molecular weight polyethylene glycol, for example. Appropriate dosages for oral administration can be determined by the skilled person though routine procedures. Typical dosage amounts are in the range of 0.1 to 1000 mg of active per subject per day. For parenteral administration, the active can be formulated as a solution, suspension, emulsion or lyophilized powder in association, or separately provided, with a

pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilised, e.g. by known or suitable techniques.

Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin,

hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods. Vehicles for injection can be a non-toxic diluting agent such as aqueous solution or a sterile injectable liquid. As the usable vehicle or solvent, water, Ringer's solution, isotonic saline, etc. are allowed; as a solvent or suspending liquid, a sterile nonvolatile oil can be used. For these purposes, any kind of non-volatile oil may be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthetic mono- or di- or tri-glycerides. The compositions for administration, therefore, can include a solution of the active dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilised by conventional, well known sterilisation techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and/or sodium lactate. The concentration of active in these formulations can vary widely, and may be selected based on fluid volumes, viscosities and/or body weight in accordance with the particular mode of administration selected and the subject's needs. A typical pharmaceutical composition for intravenous or subcutaneous administration includes about 0.1 to 1000 mg of active per subject per day, typically from 1 to 100 mg/kg body weight per day. Actual methods for preparing administrable compositions, whether for intravenous or subcutaneous administration or otherwise, will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).

The active may be provided in lyophilised form and rehydrated, e.g. with sterile water or saline, before administration, although actives may be provided in sterile solutions of known concentration. The active solution may then be added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight.

Amounts effective for therapeutic use, which may be a prophylactic use, will depend upon the severity of the disease and the general state of the patient's health. A therapeutically effective amount of the active is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. An active of the disclosure may be administered in conjunction with another active agent, whether simultaneously, separately or sequentially. The other active agent may be a second active agent of the invention or an active agent falling outside the invention.

Single or multiple administrations of the formulations of the disclosure are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of at least one of the actives disclosed herein to effectively treat the patient, bearing in mind though that it may not be possible to achieve effective treatment in every instance. The dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of treatment. The dose may be sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.

Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A. J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, Pa., (1995) incorporated herein by reference. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein as a core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μηι are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μηι so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μηι in diameter and are administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315- 339, (1992) both of which are incorporated herein by reference.

Polymers can be used for ion-controlled release of the active agents disclosed herein.

Various degradable and non-degradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has been shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxy apatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 1 12:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic

Publishing Co., Inc., Lancaster, Pa. (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Pat. No. 5,055,303; U.S. Pat. No.

5,188,837; U.S. Pat. No. 4,235,871 ; U.S. Pat. No. 4,501 ,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No.

5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268, 164; U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S. Pat. No. 5,271 ,961 ; U.S. Pat. No.

5,254,342 and U.S. Pat. No. 5,534,496).

According to a second aspect of the invention there is provided the use of an inhibitor of β- catenin, as set out above, in the manufacture of a medicament for the treatment of SMA. According to third aspect of the invention there is provided a method of treating SMA in subject, the method comprising administering a therapeutically effective amount of a composition comprising a β-catenin inhibitor, as set out above, to the subject. The term "treatment", and the therapies encompassed by this invention, include the following and combinations thereof: (1) inhibiting, e.g. delaying initiation and/or progression of, an event, state, disorder or condition, for example arresting, reducing or delaying the development of the event, state, disorder or condition, or a relapse thereof in case of maintenance treatment or secondary prophylaxis, or of at least one clinical or subclinical symptom thereof; (2) preventing or delaying the appearance of clinical symptoms of an event, state, disorder or condition developing in an animal (e.g. human) that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; and/or (3) relieving and/or curing an event, state, disorder or condition (e.g., causing regression of the event, state, disorder or condition or at least one of its clinical or subclinical symptoms, curing a patient or putting a patient into remission). The benefit to a patient to be treated may be either statistically significant or at least perceptible to the patient or to the physician. It will be understood that a medicament will not necessarily produce a clinical effect in each patient to whom it is administered; thus, in any individual patient or even in a particular patient population, a treatment may fail or be successful only in part, and the meanings of the terms "treatment", "prophylaxis" and "inhibitor" and of cognate terms are to be understood accordingly. The compositions and methods described herein are of use for therapy and/or prophylaxis of the mentioned conditions.

According to a fourth aspect of the invention there is provided a pharmaceutical composition comprising a β-catenin inhibitor, as set out above, and a pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition may be in unit dose form.

According to a fifth aspect of the invention there is provided a method of identifying an agent which is potentially useful for treating SMA comprising the steps:

a) proving β-catenin;

b) providing one or more agents;

c) bringing said one or more agents into proximity to β-catenin such that they can

interact with β-catenin;

d) detecting interaction between the agent of interest and β-catenin; and

e) selecting the agent if it interacts with β-catenin. Based upon the understanding that β-catenin is a key mediator in SMA pathogenesis, it is therefore appropriate to use β-catenin as a target in screening methods to identify agents which interact with it, and preferably which inhibit its activity. Such agents will have utility as leads for further development with the aim of delivering novel pharmaceuticals with the ability to modulate β-catenin activity in vivo, and thus potentially treat SMA.

Suitably the β-catenin is provided in solution, attached to a substrate or in/on a cell or tissue.

The agent of interest may be any agent, including, for example, a small molecule, peptide, protein or nucleic acid.

The method may further include determining whether the agent is an inhibitor of β-catenin activity. More preferably the method may involve whether the agent is an inhibitor of the induction of transcription via β-catenin/Tcf.

The method may be a high throughput (HT) screening method. The screening method may be solution-based or cell-based.

Known methodologies for screening agents of interest for interaction with a target are well known in the art. In particular, high throughput technologies are conventionally used in drug discovery processes. These would be well known to the skilled person. Exemplary reviews of HT methodologies include: Steven A Sundberg, 'High-throughput and ultra-high- throughput screening: solution- and cell-based approaches', Current Opinion in

Biotechnology 2000, 1 1 :47-53; K.P. Mishra, et al., Ά review of high throughput technology for the screening of natural products', Biomedicine & Pharmacotherapy, Volume 62, Issue 2, February 2008, Pages 94-98; and Sandra Fox, et al., 'High-Throughput Screening: Update on Practices and Success' J Biomol Screen 2006 11 :864-869.

"Inhibitor", as used herein, refer to a molecule that, when bound to a polypeptide, decreases the relevant biological activity of the polypeptide. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules that bind to the polypeptide. For example, an inhibitor may reduce the relevant activity of the polypeptide by 50% or more, more preferably 75% or more, more preferably 90% or more. The relevant inhibitory activity in the case of β-catenin inhibitors may be a reduction in the transcription of genes regulated by the β-catenin/Tcf complex. The effect on overall expression of a relevant gene will depend on whether β-catenin/Tcf is the primary or sole regulator of expression of the gene, or is one of many regulators; clearly even the complete inhibition of β-catenin will not result in complete elimination of expression where a gene's expression is regulated through one or more pathways. Accordingly, 100% inhibition would constitute and complete elimination of β-catenin regulated promotion of the relevant gene(s). Description of Embodiments of the Invention

The present invention will now be described in more detail, by way of example only, with reference to the accompanying figures: Figure 1. Perturbations in Uba1 levels and ubiquitin homeostasis in mouse and

Drosophila models of SMA. A/B - Bar charts (mean±SEM) showing significant reduction in levels of Uba1 protein in the spinal cord (A) and skeletal muscle (B) from severe SMA mice (SMA; Smn-/-;SMN2) at post-natal day 5, compared to littermate controls (CON;

Smn+/+;SMN2), quantified using fluorescent western blot (N=3 mice per genotype;

**P<0.01 , ***P<0.001 , unpaired two-tailed t test). C - Bar chart showing restoration of Uba1 protein levels in the spinal cord of severe SMA mice at P5 when treated daily from birth with the HDACi Trichostatin A (TSA; N=6 Control mice, N=5 SMA mice with DMSO, N=4 SMA mice with TSA; *P<0.05, **P<0.01 , ANOVA with Tukey's post-hoc test). D - Bar chart showing significant reduction in levels of Uba1 protein in the spinal cord from Taiwanese SMA mice at post-natal day 10, quantified using fluorescent western blot (N=3 mice per genotype; **P<0.01 , unpaired two-tailed t test). E - Representative fluorescent western blots showing reduced levels of both monomeric and multimeric ubiquitin in the central nervous system of Taiwanese SMA mice at post-natal day 10 compared to littermate controls (tubulin levels are shown as a loading control). F/G - Bar charts showing significant reductions in the level of both monomeric (F) and multimeric (G) ubiquitin in Taiwanese SMA mice at postnatal day 10 compared to littermate controls (*P<0.05, ***P<0.001 ; unpaired two-tailed t tests; N=3 mice per genotype). H - Representative confocal micrographs of striated muscle fibres from wild-type and SMA Drosophila larvae immunolabelled with an antibody which recognises mono- and polyubiquitinated proteins (stained with green label). Wild-type larvae revealed diffuse staining in muscle and muscle nuclei (stained with Hoechst blue). By contrast, SMN mutant larvae showed a distinct lack of staining in the nucleus, with increased staining in the peri-nuclear cytoplasm, suggesting that the distribution of ubiquitinated proteins is affected by loss of SMN. I - Bar chart showing no change in levels of Uba1 mRNA (or a control mRNA, Fth1; similar control data obtained for Mapt are not shown) in the spinal cord of P5 severe SMA mice compared to littermate controls, quantified using qPCR (N=3 mice per genotype; ns P>0.05, ANOVA with Tukey's post hoc test). J - Representative fluorescent western blots for SMN protein (left lane) and Uba1 protein (right lane) from co- immunoprecipitation experiments on spinal cord extracts from wild-type mice, using SMN- bound beads. Immunoblotting on the bound extract revealed that Uba1 had the ability to physically interact with SMN in vivo.

