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(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: 13.04.2016 Bulletin 2016/15

(21) Application number: 15193068.2

(22) Date of filing: 03.06.2005

(51) Int Cl.:A61K 38/50 (2006.01) A61K 38/45 (2006.01)

A61K 31/7084 (2006.01) C07H 19/20 (2006.01)

A61K 48/00 (2006.01) C12N 9/12 (2006.01)

(84) Designated Contracting States: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR

(30) Priority: 04.06.2004 US 577233 P04.01.2005 US 641330 P

(62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 05790283.5 / 1 755 391

(71) Applicant: Washington UniversitySaint Louis, MO 63130 (US)

(72) Inventors: • MILBRANDT, Jeffrey

St. Louis, MO 63105 (US)

• ARAKI, ToshiyukiKodaira, Tokyo 187-0031 (JP)

• SASAKI, YoSt. Louis, MO 63144 (US)

(74) Representative: Smaggasgale, Gillian HelenWP Thompson 138 Fetter LaneLondon EC4A 1BT (GB)

Remarks: This application was filed on 4.11.2015 as a divisional application to the application mentioned under INID code 62.

(54) METHODS AND COMPOSITIONS FOR TREATING NEUROPATHIES

(57) Methods of treating or preventing axonal degra-dation in neuropathic diseases in mammals are dis-closed. The methods can comprise administering to themammal an effective amount of an agent that acts byincreasing sirtuin activity in diseased and/or injured neu-rons. The methods can also comprise administering tothe mammal an effective amount of an agent that actsby increasing NAD activity in diseased and/or injuredneurons. Also disclosed are methods of screening agentsfor treating a neuropathies and recombinant vectors fortreating or preventing neuropathies.

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Description

GOVERNMENT INTERESTS

[0001] This work was supported at least in part withfunds from the federal government under U.S.P.H.S.5RO1 NS40745. The U.S. Government may have certainrights in the invention.

RELATED APPLICATION DATA

[0002] This application claims benefit under 35 U.S.C.§ 119(e) to United States Provisional Application SerialNo. 60/577,233, filed June 4, 2004 and United StatesProvisional Application Serial No. 60/641,330, filed Jan-uary 4,2005. These applications are incorporated hereinin their entireties by reference.

FIELD

[0003] This invention relates generally to diseases andconditions involving neurons and, more particularly, tomethods and compositions for treating or preventing neu-ropathies and other diseases and conditions involvingneurodegeneration. Also included are methods of iden-tifying agents for treating or preventing neuropathies.

BACKGROUND

[0004] Axon degeneration occurs in a variety of neu-rodegenerative diseases such as Parkinson’s and Alzhe-imer’s diseases as well as upon traumatic, toxic orischemic injury to neurons. Such diseases and conditionsare associated with axonopathies including axonal dys-function. One example of axonopathy is Wallerian de-generation (Waller, Philos Trans R. soc. Lond.140:423-429, 1850), which occurs when the distal portionof the axon is severed from the cell body. The severedaxon rapidly succumbs to degeneration. Axonopathycan, therefore, be a critical feature of neuropathic dis-eases and conditions and axonal deficits can be an im-portant component of the patient’s disability.

SUMMARY

[0005] Accordingly, the present inventors have suc-ceeded in discovering that axonal degeneration can bediminished or prevented by increasing NAD activity indiseased and/or injured neurons. It is believed that theincreased NAD activity can act to increase sirtuin activitywhich then produces a decrease in axonal degenerationof injured neuronal cells. Thus, one approach to prevent-ing axonal degeneration can be by activating sirtuin mol-ecules, i.e. SIRT1 in injured mammalian axons. The ac-tivation of SIRT1 can be through direct action on theSIRT1 molecule or by increasing the supply of nicotina-mide adenine dinucleotide (NAD) which acts as a sub-strate for the histone/protein deacetylase activity of

SIRT1. The activation of SIRT1 results in a decrease inseverity of axonal degeneration or a prevention of axonaldegeneration. It is also believed possible that the in-crease in NAD activity could act through other mecha-nisms not involving sirtuin. Thus, increasing NAD activity,which may act through increasing SIRT1 activity orthrough one or more other mechanisms or both can di-minish or prevent axonal degeneration in injured mam-malian axons.[0006] Thus, in various embodiments, the present in-vention is directed to a method of treating or preventinga neuropathy in a mammal and, in particular, in a humanin need thereof. The method can comprise administeringan effective amount of an agent that acts to increasesirtuin activity and, in particular, SIRT1 activity in dis-eased and/or injured neurons.[0007] In various embodiments, the agent can in-crease SIRT1 activity through increasing NAD activity. Itis believed that increasing NAD activity can increase sir-tuin activity because NAD can act as a substrate ofSIRT1. Such agents can include NAD or NADH, a pre-cursor of NAD, an intermediate in the NAD salvage path-way or a substance that generates NAD such as a nico-tinamide mononucleotide adenylyltransferase (NMNAT)or a nucleic acid encoding a nicotinamide mononucle-otide adenylyltransferase. The nicotinamide mononucle-otide adenylyltransferase can be an NMNAT1 protein.[0008] In various embodiments, the agent can also actto directly increase SIRT1 activity and as such, the agentcan be a sirtuin polypeptide or a nucleic acid encoding asirtuin polypeptide or a substance such as a stilbene, achalcone, a flavone, an isoflavanone, a flavanone or acatechin. Such compounds can include a stilbene select-ed from the group consisting of resveratrol, piceatannol,deoxyrhapontin, trans-stilbene and rhapontin; a chal-cone selected from the group consisting of butein, isoli-quiritigen and 3,4,2’,4’,6’-pentahydroxychalcone; a fla-vone selected from the group consisting of fisetin,5,7,3’,4’,5’-pentahydroxyflavone, luteolin, 3,6,3’,4’-tet-rahydroxyflavone, quercetin, 7,3’,4’,5’-tetrahydroxyfla-vone, kaempferol, 6-hydroxyapigenin, apigenin,3,6,2’,4’-tetrahydroxyflavone, 7,4’-dihydroxyflavone,7,8,3’,4’-tetrahydroxyflavone, 3,6,2’,3’-tetrahydroxyfla-vone, 4’-hydroxyflavone, 5,4’-dihydroxyflavone, 5,7-di-hydroxyflavone, morin, flavone and 5-hydroxyflavone; anisoflavone selected from the group consisting of daidzeinand genistein; a flavanone selected from the group con-sisting of naringenin, 3,5,7,3’,4’-pentahydroxyflavanone,and flavanone or a catechin selected from the group con-sisting of (-)-epicatechin, (-)-catechin, (-)-gallocatechin,(+)-catechin and (+)-epicatechin.[0009] In various embodiments, the invention can alsoinvolve methods of treating a neuropathy by administer-ing to a mammal and, in particular, a human, an effectiveamount of an agent that acts by increasing nuclear NADactivity in diseased and/or injured neurons and/or sup-porting cells such as, for example, glia, muscle cells, fi-broblasts, etc.

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[0010] Such agent can be NAD or NADH, nicotinamidemononucleotide, nicotinic acid mononucleotide or nico-tinamide riboside or derivatives thereof; or an enzymethat generates NAD such as a nicotinamide mononucle-otide adenylyltransferase or a nucleic acid encoding anenzyme that generates NAD such as a nucleic acid en-coding a nicotinamide mononucleotide adenylyltrans-ferase or an agent that increases expression of a nucleicacid encoding an enzyme in a pathway that generatesNAD or an agent that increases activity and/or stabilityof an enzyme in a pathway that generates NAD or anagent that increases NAD activity. The nicotinamidemononucleotide adenylyltransferase can be an NMNAT1protein.[0011] In various embodiments, the invention can alsoinvolve methods of treating or preventing an optic neu-ropathy in a mammal in need thereof. The methods cancomprise administering to the mammal an effectiveamount of an agent that acts by increasing NAD activityin diseased and/or injured neurons. Administering to themammal can comprise administering to the eye, in par-ticular by administering the agent with a sustained re-lease delivery system or by administering a sustain re-lease pellet comprising the agent to the eye.[0012] The agent can be NAD or NADH, nicotinamidemononucleotide, nicotinic acid mononucleotide or nico-tinamide riboside; or an enzyme that generates NADsuch as a nicotinamide mononucleotide adenylyltrans-ferase; or a nucleic acid encoding an enzyme that gen-erates NAD such as a nucleic acid encoding a nicotina-mide mononucleotide adenylyltransferase or an agentthat increases NAD activity. The nicotinamide mononu-cleotide adenylyltransferase can be an NMNAT1 proteinor an NMNAT3 protein.[0013] In various embodiments of the methods of thepresent invention, the neuropathy associated with axonaldegradation can be any of a number of neuropathies suchas, for example, those that are hereditary or congenitalor associated with Parkinson’s disease, Alzheimer’s dis-ease, Herpes infection, diabetes, amyotrophic lateralsclerosis, a demyelinating disease, ischemia or stroke,chemical injury, thermal injury, AIDS and the like. In ad-dition, neurodegenerative diseases not mentioned aboveas well as a subset of the above mentioned diseases canalso be treated with the methods of the present invention.Such subsets of diseases can include Parkinson’s dis-ease or non-Parkinson’s diseases, Alzheimer’s diseaseor non-Alzheimer’s diseases and so forth.[0014] In various embodiments, the present inventionis also directed to methods of screening agents for treat-ing a neuropathy in a mammal. The methods can com-prise administering to neuronal cells in vitro or in vivo, acandidate agent, producing an axonal injury to the neu-ronal cells and detecting a decrease in axonal degener-ation of the injured neuronal cells. In various embodi-ments, the method can comprise detecting an increasein NAD activity produced by a candidate agent, in a celland, in particular, in a neuronal cell. The increase in NAD

activity can be an increase in nuclear NAD activity.[0015] Methods are also provided for screening agentsthat increase sirtuin activity in neurons as well as forscreening agents that increase NAD biosynthetic activityin neurons. The methods can comprise administering tomammalian neuronal cells in vitro or in vivo a candidateagent, producing an axonal injury to the neuronal cellsand detecting a decrease in axonal degeneration of theinjured neuronal cells. Such methods can in some em-bodiments be primary screening methods in which sec-ondary assays further delineate activity as associatedwith sirtuin activity or with NAD and enzymes or compo-nents of NAD biosynthetic or salvage pathways.[0016] In various embodiments of the screening meth-ods of the present invention, axonal injury can be pro-duced by a number of methods including chemically in-juring the neuronal cells, thermally injuring the neuronalcells, oxygen-depriving the neuronal cells, and physicallyinjuring the neuronal cells.[0017] A recombinant vector is also provided in variousembodiments. The vector can comprise a promoter op-eratively linked to a sequence encoding a mammalianNMNAT1 protein or NMNAT3 protein. In various aspectsof such embodiments, the recombinant vector can be alentivirus or an adeno-associated virus.[0018] Also provided in various embodiments, is a re-combinant vector comprising a promoter operativelylinked to a sequence encoding a SIRT1 protein. In variousaspects of such embodiments, the recombinant vectorcan be a lentivirus or an adeno-associated virus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 illustrates that NMNAT1 activity of theWlds fusion protein produces a delayed degeneration ofinjured axons showing:

A) in vitro Wallerian degeneration in lentivirus-infect-ed dorsal root ganglia (DRG) neuronal explant cul-tures expressing Wlds protein or EGFP wherein tu-bulin βIII-immunoreactive neurites are shown beforetransection and 12,24,48, and 72 hr after transection(Scale Bar=1mm and the "*" denotes the location ofthe cell bodies prior to removal; andB) in vitro Wallerian degeneration in lentivirus-infect-ed DRG neurons expressing EGFP only, Wlds pro-tein, Ufd2a portion (70 residues) of Wlds proteinfused to EGFP (Ufd2a(1-70)-EGFP),Ufd2a(1-70)-EGFP with C-terminal nuclear localiza-tion signal, NMNAT1 portion of Wlds protein fusedto EGFP, dominant-negative Ufd2a(Ufd2a(P1140A)), or Ufd2a siRNA construct in whichrepresentative images of neurites and quantitativeanalysis data of remaining neurite numbers (per-centage of remaining neurites relative to pre-transection 6 S.D.) at the indicated time-point witheach construct (bottom left) are shown and the "*"indicates significant difference (p<0.0001) with

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EGFP-infected neurons; also showing EGFP signalbefore transection confirming transgene expression(bottom row; Scale bar =50mm) and immunoblotanalysis confirming protein expression by lentiviralgene transfer and siRNA downregulation of Ufd2aprotein (bottom right panels).

