The present invention relates to an Nmnat2 modulator useful as a neuroprotective medicament in the treatment of neurodegenerative disorders, in particular but not exclusively disorders involving Wallerian degeneration of neuronal tissue, to th e u se of N m n at2 as a b i o m a rker fo r Wa l l e ri a n degeneration, to a method of demonstrating Wallerian degeneration using an

Nmnat2-based biomarker, to a diagnostic kit for detecting Wallerian degeneration, to a method of screening for an Nmnat2 modulator, and to an Nmnat2 modulator identified using the aforementioned screening method.

Neurodegenerative diseases are characterised by a loss of viable nerve cells from either the peripheral or the central nervous system. In many cases this loss has been shown to be preceded by degeneration of the neuronal axon, which is invariably more pronounced at the distal rather than the proximal end of axonal processes. There are two models which attempt to explain this greater degree of distal axonal degeneration . The first is 'dying back' in which degeneration spreads retrog radely from the nerve terminals. The second is Wal lerian degeneration where degeneration spreads from the site of a lesion in either direction according to the lesion type; this ultimately results in loss of the axon distal to the lesion site, leaving the proximal portion intact. Although strictly speaking Wallerian degeneration only occurs in response to physical injury of the axon, similar mechanisms operate in diseases where no such injury has occurred. The latter is referred to as ^λ Wallerian-like' degeneration. Both types of degeneration will hereinafter be jointly referred to as 'Wallerian degeneration'.

The recently discovered WIdS mouse has led to progress in the understanding of these two processes. In these animals, Wallerian degeneration occurs at a rate roughly ten times slower than in wild-type animals. Studies have shown that this mutation also delays pathologies believed to involve 'dying back' of axonal terminals. The WIdS gene therefore provides a mechanistic link between the two models of axonal degeneration. Despite the identification and characterisation of the WIdS gene, progress towards understanding of the molecular trigger for Wallerian degeneration has been limited. Knowledge of this trigger could have a profound impact on the understanding of the early stages of ^λ dying-back' neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis and Huntington's disease. It could also substantially improve the prospects for treating conditions involving axonal death caused by neuronal injury such as multiple sclerosis, traumatic brain injury and spinal cord injury.

According to a first aspect of the invention there is provided an Nmnat2 modulator for use as a neuroprotective medicament in the treatment of a neurodegenerative disorder.

According to another aspect of the invention there is provided an Nmnat2 modulator for use as a neuroprotective medicament in the treatment of a neurodegenerative disorder involving Wallerian degeneration.

Nmnat (Nicotinamide/nicotinate mononucleotide adenylyltransferase) is the central enzyme of the NAD ⁺ (nicotinamide adenine dinucleotide) biosynthetic pathway, catalysi ng the formation of NAD ⁺ from NMN ⁺ (nicotinamide mononucleotide) and NaAD (nicotinic acid adenine dinucleotide) from NaMN (nicotinic acid mononucleotide). Three isoforms of the enzyme have been identified, expressed by three different genes in mammals: Nmnatl, Nmnat2 and Nmnat3. Other synonyms for Nmnat isoforms are KIAA0479 for the Nmnat2 protein, D4Colele for the gene encoding the Nmnatl protein or Ensadin 0625 for the gene encoding the Nmnat2 protein. Nmnat2 may exist in more than one splice form, all of which are referred to here as Nmnat2. Nmnat2 appears to be mainly expressed in brain, heart and muscle tissue whereas Nmnatl and Nmnat3 show a wider distribution pattern throughout a range of tissues. At the cellular level, Nmnatl is most abundant in the nucleus, Nmnat2 is abundant in the Golgi complex and Nmnat3 is abundant in mitochondria. Other subcellular locations are also possible in each case. The present invention is based on the hypothesis that a putative survival factor (or factors) is constitutively delivered to wild-type axons to prevent the activation of an intrinsic axon degeneration program. This is supported by studies showing that suppressing protein synthesis rapidly leads to Wallerian-like degeneration, which is significantly delayed in the presence of the WIdS gene.

Surprisingly, knock-down of Nmnat2, a different isoform from the Nmnatl incorporated into the protective WIdS protein, induced rapid Wallerian-like degeneration. By contrast, experimental reduction in expression of Nmnatl or Nmnat3 had no effect on the rate of axonal degeneration.

The term 'modulator' as used herein refers to a molecule capable of altering the function of the Nmnat2 protein, either directly or indirectly. In the present context, the goal is to increase the level of enzyme activity, or increase the activity in the location needed for neuroprotection. It will be appreciated that increased Nmnat2 activity can be achieved through numerous means, the following list of which is not exhaustive: increased expression of the protein, increased concentration of potentially important enzyme co-factors, allosteric activation of the enzyme, enhanced substrate binding to the enzyme, enhanced subcellular targeting to a key location, or increased half-life of the enzyme whether through direct interaction with Nmnat2 or interaction with a protein involved in its degradation. It will further be appreciated that modulators can however also have a negative effect on protein function.

The term 'agent' as used herein refers to a molecule capable of altering the function of Nmnat2 or of any other protein that is involved in the synthesis or degradation of NAD/NaAD. In the present context, the goal is to increase the level of NAD/NaAD, or any other product of Nmnat2 catalysis, in the location needed for neuroprotection, for example in the distal dendrites. It will be appreciated that, in addition to affecting NAD/NaAD synthesis or degradation, the agent could also act to raise the local concentration by other means, for example: translocation of NAD/NaAD to the site of interest or translocation of enzymes involved in synthesis and degradation. The term 'neuroprotective' as used herein refers to the ability to protect neurons or their axons or synapses in the central or peripheral nervous system from damage or death. Many different types of insult can lead to neuronal damage or death, for example: metabolic stress caused by hypoxia, hypoglycaemia, diabetes, loss of ionic homeostasis or other deleterious process, physical injury of neurons, exposure to toxic agents and numerous diseases affecting the nervous system including inherited disorders. It will be appreciated that this is only an illustrative list; many other examples will be found in the literature. The presence of an agent that is neuroprotective will enable a neuron to remain viable upon exposure to insults which may cause a loss of functional integrity in an unprotected neuron.

The term 'medicament' as used herein refers to a pharmaceutical formulation that is of use in treating, curing or improving a disease or in treating, ameliorating or al leviating the symptoms of a d isease . A pharmaceutical formulation comprises a pharmacologically active ingredient in a form not harmful to the subject it is being administered to and additional constituents designed to stabilise the active ingredient and affect its absorption into the circulation or target tissue.

In one aspect of the invention, there is provided a pharmaceutical composition comprising a modulator as hereinbefore defined.

The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington : The Science and Practice of Pharmacy, 19 ^th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.

Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.

In one aspect of the invention, there is provided a method of treatment of a neurodegenerative disorder involving Wallerian degeneration comprising administering to a subject an Nmnat2 modulator.

In one aspect of the invention, there is provided the use of an Nmnat2 modulator in the manufacture of a medicament for the treatment of a neurodegenerative disorder involving Wallerian degeneration.

In one aspect of the invention, there is provided a pharmaceutical composition comprising an Nmnat2 modulator for use in the treatment of a neurodegenerative disorder involving Wallerian degeneration.

Administration of Nmnat2 modulators according to the invention may be through various routes, for example oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intraperitoneal, vaginal, parenteral ( i n cl ud i n g su bcuta neous, i ntra m uscu l a r, i ntraderma l ) , i ntratheca l o r intracerebroventricular. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient chosen.

Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen- like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a formulation which may be a solution or suspension for the administration of the Nmnat2 modulator in the form of a nasal or pulmonal spray. As a still further option, the formulation containing the Nmnat2 modulator of the invention can also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, e.g. buccal, administration. Nmnat2 modulators of the current invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example, hard gelatine capsules and soft gelatine capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, infusion solution, and implants.

Nmnat2 modulators of the invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the composition, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof.

Examples of carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, poly(vinyl alcohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block co-polymers thereof, polyethylene glycols, carrier proteins, for example albumin, gels, for example, thermogelling systems, for example block co-polymeric systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticulates, liquid crystals a nd dispersions thereof, L2 phase and dispersions thereof, well known to those skilled in the art of phase behaviour in lipid-water systems, polymeric micelles, multiple emulsions, self-emulsifying, self-microemulsifying, cyclodextrins and derivatives thereof, and dendrimers.

Nmnat2 modulators of the current invention may be useful in the composition of solids, semi-solids, powder and solutions for pulmonary administration, using, for example a metered dose inhaler, dry powder inhaler and a nebulizer, all being devices well known to those skilled in the art.

Nmnat2 modulators of the current invention may be useful in the composition of controlled, sustained, protracting, retarded, and slow release drug delivery systems. More specifically, but not limited to, modulators are useful in the composition of parenteral controlled release and sustained release systems (both systems leading to a many-fold reduction in number of administrations), well known to those skilled in the art. Even more preferably, are controlled release and sustained release systems administered subcutaneously. Without limiting the scope of the invention, examples of useful controlled release system and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres and nanoparticles.

Methods to produce controlled release systems useful for compositions of the current invention include, but are not limited to, crystallization, condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenisation, en-capsulation, spray drying, microencapsulating, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Composition and Delivery (MacNally, EJ., ed. Marcel Dekker, New York, 2000).

