Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/05—Phenols
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0048—Eye, e.g. artificial tears
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5058—Neurological cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/91—Transferases (2.)
  • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • G01N2333/91205—Phosphotransferases in general
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders

Definitions

• This invention relates generally to diseases and conditions involving neurons and, more particularly, to methods and compositions for treating or preventing neuropathies and other diseases and conditions involving neurodegeneration. Also included are methods of identifying agents for treating or preventing neuropathies.
• Axon degeneration occurs in a variety of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases as well as upon traumatic, toxic or ischemic injury to neurons. Such diseases and conditions are associated with axonopathies including axonal dysfunction.
• axonopathy is Wallerian degeneration (Waller, Philos Trans R. soc. Lond. 140:423-429, 1850), which occurs when the distal portion of the axon is severed from the cell body. The severed axon rapidly succumbs to degeneration.
• Axonopathy can, therefore, be a critical feature of neuropathic diseases and conditions and neurological disorders and axonal deficits can be an important component of a patient's disability.
• the present inventors have succeeded in discovering that axonal degeneration can be diminished or prevented by increasing, separately or in combination, NAD activity, sirtuin activity, AMP activated kinase (AMPK) activity, LKB 1 activity and/or CaMKK ⁇ activity in diseased and/or injured neurons. These discoveries can also be used in various combinations with treatments employing other known mechanisms.
• one approach to preventing axonal degeneration can be by activating sirtuin molecules, i.e. SIRTl in injured mammalian axons.
• the activation of SIRTl can be through direct action on the SIRTl molecule or by increasing the supply of nicotinamide adenine dinucleotide (NAD) which acts as a substrate for the histone/protein deacetylase activity of SIRTl .
• NAD nicotinamide adenine dinucleotide
• the activation of SIRTl results in a decrease in severity of axonal degeneration or a prevention of axonal degeneration.
• NAD activity could act through other mechanisms not involving sirtuin.
• increasing NAD activity which may act through increasing SIRTl activity or through one or more other mechanisms or both can diminish or prevent axonal degeneration in injured mammalian axons.
• axonal degeneration can be prevented or decreased in severity by increasing AMPK activity, LKB 1 activity and/or calcium/calmodul in-dependent protein kinase ⁇ (CaMKK ⁇ ) activity in diseased or injured neurons.
• the polyphenol compound resveratrol is a potent stimulator of AMPK and that activity of LKBl , an upstream regulator of AMPK, is required for this AMPK stimulation.
• the inventors have found that increased AMPK activity is neuroprotective, and furthermore promotes axonal growth.
• the present teachings disclose methods of treating or preventing a neuropathy or axonopathy in a mammal and, in particular, in a human in need thereof.
• the methods can comprise administering an effective amount of an agent that acts to increase AMPK activity and/or LKBl activity and/or CaMKK ⁇ activity in diseased and/or injured neurons.
• the methods can comprise selecting an agent on the basis of having a property of effecting an increase in AMPK activity and/or LKBl activity and/or CaMKK ⁇ activity in diseased and/or injured neurons upon administration, and administering an effective amount of the agent.
• the present teachings disclose methods of treating or preventing a neuropathy in a mammal and, in particular, in a human in need thereof. These methods can comprise administering an effective amount of an agent that acts to increase sirtuin activity and, in particular, SIRTl activity in diseased and/or injured neurons.
• the agent can increase SIRTl activity through increasing NAD activity. It is believed that increasing NAD activity can increase sirtuin activity because NAD can act as a substrate of SIRT 1.
• Such agents can include NAD or NADH, a precursor of NAD, an intermediate in the NAD salvage pathway or a substance that generates NAD such as a nicotinamide mononucleotide adenylyltransferase (NMNAT) or a nucleic acid encoding a nicotinamide mononucleotide adenylyltransferase.
• NMNAT nicotinamide mononucleotide adenylyltransferase
• the nicotinamide mononucleotide adenylyltransferase can be an NMNATl protein.
• the agent can also act to directly increase SIRTl activity and as such, the agent can be a sirtuin polypeptide or a nucleic acid encoding a sirtuin polypeptide or, to increase SIRTl activity or to increase AMPK and/or LKBl and/or CaMKK ⁇ activity, a substance such as a stilbene, a chalcone, a flavone, an isoflavanone, a flavanone or a catechin.
• Such compounds can include a stilbene selected from the group consisting of resveratrol, piceatannol, deoxyrhapontin, trans-stilbene and rhapontin; a chalcone selected from the group consisting of butein, isoliquiritigen and 3,4,2',4',6'-pentahydroxychalcone; a flavone selected from the group consisting of fisetin, 5,7,3',4',5'-pentahydroxyflavone, luteolin, 3,6,3',4'- tetrahydroxyflavone, quercetin, 7,3',4',5'-tetrahydroxyflavone, kaempferol, 6-hydroxyapigenin, apigenin, 3,6,2',4'-tetrahydroxyflavone, 7,4'-dihydroxyflavone, 7,8,3',4'-tetrahydroxyflavone, 3,6,2',3'-tetrahydroxyflavone, 4'-hydroxy
• the present teachings also include methods of treating a neuropathy.
• these methods include administering to a mammal, such as a human in need of treatment, an effective amount of an agent that acts by increasing NAD activity in diseased and/or injured neurons and/or supporting cells such as, for example, glia, muscle cells and/or fibroblasts.
• an agent of these aspects can be NAD or NADH, nicotinamide mononucleotide, nicotinic acid mononucleotide or nicotinamide riboside or derivatives thereof; an enzyme that generates NAD such as a nicotinamide mononucleotide adenylyltransferase; a nucleic acid encoding an enzyme that generates NAD such as a nucleic acid encoding a nicotinamide mononucleotide adenylyltransferase; an agent that increases expression of a nucleic acid encoding an enzyme in a pathway that generates NAD or an agent that increases activity and/or stability of an enzyme in a pathway that generates NAD or an agent that increases NAD activity.
• the nicotinamide mononucleotide adenylyltransferase can be an NMNATl protein.
• the present teachings include methods of treating or preventing an optic neuropathy in a mammal in need thereof.
• the mammal can be a human, and the methods can comprise administering to the mammal an effective amount of an agent that acts at least in part by increasing AMPK activity, and/or LKB l activity and/or CaMKK ⁇ activity in diseased and/or injured neurons.
• the administering to the mammal can comprise administering to an eye.
• the administering can comprise administering the agent using a sustained-release delivery system, such as, without limitation, administering to the eye a sustained-release pellet comprising the agent.
• the present teachings also include methods of treating or preventing an optic neuropathy in a mammal in need thereof. These methods can comprise administering to the mammal an effective amount of an agent that acts by increasing NAD activity in diseased and/or injured neurons. Administering to the mammal can comprise administering to the eye, in particular by administering the agent with a sustained release delivery system or by administering a sustain release pellet comprising the agent to the eye.
• the agent can be NAD, NADH, nicotinamide mononucleotide, nicotinic acid mononucleotide or nicotinamide riboside; or an enzyme that generates NAD such as a nicotinamide mononucleotide adenylyltransferase; or a nucleic acid encoding an enzyme that generates NAD such as a nucleic acid encoding a nicotinamide mononucleotide adenylyltransferase or an agent that increases NAD activity.
• the nicotinamide mononucleotide adenylyltransferase can be an NMNATl protein or an NMNAT3 protein.
• the neuropathy associated with axonal degradation can be any of a number of neuropathies such as, without limitation, a disease that is hereditary, a congenital disease, Parkinson's disease, Alzheimer's disease, Herpes infection, diabetes, amyotrophic lateral sclerosis, a demyelinating disease such as multiple sclerosis, a seizure disorder, ischemia, stroke, chemical injury, thermal injury, or AIDS.
• the present invention is also directed to methods of screening agents for treating a neuropathy in a mammal. These methods can comprise administering a candidate agent to neuronal cells in vitro or in vivo, producing an axonal injury to the neuronal cells and detecting a decrease in axonal degeneration of the injured neuronal cells.
• the candidate agent can be an agent that acts at least in part by increasing AMPK activity, LKBl activity and/or CaMKK ⁇ activity in diseased and/or injured neurons and/or supporting cells.
• methods of screening agents can comprise detecting an increase in AMPK activity, LKBl activity, CaMKK ⁇ activity and/or activity of AMPK downstream effector acetyl Co-A carboxylase (ACC) following administration of a candidate agent to cells, tissues and/or organisms in vitro or in vivo, in particular, to one or more neuronal cells in vitro or in vivo.