[0020] Figure 2 illustrates that increased NAD supplyprotects axons from degeneration after injury showing:

A) Enzymatic activity of wild type and mutant Wlds

and NMNAT1 proteins in which lysates were pre-pared from HEK293 cells expressing the indicatedprotein were assayed for NAD production using nico-tinamide mononucleotide as a substrate and theamount of NAD generated in 1 h was converted toNADH, quantified by fluorescence intensity, and nor-malized to total protein concentration showing thatboth mutants have essentially no enzymatic activity;andB) In vitro Wallerian degeneration in lentivirus-infect-ed DRG neurons expressing NMNAT1 or Wlds pro-tein, mutants of these proteins that lack NAD-syn-thesis activity NMNAT1(W170A) and Wlds(W258A),or EGFP wherein the bar chart shows the quantita-tive analysis data of the number of remaining neur-ites at indicated time-point for each construct (per-centage of remaining neurites relative to pre-transection 6 S.D.) and the "*" indicates significantdifference (p<0.0001) with EGFP-infected neurons;C) Protein expression in lentivirus-infected cells de-tected by immunoblot analysis using antibodies tothe 6XHis tag; andD) DRG neuronal explant expressing eitherNMNAT1 or EGFP (control) cultured with 0.5 mM vin-cristine wherein representative images of neurites(phase-contrast; Bar=1mm) are shown at the indi-cated times after vincristine addition and quantifica-tion of the protective effect at the indicated timepoints is plotted as the area covered by neurites rel-ative to that covered by neurites prior to treatment.

[0021] Figure 3 illustrates that axonal protection re-quires pre-treatment of neurons with NAD prior to injuryshowing:

A) in vitro Wallerian degeneration using DRG ex-plants cultured in the presence of various concen-trations of NAD added 24 hr prior to axonal transec-tion; andB) DRG explants preincubated with 1mM NAD for 4,8, 12, 24, or 48 h prior to transection wherein the barchart shows the number of remaining neurites ineach experiment (percentage of remaining neuritesrelative to pre-transection 6 S.D.) at each of the in-dicated time points and the "*" indicates significantaxonal protection compared to control (p<0.0001).

[0022] Figure 4 illustrates that NAD-dependent AxonalProtection is mediated by SIRT1 activation showing:

A) In vitro Wallerian degeneration using DRG explantcultures preincubated with 1 mM NAD alone (control)or in the presence of either 100 mM Sirtinol (a Sir2inhibitor) or 20 mM 3-aminobenzimide (3AB, a PARPinhibitor);B) in vitro Wallerian degeneration using DRG explantcultures incubated with resveratrol (10, 50 or 100mM); andC) left: in vitro Wallerian degeneration using DRGexplant cultures infected with lentivirus expressingsiRNA specific for each member of the SIRT family(SIRT1-7) wherein the bar chart shows the quanti-tative analysis of the number of remaining neurites(percentage of remaining neurites relative to pre-transection 6 S.D.) at indicated time-point for eachcondition and the "*" indicates points significantly dif-ferent than control (<0.0001);middle table: The effectiveness of each SIRT siRNA(expressed as % of wild type mRNA level) using qRT-PCR in infected NIH3T3 cells; andright: immunoblot using antibodies to SIRT1 to showdecreased expression of SIRT1 in the presence ofSIRT1 siRNA which effectively blocked NAD de-pendent axonal protection.

[0023] Figure 5 illustrates the mammalian NAD biosyn-thetic pathway in which predicted mammalian NAD bio-synthesis is illustrated based on the enzymatic expres-sion analysis and studies from yeast and lower eukary-otes (Abbreviation used; QPRT, quinolinate phosphori-bosyltransferase; NaPRT, nicotinic acid phosphoribosyl-transferase; NmPRT, nicotinamide phosphoribosyltrans-ferase; Nrk, nicotinamide riboside kinase; NMNAT., nico-tinamide mononucleotide adenylyltransferase; QNS,NAD synthetase)[0024] Figure 6 illustrates expression analysis of NADbiosynthetic enzymes in mammal showing (A) NAD bio-synthesis enzyme mRNA levels after 1, 3, 7, and 14 daysafter nerve transection in rat DRG were determined byqRT-PCR in which the expression level was normalizedto glyceraldehydes-3-phosphate dehydrogenase ex-pression in each sample and is indicated relative to theexpression level in non-axotomized DRG; (B) neurite de-generation introduced by incubation DRG in 1 or 0.1 mMrotenone for indicated time and NAD synthesis enzymemRNA levels were determined by qRT-PCR as describedin the text.[0025] Figure7 illustrates the subcellular localization ofNMNAT enzymes and their ability to protect axon show-ing (A) in vitro Wallerian degeneration assay using len-tivirus infected DRG neuronal explant cultures express-ing NMNAT1, cytNMNAT1, NMNAT3, or nucNMNAT3 inwhich representative pictures taken at 12 and 72 hoursafter transaction are shown; (B) Subcellular localizationof NMNAT1, cytNMNAT1, NMNAT3, or nucNMNAT3 in

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HEK 293T cells using immunohistochemistry with anti-body against 6xHis tag to detect each proteins and stain-ing of the cells with the nuclear marker dye (bisbenzim-ide) for comparison to determine the nuclear vs. cyto-plasmic location of each protein (Scale bar = 25 mm); (C)enzymatic activity of wild type and mutant NMNAT1 andNMNAT3 in which 6xHis tagged each protein was purifiedfrom lysate of HEK293T cells expressing NMNAT1,cytNMNAT1, NMNAT3, nucNMNAT3 in which theamount of NAD generated after1 hour at 37 deg wasconverted NADH, quantified and normalized to proteinconcentration; (D) protein expression of NMNAT1,cytNMNAT1, NMNAT3, and nucNMNAT3 by lentivirusgene transfer confirmed by immunoblot analysis ofHEK293T cells infected with each of the virus and (E) invitro Wallerian degeneration assay using lentivirus infect-ed DRG neuronal explant cultures expressing NMNAT1,cytNMNAT1, NMNAT3, or nucNMNAT3 showing quan-titative analysis data of remaining neurite numbers at 12,24, 48, and 72 hours after axotomy.[0026] Figure 8 illustrates exogenous application ofNAD biosynthetic substrates and their ability to protectaxon showing (A) in vitro Wallerian degeneration assayusing DRG neuronal explant cultures after exogenousapplication of NAD, NmR with representative picturestaken at 12, 24, 48, and 72 hours after transaction areshown; (B) in vitro Wallerian degeneration assay usingDRG neuronal explant cultures after exogenous applica-tion of Na, Nam, NaMN, NMN, NaAD, NAD, and NmRshowing quantitative analysis data of remaining neuritenumbers at 12, 24, 48, and 72 hours after axotomy areshown; (C) DRG neuronal explants infected with NaPRTexpressing lentivirus and incubated with or without 1 mMof Na for 24 hours before axotomy, in in vitro Walleriandegeneration assay showing quantitative analysis dataof remaining neurite numbers at 12, 24, 48, and 72 hoursafter axotomy.[0027] Figure 9 illustrates optic nerve transection afterintravitreal injection of NAD biosynthetic substrates NAD,NMN, NmR, or Nam was injected into intravitreal com-partment of left rat eye and allowed to incorporate retinalganglion cells for 24 hours after which, left optic nervewas transected by eye enucleation and right and left opticnerves were collected at 4 days after transection andanalyzed by Western blotting in which optic nervestransected from mice without any treatment prior to ax-otomy were used for negative control; showing in thefigure, the quantitative analysis data of percentage of re-maining neurofilament immunoreactivity from transectedoptic nerve relative to non-transected 6 S.D.

DETAILED DESCRIPTION

[0028] The present invention involves methods andcompositions for treating neuropathies. The methods cancomprise administering to a mammal an effective amountof a substance that increases NAD activity in diseasedand/or injured neurons. It is believed that the increased

NAD activity can act to increase sirtuin activity which thenproduces a decrease in axonal degeneration of injuredneuronal cells compared to axonal degeneration that oc-curs in injured neuronal cells not treated with the agent.Such decrease in axonal degeneration can include acomplete or partial amelioration of the injury to the neu-ron. It is also believed possible that the increase in NADactivity could act through other mechanisms not involvingsirtuin molecules to produce or to contribute to the pro-duction of a decrease in axonal degeneration.[0029] Seven known sirtuin molecules referenced asSIRT’s make up the Sir2 family of histone/proteindeacetylases in mammals and all such sirtuin moleculesare included within the scope of the present invention.The seven human sirtuins, SIRT1-SIRT7, are NAD-de-pendent histone/protein deacetylases which are de-scribed more fully in connection with NCBI LocusLink IDNos. 23411, 22933, 23410, 23409, 23408, 51548 and51547, respectively (see http://www.nc-bi.nlm.hih.gov/LocusLink/). Said NCBI LocusLink refer-ence sites are hereby incorporated by reference. In var-ious embodiments, the methods and compositions of thepresent invention can increase activity of any one or moreof the sirtuins and, in particular, various methods of thepresent invention increase activity of SIRT1.[0030] By activity of a substance, reference is made toeither the concentration of the particular substance orfunctional effectiveness of the substance. Concentrationof a substance can be increased by numerous factorsincluding, for example, increasing synthesis, decreasingbreakdown, increasing bioavailability of the substance ordiminishing binding of the substance or otherwise in-creasing the available amount of free substance. Increas-ing functional effectiveness can result, for example, froma change in molecular conformation, a change in the con-ditions under which the substance is acting, a change insensitivity to the substance, and the like. Increasing ac-tivity with respect to sirtuin molecules is intended to meanincreasing concentration or enhancing functional effec-tiveness or increasing the availability of NAD or increas-ing the flux through one or more biosynthetic pathwaysfor NAD or any combination thereof.[0031] Neuropathies can include any disease or con-dition involving neurons and/or supporting cells, such asfor example, glia, muscle cells, fibroblasts, etc., and, inparticular, those diseases or conditions involving axonaldamage. Axonal damage can be caused by traumaticinjury or by non-mechanical injury due to diseases orconditions and the result of such damage can be degen-eration or dysfunction of the axon and loss of functionalneuronal activity. Disease and conditions producing orassociated with such axonal damage are among a largenumber of neuropathic diseases and conditions. Suchneuropathies can include peripheral neuropathies, cen-tral neuropathies, and combinations thereof. Further-more, peripheral neuropathic manifestations can be pro-duced by diseases focused primarily in the central nerv-ous systems and central nervous system manifestations

