WO2019104315A1 - Compositions and methods using lipids for treating neurological disease - Google Patents

Abstract

The invention relates to compositions and methods for preventing or ameliorating a neurodegenerative disease characterized by an energy deficit (e.g., amytrophic lateral syndrome (ALS), Huntington's disease, Parksinon's disease, or dementia) or disease symptoms thereof, including imbalance in lipids or lipid metabolism, in a subject by treatment with a composition that includes one or more lipids described herein. In some aspects, the invention features using an ingredient or applying to a food or a nonfood product a composition comprising a lipid described herein in order to treat a neurodegenerative disease characterized by an energy deficit (e.g., amytrophic lateral syndrome (ALS), Huntington's disease, Parksinon's disease, or dementia).

Description

COMPOSITIONS AND METHODS USING LIPIDS FOR TREATING

NEUROLOGICAL DISEASE

BACKGROUND OF THE INVENTION

This application claims the benefit under 35 U. S.C. § 119(e) of U. S. provisional application Ser. No. 62/590,912, filed November 27, 2017, the entire disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (ALS) is a devastating disease that is progressive, invariably fatal and entirely untreatable. Affected patients usually present around age 50 with weakened muscular strength in the periphery, but can also present with difficulty swallowing or breathing, and generally die within 2-5 years of diagnosis due to respiratory failure. All the while, cognitive functions remain nearly intact. Mutations in many genes can cause familial ALS (fALS), and the genes have a wide range of functions and proposed mechanisms. Yet, most cases of ALS (90%) present as sporadic ALS (sALS), meaning no family history and presumably not correlated with genetics. Nevertheless, much evidence exists to support the idea that sALS and fALS have common underlying etiologies. For instance, a common feature of ALS is hypermetabolism, dysregulation of lipids, and wasting. Many mouse models have been developed to help unravel the mechanisms of ALS motor neuron death, but to date, have resulted in candidate gene approaches (educated guesses) that do not extend lifespan of affected mice. Another important aspect of the genetics of ALS, is a strong underlying connection with mutations in some of the same genes as Frontotemporal dementia (FTD). Given these facts, there is a pressing need for fundamental insights into this intractable disease, and treatments.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions and methods useful for reducing or ameliorating an effect of a mutation causing familial amyotrophic lateral sclerosis (fALS) as well as sporadic ALS (sALS) cases that share many common features of fALS.

In one aspect, the invention provides a nutraceutical composition comprising a lipid in a nutraceutically acceptable carrier, wherein the lipid is any one or more of the lipids in Table 1.

In another aspect, the invention provides a dietary supplement comprising a lipid, the lipid is any one or more of the lipids in Table 1.

In yet another aspect, the invention provides a therapeutic composition containing an effective amount of a lipid in a pharmaceutically acceptable excipient, wherein the lipid is any one or more of the lipids in Table 1. In another aspect, the invention provides a kit containing the therapeutic composition of any one of the aspects delineated herein and instructions for the use of the composition for treating or ameliorating a symptom of or delaying progression of a neurodegenerative disease characterized by an energy deficit. In various embodiments, the kit further contains a capture reagent for detecting a level or sequence of a gene that is any one or more of SOD1, C9orf72 repeat, and TDP-43.

In still another aspect, the invention provides a method of reducing or ameliorating an effect of a mutation associated with a neurodegenerative disease characterized by an energy deficit, the method involving administering to the subject a composition comprising a lipid that is any one or more of the lipids in Table 1, thereby delaying progression of, or reducing or ameliorating an effect of a mutation associated with the neurodegenerative disease characterized by an energy deficit in the subject.

In another aspect, the invention provides a method of beating a neurodegenerative disease characterized by an energy deficit in a subject, comprising administering to the subject a composition comprising a lipid selected from the group consisting of the lipids in Table 1, thereby beating or delaying progression of the neurodegenerative disease characterized by an energy deficit in the subject.

In various embodiments of any one of the aspects delineated herein, the lipid is any one or more of lauric acid, mystiric acid, palmitic acid, oleic acid, linoleic acid, gamma-linoleic acid, arachidonic acid, EPA, and DHA, 12-hydroxy laurate, 14-hydroxy mystirate, 16-hydroxy palmitate, 19-hydroxy EPA, 20-hydroxy EPA, 21 -hydroxy DHA, and 22-hydroxy DHA.

In yet another aspect, the invention provides a nubaceutical composition containing an omega-hydroxylated fatty acid, wherein the fatty acid is any one or more of the fatty acids in Table 1.

In still another aspect, the invention provides a dietary supplement containing an omega- hydroxylated fatty acid, wherein the fatty acid is selected from the group consisting of the fatty acids in Table 1, the lipid is selected from the group consisting of the lipids in Table 1.

In another aspect, the invention provides a therapeutic composition comprising an effective amount of an omega-hydroxylated fatty acid in a pharmaceutically acceptable excipient, wherein the fatty acid is selected from the group consisting of the fatty acids in Table 1

In various embodiments of any one of the aspects delineated herein, the effective amount is an amount that ameliorates at least one symptom of a neurodegenerative disease characterized by an energy deficit. In various embodiments, the effective amount is an amount that delays onset or progression of at least one symptom of amyobophic lateral sclerosis (ALS). In some embodiments, the lipid or omega-hydroxy lated fatty acid comprises at least about 5- 75% of the weight of the composition.

In another aspect, the invention provides a method of reducing or ameliorating an effect of a mutation associated with a neurode generative disease characterized by an energy deficit, the method comprising administering to the subject a composition comprising an omega- hydroxylated fatty acid, wherein the fatty acid is selected from the group consisting of the fatty acids in Table 1, thereby delaying progression of, or reducing or ameliorating an effect of a mutation associated with the neurode generative disease characterized by an energy deficit in the subject.

In yet another aspect, the invention provides a method of treating a neurode generative disease characterized by an energy deficit in a subject, comprising administering to the subject a composition comprising an omega-hydroxy lated fatty acid, wherein the fatty acid is selected from the group consisting of the fatty acids in Table 1, thereby treating or delaying progression of the neurodegenerative disease characterized by an energy deficit in the subject.

In various embodiments of any one of the aspects delineated herein, the composition is administered to the subject by oral administration. In various embodiments, the composition is administered to the subject in a drink.

In some embodiments, the subject has a mutation and/or misexpression associated with ALS. In some other embodiments, the mutation is SOD1-G85R, TDP-43 misexpression, or C9orf72 repeat expansion. In various embodiments of any one of the aspects delineated herein, the subject is human. In various embodiments, the composition is the composition or supplement of any one of the aspects delineated herein. In some embodiments, the mutation and/or misexpression associated with ALS is measured using the kit of any one of the aspects delineated herein.

In various embodiments of any one of the aspects delineated herein the

neurodegenerative disease characterized by an energy deficit is amyotrophic lateral sclerosis (ALS). In some embodiments, the ALS is sporadic ALS.

Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By“activity of a cytochrome P450 (CYP) polypeptide” is meant lipid metabolism modulating activity. In some embodiments, the activity is omega-hydroxylation of fatty acids.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule including RNA-based molecules that can act as inhibitory molecules including siRNA or antisense RNAs, or polypeptide, or fragments thereof. In some embodiments, an agent that decreases a level or activity of Cyp4gl polypeptide is administered to a subject. In certain embodiments, the agent that decreases a level or activity of Cyp4gl polypeptide is a siRNA. In some other embodiments, the agent that decreases a level or activity of Cyp4gl polypeptide is a small molecule compound (e.g., HET0016).

By“ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally -occurring polypeptide, while having certain biochemical modifications that enhance or change the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

By“biological sample” is meant any liquid, cell, or tissue obtained from a subject.

As used herein“capture reagent” is meant a reagent specifically binding to a polynucleotide or polypeptide of interest. In some embodiments, the capture reagent is a sequencing primer, amplification primer, or hybridization probe for detecting a level or sequence of a polynucleotide. In particular embodiments, the polynucleotide is a SOD1, C9orf72 repeat, or TDP-43 polynucleotide.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

By“Cyp4gl polypeptide” is meant a polypeptide or fragment thereof having at least 30% amino acid sequence identity to NCBI Accession No. NP 525031 (Drosophila melanogaster) or NCBI Accession No. NP 997235 (human) and having an activity of a cytochrome P450 (CYP) polypeptide. Exemplary Cyp4gl polypeptide sequences at NCBI Accession No. NP 525031 and NP 997235 are provided below:

NP 525031 (Drosophila melanogaster)

1 mavevvqetl qqaasssstt vlgfspmltt lvgtlvamal yeywrrnsre yrmvanipsp

61 pelpilgqah vaaglsnaei lavglgylnk ygetmkawlg nvllvfltnp sdielilsgh

121 qhltkaeeyr yfkpwfgdgl lisnghhwrh hrkmiaptfh qsilksfvpt fvdhskawa

181 rmgleagksf dvhdymsqtt vdillstamg vkklpegnks feyaqavvdm cdiihkrqvk

241 llyrldsiyk ftklrekgdr mmniilgmts kvvkdrkenf qeesraivee istpvastpa

301 skkeglrddl ddidendvga krrlalldam vemaknpdie wnekdimdev ntimfeghdt

361 tsagssfalc mmgihkdiqa kvfaeqkaif gdnmlrdctf adtmemkyle rviletlrly

421 ppvpliarrl dydlklasgp ytvpkgttvi vlqycvhrrp diypnptkfd pdnflperma

481 nrhyysfipf sagprscvgr kyamlklkvl lstivrnyiv hstdteadfk lqadiilkle

541 ngfnvslekr qyatva

NP 997235 (human)