Nmnat2 modulators are predicted to be of uti l ity i n the treatme nt of neurodegenerative disorders involving Wallerian degeneration. Examples of disorders where such degeneration may be of importance include Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease, Canavan disease, Cerebral palsy, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Diabetic neuropathy, Frontotemporal lobar degeneration, Glaucoma, Guillain- Barre syndrome, Hereditary spastic paraplegia, Huntington's disease, HIV associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Motor neuron disease, Multiple System Atrophy, Multiple sclerosis, Narcolepsy, Neuroborreliosis, Niemann Pick disease, Parkinson's disease, Pelizaeus- Merzbacher Disease, Peripheral neuropathy, Pick's disease, Primary lateral sclerosis, Prion diseases, Progressive Supranuclear Palsy, Refsum's disease, Sandhoff's disease, Schilder's disease, Spinocerebellar ataxia, Spinal cord injury, Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Stroke and other ischaemic disorders, Tabes dorsalis or Traumatic brain injury. This list is for illustrative purposes only and is not limiting or exhaustive.

In one embodiment the modulator is intended for use as a neuroprotective medicament in the treatment of a neurodegenerative disorder resulting from neuronal injury.

In a further embodiment the modulator is intended for use as a neuroprotective medicament in the treatment of a neurodegenerative disorder involving Wallerian degeneration resulting from neuronal injury.

The term 'injury' as used herein refers to damage inflicted on the neuron, whether in the cell body or in axonal or dendritic processes. This can be a physical injury in the conventional sense i.e. traumatic injury to the brain, spinal cord or peripheral nerves caused by an external force applied to a subject. Other damaging external factors are for example environmental toxins such as mercury and other heavy metals, pesticides and solvents. Alternatively, injury can result from an insult to the neuron originating from within the subject, for example: reduced oxygen and energy supply as in ischemic stroke and diabetic neuropathy, autoimmune attack as in multiple sclerosis or oxidative stress and free-radical generation as is believed to be important in amyotrophic lateral sclerosis. Injury is also used here to refer to any defect in the mechanism of axonal transport.

In another embodiment, the modulator is intended for use as a neuroprotective medicament wherein the neurodegenerative disorder is caused by a neuronal injury resulting from a disease. In one embodiment, the modulator functions to prolong the functional half-life of Nmnat2. Experiments have shown that Nmnat2 is rapidly cleared from the cell, with a half-life of no more than 2 - 3 hours in cultured cells. The constant supply and degradation of the protein ensures that a neuron is highly responsive to environmental or cellular changes and can initiate a cascade leading to axonal degeneration within a matter of hours if necessary. A key property of a therapeutically useful modulator of Nmnat2 would therefore be to prolong its half-life, especially within terminal portions of the axon. This has been confirmed by over-expression studies where high concentrations of Nmnat2 protein are expressed in cells in order to compensate for the rapid turn-over of the protein. The result is a delay in the onset of Wallerian degeneration akin to that produced by WIdS.

An efficient way to increase the half-life of Nmnat2 would be to inhibit its degradation. Nmnat2 is known to be broken down via the ubiquitin-proteasome system whereby proteins destined for destruction undergo molecular tagging with ubiquitin which targets them for proteasomal breakdown. The enzymes that catalyze the ubiquitination step are called ubiquitin ligases. Preventing or slowing the reaction that results in the ubiquitination of Nmnat2, including inhibition of appropriate E2 ubiquitination factors or E3 or E4 ubiquitin ligase(s), therefore represents an attractive target mode of action for a potential Nmnat2 modulator.

In one embodiment, the modulator functions to increase Nmnat2 activity.

According to another aspect of the invention, there is provided an agent which functions to increase the localised concentration of products of Nmnat2 catalysis. Nmnat2 biosynthetic activity may result in the production of several different compounds, and any of these may be important in mediating the neuroprotective effect of Nmnat2 activity. It is therefore possible that a therapeutic benefit could be drawn from increasing or maintaining the concentration of these compounds at particular sites within a cell. In the case of a neuron, an important site for an increased local concentration of these compounds could be the distal neurites. In one embodiment, the agent functions to increase the localised NAD/NaAD concentration. Nmnat2 biosynthetic activity results in the localised production of NAD or NaAD; it is therefore possible that a therapeutic benefit could be drawn from increasing or maintaining the concentration of NAD or NaAD at particular sites within a cell. Enzymes other than Nmnat2 could also be envisaged as being capable of altering the local concentration of NAD/NaAD.

In one aspect of the invention, there is provided an inhibitor of the ubiquitin- proteasome system for use in the treatment of a neurodegenerative disorder involving Wallerian degeneration.

In another aspect of the invention, there is provided a method of treatment of a neurodegenerative disorder involving Wallerian degeneration comprising administering to a subject an inhibitor of the ubiquitin-proteasome system.

In another aspect of the invention, there is provided the use of an inhibitor of the ubiquitin-proteasome system in the manufacture of a medicament for the treatment of a neurodegenerative disorder involving Wallerian degeneration.

In one embodiment, the inhibitor inhibits a specific component of the ubiquitin- proteasome system.

In another aspect of the invention, there is provided a pharmaceutical composition comprising an inhibitor of the ubiquitin-proteasome system for use in the treatment of a neurodegenerative disorder involving Wallerian degeneration.

In one embodiment, the inhibitor inhibits a specific component of the ubiquitin- proteasome system.

In a further embodiment, the neurodegenerative disorder is selected from one or more of amyotrophic lateral sclerosis, multiple sclerosis, stroke or neuronal injury resulting from trauma, such as peripheral nerve injury, traumatic brain injury, spinal cord injury or neuronal injury induced by a toxic agent such as a chemotherapeutic agent.

In one embodiment, the neuronal injury results from trauma.

In one embodiment, the disorder is a neuronal injury induced by a chemotherapeutic agent. Certain drugs used in cancer chemotherapy such as Taxol or vincristine, cause peripheral neuropathy which limits the maximum doses at which they can be used. Recent studies suggest that neurons suffering from Taxol or vincristine toxicity undergo Wallerian-like changes in their morphology and in the underlying molecular events. Inhibiting Wallerian degeneration could be particularly effective in this condition as neurons are only temporarily exposed to the neurotoxic agent. Simultaneous administration of Taxol with an agent inhibiting Wallerian degeneration could therefore allow the drug to be used at substantially higher doses than is currently possible.

In one embodiment, the neurodegenerative disorder is an ophthalmic disorder such as glaucoma or macular degeneration.

In another aspect of the invention there is provided the use of Nmnat2 or a derivative, fragment or metabolite thereof as a biomarker for Wallerian degeneration . The term 'biomarker' as used herein refers to a distinctive biological or biologically-derived indicator of a process, event or condition.

Biomarkers can be used in methods of diagnosis, e.g. clinical screening and prognosis assessment, in monitoring the results of therapy and in identifying patients most likely to respond to a particular therapeutic treatment as well as in drug screening and development. They can also be used in basic and medical research. Biomarkers and uses thereof are valuable for the identification of new drug treatments and for the discovery of new targets for drug treatment. In the present context, a biomarker can be replaced by a molecule, or measurable fragments of a molecule found upstream or downstream of the biomarker in a biological pathway. In a further aspect of the invention there is provided a method for demonstrating Wallerian degeneration comprising detecting and/or quantifying in a sample from a test subject, a biomarker as hereinbefore defined.

The term "detecting" as used herein refers to confirming the presence of the biomarker present in the sample. Quantifying the amount of the biomarker present in a sample may include determining the concentration of the biomarker present in the sample. Detecting and/or quantifying may be performed directly on the sample, or indirectly on an extract therefrom, or on a dilution thereof.

Detecting and/or quantifying can be performed by any method suitable to identify the presence and/or amount of a specific protein in a biological sample from a patient or a purification or extract of a biological sample or a dilution thereof. In methods of the invention, quantifying may be performed by measuring the concentration of the biomarker in the sample or samples.

Biological samples that may be tested in a method of the invention include tissue homogenates, tissue sections and biopsy specimens from a live subject, or taken post-mortem. The samples can be prepared, diluted or concentrated where appropriate, and stored in the usual manner. Biological samples can also include cerebrospinal fluid (CSF), whole blood, blood serum, plasma, urine, saliva, or other bodily fluid.

In one embodiment, detecting and/or quantifying is performed by one or more methods selected from SELDI (-TOF), MALDI (-TO F), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC or other LC or LC-MS-based technique. Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA).

Liquid chromatography (e.g. high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin-layer chromatography, NMR (nuclear magnetic resonance) spectroscopy could also be used. The biomarker may be directly detected, e.g. by SELDI or MALDI-TOF. Alternatively, the biomarker may be detected directly or indirectly via interaction with a ligand or ligands such as an antibody or a biomarker-binding fragment thereof, or other peptide, or ligand, e.g. aptamer, or oligonucleotide, capable of specifically binding the biomarker. The ligand may possess a detectable label, such as a luminescent, fluorescent or radioactive label, and/or an affinity tag.

In one embodiment, detecting and/or quantifying is performed using a biosensor, microanalytical, microengineered, microseparation or immunochromatography system.