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can be produced by essentially peripheral or systemicdiseases.[0032] Peripheral neuropathies involve damage to theperipheral nerves and such can be caused by diseasesof the nerves or as the result of systemic illnesses. Somesuch diseases can include diabetes, uremia, infectiousdiseases such as AIDs or leprosy, nutritional deficien-cies, vascular or collagen disorders such as atheroscle-rosis, and autoimmune diseases such as systemic lupuserythematosus, scleroderma, sarcoidosis, rheumatoidarthritis, and polyarteritis nodosa. Peripheral nerve de-generation can also result from traumatic, i.e mechanicaldamage to nerves as well as chemical or thermal damageto nerves. Such conditions that injure peripheral nervesinclude compression or entrapment injuries such as glau-coma, carpal tunnel syndrome, direct trauma, penetrat-ing injuries, contusions, fracture or dislocated bones;pressure involving superficial nerves (ulna, radial, or per-oneal) which can result from prolonged use of crutchesor staying in one position for too long, or from a tumor;intraneural hemorrhage; ischemia; exposure to cold orradiation or certain medicines or toxic substances suchas herbacides or pesticides. In particular, the nerve dam-age can result from chemical injury due to a cytotoxicanticancer agent such as, for example, a vinca alkaloidsuch as vincristine. Typical symptoms of such peripheralneuropathies include weakness, numbness, paresthesia(abnormal sensations such as burning, tickling, prickingor tingling) and pain in the arms, hands, legs and/or feet.The neuropathy can also be associated with mitochon-drial dysfunction. Such neuropathies can exhibit de-creased energy levels, i.e. decreased levels of NAD andATP.[0033] The peripheral neuropathy can also be a met-abolic and endocrine neuropathy which includes a widespectrum of peripheral nerve disorders associated withsystemic diseases of metabolic origin. These diseases,some of which are mentioned earlier, include diabetesmellitus, hypoglycemia, uremia, hypothyroidism, hepaticfailure, polycythemia, amyloidosis, acromegaly, porphy-ria, disorders of lipid/glycolipid metabolism, nutritional/vi-tamin deficiencies, and mitochondrial disorders, amongothers. The common hallmark of these diseases is in-volvement of peripheral nerves by alteration of the struc-ture or function of myelin and axons due to metabolicpathway dysregulation.[0034] Neuropathies also include optic neuropathiessuch as glaucoma; retinal ganglion degeneration suchas those associated with retinitis pigmentosa and outerretinal neuropathies; optic nerve neuritis and/or degen-eration including that associated with multiple sclerosis;traumatic injury to the optic nerve which can include, forexample, injury during tumor removal; hereditary opticneuropathies such as Kjer’s disease and Leber’s hered-itary optic neuropathy; ischemic optic neuropathies, suchas those secondary to giant cell arteritis; metabolic opticneuropathies such as neurodegenerative disesases in-cluding Leber’s neuropathy mentioned earlier, nutritional

deficiencies such as deficiencies in vitamins B12 or folicacid, and toxicities such as due to ethambutol or cyanide;neuropathies caused by adverse drug reactions and neu-ropathies caused by vitamin deficiency. Ischemic opticneuropathies also include non-arteritic anterior ischemicoptic neuropathy.[0035] Neurodegenerative diseases that are associat-ed with neuropathy or axonopathy in the central nervoussystem include a variety of diseases. Such diseases in-clude those involving progressive dementia such as, forexample, Alzheimer’s disease, senile dementia, Pick’sdisease, and Huntington’s disease; central nervous sys-tem diseases affecting muscle function such as, for ex-ample, Parkinson’s disease, motor neuron diseases andprogressive ataxias such as amyotrophic lateral sclero-sis; demyelinating diseases such as, for example multiplesclerosis; viral encephalitides such as, for example,those caused by enteroviruses, arboviruses, and herpessimplex virus; and prion diseases. Mechanical injuriessuch as glaucoma or traumatic injuries to the head andspine can also cause nerve injury and degeneration inthe brain and spinal cord. In addition, ischemia and strokeas well as conditions such as nutritional deficiency andchemical toxicity such as with chemotherapeutic agentscan cause central nervous system neuropathies.[0036] The term "treatment" as used herein is intendedto include intervention either before or after the occur-rence of neuronal injury. As such, a treatment can preventneuronal injury by administration before a primary insultto the neurons occurs as well as ameliorate neuronalinjury by administration after a primary insult to the neu-rons occurs. Such primary insult to the neurons can in-clude or result from any disease or condition associatedwith a neuropathy. "Treatment" also includes preventionof progression of neuronal injury. "Treatment" as usedherein can include the administration of drugs and/or syn-thetic substances, the administration of biological sub-stances such as proteins, nucleic acids, viral vectors andthe like as well as the administration of substances suchas neutraceuticals, food additives or functional foods.[0037] The methods and compositions of the presentinvention are useful in treating mammals. Such mammalsinclude humans as well as non-human mammals. Non-human mammals include, for example, companion ani-mals such as dogs and cats, agricultural animals suchlive stock including cows, horses and the like, and exoticanimals, such as zoo animals.[0038] Substances that can increase sirtuin activity inmammals can include polyphenols some of which havebeen described earlier (see for example Howitz et al.,Nature 425:191-196, 2003 and supplementary informa-tion that accompanies the paper all of which is incorpo-rated herein by reference). Such compounds can includestilbenes such as resveratrol, piceatannol, deoxyrhapon-tin, trans-stilbene and rhapontin; chalcone such asbutein, isoliquiritigen and 3,4,2’,4’,6’-pentahydroxychal-cone and chalcone; flavones such as fisetin, 5,7,3’,4’,5’-pentahydroxyflavone, luteolin, 3,6,3’,4’-tetrahydroxyfla-

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vone, quercetin, 7,3’,4’,5’-tetrahydroxyflavone, kaemp-ferol, 6-hydroxyapigenin, apigenin, 3,6,2’,4’-tetrahydrox-yflavone, 7,4’-dihydroxyflavone, 7,8,3’,4’-tetrahydroxy-flavone, 3,6,2’,3’-tetrahydroxyflavone, 4’-hydroxyfla-vone, 5,4’-dihydroxyflavone, 5,7-dihydroxyflavone,morin, flavone and 5-hydroxyflavone; isoflavones suchas daidzein and genistein; flavanones such as naringen-in, 3,5,7,3’,4’-pentahydroxyflavanone, and flavanone orcatechins such as (-)-epicatechin, (-)-catechin, (-)-gallo-catechin, (+)-catechin and (+)-epicatechin.[0039] Additional polyphenols or other substance thatincrease sirtuin deacetylase activity can be identified us-ing assay systems described herein as well as in com-mercially available assays such as fluorescent enzymeassays (Biomol International L.P., Plymouth Meeting,Pennsylvania). Sinclair et al. also disclose substancesthat can increase sirtuin activity (Sinclair et al.,WO2005/02672 which is incorporated in its entirety byreference).[0040] In various embodiments, other substances canincrease sirtuin activity indirectly by increasing NAD ac-tivity as a result of the particular sirtuin functioningthrough NAD-dependent histone/protein deacetylase ac-tivity. NAD activity can be increased by administration ofNAD or NADH as well as by synthesizing NAD. NAD canbe synthesised through three major pathways, the denovo pathway in which NAD is synthesized from tryp-tophan, the NAD salvage pathway in which NAD is gen-erated by recycling degraded NAD products such asnicotinamide (Lin et al. Curent Opin. Cell Biol.15:241-246, 2003; Magni et al., Cell Mol. Life Sci.61:19-34, 2004) and the nicotinamide riboside kinasepathway in which nicotinamide riboside is converted tonicotinamide mononucleotide by nicotinamide ribosidekinase (Bieganowski et al., Cell 117:495-502, 2004).Thus, administering to injured neurons, a precursor ofNAD in the de novo pathway such as, for example, tryp-tophan or nicotinate and/or substances in the NAD sal-vage pathway such as, for example, nicotinamide, nico-tinic acid, nicotinic acid mononucleotide, or deamido-NAD and/or substances in the nicotinamide riboside ki-nase pathway such as, for example, nicotinamide ribo-side or nicotinamide mononucleotide, could potentiallyincrease NAD activity. As shown below, nicotinamidemononucleotide, nicotinic acid mononucleotide or nico-tinamide riboside, in addition to NAD, protected againstaxonal degeneration to a similar extent as did NAD, how-ever, nicotinic acid and nicotinamide did not. The in-creased NAD activity can then increase sirtuin his-tone/protein deacetylase activity in the injured neuronsand diminish or prevent axonal degeneration. In addition,it is believed that other substances can act by increasingenzyme activity or by increasing levels of NAD, nicotina-mide mononucleotide, nicotinic acid mononucleotide,nicotinamide riboside or sirtuin enzymes or by decreas-ing degredation of NAD, nicotinamide mononucleotide,nicotinic acid mononucleotide, nicotinamide riboside orsirtuin enzymes.

[0041] In various embodiments, NAD can be increasedin injured neurons by administering enzymes that syn-thesize NAD or nucleic acids comprising enzymes thatsynthesize NAD. Such enzymes can include an enzymein the de novo pathway for synthesizing NAD, an enzymeof the NAD salvage pathway or an enzyme of the nicoti-namide riboside kinase pathway or a nucleic acid encod-ing an enzyme in the de novo pathway for synthesizingNAD, an enzyme of the NAD salvage pathway or an en-zyme of the nicotinamide riboside kinase pathway and,in particular, an enzyme of the NAD salvage pathwaysuch as, for example, a nicotinamide mononucleotideadenylyltransferase (NMNAT) such as NMNAT1. Thus,in one non-limiting example, administration of an NMNATsuch as NMNAT1 or NMNAT3 or a nucleic acid compris-ing a sequence encoding an NMNAT such as NMNAT1or NMNAT3 can diminish or prevent axonal degenerationin injured neurons.[0042] The human NMNAT1 enzyme (E.C.2.7.7.18) isrepresented according to the GenBank Assession num-bers for the human NMNAT1 gene and/or pro-tein:NP_073624; NM_022787; AAL76934; AF459819;and NP_073624; AF314163. A variant of this gene isNMNAT-2 (KIAA0479), the human version of which canbe found under GenBank Accession numbersNP_055854 and NM_015039.[0043] As used herein, the term "percent identical" or"percent identity" or "% identity" refers to sequence iden-tity between two amino acid sequences or between twonucleotide sequences. Identity can each be determinedby comparing a position in each sequence which may bealigned for purposes of comparison. When an equivalentposition in the compared sequences is occupied by thesame base or amino acid, then the molecules are iden-tical at that position; when the equivalent site occupiedby the same or a similar amino acid residue (e.g., similarin steric and/or electronic nature), then the moleculescan be referred to as homologous (similar) at that posi-tion. Expression as a percentage of homology, similarity,or identity refers to a function of the number of identicalor similar amino acids at positions shared by the com-pared sequences. Various alignment algorithms and/orprograms may be used, including FASTA, BLAST, or EN-TREZ. FASTA and BLAST are available as a part of theGCG sequence analysis package (University of Wiscon-sin, Madison, Wis.), and can be used with, e.g., defaultsettings. ENTREZ is available through the National Cent-er for Biotechnology Information, National Library of Med-icine, National Institutes of Health, Bethesda, Md. In oneembodiment, the percent identity of two sequences canbe determined by the GCG program with a gap weightof 1, e.g., each amino acid gap is weighted as if it werea single amino acid or nucleotide mismatch between thetwo sequences. Other techniques for alignment are de-scribed in Methods in Enzymology, vol. 266: ComputerMethods for Macromolecular Sequence Analysis (1996),ed. Doolittle, Academic Press, Inc., a division of HarcourtBrace & Co., San Diego, California, USA. Preferably, an