1 maglwlglvw qklllwgaas alslagaslv lsllqrvasy arkwqqmrpi ptvarayplv

61 ghallmkpdg reffqqiiey teeyrhmpll klwvgpvpmv alynaenvev iltsskqidk

121 ssmykflepw lglglltstg nkwrsrrkml tptfhftile dfldimneqa nilvkklekh

181 inqeafncff yitlcaldii cetamgknig aqsnddseyv ravyrmsemi frrikmpwlw

241 ldlwylmfke gwehkkslqi lhtftnsvia eranemnane dcrgdgrgsa psknkrrafl

301 dlllsvtdde gnrlshedir eevdtfmfeg hdttaaainw slyllgsnpe vqkkvdheld

361 dvfgksdrpa tvedlkklry lecviketlr lfpsvplfar svsedcevag yrvlkgteav

421 iipyalhrdp ryfpnpeefq perffpenaq grhpyayvpf sagprncigq kfavmeekti

481 lscilrhfwi esnqkreelg legqlilrps ngiwiklkrr nader

By“Cyp4gl nucleic acid, gene or gene-containing fragment” or“Cyp4gl polynucleotide” is meant a nucleic acid molecule encoding a Cyp4gl polypeptide. Exemplary Cyp4gl polynucleotide sequences are provided at NCBI Accession No. NM 080292 (Drosophila melanogaster) and No. NM 207352 (human), which are reproduced below:

NM 080292 (Drosophila melanogaster)

1 tcacagttga gcactggcgg ctgatatagc aacagtgcca tcttcagaag acaaaaagga 61 tttgcaccag aggaccaggg atcaggagca aagaagcaac agcaaccatg gcagtggaag 121 tagttcagga gacgctgcaa caagcggcgt ccagttcgtc gacgacggtc ctgggattca 181 gtcctatgtt aaccaccta gtgggcaccc tggtggccat ggcatgtac gagtatggc 241 gcaggaatag ccgggaatac cgcatggttg ccaatatacc atccccaccg gagtgccta 301 ttttgggaca ggctcatgtg gccgccggct tgagcaatgc cgagatcctg gccgttggct 361 tgggtacct caacaagtac ggagaaacca tgaaggcctg gttgggcaac gtcctgtgg 421 tgtttctaac caatcccagt gacatcgagt tgatcctgag tgggcaccag cacttgacca 481 aggcggagga gtatcgctac ttcaagccct ggttcggtga tggtctactg atcagcaatg 541 gacatcatg gcgtcatcat cgtaagatga ttgcccccac cttccaccag agcatctga 601 agagcttcgt gcctacattt gtggatcact caaaggcggt agttgccagg atgggctag 661 aagcgggcaa atccttgat gttcatgact atatgtcgca gaccacggtt gacatcctgt 721 tgtctaccgc catgggtgtg aagaagcttc cggagggtaa caagagtttc gaatacgccc 781 aagccgtcgt cgacatgtgt gatatcatac ataagaggca ggttaaatta ctgtaccgcc 841 tggattccat ctacaagttt actaagcttc gcgagaaggg cgatcgcatg atgaacatca 901 tcttgggtat gaccagcaag gtggtcaagg atcgtaagga gaacttccaa gaggagtcac 961 gtgcgatgt tgaggagatt tctacacctg ttgccagcac tcccgcttcc aagaaggagg 1021 gtcttcgcga tgatctggat gatatcgatg aaaatgatgt gggcgccaag aggcgattgg 1081 ctcttctaga tgccatggtg gaaatggcta agaaccccga tatcgagtgg aacgagaagg 1141 acatcatgga tgaggtgaat acaattatgt tgagggcca cgataccacc tcggcgggat 1201 ctagtttcgc cctctgcatg atgggaatcc acaaggacat ccaggctaaa gtcttcgccg 1261 aacagaaggc catcttcggg gataatatgc tgagggattg caccttgcc gataccatgg 1321 agatgaaata tttggagcgc gtaatttag agacttgag gtgtaccca ccagtaccac 1381 ttatcgccag gcgtctggac tacgacctga agttggccag tggtccgtac acggttccca 1441 agggcactac ggtcatcgtg ctgcagtact gcgtgcacag acgtccagac atctacccca 1501 atcccaccaa attcgatccg gacaacttcc tacccgagag gatggccaac aggcattact 1561 actccttcat tcccttagc gctggaccca gaagctgtgt gggccgcaag tacgccatgc 1621 tgaagctaaa ggtcctgcta tccaccatcg tgaggaacta tatgtccac tccaccgaca 1681 cggaggcaga tttcaagctg caggctgaca tcatcctaaa gcttgagaat ggattcaatg 1741 tctcgttgga gaagcgtcag tacgccacgg tggcctagaa tccagaaatc taggaccccg 1801 actacacaca cgcaaccccg aacccgaaac cggaatccag ccctgtatat agatgatgaa 1861 taccgatgaa tatcccaaac cgaaaacttg atgacgaact tataaatcta aaacaccgaa 1921 taagaaccca acgcacaagc cagccagaga gtcaattaat ttttctttcg ttttttaact 1981 cgttactttt atatttgatt aatacctttt tgttgtgg tctttagcga gtggtgcccc

2041 tatataatgt atacgtatat actatatatc cttttaacca actattcaac gcaactgttt

2101 gtgctcttca ccttttagt actcctactt taccactat ctatactttt ttttcgtagc

2161 catgtagtgt gatttttttt cttattcta gtatttatta agtcaaatgg ttaaacgaa

2221 acccaaaaaa tatgaaaaat acacgtatgc gaggcacgta gccgatagag ctgcaaaac

NM_207352 (human)

1 gggcctccag tgcaatcact acgccctggg gcccggaaac cgttttccgg tctttcgctt 61 tcggctgggg cgtggaggcc gcggtgctgc gtaggccggg ccgggcgcag gaacagcccc 121 gtggcgccct ctctggccgc cgcccgggcg ggaaacgtcg ttccggggac cgggcgaccc 181 cgcagcgggg agcgcgccag gtccgcgcgg ggaagtgggc ggtgtgcggc cggcaccgcc 241 tcgcaccacg cccccgcggg cccgcacttt cccggagtgc accccgcggc cgccagccgg 301 ggcgatggcg gggctctggc tggggctcgt gtggcagaag ctgctgctgt ggggcgcggc 361 gagtgccctt tccctggccg gcgccagtct ggtcctgagc ctgctgcaga gggtggcgag 421 ctacgcgcgg aaatggcagc agatgcggcc catccccacg gtggcccgcg cctacccact 481 ggtgggccac gcgctgctga tgaagccgga cgggcgagaa ttttttcagc agatcattga 541 gtacacagag gaataccgcc acatgccgct gctgaagctc tgggtcgggc cagtgcccat 601 ggtggccctt tataatgcag aaaatgtgga ggtaattta actagttcaa agcaaatga 661 caaatcctct atgtacaagt ttttagaacc atggctggc ctaggacttc tacaagtac 721 tggaaacaaa tggcgctcca ggagaaagat gttaacaccc actttccatt ttaccattct 781 ggaagatttc tagatatca tgaatgaaca agcaaatata ttggttaaga aactgaaaa 841 acacataac caagaagcat ttaactgctt ttttacatc actcttgtg cctagatat 901 catctgtgaa acagctatgg ggaagaatat tggtgctcaa agtaatgatg attccgagta 961 tgtccgtgca gttatagaa tgagtgagat gatatttcga agaataaaga tgccctggct 1021 ttggcttgat ctctggtacc tatgttaa agaaggatgg gaacacaaaa agagccttca 1081 gatcctacat actttacca acagtgtcat cgctgaacgg gccaatgaaa tgaacgccaa 1141 tgaagactgt agaggtgatg gcaggggctc tgccccctcc aaaaataaac gcagggcctt 1201 tcttgacttg cttttaagtg tgactgatga cgaagggaac aggctaagtc atgaagatat 1261 tcgagaagaa gtgacacct tcatgtttga ggggcacgat acaactgcag ctgcaataaa 1321 ctggtcctta tacctgtgg gttctaaccc agaagtccag aaaaaagtgg atcatgaatt 1381 ggatgacgtg ttgggaagt ctgaccgtcc cgctacagta gaagacctga agaaacttcg 1441 gtatctggaa tgtgttatta aggagaccct tcgccttttt ccttctgttc cttattgc 1501 ccgtagtgtt agtgaagatt gtgaagtggc aggtacaga gttctaaaag gcactgaagc 1561 cgtcatcatt ccctatgcat tgcacagaga tccgagatac ttccccaacc ccgaggagtt 1621 ccagcctgag cggttcttcc ccgagaatgc acaagggcgc catccatatg cctacgtgcc 1681 cttctctgct ggccccagga actgtatagg tcaaaagttt gctgtgatgg aagaaaagac 1741 cattctttcg tgcatcctga ggcacttttg gatagaatcc aaccagaaaa gagaagagct 1801 tggtctagaa ggacagttga ttcttcgtcc aagtaatggc atctggatca agttgaagag 1861 gagaaatgca gatgaacgct aactatatta tgggtgtg ccttatcat gagaaaggtc 1921 tttattttaa gagatcctg tcattacaa tttacagatc atgagttcaa tatgcttgaa 1981 tcccctagac ctaatttttc ctgatccca ctgatcttga catcaagtct aacaaagaaa 2041 aagtttgag ttttgtattt tcttttttct tttttctta tttttttttt tgaaaccgt