The term 'biosensor' as used herein refers to something capable of detecting the presence of a biomarker. Using predictive biomarkers, appropriate diagnostic tools such as biosensors can be developed. The biosensor may incorporate an immunological method for detection of the biomarker(s), electrical, thermal, magnetic, optical (e.g. hologram) or acoustic technologies. Using such biosensors, it is possible to detect the target biomarker(s) at the anticipated concentrations found in biological samples.

In one embodiment, detecting and/or quantifying is performed by an immunological method. This may rely on an antibody, or a fragment thereof capable of specific binding to the biomarker. Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, in which the detection of the biomarker is performed using two antibodies which recognize different epitopes on a biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Fluorescence immunoassays (FIA), western blotting, immunoprecipitation, immunohistochemistry and any particle-based immunoassay (e.g. using gold, silver, or latex particles, magnetic particles, or Q- dots). Immunological methods may be performed, for example, in microtitre plate or strip format.

In one embodiment, detecting and/or quantifying is performed by an immunohistochemical method. Immunological methods in accordance with the invention may be based, for example, on any of the following methods.

Immunoprecipitation is the simplest immunoassay method; this measures the quantity of precipitate, which forms after the reagent antibody has incubated with the sample and reacted with the target antigen present therein to form an insoluble aggregate. Immunoprecipitation reactions may be qualitative or quantitative.

In particle immunoassays, several antibodies are linked to the particle, and the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows rapid and sensitive detection of the biomarker.

In immunonephelometry, the interaction of an antibody and target antigen on the biomarker results in the formation of immune complexes that are too small to precipitate. However, these complexes will scatter incident light and this can be measured using a nephelometer. The antigen, i.e. biomarker, concentration can be determined within minutes of the reaction.

Radioimmunoassay (RIA) methods employ radioactive isotopes such as I ¹²⁵ to label either the antigen or antibody. The isotope used emits gamma rays, which are usually measured following removal of unbound (free) radiolabel. The major advantages of RIA, compared with other immunoassays, are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassays.

Enzyme (EIA) immunoassays were developed as an alternative to radioimmunoassays (RIA). These methods use an enzyme to label either the antibody or target antigen. The sensitivity of EIA approaches that for RIA, without the danger posed by radioactive isotopes. One of the most widely used EIA methods for detection is the enzyme-linked immunosorbent assay (ELISA).

ELISA methods may use two antibodies one of which is specific for the target antigen and the other of wh ich is coupled to an enzyme, addition of the substrate for the enzyme results in production of a chemiluminescent or fluorescent signal.

Fluorescent immunoassay (FIA) refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement.

Chemiluminescent immunoassays utilize a chemiluminescent label, which produces light when excited by chemical energy; the emissions are measured using a light detector.

Immunological methods according to the invention can thus be performed using well-known methods. Any direct (e.g., using a sensor chip) or indirect procedure may be used in the detection of biomarkers of the invention.

The Biotin-Avidin or Biotin-Streptavidin systems are generic labelling systems that can be adapted for use in immunological methods of the invention. One binding partner (hapten, antigen, ligand, aptamer, antibody, enzyme etc) is labelled with biotin and the other partner (surface, e.g. well, bead, sensor etc) is labelled with avidin or streptavidin. This is conventional technology for immunoassays, gene probe assays and (bio)sensors, but is an indirect immobilisation route rather than a direct one. For example a biotinylated ligand (e.g. antibody or aptamer) specific for a biomarker of the invention may be immobilised on an avidin or streptavidin surface, the immobilised ligand may then be exposed to a sample containing or suspected of containing the biomarker in order to detect and/or quantify a biomarker of the invention. Detection and/or quantification of the immobilised antigen may then be performed by an immunological method as described herein.

In a further aspect of the invention there is provided a diagnostic kit for detecting Wallerian degeneration comprising a biosensor configured to detect and/or quantify the biomarker as hereinbefore defined and instructions to use said kit in accordance with the methods as hereinbefore defined.

In one embodiment, the biosensor is an antibody. The term "antibody" as used herein includes, but is not limited to: polyclonal, monoclonal, bispecific, humanised or chimeric antibodies, single chain antibodies, Fab fragments and

F(ab') ₂ fragments, fragments produced by a Fab expression library, anti-idiotypic

(anti-Id) antibodies and epitope-binding fragments of any of the above. The term "antibody" as used herein also refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. The immunoglobulin molecules of the invention can be of any class (e. g., IgG, IgE,

IgM, IgD and IgA) or subclass of immunoglobulin molecule.

In another aspect of the invention there is provided a method of screening for an Nmnat2 modulator comprising the steps of: a) blocking the synthesis of Nmnat2, or a readily detectable version of Nmnat2, in a biological sample; b) incubating said sample with a test molecule; and c) measuring the Nmnat2 - associated signal over time such that a difference from the control decay curve is indicative of an Nmnat2 modulator.

It will be appreciated that a readily detectable version of Nmnat2 comprises a modification making it suitable for rapid quantification in a high-throughput system, for example by tagging the protein with a reporter such as a fluorescent protein. The synthesis of Nmnat2 can be blocked for example by using an inducible expression system, knocking down expression or adding a general protein synthesis inhibitor. In a further aspect of the invention there is provided a method of screening for an Nmnat2 modulator comprising the steps of: a) obtaining a biological sample which is known to contain neurological tissue undergoing Wallerian degeneration; b) measuring the level of ubiquitinated Nmnat2 within said sample; c) incubating said sample with a test molecule; and d) measuring the level of ubiquitinated Nmnat2 within the sample after said incubation, such that a difference in the two levels of ubiquitinated Nmnat2 is indicative of an Nmnat2 modulator.

High-throughput screening technologies based on the biomarker, uses and methods of the invention, e.g. configured in an array format, are suitable to monitor biomarker signatures for the identification of potentially useful therapeutic compounds, e.g. ligands such as natural compounds, synthetic chemical compounds (e.g. from combinatorial libraries), peptides, monoclonal or polyclonal antibodies or fragments thereof, which may be capable of binding the biomarker.

Methods of the invention can be performed in array format, e.g. on a chip, or as a multiwell array. Methods can be adapted into platforms for single tests, or multiple identical or multiple non-identical tests, and can be performed in high throughput format. Methods of the invention may comprise performing one or more additional, different tests to confirm or exclude diagnosis, and/or to further characterise a condition.

In another aspect of the invention, there is provided an Nmnat2 modulator identified by a screening method as hereinbefore defined. Such substances may be capable of altering the activity of Nmnat2, or of suppressing degradation of Nmnat2. The term "modulator" includes substances that do not directly bind to Nmnat2 and directly modulate its function, but instead indirectly modulate the function of Nmnat2. Ligands are also included in the term modulator; ligands of the invention (e.g. a natural or synthetic chemical compound, peptide, aptamer, oligonucleotide, antibody or antibody fragment) are capable of binding, suitably specific binding, to Nmnat2.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Representative bright-field images of distal neurites from wild- type (BL/6) (A) and WId ⁵ (B) mouse SCG explant cultures treated with 1 or 10 μg/ml CHX or 10 μM emetine as indicated . Images of the same field of distal neurites were captured at the times indicated on the left. Neurites in DMSO- treated wild-type cultures continue to grow and appear morphologically normal (data not shown). (C) The fold increase in neurite blebs 12h after treatment with emetine in BL/6 cultures and 48h after treatment in WId ⁵ cultures, relative to the same neurites at the start of the treatment (Oh), was quantified from three or more independent experiments combining data from multiple fields (error bars = ± S. E. M.). Blebbing is significantly reduced in WId ⁵ cultures even at this much later timepoint (* p < 0.05, ** p = 0.01, t-test WId ⁵ at 48h versus BL/6 at 12h). (D) Anti-Neurofilament heavy chain (NF-H) and anti-β-Tubulin immunoblots of neurite-only extracts from wild-type or WId ⁵ SCG explant cultures either treated with 10 μM emetine for 8h, left untreated, or 8h after cut. Each lane represents neurites collected from SCG cultures containing three ganglia. (E) Bright-field images of a mouse SCG explant after 7 days of treatment with 1 μg/ml CHX and 7 days after CHX removal . Images are representative of three independent experiments.

Figure 2 : (A) Diagram showing the organization of compartmented wild- type mouse SCG cultures. (B) Representative bright-field images of d istal neurites from the side chamber of a compartmented culture in which 10 μ M emetine was added to either the central chamber (cell bodies treated) or side chamber (neurites treated). Images of the same field of neurites were captured just after emetine addition (Oh) and 24h later. The fold increase in blebbing at 24h relative to Oh (of the same neurites) was quantified from three independent experiments combining data from multiple fields (error bars = ± S. E. M .) and is shown on the right. Treatment of cell bodies (CB) alone induces significantly more blebbing of distal neurites than treatment of the distal neurites (N) themselves (* p = 0.026, t-test) . Comparable results were obtained with 1 μg/ml CHX (data not shown).