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alignment program that permits gaps in the sequence isutilized to align the sequences. The Smith-Waterman isone type of algorithm that permits gaps in sequence align-ments. See Meth. Mol. Biol. 70: 173-187 (1997). Also,the GAP program using the Needleman and Wunschalignment method can be utilized to align sequences. Analternative search strategy uses MPSRCH software,which runs on a MASPAR computer. MPSRCH uses aSmith-Waterman algorithm to score sequences on amassively parallel computer. This approach improvesability to pick up distantly related matches, and is espe-cially tolerant of small gaps and nucleotide sequence er-rors. Nucleic acid-encoded amino acid sequences canbe used to search both protein and DNA databases. Da-tabases with individual sequences are described in Meth-ods in Enzymology, ed. Doolittle, supra. Databases in-clude Genbank, EMBL, and DNA Database of Japan(DDBJ).[0044] A "variant" of a polypeptide refers to a polypep-tide having the amino acid sequence of the polypeptidein which is altered in one or more amino acid residues.The variant may have "conservative" changes, whereina substituted amino acid has similar structural or chem-ical properties (e.g., replacement of leucine with isoleu-cine). A variant may have "nonconservative" changes(e.g., replacement of glycine with tryptophan). Analogousminor variations may also include amino acid deletionsor insertions, or both. Guidance in determining whichamino acid residues may be substituted, inserted, or de-leted without abolishing biological or immunological ac-tivity may be found using computer programs well knownin the art, for example, LASERGENE software (DNAS-TAR).[0045] The term "variant," when used in the context ofa polynucleotide sequence, may encompass a polynu-cleotide sequence related to that of a particular gene orthe coding sequence thereof. This definition may alsoinclude, for example, "allelic," "splice," "species," or "pol-ymorphic" variants. A splice variant may have significantidentity to a reference molecule, but will generally havea greater or lesser number of polynucleotides due to al-ternate splicing of exons during mRNA processing. Thecorresponding polypeptide may possess additional func-tional domains or an absence of domains. Species var-iants are polynucleotide sequences that vary from onespecies to another. The resulting polypeptides generallywill have significant amino acid identity relative to eachother. A polymorphic variation is a variation in the poly-nucleotide sequence of a particular gene between indi-viduals of a given species. Polymorphic variants also mayencompass "single nucleotide polymorphisms" (SNPs)in which the polynucleotide sequence varies by one base.The presence of SNPs may be indicative of, for example,a certain population, a disease state, or a propensity fora disease state.[0046] An agent that can be used in treating or pre-venting a neuropathy in accordance with the methodsand compositions of the present invention can be com-

prised by a nicotinamide mononucleotide adenylyltrans-ferase (NMNAT) or a polynucleotide encoding anNMNAT. In particular, the agent can be an enzyme hav-ing NMNAT activity and at least 50% identity with a hu-man NMNAT1 or at least 50% identity with a humanNMNAT3, at least 60% identity with a human NMNAT1or at least 60% identity with a human NMNAT3, at leastidentity with a human NMNAT1 or at least 70% identitywith a human NMNAT3, at least 80% identity with a hu-man NMNAT1 or at least 80% identity with a humanNMNAT3, at least 90% identity with a human NMNAT1or at least 90% identity with a human NMNAT3, at least95% identity with a human NMNAT1 or at least 95% iden-tity with a human NMNAT3. Moreover, the agent can becomprised by a human NMNAT1, a human NMNAT3 ora conservatively substituted variants thereof.[0047] The agent can also be comprised by a polynu-cleotide having at least 50% identity with a nucleic acidencoding a human NMNAT1 or a polynucleotide havingat least 50% identity with a nucleic acid encoding a hu-man NMNAT3, a polynucleotide having at least 60%identity with a nucleic acid encoding a human NMNAT1or a polynucleotide having at least 60% identity with a.nu-cleic acid encoding a human NMNAT3, a polynucleotidehaving at least 70% identity with a nucleic acid encodinga human NMNAT1 or a polynucleotide having at least70% identity with a nucleic acid encoding a humanNMNAT3, a polynucleotide having at least 80% identitywith a nucleic acid encoding a human NMNAT1 or a poly-nucleotide having at least 80% identity with a nucleic acidencoding a human NMNAT3, a polynucleotide having atleast 90%.identity with a nucleic acid encoding a humanNMNAT1 or a polynucleotide having at least 90% identitywith a nucleic acid encoding a human NMNAT3, a poly-nucleotide having at least 95% identity with a nucleic acidencoding a human NMNAT1 or a polynucleotide havingat least 95% identity with a nucleic acid encoding a hu-man NMNAT3. The agent can also be a polynucleotideencoding a human NMNAT1, a human NMNAT3 or avariant thereof.[0048] The agent can also be comprise by a sirtuinpolypeptide or a nucleic acid encoding a sirtuin polypep-tide. In particular, the agent can comprise an enzymehaving SIRT activity and at least 50% identity with a hu-man SIRT1, at least 60% identity with a human SIRT1,atleast 70% identity with a human SIRT1,at least 80% iden-tity with a human SIRTl,at least 90% identity with a humanSIRT1, or at least 95% identity with a human SIRT1.Moreover, the agent can be comprised by a humanSIRT1 or a conservatively substituted variants thereof.The agent can also be comprised by a polynucleotidehaving at least 50% identity with a nucleic acid encodinga human SIRT1, a polynucleotide having at least 60%identity with a nucleic acid encoding a human SIRT1, apolynucleotide having at least 70% identity with a nucleicacid encoding a human SIRT1, a polynucleotide havingat least 80% identity with a nucleic acid encoding a hu-man SIRT1, a polynucleotide having at least 90% identity

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with a nucleic acid encoding a human SIRT1 or a poly-nucleotide having at least 95% identity with a nucleic acidencoding a human SIRT1. Moreover, the agent can com-prise a polynucleotide encoding a human SIRT1 or a var-iant thereof.[0049] Administration can be by any suitable route ofadministration including buccal, dental, endocervical, in-tramuscular, inhalation, intracranial, intralymphatic, in-tramuscular, intraocular, intraperitoneal, intrapleural, in-trathecal, intratracheal, intrauterine, intravascular, intra-venous, intravesical, intranasal, ophthalmic, oral, otic,biliary perfusion, cardiac perfusion, priodontal, rectal,spinal subcutaneous, sublingual, topical, intravaginal,transermal, ureteral, or urethral. Dosage forms can beaerosol including metered aerosol, chewable bar, cap-sule, capsule containing coated pellets, capsule contain-ing delayed release pellets, capsule containing extendedrelease pellets, concentrate, cream, augmented cream,suppository cream, disc, dressing, elixer, emulsion, en-ema, extended release fiber, extended release film, gas,gel, metered gel, granule, delayed release granule, ef-fervescent granule, chewing gum, implant, inhalant, in-jectable, injectable lipid complex, injectable liposomes,insert, extended release insert, intrauterine device, jelly,liquid, extended release liquid, lotion, augmented lotion,shampoo lotion, oil, ointment, augmented ointment,paste, pastille, pellet, powder, extended release powder,metered powder, ring, shampoo, soap solution, solutionfor slush, solution/drops, concentrate solution, gel form-ing solution/drops, sponge, spray, metered spray, sup-pository, suspension, suspension/drops, extended re-lease suspension, swab, syrup, tablet, chewable tablet,tablet containing coated particles, delayed release tablet,dispersible tablet, effervescent tablet, extended releasetablet, orally disintegrating tablet, tampon, tape or tro-che/lozenge.[0050] Intraocular admistration can include adminis-tration by injection including intravitreal injection, by eye-drops and by trans-scleral delivery.[0051] Administration can also be by inclusion in thediet of the mammal such as in a functional food for hu-mans or companion animals.[0052] It is also contemplated that certain formulationscontaining the compositions that increase sirtuin activityare to be administered orally. Such formulations are pref-erably encapsulated and formulated with suitable carri-ers in solid dosage forms. Some examples of suitablecarriers, excipients, and diluents include lactose, dex-trose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, calcium silicate, microc-rystalline cellulose, polyvinylpyrrolidone, cellulose, gela-tin, syrup, methyl cellulose, methyl- and propylhydroxy-benzoates, talc, magnesium, stearate, water, mineral oil,and the like. The formulations can additionally includelubricating agents, wetting agents, emulsifying and sus-pending agents, preserving agents, sweetening agentsor flavoring agents. The compositions may be formulatedso as to provide rapid, sustained, or delayed release of

the active ingredients after administration to the patientby employing procedures well known in the art. The for-mulations can also contain substances that diminish pro-teolytic degradation and promote absorption such as, forexample, surface active agents.[0053] The specific dose can be calculated accordingto the approximate body weight or body surface area ofthe patient or the volume of body space to be occupied.The dose will also depend upon the particular route ofadministration selected. Further refinement of the calcu-lations necessary to determine the appropriate dosagefor treatment is routinely made by those of ordinary skillin the art. Such calculations can be made without undueexperimentation by one skilled in the art in light of theactivity in assay preparations such as has been de-scribed elsewhere for certain compounds (see for exam-ple, Howitz et al., Nature 425:191-196, 2003 and supple-mentary information that accompanies the paper). Exactdosages can be determined in conjunction with standarddose-response studies. It will be understood that theamount of the composition actually administered will bedetermined by a practitioner, in the light of the relevantcircumstances including the condition or conditions to betreated, the choice of composition to be administered,the age, weight, and response of the individual patient,the severity of the patient’s symptoms, and the chosenroute of administration.[0054] In various embodiments, the present inventionalso provides methods of screening candidate agents.In one such assay method, agents are tested for effec-tiveness in decreasing or preventing axonal degenera-tion of injured neuronal cells. Candidate agents are thusadministered to neuronal cells subjected to injury and adecrease in axonal degeneration of the injured neuronalcells is detected. Typically, the agent is added prior toproducing the injury, however, in some instances, theinjury can be produced before addition of the candidatecompound. The method can be performed in vitro or invivo. The in vitro tests can be performed using any of anumber of mammalian neuronal cells under a variety ofexperimental conditions in which injury is elicited. An ex-ample of mammalian neuronal cell-types that can beused are primary dorsal root ganglion cells injured byeither transection and removal of the neuronal cell bodyor growth in media containing vincristine as describedbelow. The in vivo tests can be performed in intact ani-mals such as, for example, a mouse model of peripheralnerve regeneration (Pan et al., J. Neurosci.23:11479-11488, 2003) or mouse model of progressivemotor neuronopathy (Schmalbruch et al., J. Neuropathol.Exp. Neurol. 50:192-204, 1991; Ferri et al., Current Biol.13:669-673, 2003).[0055] Because, the mechanism of decreasing or pre-venting neuronal injury results from an increase in NAD-dependent histone/protein deacetylase activity of sirtuinmolecules, the assay method can also be used as a pri-mary screen for substances that either increase sirtuinactivity directly or through increasing NAD activity. Thus

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the methods above can be used to screen for agents thatinrease NAD biosynthetic activity or agents that increasesirtuin activity in neurons.[0056] Recombinant vectors that serve as carriers fora nucleic acid encoding a sirtuin molecule or an enzymefor biosynthesis of NAD are also within the scope of thepresent invention. Such recombinant vectors can com-prise a promoter operatively linked to a sequence encod-ing a mammalian NMNAT1 protein or a mammalian sir-tuin protein such as a SIRT1 protein. Such recombinantvectors can be any suitable vector such as, for examplea lentivirus or an adeno-associated virus. Any suitablepromoter can be also used such as, for example a ubiq-uitin promoter, a CMV promoter or a β-actin promoter.[0057] The invention can be further understood by ref-erence to the examples which follow.

EXAMPLE 1

[0058] This example demonstrates that transected ax-ons from neurons tranfected with a vector expressingWlds protein show a delayed degeneration compared tocontrol neurons.[0059] In wlds mice, Wallerian degeneration in re-sponse to axonal injury has been shown to be delayed(Gillingwater, et al., JPhysiol, 534:627-639, 2001). Ge-netic analysis has shown that the wlds mutation compris-es an 85 kb tandem triplication, which results in overex-pression of a chimeric nuclear molecule (Wlds protein).This protein is composed of the N-terminal 70 AAs of Ufd(ubiquitin fusion degradation protein)2a, a ubiquitin chainassembly factor, fused to the complete sequence of nico-tinamide mononucleotide adenylyltransferase1(NMNAT1), an enzyme in the NAD salvage pathway thatgenerates NAD within the nucleus. The Wlds protein hasNMNAT activity but lacks ubiquitin ligase function, sug-gesting that axonal protection is derived from either in-creased NMNAT1 activity or a ’dominant negative’ inhi-bition of Ufd2a function.[0060] To identify the mechanism of delayed axonaldegeneration mediated by the Wlds protein, we employedan in-vitro Wallerian degeneration model. Primary DRGexplant neurons were infected with lentivirus expressingthe appropriate proteins, and axons were injured by ei-ther removal of the neuronal cell body (transection) orgrowth in vincristine (toxic).[0061] Lentiviral expression constructs were kindlyprovided by D. Baltimore (Lois, et al., Science295:868-72, 2002). We modified the FUGW vector togenerate a general expression shuttle FUIV (ubiquitinpromoter - gene of interest-IRES-enhanced YFP (Ve-nus)) vector that enables enhanced YFP expression incells that express the gene-of-interest. The following pro-teins, each with a hexahistidine tag at the C-terminus,were cloned into the FUIV vector: Wlds chimeric mutantprotein; Ufd2a containing a point mutation (P1140A),which has previously been shown to inhibit wild-typeUfd2a function as a "dominant-negative"(Ufd2a(P