Figure 1a. SMN protein localizes to synaptic compartments of neurons in the central and peripheral nervous system in vivo. A - Representative confocal micrographs of NMJs in Drosophila larval muscles 6/7 from segments A3 or A4. Pan-neurally expressed YFP- tagged Drosophila SMN (YFP-dSMN; upper panels; stained with green label) was readily identifiable in the majority of pre-synaptic boutons (arrows; A). GFP fluorescence was not observed in boutons in control larvae expressing YFP-dSMN in muscle (lower panels), but was present in puncta within muscle nuclei (arrowheads; lower panels). NMJs were counter stained with anti-HRP (red) to enable visualisation of axons and with Hoechst to visualise nuclei. Scale bar = 10μηι. B - Representative fluorescent western blots showing the purity of synaptosome preparations from a wild-type mouse, and the localisation of SMN in both synaptic preparations without mitochondria and the synaptic mitochondria. The purity of the synaptosomes was demonstrated by the presence of a nuclear protein (Histone H2B) only in the non-synaptic fraction, with complete absence from the two synaptic fractions

(synaptosomes without mitochondria and synaptic mitochondria). Synaptophysin was used as a marker of synaptic vesicles and COX IV was used as a marker of mitochondria. Tubulin is shown as a loading control. C - Bar chart (mean±SEM) showing quantification of SMN protein levels in subcellular fractions (as shown in SMN bands in panel B; N=3 mice per fraction). Total protein loaded was 3C^g per lane. D - Bar chart showing relative expression levels of SMN protein in brain synaptosomes from wild-type mice (WT; Smrf^iSMN?^{9 9}), heterozygous 'US' SMA mice (Het; Smn^+/';SMN2f^{9 ta}) and homozygous 'US' SMA mice (KO; Smn^iSMN^⁹) at post-natal day 5, quantified using fluorescent western blot (N=3 mice per genotype). Note that SMN protein was still present, albeit at very low levels, in homozygous SMA mouse synapses, as a result of expression from the human SMN2 transgene. E - Representative bands from a standard western blot on whole brain synaptosomes from wild- type mice showing strong expression of SMN and gemin5. F - Representative bands from an IP experiment where whole synaptosome extracts were incubated with SMN, gemin5 or neurofilament (as a non-specific control) beads and bound proteins were eluted, subject to separation by SDS-PAGE and transferred to nitrocellulose by western blotting. The blot was developed with anti-SMN antibodies. All lanes loaded with SDS extracts of beads contain a 50kDa band of mouse Ig heavy chain which reacts with the HRP anti-mouse Ig used to develop the blot. Only the lanes with SMN or gemin5 beads bound SMN, showing that gemin5 and SMN retained the ability to physically interact in the synaptic proteome. G - Representative bands from a standard western blot on fractionated extracts obtained from wild-type mouse brain synaptosomes, revealing a matched pattern of enrichment of SMN and gemin5.

Figure 2. Genetic and pharmacological suppression of ubal in zebrafish leads to dose-dependent motor axon pathology. A - Representative fluorescence micrographs of motor axons growing out from the spinal cord in a control zebrafish 34 hours after fertilization (Control), and in animals injected with either 4ng or 6ng of a morpholino (MO) suppressing ubal levels (see Fig. 3a). Note how the gross development of motor neurons was not impaired in the MO-treated zebrafish. B - Representative higher-magnification confocal micrographs showing perturbations in motor axon morphology in MO-treated zebrafish. Note the large number of abnormal sprouts and axonal extensions in the MO-treated zebrafish. Scale bars = 50μηι. C - Bar chart (mean±SEM) showing levels of abnormal motor axon branching in MO-treated zebrafish. Only motor axons with modest (type 2; see Fig. 3a) or severely abnormal branching (type 3; see Fig. 3a), were quantified as having abnormal branching. Note the dose-dependent increase in the occurrence of abnormal branching (*** P<0.001 ; Kruskall-Wallis test with Dunn's post hoc test; uninjected, n=310 motor neurons, N=31 animals; 4ng, n=360, N=36 animals; 6ng, n=360, N=36 animals). D - Representative confocal micrographs showing perturbations in motor axon morphology in Tg(hb9:gfp) zebrafish embryos treated with 50μΜ of the Ubal inhibitor UBEI-41. Note the presence of a 'double exit' motor axon (right hand side of image) in the UBEI-41 example, with the axon branch emerging on the right side of the pair showing stunted outgrowth. Additional examples of disrupted axon branching and outgrowth in UBEI-41 treated animals are shown in Fig. 5A. Scale bars = 40μηι. E - Bar chart showing levels of abnormal motor axon branching and axon outgrowth in UBEI-41 treated zebrafish. Note the dose-dependent increase of numbers of aberrant motor axons in the UBEI-41 group compared to DMSO controls (*** P<0.001 ; 10μΜ UBEI-41 n=258 nerves, N=1 1 animals; 50μΜ UBEI-41 n=280 nerves N=12 animals; Kruskal Wallis test with Dunn's post hoc test).

Figure 2a. Confirmation of SMN expression in mouse brain synapses using a panel of 23 anti-SMN antibodies. Representative western blots for a panel of 23 SMN antibodies on extracts from HeLa cells (human origin; top strip), whole mouse cerebellum (not fractionated; middle strip) and whole mouse hippocampal synaptosomes (bottom strip). Lanes 1-12:

MANSMA1-MANSMA12; Lane 13: dilution buffer (no antibody); Lanes 14-23: MANSMA13- MANSMA22 (see methods for details of antibodies used). The blots have been purposefully overexposed in order to indicate presence or absence of signal. As expected, not all antibodies recognised mouse SMN. However, note how all of the antibodies that did detect SMN in mouse tissue (whole cerebellum) also detected SMN in synaptosome preparations from mouse brain (hippocampal synaptosomes). Figure 3. β-catenin is a downstream target of ubiquitin-dependent pathways in the neuromuscular system and accumulates in SMA. A - Representative fluorescent western blots showing levels of β-catenin (92 kDa) in the spinal cord of P5 severe SMA mice (KO), heterozygous SMA mice (Het), and control littermate mice (CON), alongside SMN protein levels (38 kDa) and a Tubulin loading control (50 kDa). Note how levels of β-catenin increased as levels of SMN decreased. B - Bar chart (mean±SEM) showing a significant increase in levels of β-catenin in the spinal cord of P10 Taiwanese SMA mice compared to littermate controls, quantified using fluorescent western blot (N=6 mice per genotype;

*P<0.05, unpaired two-tailed t test). C - Bar chart showing a significant reduction in levels of stabilized β-catenin (ABC) in the spinal cord of P10 Taiwanese SMA mice compared to littermate controls, quantified using fluorescent western blot (N=6 mice per genotype;

*P<0.05, unpaired two-tailed t test). D - Bar chart showing a significant reduction in levels of Tcf-4 protein, a key β-catenin interacting protein required for activation of downstream response genes, in the spinal cord of P10 Taiwanese SMA mice compared to littermate controls, quantified using fluorescent western blot (N=6 mice per genotype; **P<0.01 , unpaired two-tailed t test). E - Representative standard western blots showing levels of β- catenin and an actin loading control (42 kDa) in control zebrafish 48 hours post fertilization (Con), and in animals injected with either 4 ng or 6 ng of a morpholino (MO) suppressing ubal levels. Note the dose-dependent increase in levels of β-catenin in the MO-treated animals. F - Bar chart showing levels of β-catenin in primary cultures of rat hippocampal neurons and a motor neuron-like cell line (NSC-34) treated with 50 μΜ UBEI-41 (a specific Uba1 inhibitor) or DMSO control for 2 hours, quantified using fluorescent western blot (***p<0.001 , ANOVA with Tukey's post hoc test; N=12 coverslips DMSO, N=15 coverslips UBEI-41 for hippocampal neurons; N=19 coverslips DMSO, N=18 coverslips UBEI-41 for NSC-34 cells). UBEI-41 treatment led to a rapid and significant increase in β-catenin levels in both types of neuron, although motor neurons were significantly more susceptible than hippocampal neurons (**P<0.01 , ANOVA with Tukey's post hoc test). G - Representative fluorescent western blots for β-catenin (left panel - green bands) and ubiquitin (right panel - red bands) from co-immunoprecipitation experiments on synaptic extracts from wild-type mice. Immunoblotting on the bound extract revealed the presence of ubiquitinated catenin (right hand side - upper arrow) at the synapse.

Figure 3a. Knockdown of ubal protein expression in zebrafish and overview of methodology used for quantifying axon branching defects. A - Representative standard western blots showing reduced levels of ubal protein in zebrafish treated with either 4 ng or 6 ng ubal MO compared to controls. Actin is shown as a loading control. B - Representative fluorescence micrographs showing motor axons growing out from the zebrafish spinal cord, depicting categories used to quantify levels of abnormal motor axon branching. The upper two panels (no branching and minor, type 1 , branching) are taken from a control animal. The bottom two panels (showing moderate and severe branching phenotypes) are taken from a 6 ng MO-treated animal.

Figure 4. Rescue of u£>a7-dependent motor axon defects in zebrafish by

pharmacological inhibition of β-catenin signalling. A - Representative confocal micrographs showing three segments of Jg(hb9:gfp) zebrafish embryos from the trunk region. Note the severe branching phenotype of the motor nerves in the UBEI-41 treated animal. This phenotype was rescued by quercetin treatment. Scale bar = 30 μηι. B - Bar chart (mean±SEM) showing a dose dependent increase in numbers of aberrant motor axons in the UBEI-41 group compared to DMSO controls. This phenotype was rescued by quercetin in a dose dependent manner (*** P<0.001 ; DMSO controls n=331 nerves, N=14 animals; 10 μΜ UBEI-41 n=258 nerves, N=1 1 animals; 50 μΜ UBEI-41 n=280 nerves N=12 animals; 50 μΜ UBEI-41 + 25 μΜ Quercetin n=143 nerves, N=6 animals; 50 μΜ UBEI-41 + 50 μΜ Quercetin n=168 nerves, N=7 animals; Kruskal Wallis test with Dunn's post hoc test). C - Representative overview images showing body axis defects in zebrafish embryos after UBEI-41 treatment compared to DMSO control. Note the rescue of this gross phenotype following application of 50 μΜ quercetin.