Figure 3: (A) Each Nmnat siRNA pool specifically blocks expression of a FLAG-tagged version of its intended target in transfected HEK 293T cells. Representative FLAG immunoblot of cells 24h after co-transfection with expression vectors for each FLAG-Nmnat isoform together with a non-targeting pool of siRNA (siControl) or one of the siNmnat siRNA pools as indicated. Quantification of band intensities relative to bands in the s\Control lane in three independent experiments is shown on the right (error bars = ± S. E. M.). Each siRNA pool specifically and significantly reduces expression of its target (*** p < 0.001, t-test of respective siNmnat versus siControl). Each FLAG-Nmnat acts as internal control of transfection efficiency for the others. Only 3/4 of the pooled Nmnat2 siRNAs and 2/4 of the pooled Nmnat3 siRNAs target sequences in the respective expression vectors (compared to 4/4 for the Nmnatl siRNA pool). All should target the endogenous mRNAs. (B) Each Nmnat siRNA pool specifically blocks expression of a FLAG-tagged version of its intended target in injected SCG neurons. Representative fluorescent images of SCG neurons 48h after co- injection with one FLAG-Nmnat expression vector, pEGFP-Cl and the relevant siNmnat pool or non-targeting siRNA pool (siControl) . eGFP fluorescence identifies injected neurons, FLAG immunostaining shows expression levels of each FLAG-Nmnat, and DAPI labels nuclei. Each s\Nmnat siRNA pool significantly reduces expression of its target isoform compared to s\Control as indicated on the right by quantification of FLAG immunostaining relative to eGFP fluorescence in individual neurons (** p < 0.01, *** p < 0.001, t-test of respective siNmnat versus s\Control). Data is from three independent experiments in which a total of 36-55 injected neurons were analysed (error bars = ± S. E. M .) Localization of each tagged isoform is consistent with their expected distribution; Nmnatl in nuclei and Nmnat2 and Nmnat3 in cytoplasmic compartments.

Figure 4: (A) Representative fluorescent images of the distal ends of (DsRed2-labeled) neurites of wild-type (BL/6) or WId ⁵ SCG neurons 24 and 72h after injection with non-targeting siRNA (s\Control) or siRNA targeting each Nmnat isoform (siNmnatl, 2 or J), as indicated, together with pDsRed2-Nl (50 ng/μl) (B) Relative survival of (DsRed2-labeled) neurites of wild-type SCG neurons injected with siControl, siNmnatl, s\Nmnat2, or s\Nmnat3. The number of healthy DsRed2-labeled neurites remaining at each timepoint is shown as a percentage of the total number (both healthy and abnormal) 24h after injection and was quantified from three independent experiments combining data from multiple fields (error bars = ± S. E. M.). Only s\Nmnat2 causes significant neurite loss (**p < 0.01, ***p < 0.001, t-test s\Nmnat2 versus siControl at equivalent timepoints). (C) Co-injection of siNmnatl, 2 and 3 (each at 100 ng/μl) does not accelerate (wild-type) neurite loss compared to injection of s\Nmnat2 alone (100 ng/μl). Data was quantified as in (B) from three independent experiments (error bars = ± S. E. M.). (D) Neurite loss caused by s\Nmnat2 injection is abolished in WId ⁵ SCG neurons up to 72h after injection (** p < 0.01, *** p < 0.001, t-test WId ⁵ versus BL/6 at equivalent timepoints). Data was quantified as in (B) from four independent experiments (error bars = ± S. E. M .). (E-G) Representative fl uorescent i mages sh owi n g the typica l ch a racteristics of the neu rite degeneration caused by s\Nmnat2 injection. Multiple DsRed2-positive neuritic swellings are only observed after s\Nmnat2 injection (E) and these precede a characteristic progressive distal-to-proximal ^λ dying-back' neurite degeneration (F). Neurites showing 'dying-back' degeneration can be followed back to morphologically normal cell bodies. This contrasts the more rapid, catastrophic neurite degeneration that coincides with cell body death (G) and is seen following injection of all siRNA pools (including s\Control). Images in (E) were captured 48h after injection and show injected cell bodies at the top of each panel.

Figure 5: (A) Viability of wild-type SCG neurons injected with non- targeting siRNA (slControl) or Nmnat2 siRNA (s\Nmnat2), together with pEGFP- C l ( 10 n g/μ l ) , and treated with 50 μ M z-VAD-fmk or vehicle (DMSO) as indicated. The number of neurons with normal gross morphology remaining at each timepoint is shown as a percentage of those present at 24h and was quantified from three independent experiments (error bars = ± S. E. M.). There is a small but significant decrease in the percentage of viable neurons 48 and 72h after injection with s\Nmnat2 ( + DMSO) relative to siControl (*** p < 0.001, t- test s\Nmnat2 versus siControl at equivalent timepoints) but this is reduced to control levels following treatment with z-VAD-fmk. (B) Assessment of neuronal viability based on gross morphology matches that based on other indicators of viability. At the end of experiments in (A), neurons were counter-stained with propidium iodide (PI), a DNA stain that can only penetrate the membranes of non-viable cells, and DAPI. Abnormal gross morphology seen with bright-field and eGFP imaging correlated precisely with PI staining and nuclear condensation / fragmentation (revealed by DAPI staining) typical of cell death. An arrowhead indicates an abnormal neuron (rarely seen in these experiments) and an arrow a healthy neuron. (C) Quantification of neurite survival corresponding to the analysis of neuronal viability in (A). The number of healthy eGFP-labeled neurites remaining at each timepoint (quantified from multiple fields in each experiment) is shown as a percentage of the total number (both healthy and abnormal) 24h after injection (error bars = ± S. E. M .). Significant neurite loss is seen following s\Nmnat2 injection irrespective of treatment with z-VAD-fmk (* p < 0.05, ** p < 0.01 , ** * p < 0.001 , t-test s\Nmnat2 ± z-VAD-fmk versus siControl ± z-VAD-fmk at equivalent timepoints) with no significant difference between the two. (D) Representative fluorescent images of the distal ends of eGFP-labeled neurites of wild-type (BL/6) SCG neurons 24 and 72h after injection with s\Control or s\Nmnat2 and treated with z-VAD-fmk.

Figure 6: (A) Relative stabilities of FLAG-tagged Nmnat isoforms and Wld ^s in H EK 293T cel ls co-transfected with expression vectors for each and treated 24h after transfection with 10 μM emetine to block translation for the times indicated. A representative FLAG immunoblot is shown (top panel). The same blot was re-probed with an Nmnat2 antibody (bottom panel) to show that loss of anti-FLAG signal is primarily due to protein turnover rather than cleavage of the FLAG epitope from the protein. Quantification of band intensities for each protein from three independent experiments is shown below as a percentage of untreated (Oh) band intensities (error bars = ± S. E. M.). Co-transfection allows direct comparison of stabilities of each protein in the same cells. (B) FLAG- Nmnat2 turnover in transfected HEK 293T cells is prevented by proteasome inhibition. Cells were transfected as in (A) and treated with 10 μM emetine for 0 or 24h , ± 20 μ M MG-132. A FLAG immunoblot blot representative of three independent experiments is shown. (C) Endogenous Nmnat2 is rapidly turned over in SCG explant cultures after blocking translation with CHX (1 μg/ml) or emetine (10 μM). Representative immunoblots are shown comparing steady- state levels of N m n at2 (O h ) with levels after 4h of protei n synthesis suppression). β-Tubulin acts as a loading control. Nmnat2 band intensity at 4h is shown as a fraction of that at Oh after normalization to β-Tubulin and was quantified from two independent experiments each (error bars = ± S. E. M .) . These values are consistent with the different rates at which CHX (1 μg/ml) and emetine induce neurite degeneration (Figure 1).

Figure 7: (A-C) Relationship between Nmnat2 turnover in neurites and other parameters of neurite health in wild-type cultures (A), wild-type cultures ± 20 μM MG-132 (B), and Wld ^s cultures (C). Representative immunoblots show detection of Nmnat2, Wld ^s (where applicable), NF-H, 16 kDa core Histones, and β-Tubulin just after cut (Oh) and 4 and/or 8h later. Material collected from SCG explant cultures derived from 15-20 ganglia was needed to detect Nmnat2 in each lane. Loss of the NF-H band is an early consequence of axon degeneration and absence of the 16 kDa core Histones band in neurite extracts confirms there is no detectable contamination with SCG cell bodies or non-neuronal cells, β- Tu bul in represents a loading control. Relative Nmnat2 band intensities in neurite-only lanes are shown as a fraction of levels at Oh after normalisation to β-Tubulin and were quantified from two or three independent experiments (error bars = ± S. E. M .). Nmnat2 is significantly depleted in untreated wild-type and WId ⁵ neurites shortly after separation from their ganglia (* p < 0.05, ** p < 0.01, *** p < 0.001, t-test 4h or 8h versus Oh). Proteasome inhibition with MG- 132 significantly reduces this Nmnat2 loss at 8h after transection ( ^§§ p < 0.01, t- test 8h +MG-132 versus 8h untreated). Images show representative transected neurite morphology (in the same field where applicable) at the indicated times after cut. (D) Kymograph of a bottom axon showing fast axonal transport of particles containing Nmnat2-eGFP with a bias in the anterograde direction. An image series from the indicated region of the kymograph is shown on the right. It highlights a particle moving anterogradely (filled arrowhead), one moving retrogradely (empty arrowhead) and several stationary particles (asterisks).