2101 gtctcactct gtcgcccagg ctggaggagt gcagtggtgt gatctcagct cactgcaacc 2161 tccacctccc aggttcaagc aattcttctg cctcagcctc ccaagtagct gggatacag 2221 gtgcctgcca ccatgcctgg ctaatttttt tgtatttta gtagaaacag ggtgtcacca 2281 tgtggccag actggtctca aactcctgac ctcaagtgat ccacctgcct cagcctccca 2341 aagtgctggg attatagtcg tgagccacca cgcctggcca gagttttta ttttatcac 2401 caccatagat gttacagttg gctgtggtca caaaagtagt taattgtgtc agcacccaaa 2461 taaacatcta acaggtttct caacagagga atccacagtc caattccact tcaatgata 2521 gacccaaaaa atataattta atcaaagttc tagagttttt gtttgttgt ttgagatgga 2581 gtctgctct gtcgcccagg ctggaacgca gtggtgacat ctcggctcac tgcaacctcc 2641 acctcccagg ttcaagtgat tctcctgcct cagcctcctg agtagctggg actacaggcg 2701 cctgccacca cgcccagcta atttttgtat ttttagtaga gatggggttt caccatgtg 2761 gccaggatgg tcttgatctc tgacctcgt gatctgcctg cctcggcctc ccaaagtgct 2821 ggcattacag gcatgagcca ccatgcctgg cccaaagttc tagaattttt taaaggtatt 2881 catggtgact caggaataca cacatacaca cacacacaca cacacacaca cacacacata 2941 cacacatata atttgaaaga ggtgagtatg tactctgact tcagctctca ggttttaaaa 3001 attatattag tgggaccagt tatgacaaga ataatcatta tagtactttt cagatttat 3061 aacctggagc agattatttt aagtgata gtaggttctg tacagtttt tctttgatc 3121 gtgcacttat agtcttcatt taattcctca tagaatccca gtcaccttta tatatcatat 3181 tatggaaga gattcatctt cataatctcc agttttttca cagtgcctca cagagtaat 3241 catgcctttt ggagctagaa ggacttaga actatctag tatgctcct ttatattata 3301 agtaagggaa tagaatcaat aagacagttt ctgcccaaag tcatgttacc agtggtgac 3361 agagctggaa atacgtagag atctataccc ttaaatctct ccactcacat gctgatatac 3421 tttctactac aatatgctat agcttatgg aactcagggt gatgatcaga cgtgtcatta 3481 gaacatgagt cctctgcttc tgattcaggc atactttgg gattcttcca tcttaaagg 3541 aaaaaggaag ccattcatct atattagta acccagtaat atctcacta gtttagggtt 3601 agatcttag taattcaac ctatagatc atacttatga aggtgataac tgacacgtgt 3661 tcactgaatt ttaatttgat aggcaataca tctacccact ccattatttt taaaacttc 3721 atttaatagt taaacaaga tggtttgt ttcaatttt tattcactct tcatagaatc

3781 acaatacct tatatatca tatgttattg gaagagattc ctcagtaatc tccaatctct 3841 catagtgcct cacagggttg gtcaatggct ttggaactg gaaggacctt agaacttatc 3901 tgtatgctc ctgatagcca atagcagata gaagcttgca atcaagaggg taggacatgt 3961 gttcttcaat ggatatcaaa ggaagaggtt gcaaaccaaa gccattggc aagccctgta 4021 gcctgggcca tttaagacag gggcggtctc agccaaatg cacccattta actatcccaa 4081 agagccacag tgcctacaac ccaggcccta agttgatgaa gaaaaagtca aggaaggagg 4141 tgatacaatt ggaaatattc ccatcaaatg gtaatcta ttagaaaat gggcatatta 4201 gaaaaagtcc ttccaagatg atttggata ataaaagttg tattgtgga aatggtatt 4261 atctctgttt tatgcactta cattatccc tacatttg ttttagtga ccctacatga 4321 cattaaattt aaagtaaaac atgttaat gtacctttt ggcttgagaa tgtctttcag 4381 ctccagaatt atgtactc atattttaat cagtaagtca ttaagctat gacagagtag 4441 gaattgagaa atatttcat atgctacagt atgaaatgt ggatgctgcc ttgttttata 4501 agaagatgat caaggtttgt gtgcccatta cctttcctct gcctgaaaga cgtgtctcaa 4561 gaaaaataaa ttctatttta gatgcaggta ctgcatttta ttctaagaat tgatatcaat

4621 tcaaaacata gaaaactgta aaagataaat caggagatgg ctgattcata atgggtaata

4681 aaataaatag cactttcgag ctgaaaaaaa aaa

By“C9orf72 repeat expansion” is meant an expansion of the hexanucleotide

(GGGGCC) repeat in the C9orf72 gene, which encodes the C9orf72 polypeptide. In a healthy subject, or a subject that does not have ALS or a neurological disease, there are usually a few repeats of the hexanucleotide. However, in subject having fALS or sALS, there may be up to hundreds or thousands of repeats of the hexanucleotide. An exemplary sequence of the C9orf72 gene is provided at NCBI Accession No. NG 031977 (Homo sapiens).

“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

By“disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neurological diseases, including neurodegenerative diseases associated with or characterized by an energy deficit, such as amyotrophic lateral sclerosis (ALS), fronto-temporal dementia (FTD), Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease.

By "effective amount" is meant the amount that is required to ameliorate the symptoms and/or progression of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, stage of disease, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

The invention provides a number of targets that are useful for the development of highly specific drugs to beat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

By“fatty acid” is meant a carboxylic acid with a long aliphatic“tail” (i.e., hydrocarbon chain). In some embodiments, the fatty acid has 10 carbon atoms, 12 carbon atoms, 14 carbon atoms, 18 carbon atoms, 20 carbon atoms, 22 carbon atoms, 24 carbon atoms, 26 carbon atoms, or 28 carbon atoms. In some embodiments, the fatty acid is saturated or unsaturated. In some embodiments, the unsaturated fatty acid is an omega-3, omega-6, or omega-9 fatty acid.

By“omega-hydroxylated” fatty acid or“omega-hydroxylation” of a fatty acid is meant addition of a hydroxy (-OH) group to an“omega” carbon. An“omega” carbon is a carbon atom that is not an alpha or beta carbon atom in the fatty acid (e.g., a carbon atom at or near the end of the carbon chain, away from the carboxylic acid end). The“alpha” carbon atom in a fatty acid is the first carbon next to the -COOH group; the carbon atom next to the alpha carbon is the“beta” carbon. As used herein, any carbon atom farther away from the -COOH group than the beta carbon is an“omega” carbon.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally -occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally -occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By“lipid” is meant an organic compound that is insoluble in water. Exemplary lipids include fatty acids, oils, waxes, sterols, and triglycerides. As used herein, the term“lipid” includes any lipid in the human lipidome as described by Oswald Quehenberger, Ph.D., and Edward A. Dennis, Ph.D. N Engl J Med 2011; 365: 1812-1823 November 10, 2011 DOI:

10.1056/NEJMral 10490 and J Lipid Res. 2010 Nov; 51(11): 3299-3305., including the references cited therein. Also encompassed in this definition are compounds and molecules that regulate the abundance, distribution and/or activity of lipids.

By“marker” is meant any lipid, protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By“misexpression” is meant an alteration in expression level of a polypeptide or polynucleotide in a cell relative to expression level in a control (e.g., cells from a healthy subject or cells from a subject that does not have amyotrophic lateral sclerosis (ALS) or a neurological disease). Misexpression may be an overexpression (i.e., positive alteration in expression level) or underexpression (i.e., negative alteration in expression level) of a polynucleotide or polypeptide. In some embodiments, overexpression of TDP-43 is a misexpression associated with ALS.

By“mutation” is meant a change in a polypeptide or polynucleotide sequence relative to a wild-type reference sequence. Exemplary mutations include point mutations, missense mutations, amino acid substitutions, and frameshift mutations. In some embodiments, a missense mutation in a Cyp4gl polynucleotide causes an amino acid substitution in a Cyp4gl polypeptide (e.g., a human Cyp4gl polypeptide) . In other embodiments, a mutation in Cyp4gl polypeptide or polynucleotide suppresses an effect of a mutation causing or associated with amyotrophic lateral sclerosis (ALS) (in particular, an effect of a SOD1 G85R allele). A“loss-of- function mutation” is a mutation that decreases or abolishes an activity or function of a polypeptide. A“gain-of-function mutation” is a mutation that enhances or increases an activity or function of a polypeptide. For example, in some embodiments, a gain-of-function mutation in a Cyp4gl polypeptide increases enzymatic activity of the Cyp4gl polypeptide (e.g., increases its ability to perform omega-hydroxylation of fatty acids) or causes an alternative product lipid to be synthesized as opposed to the normal product.

By“mutation and/or misexpression associated with amyotrophic lateral sclerosis (ALS)” or“mutation associated with a neurological disease” is meant any mutation causing or correlated with ALS or a neurological disease in a subject. Exemplary mutations associated with ALS or a neurological disease include, without limitation, mutations in SOD1 (e.g., SOD1 G85R, SOD1 H71Y) or expansion of C9orf72 repeat.

By“neurodegenerative disease characterized by an energy deficit” is meant a disease characterized by or associated with progressive loss of function or structure of neurons, including death of neurons. In particular embodiments, the neuron is a motor neuron. Examples of neurodegenerative disease characterized by an energy deficit include, without limitation, amyotrophic lateral sclerosis (ALS), Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease.

Exemplary neurodegenerative diseases amenable to treatment with a composition delineated herein include, but are not limited to, ALS, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease.

By“nutraceutical” is meant a food or food component that provides a health or medicinal benefit, such as prevention, amelioration, or treatment of a disease. In some embodiments, a nutraceutical herein for treating a neurological disease (e.g., ALS) comprises one or more lipids. In some embodiments, the lipid is a fatty acid. In some other embodiments, the lipid or fatty acid is a substrate or product of fatty acid omega-hydroxylation catalyzed by Cyp4gl polypeptide (e.g., human Cyp4V2). As used herein,“obtaining” as in“obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

By“phenotype associated with amyotrophic lateral sclerosis (ALS)” or“ALS phenotype” is meant any observable or measurable characteristic exhibited by an organism having ALS. Examples of ALS phenotypes include, without limitation, weakened muscles, motor neuron death, metabolic wasting and shortened lifespan.