Figure 5. Amelioration of neuromuscular pathology in zebrafish and Drosophila models of SMA following pharmacological inhibition of β-catenin signalling. A -

Representative confocal micrographs of motor axons growing out from the spinal cord in a SMA zebrafish 34 hours after fertilization (top panel) as well as a SMA zebrafish treated with 50μΜ quercetin (bottom panel). Note how the abnormal outgrowth and branching of motor axons in the SMA zebrafish and how these defects are absent in the SMA zebrafish treated with quercetin. B/C - Bar charts (mean±SEM) showing a significant improvement in the number of truncated motor axons (B) and abnormally branched motor axons (C) in SMA zebrafish treated with 50 μΜ quercetin (* P<0.05, ** P<0.01 , Kruskal Wallis test with Dunn's post hoc test; N=31 animals, control; N=32 animals SMA; N=30 animals SMA+50 μΜ quercetin). D - Representative confocal micrographs of neuromuscular junctions in wild-type Drosophila (w¹¹¹⁸), SMA Drosophila without quercetin and SMA Drosophila fed 50 μΜ quercetin. Neuromuscular junctions were stained with anti-HRP to visualise axons and anti- cysteine string protein to identify synaptic boutons. E/F - Feeding SMA Drosophila 50 μΜ quercetin restored bouton size (E) and rescued synaptic overgrowth (F) (N=8 larvae per treatment; ***P<0.001 ANOVA with Tukey's post test).

Figure 6. Amelioration of neuromuscular, but not systemic, pathology in SMA mice following pharmacological inhibition of β-catenin signalling. A - Bar chart (mean±SEM) showing a significant improvement in neuromuscular function (measured using the righting test) in symptomatic (P6) Taiwanese SMA (KO) mice treated with 10 mg/kg (daily i.p.

injection) quercetin from birth compared to SMA mice treated with DMSO alone. Note how quercetin treatment had no significant effect on neuromuscular function at pre-symptomatic stages (P3) (ns not significant, ** P<0.01 , *** P<0.001 , Kruskal Wallis test with Dunn's post hoc test; N=30 tests, Het DMSO P3; N=27 tests KO DMSO P3; N=21 tests KO quercetin P3; N=31 tests, Het DMSO P6; N=28 tests KO DMSO P6; N=27 tests KO quercetin P6). B - Representative micrographs of single teased skeletal muscle fibres from the levator auris longus muscle of a control (Het DMSO) and a Taiwanese SMA (KO DMSO) mouse at P10 following daily injection with DMSO, as well as from Taiwanese SMA mouse treated with 10 mg/kg quercetin (KO Quercetin). Note the smaller muscle fibre diameter and loss of clear striations in the Taiwanese SMA (KO DMSO) mouse compared to the control (Het DMSO), and how the treatment with 10 mg/kg quercetin restored muscle fibre diameter and striations (KO Quercetin). C - Bar chart showing significant amelioration of skeletal muscle fibre atrophy in SMA mice treated with daily injections of 10mg/kg quercetin from birth (* P<0.05, unpaired two-tailed t-test; N=8 muscles Het DMSO, N=9 muscles KO DMSO, N=4 muscles KO quercetin). D - Bar chart showing significant amelioration of neuromuscular junction denervation in SMA mice treated with daily injections of 10mg/kg quercetin from birth (* P<0.05, unpaired two-tailed t-test; N=8 muscles Het DMSO, N=8 muscles KO DMSO, N=4 muscles KO quercetin). E - Representative photographs of a Taiwanese SMA mouse at P10 (left panel) as well as a Taiwanese SMA mouse at P10 following daily treatment with 10 mg/kg quercetin (right panel). F - Survival curve for Taiwanese SMA mice treated with daily injections of DMSO (KO DMSO) or 10 mg/kg quercetin (KO Quercetin). There was no significant difference between the two groups (P=0.9897 Chi-square test; N=10 mice DMSO; N=13 mice quercetin). G - Representative photographs of hearts (top row) and livers (bottom row) from P10 control mice (Het DMSO) and Taiwanese SMA (KO DMSO) mice treated with daily injections of DMSO as well as P10 Taiwanese SMA mice treated with daily injections of 10mg/kg quercetin (KO quercetin). Note how the heart and liver were much smaller in the SMA mice (as previously reported in the literature), with no improvement following treatment with quercetin. H - Bar chart (mean±SEM) showing levels of β-catenin protein in the spinal cord, liver and heart of Taiwanese SMA mice at P10 (KO) compared to littermate controls (Het), quantified using fluorescent western blot. Note how levels of β- catenin were significantly increased in the spinal cord, but not in the liver or heart (N=3 mice per genotype; ***P<0.001 , ANOVA with Tukey's post-hoc test).

Figure 7. shows the molecular structure of quercetin.

Figure 8. Uba1 splicing is dysreguiated at late symptomatic time-points in SMA mouse spinal cord. A - Graphic overview of the exon structure of Uba1. Two Uba1 splice variants are generated with unique first exons. The position of primers used to amplify each splice variant is highlighted. Note that the coding sequence of Uba1 starts in exon 2. B-D - Bar charts showing relative expression levels of Uba la and Ubalb, as well as the ratio of Ubala to Uba lb, in SMA (Taiwanese') and control spinal cord at P3 (D; pre-symptomatic), P7 (E; early-symptomatic) and P1 1 (late-symptomatic) (N=3 mice per genotype, 3 independent amplifications per sample; two-tailed, unpaired t-tests). Uba1 splicing was significantly dysregulated in the late-symptomatic mice.

Figure 9. Decreased levels of Uba1 protein at pre/early- and late-symptomatic time points in SMA mice. A - Representative fluorescent Li-Cor western blots showing Uba1 and beta-tubulin (loading control) levels in synaptosome preparations from wild-type (WT), heterozygous SMA controls (Het) and homozygous 'severe' SMA (KO) mice at postnatal day 1 (pre/early-symptomatic). The reduced levels of Uba1 in the SMA mice validate the proteomics data where low levels of Uba1 were initially identified. B - Representative fluorescent Li-Cor western blots showing Uba1 and beta-tubulin (loading control) levels in the spinal cord from wild-type (WT) and homozygous 'severe' SMA (KO) mice at postnatal day 5 (late-symptomatic).

Figure 10. Pharmacological inhibition of β-catenin did not ameliorate the loss of body weight in SMA mice. No significant improvement in body weight at any age in 'Taiwanese' SMA mice treated with 10 mg/kg quercetin daily from birth.

Figure 11. Perturbations in Uba1 levels and ubiquitin homeostasis in mouse models of SMA. A/B - Uba1 (green) and NeuN (red) immunolabelling of motor neurons from 'Taiwanese' SMA and littermate control mouse spinal cords at P3 (A) and P7 (B). Note how Uba1 was predominantly cytoplasmic at P3 but nuclear at P7. C - Significant increase in the ratio of nuclear to cytoplasmic Uba1 in SMA motor neurons at P7 compared to littermate controls (N=24 motor neurons per genotype). D - Uba1 levels in whole spinal cord of

'Taiwanese' SMA mice remained unchanged at P3 and P7, but were significantly reduced by P10 (N>3 mice per time-point/genotype; ANOVA with Tukey's post-hoc test). E - Levels of Uba1 in skeletal muscle were significantly reduced at an early-symptomatic age (P7) in 'Taiwanese' SMA mice (N=4 mice per genotype).

Figure 12. β-catenin is a downstream target of Uba1 and accumulates in SMA. . A - Increased β-catenin protein in muscle biopsies from 3 human SMA patients (pooled data on right of dotted line). B - Increased β-catenin signalling activity in NSC-34 cells treated with 50 μΜ UBEI-41 measured with a luciferase reporter construct (N=3 coverslips per treatment). C - The majority of proteins modified in SMA synapses (66 out of 1 15 analysed; see Tables S1 &S2) are known β-catenin target genes identified by ChlP-Seq analyses. D - β-catenin was increased in P10 'Taiwanese' SMA mouse spinal cord (N=3 CON mice, N=4 SMA; unpaired two-tailed t-test). Figure 13. Uba1 levels were reduced in all tissues from SMA mice at P10 (N>3 mice per genotype; ANOVA with Tukey's post-hoc).

Figure 14. Amelioration of neuromuscular pathology in SMA mice following

pharmacological inhibition of β-catenin signalling. Quercetin treatment preserves motor neurons in the spinal cord of SMA mice. A and B show reduced motor neuron loss from spinal cord of quercetin-treated SMA mice at P10.

Investigations into the Molecular Mechanisms of SMA The studies described here were initiated to identify key molecular pathways acting downstream of SMN that are responsible for regulating neuromuscular and systemic pathology in SMA. Combining proteomic screens with cellular and molecular approaches in mouse, Drosophila and zebrafish models, we reveal a novel role for SMN in regulating ubiquitin homeostasis in vivo, likely mediated via physical interactions between SMN and key proteins involved in ubiquitination pathways. We demonstrate that both genetic and pharmacological targeting of one major E1 enzyme (ubiquitin-like modifier activating enzyme 1 ; Uba1) is sufficient to induce SMA-like neuromuscular pathology in zebrafish. We also identify perturbations in β-catenin signalling pathways as a major downstream consequence of disrupted ubiquitin homeostasis in SMA, and show that pharmacological suppression of β- catenin ameliorated neuromuscular pathology in zebrafish, Drosophila and mouse models of SMA. Surprisingly, however, we demonstrate that β-catenin-dependent pathways only contribute to SMN-dependent pathology in the neuromuscular system, with pathological events disrupting other tissues and organs in SMA occurring via β-catenin-independent pathways. Taken together, these data provide a direct link between SMN protein and the regulation of ubiquitin homeostasis and β-catenin signalling in the neuromuscular system.

