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WO2010092317A1 - Modulateur nnmnat2 - Google Patents

Modulateur nnmnat2 Download PDF

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Publication number
WO2010092317A1
WO2010092317A1 PCT/GB2009/051765 GB2009051765W WO2010092317A1 WO 2010092317 A1 WO2010092317 A1 WO 2010092317A1 GB 2009051765 W GB2009051765 W GB 2009051765W WO 2010092317 A1 WO2010092317 A1 WO 2010092317A1
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nmnat2
modulator
degeneration
neurites
agent
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Michael Philip Coleman
Jonathan Nicholas Gilley
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Babraham Institute
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Babraham Institute
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • 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.
  • Wallerian degeneration 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 ⁇ 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.
  • 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 ⁇ 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.
  • an Nmnat2 modulator for use as a neuroprotective medicament in the treatment of a neurodegenerative disorder.
  • 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).
  • NAD + nicotinamide adenine dinucleotide
  • NaAD nicotinic acid adenine dinucleotide
  • Three isoforms of the enzyme have been identified, expressed by three different genes in mammals: Nmnatl, Nmnat2 and Nmnat3.
  • 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.
  • 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.
  • modulator' refers to a molecule capable of altering the function of the Nmnat2 protein, either directly or indirectly.
  • the goal is to increase the level of enzyme activity, or increase the activity in the location needed for neuroprotection.
  • 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.
  • 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.
  • 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.
  • neuroprotective 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.
  • a pharmaceutical formulation 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.
  • a pharmaceutical composition comprising a modulator as hereinbefore defined.
  • 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.
  • solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and lower alkyl ethers of cellulose.
  • liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.
  • a method of treatment of a neurodegenerative disorder involving Wallerian degeneration comprising administering to a subject an Nmnat2 modulator.
  • an Nmnat2 modulator in the manufacture of a medicament for the treatment of a neurodegenerative disorder involving Wallerian degeneration.
  • a pharmaceutical composition comprising an Nmnat2 modulator for use in the treatment of a neurodegenerative disorder involving Wallerian degeneration.
  • Nmnat2 modulators 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.
  • 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.
  • the preferred route will depend on the general condition and age of
  • Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen- like syringe.
  • 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.
  • 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.
  • solutions for example, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated
  • 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.
  • 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.
  • polymers for example cellulose and derivatives, polysaccharides, for example dextran
  • 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.
  • 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.
  • 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 neuro
  • the modulator is intended for use as a neuroprotective medicament in the treatment of a neurodegenerative disorder resulting from neuronal injury.
  • the modulator is intended for use as a neuroprotective medicament in the treatment of a neurodegenerative disorder involving Wallerian degeneration resulting from neuronal injury.
  • 'injury' 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.
  • 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.
  • the modulator is intended for use as a neuroprotective medicament wherein the neurodegenerative disorder is caused by a neuronal injury resulting from a disease.
  • the modulator functions to prolong the functional half-life of Nmnat2.
  • 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.
  • 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.
  • the modulator functions to increase Nmnat2 activity.
  • 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.
  • 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.
  • an inhibitor of the ubiquitin- proteasome system for use in the treatment of a neurodegenerative disorder involving Wallerian degeneration.
  • a method of treatment of a neurodegenerative disorder involving Wallerian degeneration comprising administering to a subject an inhibitor of the ubiquitin-proteasome system.
  • an inhibitor of the ubiquitin-proteasome system in the manufacture of a medicament for the treatment of a neurodegenerative disorder involving Wallerian degeneration.
  • the inhibitor inhibits a specific component of the ubiquitin- proteasome system.
  • composition comprising an inhibitor of the ubiquitin-proteasome system for use in the treatment of a neurodegenerative disorder involving Wallerian degeneration.
  • the inhibitor inhibits a specific component of the ubiquitin- proteasome system.
  • 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.
  • the neuronal injury results from trauma.
  • the disorder is a neuronal injury induced by a chemotherapeutic agent.
  • chemotherapeutic agents used in cancer chemotherapy such as Taxol or vincristine, cause peripheral neuropathy which limits the maximum doses at which they can be used.
  • 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.
  • the neurodegenerative disorder is an ophthalmic disorder such as glaucoma or macular degeneration.
  • 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.
  • 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.
  • a method for demonstrating Wallerian degeneration comprising detecting and/or quantifying in a sample from a test subject, a biomarker as hereinbefore defined.
  • detecting 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.
  • 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.
  • CSF cerebrospinal fluid
  • 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.
  • SELDI SELDI
  • MALDI MALDI
  • MS mass spec
  • RP reverse phase
  • size permeation gel filtration
  • ion exchange affinity
  • HPLC HPLC
  • UPLC UPLC or other LC or LC-MS-based technique.
  • LC MS techniques include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA).
  • the biomarker may be directly detected, e.g. by SELDI or MALDI-TOF.
  • 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.
  • detecting and/or quantifying is performed using a biosensor, microanalytical, microengineered, microseparation or immunochromatography system.
  • biosensor' 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.
  • detecting and/or quantifying is performed by an immunological method.
  • 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.
  • 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.