By“reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

By“reference” is meant a standard or control condition.

A "reference sequence" is a defined sequence used as a basis for sequence comparison.

A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

By "siRNA" is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity.

By“SOD1 polypeptide” is meant a polypeptide or fragment thereof having at least 85% amino acid sequence identity to GenBank Accession No. CAG46542 (Homo sapiens), NCBI Accession No. NP 001261700, or NCBI Accession No. NP 476735 (various isoforms in Drosophila melanogaster), and having an activity of superoxide dismutase (e.g., antioxidant activitiy or catalysis of dismutation of a superoxide radical). The exemplary SOD1 polypeptide sequence at GenBank Accession No. CAG46542 is provided below:

1 matkavcvlk gdgpvqgiin feqkesngpv kvwgsikglt eglhgfhvhe fgdntagcts

61 agphfnplsr khggpkdeer hvgdlgnvta dkdgvadvsi edsvislsgd hciigrtlvv

121 hekaddlgkg gneestktgn agsrlacgvi giaq

By“SOD1 polynucleotide” is meant a nucleic acid molecule encoding a SOD1 polypeptide. An exemplary SOD1 polynucleotide sequence is provided at GenBank Accession No. CR541742, which is reproduced below:

1 atggcgacga aggccgtgtg cgtgctgaag ggcgacggcc cagtgcaggg catcatcaat 61 ttcgagcaga aggaaagtaa tggaccagtg aaggtgtggg gaagcattaa aggactgact

121 gaaggcctgc atggattcca tgttcatgag ttggagata atacagcagg ctgtaccagt

181 gcaggtcctc acttaatcc tctatccaga aaacacggtg ggccaaagga tgaagagagg

241 catgttggag actgggcaa tgtgactgct gacaaagatg gtgtggccga tgtgtctatt

301 gaagattctg tgatctcact ctcaggagac catgcatca tggccgcac actggtggtc

361 catgaaaaag cagatgactt gggcaaaggt ggaaatgaag aaagtacaaa gacaggaaac

421 gctggaagtc gtttggcttg tggtgtaatt gggatcgccc aataa

By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.

Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e ³ and e ¹⁰⁰ indicating a closely related sequence.

By“TDP-43 polypeptide” is meant a polypeptide or fragment thereof having at least 85% amino acid sequence identity to NCBI Accession No. NP_031401.1 and DNA-binding activity. The exemplary TDP-43 polypeptide sequence at NCBI Accession No. NP 031401.1 is provided below:

1 mseyirvted endepieips eddgtvllst vtaqfpgacg lrympvsqc mrgvrlvegi

61 lhapdagwgn lvywnypkd nkrkmdetda ssavkvkrav qktsdlivlg lpwktteqdl

121 keyfstfgev lmvqvkkdlk tghskgfgfv rfteyetqvk vmsqrhmidg rwcdcklpns

181 kqsqdeplrs rkvfvgrcte dmtedelref fsqygdvmdv fipkpfrafa fvtfaddqia

241 qslcgedlii kgisvhisna epkhnsnrql ersgrfggnp ggfgnqggfg nsrgggaglg

301 nnqgsnmggg mnfgafsinp ammaaaqaal qsswgmmgml asqqnqsgps gnnqnqgnmq 361 repnqafgsg nnsysgsnsg aaigwgsasn agsgsgfngg fgssmdskss gwgm

By“TDP-43 polynucleotide” is meant a nucleic acid molecule encoding a TDP-43 polypeptide. An exemplary TDP-43 polynucleotide sequence is provided at NCBI Accession No. NM 007375.3, which is reproduced below:

1 ggtgggcggg gggaggaggc ggccctagcg ccattttgtg ggagcgaagc ggtggctggg

61 ctgcgcttgg gtccgtcgct gcttcggtgt ccctgtcggg cttcccagca gcggcctagc

121 gggaaaagta aaagatgtct gaatatattc gggtaaccga agatgagaac gatgagccca

181 ttgaaatacc atcggaagac gatgggacgg tgctgctctc cacggttaca gcccagtttc

241 caggggcgtg tgggcttcgc tacaggaatc cagtgtctca gtgtatgaga ggtgtccggc

301 tggtagaagg aattctgcat gccccagatg ctggctgggg aaatctggtg tatgttgtca

361 actatccaaa agataacaaa agaaaaatgg atgagacaga tgcttcatca gcagtgaaag

421 tgaaaagagc agtccagaaa acatccgatt taatagtgtt gggtctccca tggaaaacaa

481 ccgaacagga cctgaaagag tattttagta cctttggaga agttcttatg gtgcaggtca

541 agaaagatct taagactggt cattcaaagg ggtttggctt tgttcgtttt acggaatatg

601 aaacacaagt gaaagtaatg tcacagcgac atatgataga tggacgatgg tgtgactgca

661 aacttcctaa ttctaagcaa agccaagatg agcctttgag aagcagaaaa gtgtttgtgg

721 ggcgctgtac agaggacatg actgaggatg agctgcggga gttcttctct cagtacgggg

781 atgtgatgga tgtcttcatc cccaagccat tcagggcctt tgcctttgtt acatttgcag

841 atgatcagat tgcgcagtct ctttgtggag aggacttgat cattaaagga atcagcgttc

901 atatatccaa tgccgaacct aagcacaata gcaatagaca gttagaaaga agtggaagat 961 ttggtggtaa tccaggtggc tttgggaatc agggtggatt tggtaatagc agagggggtg 1021 gagctggttt gggaaacaat caaggtagta atatgggtgg tgggatgaac tttggtgcgt 1081 tcagcataa tccagccatg atggctgccg cccaggcagc actacagagc agttggggta 1141 tgatgggcat gtagccagc cagcagaacc agtcaggccc atcgggtaat aaccaaaacc 1201 aaggcaacat gcagagggag ccaaaccagg ccttcggttc tggaaataac tcttatagtg 1261 gctctaattc tggtgcagca atggtggg gatcagcatc caatgcaggg tcgggcagtg 1321 gttttaatgg aggctttggc tcaagcatgg attctaagtc ttctggctgg ggaatgtaga 1381 cagtggggtt gtggtggtt ggtatagaat ggtgggaatt caaatttttc taaactcatg 1441 gtaagtatat tgtaaaatac atatgtacta agaattttca aaatggttt gttcagtgtg 1501 gagtatattc agcagtattt tgacatttt tcttagaaa aaggaagagc taaaggaatt 1561 tataagttt tgtacatga aaggtgaaa tattgagtgg tgaaagtga actgctgttt 1621 gcctgatgg taaaccaaca cactacaatt gatatcaaaa ggtttctcct gtaatatttt 1681 atccctggac ttgtcaagtg aattcttgc atgttcaaaa cggaaaccat tgattagaac 1741 tacattcttt acccctgtt ttaatttgaa ccccaccata tggatttttt tcctaagaa 1801 aatctccttt taggagatca tggtgtcaca gtgttggtt ctttgtttt gttttttaac 1861 actgtctcc cctcatacac aaaagtacaa tatgaagcct tcatttaatc tctgcagttc 1921 atctcatttc aaatgtttat ggaagaagca cttcatgaa agtagtgctg taaatattct 1981 gccataggaa tactgtctac atgctttctc attcaagaat tcgtcatcac gcatcacagg 2041 ccgcgtcttt gacggtgggt gtcccatttt tatccgctac tctttatttc atggagtcgt 2101 atcaacgcta tgaacgcaag gctgtgatat ggaaccagaa ggctgtctga actttgaaa 2161 ccttgtgtgg gatgatggt ggtgccgagg catgaaaggc tagtatgagc gagaaaagga 2221 gagagcgcgt gcagagactt ggtggtgcat aatggatatt tttaactg gcgagatgtg 2281 tctctcaatc ctgtggcttt ggtgagagag tgtgcagaga gcaatgatag caaataatgt 2341 acgaatgttt ttgcattca aaggacatcc acatctgtg gaagactttt aagtgagttt 2401 tgttctag ataacccaca ttagatgaat gtgtaagtg aaatgatact tgtactcccc 2461 ctaccccttt gtcaactgct gtgaatgctg tatggtgtgt gttctcttct gttactgata 2521 tgtaagtgtg gcaatgtgaa ctgaagctga tgggctgaga acatggactg agcttgtggt 2581 gtgcttgca ggaggacttg aagcagagtt caccagtgag ctcaggtgtc tcaaagaagg 2641 gtggaagttc taatgtctgt tagctaccca taagaatgct gtttgctgca gttctgtgtc 2701 ctgtgctgg atgctttta taagagttgt catgtgga aattctaaa taaaactgat 2761 ttaaataata tgtgtcttg tttgcagcc ctgaatgcaa agaattcata gcagttaatt 2821 cccctttttt gaccctttg agatggaact ttcataaagt ttctggcag tagttattt 2881 tgcttcaaat aaacttattt gaaaagtgt ctcaagtcaa atggattcat cacctgtcat 2941 gcattgacac ctgataccca gactaatg gtattgttc ttgcattggc caaagtgaaa 3001 attttttttt ttctttgaa atctagtttt gaataagtct gggtgaccgc acctaaaatg 3061 gtaagcagta ccctccggct ttttcttagt gcctctgtgc attgggtga tgttctattt 3121 acatggcctg tgtaaatctc catgggaag tcatgccttc taaaaagatt ctatttggg 3181 ggagtgggca aaatgttgat tattttctaa tgcttgtag caaagcatat caatgaaaa 3241 gggaatatca gcaccttcct agttgggat tgaaaagtg gaattaattg cagtagggat 3301 aaagtagaag aaaccacaaa ttatcttgtg cctgaaatcc ataagaggc ctgatagctt 3361 taagaattag ggtgggtgt ctgtctggaa gtgtaagtg gaatgggct tgtcctccag 3421 gaggtggggg aatgtggtaa catgaatac agtgaataa aatcgctac aaaactcaca 3481 ctctcacaat gcatgtaa gtatgtaaaa gcaataacat tgatctctg tgtacttt 3541 tgtaactaa tctgtgaga gtgagctca ttctagt ggaagaatgt gatattgt 3601 gtgttggtag tttacctaat gcccttacct aattagatta tgataaatag gtttgtcatt