Further, we demonstrate that distinct molecular pathways drive neuromuscular and systemic pathology in SMA. EXPERIMENTAL PROCEDURES

Mice

Breeding pairs of mice required to establish colonies of 'US' or 'severe' (Smn^iSMN?^{9 9}; Monani et al., 2000) and 'Taiwanese' {Smn^; SMN?⁹'⁰; Hsieh-Li et al 2000; Riessland et al., 2010) SMA mice, both on a congenic FVB background, were obtained from Jackson

Laboratories and maintained at the University of Edinburgh. Littermate animals either wild- type or heterozygous for Smn (Smn^+/+ or Smn^+/~) were used for controls. Mice were bred and sacrificed under license from the UK Home Office, and were genotyped using standard PCR methods (Murray et al., 2008; Riessland et al., 2010). For quercetin injection experiments, Taiwanese' mice and littermate controls were dosed daily with either 10 mg/kg quercetin (Sigma) or DMSO alone for a vehicle-only control via intra-peritoneal injection.

Skeletal muscle (quadriceps femoris) biopsy samples were obtained from two different biobanks in Italy (Fondazione IRCCS Istituto Neurologico 'C Besta' in Milan and Fondazione Ospediale Maggiore Polclinico Mangiagalli en Regina Elena, IRCCS in Milan) through EuroBioBank (http://www.eurobiobank.org/). Biopsies were obtained from three type I l/l 11 SMA patients (aged between 3 and 25 years old), with a confirmed homozygous deletion of the SMN1 gene confirming a genetic diagnosis of SMA (47). Three age-matched control samples were also obtained, genetically confirmed to have no mutations in the SMN1 gene. All required ethical approvals to acquire and distribute human patient tissue samples were obtained by the host biobanks. Tissue was provided in an anonymous fashion, with no identifying details apart from the age, gender and genetic status of the patient.

iTRAQ quantitative proteomics

iTRAQ proteomics was performed on freshly isolated synaptosome preparations from the hippocampus of P1 Smn^^SMN^⁹ ('US' SMA) and Smn^+/+;SMN2^m (littermate control) mice (N=9 mice per genotype), as previously described (Wishart et al., 2012). Samples were run in duplicate using the 4-plex system (114 &116 Smn^.SMN^⁹ and 1 15 & 117

Smn^iSMN?^{9 9}). Only proteins identified by 2 or more unique peptides were taken forward for subsequent analysis. A stringent cut off threshold of 20% change (increase or decrease) was used to identify proteins with modified expression. In silico functional pathways analyses were performed using Ingenuity Pathway Analysis software (Wishart et al., 2012).

Mouse western blot analyses

Quantitative Li-Cor fluorescent western blotting was performed as previously described (Mutsaers et al., 201 1) using primary antibodies detailed below.

Antibodies used for mouse western blot analyses

The following primary antibodies were used for quantitative fluorescent western blotting: SMN, synaptophysin - Santa Cruz; SMN, Beta catenin - BD Biosciences; βΙΙΙ tubulin, Uba1 - Abeam; Histone H2B - Active motif; Cox IV - Mitosciences; Ubiquitin (UBI 1) - Millipore. Standard western blotting was performed using the following primary antibodies: SMN: all MANSMA antibodies (Young et al., 2000), gemin5: GEM5M, GEM50, GEM5Q (Hao et al. 2007). All protein levels reported on graphs represent arbitrary fluorescence units.

Gemin5 immunoprecipitation experiments

Anti-mouse Pan Ig-coated magnetic beads (50μΙ) (Dynal, Oslo) were washed in 4% BSA / PBS and incubated for 30 minutes with a monoclonal SMN antibody (MANSMA12; 10Mg), GEM5M antibody against Gemin 5, or 150kDa neurofilament antibodies as a control. After washing, beads were incubated with synaptosome extract for 1 hour, followed by washing five times with PBS. Proteins bound to beads were eluted by boiling in 2 x SDS sample buffer (2% sodium dodecyl sulphate- SDS, 5% 2-mercaptoethanol, 62.5 mM Tris-HCI, pH 6.8) as previously described (Fuller et al., 2010).

Immunoprecipitation (IP) experiments

IP was performed as previously described (Hanafusa et al., 201 1). Briefly, protein was extracted in NP-40 lysis buffer (Novex, Life Technologies) containing 1 % protease inhibitor cocktail (Roche). Following centrifugation at 16000g the supernatant was added to dynabead protein G anti-mouse beads bound with pre-selected antibodies (beta-catenin, Uchl1 and Smn) and subjected to quantitative fluorescent immunoblotting. RNA extraction, qPCR and Uba 1 splicing assays

mRNA was extracted from synaptosomes using a RNeasy Microkit (Qiagen). Samples were checked for DNA contamination and concentration was determined using a nanodrop spectrophotometer (Themo Scientific). qRT-PCR was carried out using a Sybr-Green'1 step qRT - PCR kit' (Invitrogen) on a Model 7700 instrument (Applied Biosystems). The following primers were used:

Uba1 - Forward GAGCGGGGACTTTGTCTCCT (SEQ ID NO 1)

Uba1 - Reverse CTTTGACCTGACTGACGAT (SEQ ID NO 2)

Fth1 - Forward AATTTCTTGACCCACTGGTGCACT (SEQ ID NO 3)

Fth1 - Reverse TCGAATCGAGAGTAGTGGCACA (SEQ ID NO 4)

Mapt - Forward GATTCTCGTGAGGTTGACGACT (SEQ ID NO 5)

Mapt - Reverse TACGGACGAAGAAGCCGACATT (SEQ ID NO 6) mRNA was extracted from spinal cord using an RNeasy Microkit (Qiagen). Samples were checked for DNA contamination and concentration was determined using a nanodrop spectrophotometer (Thermo Scientific). RNA integrity was checked visually by resolution on agarose gels. cDNA was made from 2 μg RNA using the High Capacity cDNA Reverse Transcription kit (Invitrogen). Primers were designed that amplified the two mouse Uba1 transcripts, using a unique forward primer with a common reverse primer (Uba la - Forward GCTTGTCTCCAGAAGGAAGG (SEQ ID NO 7); Uba lb - Forward

CTTGACTTCGGCTCCTTGAG (SEQ ID NO 8); Uba1a/Uba1b - R

CACTGAGGACACTTCGGACA (SEQ ID NO 9)). Two mouse housekeeping genes (GAPDH and OAZ1) were used (GAPDH - Forward CGTCCCGTAGACAAAATGGT (SEQ ID NO 10); GAPDH - Reverse GAATTTGCCGTGAGTGGAGT (SEQ ID NO 11); OAZ1 - Forward ATCCTCAACAGCCACTGCTT (SEQ ID NO 12); OAZ1 - Reverse

CGGACCCAGGTTACTACAGC (SEQ ID NO 13)). For real-time detection an ABI7000 machine (Applied Biosystems) was used. cDNA was amplified using 0.5-1 μΜ primer with the DyNAmo Flash SYBR Green qPCR kit (Thermo Scientific) and using a standard PCR program with amplification at 60°C. Each cDNA sample was amplified in triplicate with all primer pairs. Experimental CT values for each sample were compared and sample representing CT minimum for each primer pair was identified. The equation where raw data = 1+E^A(Ctmin-Ct) was used to determine relative expression levels for each sample. Raw values obtained for Ubala and Uba lb were normalised using the geomean of the 2 housekeeping genes.

SMA Drosophila model experiments

To examine levels of mono- and poly-ubiquitinated proteins in SMA skeletal muscle we used wandering third instar larvae null for SMN (Chan et al., 2003). Ubiquitinated proteins were detected using an anti-mono- and poly-ubiquitinylated primary antibody (1 : 100; FK2, Enzo Life Sciences BML-PW8110). Nuclei were visualised using Hoechst (1 :500). For the quercetin rescue experiments, SMN or wildtype flies (w¹¹¹⁸) were maintained on a diet of Drosophila Quick Mix Medium (Blades Biological) containing 0-50 μΜ quercetin hydrate (Sigma-Aldrich). Third instar smn null larvae were dissected and fixed. NMJ were visualised using Dylight 649-conjugated anti-HRP (1/200, Jackson Immuno Research), and the presynaptic marker cysteine string protein (1/200, Developmental Studies Hybridoma Bank) was used to identify synaptic boutons. The average number of boutons (normalised to mucle area) per NMJ at muscle 6/7 was calculated. Maximum bouton diameters were averaged per NMJ at muscle 6/7.

Additional Drosophila experiments

To examine localization of SMN, pan-neural expression of UAS-YFP-dSMN was driven by Elav-Gal4^C155. MHC82-Gal4C crossed to UAS-YFP-dSMN flies were used as controls.

Wandering third instar larvae were dissected in PBS and fixed for 10mins in 4%

paraformaldehyde. GFP staining at NMJs was detected by blocking larval fillets in 3% BSA, 0.1 % Triton-X-100 in PBS overnight, incubating in primary antibody, 1 :400 (chicken anti- GFP, Invitrogen A10262), followed by secondary antibody, 1 :250 (goat anti-chicken Alexa Fluor 488, Invitrogen A11039). NMJs and motor neurons were visualised with Dylight 649- conjugated anti-HRP, 1 :200 (Jackson Immuno Research). Images were acquired on a Zeiss LSM510 Meta using a Pan Apochromat 63X 1.4 NA oil immersion objective.

UBEI-41 in vitro experiments

Primary rat hippocampal cultures were established from E18 Sprague-Dawley rat embryos as previously described (Bekkers and Stevens, 1989). NSC-34 cells (Cashman et al., 1992) were generated from existing stocks held at the University of Edinburgh. UBEI-41

(Biogenova), a cell-permeable Uba1 inhibitor with an IC50 ~ 5 μΜ, was added to the culture medium (50μΜ) for 2 hours, β-catenin levels (and β-ΙΙΙ tubulin loading control levels) were quantified using fluorescent western blotting (see above) with anti^-catenin (1 :1000, BD Transduction Laboratories) and anti-β III tubulin (1 :1600, Abeam) primary antibodies. To quantify β-catenin activity, NSC-34 cells were transiently transfected with a TOPflash reporter plasmid containing a luciferase reporter under the control of three copies of the TCF/LEF-binding element upstream of the thymidine kinase minimal reporter, specifically regulated by Wnt^-catenin signaling. NSC-34 cells were seeded in a 24-well plate and at 70-80% confluency, cells were transfected, in triplicate, with 350 ng TOPflash plasmid using Lipofectamine 2000 (Invitrogen). To control for transfection efficiency, 20 ng phTKRenilla luciferase plasmid was used. Cells were treated with 50 μΜ UBEI-41 28 h post-transfection. Transfected samples were analysed for firefly and renilla luciferase activities 30 h post- transfection using the Dual-Luciferase Reporter Assay System (Promega) and measured using a FLUOstar OPTIMA Microplate Reader (BMG LABTECH). All values were corrected to blank wells and normalized to expression from the phTKRenilla plasmid.