• a method can comprise detecting an increase in NAD activity produced by a candidate agent, in one or more cells and, in particular, in one or more neuronal cells, in vitro or in vivo.
• an increase in NAD activity can be an increase in nuclear NAD activity.
• Methods are also provided for screening agents that increase sirtuin activity in neurons as well as for screening agents that increase NAD biosynthetic activity in neurons.
• the methods can comprise administering to mammalian neuronal cells in vitro or in vivo a candidate agent, producing an axonal injury to the neuronal cells and detecting a decrease in axonal degeneration of the injured neuronal cells.
• Such methods can further comprise, in some aspects, secondary assays which further delineate AMPK activity, LKB 1 activity and/or CaMKK ⁇ activity, with sirtuin activity, NAD and enzymes or components of NAD biosynthetic or salvage pathways, or various combinations thereof.
• axonal injury can be produced by various methods of injuring neuronal cells, such as chemical injury, metabolic injury, genetic impairment, altering mitochondrial activity, thermal injury, oxygen-deprivation, and/or mechanical injury.
• a recombinant vector is also provided in various aspects of the present teachings.
• a vector of these aspects can comprise a promoter operatively linked to a sequence encoding a mammalian NMNATl protein or NMNAT3 protein, such as a human NMNATl protein or NMNAT3 protein.
• the recombinant vector can be a lentivirus or an adeno-associated virus.
• a recombinant vector comprising a promoter operatively linked to a sequence encoding a SIRTl protein.
• a recombinant vector can be a lentivirus or an adeno-associated virus.
• Figure 2 illustrates that increased NAD supply protects axons from degeneration after injury showing: A) Enzymatic activity of wild type and mutant Wlds and NMNATl proteins in which lysates were prepared from HEK293 cells expressing the indicated protein were assayed for NAD production using nicotinamide mononucleotide as a substrate and the amount of NAD generated in 1 h was converted to NADH, quantified by fluorescence intensity, and normalized to total protein concentration showing that both mutants have essentially no enzymatic activity; and B) In vitro Wallerian degeneration in lentivirus-infected DRG neurons expressing NMNATl or Wlds protein, mutants of these proteins that lack NAD-synthesis activity NMNAT1(W17OA) and Wlds(W258A), or EGFP wherein the bar chart shows the quantitative analysis data of the number of remaining neurites at indicated time-point for each construct (percentage of remaining neurites relative to pre-transection
• D. and the "*" indicates significant difference (p ⁇ 0.0001) with EGFP-infected neurons;
• C) Protein expression in lentivirus-infected cells detected by immunoblot analysis using antibodies to the 6XHis tag; and
• D) DRG neuronal explant expressing either NMNATl or EGFP (control) cultured with 0.5 ⁇ M vincristine wherein representative images of neurites (phase-contrast; Bar lmm) are shown at the indicated times after vincristine addition and quantification of the protective effect at the indicated time points is plotted as the area covered by neurites relative to that covered by neurites prior to treatment.
• Figure 3 illustrates that axonal protection requires pre-treatment of neurons with NAD prior to injury showing: A) in vitro Wallerian degeneration using DRG explants cultured in the presence of various concentrations of NAD added 24 hr prior to axonal transection; and B) DRG explants preincubated with ImM NAD for 4, 8, 12, 24, or 48 h prior to transection wherein the bar chart shows the number of remaining neurites in each experiment (percentage of remaining neurites relative to pre-transection ⁇ S. D.) at each of the indicated time points and the "*" indicates significant axonal protection compared to control (pO.0001).
• FIG. 4 illustrates that NAD-dependent Axonal Protection is mediated by SIRTl activation showing: A) In vitro Wallerian degeneration using DRG explant cultures preincubated with 1 mM NAD alone (control) or in the presence of either 100 ⁇ M Sirtinol (a Sir2 inhibitor) or 20 mM 3-aminobenzimide (3AB, a PARP inhibitor); B) in vitro Wallerian degeneration using DRG explant cultures incubated with resveratrol (10, 50 or 100 ⁇ M); and C) left: in vitro Wallerian degeneration using DRG explant cultures infected with lentivirus expressing siRNA specific for each member of the SIRT family (SIRT 1-7) wherein the bar chart shows the quantitative analysis of the number of remaining neurites (percentage of remaining neurites relative to pre-transection ⁇ S.
• middle table The effectiveness of each SIRT siRNA (expressed as % of wild type mRNA level) using qRT-PCR in infected NIH3T3 cells; and right: immunoblot using antibodies to SIRTl to show decreased expression of SIRTl in the presence of SIRTl siRNA which effectively blocked NAD dependent axonal protection.
• Figure 5 illustrates the mammalian NAD biosynthetic pathway in which predicted mammalian NAD biosynthesis is illustrated based on the enzymatic expression analysis and studies from yeast and lower eukaryotes (Abbreviation used; QPRT, quinolinate phosphoribosyltransferase; NaPRT, nicotinic acid phosphoribosyltransferase; NmPRT, nicotinamide phosphoribosyltransferase; Nrk, nicotinamide riboside kinase; NMNAT, nicotinamide mononucleotide adenylyltransferase; QNS, NAD synthetase)
• Figure 6 illustrates expression analysis of NAD biosynthetic enzymes in mammal showing (A) NAD biosynthesis enzyme mRNA levels after 1, 3, 7, and 14 days after nerve transection in rat DRG were determined by qRT-PCR in which the expression level was normalized to glyceraldehydes-3 -phosphate dehydrogenase expression in each sample and is indicated relative to the expression level in non-axotomized DRG; (B) neurite degeneration introduced by incubation DRG in 1 or 0.1 ⁇ M rotenone for indicated time and NAD synthesis enzyme mRNA levels were determined by qRT-PCR as described in the text.
• Figure7 illustrates the subcellular localization of NMNAT enzymes and their ability to protect axon showing (A) in vitro Wallerian degeneration assay using lentivirus infected DRG neuronal explant cultures expressing NMNATl, cytNMNATl, NMNAT3, or nucNMNAT3 in which representative pictures taken at 12 and 72 hours after transaction are shown; (B) Subcellular localization of NMNATl, cytNMNATl , NMNAT3, or nucNMNAT3 in HEK 293T cells using immunohistochemistry with antibody against 6xHis tag to detect each proteins and staining of the cells with the nuclear marker dye (bisbenzimide) for comparison to determine the nuclear vs.
• A in vitro Wallerian degeneration assay using lentivirus infected DRG neuronal explant cultures expressing NMNATl, cytNMNATl, NMNAT3, or nucNMNAT3 in which representative pictures taken at 12 and 72 hours after transaction are shown
• Figure 8 illustrates exogenous application of NAD biosynthetic substrates and their ability to protect axon showing
• A in vitro Wallerian degeneration assay using DRG neuronal explant cultures after exogenous application of NAD, NmR with representative pictures taken at 12, 24, 48, and 72 hours after transaction are shown;
• B in vitro Wallerian degeneration assay using DRG neuronal explant cultures after exogenous application of Na, Nam, NaMN, NMN, NaAD, NAD, and NmR showing quantitative analysis data of remaining neurite numbers at 12, 24, 48, and 72 hours after axotomy are shown;
• C DRG neuronal explants infected with NaPRT expressing lentivirus and incubated with or without 1 mM of Na for 24 hours before axotomy, in in vitro Wallerian degeneration assay showing quantitative analysis data of remaining neurite numbers at 12, 24, 48, and 72 hours after axotomy.
• Figure 9 illustrates optic nerve transection after intravitreal injection of NAD biosynthetic substrates NAD, NMN, NmR, or Nam was injected into intravitreal compartment of left rat eye and allowed to incorporate retinal ganglion cells for 24 hours after which, left optic nerve was transected by eye enucleation and right and left optic nerves were collected at 4 days after transection and analyzed by Western blotting in which optic nerves transected from mice without any treatment prior to axotomy were used for negative control; showing in the figure, the quantitative analysis data of percentage of remaining neurofilament immunoreactivity from transected optic nerve relative to non-transected ⁇ S. D.
• FIG 10 illustrates resveratrol activation of AMP kinase in Neuro2a cells.
• Neuro2a cells were switched to serum-starvation medium (medium containing 0.2% FCS) and treated with DMSO (vehicle control), resveratrol (10 ⁇ M) or 5-aminoimidazole-4-carboxamide- 1- ⁇ -D-ribofuranoside (AICAR) (ImM).
• DMSO vehicle control
• resveratrol 10 ⁇ M
• AICAR 5-aminoimidazole-4-carboxamide- 1- ⁇ -D-ribofuranoside
• Figure 1 1 illustrates the resveratrol and AICAR stimulation of neurite outgrowth in Neuro2a cells.