  • particle immunoassays 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.
  • 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.
  • EIA Enzyme 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.
  • ELISA enzyme-linked immunosorbent assay
  • 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 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.
  • 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
  • biotin is labelled with avidin or streptavidin.
  • avidin or streptavidin is conventional technology for immunoassays, gene probe assays and (bio)sensors, but is an indirect immobilisation route rather than a direct one.
  • a biotinylated ligand e.g.
  • an 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.
  • 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.
  • the biosensor is an antibody.
  • antibody 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
  • 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 immunoglobulin molecule
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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/ ⁇ l) (B) Relative survival of (DsRed2-labeled) neurites of wild-type SCG neurons injected with siControl, siNmnatl, s ⁇ Nmnat2, or s ⁇ Nmnat3.
  • 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/ ⁇ l ) , and treated with 50 ⁇ M z-VAD-fmk or vehicle (DMSO) as indicated.
  • FIG. 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 ⁇ M 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.
  • 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 ⁇ M 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 ⁇ -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, ⁇ - Tu bul in represents a loading control.
  • 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.
  • Figure 8 (A) Protection of transected neurites by exogenous expression of FLAG-Nmnat2 or FLAG-Wld s .
  • 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/ ⁇ l vector concentration (*** p ⁇ 0.001 , t-test). There is no significant difference in protection 24h after transection at the 50 ng/ ⁇ l 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.
  • 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 ⁇ g 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.
  • DsRed2 Discosoma red fluorescent protein
  • eGFP enhanced green fluorescent protein
  • Dharmacon ON-TARGETp/us SMART pools of siRNA 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.
  • 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 ⁇ M aphidicolin (Calbiochem) was used to reduce proliferation and viability of small numbers of non-neuronal cells. Cultures were used after 5-7 days.
  • DMEM Dulbecco's Modified Eagle's Medium
  • 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
  • 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.
  • 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).
  • PAA sod i u m pyruvate
  • mice 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)
  • RNA from dissociated SCG neuronal cultures was isolated using RNeasy columns (Qiagen). 1 ⁇ g 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).
  • 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/ ⁇ l and individual siRNAs or sub-pools as indicated in the text, pDsRed2-N l at 50 ng/ ⁇ l , p EG FP-Cl at 10 ng/ ⁇ l , the N m n at2-eGFP expression construct at 50 ng/ ⁇ l, and FLAG-Nmnat/Wld s expression constructs or FLAG-empty control (pCMV Tag-2B) at 10 ng/ ⁇ l for siRNA-mediated knock- down analysis by immunostaining (Figure 3B) and at 1, 2.5 or 50 ng/ ⁇ l 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.
  • 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).
  • 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.
  • DMSO inhibitors of translation or vehicle
  • 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 ⁇ g 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.
  • 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.
  • mouse monoclonal anti-FLAG (1 : 2000-1 : 5000, Sigma, M2)
  • rabbit polyclonal anti-Wld s (1 :4000, Wld l ⁇
  • mouse monoclonal anti- neurofilament heavy chain NF-H
  • mouse monoclonal anti-Nmnat2 2.0 ⁇ g/ml, Abeam, ab56980
  • mouse monoclonal anti-neuronal class ⁇ lll-Tubulin 1 : 2000-1 : 10,000, Covance, M MS-435P
  • 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.
  • 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.
  • Neurite blebbing Membrane blebs more than twice the width of the associated neurite or neurite bundle were counted in a 100 x 100 ⁇ m 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.
  • Example 1 Somatic protein synthesis suppression induces Wallerian- like degeneration
  • 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).
  • NF-H neurofilament heavy chain
  • Nmnat2 knock-down is sufficient to induce neurite degeneration, whereas knockdown of the other Nmnat isoforms has no clear effect on neurite survival .
  • WId 5 neurons were microinjected with s ⁇ Nmnat2 and found degeneration was completely blocked for at least 72 hours ( Figure 4A and 4D).
  • 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).
  • the small amount of background neurite degeneration seen with all the siRNA pools coincides with cell death and is faster and morphologically distinct (Figure 4G).
  • Nmnat2 is the most labile Nmnat isoform
  • 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.
  • Nmnat2 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.
  • 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).
  • 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.
  • Nmnat2 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 ⁇ -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).
  • 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).
  • 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.
  • cleavage of NF-H was prevented, showing that proteins that degrade as a consequence of degeneration are stabilized in WId 5 neurites.
  • 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).
  • 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.
  • Example 5 Net anterograde delivery of Nmnat2 by fast axonal transport
  • Nmnat2-eGFP fusion protein localizes to SCG neurites in highly defined particles shortly after being expressed ( Figure 7D).
  • 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 ⁇ m/sec) and retrogradely (0.29 ⁇ 0.06 and 1.18 ⁇ 0.10 ⁇ m/sec) are consistent with fast axonal transport.
  • 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).
  • Nmnat2 is an endogenous axon survival factor
  • overexpression should protect transected neurites by preloading them with increased amounts of the protein.
  • protection should be highly dose- dependent and prolonged protection might only be achieved with very high levels of Nmnat2.
  • relatively long-lived Wld s should also confer protection at much lower levels.
  • 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 lO ⁇ M 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 lO ⁇ M emetine for the times indicated ⁇ 20 ⁇ M 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
  • 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 ⁇ g/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 ⁇ g/ml vector concentration, or approximately 250-1000 vector molecules injected per neuron) should confer some protection.
  • Nmnat2 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.
  • proteasome inhibition slows both Nmnat2 turnover and neurite degeneration.
  • 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.