Zebrafish ubaland smn knock-down

An antisense Morpholino oligonucleotide (MO) was designed against the translational start codon of the uba l gene (Gene Tools, LLC): 5'-ACAGCGGCGAGCTGGACATCGTTTC-3' (SEQ ID NO 14). The previously published smn-MO was designed against the 5' start sequence of the smn gene (Gene Tools, LLC); 5 -CGACATCTTCTGCACCATTGGC-3' (SEQ ID NO 15) (McWhorter et al., 2003). Zebrafish embryos were injected between the one- and four-cell stage. For direct evaluation of motor axon phenotype, we used embryos obtained from crossing TL/EK wild-type and Tg(mnx:GFP)ml2 transgenic animals (Flanagan- Steet et al., 2005). Embryos were injected with either 4 or 6 ng of uba1-MO or 4 ng of smn- MO in aqueous solution containing 0.05% phenol red and 0.05% rhodamine-dextran. Six hours after injection embryos were sorted according to homogeneity of rhodamine fluorescence reflecting equal distribution of the injected MO solution. Quercetin (Sigma) treatment was performed at 6 hpf. In brief, quercetin solution (in DMSO) or DMSO (Sigma) was added to buffered embryo medium (final volume: 2 l_DMso ml_medium) to obtain a final concentration of 50 μΜ. See below for details of immunolabelling and microscopic analysis.

Western blotting on zebrafish tissue following uba 1 knock-down

Western blots for were performed using the following antibodies: anti-beta-actin (zebrafish) (rabbit polyclonal, 1 : 1 ,000, Anaspec); anti-Ubal (mouse monoclonal, 1 : 1 ,000, Santa Cruz); anti-rabbit-HRP (GE Healthcare, 1 : 10,000); anti-mouse-HRP (Sigma, 1 : 10,000); anti-beta- catenin (polyclonal rabbit, 1 :1 ,000, Sigma C2206).

Zebrafish immunohistochemistry

For immunolabelling, zebrafish were dechorionated and fixed in 4% paraformaldehyde at 34 hours post fertilization (hpf). After collagenase treatment (Sigman C-9891 ; 1 μg/ml) for 10 min at room temperature, embryos were blocked in PBST+1 %DMSO+10%FCS, followed by overnight incubation in 500μΙ blocking solution containing monoclonal mouse anti-zebrafish Synaptotagmin (znp-1) antibody (1 :300, Developmental Studies Hybridoma Bank). After washing fish were incubated in donkey anti-mouse secondary antibody labelled with

Alexafluor 488 (1 :200, Invitrogen). Microscopical analysis was performed in 80% glycerol on micro slides using an Axioskop 2 fluorescence microscope (Zeiss). The length of each of the first ten motor axons behind the yolk was analyzed and evaluated, as was the extent of abnormal branching (Fig. 3a).

Zebrafish UBEI-41 experiments

The drugs UBEI-41 (BioGenova), on its own or with quercetin (Sigma), were dissolved in DMSO and added at 6 hpf to Tg(mnx:GFP)ml2 embryos. The final amount of DMSO in the test groups was always 2 Ι/ml in buffered embryo medium. Western blots for were performed using the following antibodies: anti-beta-actin (zebrafish) (rabbit polyclonal, 1 :1 ,000, Anaspec); anti-Ubal (mouse monoclonal, 1 : 1 ,000, Santa Cruz); anti-rabbit-HRP (GE Healthcare, 1 : 10,000); anti-mouse-HRP (Sigma, 1 : 10,000); anti-beta-catenin (polyclonal rabbit, 1 :1 ,000, Sigma C2206). At 27 hpf, embryos were dechorionated, fixed in 4% PFA for 2 hours and mounted in 70% glycerol. Motor nerves were assessed for abnormal trajectories using a Zeiss Axioscope A1 fluorescence microscope and image stacks were taken using a Zeiss LSM710. Per embryo, 24 axons in 12 segments were analyzed. The observer was always blinded to the treatment.

SMA mouse quercetin experiments Taiwanese' mice and littermate controls were dosed daily with either 10 mg/kg quercetin (Sigma) or DMSO alone for a vehicle-only control via intra-peritoneal injection. Mice were randomly assigned to treatment groups. Righting reflex tests were performed in order to assess neuromuscular function, as previously described (Butchbach et al., 2007). Muscle fibre diameter measurements were taken from phase contrast micrographs of teased muscle fibre preparations using Image J software, as previously described (Mutsaers et al., 201 1). Neuromuscular junction pathology was assessed on whole-mount preparations of levator auris longus muscles, as described previously (Comley et al., 2011). Kaplan-Meier survival analyses were performed on DMSO and quercetin treated mice as previous described (Lee et al., 2012). Organs were dissected and either fixed in 4% paraformaldehyde overnight for subsequent imaging, or were prepared for quantitative western blotting (see main methods section).

Statistical analysis

All statistical analyses were completed using GraphPad Prism software. P values <0.05 were considered to be statistically significant for all analyses (*P<0.05; **P<0.01 ;

***P<0.001).

RESULTS

Widespread perturbations of ubiquitin homeostasis in SMA

In order to identify novel molecular pathways underlying SMN-dependent pathology in SMA, we initially performed an unbiased iTRAQ comparative proteomics experiment to quantify alterations in the molecular composition of synapses isolated from the hippocampus of pre/early-symptomatic (P1) 'US' SMA mice {Smn^iSMN?^{9 9}) compared to littermate controls (Smn^iSMN?^{9 9}; n=9 per genotype). We chose to use hippocampal synaptosomes for these experiments due to the known susceptibility of the hippocampus in mouse models of SMA (Wishart et al., 2010), with isolated synaptic preparations used in order to provide data on molecular events occurring in cytoplasmic/synaptic compartments of neurons targeted during the early stages of disease (Ling et al., 2010; Mentis et al., 201 1). Biochemical analysis of isolated synaptic preparations from mice confirmed the presence of SMN protein and its known interacting proteins (e.g. gemin5) in the synaptic cytoplasm (Fig. 1A-2A). SMN protein was also present in synaptic boutons at the Drosophila NMJ (Fig. 1A-2A), confirming the relevance of using synaptic preparations for a proteomics screen.

The iTRAQ screen revealed that the synaptic proteome was robustly disrupted in SMA mice, where 52 out of 150 (35%) unique synaptic proteins identified in the proteomics analysis had expression levels modified >20% (Tables 1 to 3). Functional clustering analyses, using IPA software (see methods), linked these changes with significant modifications in a range of core functional pathways (Table 1). Interestingly, we identified significant disruption of protein ubiquitination pathways in SMA mice, with modified expression levels of ubiquitin-like modifier activating enzyme 1 (Uba1) and ubiquitin carboxyl-terminal esterase L1 (Uchl 1) (Table 1 -3). Ubiquitination pathways, and Uba1 in particular, were of interest in the context of SMA because mutations in the gene coding for human UBA 1 (UBE1) are sufficient to cause a genetically-distinct form of the disease, known as X-linked infantile spinal muscular atrophy (Ramser et al., 2008). Moreover, ubiquitination pathways are known to regulate axonal and synaptic stability (Korhonen and Lindholm, 2004), as well as the stability and degradation of SMN protein itself (Chang et al. , 2004; Burnett et al. , 2009; Hsu et al. , 2010).

Table 1 : Clustering analysis of proteomics data revealing functional pathways modified in P1 SMA mouse hippocampus

Table 2: Proteins with increased expression >20% in synapses from SMA mice vs littermate controls

IPI00123494.3 Psmd2 26S proteasome non- 168 108430 3 0.03 1.93 ATPase regulatory subunit 2

IPI00407692.3 Atp6v1 a Isoform 1 of V-type 122 73812 3 0.04 1.77

proton ATPase catalytic

subunit A

IPI00323179.3 Gdi1 Rab GDP dissociation 396 55382 8 0.18 1.73

inhibitor alpha

IPI00313962.3 Uchl1 Ubiquitin carboxyl- 296 27614 11 0.24 1.67

terminal hydrolase isozyme L1

IPI00312527.4 Crmpl Crmpl protein 205 79818 7 0.12 1.65

IPI00555069.3 Pgk1 Phosphoglycerate kinase 112 50973 3 0.13 1.64

1

IPI00222430.5 Dbi acyl-CoA-binding protein 122 17726 3 0.18 1.62

isoform 1

IPI00321 190.1 Psap Sulfated glycoprotein 1 193 68883 5 0.09 1.60

IPI00221402.7 Aldoa Fructose-bisphosphate 77 43678 2 0.07 1.59

aldolase A

IPI001 17896.3 Maprel Microtubule-associated 69 33627 2 0.09 1.59

protein RP/EB family member

1

IPI00407130.4 Pkm2 Isoform M2 of Pyruvate 158 63854 7 0.15 1.55

kinase isozymes M1/M2

IPI00120030.1 Crym Mu-crystallin homolog 86 36123 2 0.09 1.54

IPI00127987.1 Arpd a Actin-related protein 76 45628 2 0.07 1.52

2/3 complex subunit 1 A

IPI001 191 13.3 Atp6v1 b2 V-type proton 299 60171 6 0.05 1.47

ATPase subunit B, brain

isoform

IPI00462072.3 Eno1 ;Gm5506 Alpha-enolase 146 52497 5 0.26 1.46

IPI001 18899.1 Actn4 Alpha-actinin-4 83 1 13581 3 0.06 1.46

IPI00230707.6 Ywhag 14-3-3 protein gamma 925 31050 29 0.79 1.45

IPI00129685.3 Tpt1 Translationally-controlled 155 21725 4 0.15 1.42

tumor protein

IPI00133903.1 Hspa9 Stress-70 protein, 82 81549 2 0.04 1.42

mitochondrial

IPI001 13141 .1 Cs Citrate synthase, 774 56023 20 0.18 1.42

mitochondrial

IPI0028101 1 .7 MarcksH MARCKS-related 176 22948 5 0.3 1.40

protein

IPI001 18821 .2 Pafah1 b2 Platelet-activating 136 27898 3 0.1 1 1.36

factor acetylhydrolase IB

subunit beta

IPI00624192.3 Dpysl5 Dihydropyrimidinase- 207 66659 8 0.15 1.34

related protein 5

IPI001 19762.4 Dclk1 Doublecortin-like protein 150 65687 3 0.05 1.34

IPI00330804.4 Hsp90aa1 Heat shock protein 145 96806 4 0.07 1.32

HSP 90-alpha

IPI001 10684.1 Ppa1 Inorganic 91 37425 3 0.08 1.29

pyrophosphatase

IPI00229080.7 Hsp90ab1 Putative 134 94523 5 0.1 1.28

uncharacterized protein

IPI001 10753.1 Tubal a Tubulin alpha-1A chain 5994 53670 160 3.63 1.26

IPI001 16283.1 Cct3 T-complex protein 1 43 66349 2 0.1 1.25

subunit gamma IPI001 17348.4 Tubal b Tubulin alpha-1 B chain 5770 53686 153 3.13 1.25