• Neuro2a cells were switched to serum-starvation medium (medium containing 0.2% FCS) and treated with DMSO (vehicle control), resveratrol (10 ⁇ M) or AICAR (ImM).
• DMSO vehicle control
• resveratrol 10 ⁇ M
• AICAR ImM
• A Serum deprivation of Neuro2a cells results in growth of short neurites that increase in length over 72 h.
• B Resveratrol induced rapid neurite outgrowth resulting in elaborate neurite network formation by 48 h.
• AICAR stimulated extensive neurite outgrowth similar to that observed with resveratrol.
• D Quantification of neurite length showed significantly longer neurites in resveratrol or AICAR treated cells compared with Neuro2a cells grown in 0.2% serum alone (p ⁇ 0.001).
• Figure 12 illustrates the dependency of resveratrol-induced neurite outgrowth and mitochondrial biogenesis on AMPK.
• Neuro2a cells were infected with lentivirus expressing GFP only (FUGW, control), dominant negative AMPK (dnAMPK) or constitutively active AMPK (caAMPK). Three days later cells were shifted to serum-starvation medium containing DMSO (control) or resveratrol (10 ⁇ M). In addition, uninfected Neuro2a cells in serum- starvation medium were treated with resveratrol alone, the AMPK inhibitor Compound C (CC, Biomol International L. P. (Plymouth Meeting, PA)) (10 ⁇ M) alone or with resveratrol and CC together.
• FUGW lentivirus expressing GFP only
• dnAMPK dominant negative AMPK
• caAMPK constitutively active AMPK
• Quantitative analysis of average neurite length (I) showed significant (denoted by asterisks) neurite outgrowth inhibition by AMPK inhibition (p ⁇ 0.001) and neurite outgrowth promotion by caAMPK (p ⁇ 0.005).
• Quantitative RT-PCR analysis of markers of mitochondrial biogenesis demonstrated that resveratrol treatment resulted in an 18-fold increase in Tfam (K) and 2-fold increases in PGC- l ⁇ and MFN2 mRNA levels (J). Values were normalized to the 18S transcript. Data shown is representative of two independent experiments.
• FIG. 13 illustrates that resveratrol-mediated AMPK activation and neurite outgrowth is independent of SlRTl and CaMKK ⁇ in Neuro2a cells.
• Phospho-specific antibodies were used to assess activation of AMPK and ACC in lysates of Neuro2a cells treated with DMSO (control) or resveratrol (10 ⁇ M) in the presence or absence of three SIRTl inhibitors (Sirtinol, splitomycin, nicotinamide) or the CaMKK ⁇ inhibitor STO 609 for 2 hr.
• Resveratrol induces rapid activation of AMPK (A) that occurs in concurrence with phosphorylation of ACC (B) and is not prevented by SIRTl or CaMKK ⁇ inhibitors.
• AICAR is included as a positive control for AMPK and ACC phosphorylation. Total AMPK and ACC are shown in the lower panels of (A) and (B).
• C Neuro2a cells were allowed to differentiate in serum-starvation medium containing resveratrol in the absence or presence of the SIRTl inhibitors splitomycin (10 ⁇ M), nicotinamide (10 mM), sirtinol (10 ⁇ M, data not shown) or STO 609 (2.5 ⁇ M). No inhibition of neurite outgrowth was observed in the presence of either SIRTl or CaMKK ⁇ inhibitors.
• Figures 14 illustrates that AMPK activation by resveratrol in dorsal root ganglia sensory neurons requires Lkbl.
• Embryonic DRG neurons from Lkbl flox/flox mice were infected with lentivirus expressing Cre recombinase (FCIV-Cre) or GFP only (FUGW control). Lentiviral infection was monitored by GFP fluorescence (A). A western blot using antibody against LKBl demonstrates complete loss of Lkbl in Cre-expressing neurons (B). Lkbl flox/flox DRG neurons were infected with FUGW or FCIV-Cre and treated as indicated. AMPK or ACC was immunoprecipitated from neuronal lysates and western blots were probed with the respective phospho-specific antibodies.
• Resveratrol-mediated AMPK (C) and ACC phosphorylation (D) was significantly reduced upon Lkbl excision by FCIV-Cre. No inhibition of AMPK or ACC phosphorylation was observed by treatment with SIRTl or CaMKK ⁇ inhibitors. Lysate from AICAR treated DRG neurons was included as a positive control.
• E, F Embryonic DRG neurons were cultured from wild type and SIRTl -deficient littermates derived from SIRTl heterozygous matings. Western blot analysis with phospho-specific antibodies revealed that resveratrol stimulated AMPK (E) and ACC phosphorylation (F) equivalently in wild type and SIRTl -deficient neurons.
• Levels of total AMPK and ACC are shown in the bottom panels of C, D, E and F. Densitometric analysis of changes in levels of phospho AMPK (G) and phospho ACC (H) in SIRTl +/+ and SIRTl-/- DRG neurons in presence and absence of resveratrol is shown. Note: Resv, resveratrol.
• FIG. 15 illustrates that resveratrol treatment causes AMPK phosphorylation in the brain.
• Western analysis with AMPK and ACC phospho-specific antibodies showed increased levels of phosphorylated AMPK (A) and ACC (C) in brains of resveratrol treated animals. Total AMPK and ACC levels are shown as loading controls in B and D.
• E Densitometry was used to quantify the increased level of AMPK and ACC phosphorylation in the brain of resveratrol treated animals (* p ⁇ 0.005).
• FIG. 16 illustrates that resveratrol activates AMP kinase in cortical neurons through Lkbl and CaMKK ⁇ .
• A Cortical neuron cultures were established from E 13.5 Lkbl flox/flox embryos and infected with lentivirus expressing Cre recombinase (FCIV-Cre) or GFP only (FUGW control). Lentiviral infection of cortical neurons was monitored by GFP fluorescence (A). A western blot using LKBl antibody demonstrates complete loss of Lkbl in Cre-expressing cortical neurons (B). Lkbl flox/flox cortical neurons were infected with FUGW or FClV-Cre and treated as indicated.
• AMPK or ACC was immunoprecipitated from neuronal lysates and western blots were probed with the respective phospho-specific antibodies.
• Resveratrol-mediated AMPK (C) and ACC phosphorylation (D) was significantly reduced upon Lkbl excision by FCIV-Cre and in neurons treated with the CaMKK ⁇ inhibitor STO 609 (2.5 ⁇ M).
• No inhibition of resveratrol-stimulated AMPK or ACC phosphorylation was observed in neurons treated with SIRTl inhibitors. Lysates from AICAR treated cortical neurons is included as a positive control.
• E, F Embryonic cortical neurons were cultured from wild type and SIRTl -deficient littermates derived from SIRTl heterozygous matings. Western blot analysis with phospho-specific antibodies revealed that resveratrol-stimulated AMPK (E) and ACC phosphorylation (F) equivalently in wild type and SIRTl -deficient cortical neurons. Levels of total AMPK and ACC are shown in the bottom panels of C, D, E and F.
• Figure 17 illustrates that activation of the AMPK pathway by resveratrol or AICAR promotes the survival of neuronal cells in nutrient-deprived conditions.
• FCS 10% FCS
• cells proliferate and ultimately die if cells are not passaged.
• FCS 0.2% FCS
• neuro2A cells differentiate and project neurites but ultimately die within one week.
• neuro2A cells are treated with either resveratrol or AICAR, regardless of the FCS concentration, these cells are resistant to death for a prolonged period of time. A subset of the surviving cells also exhibit robust neurite projections.
• FIG 18 illustrates that activation of AMPK protects axons while inhibiton of AMPK function enhances axonal injury during oxygen deprivation.
• DRG dorsal root ganglia
• AMPK function was genetically inhibited by expressing a dominant negative AMPK (DnAMPK; which perturbs endogenous AMPK function) in DRG neurons by lentiviral transduction or was pharmacologically inhibited by pre-incubating DRG neurons with the AMPK inhibitor Compound C for 45 min prior to hypoxia (0.1% 02).
• DnAMPK dominant negative AMPK
• the present invention involves methods and compositions for treating neuropathies, neurodegenerative diseases, and other neurological disorders in which axonal degeneration is a component.
• the methods in various embodiments, comprise administering to a mammal such as a human an effective amount of a substance that increases AMPK activity, LKBl activity and/or CaMKK ⁇ activity in diseased and/or injured neurons or supporting cells.
• the methods can comprise administering to a mammal an effective amount of an agent that effects an increase in NAD activity in diseased and/or injured neurons or supporting cells.