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Abstract

La présente invention porte sur un modulateur nNmnat2 utile en tant que médicament neuroprotecteur dans le traitement de troubles neurodégénératifs, en particulier, mais non exclusivement, des troubles impliquant la dégénérescence de Wallerian du tissu neuronal, sur l'utilisation de nNmnat2 en tant que biomarqueur pour la dégénérescence de Wallerian, sur un procédé consistant à démontrer une dégénérescence de Wallerian en utilisant un biomarqueur à base de nNmnat2, sur un kit de diagnostic destiné à détecter une dégénérescence de Wallerian, sur un procédé de criblage d'un modulateur nNmnat2, et sur un modulateur nNmnat2 identifié à l'aide du procédé de criblage mentionné ci-dessus.
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WO2011135332A3 (fr) * 2010-04-27 2011-12-22 Babraham Institute Modulateur de la nmn
US20120328526A1 (en) * 2011-06-27 2012-12-27 University Of Maryland, Baltimore Modulation of Nad+ Activity in Neuropathophysiological Conditions and Uses Thereof
WO2020172565A1 (fr) * 2019-02-22 2020-08-27 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Procédés et matériaux permettant d'augmenter ou de maintenir des niveaux de polypeptide d'adénylyl transférase-2 (nmnat2) de nicotinamide mononucléotide

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WO2011135332A3 (fr) * 2010-04-27 2011-12-22 Babraham Institute Modulateur de la nmn
US20120328526A1 (en) * 2011-06-27 2012-12-27 University Of Maryland, Baltimore Modulation of Nad+ Activity in Neuropathophysiological Conditions and Uses Thereof
WO2020172565A1 (fr) * 2019-02-22 2020-08-27 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Procédés et matériaux permettant d'augmenter ou de maintenir des niveaux de polypeptide d'adénylyl transférase-2 (nmnat2) de nicotinamide mononucléotide

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