1140)). The following genes were cloned into FUGW vec-tor: 1) The first 70 AAs of Ufd2a (the portion containedin Wlds protein) fused to the N-terminus of EGFP(Ufd2a(1-70)-EGFP) or EGFP with nuclear localizationsignal at the C-terminal (Ufd2a(1-70)-nucEGFP). 2) TheNMNAT1 portion of Wlds protein fused to the C-terminusof EGFP (EGFP-NMNAT1).[0062] The murine cDNA for Ufd2a/Ube4b(mKIAA0684) was provided by Kazusa DNA ResearchInstitute. Murine cDNAs for NMNAT1 (accessionnumber: BC038133) were purchased from ATCC. PCR-mediated mutagenesis was used to generate point mu-tations in Ufd2a, NMNAT1 and Wlds.[0063] We generated siRNA constructs in the FSP-sivector generated from the FUGW backbone by replacingthe ubiquitin promoter and GFP cDNA with the humanU6 promoter and Pol I termination signal followed by theSV40 promoter-puromycin-N-acetyl-transferase gene.Cloning of siRNA construct was performed as describedpreviously, so that the siRNA is transcribed from the U6promoter (Castanotto, et al., RNA, 8:1454-60, 2002). Se-quences used for siRNA downregulation of protein ex-pression were 1692-1710 of SIRT1, 1032-1050 of SIRT2,538~556 of SIRT3, 1231~1249 of SIRT4, 37~55 ofSIRT5, 1390~1408 of SIRT6, and 450-468 of SIRT7. Theintegrity of each lentiviral expression and siRNA con-struct was confirmed by DNA sequencing.[0064] Mouse DRG explants from E12.5 embryos werecultured in the presence of 1 nM nerve growth factor.Non-neuronal cells were removed from the cultures byadding 5-fluorouracil to the culture medium. Transectionof neurites was performed at 10-20 DIV using an 18-gauge needle to remove the neuronal cell bodies. Incu-bation with β-nicotinamide adenine dinucleotide (Sigma)or Sirtinol (Calbiochem) was performed using conditionsindicated in the text or figures.[0065] Lentiviral expression vectors were generatedusing HEK293T cells as described above. For confirma-tion of lentivirus-derived protein expression, HEK293Tcells were infected with lentivirus and cells were lysed 3days after infection. These lysates were analyzed by im-munoblot to using anti-His tag monoclonal antibody (Qia-gen) to detect expression of the respective hexahistidine-tagged proteins. Lentiviral infection of DRG neurons wasperformed by incubating ~106-107 pfu/ml virus with theDRG explant for 24 h beginning 3-7 days prior to axonaltransection. The infected neurons were examined underan inverted fluorescent microscope to insure detectablelentivirus-mediated transgene expression in >95% ofneurons.[0066] Quantitative analysis of axonal degenerationwas performed as previously described (Zhai, et al., Neu-ron 39:217-25, 2003). Briefly, the cultures were exam-ined using phase contrast microscopy at the indicatedtimes. Axons with a fragmented, non-refractile appear-ance were designated as "degenerated." At each timepoint, at least 200 singly distinguishable axons wereblindly scored from several randomly taken images of

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each culture. Each condition was tested in triplicate ex-plants in each experiment. Results were obtained from2-4 independent experiments for each condition. Statis-tical analysis was performed by Student’s T test. For cal-culations of neurite-covered area, digitally captured im-ages from quadruplicate samples of two independent ex-periments were analyzed using analysis 3.1 software(Soft Imaging System, Lakewood, CO).[0067] We found that transected axons from neuronsexpressing the Wlds protein degenerated with the de-layed kinetics characteristic of neurons derived from wlds

(Buckmaster, et al., Eur J Neurosci 7:1596-602,1995)mice as shown in Figure 1A.[0068] Next, we compared axonal degeneration aftertransection in neurons that overexpress Wlds protein withthose that express the Ufd2a or NMNAT1 portions thatmake up the Wlds protein linked to EGFP. Results areshown in Figure 1B.[0069] We found that expression of EGFP-NMNAT1delayed axonal degeneration comparable to Wlds proteinitself, whereas the N-terminal 70 AA of Ufd2a (fused toEGFP), either targeted to the nucleus or cytoplasm, didnot affect axonal degeneration. Quantification of theseeffects was performed by counting the percentage of re-maining neurites at various times after removal of neu-ronal cell bodies. This analysis showed that EGFP-NMNAT1, like Wlds protein itself, resulted in a >10-foldincrease in intact neurites 72 hr after injury. To furtherexclude direct involvement of the UPS in Wlds protein-mediated axonal protection, we examined the effect ofUfd2a inhibition using either a dominant-negative Ufd2amutant or an Ufd2a siRNA construct. However, neitherof these methods resulted in delayed axonal degradationin response to axotomy. Together, these experimentsdemonstrated that the NMNAT1 portion of the Wlds pro-tein is responsible for the delayed axonal degenerationobserved in wlds mice.

EXAMPLE 2

[0070] This example shows that mutations in the fulllength NMNAT1 and in Wlds protein abolish the axonalprotective effects of the proteins.[0071] NMNAT1 is an enzyme in the nuclear NAD sal-vage pathway that catalyzes the conversion of nicotina-mide mononucleotide (NMN) and nicotinate mononucle-otide (NaMN) to NAD and nicotinate adenine mononu-cleotide (NaAD), respectively. The axonal protection ob-served in NMNAT1 overexpressing neurons could bemediated by its ability to synthesize NAD (i.e. its enzy-matic activity), or perhaps, by other unknown functionsof this protein. To address this question, we used theNMNAT1 crystal structure to identify several residuespredicted to participate in substrate binding. A mutationin one of these residues (W170A) was engineered intofull length NMNAT1 and Wlds protein. In vitro enzymaticassays confirmed that both of these mutant proteins wereseverely limited in their ability to synthesize NAD (Fig.

2A). Each of these mutants and their respective wild typecounterparts were introduced into neurons to assesstheir ability to protect axons from degradation. We foundthat neurons expressing these enzymatically inactivemutants had no axonal protective effects (Fig. 2A), indi-cating that NAD/NaAD-production is responsible for theability of NMNAT1 to prevent axonal degradation.

EXAMPLE 3

[0072] This example illustrates that increased NMNATactivity in neurons injured with vincristine also show adelayed axonal degradation.[0073] In addition to mechanical transection, axonalprotection in wlds mice is also observed against otherdamaging agents such as ischemia and toxins (Coleman,et al., Trends Neurosci 25:532-37, 2002; Gillingwater, etal., J Cereb Blood Flow Metab 24:62-66, 2004). Wesought to determine whether increased NMNAT activitywould also delay axonal degradation in response to othertypes of axonal injury such as vincristine, a cancer chem-otherapeutic reagent with well-characterized axonal tox-icity. Neurons expressing either NMNAT1 or EGFP (con-trol) were grown in 0.5mM vincristine for up to 9 d. Wefound that axons of neurons expressing NMNAT1 main-tained their original length and refractility, whereas axonsemanating from neurons expressing EGFP gradually re-tracted and had mostly degenerated by day 9 (Fig. 2B).These results indicate that NMNAT activity by itself canprotect axons from a number of insults and mediate theprotective effects observed in wlds mice.

EXAMPLE 4

[0074] This example shows that exogenously admin-istered NAD can protect injured neurons from axonal de-generation.[0075] Previous experiments have shown that neuro-nal cells express membrane proteins that can bind andtransport extracellular NAD into the cell (Bruzzone, et al.,Faseb J 15:10-12, 2001). This encouraged us to inves-tigate whether exogenously administered NAD couldprevent axonal degeneration. We added various concen-trations of NAD to neuronal cultures prior to axonaltransection and examined the extent of axonal degrada-tion. We found that 0.1-1 mM NAD added 24 hr prior toaxotomy significantly delayed axonal degeneration, al-though exogenously applied NAD was slightly less effec-tive in protecting axons than lentivirus mediatedNMNAT1 expression (Fig. 3A). These results provide di-rect support for the idea that increased NAD supply canprevent axonal degradation.

EXAMPLE 5

[0076] This example illustrates that NAD was requiredprior to the removal of the neuronal cell bodies to protectthe injured neurons from axonal degeneration.

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[0077] To gain insights into the mechanism of NAD-dependent axonal protection (NDAP), we examinedwhether NAD was required prior to the removal of theneuronal cell bodies, or whether direct exposure of thesevered axons to high levels of NAD was sufficient toprovide protection (Fig. 3B). Neuronal cultures were pre-pared and 1 mM NAD was added to the culture mediumat the time of axonal transection or at various times (4 to48 hr) prior to injury.[0078] We found that administering NAD at the time ofaxonal transection or, for up to 8 hr prior to injury, hadno protective effects on axons. However, significant axonsparing was observed when neurons were incubated withNAD for longer periods of time prior to injury, with thegreatest effects occurring after at least 24 h of NAD pre-treatment, These results indicate that NAD dependentaxonal protection is not mediated by a rapid post-trans-lational modification within the axons themselves.[0079] The requirement for extended exposure to NADof the intact neurons to prevent axonal degradation inresponse to injury suggests that the protective processrequires de novo transcriptional and/or translationalevents. Interestingly, both the Wlds protein and NMNAT1are located within the nucleus (data not shown). Similarly,most enzymes that make up the NAD salvage pathwayin yeast are also compartmentalized in the nucleus. Wecompared NAD levels in wild type and NMNAT1 express-ing DRG neurons using sensitive microscale enzymaticassays (Szabo, et al., Proc Natl Acad Sci USA,93:1753-58 ,1996), however no changes in overall cel-lular NAD levels were found (data not shown). This issimilar to observations in yeast, in which activation of thisnuclear pathway did not change overall levels of NAD(Anderson, et al., J Biol Chem, 277:18881-90,2002; Huh,et al., Nature, 425:686-91, 2003). Furthermore, levels oftissue NAD in the brains of wild type and wlds mice aresimilar despite the increased levels of NMNAT activity inwlds mice (Mack, et al., Nat Neurosci, 4:1199-206, 2001).These data suggest that an NAD-dependent enzymaticactivity in the nucleus, as opposed to cytoplasmic NAD-dependent processes, is likely to mediate the axonal pro-tection observed in response to increased NMNAT ac-tivity.

EXAMPLE 6

[0080] This example shows that inhibition of Sir2 is in-volved in NAD-dependent axonal protection.[0081] The Sir2 family of protein deacetylases and po-ly(ADP-ribose) polymerase (PARP) are the major NAD-dependent nuclear enzymatic activities. Sir2 is an NAD-dependent deacetylase of histones and other proteins,and its activation is central to promoting increased lon-gevity in yeast and C. elegans (Bitterman, et al., MicrobiolMol Biol Rev, 67:376-99, 2003; Hekimi, et al., Science299:1351-54, 2003). PARP is activated by DNA damageand is involved in DNA repair (S.D. Skaper, Ann NYAcadSci, 993:217-28 and 287-88, 2003). These enzymes, in

particular the Sir2 proteins, have generated great interestin recent years as they provide a potential link betweencaloric restriction and its effects on the ageing process.The importance of these NAD-dependent enzymes inregulating gene activity, prompted us to investigate theirrole in the self-destructive process of axonal degradation.We therefore tested whether inhibitors of Sir2 (Sirtinol)and PARP (3-aminobenzamide (3AB)) could affect NAD-dependent axonal protection (NDAP) (Fig. 4A). Neuronswere cultured in the presence of 1 mM NAD and eitherSirtinol (100 mM) or 3AB (20 mM). Axonal transectionwas performed by removal of the neuronal cell bodiesand the extent of axonal degradation was assessed 12to 72 hr later. We found that Sirtinol effectively blockedNDAP, indicating that Sir2 proteins are likely effectors ofthis process. In contrast, 3AB had no effect on NDAP,indicating that PARP does not play a role in axonal pro-tection. To further examine the role of Sir2 proteins inNDAP, we tested the effects of resveratrol (10~100mM),a polyphenol compound that enhances Sir2 activity(Howitz, et al., Nature, 425:191-96, 2003). We found thatneurons treated with resveratrol prior to axotomy showeda decrease in axonal degradation that was comparableto that obtained using NAD (Fig. 4A), providing furthersupport for the idea that Sir2 proteins are effectors of theaxonal protection mediated by increased NMNAT activ-ity.