• an increase in NAD activity can act to increase sirtuin activity which then produces a decrease in axonal degeneration of injured neuronal cells compared to axonal degeneration that occurs in injured neuronal cells not treated with the agent. Such a decrease in axonal degeneration can include a complete or partial amelioration of the injury to the neuron.
• an increase in NAD activity could act through mechanisms not involving sirtuin molecules to produce or to contribute to the production of a decrease in axonal degeneration.
• an agent effective for treatment of diseased and/or injured neurons or supporting cells may act via several mechanisms, such as, for example, in the case of resveratrol, as shown below.
• SIRT1-SIRT7 Seven known sirtuin molecules referenced as SIRT's make up the Sir2 family of histone/protein deacetylases in mammals and all such sirtuin molecules are included within the scope of the present teachings.
• the seven human sirtuins, SIRT1-SIRT7, are NAD-dependent histone/protein deacetylases which are described more fully in connection with NCBI LocusLink ID Nos. 2341 1, 22933, 23410, 23409, 23408, 51548 and 51547, respectively (see http://www.ncbi.nlm.hih.gov/LocusLink/). Said NCBI LocusLink reference sites are hereby incorporated by reference.
• Amino acid sequences of human SIRT1-SIRT7 are set forth herein as SEQ ID NO: 1- SEQ ID NO: 7, respectively.
• the methods and compositions of the present invention can increase activity of any one or more of the sirtuins and, in particular, various methods of the present teachings can lead to an increase of activity of SIRTl .
• activity of a particular substance can depend upon the concentration of the substance and the functional effectiveness of the substance. Activity of a substance can be increased by numerous factors including, for example, increasing synthesis, decreasing breakdown, increasing bioavailability of the substance or diminishing binding of the substance or otherwise increasing the available amount of free substance. Increasing functional effectiveness can result, for example, from a change in molecular conformation, a change in the conditions under which the substance is acting, or a change in sensitivity to the substance. Increasing activity with respect to sirtuin molecules is intended to mean increasing concentration or enhancing functional effectiveness or increasing the availability of NAD, increasing the flux through one or more biosynthetic pathways for NAD or any combination thereof . Reference to an agent or substance acting "at least in part" by a certain activity or mechanism indicates that the activity or mechanism represents at least one effect of administration of the agent or substance.
• Neuropathies in various aspects of the present teachings can include any disease or condition involving neurons and/or supporting cells, such as, for example, glia, muscle cells, or fibroblasts.
• neuropathies include diseases or conditions involving axonal damage, i.e., axonopathies.
• Axonal damage can be caused by traumatic injury or by non-mechanical injury due to diseases or conditions and the result of such damage can be degeneration or dysfunction of an axon and loss of functional neuronal activity.
• Disease and conditions producing or associated with such axonal damage are among a large number of neuropathic diseases and conditions.
• Such neuropathies can include peripheral neuropathies, central neuropathies, and combinations thereof.
• peripheral neuropathic manifestations can be produced by diseases focused primarily in the central nervous systems, and central nervous system manifestations can be produced by essentially peripheral or systemic diseases.
• Peripheral neuropathies involve damage to the peripheral nerves and such can be caused by diseases of the nerves or as the result of systemic illnesses. Some such diseases can include diabetes, uremia, infectious diseases such as AIDs or leprosy, nutritional deficiencies, vascular or collagen disorders such as atherosclerosis, and autoimmune diseases such as systemic lupus erythematosus, scleroderma, sarcoidosis, rheumatoid arthritis, and polyarteritis nodosa. Peripheral nerve degeneration can also result from traumatic, i.e., mechanical damage to nerves as well as chemical or thermal damage to nerves.
• Such conditions that injure peripheral nerves include compression or entrapment injuries such as glaucoma, carpal tunnel syndrome, direct trauma, penetrating injuries, contusions, fracture or dislocated bones; pressure involving superficial nerves (ulna, radial, or peroneal) which can result from prolonged use of crutches or staying in one position for too long, or from a tumor; intraneural hemorrhage; ischemia; exposure to cold or radiation or certain medicines or toxic substances such as herbacides or pesticides.
• the nerve damage can result from chemical injury due to a cytotoxic anticancer agent such as, for example, a vinca alkaloid such as vincristine.
• Typical symptoms of such peripheral neuropathies include weakness, numbness, paresthesia (abnormal sensations such as burning, tickling, pricking or tingling) and pain in the arms, hands, legs and/or feet.
• the neuropathy can also be associated with mitochondrial dysfunction.
• Such neuropathies can exhibit decreased energy levels, i.e., decreased levels of NAD and ATP.
• a peripheral neuropathy can also be a metabolic and endocrine neuropathy which includes a wide spectrum of peripheral nerve disorders associated with systemic diseases of metabolic origin.
• Some non-limiting examples of these diseases include diabetes mellitus, hypoglycemia, uremia, hypothyroidism, hepatic failure, polycythemia, amyloidosis, acromegaly, porphyria, disorders of lipid/glycolipid metabolism, nutritional/vitamin deficiencies, and mitochondrial disorders, among others.
• the common hallmark of these diseases is involvement of peripheral nerves by alteration of the structure or function of myelin and axons due to metabolic pathway dysregulation.
• Neuropathies also include optic neuropathies such as glaucoma; retinal ganglion degeneration such as r those associated with retinitis pigmentosa and outer retinal neuropathies; optic nerve neuritis and/or degeneration including that associated with multiple sclerosis; traumatic injury to the optic nerve which can include, for example, injury during tumor removal; hereditary optic neuropathies such as Kjer's disease and Leber's hereditary optic neuropathy; ischemic optic neuropathies, such as those secondary to giant cell arteritis; metabolic optic neuropathies such as neurodegenerative disesases including Leber's neuropathy mentioned earlier, nutritional deficiencies such as deficiencies in vitamins B12 or folic acid, and toxicities such as due to ethambutol or cyanide; neuropathies caused by adverse drug reactions and neuropathies caused by vitamin deficiency. Ischemic optic neuropathies also include non- arteritic anterior ischemic optic neuropathy.
• Neurodegenerative diseases that are associated with neuropathy or axonopathy in the central nervous system include a variety of diseases. Such diseases include those involving progressive dementia such as, for example, Alzheimer's disease, senile dementia, Pick's disease, and Huntington's disease; central nervous system diseases affecting muscle function such as, for example, Parkinson's disease; motor neuron diseases and progressive ataxias such as amyotrophic lateral sclerosis; demyelinating diseases such as, for example multiple sclerosis; viral encephalitides such as, for example, those caused by enteroviruses, arboviruses, and herpes simplex virus; and prion diseases.
• progressive dementia such as, for example, Alzheimer's disease, senile dementia, Pick's disease, and Huntington's disease
• central nervous system diseases affecting muscle function such as, for example, Parkinson's disease
• motor neuron diseases and progressive ataxias such as amyotrophic lateral sclerosis
• demyelinating diseases such as, for example
• Mechanical injuries such as glaucoma or traumatic injuries to the head and spine can also cause nerve injury and degeneration in the brain and spinal cord.
• ischemia and stroke as well as conditions such as nutritional deficiency and chemical toxicity such as with chemotherapeutic agents can cause central nervous system neuropathies.
• Additional manifestations within the scope of the neurological conditions which can be treated or ameliorated by the methods of the present teachings include convulsions and seizures, e.g., those associated with epilepsy, migraine, syncope, bipolar disorder, psychosis, anxiety, a stress-inducing disorder, or other neuropsychiatric disorders having paroxysmal or periodic features.
• convulsions and seizures e.g., those associated with epilepsy, migraine, syncope, bipolar disorder, psychosis, anxiety, a stress-inducing disorder, or other neuropsychiatric disorders having paroxysmal or periodic features.
• treatment is intended to include intervention after the occurrence of neuronal injury.
• a treatment can ameliorate neuronal injury by administration after a primary insult to the neurons occurs.
• Such primary insult to the neurons can include or result from any disease or condition associated with a neuropathy.
• Treatment also includes prevention of progression of neuronal injury.
• Treatment as used herein can include the administration of drugs and/or synthetic substances, the administration of biological substances such as proteins, nucleic acids, viral vectors and the like as well as the administration of substances such as neutraceuticals, food additives or functional foods.
• the methods and compositions of the present invention can be useful in treating mammals.
• mammals include humans as well as non-human mammals.
• Non-human mammals include, for example, companion animals such as dogs and cats, agricultural animals such live stock including cows, horses and the like, and exotic animals, such as zoo animals.