3661 tgcaagta cataaacatt tatcaatgaa gtcatccttt agactgtaa tcgccacatt

3721 gtttcattat tcagtttcct ctgtaaaggg atctgagtt gttttaattt tttttttctg

3781 catctgaatc tgcatgattt ccaaaccctg taccatctga attttgcatt tagcactg

3841 cactattact cagcagcagt aacatggtaa cactaaaat ggtactcggg gacctccaaa

3901 gactaaactg acaagccttc aaggagccca ggggtaagtt aactgtcaa cggcatggtt

3961 taatcccttc tttacacttg tgtaaatttc agtactggt catagaaggc tttcaatgtt

4021 gagtggcctt tataacat gttatggta ctgcatagat acgggtattt atttaccct

4081 aagaagattt tgaagttaa aagtacttaa actattggc aaagattgt tttaaaaat

4141 ctatttggtc aatctaaatg cattcattct aaaaaatttt tgaaccaga taaataaaat

4201 ttttttttga caccacaaaa aaaaaaaaaa aaaaaa

By "subject" is meant a multicellular organism, including, but not limited to, a vertebrate (e.g. a human or a non-human mammal such as bovine, equine, canine, ovine, feline) or invertebrate organism (e.g., a fly or worm). In some embodiments, the subject is a vertebrate or invertebrate. In other embodiments, the subject is human. In particular embodiments, the subject is Drosophila melanogaster.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%,

3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of an unbiased forward genetic screen described herein. Forward genetics is the process of identifying a phenotype with a genotype. A fly (Drosophila melanogaster) model of amyotrophic lateral sclerosis (ALS) was generated by introduction of SOD1 ALS-causing mutations into the fly genome. Such human rapid- progressing mutations are lethal in the fly, in particular, the G85R SOD1 allele. Flies die late in development or early in adult life with a profound nerve degeneration. A forward genetic screen using random chemical mutagenesis was performed. The goal was to identify second-site mutations in genes that would restore viability of G85R when mutated.

FIGS. 2A-2C are schematic representations of a forward genetic screen designed to identify suppressors for SOD1 lethality. Shown in FIG. 2A is a screen designed such that all progeny of the mutagenesis cross have a genotype that is ultimately lethal, unless the progeny carries a mutation in another gene that suppresses the lethal effect of the SOD1 G85R allele. In FIG. 2B, males heterozygous for G85R (line A) are starved, fed 25mM EMS, and mated to another line of heterozygous G85R (line B) unmutagenized females. The mated females are allowed to lay eggs, then the progeny larvae are exposed to 37°C heat shock to kill any offspring carrying the TM3,hs-hid balancer to yield homozygous G85R flies. FIG. 2C shows

Representative progeny as an outcome of this cross. G85R homozygotes will only survive this treatment if they carry a potential dominant suppressor mutation (asterisk). The balancer homozygous flies will die due to the recessive lethal gene on the TM3,hs-hid balancer chromosome, and G85R homozygous flies will die due to the lethality of the G85R allele. The survivors are mated back to the original balanced stock of generation 1 (Line B), to show that the suppressor behaves in a Mendelian fashion and to generate a stock.

FIG. 3A-3E are schematics and diagrams depicting results of the screen performed and mapping of the mutations as described herein. FIG. 3A is diagram showing a summary of the results of the screen performed. From the screen, five (5) true-breeding suppressed stocks of G85R/G85R were obtained. All five rescuing mutations mapped to the same gene (Cyp4gl). FIGS. 3B-3C show genetic mapping of the lethality of EMS 81 and EMS 102. Genetic crosses were performed to combine the suppressor mutants with a standard mapping chromosome in females. In FIG. 3B, since it was clear that the lethal mutants both map to between the p and q loci (where p and q are the recessive alleles, and + indicated wild type), recombinants on the p-q interval were obtained by recombination on either interval I or interval II. The lethal suppressor mutations are indicated by let for mutant, or + for wild type alleles. In FIG. 3C, Only three classes of viable males (non-let containing) chromosomes could be obtained from this configuration in recombinogenic females, and all are distinguishable by visible markers. The relative distance on the p-q interval was obtained by [RI/(RI+RII)] x lcM. FIGS. 3D-3E show molecular mapping of the lethality of EMS 81 and EMS 102. In FIG. 3D, EMS 81- PR males were crossed to females of five balanced deficiency stocks for the region determined by genetic mapping (BSCdfl-5). Deficiencies that complement EMS 81 are shown in green . Only BSCdf2 (red) failed to complement EMS 81 lethality, mapping it to a ~260kb region. In FIG. 3E, both EMS 81 and EMS 102 balanced females were crossed to males of seven stocks carrying the w⁺-marked duplications on the third chromsome. Duplications that failed to rescue male lethality are shown in pink. Duplications 2-4 rescued male lethality of both EMS 81 and EMS 102, and limit the molecular interval where they are located. The refined interval contained two genes, A and B (shown at the bottom of FIG. 3E). Gene B had a previously molecularly uncharacterized lethal mutation, l(l)x, which was sequence verified as a 13bp deletion in coding sequence, and EMS 81 failed to complement l(l)x, thus confirming the identity of the gene. Sequence analysis also revealed mutations in both alleles EMS 81 and EMS 102 of the Su(G85R) gene. The Su(G85R) gene is Cyp4gl.

FIG. 4 is a sequence diagram showing the mutations in the protein sequence of Cyp4gl. The mutated amino acids are in bold. The amino acid substitutions are listed at the bottom of FIG. 4.

FIG. 5 is a sequence diagram showing the mutations in the nucleotide sequence of Cyp4gl. The nucleotide sequence encoding the protein sequence of Cyp4gl is in bold. The mutated codons are underlined. The nucleotide mutations are indicated at the bottom of FIG. 5.

FIG. 6 is a diagram depicting the structure of the dCyp4gl (Drosophila melanogaster Cyp4gl) ALS-suppressor gene. The dCyp4gl (CG3972) gene in Drosophila is an intronless gene.

FIG. 7 is an alignment of dCyp4gl and hCYP4V2 (closest human homologue). The box labeled“EMS81” indicates a position of mutation in the mutant line EMS81 obtained in a screen performed herein. The mutation suppressed SOD1 is G85R lethality. The amino acid in this position is conserved between dCyp4gl and hCYP4V2.

FIG. 8 is an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in other invertebrates. The boxes labeled“EMS35/130,”“EMS102,” and“EMS81” indicate positions of suppressor mutations in mutant lines EMS35/130, EMS102, and EMS81 obtained in the screen performed herein, which suppressed SOD1 G85R lethality. Alignment was performed using T-COFFEE, Version_11.00.8cbe486.

The Cyp4gl allele l(l)lBb[19] is a previously identified lethal mutation attributed to the wrong gene, but has a phenotype similar to EMS81 and EMS102. Sequence analyses of Cyp4gl revealed a 13nt deletion in the (1) lBb[19] line which leads to an early truncation of the Cyp4gl gene at the position indicated by an arrow. (1) lBb[19] is almost certainly a null allele of Cyp4gl. The (1) lBb[19] mutation weakly suppresses Sodl-G85R lethality as a heterozygote. Thus, without being bound by theory, it is believed that there are loss-of-function and gain-of- function components to the EMS35/130, EMS102, and EMS81 suppressor alleles, which make them novel.

FIG. 9 is an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in vertebrates. The alignment was performed using T-COFFEE,

Version_11.00.8cbe486. “Dmel” is Drosophila. All other sequences are vertebrate sequences. Suppressor alleles occurred in regions of high conservation, but only EMS81 serine(S) was conserved from flies through mammals

FIG. 10 is an alignment of dCyp4gl and hCYP4V2 showing conserved residues lining a substrate binding pocket. Modeling of the active site of the CYP4A11 protein (related to CYP4V2) showed residues lining the substrate binding pocket and active site involved in omega-hydroxy lation. The EMS81 mutation lies very near residues deep in the substrate binding pocket near the active site.

FIG. 11 is a schematic showing a structure of the compound HET0016. HET0016 (N- hydroxy-N'-(4-n-butyl-2-methylphenyl)Formamidine (CAS 339068-25-6)) is a potent inhibitor of CYP4V2.

FIG. 12 is a table showing the Cyp4 class of enzymes and their interfamily homology.

A Blast search with Cyp4V2[NP_997235(human)] revealed that homology within family Cyp4 is >30% for all homologues. Thus, the Cyp4 class (paralogues) in humans and flies (across families) is conserved at a level of about >30%. FIG. 12 also shows a sharp drop in E-value in non-Cyp4 family members. These non-Cyp4 family members are other Cyp classes of enzymes (e.g., Cyp3), which are quite different from Cyp4 in terms of substrates. Thus, a Cyp4gl polypeptide herein may be any polypeptide having at least about 30% sequence identity to a human Cyp4gl (Cyp4V2) or Drosophila Cyp4gl and having an activity of a Cyp4 enzyme (e.g., omega hydroxy lation of a fatty acid).