EXAMPLE 7

[0082] This example shows that SIRT1 is involved inNAD-dependent axonal protection.[0083] In humans and rodents, seven molecules shar-ing Sir2 conserved domain (sirtuin (SIRT)1 through 7)have been identified, although some of these proteins donot appear to have histone/protein deacetylase activity(Buck, et al., J Leukoc Biol, S0741-5400, 2004). SIRT1is located in the nucleus and is involved in chromatinremodeling and the regulation of transcription factorssuch as p53 (J. Smith, Trends Cell Biol, 12:404-406,2002). The cellular location of other SIRT proteins is lessclear, but some have been found in the cytoplasm andin mitochondria. To determine which SIRT protein(s) isinvolved in NAD-dependent axonal protection, we per-formed knockdown experiments using siRNA constructsto specifically target each member of the SIRT family.Neurons were infected with lentiviruses expressing spe-cific SIRT siRNA constructs that effectively suppressedexpression of their intended target (Fig. 4B). The infectedneurons were cultured in 1 mM NAD and axonal transec-tion was performed by removing the cell bodies. Wefound that the SIRT1 siRNA construct was just as effec-tive at blocking the axonal protective effects of NAD asthe Sirtinol inhibitor. In contrast, inhibition of the otherSIRT proteins did not have significant effects on NDAP(Fig. 4B). These results indicate that SIRT1 is the majoreffector of the increased NAD supply that effectively pre-vents axonal self destruction. Although, SIRT1 may

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deacetylate proteins directly involved in axonal stability,its predominantly nuclear location, along with the require-ment for NAD -24 hr prior to injury for effective protection,suggest that SIRT1 regulates a genetic program thatleads to axonal protection.[0084] Axonal degeneration is an active, self-destruc-tive phenomenon observed not only after injury and inresponse to chemotherapy, but also in association withaging, metabolic diseases such as diabetic neuropathy,and neurodegenerative diseases. Our results indicatethat the molecular mechanism of axonal protection in thewlds mice is due to the increased supply of NAD resultingfrom enhanced activity of the NAD salvage pathway andconsequent activation of the histone/protein deacetylaseSIRT1.

EXAMPLES 8-11

[0085] The following Materials and Methods were usedin Examples 8-11.[0086] Construction of expression plasmids andmutagenesis. Coding regions of the NAD biosyntheticenzymes were PCR amplified from EST clonesBC038133 for murine NMNAT1 and BC005737 formurine nicotinamide mononucleotideadenylyltransferase3 (NMNAT3), using Herculase(Stratagene). Human NAD synthetase (QNS) hexahisti-dine-tagged cDNA was kindly provided by Dr. N. Hara(Shimane University, Shimane, Japan). Hexahistidinetag was added at the 3’-end of each cDNA. NMNAT1cytosolic mutant (cytNMNAT1) was generated by PCR-mediated site-directed mutagenesis. Nuclear form ofNMNAT3 (nucNMNAT3) was generated by adding a nu-clear localization signal to the C-terminal end ofNMNAT3. Each PCR amplified NAD synthetic enzymefragment was cloned into FCIV lentiviral shuttle vectoras previously described. The integrity of all the constructswas sequenced using Taq DyeDeoxy Terminator cyclesequencing kits (Applied Biosystems) and an Applied Bi-osystems 373 DNA sequencer.[0087] NAD biosynthetic substrates. All substratesfor NAD biosynthetic enzymes were purchased from Sig-ma (Na, Nam, NMN, NaMN, nicotininc acid adenine di-nucleotide (NaAD), and NAD). NmR was synthesizedfrom NMN. Conversion of NMN to NmR was confirmedby HPLC (Waters) using reverse phase column LC-18T(Supelco). NmR is eluted 260 6 10 seconds and NMNis eluted 150 6 10 seconds under 1 ml/min flow rate ofbuffer containing 50mM K2HPO4 and 50mM KH2PO4 (pH7.0). Biological activity of NmR was accessed as previ-ously described by using yeast strains kindly providedfrom Dr. Charles Brenner (Dartmouth Medical School,New Hampshire, USA).[0088] Real-time quantitative reverse transcrip-tion-PCR analysis. All the surgical procedures were per-formed according to National Institute of Health guide-lines for care and use of laboratory animals at Washing-ton University. For the expression analysis following

nerve injury, the sciatic nerves of a C57BL/6 mouse wastransected and L4 to L5. DRGs were collected at indicat-ed time points and pooled to extract RNA. Rat DRG ex-plants from E14.5 embryo were cultured for 14 days ac-cording to the method desctribed and cultured with mediacontaining 10 nM vincristin for indicated period and ex-tracted RNA. Total RNAs from pooled tissue sources orDRG explant cultures were prepared. First-strand cDNAtemplates were prepared from 1 mg of each RNA usingstandard methods. Two independent cDNA syntheseswere performed for each RNA sample. Quantitative re-verse transcription (RT)-PCR was performed by moni-toring in real-time the increase in fluorescence of theSYBR-GREEN dye on a TaqMan 7700 Sequence De-tection System (Applied Biosystems).[0089] Cell culture, in vitro axotomy, and quantifi-cation of axonal degeneration. Mouse DRG explantsfrom E12.5 embryos were cultured in the DMEM contain-ing 10% FCS and 1 nM nerve growth factor. Non-neuro-nal cells were removed from the cultures by adding 5-fluorouracil to the culture media. Transection of neuriteswas performed at 14-21 DIV using an 18-gauge needleto remove the neuronal cell bodies. Lentiviral expressionvectors were generated. Lentiviral infection was per-formed 3-7 days prior to axonal transection for 24 hr.Quantitative analysis of neurite degeneration was per-formed.[0090] Determination of protein expression and lo-calization. For confirmation of protein expression,HEK293T cells were infected with a virus that expresseseach of NAD biosynthetic enzymes. Cells were lysed 5days after infection to be analyzed by immunoblot to de-tect expression of each protein with a hexa-histidine tagby anti-6xHis tag monoclonal antibody (R&D Systems).Subcellular localization of each protein was analyzed us-ing HEK293T cells transiently transfected with a viralshuttle vector for each NAD biosynthetic enzymes. Cellswere fixed in 4% paraformaldehyde in PBS containing0.1% tween-20 (PBS-T) and incubated with PBS-T con-taining 5% BSA for 1 hour, and then covered with 1:1000diluted anti-6xHis tag antibody (R&D Systems) in PBS-T containing 1% BSA and for 16 hours at 4°C. Cells werewashed with PBS-T and incubated with Alexa Fluor 594-conjugated secondary antibody (Molecular Probes) inTBS-T for 1 hour and examined by fluorescence micro-scopy (Nikon).[0091] NMNAT protein overexpression, affinity pu-rification and enzymatic assay. HEK293T cells weretransfected with an expression plasmid for each enzymeby using calcium phosphate precipitation. Three days lat-er, cells were washed with PBS twice and then suspend-ed in the buffer containing 50 mM Sodium Phosphate(pH8.0), and 300 mM NaCl (buffer A). Cells were thenhomogenized by SONIFIRE 450 (BRANSON) and su-pernatant was collected by centrifugation at 10,000 g for10 min. His-select Nickel Affinity Gel (Sigma) waswashed with buffer A and 0.1 ml of 50% gel suspensionwas added to 1 ml of supernatant and incubated for 10

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min at 4°C, then beads binding hexa-histidine -taggedprotein was extensively washed with the buffer A. Pro-teins were eluted by adding 100 ml of the solution con-taining 50 mM Sodium Phosphate (pH 8.0), 300 mM Na-Cl, and 250 mM imidazole. Relative NMNAT enzymaticactivity was measured by using affinity purified proteinsas described before and subtracted the value obtainedfrom mock transfected cells and normalized by theamount of recombinant protein determined by densitom-etry.[0092] Administration of NAD biosynthetic sub-strates and optic Nerve transection. Nam, NMN, NmR,or NAD was dissolved in PBS at the concentration of 100mM or 1 M. Each of 5 ml solution was injected into leftintravitreal component under the anesthesia at a rate of0.5 ml ml per second. The left optic nerve was transectedat 24 hours after intravitreal injection and optic nerve wasrecovered at indicated time. Optic nerve tissue was ho-mogenized in 100 ml of a buffer containing 100mM tris-HCl (pH 6.8), 1 % SDS, and 1mM DTT. Fifty mg of proteinfor each sample was analyzed by the Western blottingusing anti-neurofilament antibody 2H3 (DevelopmentalStudies Hybridoma Center) and peroxidase-conjugatedsecondary antibody (Jackson ImmunoResearch). Thedegeneration rate was calculated from the ratio of theneurofilament immunoreactivity of transected vs. contral-ateral nerves.

EXAMPLE 8

[0093] This example illustrates the NAD biosyntheticpathway and expression analysis of mammalian NADbiosynthetic enzymes.[0094] NAD is synthesized via three major pathwaysin both prokaryotes and eukaryotes. In the de novo path-way, NAD is synthesized from tryptophan (Fig.5). In thesalvage pathway, NAD is generated from vitamins includ-ing nicotinic acid and nicotinamide. A third route fromnicotinamide riboside called Preiss-Handler independentpathway has recently been discovered. The last enzy-matic reaction of the de novo pathway involves the con-version of quinolinate to NaMN by QPRT (EC 2.4.2.19).At this point, the de novo pathway converges with thesalvage pathway. NaPRT (EC 2.4.2.11) converts Na toNaMN, which is then converted to NaAD by NMNAT (EC2.7.7.1). QNS1 (EC 6.3.5.1) converts NaAD to NAD.NmPRT (EC 2.4.2.12); also reported as visfatin) convertsNam to NMN. NMN is also converted to NAD by NMNAT.Nicotinamidase (PNC, EC 3.5.1.19), which convertsNam to Na in yeast and bacteria salvage pathway hasnot been identified in mammals. In the Preiss-Handlerindependent pathway, Nrk (EC 2.7.1.22) converts NmRto NMN and converge to salvage pathway. Most of thesemammalian enzymes including QPRT, NmPRT, QNS1,Nrk1/2 and NMNAT1/2/3 have previously cloned andcharacterized. A mammalian homologue of NaPRT wasalso identified as an EST annotated as a mammalianhomolog of a bacterial NaPRT.

[0095] To investigate the expression of mammalianNAD biosynthetic enzymes in the nervous system, weperformed quantitative RT-PCR using RNA from mousebrain, retina, spinal code, and DRG at age of E14, P0,P7, P14 and P21. All enzymes are expressed ubiquitous-ly in the nervous system throughout the development andin adulthood, with an exception of Nrk2, whose expres-sion is very low in all examined tissues (data not shown).To identify inducibility of NAD-synthesizing enzymes inresponse to neuronal insults, we compared the RNA ex-pression of each enzyme in DRGs at 1, 3, 7, and 14 daysafter sciatic nerve transection against non-injured DRG.As shown in Fig. 6A, most of the enzymes were up-reg-ulated 2 to 8-fold after injury. Among those, Nrk2 expres-sion is exceptionally highly induced (more than 20-fold)at 14 days after axotomy. We also analyzed expressionof NAD synthetic enzymes during the axonal degenera-tion caused by neurotoxin in cultured rat DRG neuron.DRG neurons were treated with 0.1 mM and 1 mM roten-one to cause axonal degeneration and collected RNA at24 hours after the addition of rotenone. The expressionof Nrk2 was increased more than 6 folds after rotenonetreatment (Fig. 6B). These results suggest that, while allenzymatic activities in NAD synthesis pathway is ubiqui-tously present, Nrk2 may be responsible for supplyingNAD synthesizing substrate after neuronal insults.