• Substances that can increase sirtuin activity in mammals can include polyphenols, some of which have been described earlier (see for example Howitz et al., Nature 425: 191-196, 2003 and supplementary information that accompanies the paper all of which is incorporated herein by reference).
• Polyphenol compounds of the present teachings can include stilbenes such as resveratrol, piceatannol, deoxyrhapontin, trans-stilbene and rhapontin; chalcones such as butein, isoliquiritigen and 3,4,2',4',6'-pentahydroxychalcone and chalcone; flavones such as fisetin, 5,7,3',4',5'-pentahydroxyflavone, luteolin, 3,6,3',4'-tetrahydroxyflavone, quercetin, 7,3',4',5'-tetrahydroxyflavone, kaempferol, 6-hydroxyapigenin, apigenin, 3,6,2',4'- tetrahydroxyflavone, 7,4'-dihydroxyflavone, 7,8,3',4'-tetrahydroxyflavone, 3,6,2', 3'- tetrahydroxyflavone, 4'-hydroxyflavone, 5,
• a polyphenol can be resveratrol, fisetin, butein, piceatannol or quercetin.
• resveratrol and other polyphenol compounds as discussed above can be used to treat or prevent a neuropathy or axonopathy in a mammal in need thereof.
• other substances which increase AMPK activity, LKBl activity and/or CaMKK ⁇ activity can be used to treat or prevent a neuropathy or axonopathy in a mammal in need thereof.
• an activator of AMPK such as AICAR, metformin or phenformin
• additional polyphenols or other substances that increase AMPK activity, LKB 1 activity and/or CaMKK ⁇ activity can be identified using the assay systems described herein, as well as in commercially available assays known to those skilled in the art. Additional assays are disclosed, e.g., in U.S. published patent applications 2005026233 (Carling et al.) and 20060035301 (Corvera et al.).
• additional polyphenols or other substances that increase sirtuin deacetylase activity can be identified using assay systems described herein as well as in commercially available assays such as fluorescent enzyme assays (Biomol International L.P., Plymouth Meeting, Pennsylvania).
• Sinclair et al. also disclose substances that can increase sirtuin and/or AMPK activity (Sinclair et al., WO2005/02672; and Sinclair et al., Publication No. 200601 1 1435, which are incorporated in their entirety by reference).
• NAD activity can be increased by administration of NAD or NADH as well as by synthesizing NAD.
• NAD can be synthesised through three major pathways, the de novo pathway in which NAD is synthesized from tryptophan, the NAD salvage pathway in which NAD is generated by recycling degraded NAD products such as nicotinamide (Lin et al. Curent Opin. Cell Biol. 15:241-246, 2003; Magni et al., Cell MoI. Life Sci.
• a precursor of NAD in the de novo pathway such as, for example, tryptophan or nicotinate and/or substances in the NAD salvage pathway such as, for example, nicotinamide, nicotinic acid, nicotinic acid mononucleotide, or deamido-NAD and/or substances in the nicotinamide riboside kinase pathway such as, for example, nicotinamide riboside or nicotinamide mononucleotide, could potentially increase NAD activity.
• nicotinamide mononucleotide, nicotinic acid mononucleotide or nicotinamide riboside in addition to NAD, can protect against axonal degeneration to a similar extent as NAD, however, nicotinic acid and nicotinamide do not.
• the increased NAD activity can then increase sirtuin histone/protein deacetylase activity in the injured neurons and diminish or prevent axonal degeneration.
• NAD can be increased in injured neurons by administering enzymes that synthesize NAD or nucleic acids comprising enzymes that synthesize NAD.
• Such enzymes can include an enzyme in the de novo pathway for synthesizing NAD, an enzyme of the NAD salvage pathway or an enzyme of the nicotinamide riboside kinase pathway or a nucleic acid encoding an enzyme in the de novo pathway for synthesizing NAD, an enzyme of the NAD salvage pathway or an enzyme of the nicotinamide riboside kinase pathway and, in particular, an enzyme of the NAD salvage pathway such as, for example, a nicotinamide mononucleotide adenylyltransferase (NMNAT) such as NMNATl .
• NMNAT nicotinamide mononucleotide adenylyltransferase
• administering can diminish or prevent axonal degeneration in injured neurons.
• the human NMNATl enzyme (E.C.2.7.7.18) is represented according to the GenBank Assession numbers for the human NMNATl gene and/or protein:NP_073624; NM_022787; AAL76934; AF459819; and NP_073624; AF314163.
• a variant of this gene is NMNAT-2 (KIAA0479), the human version of which can be found under GenBank Accession numbers NP_055854 and NM_015039.
• percent identical refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position.
• FASTA FASTA
• BLAST BLAST
• ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.
• the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
• a gap weight e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
• Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA.
• an alignment program that permits gaps in the sequence is utilized to align the sequences.
• the Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. MoI. Biol. 70: 173-187 (1997).
• the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences.
• An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer.
• MPSRCH uses a Smith- Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors.
• Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases. Databases with individual sequences are described in Methods in Enzymology, ed. Doolittle, supra. Databases include Genbank, EMBL, and DNA Database of Japan (DDBJ).
• a "variant" of a polypeptide refers to a polypeptide having the amino acid sequence of the polypeptide in which is altered in one or more amino acid residues.
• the variant may have "conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine).
• a variant may have "nonconservative” changes (e.g., replacement of glycine with tryptophan).
• Analogous minor variations may also include amino acid deletions or insertions, or both.
• Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
• variants when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to that of a particular gene or the coding sequence thereof. This definition may also include, for example, "allelic,” “splice,” “species,” or “polymorphic” variants.
• a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
• the corresponding polypeptide may possess additional functional domains or an absence of domains.
• Species variants are polynucleotide sequences that vary from one species to another.
• polymorphic variation is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
• Polymorphic variants also may encompass "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 for a disease state.
• SNPs single nucleotide polymorphisms
• An agent that can be used in treating or preventing a neuropathy in accordance with the methods and compositions of the present invention can be comprised by a nicotinamide mononucleotide adenylyltransferase (NMNAT) or a polynucleotide encoding an NMNAT.
• NMNAT nicotinamide mononucleotide adenylyltransferase
• the agent can be an enzyme having NMNAT activity and at least 50% identity with a human NMNATl or at least 50% identity with a human NMNAT3, at least 60% identity with a human NMNATl or at least 60% identity with a human NMNAT3, at least identity with a human NMNATl or at least 70% identity with a human NMNAT3, at least 80% identity with a human NMNATl or at least 80% identity with a human NMN AT3, at least 90% identity with a human NMNATl or at least 90% identity with a human NMNAT3, at least 95% identity with a human NMNATl or at least 95% identity with a human NMN AT3.
• the agent can be comprised by a human NMNATl, a human NMNAT3 or a conservatively substituted variants thereof.
• the agent can also be comprised by a polynucleotide having at least 50% identity with a nucleic acid encoding a human NMNATl or a polynucleotide having at least 50% identity with a nucleic acid encoding a human NMNAT3, a polynucleotide having at least 60% identity with a nucleic acid encoding a human NMNATl or a polynucleotide having at least 60% identity with a nucleic acid encoding a human NMNAT3, a polynucleotide having at least 70% identity with a nucleic acid encoding a human NMNATl or a polynucleotide having at least 70% identity with a nucleic acid encoding a human NMNAT3, a polynucleotide having at least 80% identity with a nucleic acid encoding a human NMNATl or a polynucleotide having at least 80% identity with a nucleic acid en
• the agent can also be comprised by a sirtuin polypeptide or a nucleic acid encoding a sirtuin polypeptide.
• the agent can comprise an enzyme having SIRT activity and at least 50% identity with a human SIRTl, at least 60% identity with a human SIRTl,at least 70% identity with a human SIRTl,at least 80% identity with a human SIRTl,at least 90% identity with a human SIRTl, or at least 95% identity with a human SIRTl .
• the agent can be comprised by a human SIRTl or a conservatively substituted variants thereof.
• the agent can also be comprised by a polynucleotide having at least 50% identity with a nucleic acid encoding a human SIRTl, a polynucleotide having at least 60% identity with a nucleic acid encoding a human SIRTl, a polynucleotide having at least 70% identity with a nucleic acid encoding a human SIRTl, a polynucleotide having at least 80% identity with a nucleic acid encoding a human SIRTl, a polynucleotide having at least 90% identity with a nucleic acid encoding a human SIRTl or a polynucleotide having at least 95% identity with a nucleic acid encoding a human SIRTl .
• the agent can comprise a polynucleotide encoding a human SIRTl or a variant thereof.