Figure 8: (A) Protection of transected neurites by exogenous expression of FLAG-Nmnat2 or FLAG-Wld ^s . SCG neurons injected with 1, 2.5 or 50 ng/μl empty vector (FLAG-empty) or FLAG-Nmnat2 or FLAG-Wld ^s expression vectors, together with pDsRed2-N l (50 ng/μl), were transected 48h later. Survival of DsRed2-labeled neurites 24h after transection is shown as a percentage of the number of labeled neurites with normal morphology just after cut (Oh) and was quantified from two to four independent experiments combining data from multiple fields (error bars ±S. E. M) . FLAG-Wld ^s protects neurites significantly better than FLAG-Nmnat2 24h after cut at the 1 ng/μl vector concentration (*** p < 0.001 , t-test). There is no significant difference in protection 24h after transection at the 50 ng/μl vector concentration. (B) Representative fluorescent images of transected DsRed2-labeled neurites of SCG neurons injected with selected concentrations of FLAG-Nmnat2 or FLAG-Wld ^s expression vectors (as indicated). The same field of neurites is shown at the time of cut (Oh) and 24h later. The cut site is located to the left of each image. Increased magnification of the framed region in each panel is shown for better visualization of neurite morphology.

Figure 9 shows the effect of Nmnat2 siRNA on inducing Wallerian-like degeneration of uncut SCG neuritis.

Figure 10 shows the difference between sil\lmnat2 - induced neurite degeneration in wild-type and WIdS SCG neuron.

Figure 11 shows the characteristics of neurite degeneration after injection with sil\lmnat2.

Figure 12 compares the relative turn-over rates of the FLAG-Nmnat isoforms as well as FLAG-WIdS. Figure 13 shows the reduced turn-over of FLAG-Nmnat2 when the ubiquitin proteasome system is blocked .

Figure 14 shows the effect of varying levels of FLAG-Nmnat2 expression on the survival of transected SCG neurites. The invention will now be described, by way of example only, with reference to the accompanying examples:

EXAMPLES

MATERIALS AND METHODS

1. Plasmids constructs and siRNA reagents

Expression vectors encoding FLAG-tagged murine Nmnat isoforms and Wld ^s were generated by amplification of the full coding region of each gene by RT-PCR (see below) from 1 μg total RNA from wild-type and Wld ^s mouse brain. Products were cloned into pCMV Tag-2B (Stratagene) to generate FLAG-Nmnat/Wld ^s expression vectors, or pEGFP-Nl (BD Biosciences Clontech) to generate an Nmnat2-eGFP expression vector. Sequencing (Cogenics) was performed to confirm the absence of PCR errors. Other plasmids used were pDsRed2-Nl for expression of variant Discosoma red fluorescent protein (DsRed2) and pEGFP-Cl for expression of enhanced green fluorescent protein (eGFP) (both BD Biosciences Clontech). Dharmacon ON-TARGETp/us SMART pools of siRNA (Thermo Scientific) specifically targeted against mouse Nmnatl (L-051136-01), Nmnat2 (L-059190-01), or Nmnat3 (L-051688-01 ) were used in this study. Dharmacon ON-TARGETp/us siControl non-targeting siRNA pool (D-001810-10) was used as a control in experiments. Each pool consists of 4 individual siRNAs. The siRNAs making up the ON-TARGETp/us Nmnat2 SMART pool (J-059190-09, - 10, -11 and -12) were also tested individually or in sub-pools.

2. Cell culture

(a) Explant cultures: Superior cervical ganglia were dissected from Pl or P2 mouse or rat pups and dorsal root ganglia were dissected from E15.5 mouse embryos. Cleaned explants were placed in the centre of 3.5 cm tissue culture dishes pre-coated with poly-L-lysine (20 μg/ml for 1-2 hours; Sigma) and laminin (20 μg/ml for 1-2 hours; Sigma). Explants were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 4500 mg/L glucose and 110 mg/L sodium pyruvate (Sigma), 2 mM glutamine, 1% penicillin/streptomycin, 100 ng/ml 7S NGF (all Invitrogen), and 10% fetal bovine serum (Sigma). 4 μM aphidicolin (Calbiochem) was used to reduce proliferation and viability of small numbers of non-neuronal cells. Cultures were used after 5-7 days.

(b) Dissociated SCG cultures: Dissected SCG ganglia were incubated in 0.025% trypsin (Sigma) in PBS (without CaCI ₂ and MgCI ₂ ) for 30 minutes followed by

0.2% collagenase type II (Gibco) in PBS for 30 minutes. Ganglia were then gently triturated using a pipette. After a 2 hour pre-plating stage to remove non- neuronal cells, 5-10,000 dissociated neurons were plated in a 1 cm ² poly-L- lysine and laminin-coated area of normal 3.5 cm dishes (Nunc), or ibidi μ-dishes (Thistle Scientific) for microinjection experiments. Dissociated cultures were maintained as explant cultures except that 20 μM uridine and fluorodeoxyuridine was used to reduce proliferation and viability of non-neuronal cells (Sigma).

(c) Compartmented cultures: Dissected SCG explants were broken into small pieces using forceps and then placed into the central compartment of three- compartment Campenot Teflon divider (Tyler Research) essentially as described previously (Campenot RB (1992) Construction and Use of Compartmented Cultures. Protocols for Neural Cell Culture: Humana Press Inc. pp. 53-63). The ability of the barriers to prevent diffusion of bromophenol blue between the independent compartments containing the cell bodies and distal neurites for at least 24 hours after completion of the experiment was assessed to confirm their integrity. Compartmented cultures were maintained as explant cultures.

(d) HEK 293 culture: HEK 293 cells were cultured under standard conditions in D M EM with 4500 mg/L g l u cose a nd 110 mg/L sod i u m pyruvate ( PAA) , supplemented with 2 mM glutamine and 1% penicillin/streptomycin (both Invitrogen), and 10% fetal bovine serum (Sigma).

(e) Animals: C57BL/6JOIaHsd and homozygous C57BL/6OlaHsd-Wld (WId ⁵ ) mice and Sprag ue Dawley rats were obtained from Harlan U K ( Bicester, U K) .

Transgenic Wld ^s rat line 79 has been described previously (Adalbert R et al. (2005) Eur J Neurosci 21 : 271-277). All animal work was carried out in accordance with the Animals (Scientific Procedures) Act, 1986, under Project Licenses PPL 80/1778 and 80/2254. 3. Reverse transcriptase PCR (RT-PCR)

Total brain RNA was extracted using TRIzol reagent (Invitrogen) and RNA from dissociated SCG neuronal cultures was isolated using RNeasy columns (Qiagen). 1 μg of brain RNA and 30% of that recovered from SCG cultures was reverse transcribed into cDNA using Superscript II (both Invitrogen). Control samples without reverse transcriptase were processed simultaneously to rule out DNA contamination of samples. Standard PCR amplification was performed using REDTaq DNA polymerase (Sigma). Primers used for detection of Nmnat isoform transcripts in SCG neuron RNA were as follows: Nmnatl 5'- ttcaaggcctgacaacatcgc-3' (SEQ ID NO: 1) and 5'-gagcaccttcacagtctccacc-3' (SEQ ID NO: 2), Nmnat2 5'-cagtgcgagagacctcatccc-3' (SEQ ID NO: 3) and 5'- acacatgatgagacggtgccg-3' (SEQ ID NO: 4), Nmnat3 5'-ggtgtggaggtgtgtgacagc-3' (SEQ ID NO: 5) and 5'-gccatggccactcggtgatgg-3' (SEQ ID NO: 6). Products were sequenced to confirm correct amplification.

4. Inhibitor treatments

100Ox aqueous stock solutions of emetine (dihydrochloride hydrate) and cycloheximide (CHX) in DMSO (both Sigma) were diluted 1 : 1000 in culture media to give final concentrations indicated (1 μg/ml CHX = 3.5 μM). InSolution MG-132 (Calbiochem) was diluted to 20 μM. MG-132 was added to SCG explant cultures 3 hours prior to neurite transection. This pre-treatment is required to see neurite protection in these cultures (Zhai Q et al (2003) Neuron 39 : 217- 225). Media was changed once with addition of fresh inhibitors when cultures were treated for more than 5 days. CHX-containing media was completely removed and replaced with media containing no CHX in experiments involving temporary suppression of protein synthesis.

5. Microinjection and immunostaining Microinjection was performed on a Zeiss Axiovert 200 microscope with an Eppendorf 5171 transjector and 5246 micromanipulator system and Eppendorf Femtotips. Plasmids and siRNAs were diluted in 0.5x PBS and passed through a Spin-X filter (Costar). The mix was injected directly into the nuclei of SCG neurons in dissociated cultures. ON-TARGETp/us siRNA pools were injected at a concentration of 100 ng/μl and individual siRNAs or sub-pools as indicated in the text, pDsRed2-N l at 50 ng/μ l , p EG FP-Cl at 10 ng/μ l , the N m n at2-eGFP expression construct at 50 ng/μl, and FLAG-Nmnat/Wld ^s expression constructs or FLAG-empty control (pCMV Tag-2B) at 10 ng/μl for siRNA-mediated knock- down analysis by immunostaining (Figure 3B) and at 1, 2.5 or 50 ng/μl in neurite transection experiments (Figure 8). 70-150 neurons were injected per dish. Injection of relatively few neurons per dish facilitated visualization of individual labeled neurites as neurites tend to cluster together in bundles. For detection of FLAG-tagged protein expression by immunostaining, neurons were fixed with 4% paraformaldehyde (20 min), permeabilized with 1% Triton X-IOO in PBS (10 min), blocked in 50% goat serum in PBS containing 1% BSA (30 min) and stained using monoclonal M2 anti-FLAG (Sigma) (1 :400 in PBS, 1% BSA for 1 hour) and an Alexa568-conjugated secondary antibody (1 : 200 in PBS, 1% BSA for 1 hour). Cells were mounted in Vectashield containing DAPI (Vector Laboratories) for counterstaining of nuclei. For comparing the quantification of neuronal viability based on gross morphology with other indicators of health (Figure 5B), cultures were incubated with 1 μg/ml propidium iodide (Invitrogen) for 15 mins and were then fixed with 4% paraformaldehyde (20 min) before being mounted in Vectashield containing DAPI.