IPI00405986.3 Epb4.111 Erythrocyte protein 81 127928 2 0.02 1.24

band 4.1 -like 1

IPI001 18986.1 Atp5o;LOC100047429 ATP 64 26576 2 0.12 1.22

synthase subunit O,

mitochondrial

IPI00330754.1 Bdh1 D-beta-hydroxybutyrate 51 42061 2 0.07 1.21

dehydrogenase, mitochondrial

Table 3: Proteins with decreased expression >20% in synapses from SMA mice vs littermate controls

In order to establish whether this disruption of ubiquitin-dependent pathways extended across all cells and tissues in the neuromuscular system, Uba1 protein levels were examined in preparations of spinal cord (Fig. 1A) and skeletal muscle (Fig. 1 B) from 'US' 'severe' SMA mice. Uba1 protein levels were reduced -50% in SMA mouse spinal cord and >60% in skeletal muscle (gastrocnemius), compared to littermate controls.

Levels of Uba1 were also significantly reduced in spinal cords from 'Taiwanese' SMA mice (Fig. 1 D; Smn-/-;SMN2tg/0 mice carrying two SMN2 copies on one allele on a null murine Smn background; Riessland et al., 2010), which display a slightly milder phenotype than 'US' SMA mice (mean survival of -10-11 days compared to -5-6 days). Treatment with the HDAC inhibitor trichostatin A (TSA) robustly elevates SMN protein levels in the spinal cord, resulting in amelioration of neuromuscular and CNS synaptic pathology in SMA mice (Mentis et al., 201 1 ; Ling et al., 2012). We therefore used TSA treatment to establish whether perturbations in Uba1 were directly correlated with SMN levels and therefore amenable to pharmacological intervention. Severe SMA mice and littermate controls were treated with either TSA (10 mg/kg daily) or a vehicle only control (DMSO) between P1 and P5. SMN protein levels were elevated -40% in the spinal cord of treated mice at P5 (data not shown). Strikingly, levels of Uba1 were also significantly increased in TSA-treated SMA mice, almost returning to levels found in littermate controls (Fig. 1C). Thus, the perturbations in Uba1 observed in SMA mice were highly sensitive to SMN levels and could also be reversed in post-natal mice.

Given that Uba1 regulates the first stages of an enzymatic cascade in which ubiquitin molecules are ultimately conjugated to target proteins, disruption of Uba1 (alongside concomitant changes in other ubiquitin-related proteins such as Uchl1) would be predicted to affect ubiquitin homeostasis. To examine whether this was occurring in SMA, we measured levels of monomeric and multimeric ubiquitin in the spinal cord from Taiwanese SMA mice. Levels of both monomeric and multimeric ubiquitin were significantly decreased in SMA mice compared to littermate controls (Fig. 1 E-G). SMN-dependent perturbations in ubiquitin homeostasis were conserved across species, as a dramatic redistribution of mono- and polyubiquitinated proteins was observed in striated muscle from an established Drosophila model of SMA (Fig. 1 H; Chan et al., 2003).

In order to investigate potential pathways through which SMN may regulate Uba1 and ubiquitin homeostasis, we first investigated whether low levels of SMN resulted in modified expression of Uba1 mRNA. Interestingly, mRNA levels for Uba1 remained unchanged in the spinal cord of SMA mice (Fig. 11). It was unlikely, therefore, that Uba1 protein levels were reduced in SMA as a result of global deficiencies in transcription of Uba1 mRNA. Given that SMN protein has been shown to interact with other proteins in vitro, including members of the ubiquitin-proteasome system such as Uchl1 (Burnett et al., 2009; Hsu et al., 2010), we next wanted to determine whether SMN could be influencing Uba1 levels as a result of direct physical interactions in vivo. We therefore performed co-immunoprecipitation experiments on spinal cord extracts from wild-type mice, using anti-SMN beads. Immunoblotting on the bound extract with antibodies against Uba1 revealed that Uba1 had the ability to physically interact with SMN in vivo (Fig. 1J). To determine the time-course of Uba1 changes in SMA, we examined Uba1 expression in Taiwanese' SMA mice. We examined Uba1 protein expression in motor neuron cell soma located in the ventral horn of spinal cord at pre- symptomatic (P3) and early-symptomatic (P7) stages of disease (Figure 11A-C). In control mice, Uba1 was almost exclusively localised to the neuronal cytoplasm at P3, before undergoing a dramatic sub-cellular relocalisation to the nucleus by P7 (Figure 11A-B). There was no overt difference in Uba1 localisation in SMA mice at P3. However, at P7 we noted an almost complete absence of Uba1 from the motor neuron cytoplasm in SMA mice, when low levels persisted in littermate control mice (Figure 11 B). Quantification of the ratio of nuclearcytoplasmic Uba1 levels in SMA mice revealed a significant reduction in the cytoplasm compared to controls (Figure 11 C). Thus, redistribution of Uba1 occurring during the early postnatal period was perturbed in SMA mice at early-symptomatic stages of the disease. Interestingly, quantification of total Uba1 levels in spinal cord did not reveal a significant reduction in SMA mice until late- symptomatic stages (P10; Figure 11 D), suggesting that subtle changes in the sub-cellular distribution of Uba1 were perturbed in motor neurons in advance of a widespread reduction in protein levels throughout the spinal cord. Comparable examination of Uba1 levels in whole muscle showed a significant loss by P7 in SMA (Figure 11 E), suggesting that muscle may be more severely affected than spinal cord.

Taken together, these data demonstrate that SMN protein plays a key role in maintaining Uba1 levels and ubiquitin homeostasis throughout the nervous system and neuromuscular system, disruption of which appears to be a significant feature of SMA pathology.

SMN-Uba 1 interactions and dys regulation of Uba 1 splicing in SMA

To investigate potential pathways through which Uba1 and ubiquitin homeostasis are targeted in SMA, we first asked whether changes were occurring due to reduced expression of Uba1 mRNA. As mRNA levels for Uba1 remained unchanged in the spinal cord of SMA mice (Figure 11), it was unlikely that Uba1 protein levels were reduced as a result of global deficiencies in transcription of Uba1 mRNA. Given that SMN protein interacts with other proteins in vitro, including members of the ubiquitin-proteasome system such as Uchl1 (Burnett et al., 2009; Hsu et al., 2010), we next wanted to determine whether SMN could be influencing Uba1 levels as a result of direct physical interactions in vivo. We therefore performed co-immunoprecipitation experiments on spinal cord extracts from wild-type mice, using anti-SMN beads. Immunoblotting on the bound extract with antibodies against Uba1 revealed that Uba1 and SMN physically interact in the neuronal cytoplasm in vivo (Figure 1J).

Several recent studies have also implicated SMN in the regulation of splicing, therefore we next analysed splicing patterns for Uba1 in the spinal cord of Taiwanese' SMA mice at pre- symptomatic (P3), early-symptomatic (P7) and late-symptomatic (P10) time-points (Figure 8A-D). Two Uba1 splice variants are generated with unique first exons (Ubala and Ubal b; Figure 8A). Quantification of relative expression levels of Ubal a and Ubal b in SMA and control spinal cord at P3 and P7 showed no significant differences between the genotypes (Figure 8B-C). In contrast, there was a significant reduction in relative expression levels of Ubal a (but not Ubal b) and a significant alteration in the ratio of Ubal a to Ubal b in SMA mice at P10 (Figure 8D). Thus, dysregulation of Uba1 splicing may contribute to the alterations in Uba1 protein levels observed in SMA, with the disruption in splicing in spinal cord temporally correlating with the reduction in Uba1 protein levels in spinal cord (see Figure 11 D). Taken together with data showing Uba1 sub-cellular redistribution and Uba1- SMN interaction, these findings suggest that SMN-dependent regulation of Uba1 and its disruption in SMA is a complex process, with combinatorial effects of disruption to multiple regulatory pathways likely to be responsible. Suppression of Uba 1 is sufficient to induce motor neuron pathology in vivo