• Administration can be by any suitable route of administration including buccal, dental, endocervical, intramuscular, inhalation, intracranial, intralymphatic, intramuscular, intraocular, intraperitoneal, intrapleural, intrathecal, intratracheal, intrauterine, intravascular, intravenous, intravesical, intranasal, ophthalmic, oral, otic, biliary perfusion, cardiac perfusion, priodontal, rectal, spinal subcutaneous, sublingual, topical, intravaginal, transermal, ureteral, or urethral.
• routes of administration including buccal, dental, endocervical, intramuscular, inhalation, intracranial, intralymphatic, intramuscular, intraocular, intraperitoneal, intrapleural, intrathecal, intratracheal, intrauterine, intravascular, intravenous, intravesical, intranasal, ophthalmic, oral, otic, biliary perfusion, cardiac perfusion, priodontal, rec
• Dosage forms can be aerosol including metered aerosol, chewable bar, capsule, capsule containing coated pellets, capsule containing delayed release pellets, capsule containing extended release pellets, concentrate, cream, augmented cream, suppository cream, disc, dressing, elixer, emulsion, enema, extended release fiber, extended release film, gas, gel, metered gel, granule, delayed release granule, effervescent granule, chewing gum, implant, inhalant, injectable, 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, solution for slush, solution/drops, concentrate solution, gel forming solution/drops, sponge, spray, metered spray, suppository, suspension, suspension/drops, extended
• Intraocular admistration can include administration by injection including intravitreal injection, by eyedrops and by trans-scleral delivery.
• Administration can also be by inclusion in the diet of the mammal such as in a functional food for humans or companion animals.
• compositions that increase sirtuin activityof the invention can be administered orally.
• such formulations can be encapsulated and formulated with suitable carriers in solid dosage forms.
• suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.
• the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
• the compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art.
• the formulations can also contain substances that diminish proteolytic degradation and promote absorption such as, for example, surface active agents.
• the specific dose can be calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also depend upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity in assay preparations such as has been described elsewhere for certain compounds (see for example, Howitz et al., Nature 425:191-196, 2003 and supplementary information that accompanies the paper). Exact dosages can be determined in conjunction with standard dose-response studies.
• the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, 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 chosen route of administration.
• the present teachings demonstrate several mechanisms (such as increased activity of AMPK, LKBl, CaMKK ⁇ , NAD, and/or sirtuin) for treating neuropathies, and other mechanisms are known, included among the methods of the present teachings are methods of treating or preventing a neuropathy or axonopathy in a mammal in need thereof, involving administering to the mammal an effective amount, in combination, of two or more of: (a) an agent that acts at least in part by increasing AMPK activity, LKBl activity and/or CaMKK ⁇ activity in diseased and/or injured neurons and supporting cells; (b) an agent that acts at least in part by increasing sirtuin activity in diseased and/or injured neurons and supporting cells; (c) an agent that acts at least in part by increasing NAD activity in diseased and/or injured neurons and supporting cells; and (d) an agent that acts at least in part by another mechanism in diseased and/or injured neurons and supporting cells.
• antidepressants e.g., tricyclics
• anticonvulsants e.g., gabapentin and carbamazepine
• sodium channel blockers e.g., mexiletine
• Some methods of the present teachings in addition to a primary step of administering therapeutically effective amounts of an agent, or combination of agents, also include methods for treatment or prevention of a neuropathy which involve identifying a subject in need of such administration.
• identification of such a subject can be accomplished by diagnosing an individual as having, or being at risk of developing, a clinically diagnosable neurodegenerative disease or neurological condition wherein the disease or condition is believed to be treatable or preventable by increasing AMPK activity, LKBl activity and/or CaMKK ⁇ activity, as described herein.
• these methods can involve assessment of the levels of AMPK activity, LKBl activity, CaMKK ⁇ and/or ACC activity, assessment of the effects low levels of AMPK activity, LKBl activity, CaMKK ⁇ and/or ACC activity, or assessment of the neurological symptoms or effects associated therewith related to the disease or condition in question.
• the methods can also involve monitoring of the subject before, during or after a course of treatment to assess the effectiveness of the regimen to increase AMPK and/or LKBl and/or CaMKK ⁇ activity, other activities disclosed herein, or to determine the need for, or appropriate modifications to, further treatment or prophylaxis.
• an assessment may include genetic analysis of mutations or alterations in a sample of a subject's DNA for AMPK, LKBl, CaMKK ⁇ or related proteins. Assessment may be accomplished by various sequencing procedures well know to those in this art.
• the present teachings include methods of screening candidate agents.
• agents can be tested for effectiveness in decreasing or preventing axonal degeneration of injured neuronal cells.
• a candidate agent can be administered to neuronal cells subjected to injury; the injured neuronal cells can then be assayed for a decrease in axonal degeneration.
• a candidate agent can be added prior to producing the injury.
• an injury can be introduced prior to addition of the candidate compound.
• the method can be performed in vitro or in vivo. The in vitro tests can be performed using any of a number of mammalian neuronal cells, or neuronal cell lines (e.g.
• Neuro2a under a variety of experimental conditions in which injury is elicited.
• mammalian neuronal cell-types that can be used are primary dorsal root ganglion cells injured by either transection and removal of the neuronal cell body or growth in media containing vincristine as described below.
• the in vivo tests can be performed in intact animals such as, for example, a mouse model of peripheral nerve regeneration (Pan et al., J. Neurosci. 23:1 1479-1 1488, 2003) or mouse model of progressive motor neuronopathy (Schmalbruch et al., J. Neuropathol. Exp. Neurol. 50: 192-204, 1991; Ferri et al., Current Biol. 13:669-673, 2003).
• an increase in AMPK activity, LKB 1 activity and/or CaMKK ⁇ activity can lead to a decrease or prevention of neuronal injury
• assays which measure, directly or indirectly, increases in such activities can also be used in screens for therapeutic agents.
• methods described above can be used as part of a system to screen for agents that increase AMPK activity, LKB 1 activity and/or CaMKK ⁇ activity, or candidate agents can be screened directly for their impact on such activity, or some combination can be used.
• the assay method can also be used as part of a primary screen for substances that either increase sirtuin activity directly or through increasing NAD activity.
• the methods above also can be used to screenassist in screens for agents that increase NAD biosynthetic activity or agents that increase sirtuin activity in neurons.
• Recombinant vectors that serve as carriers for a nucleic acid encoding a sirtuin molecule or an enzyme for biosynthesis of NAD are also within the scope of the present invention.
• Such recombinant vectors can comprise a promoter operatively linked to a sequence encoding a mammalian NMNATl protein or a mammalian sirtuin protein such as a SIRTl protein.
• Such recombinant vectors can be any suitable vector such as, for example a lentivirus or an adeno-associated virus.
• Any suitable promoter can be also used such as, for example a ubiquitin promoter, a CMV promoter or a ⁇ -actin promoter.
• This protein is composed of the N-terminal 70 AAs of Ufd (ubiquitin fusion degradation protein)2a, a ubiquitin chain assembly factor, fused to the complete sequence of nicotinamide mononucleotide adenylyltransferasel (NMNATl), an enzyme in the NAD salvage pathway that generates NAD within the nucleus.
• Ufd ubiquitin fusion degradation protein
• NMNATl nicotinamide mononucleotide adenylyltransferasel
• the Wlds protein has NMNAT activity but lacks ubiquitin ligase function, suggesting that axonal protection is derived from either increased NMNATl activity or a 'dominant negative' inhibition of Ufd2a function.
• the following genes were cloned into FUGW vector: 1) The first 70 AAs of Ufd2a (the portion contained in Wlds protein) fused to the N-terminus of EGFP (Ufd2a(l -7O)-EGFP) or EGFP with nuclear localization signal at the C-terminal (Ufd2a(l-70)- nucEGFP). 2) The NMNATl portion of Wlds protein fused to the C-terminus of EGFP (EGFP- NMNATl).
• the murine cDNA for Ufd2a/Ube4b was provided by Kazusa DNA Research Institute.
• Murine cDNAs for NMNATl (accession number: BC038133) were purchased from ATCC. PCR-mediated mutagenesis was used to generate point mutations in Ufd2a, NMNATl and Wlds.
• siRNA constructs in the FSP-si vector generated from the FUGW backbone by replacing the ubiquitin promoter and GFP cDNA with the human U6 promoter and Pol I termination signal followed by the SV40 promoter-puromycin-N-acetyl-transferase gene. Cloning of siRNA construct was performed as described previously, so that the siRNA is transcribed from the U6 promoter (Castanotto, et al., RNA, 8: 1454-60, 2002).