6. Neurite transection for imaging and quantification of degeneration

Neurites were cut with a disposable scalpel roughly half-way between their cell bodies and their most distal ends. Where applicable, inhibitors of translation or vehicle (DMSO) were added to the media less than 10 minutes before transection. Uncut neurites treated with DMSO continue to grow normally (data not shown). Microinjection of a row of cell bodies in dissociated SCG cultures enabled neurites to be cut so that all injected cell bodies and their proximal neurites were located on the opposite side of the cut site to their distal stumps.

7. Western blot analysis

(a) HEK 293 transfection: Cells were plated so that they reached 60-80% confluence on the day of transfection and were transfected using Lipofectamine 2000 reagent (Invitrogen). For turnover experiments (Figure 6), cells in a 12- well dish format were co-transfected with 100 ng each of the FLAG-Nmnatl, FLAG-Nmnat-3 and FLAG-Wld ^s expression constructs and 250 ng of the FLAG- Nmnat-2 expression construct. For specificity experiments (Figure 3), 1 μg of one of the si RNA pools was also added as indicated. After the treatments described in the text, cells were lysed directly into 2x Laemmli sample buffer after washing with PBS.

(b) SCG neurite extract preparation: Following treatment (as indicated), ganglia in 6- or 7-day-old SCG explant cultures were separated from their neurites with a scalpel. Ganglia (including proximal neurite stumps) and neurites were collected separately, washed in PBS, and lysed and processed as above.

(c) Immunoblotting: Extracts were separated by standard SDS-PAGE on SDS polyacrylamide gels (6-13% depending on the proteins being detected) and transferred to Immobilon-P membrane (Millipore) using the Bio-Rad Mini- PROTEAN III wet transfer system . Blots were blocked and incubated with primary antibodies overnight (in Ix TBS p. H. 8.3, with 0.05% Tween 20 and 5% milk powder or 5% BSA) followed by the appropriate HRP-conjugated secondary antibody (1 hour at 1 : 2000-1 : 5000 dilution) and detection by ECL (Amersham Pharmacia Biotech) with washes between each stage. The following primary antibodies were used; mouse monoclonal anti-FLAG (1 : 2000-1 : 5000, Sigma, M2), rabbit polyclonal anti-Wld ^s (1 :4000, Wld lδ), mouse monoclonal anti- neurofilament heavy chain (NF-H) (1 : 2000, Sigma, N52), mouse monoclonal anti-Nmnat2 (2.0 μg/ml, Abeam, ab56980), mouse monoclonal anti-neuronal class βlll-Tubulin ( 1 : 2000-1 : 10,000, Covance, M MS-435P), and mouse monoclonal anti-Histones (1 : 1000, Millipore, MAB052). Relative band intensities on scanned autoradiographs were determined using ImageJ software. Statistical analysis was performed using a two-tailed t-test.

8. Microscopy and imaging Bright-field and fl uorescence images were captured on an Olympus 1X81 inverted fluorescence microscope using a Soft Imaging Systems (SIS) F-View camera linked to a PC running the appropriate SIS imaging software. Wherever possible, images of the same field of neurites or neuronal cell bodies were captured at the indicated time points after initial manipulation. Images were processed using Adobe Photoshop Elements 4.0. The intensity of FLAG immunostaining relative to eGFP fluorescence in individual injected neurons (Figure 3) was quantified using ImageJ software. Images were captured for analysis using identical microscope settings between samples for each channel. Time-lapse images of Nmnat2-eGFP transport were acquired 6 hours after injection of the expression vector using an Olympus Cell ^R imaging system comprising 1X81 microscope linked to a Hamamatsu ORCA ER camera and a 10Ox 1.45 NA apochromat objective. Cultures were maintained at 37°C in a Solent Scientific environment chamber. Wide-field epifluorescence images were captured at 2 Hz for 1 minute. ImageJ software plug-ins were used for analysis of the stacks (kymograph generation, and analysis of particle velocities) and conversion of an image stack into an annotated movie.

9. Quantification of neurite degeneration (a) Neurite blebbing: Membrane blebs more than twice the width of the associated neurite or neurite bundle were counted in a 100 x 100 μm box in bright-field images of the same neurites just after treatment (0 hours) and the indicated times afterwards. Bleb numbers are likely to be under-scored on highly degenerated neurites due to clustering of multiple blebs that cannot easily be individually differentiated or fragment loss. Statistical analysis was performed using a two-tailed t-test.

(b) Degeneration of fluorescent marker-labeled neurites: Numbers of morphologically normal and continuous Ds-Red2- or eGFP-labeled neurites were counted in the same field of distal neurites at various times after manipulation / injection. In siRNA injection experiments (Figure 4 and 5) and transection experiments (Figure 8) the percentage of healthy neurites remaining relative to the first timepoint was determined. Neurites were deemed unhealthy if they displayed abnormal morphology (including those with multiple swellings), or had undergone fragmentation. Neurite outgrowth still occurred from neurons injected with s\Control, s\Nmnatl or s\Nmnat3 but any neurites that grew into the analyzed field during the time course were not counted. In each case statistical analysis was performed using a two-tailed t-test. RESULTS

Example 1: Somatic protein synthesis suppression induces Wallerian- like degeneration

In this study, all protein translation in mouse superior cervical ganglia (SCG) explant cultures were inhibited, using two unrelated inhibitors, cycloheximide (CHX) and emetine to rule out non-specific effects. 1 μg/ml CHX, which suppresses global protein synthesis by more than 95% (Martin DP et al (1992) J Neurobiol 23 : 1205-1220; Kirkland RA, Franklin JL (2007) Neurosci Lett 411 : 52-55), not only stopped neurite outg rowth, but also induced widespread blebbing of distal neurites (Figure IA and 1C). 10 μg/ml CHX or 10 μM emetine caused more rapid and extensive blebbing of neurites, presumably due to more complete suppression of protein synthesis, followed by fragmentation and detachment shortly afterwards (Figure IA and 1C), similar to the degeneration of transected neurites. To test whether the degeneration is Wallerian-like, cultures were used from slow Wallerian degeneration (WId ⁵ ) mice and found a delay of over 48 hours (Figure IB and 1C). Similar results with rat SCG cultures and mouse dorsal root ganglion (DRG) cultures indicate that these events are not restricted to one species or neuron type (data not shown) . Delayed degeneration in Wld ^s cultures after inhibition of translation also shows that local translation of mRNAs in neurites is unlikely to underlie Wld ^s -mediated axon protection. Similarly, localized translation is not required in injured neurites for Wld ^s -mediated protection, and it is also not needed for Wallerian degeneration itself (data not shown).

Rapid cleavage of neurofilament heavy chain (NF-H) is an early molecular change that occurs as injury-induced Wallerian degeneration is initiated after the latent phase both in vitro and in vivo (Mack TG et al. (2001) Nat Neurosci 4: 1199-1206; Zhai Q et al. (2003) Neuron 39: 217-225). It was found that this also occurs after protein synthesis suppression in wild-type cultures but not in WId ⁵ cultures (Figure ID). Thus, molecular assays also indicate this degeneration is Wallerian-like.

Importantly, degeneration induced by protein synthesis suppression is not due to loss of neuronal viability but is a much earlier event independent of cell death. Even 7 days after treatment with 1 μg/ml CHX, long after complete degeneration of neurites, many SCG cell bodies retain the ability to re-grow neurites when this reversible inhibitor is removed (Figure IE). Most cell bodies in 7 day CHX-treated dissociated cultures also excluded Trypan Blue, further indicating neuron viability (data not shown).

To test directly whether a critical axon survival factor(s) has to be synthesized and delivered from cell bodies, compartmented cultures were used where distal neurites can be treated separately from cell bodies and proximal neurites (Figure 2). Neurites degenerated only when inhibitors were applied to the compartment containing neuronal cell bodies and proximal neurites. Tanslation inhibitors applied only to distal neurites caused no significant degeneration within this timeframe . Indeed , neurites continued to g row (data not shown ) . Th us, suppression of protein synthesis in the cell body triggers Wallerian-like neurite degeneration, providing strong support for the survival factor delivery hypothesis and suggesting the survival factor(s) is proteinaceous.

Example 2: Nmnat2 knock-down induces Wallerian-like degeneration

Pools of siRNAs (s\Nmnatl, 2 or 3) were used to knock down expression of the murine Nmnat isoforms and confirmed specificity for the appropriate isoform by assessing their ability to prevent expression of N-terminal FLAG-tagged Nmnat (FLAG-Nmnat) proteins in transfected HEK 293T cells and SCG neurons (Figure 3).