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods that are useful for suppressing a neurological disease, in particular, mutations causing amyotrophic lateral sclerosis (ALS) as well as sporadic cases and related dementias. The invention is based, at least in part, on the discovery of mutations in Cyp4gl in a Drosophila melanogaster model of ALS that suppressed lethal effects of SOD1-G85R (an ALS-causing mutation). The dCyp4gl gene is known to play a key role in lipid metabolism, in particular, in regulation of triacylglyceride (TAG) content of Drosophila fat storage cells. Interestingly, dCyp4gl itself is expressed exclusively in oenocytes, the Drosophila equivalent of the mammalian liver (Gutierrez et al., Nature (2007) 445:275-280). Fatty acids have been implicated in ALS, as a system-wide metabolic defect called wasting (or hypermetabolism) is seen in patients. In addition, a very early event in disease progression has been demonstrated in mice, namely, a switch of muscles to utilizing lipids versus glucose. In addition, motor neurons (MNs), in particular those involved in voluntary motion, are the most energy consumptive cells in an organism. Modeling studies and other experiments have strongly suggested that a key determinant of MN health may likely be the ability of the cell to keep up with energy demands. Thus, there is ample precedent in the ALS, as well as dementia, Himtington’s disease,

Parkinson’s disease, and Alzheimer’s disease fields, that energy metabolism, in particular lipid metabolism may be a key pathological target.

CYP4 proteins and fatty acid metabolism in neurological disease

A forward genetic screen described herein revealed four (4) novel missense mutations causing amino acid substitutions in Drosophila melanogaster Cyp4gl that suppressed SOD1 G85R mutant lethality. The suppressor mutations found in Cyp4gl were G92E, D112N, S143N, and M333I. Mapping and molecular studies have revealed the nature of the Cyp4gl gene in flies, a member of the ubiquitous cytochrome P450 (CYP) class of proteins found in all life forms, including bacteria. CYP proteins play two major roles in biology; detoxifying

(biodefense) against many xenobiotics and antibiotics, and biosynthesis of endogenous molecules such as steroids and lipids. There are as many as 37 families of CYP genes. The CYP4 family has a role in the metabolism of fatty acids mobilized from fat stores in animals.

The novel chemistry at its active site (found only in the CYP4 family), allows it to perform omega-hydroxylation of fatty acids, a necessary function to utilize excess free fatty acids.

Accordingly, in some aspects, the invention provides Cyp4gl polypeptides, or fragments thereof having an activity of a cytochrome P450 (CYP) polypeptide, comprising a mutation in at least one amino acid position selected from the group consisting of positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333. In some embodiments, the mutation is G92E, D112N, S143N, or M333I. In some aspects, the invention provides polynucleotides or expression vectors encoding Cyp4gl polypeptides comprising the suppressor mutations described herein. In other aspects, cells or organisms (in particular, Drosophila melanogaster mutant lines) harboring suppressor Cyp4gl mutations are provided.

In some aspects, the suppressor Cyp4gl mutation is a gain-of-function or a loss-of- function mutation. The Cyp4gl allele l(l)lBb[19] is a previously identified lethal mutation attributed to the wrong gene, but has a phenotype similar to EMS81 and EMS102 (i.e., Drosophila lines harboring Cyp4gl S143N and D112N mutations). Sequence analyses of Cyp4gl revealed a 13nt deletion in the (1) lBb[19] line which leads to an early truncation of the Cyp4gl gene at the position indicated by an arrow. (1) lBb[19] is almost certainly a null allele of Cyp4gl. The (1) lBb[19] mutation weakly suppresses Sodl-G85R lethality as a heterozygote. Thus, without being bound by theory, it is believed that there are loss-of-function and gain-of- function components to the EMS35/130, EMS102, and EMS81 suppressor alleles, which make them novel.

The dCyp4gl gene is known to play a key role in lipid metabolism, in particular, in regulation of triacylglyceride (TAG) content of Drosophila fat storage cells. Interestingly, dCyp4gl itself is expressed exclusively in oenocytes, the Drosophila equivalent of the mammalian liver (Gutierrez et al., Nature (2007) 445:275-280). Fatty acids have been implicated in ALS, as a system-wide metabolic defect called wasting (or hypermetabolism) is seen in patients. In addition, a very early event in disease progression has been demonstrated in mice, namely, a switch of muscles to utilizing lipids versus glucose. In addition, motor neurons (MNs), in particular those involved in voluntary motion, are the most energy consumptive cells in an organism. Modeling studies and other experiments have strongly suggested that a key determinant of MN health may likely be the ability of the cell to keep up with energy demands. Thus, there is ample precedent in the ALS, as well as dementia, Huntington’s disease,

Parkinson’s disease, and Alzheimer’s disease fields, that energy metabolism, in particular lipid metabolism may be a key pathological target. Without intending to be bound by theory, it is hypothesized, based on phenotypes and RNAseq data indicating systemic metabolic dysregulation (especially lipid metabolism), that SOD-based ALS model animals die because MNs are not sufficiently“powered” by the animal’s metabolism. The suppressor mutations in the Cyp4gl gene found in the forward genetic screen described herein point to key and conserved changes in protein sequence in this CYP4 family member leading to nearly complete suppression of these SOD1 ALS-causing mutations. In all, 4 novel missense mutations causing amino acid substitutions in Cyp4gl were found. It is worth emphasizing that all genes of the genome were mutagenized without bias and at random, and four independent changes in Cyp4gl that all suppress ALS-mutant lethality were obtained. It is proposed that these Cyp4gl suppressor mutations alter the ability of flies harboring these mutations to mobilize or utilize fat in such a way that the“wasting” of the fly is suppressed or greatly alleviated. This benefits MNs in the fly most, as like most animals, MNs demand the most energy. Thus, in some aspects, the invention features methods of modulating fatty acid or lipid metabolism in a subject, comprising administering to the subject a Cyp4gl polypeptide comprising the suppressor mutations described herein (e.g., mutations in positions corresponding to Drosophila melanogaster Cyp4gl amino acid positions 92, 112, 143, and 333).

In some other aspects, the invention provides methods of identifying a modulator of neurological diseases (particularly ALS, Parkinson’s, dementia, and Huntington’s), comprising screening of candidate agents that modulate the level or activity of a Cyp4gl polypeptide. Without being bound by theory, given current understanding of results obtained in the studies described herein, which are quite novel, it is foreseen that ALS and other age-related cognitive, neuromuscular, or neurological disorders may result from the aberrant“wasting” of resources, especially stored fats. This hypermetabolic effect may likely be a result of a chronic induction of innate immunity, through currently unknown toxic insults, and the bodies’ response to mounting a response to a pathogen that is“unreal.” It is motor neurons in particular that rely most on systemic energetics for their function. The CYP4 class of enzymes play a unique biochemical role in regulation of body fat mobilization and utilization.

It was not expected that the Cyp4gl protein would be involved in the metabolic pathway, considering that motor neuron specific genes were the first candidates expected from an ALS-related suppressor screen. Previous human studies have shown that ALS patients show an early and persistent hypermetabolic signature associated with energy wasting (Desport et al., 2001; Bouteloup et al., 2009). Moreover, a strong transcriptomic signature was observed that indicated that metabolism was altered in the ALS flies. Furthermore, a hSODlG93A transgenic mouse model of ALS shows defective energy homeostasis that benefits from a high energy diet (Dupuis et al., 2004) or lowered amounts of leptin, a regulator of whole-animal energy expenditure (Lim et al., 2014). In addition, the hSODlG85R transgenic mouse model has also been shown to have several metabolic changes consistent with a metabolic switch occurring as an early pathological event (Palamiuc et al., 2015). Thus, without being bound by theory, Cyp4gl a suppressor gene of dSodlG85R lethality and a gene involved in metabolism, is believed to a role in energy balance in cells at a whole-animal level.

Thus, the Cyp4gl mutants obtained herein define the CYP4 class of enzymes (FIG. 12) as a potential target in regulating energy metabolism in disease, particularly neurological diseases associated with or characterized by an energy deficit (e.g., ALS, Parksinon’s, dementia, and Huntington’s). Without being bound by theory, it is believed common cellular mechanisms underlie the motor neuron death that links all of ALS and other neurological diseases characterized by an energy deficit, such as Parksinon’s, dementia, or Huntington’s disease. Agents that modulate the level or activity of a Cyp4gl polypeptide, therefore, are expected to modulate energy balance at a whole-animal level and thereby“correct” the energy deficit in a subject having a neurological disease such as ALS, Parksinon’s, dementia, or Huntington’s disease. In addition to agents that modulate the level or activity of a Cyp4gl polypeptide, a Cyp4gl polypeptide or polynucleotide comprising the suppressor mutations herein is also expected to ameliorate the energy deficit in a subject having a neurological disease.

Accordingly, in some aspects, the invention additionally provides methods of suppressing or beating a neurological disease in a subject (particularly a neurological disease characterized by an energy deficit, such as ALS, Parkinson’s, dementia, or Huntington’s disease), comprising administering to a subject a Cyp4gl polynucleotide or polypeptide comprising suppressor mutations described herein.

In other aspects, the invention further provides Cyp4gl polypeptides in other organisms (e.g., human Cyp4gl polypeptide), comprising the suppressor mutations. In some embodiments, the Cyp4gl polypeptide is from an invertebrate or vertebrate. In some other embodiments, the polypeptide has at least 30%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to a Drosophila melanogaster Cyp4gl polypeptide. In particular

embodiments, the Cyp4gl polypeptide is human Cyp4gl polypeptide. Interestingly, a human homologue of Cyp4gl is the CYP4V2 gene, mutations in which cause a retinal degenerative disease called Biebi’s Crystalline Dysbophy (BCD). Patients with BCD develop a late onset degeneration of the retina with deposits, but more interestingly, BCD patients have a systemic increase in fatty acids/lipids. Many mutations that cause BCD map to very near the suppressor mutations found in Cyp4gl. A mouse model of BCD also recapitulates key feature of the disease, including altered lipid profdes. Thus, without intending to be bound by theory, it is hypothesized that mutations in CYP4V2 could possibly suppress mouse ALS models, as in the fly system.