• Mouse DRG explants from E12.5 embryos were cultured in the presence of 1 nM nerve growth factor. Non-neuronal cells were removed from the cultures by adding 5- fluorouracil to the culture medium. Transection of neurites was performed at 10-20 DIV using an 18-gauge needle to remove the neuronal cell bodies. Incubation with ⁇ -nicotinamide adenine dinucleotide (Sigma) or Sirtinol (Calbiochem) was performed using conditions indicated in the text or figures.
• Lentiviral expression vectors were generated using HEK293T cells as described above.
• HEK293T cells were infected with lentivirus and cells were lysed 3 days after infection. These lysates were analyzed by immunoblot to using anti-His tag monoclonal antibody (Qiagen) to detect expression of the respective hexahistidine-tagged proteins.
• Lentiviral infection of DRG neurons was performed by incubating -106-107 pfu/ml virus with the DRG explant for 24 h beginning 3-7 days prior to axonal transection. The infected neurons were examined under an inverted fluorescent microscope to insure detectable lentivirus-mediated transgene expression in >95% of neurons.
• NMNATl is an enzyme in the nuclear NAD salvage pathway that catalyzes the conversion of nicotinamide mononucleotide (NMN) and nicotinate mononucleotide (NaMN) to NAD and nicotinate adenine mononucleotide (NaAD), respectively.
• NMN nicotinamide mononucleotide
• NaMN nicotinate mononucleotide
• NaAD nicotinate adenine mononucleotide
• EXAMPLE 3 This example illustrates that increased NMNAT activity in neurons injured with vincristine also show a delayed axonal degradation.
• NMNAT activity by itself can protect axons from a number of insults and mediate the protective effects observed in wlds mice.
• NAD-dependent axonal protection To gain insights into the mechanism of NAD-dependent axonal protection (NDAP), we examined whether NAD was required prior to the removal of the neuronal cell bodies, or whether direct exposure of the severed axons to high levels of NAD was sufficient to provide protection (Fig. 3B). Neuronal cultures were prepared and 1 mM NAD was added to the culture medium at the time of axonal transection or at various times (4 to 48 hr) prior to injury.
• Sir2 family of protein deacetylases and poly(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 longevity in yeast and C. elegans (Bitterman, et al., Microbiol MoI Biol Rev, 67:376- 99, 2003; Hekimi, et al., Science 299:1351-54, 2003).
• PARP is activated by DNA damage and is involved in DNA repair (S.D. Skaper, Ann NY Acad Sci, 993:217-28 and 287-88, 2003).
• Axonal transection was performed by removal of the neuronal cell bodies and the extent of axonal degradation was assessed 12 to 72 hr later.
• Sirtinol effectively blocked NDAP, indicating that Sir2 proteins are likely effectors of this process.
• 3AB had no effect on NDAP, indicating that PARP does not play a role in axonal protection.
• resveratrol 10 ⁇ 100 ⁇ M
• a polyphenol compound that enhances Sir2 activity Howitz, et al., Nature, 425: 191-96, 2003.
• SIRT conserved domain
• SIRTl is the major effector of the increased NAD supply that effectively prevents axonal self destruction.
• SIRTl may deacetylate proteins directly involved in axonal stability, its predominantly nuclear location, along with the requirement for NAD ⁇ 24 hr prior to injury for effective protection, suggest that SIRTl regulates a genetic program that leads to axonal protection.
• Axonal degeneration is an active, self-destructive phenomenon observed not only after injury and in response to chemotherapy, but also in association with aging, metabolic diseases such as diabetic neuropathy, and neurodegenerative diseases.
• Our results indicate that the molecular mechanism of axonal protection in the wlds mice is due to the increased supply of NAD resulting from enhanced activity of the NAD salvage pathway and consequent activation of the histone/protein deacetylase SIRTl .
• NMNATl cytosolic mutant (cytNMNATl) was generated by PCR-mediated site-directed mutagenesis.
• Nuclear form of NMNAT3 (nucNMNAT3) was generated by adding a nuclear localization signal to the C-terminal end of NMNAT3.
• Each PCR amplified NAD synthetic enzyme fragment was cloned into FCIV lentiviral shuttle vector as previously described. The integrity of all the constructs was sequenced using Taq DyeDeoxy Terminator cycle sequencing kits (Applied Biosystems) and an Applied Biosystems 373 DNA sequencer.
• NAD biosynthetic substrates All substrates for NAD biosynthetic enzymes were purchased from Sigma (Na, Nam, NMN, NaMN, nicotininc acid adenine dinucleotide (NaAD), and NAD).
• NmR was synthesized from NMN. Conversion of NMN to NmR was confirmed by HPLC (Waters) using reverse phase column LC- 18T (Supelco). NmR is eluted 260 ⁇ 10 seconds and NMN is eluted 150 ⁇ 10 seconds under 1 ml/min flow rate of buffer containing 5OmM K 2 HPO 4 and 5OmM KH 2 PO 4 (pH 7.0). Biological activity of NmR was accessed as previously described by using yeast strains kindly provided from Dr. Charles Brenner (Dartmouth Medical School, New Hampshire, USA).
• HEK293T cells were infected with a virus that expresses each of NAD biosynthetic enzymes. Cells were lysed 5 days after infection to be analyzed by immunoblot to detect expression of each protein with a hexa-histidine tag by anti-6xHis tag monoclonal antibody (R&D Systems). Subcellular localization of each protein was analyzed using HEK293T cells transiently transfected with a viral shuttle vector for each NAD biosynthetic enzymes.
• NMNAT protein overexpression, affinity purification and enzymatic assay HEK293T cells were transfected with an expression plasmid for each enzyme by using calcium phosphate precipitation. Three days later, cells were washed with PBS twice and then suspended in the buffer containing 50 tnM Sodium Phosphate (pH 8.0), and 300 mM NaCl (buffer A). Cells were then homogenized by SONIFIRE 450 (BRANSON) and supernatant was collected by centrifugation at 10,000 g for 10 min.
• BRANSON SONIFIRE 450
• His-select Nickel Affinity Gel (Sigma) was washed with buffer A and 0.1 ml of 50% gel suspension was added to 1 ml of supernatant and incubated for 10 min at 4°C, then beads binding hexa-histidine -tagged protein was extensively washed with the buffer A. Proteins were eluted by adding 100 ⁇ l of the solution containing 50 mM Sodium Phosphate (pH 8.0), 300 mM NaCl, and 250 mM imidazole. Relative NMNAT enzymatic activity was measured by using affinity purified proteins as described before and subtracted the value obtained from mock transfected cells and normalized by the amount of recombinant protein determined by densitometry.
• Nerve transection Nam, NMN, NmR, or NAD was dissolved in PBS at the concentration of 100 mM or 1 M. Each of 5 ⁇ l solution was injected into left intravitreal component under the anesthesia at a rate of 0.5 ⁇ l ml per second. The left optic nerve was transected at 24 hours after intravitreal injection and optic nerve was recovered at indicated time. Optic nerve tissue was homogenized in 100 ⁇ l of a buffer containing 10OmM tris-HCl (pH 6.8), 1 % SDS, and ImM DTT.
• NAD is synthesized via three major pathways in both prokaryotes and eukaryotes.
• NAD is synthesized from tryptophan (Fig.5).
• the salvage pathway NAD is generated from vitamins including nicotinic acid and nicotinamide.
• a third route from nicotinamide riboside called Preiss-Handler independent pathway has recently been discovered.
• the last enzymatic reaction of the de novo pathway involves the conversion of quinolinate to NaMN by QPRT (EC 2.4.2.19). At this point, the de novo pathway converges with the salvage pathway.
• NaPRT (EC 2.4.2.1 1) converts Na to NaMN, which is then converted to NaAD by NMNAT (EC 2.7.7.1).
• QNSl (EC 6.3.5.1) converts NaAD to NAD.
• NmPRT (EC 2.4.2.12); also reported as visfatin) converts Nam to NMN.
• NMN is also converted to NAD by NMNAT.
• Nicotinamidase (PNC, EC 3.5.1.19), which converts Nam to Na in yeast and bacteria salvage pathway has not been identified in mammals.
• Nrk (EC 2.7.1.22) converts NmR to NMN and converge to salvage pathway.
• a mammalian homologue of NaPRT was also identified as an EST annotated as a mammalian homolog of a bacterial NaPRT.
• Nrk2 is exceptionally highly induced (more than 20-fold) at 14 days after axotomy.