To assess the effect of Nmnat isoform knock-d own i n SCG ne u ro ns a microinjection-based strategy was used, which enabled consistent introduction of similar amounts of siRNA. Neurons in wild-type dissociated cultures were first injected with each siRNA pool, with DsRed2 expression allowing visualization of injected neurons and their neurites. Of the three Nmnat siRNA pools, only injection of s\Nmnat2 caused a significant reduction in the percentage of healthy neurites compared to the non-targeting siRNA pool (s\Control) (Figure 4A and 4B). Some of the neurites of the si/Vmnat2-injected neurons already appeared abnormal 24 hours after injection, when the entire lengths of the DsRed2- labeled neurites could first be clearly visualized, and almost all showed abnormal morphology or had completely degenerated 72 hours after injection. In contrast, injection of siControl, siNmnatl and s\Nmnat3 all caused relatively little degeneration (Figure 4A and 4B), and neurites continued to grow (data not shown). Combined injection of all three Nmnat siRNA pools did not significantly accelerate neurite degeneration relative to s\Nmnat2 alone (Figure 4C). Thus, Nmnat2 knock-down is sufficient to induce neurite degeneration, whereas knockdown of the other Nmnat isoforms has no clear effect on neurite survival . To confirm that the si/Vmnat2-induced neurite degeneration is Wallerian-like, WId ⁵ neurons were microinjected with s\Nmnat2 and found degeneration was completely blocked for at least 72 hours (Figure 4A and 4D).

To rule out a contribution from any off-target effect of the four individual siRNAs within the s\Nmnat2 pool, it was tested whether they could cause neurite degeneration when injected individually or in non-overlapping sub-pools (data not shown). One siRNA alone (J-059190-11), and two others in combination (J- 059190-10 and J-059190-12) triggered significant neurite degeneration that was similar to that induced by the complete pool. A clear combinatorial effect was also seen as J-059190-11 injected at the concentration it contributes to the s\Nmnat2 pool caused significantly less neurite degeneration than the pool itself. Together, these observations show that si/Vmnat2-induced neurite degeneration is due to knock-down of Nmnat2.

The si/Vm/7at2-induced neurite degeneration is distinctive, characterized by the appearance of multiple neuritic DsRed2-containing swellings and a distal-to- proximal 'dying-back' progression that appears to be independent of neuronal viability (Figure 4E and 4F). In contrast, the small amount of background neurite degeneration seen with all the siRNA pools (including s\Control) coincides with cell death and is faster and morphologically distinct (Figure 4G).

Some loss of neuronal viability occurred in these experiments, irrespective of the siRNA injected, but a small, additional decrease in neuronal viability following s\Nmnat2 knock-down was also apparent (data not shown). Even though this reduction in neuronal viability, relative to siControl, was proportionately much smaller than the reduction in neurite survival (data not shown), it was sought to completely exclude the possibility that cell death might be responsible for the si/Vm/7at2-associated neurite degeneration. It was possible to almost completely eliminate neuronal cell death in the s\Nmnat2 injection experiments in two ways (Figure 5). First, expression of the fluorescent marker was reduced after finding that toxicity was causing the (caspase-independent) background cell death . Second, it was found that the small si/Vm/7at2-associated decrease in neuronal viability could be prevented by the pan-caspase inhibitor z-VAD-fmk (Figure 5A), indicating that this death is caspase-dependent. Importantly, the amount of si/Vm/7at2-induced neurite degeneration was unchanged when cell death was reduced in these ways (compare Figure 5C and 5D to Figure 4A and 4B). This clearly shows that neurite degeneration precedes any associated loss of neuron viability in these experiments. It is also consistent with WId ⁵ - mediated protection of neurites (Figure 4D) being able to reduce si/Vm/7at2-associated neuronal loss to control levels (data not shown), despite the fact that Wld ^s cannot directly prevent neuronal cell death in SCG cultures (Deckwerth TL, Johnson EM, Jr. (1994) Dev Biol 165: 63-72). In addition, failure of z-VAD-fmk to prevent si/Vm/7at2-induced neurite degeneration provides further evidence that it is Wallerian-like as Wallerian degeneration has been shown to be unaffected by a range of anti-apoptotic interventions (Burne JF et a/ (1996) J Neurosci 16: 2064- 2073; Finn JT et al (2000) J Neurosci 20: 1333-1341; Whitmore AV et al (2003) Cell Death Differ 10: 260-261).

Thus, constitutive expression of endogenous Nmnat2 in SCG neurons is required to prevent spontaneous 'dying-back' Wallerian-like neurite degeneration. Importantly, these data also indicate that endogenous Nmnatl and Nmnat3 cannot compensate for loss of Nmnat2, despite the ability of these proteins to protect injured neurites when sufficiently overexpressed (Araki T et a/ (2004) Science 305: 1010-1013; Sasaki Y et a/ (2006) Neurosci 26: 8484-8491).

Example 3: Nmnat2 is the most labile Nmnat isoform

In the model of the invention, axon degeneration is initiated when survival factor levels drop below a critical threshold after synthesis or delivery is blocked. If Nmnat2 depletion acts as a trigger for Wallerian degeneration, Nmnat2 half-life should be compatible with the short latent phase of 4-6 hours before transected SCG neurites degenerate. Wld ^s , on the other hand, should be more stable to directly substitute for loss of endogenous Nmnat2. A direct comparison of the relative turnover rates of the FLAG-tagged murine Nmnat isoforms and Wld ^s in co-transfected HEK 293T cells (Figure 6A) showed that FLAG-tagged Nmnat2 is turned over rapidly when protein synthesis is blocked with an in vitro half-life of less than 4 hours. In contrast, there was minimal turnover of FLAG-tagged Wld ^s , Nmnatl and Nmnat3 up to 72 hours. Similar results were also obtained with C- terminal FLAG-tagged proteins (data not shown). It was also found that proteasome inhibition with MG-132 largely prevented turnover of FLAG-tagged Nmnat2 in these cells for at least 24 hours (Figure 6B). Importantly, turnover of endogenous Nmnat2 in SCG explants following protein synthesis inhibition was found to be similarly rapid (Figure 6C).

The half-life of Nmnat2 is also consistent with the time when wild-type SCG neurites become committed to degenerate after inhibition of translation (data not shown). Neurites exposed to CHX for just 4 hours remain healthy and continue to grow for over 5 days, but they become irreversibly committed to degenerate when exposed to CHX for just 8 hours, despite only minimal evidence of degeneration when CHX is removed. Intermediate treatment for 6 hours gave a mixed outcome. This suggests that degeneration of these neurites can be prevented by re-establishing synthesis of the labile survival factor(s) providing levels have not dropped below a critical threshold. The precise threshold can only be determined when the duration of downstream events leading to activation and execution of degeneration are better understood. Importantly, Wld ^s expression not only delays the onset of neurite degeneration following protein synthesis suppression, it also delays their commitment to degenerate at least 3-fold (data not shown).

Therefore, the half-life of Nmnat2, but not Nmnatl and Nmnat3, is compatible with its turnover being a trigger for Wallerian degeneration. Furthermore, the longer half-life of Wld ^s is consistent with it substituting for Nmnat2 loss for a prolonged period. Example 4: Endogenous Nmnat2 degrades rapidly and spontaneously in injured neurites

According to the model of the invention, the putative axon survival factor should also be present in neurites under normal conditions and its level in transected neurites should drop significantly prior to initiation of degeneration at 4-6 hours. Therefore, Nmnat2 levels were assessed in neurite-only extracts from SCG explant cultures at the time of transection, and 4 hours afterwards when the gross morphology of the transected neurites still appears relatively normal (Figure 7A). Neurite extracts contained significant amounts of Nmnat2 at the time of transection and this fell to ~30% of steady-state levels within 4 hours. Furthermore, loss of endogenous Nmnat2 occurs before cleavage of NF-H, which accompanies physical break-down of SCG neurites after injury (Zhai Q et al. (2003) Neuron 39: 217-225) or protein synthesis suppression (Figure ID), and before β-Tubulin degradation. An increase in Nmnat2 levels in the corresponding cell body / proximal neurite extracts 4 hours after separation of their transected distal neurites is also seen. This probably represents accumulation of Nmnat2 in a greatly reduced cellular volume.