In addition, adminisbation of lipids, fatty acids, or omega-hydroxy lated fatty acids described herein is expected to ameliorate the energy deficit in a subject having a neurological disease. Such lipids, fatty acids, or omega-hydroxylated fatty acids are subsbates or products of Cyp4-catalyzed omega-hydroxy lation of fatty acids or lipid subsbates or products of metabolic reactions that involve (e.g. downsbeam ol) the fatty acid omega-hydroxylation reaction.

Accordingly, in some aspects, the invention additionally provides methods of suppressing or beating a neurological disease in a subject (particularly a neurological disease characterized by an energy deficit, such as ALS, Parkinson’s, dementia, or Huntington’s disease), comprising administering to a subject one or more lipids, fatty acids, or omega-hydroxylated fatty acids (or any combination thereol) described herein. In some embodiments, the lipid is a glycerolipid, glycerophospholipid, sphingolipid, sterol, or a prenol. In particular embodiments, the lipid is a fatty acid. In still other embodiments, the lipid is a lipid in Table 1. In other embodiments, the lipid or fatty acid is a subsbate or product of Cyp4gl polypeptide (e.g., human Cyp4V2) catalyzed omega- hydroxylation of a fatty acid. In some other embodiments, the fatty acid is saturated, unsaturated, or branched. In still other embodiments, the fatty acid comprises a C-10, C-12, C-13, C-14, C-16, C-18, C-20, C-22, or C-24 carbon chain. In various embodiments, the fatty acid is an omega-3, omega-6, or omega-9 fatty acid. In some embodiments, the fatty acid is eicosapentanoic acid (EPA) or docosahexanoic acid DHA. In some other embodiments, the lipid product of omega-hydroxylation of any one of the fatty acids described or delineated herein (e.g., 12-hydroxy laurate, 14-hydroxy myristate, 16-hydroxy palmitate, 18-hydroxy stearate, 19- hydroxy EPA, 20-hydroxy-EPA, 21 hydroxy DHA, 22-hydroxy DHA). In still other embodiments, the lipid or fatty acid is a signaling molecule. In other embodiments, the lipid or fatty acid is a substrate or product of a reaction or metabolic pathway downstream of omega- hydroxylation of a fatty acid by Cyp4gl (e.g., human Cyp4V2 or other Cyp4 enzyme). For example, the lipid or fatty acid may be a product or substrate in a reaction synthesizing or breaking down fatty acids. In particular embodiments, modulating (e.g., increasing or decreasing) the level of any one or more of a lipid or fatty acid delineated herein is useful in treatment of ALS and/or another neurological disease described herein

Methods of treatment

The present invention provides methods of treating a neurological disease (in particular, ALS) and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a Cyp4gl polynucleotide or polypeptide comprising a suppressor mutation described herein to a subject (e.g., a mammal such as a human). The present invention further methods of treating a neurological disease (in particular, ALS) and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical or nutraceutical composition comprising one or more lipids described herein (e.g., any one or more of the lipids in Table 1) to a subject.

Thus, one embodiment is a method of treating a subject suffering from or susceptible to a neurological disease or disorder or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of one or more lipids described herein (e.g., any one or more of the lipids in Table 1) sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated. In some embodiments, the lipid is a fatty acid. In other embodiments, the lipid or fatty acid is a substrate or product of Cyp4gl polypeptide (e.g., human Cyp4V2) catalyzed omega-hydroxylation of a fatty acid. In some other embodiments, the fatty acid is saturated, unsaturated, or branched. In still other embodiments, the fatty acid comprises a C-10, C-12, C-13, C-14, C-16, C-18, C-20, C-22, or C- 24 carbon chain. In various embodiments, the fatty acid is an omega-3, omega-6, or omega-9 fatty acid. In some embodiments, the fatty acid is eicosapentanoic acid (EPA) or

docosahexanoic acid DHA. In some other embodiments, the lipid product of omega- hydroxylation of any one of the fatty acids described or delineated herein (e.g., 12-hydroxy laurate, 14-hydroxy myristate, 16-hydroxy palmitate, 18-hydroxy stearate, 19-hydroxy EPA, 20- hydroxy-EPA, 21 hydroxy DHA, 22 -hydroxy DHA). In still other embodiments, the lipid or fatty acid is a signaling molecule. In other embodiments, the lipid or fatty acid is a substrate or product of a reaction or metabolic pathway downstream of omega-hydroxy lation of a fatty acid by Cyp4gl (e.g., human Cyp4V2 or other Cyp4 enzyme). In still other embodiments, the lipid is a lipid in Table 1. For example, the lipid or fatty acid may be a product or substrate in a reaction synthesizing or breaking down fatty acids. In particular embodiments, modulating (e.g., increasing or decreasing) the level of anyone or more of a lipid or fatty acid delineated herein ameliorates an imbalance in lipids and/or reduces or eliminates an energy deficit in a cell or organism.

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a lipid or fatty acid described herein (e.g., any one or more of the lipids in Table 1), or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

As used herein, the terms“beat,” beating,”“beatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, beating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms“prevent,”“preventing,”“prevention,”“prophylactic beatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

The therapeutic methods of the invention (which include prophylactic beatment) in general comprise adminisbation of a therapeutically effective amount of the lipid to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such beatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a neurological disease (particularly ALS), disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, ALS causing or ALS associated mutations or misexpression (e.g., a SOD1 mutation (such as SOD1 G85R), C9orf72 repeat expansion, or TDP-43 misexpression), family history, and the like). The lipids herein may be also used in the beatment of any other disorders in which ALS causing or ALS associated mutations (e.g. mutations in SOD1) may be implicated. For instance, many sALS patients have been shown to possess expanded C90rf72 repeats even though there is no history in the family, thus, repeats may expand de novo. Likewise, even though only a small proportion of fALS patients have mutations in the TDP-43 gene, almost all sALS patients display pathological aggregation of TDP-43 protein upon analyses of post-mortem tissue.

In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of a neurological disease marker (e.g.,

SOD1 mutation (such as SOD1 G85R), C9orf72 repeat expansion, or TDP-43 misexpression, or a level of any one of the lipids or fatty acids described herein) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with a neurological disease, in which the subject has been administered a therapeutic amount of a composition herein sufficient to treat the disease or symptoms thereof. The level of a neurological disease marker determined in the method can be compared to known levels of the neurological disease marker in either healthy normal controls or in other afflicted patients to establish the subject’s disease status. In particular embodiments, a second level of a neurological al disease marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain embodiments, a pre-treatment level of a neurological disease marker in the subject is determined prior to beginning treatment according to this invention; this pre -treatment level of the neurological disease marker can then be compared to the level of the marker in the subject after the treatment commences, to determine the efficacy of the treatment.

In some embodiments, a subject identified as having increased level of a ALS or neurological disease marker polynucleotide or polypeptide (e.g. C9orf72 repeat or TDP-43, or a mutation in SOD1 (particularly, SOD1 G85R)) relative to a reference is administered a therapeutic composition of the invention. Levels of neurological disease marker

polynucleotides or polypeptides are measured in a subject sample and used as an indicator of an ALS or neurological disease that is responsive to treatment with a suppressor Cyp4gl polynucleotide or polypeptide of the invention. Levels of neurological disease marker polynucleotides may be measured by standard methods, such as quantitative PCR, Northern Blot, microarray, mass spectrometry, and in situ hybridization. Standard methods may be used to measure levels of neurological disease marker polypeptides in a biological sample derived from a subject. Such methods include immunoassay, ELISA, western blotting using an antibody that binds the marker polypeptide, and radioimmunoassay. Methods for detecting a mutation in a marker polypeptide (e.g., SOD1 mutation) include immunoassay, direct sequencing, and probe hybridization to a polynucleotide encoding the mutant polypeptide. Elevated levels of neurological disease marker polynucleotides or polypeptides and/or a mutation in a neurological disease polynucleotide or polypeptide are considered a positive indicator of ALS or a neurological disease that is responsive to treatment with a suppressor Cyp4gl polynucleotide or polypeptide, or a lipid (e.g., any one or more of the lipids in Table 1) of the invention.

Pharmaceutical and nutraceutical compositions

The present invention features compositions useful for treating a neurological disease or suppressing effects of mutations causing a neurological disease (particularly ALS) in a subject.

In some embodiments, the composition comprises a lipid or fatty acid described herein (e.g., any one or more of the lipids in Table 1). In some embodiments, the lipid is a fatty acid. In other embodiments, the lipid or fatty acid is a substrate or product of Cyp4gl polypeptide (e.g., human Cyp4V2) catalyzed omega-hydroxylation of a fatty acid. In some other embodiments, the fatty acid is saturated, unsaturated, or branched. In still other embodiments, the fatty acid comprises a C-10, C-12, C-13, C-14, C-16, C-18, C-20, C-22, or C-24 carbon chain. In various embodiments, the fatty acid is an omega-3, omega-6, or omega-9 fatty acid. In some embodiments, the fatty acid is eicosapentanoic acid (EPA) or docosahexanoic acid DHA. In some other embodiments, the lipid product of omega-hydroxylation of any one of the fatty acids described or delineated herein (e.g., 12-hydroxy laurate, 14-hydroxy myristate, 16-hydroxy palmitate, 18-hydroxy stearate, 19-hydroxy EPA, 20-hydroxy -EPA, 21 hydroxy DHA, 22- hydroxy DHA). In some embodiments, the lipid is a lipid selected from the lipids in Table 1. In still other embodiments, the lipid or fatty acid is a signaling molecule. In other embodiments, the lipid or fatty acid is a substrate or product of a reaction or metabolic pathway downstream of omega-hydroxylation of a fatty acid by Cyp4gl (e.g., human Cyp4V2 or other Cyp4 enzyme). For example, the lipid or fatty acid may be a product or substrate in a reaction synthesizing or breaking down fatty acids. In particular embodiments, modulating (e.g., increasing or decreasing) the level of anyone or more of a lipid or fatty acid delineated herein ameliorates an imbalance in lipids and/or reduces or eliminates an energy deficit in a cell or organism.