• NAD synthetic enzymes during the axonal degeneration caused by neurotoxin in cultured rat DRG neuron. DRG neurons were treated with 0.1 ⁇ M and 1 ⁇ M rotenone to cause axonal degeneration and collected RNA at 24 hours after the addition of rotenone. The expression of Nrk2 was increased more than 6 folds after rotenone treatment (Fig. 6B).
• Nmat enzymes save axons from degeneration.
• NMNATl nuclear localization of NMNATl
• PGRKRKW nuclear localization signal
• NMNATl and its mutant cytNMNATl were purified from the cell lysate expressing either of proteins by using affinity gel.
• the enzymatic activity of affinity purified proteins was measured as described above and we found that cytNMNATl activity did not altered by its mutation (Fig. 7C).
• Fig.7A, E we observed strong neurite protection as well as nuclear wild NMNATl.
• NMNATl isoenzyme that lacks nuclear localization signal.
• NMNAT3 is previously reported to locate outside nucleus and mitochondria, and have comparable enzymatic activity to NMNATl.
• NMNAT3 nuclear localization signal KPKKIKTED of human topoisomerase I to the C-terminal of NMNAT3 to generate nuclear NMNAT3.
• KPKKIKTED nuclear localization signal
• NMNAT3 was expressed hexa-histidine tagged NMNAT3 or nucNMNAT3 in HEK293T cells and analyzed subcellular localization and its enzymatic activity.
• NMNAT3 was distributed outside the nucleus including bright punctuate staining as reported before and nucNMNAT3 mainly localized in the nucleus with some punctuate staining in the cytosol (Fig. 7B).
• the enzymatic activity of NMNAT3 and nucNMNAT3 were measured and both proteins have comparable enzymatic activity compared with NMNATl (Fig. 7C).
• NAD biosynthetic substrates delays the axonal degeneration of retinal ganglion cells.
• Transection of optic nerve is an in vivo model which can be used to investigate mechanisms leading to Wallerian degeneration and following retinal ganglion cell (RGC) death observed in human diseases such as glaucoma.
• RGC retinal ganglion cell
• intravitreal injection is used for screening of compounds that protect RGC axon from degeneration in vivo and thus we can asses the axon protective effect of each NAD biosynthetic substrates in vivo by intraocular injection of compounds including NAD, NMN, NmR, and Nam.
• mice Lkbl floxed mice were the gift of Dr. Ronald A
• Dorsal root ganglia sensory and cortical neurons were established from El 3.5 mouse embryos, maintained in neurobasal medium supplemented with B27 (and NGF for DRG neurons) and infected with lenti viruses as described (Araki et al., 2004, Science 305:1010-1013; Hawley et al., 2005, Cell Metab 2:21-23).
• Resveratrol was a gift from Sirtris Pharmaceuticals (Cambridge MA) and AICAR was obtained from Toronto Research (North York, Canada).
• Splitomycin and Compound C were purchased from Biomol (Plymouth Meeting, PA) and Calbiochem (San Diego, CA).
• Sirtinol and nicotinamide were purchased from Sigma (Saint Louis, MO)
• Plasmids and viruses Plasmids and viruses.
• the dominant negative (dnAMPK) and constitutively active (caAMPK) plasmids were gifts from Russell Jones (University of Pennsylvania, Philadelphia PA), and pCXN-Cre was a gift of Inder Verma (SaIk Institute, San Diego, CA). All constructs were subcloned into the lentiviral shuttle vector FCIV and verified by nucleotide sequence analysis. Viruses were prepared as previously described (Araki et al., 2004, Science 305:1010-1013).
• Protein and mRNA analysis were performed by standard methods using antibodies directed against total AMPK, phosphorylated AMPK, total ACC or phosphorylated ACC that were obtained from Cell Signaling Technology (Beverley, MA).
• the phospho-AMPK antibody detects endogenous AMPK ⁇ l and cc2 when phosphorylated at threonine 172.
• the LKB 1 antibody was purchased from Upstate (Lake Placid, NY).
• Quantitative RT-PCR analysis was performed using Sybr-Green methodology on a model 7700 instrument (Applied Biosystems) as previously described (Araki et al., 2004, Science 305:1010-1013). Primer sequences were those used in previous studies (Motoshima, et al., 2006, J Physiol 574:63-71).
• resveratrol activated AMPK to a similar extent as AICAR (5-aminoimidazole-4- carboxamide 1- ⁇ -D-ribofuranoside), a well characterized activator of AMPK that is converted to ZMP, an AMP mimetic (Culmsee, et al., 2001, J MoI Neurosci 17:45-48; Terai, et al., 2005, MoI Cell Biol 25:9554-9575).
• AMPK activation by resveratrol results in typical AMPK-mediated downstream responses, we monitored phosphorylation of ACC (acetyl Co-A carboxylase), a primary target of activated AMPK.
• resveratrol stimulation lead to robust phosphorylation of ACC both acutely and chronically to a similar degree as that observed with AICAR stimulation (Fig. 10B).
• AMP ratio increases the phosphorylation of AMPK.
• ATP levels were monitored using a luciferase-based assay in neuro2A cells treated with resveratrol or Oligomycin, an inhibitor of mitochondrial oxidative phosphorylation and therefore ATP production.
• Neuro2a cells are a widely used as in vitro model of neuronal differentiation. These cells cease to proliferate and begin to differentiate, as evidenced by neurite outgrowth, in response to serum starvation, retinoic acid, or growth factors like neurotrophins and GDNF family ligands. As AMPK activation inhibits proliferation of a number of cell types, we first tested whether AMPK activation also inhibits Neuro2a cell proliferation. Neuro2a cells grown under serum starvation conditions (0.2% fetal calf serum) were treated with AICAR (1 mM) or resveratrol (10 ⁇ M).
• AMPK activation by resveratrol could promote mitochondrial biogenesis.
• MFN2 mitofusin 2
• POC l ⁇ peroxisome proliferator activated receptor ⁇ coactivator l ⁇
• Tfam mitochondrial transcription factor A
• SIRTl is expressed in Neuro2a cells by western blot analysis and immunocytochemistry (data not shown).
• resveratrol 10 ⁇ M
• nicotinamide 10 mM
• SIRTl inhibitors None of the SIRTl inhibitors attenuated the robust activation of AMPK by resveratrol as judged by the increased phosphorylation of AMPK and its downstream target ACC (Fig. 13 A, B). Similarly, SIRTl inhibitors had no effect on the ability of resveratrol to stimulate Neuro2a neurite outgrowth (Fig. 13C). These results suggested that resveratrol effects on AMPK are independent of SIRTl activity within the time period examined.
• LKB 1 and CaMKK ⁇ Two kinases, LKB 1 and CaMKK ⁇ , have been identified as upstream activators of AMPK. While no pharmacological inhibitors of LKBl are presently available, we were able to use a selective CaMKK ⁇ inhibitor STO 609 (2.5 ⁇ M) to test whether resveratrol activates AMPK in Neuro2a cells through CaMKK ⁇ . We found that inhibition of CaMKK ⁇ had no effect on resveratrol-mediated AMPK activation or neurite outgrowth (Fig. 13). Together these results suggest that resveratrol-stimulated AMPK activation in Neuro2a cells is independent of rapid deacetylation by SIRT proteins or CaMKK ⁇ function and predict the involvement of other upstream activators of AMPK.
• STO 609 2.5 ⁇ M
• EXAMPLE 16 [00137] This example shows that LKB 1 , but not SIRT 1 is required for resveratrol-stimulated AMPK activation in cortical and dorsal root ganglia sensory neurons.
• LKBl is likely to be the major effector of AMPK activation in neurons.
• DRG dorsal root ganglia
• AMPK activation that we observed in Neuro2a cells and in primary neurons, we performed experiments with pharmacological inhibitors as well as genetic experiments using neurons from SIRTl -deficient mice. Similar to our observations in Neuro2a cells, none of the SIRT inhibitors (Sirtinol, splitomycin and nicotinamide) inhibited resveratrol-stimulated AMPK phosphorylation in DRG or cortical neurons (Fig. 14C, Fig. 16). We also treated embryonic DRG and cortical neurons from SIRTl -deficient mice with resveratrol.
• mice with resveratrol activates AMPK in the brain.
• resveratrol 20 mg/kg body weight
• DMSO DMSO
• a single intraperitoneal injection of resveratrol resulted in increased AMPK (2.5-fold) and ACC (2.1-fold) phosphorylation in the brain within 2 h (Fig 15).
• Fig 15 Western blot analysis revealed that intraperitoneal administration of resveratrol acutely activates AMPK in the brain and, that this activation translates into the phosphorylation and presumed inhibition of ACC, an important downstream target.

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