EXAMPLE 9

[0096] This example illustrates that both nuclear andcytoplasmic Nmat enzymes save axons from degenera-tion.[0097] To determine whether nuclear localization ofNMNAT1 is essential to provide the axonal protection,we analyzed the effect of subcellular distribution ofNMNAT enzyme in the in vitro Wallerian degenerationassay and compared the extent of axonal protection be-tween overexpression of cytoplasmic and nuclearNMNAT. NMNAT1 has putative nuclear localization sig-nal PGRKRKW in the 211-217 amino-acids of NMNAT1protein. We generated a mutant NMNAT1 designated ascytNMNAT1 in which this nuclear localization signal wasaltered as PGAAAAW and examined subcellular distri-bution. As shown in Fig. 7B, the majority of cytNMNAT1located in the cytosol as we expected.[0098] Next we confirmed enzymatic activity ofcytNMNAT1, NMNAT1 and its mutant cytNMNAT1 werepurified from the cell lysate expressing either of proteinsby using affinity gel. The enzymatic activity of affinity pu-rified proteins was measured as described above andwe found that cytNMNAT1 activity did not altered by itsmutation (Fig. 7C). After the overexpression ofcytNMNAT1 in DRG neurons, we observed strong neu-rite protection as well as nuclear wild NMNAT1 (Fig.7A,E). We further confirmed this result by using NMNAT1isoenzyme that lacks nuclear localization signal. Amongtwo NMNAT isoenzymes, NMNAT3 is previously report-ed to locate outside nucleus and mitochondria, and have

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comparable enzymatic activity to NMNAT1. We addednuclear localization signal KPKKIKTED of human topoi-somerase I to the C-terminal of NMNAT3 to generatenuclear NMNAT3. We expressed hexa-histidine taggedNMNAT3 or nucNMNAT3 in HEK293T cells and ana-lyzed subcellular localization and its enzymatic activity.NMNAT3 was distributed outside the nucleus includingbright punctuate staining as reported before andnucNMNAT3 mainly localized in the nucleus with somepunctuate staining in the cytosol (Fig. 7B). The enzymaticactivity of NMNAT3 and nucNMNAT3 were measuredand both proteins have comparable enzymatic activitycompared with NMNAT1 (Fig. 7C). Then, in vitro Walle-rian degeneration assay was performed after overex-pression of these two NMNAT3 enzymes, and we foundthat overexpression of both NMNAT3 and nucNMNAT3showed same extent of delay in neurite degeneration aswell as NMNAT1 (Fig. 7A, E). The lentivirus mediatedexpression of each enzyme was confirmed by Westernblotting (Fig. 7D). These experiments confirmed thatNMNAT targeted to either the nucleus or cytosol protectsneurite from degeneration.

EXAMPLE 10

[0099] This example illustrates that exogenous appli-cation of substrates for NAD biosynthetic enzymes pro-tects axon from degeneration.[0100] We have previously shown that exogenouslyapplied NAD in the culture medium shows axonal savingeffect in vitro. Here we showed that expression of NmPRTalso shows axonal protection suggesting that Nam isused as a substrate for NAD synthesis in neurons. Todetermine which substrate shown in Fig. 5 is used forNAD synthesis in neurons and to identify whether any ofNAD precursors may be able to save axons similar to orpossibly better than NAD, we applied Na, Nam, NmR,NaMN, NMN, or NaAD in the culture media and per-formed in vitro Wallerian degeneration assay. An appli-cation of 1 mM NMN for 24 hours before neurite transec-tion successfully saved neurites from degeneration.Quantitative analysis revealed that NMN treatment re-sults in neurite protection to an extent similar to thatachieved by exogenously applied NAD (Fig. 8B). Theseresults further suggested the possibility that increasedsupply of other NAD biosynthetic substrates have an abil-ity to save neurites from degeneration. We then exoge-nously applied 1 mM of NAD biosynthetic substrates in-cluding Na, Nam, NaMN, NaAD, and NmR to the DRGneurons for 24 hours and performed neurite transection.As shown in Fig. 8A and B, NaMN or NmR treatment alsosaved neurites as well as NAD. NaAD showed slight pro-tection but Na failed to save neurites, while Na and Namhad no effect. Quantitative analysis revealed that exog-enous application of 1mM NaMN, NMN, NmR, or NADcaused comparable increase in intact neurites at 48hours after transection (Fig. 8B). Because the protectiveeffect of NaMN is equal to NMN, a step synthesize NAD

from NaAD by QNS is active enough to save neuritesunder the increased supply of NaAD. Nevertheless, ex-ogenous application of NaAD shows less increase in in-tact neurites at 48 hours compared with NAD (Fig. 8B).This indicates insufficient incorporation into the cell orinstability of NaAD in our assay condition. These exper-iments suggest that there are several different ways tosave neurites including exogenous application of NMN,NaMN, and NmR. All of these treatments seem to causeincreased supply of NAD and it is consistent to the pre-vious experiments showing NAD application or NMNAT1overexpression save neurites from degeneration.

EXAMPLE 11

[0101] This example demonstrates that intraviteal ap-plication of NAD biosynthetic substrates delays the ax-onal degeneration of retinal ganglion cells.[0102] Transection of optic nerve is an in vivo modelwhich can be used to investigate mechanisms leading toWallerian degeneration and following retinal ganglion cell(RGC) death observed in human diseases such as glau-coma. In the C57BL/Wlds mouse strain, optic nerve de-generation during Wallerian degeneration after axotomyis dramatically slowed. In addition, intravitreal injectionis used for screening of compounds that protect RGCaxon from degeneration in vivo and thus we can assesthe axon protective effect of each NAD biosynthetic sub-strates in vivo by intraocular injection of compounds in-cluding NAD, NMN, NmR, and Nam. From in vitro Walle-rian degeneration assay, 1mM of NAD, NMN, and NmRin the culture media is enough to protect axon from de-generation. We initially injected 5 ml of 100 mM or 1 MNAD solution into left intravitreal compartment. After 24hours incubation, left optic nerve was transected andcontrol (right) and axotomized (left) optic nerve were col-lected at 3, 4, and 5 days after transection. Neurofilamentimmunoreactivity from the axotomized optic nerve wasmeasured and normalized against the value obtainedfrom the right side of the optic nerve. We found that theimmunoreactivity at 4days after transection was77627% and 78622% of non-axotomized optic nerve in1 M and 100 mM NAD injected rats respectively, whilecontrol animal showed only 7616 % (Fig. 9)[0103] We then injected 5 ml of 100 mM NMN, NmR,and Nam into left intravitreal compartment and collectedoptic nerves at 4 days after left optic nerve transaction.The immunoreactivity obtained from NMN and NmR in-jected optic nerve was 60625 and 72619 % of non-ax-otomized nerve. Nam injected animals did not show anydifference from the control animals. These results areconsistent with the in vitro study that showed NAD, NMN,and NmR have axon saving activity but Nam does not.Our in vivo study revealed that these small moleculesthat are involved in the NAD biosynthetic pathway areuseful tools to save axon from degeneration.[0104] All references cited in this specification arehereby incorporated by reference. Any discussion of ref-

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erences cited herein is intended merely to summarizethe assertions made by their authors and no admissionis made that any reference or portion thereof constitutesrelevant prior art. Applicants reserve the right to chal-lenge the accuracy and pertinency of the cited referenc-es.

CLAUSES:

[0105]

1. A method of treating or preventing a neuropathyor axonopathy in a mammal in need thereof, themethod comprising administering to the mammal aneffective amount of an agent that acts by increasingsirtuin activity in diseased and/or injured neuronsand supporting cells.

2. A method according to clause 1, wherein the agentacts by increasing SIRT1 activity.

3. A method according to clause 1, wherein the agentis NAD, NADH, an intermediate of a de novo pathwayfor synthesizing NAD, an intermediate of a NAD sal-vage pathway, an intermediate of a nicotinamide ri-boside kinase pathway or a combination thereof.

4. A method according to clause 1, wherein the agentis NAD, nicotinamide mononucleotide, nicotinic acidmononucleotide or nicotinamide riboside.

5. A method according to clause 1, wherein the agentcomprises an enzyme of a de novo pathway for syn-thesizing NAD, an enzyme of a NAD salvage path-way or an enzyme of a nicotinamide riboside kinasepathway; a nucleic acid encoding an enzyme of a denovo pathway for synthesizing NAD, an enzyme ofa NAD salvage pathway or an enzyme of a nicotina-mide riboside kinase pathway; an agent that increas-es expression of an enzyme of a de novo pathwayfor synthesizing NAD, an enzyme of a NAD salvagepathway or an enzyme of a nicotinamide ribosidekinase pathway; or an agent that increases catalyticactivity and/or stability of an enzyme of a de novopathway for synthesizing NAD, an enzyme of a NADsalvage pathway or an enzyme of a nicotinamideriboside kinase pathway.

6. A method according to clause 5, wherein the agentcomprises a nicotinamide mononucleotide adenylyl-transferase (NMNAT) or a nucleic acid encoding anNMNAT.

7. A method according to clause 6, wherein the agentcomprises an enzyme having NMNAT activity andat least 50% identity with a human NMNAT1 or atleast 50% identity with a human NMNAT3.

8. A method according to clause 7, wherein the agenthas at least 70% identity with a human NMNAT1 orat least 70% identity with a human NMNAT3.

9. A method according to clause 6, wherein the agentis selected from the group consisting of a humanNMNAT1, a human NMNAT3 and conservativelysubstituted variants thereof.

10. A method according to clause 6, wherein theagent comprises a nucleic acid having at least 50%identity with a nucleic acid encoding a human NMNA-Tor a nucleic acid having at least 50% identity witha nucleic acid encoding a human NMNAT3.

11. A method according to clause 10, wherein theagent comprises a nucleic acid having at least 70%identity with a nucleic acid encoding a humanNMNAT1 or a nucleic acid having at least 70% iden-tity with a nucleic acid encoding a human NMNAT3.

12. A method according to clause 11 wherein theagent comprises a nucleic acid encoding a humanNMNATor a human NMNAT3 or a nucleic acid var-iant thereof.

13. A method according to clause 1, wherein theagent comprises a sirtuin polypeptide or a nucleicacid encoding a sirtuin polypeptide.

14. A method according to clause 6, wherein theagent comprises an enzyme having SIRT activityand at least 50% identity with a human SIRT1.

15. A method according to clause 14, wherein theagent has at least 70% identity with a human SIRT1.

16. A method according to clause 15, wherein theagent is selected from the group consisting of a hu-man SIRT1 and conservatively substituted variantsthereof.

17. A method according to clause 6, wherein theagent comprises a nucleic acid having at least 50%identity with a nucleic acid encoding a human SIRT1.

18. A method according to clause 17, wherein theagent comprises a nucleic acid having at least 70%identity with a nucleic acid encoding a human SIRT1.

19. A method according to clause 18 wherein theagent comprises a nucleic acid encoding a humanSIRT1 or a nucleic acid variant thereof.

20. A method according to clause 1, wherein theagent is a stilbene, a chalcone, a flavone, an isofla-vanone, a flavanone or a catechin.

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21. A method according to clause 20, wherein theagent is a stilbene selected from the group consistingof resveratrol, piceatannol, deoxyrhapontin,trans-stilbene and rhapontin; a chalcone selectedfrom the group consisting of butein, isoliquiritigenand 3,4,2’,4’,6’-pentahydroxychalcone; a flavoneselected from the group consisting of fisetin,5,7,3’,4’,5’-pentahydroxyflavone, luteolin, 3,6,3’,4’-tetrahydroxyflavone, quercetin, 7,3’,4’,5’-tetrahy-droxyflavone, kaempferol, 6-hydroxyapigenin, api-genin, 3,6,2’,4’-tetrahydroxyflavone, 7,4’-dihydroxy-flavone, 7,8,3’,4’-tetrahydroxyflavone, 3,6,2’,3’-tet-rahydroxyflavone, 4’-hydroxyflavone, 5,4’-dihydrox-yflavone, 5,7-dihydroxyflavone, morin, flavone and5-hydroxyflavone; an isoflavone selected from thegroup consisting of daidzein and genistein; a fla-vanone selected from the group consisting of narin-genin, 3,5,7,3’,4’-pentahydroxyflavanone, and fla-vanone or a catechin selected from the group con-sisting of (-)-epicatechin, (-)-catechin, (-)-gallocate-chin, (+)-catechin and (+)-epicatechin.