Although suppression of Uba1 expression and disruption of ubiquitin homeostasis was a robust correlate of neuromuscular pathology in SMA models, it remained unclear whether these changes directly contributed to disease pathogenesis. To examine this possibility, we designed experiments suppressing uba l in an established zebrafish model system previously shown to be ideal for assessing the effects of genetic manipulations on motor neuron stability (McWhorter et al., 2003; Oprea et al., 2008). Uba1 protein expression was targeted using an antisense Morpholino oligonucleotide (MO) designed against the translational start codon of the uba l gene. Embryos were injected with either 4ng or 6ng of MO and examined 34 hours after fertilization. Levels of ubal protein were robustly reduced in fish treated at both doses (Fig. 3aA). Suppression of ubal using MO had no effect on the gross development of motor neurons in the spinal cord (Fig. 2A). By contrast, uba l suppression led to a profound, dose-dependent disruption of motor axon

outgrowth/branching. Whereas the vast majority of axons in control animals showed a simple, unbranched morphology, axons in both 4 ng and 6 ng MO-injected animals revealed grossly abnormal branching patterns (Fig. 2A-B). The percentage of motor axons with branching abnormalities (see Fig. 3aB) increased from <5% in controls to -50% in 4 ng MO animals and -75% in 6 ng MO animals (Fig. 2C; P<0.001 for both 4 ng and 6 ng MO compared to controls; Kruskall-Wallis test with Dunn's post-hoc test; control, n=310 axons; 4 ng, n=360; 6 ng, n=360). Importantly, these findings phenocopied branching defects previously reported in zebrafish models of SMA (McWhorter et al., 2003; Oprea et al., 2008). In order to confirm these findings using a pharmacological approach to suppress Uba1 , we next treated zebrafish with UBEI-41 , a cell-permeable Uba1 inhibitor (Balut et al., 2011). Pharmacological inhibition of Uba1 revealed a similar, dose-dependent disruption of motor axon branching to that described above in our MO experiments (Fig. 2D-E). At a dose of 10 μΜ UBEI-41 , the majority of motor nerve abnormalities were due to aberrant axonal branching (data not shown). At 50 μΜ, however, half of the motor nerve abnormalities observed (47.2%) were a result of aberrant nerve branching, with the remaining motor nerves revealing a more severe phenotype: truncated or missing motor axons (Fig. 2D). Once again, these pathological events phenocopied motor axon defects observed in zebrafish models of SMA (McWhorter et al., 2003; Oprea et al., 2008). Taken together with the data from MO experiments, these findings confirm that suppression of uba l is sufficient to cause SMA-like motor neuron pathology in vivo. In addition to the observed motor axon defects, ubal suppression led to body axis defects in zebrafish, both following UBEI-41 treatment (see Figure 4) and following MO treatment (data not shown). However, the viability of the fish was not compromised as there was not a significant increase in the numbers of fish dying after ubal suppression.

SMN-dependent suppression of Uba l leads to accumulation οίβ-catenin

Given that Ubal is a key component of ubiquitination pathways, through which substrate proteins are 'tagged' for targeting to the proteasome, we next wanted to identify specific substrate proteins affected as a downstream consequence of the disruption of ubiquitin homeostasis in SMA. Perturbations in ubiquitin pathways are often associated with a failure to degrade target proteins, resulting in a characteristic accumulation within the cell. Reexamination of our original proteomics dataset revealed one notable protein accumulation: β- catenin levels were robustly increased by more than 400% in synapses from SMA mice compared to littermate controls (Table 1-3). Examination of β-catenin levels in freshly- prepared synaptosomes from control, heterozygous and homozygous 'US' SMA mice validated the original proteomics data, revealing that increased levels of β-catenin corresponded to reduced levels of SMN protein in a dose-dependent manner (Fig. 3A). Similarly, β-catenin levels were significantly increased in the spinal cord from 'Taiwanese' SMA mice (Fig. 3B). Alongside changes in β-catenin levels, levels of stabilised β-catenin (ABC) were reduced in the spinal cord from 'Taiwanese' SMA mice (Fig. 3C). Similarly, levels of transcription factor 4 (Tcf-4), a key β-catenin interacting protein required for activation of downstream response genes (Cadigan, 2012), were significantly reduced in the spinal cord from Taiwanese' SMA mice (Fig. 3D). Importantly, β-catenin protein levels in muscle biopsy samples from SMA patients suggested that elevated levels were also a major feature of neuromuscular pathology in human patients (Figure 12A)

To confirm that increased levels of β-catenin occurred downstream of perturbations in Uba1 , we first examined global β-catenin levels in zebrafish treated with 4ng or 6ng MO against ubal. β-catenin levels were increased in a dose-dependent manner, reaching -250% of control levels in zebrafish treated with 6ng MO against uba l (Fig. 3E). To further confirm a link between Ubal levels and the regulation of β-catenin in neurons, cultures of primary hippocampal neurons were exposed to the Ubal inhibitor UBEI-41 for 2 hours. UBEI-41 treatment led to a rapid and significant increase in levels of β-catenin (-50%; Fig. 3F).

Similar experiments using an NSC-34 motor neuron-like cell line (Cashman et al., 1992) revealed an even more robust increase in β-catenin levels (-90%; Fig. 3F) suggesting that motor neurons were particularly susceptible to suppression of Ubal To establish whether increased levels of β-catenin protein led to a corresponding increase in β-catenin signalling activity, we performed luciferase activity reporter experiments, β-catenin activity was significantly increased in NSC-34 cells treated with UBEI-41 for 2 hours (Figure 12B). In order to ascertain whether such changes in β-catenin signalling activity could explain changes in levels of other proteins found to be altered in SMA synaptosomes, we compared our proteomics dataset to published β-catenin ChlP-Seq data revealing potential

downstream targets of β-catenin. This analysis revealed that the majority of proteins modified in SMA synapses (66 out of 115 analysed) were also present in the ChlP-seq data (Figure 12C), indicative of them being putative β-catenin targets.

Although β-catenin is a known target of the ubiquitin-proteasome system (Aberle et al., 1997), we next wanted to establish whether β-catenin is ubiquitinated specifically in neurons. We therefore performed immunoprecipitation experiments on protein isolated from synaptosomes generated from wild-type mice (as in our initial proteomics screen where increased levels of β-catenin were identified) using either β-catenin beads or pan-ubiquitin beads. Western blotting with antibodies against β-catenin and ubiquitin demonstrated that ubiquitinated forms of β-catenin were present in neurons and their synaptic compartments (Fig. 3G).

Inhibition οίβ-οθίβηίη signalling reverses Ubal -dependent destabilization of motor neurons To test whether the Uba1 -dependent effects on motor nerve branching we observed previously were mediated by downstream effects on β-catenin signalling pathways, we exposed zebrafish embryos treated with the cell-permeable Uba1 inhibitor UBEI-41 to quercetin, a plant-derived flavonoid that robustly inhibits β-catenin signalling pathways by disrupting transcriptional activity of the β-catenin-Tcf complex (Park et al., 2005; Gelebart et al., 2008; Asuthkar et al., 2012). Treatment of fish with motor neuron defects resulting from the addition of 50μΜ UBEI-41 with 25μΜ or 50μΜ quercetin revealed a statistically- significant, dose-dependent rescue of the motor axon branching phenotype (Fig. 4A,B). Strikingly, in the fish treated with 50μΜ quercetin, the number of abnormal motor nerves was reduced to that observed in control fish not exposed to the Uba1 inhibitor. Treatment with 50 μΜ quercetin also ameliorated motor axon branching defects in zebrafish treated with morpholinos against ubal (data not shown). Thus, motor nerve abnormalities induced by inhibition of ubal in zebrafish were fully rescued by simultaneous inhibition of β-catenin signalling. In addition to the motor nerve disruptions observed, we also noted that exposure to UBEI-41 caused dose-dependent alteration of the gross morphology of zebrafish embryos, manifesting as a bent body axis (Fig. 4C). These deficits were also corrected by treatment with quercetin (Fig. 4C).

Inhibition οίβ-catenin signalling ameliorates neuromuscular pathology in zebrafish and Drosophila models of SMA

Given that pharmacological inhibition of β-catenin signalling with quercetin was sufficient to block motor axon defects resulting from targeting of Ubal , we next wanted to know whether pharmacological inhibition of β-catenin signalling would have similar beneficial effects on neuromuscular pathology in animal models of SMA. First, we treated SMA model zebrafish (McWhorter et al., 2003) with 50 μΜ quercetin. Quercetin treatment robustly and significantly reduced the incidence of both truncated motor axons and motor axon branching defects in SMA fish (Fig. 5A-C). Similarly, quercetin treatment reversed morphological defects associated with the neuromuscular junction in a dose-dependent manner in an established Drosophila model of SMA (Chan et al., 2003) (Fig. 5D-F). Thus, pharmacological inhibition of β-catenin signalling with quercetin ameliorated key markers of neuromuscular pathology in two distinct animal models of SMA.

Inhibition offi-catenin signalling ameliorates neuromuscular pathology, but not systemic, pathology in 'Taiwanese' SMA mice

Finally, we wanted to establish whether pharmacological inhibition of β-catenin signalling with quercetin would have any effect on the disease phenotype in a mouse model of SMA. Taiwanese' SMA mice and littermate controls were treated with 10 mg/kg quercetin daily (i.p. injection) from birth. A parallel group of mice received injections of DMSO vehicle only. Treatment with 10 mg/kg quercetin had no effect on the health or weight of healthy littermate controls (data not shown - an increased dose of 50 mg/kg quercetin was found to have toxic effects after several days of administration) and had no significant detrimental effect on neuromuscular function in SMA mice at pre-symptomatic ages (P3; Fig. 6A). By contrast, quercetin treatment significantly improved the performance of symptomatic (P6) SMA mice a behavioural test of neuromuscular function (the righting test; Fig. 6A). Quercetin treatment increased and restored muscle fibre diameters of SMA mice to the same size as littermate control mice at late-symptomatic stages (P1 1 ; Fig. 6B-C) and also ameliorated

neuromuscular junction pathology, restoring the average number of axonal inputs in SMA mice to the same levels observed in littermate controls (Fig. 6D). Furthermore, quercetin treatment ameliorated motor neuron cell body loss from the spinal cord (Figure 14A-B). Thus, as in zebrafish and Drosophila models, pharmacological inhibition of β-catenin signalling with quercetin ameliorated neuromuscular dysfunction and pathology in SMA mice.