Proteasome inhibition modestly extends the latent phase of Wallerian degeneration in SCG explant cultures (Zhai Q et al. (2003) Neuron 39: 217- 225), so it was tested whether this correlates with reduced turnover of endogenous Nmnat2 given that FLAG-tagged Nmnat2 is degraded via the proteasome in HEK cells (Figure 6B). Neurites treated with the proteasome inhibitor MG-132 appear relatively normal 8 hours after transection, with no associated NF-H cleavage, whereas untreated neurites show extensive physical and molecular signs of degeneration (Figure 7B). It was found that loss of Nmnat2 was also significantly reduced by MG-132 at this time (Figure 7B), consistent with depletion of endogenous Nmnat2 being a critical trigger for axon degeneration. The fact that Nmnat2 turnover was not completely prevented might explain why the duration of neurite protection by MG-132 is fairly limited (Zhai Q et al. (2003) Neuron 39: 217-225), although prolonged proteasome inhibition is also toxic to axons (Laser H et al (2003) J Neurosci Res 74: 906- 916). Nmnat2 loss within 4 hours in transected wild-type neurites seems unlikely to be a consequence of axon degeneration, as cytoskeletal proteins and neurite morphology are little altered at this timepoint (Figure 7A). However, to rule this out conclusively, Nmnat2 turnover was assessed in transected WId ⁵ neurites (Figure 7C), which do not degenerate for several days. Nmnat2 levels in WId ⁵ neurites fell with a remarkably similar timecourse to those in wild-type neurites. In contrast, cleavage of NF-H was prevented, showing that proteins that degrade as a consequence of degeneration are stabilized in WId ⁵ neurites. As predicted, Wld ^s levels in neurites also remained relatively constant. Indeed, levels of Wld ^s protein are only moderately reduced in neurites 48 hours after transection (data not shown).

Thus, Nmnat2 is rapidly depleted in distal stumps of injured neurites, as a result of natural turnover rather than a consequence of degeneration. This is consistent with Nmnat2 loss triggering Wallerian degeneration. The continued presence of Wld ^s in transected WId ⁵ neurites long after Nmnat2 is lost shows that Wld ^s does not act by stabilizing Nmnat2, but instead supports a model in which Wld ^s substitutes for the functionally-related Nmnat2.

Example 5: Net anterograde delivery of Nmnat2 by fast axonal transport

It was also found that an Nmnat2-eGFP fusion protein localizes to SCG neurites in highly defined particles shortly after being expressed (Figure 7D). In contrast, eGFP alone showed uniform distribution in neurites (data not shown). Particles containing Nmnat2-eGFP travel bi-directionally, but the majority move in an anterograde direction (72.2 ± 3.8% based on particle movements in 18 neurites). The average and maximal velocities of particles moving anterogradely (0.58 ± 0.09 and 1.52 ± 0.12 μm/sec) and retrogradely (0.29 ± 0.06 and 1.18 ± 0.10 μm/sec) are consistent with fast axonal transport. This indicates that Nmnat2 undergoes rapid net anterograde delivery from the cell body to neurites. This is another important prediction of the model of the invention, as rapid delivery is needed to replenish constant turnover of Nmnat2 in distal neurites (above).

Example 6: Nmnat2 protects transected neurites when highly overexpressed

If Nmnat2 is an endogenous axon survival factor, overexpression should protect transected neurites by preloading them with increased amounts of the protein. However, due to its relatively short half-life, protection should be highly dose- dependent and prolonged protection might only be achieved with very high levels of Nmnat2. In contrast, relatively long-lived Wld ^s should also confer protection at much lower levels.

The ability of exogenous expression of tagged Nmnat2 and Wld ^s to protect transected neurites was tested in a microinjection-based assay (data not shown). Dilution of the injected construct allowed controlled amounts to be reproducibly introduced into neurons. At low vector concentration (1 ng/ml), Wld ^s conferred robust protection to neurites for 24 hours after cutting, whereas Nmnat2 provided almost no protection (Figure 8A and 8B). In contrast, at 50- fold higher construct concentrations, both Nmnat2 and Wld ^s conferred protection to almost all cut neurites at 24 hours (Figure 8A and 8B). Although identical expression cassettes were used to give the best chance of equal expression of the two proteins in this assay, the shorter half-life of FLAG-Nmnat2 probably manifests as a lower steady-state level at the time of cut relative to FLAG-Wld ^s . Indeed, in transfected HEK 293T cells it was found that 2.5 times more FLAG- Nmnat2 construct was required to give steady-state protein levels approximately equal to FLAG-Wld ^s (and the other Nmnat isoforms). Importantly, whilst it was found that injection of the FLAG-Nmnat2 construct at 2.5 ng/ml gave slightly increased protection 24 hours after cut relative to 1 ng/ml, this was still greatly reduced protection compared to the FLAG-Wld ^s construct at the l ower concentration (Figure 8A). Thus exogenous Nmnat2 only confers significant protection of cut neurites when expressed at high levels, consistent with its short half-life, whilst more stable Wld ^s protects even at low levels.

Example 7: Degeneration Analysis using uncut wild-type SCG neurites

Injection of s\Nmnat2, into dissociated SCG neurons induced significant degeneration of uninjured distal neurites which was already apparent 24hr after injection (the first timepoint at which all distal neurites contained enough DsRed to be clearly imaged) and worsened over the next 48 hr (Figure 9A). In contrast to this, injection of s\Nmnatl and s\Nmnat3 had no significant effect on distal neurite survival relative to injection of a non-targeting pool of siRNA denoted as siControl (Figure 9B). These results indicate that Nmnat2 siRNA, but not Nmnatl or Nmnat3 siRNA, triggers Wallerian-like degeneration of SCG neuritis prior to a small but significant decrease in neuron survival.

Example 8: Degeneration Analysis using WIdS SCG neurites

This experiment was performed in an analogous manner to Example 7, with the exception of using WIdS rather than wild-type neurons. The data shown in Figure 10 demonstrates that Nmnat2-s\RNA induced neurite degeneration is virtually abolished for up to 72h in WIdS SCG neurons. This indicates that siNmnat2- induced degeneration in these cells is Wallerian in nature.

Example 9: Characteristics of neurite degeneration after injection with Nmnat2-s\RNA

Progressive distal-to-proximal dying-back degeneration of neurites over a 48 hr period characteristic of neurons injected with sil\lmant2 pool, is shown in Figure 11. Neurites run proximal to distal from top to bottom of the images. The images demonstrate that degeneration begins in distal portions of the neurite and spreads toward the neuronal cell body.

Example 10: Relative turn-over rates of FLAG-Nmnats and FLAG- WIdS in HEK 293 cells

Figure 12 shows summary data of band intensities from an immunoblot experiment of extracts of HEK 293 cells co-transfected with expression vectors for all FLAG-Nmnats and FLAG- WIdS. Twenty-four hours after transfection, cells were treated with lOμM emetine to block protein synthesis from the time points indicated. The data show that FLAG-Nmnat2 was rapidly lost after suppression of protein synthesis, while FLAG-WIdS and the other Nmnat isoforms are relatively stable.

Example 11: Reduced turn-over of FLAG-N mnat2 when the ubiquitin proteasome system is blocked Figure 13 shows summary data of band intensities from an immunoblot experiment of extracts of HEK 293 cells transfected as in Example 10. Cells were treated with lOμM emetine for the times indicated ± 20μM MG 132 to block proteasome-mediated degradation. Nmnat2 degradation is virtually abolished in the presence of MG132 confirming that Nmnat2 turn-over is mediated by the ubiquitin-proteasome system.

Example 12: Effect of expression of FLAG-tagged Nmnat2 on the survival of transected neu rites

Representative fluorescent images of transected DsRed-labeled neurites of wildtype

SCG neurons injected with 0.001 μg/μl or 0.05 μg/μl FLAG-Nmnat2 or FLAG-

WIdS expression vectors, together with DsRed expression vector 48 hr before cut. Images of neurites were captured at the indicated times after cut. Increased magnification of the framed region in each panel is shown for better visualisation of cut neurite morphology. The cut site is located at the top of each image. It was found that at a concentration of 0.001 μg/ml (estimated to represent injection of approximately 5-20 vector molecules per neuron) injection of the FLAG-Nmnat2 expression vector provided only minimal protection (Figure 14, left hand panels). However we predicted that a 50-fold higher concentration of FLAG-Nmnat2 (0.05 μg/ml vector concentration, or approximately 250-1000 vector molecules injected per neuron) should confer some protection. Injection of FLAG-Nmnat2 at the higher concentration conferred strong protection for at least 72 hr after cut (Figure 14, right-hand panels). These data show that FLAG- tagged Nmnat2 can protect neurites in a dose-dependent manner and provide strong support for the model of the invention in whichl\lmnat2 is a highly labile neurite survival factor.

SUMMARY

The data presented herein identifies endogenous Nmnat2 as a labile axon survival factor whose constant replenishment by anterograde axonal transport is a limiting factor for axon survival. Specific depletion of Nmnat2 is sufficient to induce Wallerian-like degeneration of uninjured axons which endogenous Nmnatl and Nmnat3 cannot prevent. Nmnat2 is by far the most labile Nmnat isoform and is depleted in distal stumps of injured neurites before Wallerian degeneration begins. Nmnat2 turnover is equally rapid in injured WId ⁵ neurites, despite delayed neurite degeneration, showing it is not a consequence of degeneration and also that Wld ^s does not stabilize Nmnat2. Depletion of Nmnat2 below a threshold level is necessary for axon degeneration since exogenous Nmnat2 can protect injured neurites when expressed at high enough levels to overcome its short half-life. Furthermore, proteasome inhibition slows both Nmnat2 turnover and neurite degeneration. The data presented herein concludes that endogenous Nmnat2 prevents spontaneous degeneration of healthy axons and without being bound by theory it is proposed that, when present, the more long-lived, functionally-related Wld ^s protein substitutes for Nmnat2 loss after axon injury. Therefore, endogenous Nmnat2 represents an exciting new therapeutic target for axonal disorders.