22. A method according to clause 1, wherein the neu-ropathy or axonopathy is hereditary or congenital orassociated with neurodegenerative disease, motorneuron disease, neoplasia, endocrine disorder, met-abolic disease, nutritional deficiency, atherosclero-sis, an autoimmune disease, mechanical injury,chemical or drug-induced injury, thermal injury, ra-diation injury, nerve compression, retinal or opticnerve disorder, mitochondrial dysfunction, progres-sive dementia demyelinating diseases ischemiaand/or stroke infectious disease; or inflammatorydisease.

23. A method according to clause 22, wherein theneuropathy or axonopathy is induced by a cytotoxicanticancer agent.

24. A method according to clause 22, wherein theoptic neuropathy is glaucoma, retinal ganglion de-generation, optic neuritis and/or degeneration, mac-ular degeneration, ischemic optic neuropathy, trau-matic injury to the optic nerve, hereditary optic neu-ropathy, metabolic optic neuropathy, neuropathydue to a toxic agent or that caused by adverse drugreactions or vitamin deficiency.

25. A method according to clause 22, wherein theneuropathy associated with mitochondrial dysfunc-tion results from oxidative damage, from mutationsin mitochondrial proteins encoded either in the mito-chondrial genome or nuclear genome, from expo-sure to toxins, or from the process of aging.

26. A method according to clause 1, wherein themammal is a human.

27. A method of treating or preventing a neuropathyor axonopathy in a mammal in need thereof, themethod comprising administering to the mammal aneffective amount of an agent that acts by increasingNAD activity in diseased and/or injured neuronsand/or supporting cells.

28. A method according to clause 27, wherein theagent is NAD, NADH, an intermediate of a de novopathway for synthesizing NAD, an intermediate of aNAD salvage pathway, an intermediate of a nicoti-namide riboside kinase pathway or a combinationthereof.

29. A method according to clause 28, wherein theagent is NAD, nicotinamide mononucleotide, nico-tinic acid mononucleotide or nicotinamide riboside.

30. A method according to clause 27, wherein theagent comprises an enzyme of a de novo pathwayfor synthesizing NAD, an enzyme of a NAD salvagepathway or an enzyme of a nicotinamide ribosidekinase pathway; a nucleic acid encoding an enzymeof a de novo pathway for synthesizing NAD, an en-zyme of a NAD salvage pathway or an enzyme of anicotinamide riboside kinase pathway; an agent thatincreases expression of an enzyme of a de novopathway for synthesizing NAD, an enzyme of a NADsalvage pathway or an enzyme of a nicotinamideriboside kinase pathway; or an agent that increasescatalytic activity and/or stability of an enzyme of ade novo pathway for synthesizing NAD, an enzymeof a NAD salvage pathway or an enzyme of a nico-tinamide riboside kinase pathway.

31. A method according to clause 30, wherein theagent comprises a nicotinamide mononucleotideadenylyltransferase (NMNAT) or a nucleic acid en-coding an NMNAT.

32. A method according to clause 31, wherein theagent comprises an enzyme having NMNAT activityand at least 50% identity with a human NMNAT1 orat least 50% identity with a human NMNAT3.

33. A method according to clause 32, wherein theagent has at least 70% identity with a humanNMNAT1 or at least 70% identity with a humanNMNAT3.

34. A method according to clause 31, wherein theagent is selected from the group consisting of a hu-man NMNAT1, a human NMNAT3 and conserva-tively substituted variants thereof.

35. A method according to clause 31, wherein theagent comprises a nucleic acid having at least 50%identity with a nucleic acid encoding a human

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NMNAT1 or a nucleic acid having at least 50% iden-tity with a nucleic acid encoding a human NMNAT3.

36. A method according to clause 35, wherein theagent comprises a nucleic acid having at least 70%identity with a nucleic acid encoding a humanNMNAT1 or a nucleic acid having at least 70% iden-tity with a nucleic acid encoding a human NMNAT3.

37. A method according to clause 36 wherein theagent comprises a nucleic acid encoding a humanNMNAT1 or a human NMNAT3 or a nucleic acid var-iant thereof.

38. A method according to clause 27, wherein theneuropathy or axonopathy is hereditary or congenitalor associated with neurodegenerative disease, mo-tor neuron disease, neoplasia, endocrine disorder,metabolic disease, nutritional deficiency, athero-sclerosis, an autoimmune disease, mechanical inju-ry, chemical or drug-induced injury, thermal injury,radiation injury, nerve compression, retinal or opticnerve disorder, mitochondrial dysfunction, progres-sive dementia demyelinating diseases ischemiaand/or stroke infectious disease; or inflammatorydisease.

39. A method according to clause 38, wherein theneuropathy or axonopathy is induced by a cytotoxicanticancer agent.

40. A method according to clause 22, wherein theoptic neuropathy is glaucoma, retinal ganglion de-generation, optic neuritis and/or degeneration, mac-ular degeneration, ischemic optic neuropathy, trau-matic injury to the optic nerve, hereditary optic neu-ropathy, metabolic optic neuropathy, neuropathydue to a toxic agent or that caused by adverse drugreactions or vitamin deficiency.

41. A method according to clause 38, wherein theneuropathy associated with mitochondrial dysfunc-tion results from oxidative damage, from mutationsin mitochondrial proteins encoded either in the mito-chondrial genome or nuclear genome, from expo-sure to toxins, or from the process of aging.

42. A method according to clause 27, wherein themammal is a human.

43. A method of screening agents for treating a neu-ropathy in a mammal, the method comprising admin-istering to mammalian neuronal cells in vitro or invivo, a candidate agent; producing an axonal injuryto the neuronal cells; and detecting a decrease inaxonal degeneration of the injured neuronal cells.

44. A method according to clause 43, wherein pro-

ducing an axonal injury to the neuronal cells com-prises chemically injuring the neuronal cells, ther-mally injuring the neuronal cells, oxygen-deprivingthe neuronal cells, physically injuring the neuronalcells, inhibiting energy metabolism or a combinationthereof.

45. A method of screening agents for treating a neu-ropathy in a mammal, the method comprising detect-ing an increase in NAD activity produced by a can-didate agent, in a cell.

46. A method of screening for agents that increasesirtuin activity in neurons, the method comprising ad-ministering to mammalian neuronal cells in vitro orin vivo, a candidate agent; producing an axonal injuryto the neuronal cells; and detecting a decrease inaxonal degeneration of the injured neuronal cells.

47. A method of screening for agents that increaseNAD activity in neurons, the method comprising ad-ministering to mammalian neuronal cells in vitro orin vivo, a candidate agent; producing an axonal injuryto the neuronal cells; and detecting a decrease inaxonal degeneration of the injured neuronal cells.

48. A recombinant vector comprising a promoter op-eratively linked to a polynucleotide having at least50% identity with a nucleic acid encoding a humanNMNAT1 or a polynucleotide having at least 50%identity with a nucleic acid encoding a humanNMNAT3.

49. A recombinant vector according to clause 48comprising a polynucleotide having at least 70%identity with a nucleic acid encoding a humanNMNAT1 or a polynucleotide having at least 70%identity with a nucleic acid encoding a humanNMNAT3.

50. A recombinant vector according to clause 48comprising a polynucleotide encoding a humanNMNAT1 or a human NMNAT3 or a polynucleotidevariant thereof.

51. A recombinant vector according to clause 48comprising a lentivirus or an adeno-associated virus.

52. A recombinant vector comprising a promoter op-eratively linked to a poloynucleotide having at least50% identity with a nucleic acid encoding a humanSIRT1.

53. A recombinant vector according to clause 52comprising a polynucleotide having at least 70%identity with a nucleic acid encoding a human SIRT1.

54. A recombinant vector according to clause 54

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comprising a polynucleotide acid encoding a humanSIRT1 or a polynucleotide variant thereof.

55. A recombinant vector according to clause 52comprising a lentivirus or an adeno-associated virus.

56. A method of treating or preventing an optic neu-ropathy in a mammal in need thereof, the methodcomprising administering to the mammal an effectiveamount of an agent that acts by increasing NAD ac-tivity in diseased and/or injured neurons.

57. A method according to clause 56, wherein ad-ministering to the mammal comprises intraocular ad-ministering.

58. A method according to clause 57, wherein in-traocular administering comprises intraocular ad-ministering of a sustained release delivery system.

59. A method according to clause 57, wherein in-traocular administering comprises intravitrial injec-tion, administration by eyedrops or administration bytrans-scleral delivery.

60. A method according to clause 56, wherein theagent is NAD, NADH, an intermediate of a de novopathway for synthesizing NAD, an intermediate of aNAD salvage pathway, an intermediate of a nicoti-namide riboside kinase pathway or a combinationthereof.

61. A method according to clause 60, wherein theagent is NAD, nicotinamide mononucleotide, nico-tinic acid mononucleotide or nicotinamide riboside.

62. A method according to clause 56, wherein theagent comprises an enzyme of a de novo pathwayfor synthesizing NAD, an enzyme of a NAD salvagepathway or an enzyme of a nicotinamide ribosidekinase pathway; a nucleic acid encoding an enzymeof a de novo pathway for synthesizing NAD, an en-zyme of a NAD salvage pathway or an enzyme of anicotinamide riboside kinase pathway; an agent thatincreases expression of an enzyme of a de novopathway for synthesizing NAD, an enzyme of a NADsalvage pathway or an enzyme of a nicotinamideriboside kinase pathway; or an agent that increasescatalytic activity and/or stability of an enzyme of ade novo pathway for synthesizing NAD, an enzymeof a NAD salvage pathway or an enzyme of a nico-tinamide riboside kinase pathway.

63. A method according to clause 62, wherein theagent comprises a nicotinamide mononucleotideadenylyltransferase (NMNAT) or a nucleic acid en-coding an NMNAT.

64. A method according to clause 62, wherein theagent comprises a nucleic acid having at least 50%identity with a nucleic acid encoding a humanNMNAT1 or a nucleic acid having at least 50% iden-tity with a nucleic acid encoding a human NMNAT3.

65. A method according to clause 64, wherein theagent comprises a nucleic acid having at least 70%identity with a nucleic acid encoding a humanNMNAT1 or a nucleic acid having at least 70% iden-tity with a nucleic acid encoding a human NMNAT3.

66. A method according to clause 65 wherein theagent comprises a nucleic acid encoding a humanNMNAT1 or a human NMNAT3 or a nucleic acid var-iant thereof.

67. A method according to clause 22, wherein theoptic neuropathy is glaucoma, retinal ganglion de-generation, optic neuritis and/or degeneration, mac-ular degeneration, ischemic optic neuropathy, trau-matic injury to the optic nerve, hereditary optic neu-ropathy, metabolic optic neuropathy, neuropathydue to a toxic agent or that caused by adverse drugreactions or vitamin deficiency.

68. A method according to clause 56, wherein themammal is a human.

Claims

1. An agent for use in the treatment or prevention of anaxonopathy in a mammal in need thereof, whereinthe agent is NAD, NADH, an intermediate of a denovo pathway for synthesizing NAD, an intermediateof a NAD salvage pathway or an intermediate of thePreiss-Handler independent pathway.

2. An agent in accordance with claim 1, wherein theintermediate of a de novo pathway for synthesizingNAD is quinolinic acid or nicotinate adenine dinucle-otide (NaAD).

3. An agent in accordance with claim 1, wherein theintermediate of a NAD salvage pathway is nicotinicacid.

4. An agent in accordance with claim 1, wherein theintermediate of a NAD salvage pathway is nicotina-mide.

5. An agent in accordance with claim 1, wherein theagent is nicotinic acid mononucleotide (NMN).

6. An agent in accordance with claim 1, wherein theagent is nicotinate mononucleotide (NaMN).

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7. An agent in accordance with claim 1, wherein theagent is nicotinamide riboside (NmR)

8. An agent in accordance with claim 1, wherein themammal is a companion animal, an agricultural an-imal, or an exotic animal.

9. An agent in accordance with claim 8, wherein themammal is a dog, a cat, a cow or a horse.

10. An agent in accordance with claim 1, wherein themammal is mouse.

11. An agent in accordance with claim 1, wherein themammal is a human.

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