Although quercetin treated mice often appeared much healthier than their DMSO-treated counterparts at late symptomatic stages (Fig. 6E), treatment with quercetin did not increase survival (Fig. 6F) or average body weight (Fig 10). Examination of quercetin-treated SMA mice post-mortem suggested death was still occurring due to widespread organ defects (Fig. 6G), and SMA systemic pathology is known to target organs including the heart and liver. The contribution of non-neuromuscular, systemic pathology, affecting tissues and organs such as the heart, liver, lungs, intestine and vasculature, has only recently been fully appreciated in SMA animal models (Hamilton & Gillingwater, 2013). As a result, several studies have demonstrated that a failure to target peripheral tissues and organs minimises the effectiveness of therapeutics (Hua et al., 201 1 ; SchremI et al., 2012). In order to examine the possible causes of quercetin failing to ameliorate non-neuromuscular, systemic pathology (even when administered systemically via i.p. injection), we quantified Uba1 and β-catenin levels in the heart and liver from SMA mice. Levels of Uba1 in the heart and liver were reduced in SMA mice to a similar level observed in spinal cord (Figure 13).

Surprisingly, β-catenin levels remained unchanged in heart and liver (Figure 6H). Thus, perturbations in β-catenin signalling pathways occurring downstream of disrupted ubiquitin homeostasis were restricted to the neuromuscular system, thereby revealing distinct mechanisms driving pathology in the neuromuscular system compared to other tissue and organ systems in SMA. This suggests that distinct mechanisms drive pathology in the neuromuscular system compared to other tissue and organ systems in SMA and may explain, at least in part, why pharmacological inhibition of β-catenin selectively ameliorated neuromuscular pathology in SMA mice. DISCUSSION

The studies described here were initiated to identify pathways downstream of SMN that contribute to SMA pathogenesis. Using rodent, Drosophila and zebrafish models we have revealed a novel role for SMN in regulating ubiquitin homeostasis, mediated largely via interactions with Ubal Suppression of Uba1 in SMA is likely to result from a complex series of events, including disruptions in both splicing of Uba1 mRNA and physical interactions between SMN and Uba1 protein in the cytoplasm. Targeting of Uba1 expression was sufficient to generate motor neuron pathology in vivo, phenocopying the severe axonal defects previously reported in zebrafish models of SMA. We have identified β-catenin as a downstream target of Uba1/ubiquitination pathways in the neuromuscular system, with disruption of β-catenin pathways in SMA. Pharmacological inhibition of β-catenin signalling using quercetin ameliorated neuromuscular pathology in zebrafish, Drosophila and mouse models of SMA. Surprisingly, we also found that disruption of β-catenin was restricted to the neuromuscular system, and was not responsible for regulating SMN-dependent pathology in other tissues and organs. Our findings provide experimental evidence directly linking SMN protein to the regulation of ubiquitin homeostasis and β-catenin signalling in the

neuromuscular system and also reveal fundamental molecular differences between pathways underlying neuromuscular and systemic pathology in SMA.

Although it is known that SMN protein interacts with the ubiquitin-proteasome system in order to regulate its own stability (Chang et al., 2004; Burnett et al., 2009; Hsu et al., 2010), our study significantly extends our understanding of the importance of these interactions to include a direct role for ubiquitin homeostasis in regulating SMA disease pathogenesis. When taken together with human genetic data showing that mutations in UBA1 cause similar pathological changes to those found in SMN-dependent SMA (Ramser et al., 2008), our findings suggest that perturbations in ubiquitin homeostasis, and Uba1 in particular, may represent a common molecular pathway underlying neuromuscular pathology across genetically-distinct forms of the disease. Moreover, the finding that perturbations in ubiquitin homeostasis in response to SMN deficiency, as well as dysregulation of β-catenin downstream of perturbations in Uba1 , are evolutionarily conserved between mouse, zebrafish and Drosophila models, suggests that regulation of ubiquitin homeostasis represents a key biological role for SMN. Our finding that levels of Uba1 mRNA remained unchanged in SMA mouse spinal cord, when Uba1 protein levels were concomitantly reduced by -50%, suggests that SMN is not regulating Uba1 protein levels simply by targeting global Uba1 transcription. In contrast, our biochemical evidence showing interactions between Uba1 and SMN in vivo suggests that SMN likely regulates Uba1 levels through direct physical interaction at the protein level. Similar direct interactions between SMN and proteins belonging to ubiquitin pathways have previously been shown for Uchl1 (Hsu et al., 2010).

Our finding that loss of Uba1 protein in SMA likely resulted from perturbations to both Uba1 mRNA mis-splicing and disruption to physical interactions between SMN and Uba1 , as well as modifications to normal postnatal sub-cellular redistribution of the protein, suggests that molecular pathways responsible for controlling Uba1 levels in vivo are complex and multi- faceted. However, the demonstration of interactions between Uba1 and SMN provides additional evidence to support the hypothesis that interactions between SMN and ubiquitination pathways are key for the normal form and function of the neuromuscular system, not only with regards to the regulation of SMN protein stability, but also with respect to modulating ubiquitin homeostasis and cell viability.

Our identification of β-catenin as a key downstream target of ubiquitin pathways disrupted in SMA provides mechanistic insights into the pathways through which defects in ubiquitin homeostasis are transferred into pathological changes in the neuromuscular system.

Although these pathways have not previously been linked to neuromuscular pathology in SMA, β-catenin signalling pathways are known to play an important role in regulating motor neuron differentiation and stability, including regulating synaptic structure and function (Murase et al., 2002; Li et al., 2008; Ojeda et al., 201 1). Interestingly, Li and colleagues showed that motor neuron differentiation was regulated by retrograde signalling through β- catenin from skeletal muscle (Li et al., 2008). Our demonstration of robust amelioration of neuromuscular pathology in zebrafish, Drosophila and mouse models of SMA treated with quercetin highlights the fact that β-catenin pathways in the neuromuscular system are amenable to pharmacological targeting in vivo. Thus, targeting β-catenin signalling pathways during the early stages of disease may represent an attractive therapeutic option for stabilizing the neuromuscular system in SMA, and possibly also related conditions.

Given that β-catenin is such a well-established target for the ubiquitin-proteasome system, and the magnitude of changes in β-catenin pathways observed in the neuromuscular system in SMA mice, it was surprising to find that quercetin treatment did not target systemic pathology in SMA. Our finding that β-catenin signalling pathways remained stable in tissues and organs outside the neuromuscular system explains the quercetin result, but also served to highlight an underappreciated complexity in molecular pathways underlying disease pathogenesis in SMA, implicating both β-catenin-dependent and β-catenin-independent pathways. These findings add significant additional support to the hypothesis that SMA is a multi-system disorder (Hamilton & Gillingwater, 2013), adding a layer of complexity with regards to distinct molecular pathways driving pathology in different tissues. Moreover, they further highlight the likely requirement to deliver therapeutics targeting either the SMN1 or SMN2 gene systemically in order to fully rescue SMA symptoms (Hua et al., 201 1).

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Claims

Claims

1. A composition comprising an inhibitor of β-catenin for the treatment of spinal muscular atrophy (SMA) or a related neurological condition in a subject.

2. A composition according to claim 1 for the treatment of autosomal recessive proximal spinal muscular atrophy.

3. A composition according to claim 1 for the treatment of at least one of: X-linked spinal muscular atrophy (XLSMA); Distal spinal muscular atrophy (DMSA); Autosomal dominant distal spinal muscular atrophy (ADSMA); Scapuloperoneal spinal muscular atrophy (SPSMA); Juvenile asymmetric segmental spinal muscular atrophy (JASSMA); Spinal muscular atrophy with lower extremity predominance (SMA-LED); Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME); Spinal muscular atrophy with congenital bone fractures (SMA-CBF); and Spinal muscular atrophy with pontocerebellar hypoplasia (SMA-PCH).

4. The composition claim 1 wherein the composition is for the treatment of a neurological condition related to SMA.

5. The composition of claim 4, wherein the neurological condition is at least one of:

- Hereditary motor and sensory neuropathies (HMSN) - also known as Charcot- Marie-Tooth disease (CMT) and peroneal muscular atrophy (PMA);

- Spinal and bulbar muscular atrophy (SBMA);

- Peripheral neuropathies; and

- Motor neuron disease.

6. A composition according to any preceding claim, wherein the inhibitor of β-catenin inhibits the transcriptional activity of β-catenin, preferably wherein the inhibitor of β- catenin inhibits the transcriptional activity by disrupting the interaction of β-catenin with the Tcf complex.

7. A composition according to any preceding claim, wherein the inhibitor of β-catenin is a flavonoid, or a derivative or variant thereof.

8. A composition according to claim 7, wherein the inhibitor of β-catenin is a flavonol, or a derivative or variant thereof.

9. A composition according to claim 8, wherein the inhibitor of β-catenin is quercetin, or a derivative or variant thereof.

10. A composition according to claim 9, wherein the inhibitor of β-catenin is an aglycone form of quercetin.

11. A composition according to claim 1 , wherein the inhibitor of β-catenin is one or more of: rutin, quercitrin, spiraeoside, troxerutin, isoquercitin, hyperoside, quercetin 3-0- sulphate, myrestin, kaempferol, genistein, isorhamnentin, baicalein, PKF1 15-584, PNU-74654, PKF1 18-744, CGP049090, PKF118-310, ZTM000990, and BC21.

12. A composition for use according to any preceding claim, wherein the subject is a

human.

13. The use of an inhibitor of β-catenin as set out in any preceding claim in the

manufacture of a medicament for the treatment of SMA.

14. A method of treating SMA in a subject, the method comprising administering a

therapeutically effective amount of a composition comprising a β-catenin inhibitor according to any one of claims 1 to 11 to the subject.

15. A pharmaceutical composition comprising a β-catenin inhibitor according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier or excipient.

16. A pharmaceutical composition according to claim 15 in unit dose form.

17. A method of identifying an agent which is potentially useful for treating SMA

comprising the steps:

- providing β-catenin;

- providing one or more agents;

- bringing said one or more agents into proximity to the β-catenin such that they can interact with β-catenin;

- detecting interaction between the agent of interest and β-catenin; and - selecting the agent if it interacts with β-catenin.

18. A method according to claim 17, wherein it is determined whether a selected agent is an inhibitor of β-catenin activity.

19. A method according to either one of claims 17 or 18, wherein the one or more agents may be a small molecule, peptide or nucleic acid.

20. A method according to any one of claims 17 to 19, wherein the β-catenin is provided in solution, attached to a substrate or in/on a cell or tissue.