The administration of a composition comprising a lipid or fatty acid herein for the treatment of a neurological disease such as ALS may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing the disease symptoms in a subject. The composition may be administered systemically, for example, formulated in a pharmaceutically -acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the agent in the patient. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the neurological disease. Generally, amounts will be in the range of those used for other agents used in the treatment of neurological diseases such as ALS (or other diseases associated with ALS causing mutations, such as SOD1 G85R), although in certain instances lower amounts will be needed because of the increased specificity of the agent. A composition is administered at a dosage that suppresses effects of the neurological disease causing mutation or that decreases effects or symptoms of ALS or neurological disease (e.g., wasting, weakened muscular strength, or shortened lifespan) as determined by a method known to one skilled in the art.

The therapeutic composition comprising one or more lipids or fatty acids herein may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an organ, such as the liver; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a cancer using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., liver cell). For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner.

Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, nontoxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms (e.g., in singledose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a neurological disease, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) (e.g., a Cyp4gl polypeptide or polynucleotide or lipid (e.g., any one or more of the lipids in Table 1) described herein) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

In some embodiments, the composition comprising the active therapeutic (i.e., a lipid or fatty acid described herein, such as a lipid in Table 1) is formulated for intravenous delivery. As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable therapeutic (s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p- hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10- 60% w/w of propylene glycol or the like.

The term“pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the lipid.

Pharmaceutically and nutraceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-D -tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium

carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins, such as□-,□-, and□ -cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3- hydroxypropyl-P -cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.

The preparations containing lipids or fatty acids are manufactured by an ordinary method using ordinary recipients and food additives. As an oral preparation, it can be formulated in the form of ordinary tablets, capsules, fine granules or powders. The food product can be a solid, a paste, or a liquid food product, such as milk, tea, soft drinks, juices, coffee, seasonings, cereals, water, cookies, yogurt, chewing gum, chocolate, or soups. The food product can be a "non-alcoholic" food product, that is a food product having low (e.g., <3%, <2%, <1%, <0.5%, <0.25%, ,<0.1%, <0.05%) or no (e.g., essentially zero) alcohol content. In the invention, the nutraceutical carrier for the compositions herein may include, a base of fruit, vegetables or fruit or vegetable juice or puree, a base of vegetable soup or bouillon, a soya-milk drink, a tea or coffee drink, or a nutritive supplement.

Additionally the components can be fortified with electrolytes, flavors, other plant extracts, preservatives, and other additives, (e.g., vitamin supplements and maltodextrin).

Examples of preservatives include, but are not limited to, ascorbic acid and propyl gallate. Examples of electrolytes include, but are not limited to, magnesium sulfate and potassium chloride. Recombinant polypeptide expression

Cyp4gl polypeptides of the invention, comprising mutations suppressing effects of ALS-causing mutations (e.g., SOD1 G85R), are useful for treating or suppressing a neurological disease such as ALS in a subject. Recombinant Cyp4gl polypeptides of the invention are produced using virtually any method known to the skilled artisan. Typically, recombinant polypeptides are produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle. Accordingly, the invention provides methods of producing a polypeptide of the invention, the method comprising (a) heterologously expressing an expression vector comprising a polynucleotide encoding the polypeptide in a host cell; and (b) isolating the polypeptide from the host cell.

Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. A Cyp4gl polypeptide of the invention may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et ak, Current Protocol in Molecular Biology, New York: John Wiley and Sons, 1997). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et ak, 1985,

Supp. 1987).

A variety of expression systems exist for the production of the polypeptides of the invention. Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof.

In some embodiments, the polypeptides of the invention are produced in a bacterial expression system. One particular bacterial expression system for polypeptide production is the E. coli pET expression system (e.g., pET-28) (Novagen, Inc., Madison, Wis). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains that express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those described herein.

Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system that is designed for high- level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX- 2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa.

Alternatively, recombinant polypeptides of the invention are expressed in Pichia pastoris, a methylotrophic yeast. Pichia is capable of metabolizing methanol as the sole carbon source. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde by the enzyme, alcohol oxidase. Expression of this enzyme, which is coded for by the AOX1 gene is induced by methanol. The AOX1 promoter can be used for inducible polypeptide expression or the GAP promoter for constitutive expression of a gene of interest.

Once the recombinant Cyp4gl polypeptide of the invention is expressed, it is isolated, for example, using affinity chromatography. In one example, an antibody (e.g., produced as described herein) raised against a polypeptide of the invention may be attached to a column and used to isolate the recombinant polypeptide. In some embodiments, to facilitate purification of the recombinant polypeptide, the polypeptide comprises an epitope tag fused to the Cyp4gl polypeptide. The polypeptide is then isolated using an antibody against the epitope tag. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra). Alternatively, the polypeptide is isolated using a sequence tag, such as a hexahistidine tag, that binds to nickel column. Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980). Polypeptides of the invention, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.)· These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (described herein).

Methods of Delivery

Suppressor Cyp4gl polypeptides or polynucleotides of the invention, which are useful for suppressing a neurological disease (particularly ALS) in an organism, may be delivered to an organism (particularly liver cells of the organism) in any manner such that the polypeptide is in functional form in the cell. The Cyp4gl polypeptide comprising suppressor mutations may be delivered to cells as a polypeptide. Alternatively, a polynucleotide encoding an amino acid sequence of the Cyp4gl polypeptide may be delivered to cells for heterologous expression of a suppressor Cyp4gl polypeptide in the cells. Thus, the present invention features polypeptides delivered to a cell by contacting the cell with a composition comprising the polypeptide or by heterologously expressing the polypeptide in the cell.

Intracellular Delivery of Polypeptides

Polypeptides of the invention, such as Cyp4gl polypeptides comprising mutations suppressing ALS, may be delivered intracellularly to cells. The polypeptide must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of the polypeptide, or fragment thereof, is in functional form in the cells.

Methods of intracellular delivery of polypeptides are known to one of skill in the art. Exemplary methods of intracellular delivery of polypeptides include, without limitation, incorporation of the polypeptide into a liposome. Liposomes are phospholipid vesicles with sizes varying from 50 to 1000 nm, which can be loaded with polypeptides or other agents. Liposomal intracellular delivery of polypeptides into cells typically relies on endocytosis of the liposome-encapsulated polypeptide into the cell. Examples of suitable liposomes for intracellular delivery of polypeptides may be pH-sensitive liposomes. Such liposomes are made of pH-sensitive components; after being endocytosed in intact form, the liposome fuses with the endovacuolar membrane under lowered pH inside the endosome and destabilizes it, thereby releasing the contents (including the polypeptides encapsulated in the liposome) into the cytoplasm. The liposomes may also be further modified to enhance their stability or lifetime during circulation (e.g., by PEGylated liposomes). Liposomes may also be modified to specifically target antigens (e.g.,“immunoliposomes” or liposomes embedded with antibodies to an antigen). Antibody -bearing liposomes may have the advantages of targetability and facilitated uptake via receptor-mediated endocytosis.

Other methods of intracellular delivery of polypeptides include, without limitation, use of cell penetrating peptides (CPPs). A cell penetrating peptide or“CPP” is a protein or peptide that can translocate through cellular membranes. A polypeptide for delivery into a cell is fused with a CPP, thereby enabling or enhancing delivery of the polypeptide fusion into the cell. Cell penetrating peptides include, for example, a trans-activating transcriptional activator (TAT) from HIV-1, Antenapedie (Antp, a transcription factor in Drosophila), and VP22 (a herpes virus protein).

Another exemplary method for intracellular delivery of polypeptides of the invention is the use of supercharged proteins. Supercharged proteins or supercharged polypeptides are a class of engineered or naturally existing polypeptides having an unusually high positive or negative net theoretical charge. Membranes of cells are typically negatively charged.

Superpositively charged polypeptides are able to penetrate cells (particularly mammalian cells), and associating cargo with superpositively charged polypeptides (e.g., polypeptides or polynucleotides) can enable functional delivery of these macromolecules into cells, in vitro or in vivo. Methods of generating supercharged polypeptides and using supercharged polypeptides for intracellular polypeptide delivery are described in further detail in, for example, Zuris et al. Nat. Biotechnol. (2015) 33:73-80 and Liu et al. Methods Enzymol. 2012, 503: 293-319.

Accordingly, in some aspects, the invention provides a suppressor Cyp4gl polypeptide fused to a polypeptide enabling intracellular delivery of the suppressor Cyp4gl polypeptide (e.g., a cell penetrating peptide or supercharged polypeptide).

Another therapeutic approach for treating or suppressing a neurological disease is polynucleotide therapy using a polynucleotide encoding a suppressor Cyp4gl polypeptide of the invention, or fragment thereof. Thus, provided herein are isolated polynucleotides encoding a Cyp4gl polypeptide of the invention, or fragment thereof. Expression of such polynucleotides or nucleic acid molecules in a cell or organism is expected to suppress effects of mutations causing neurological disease (particularly, ALS) in the subject. Such nucleic acid molecules can be delivered to cells of a subject having a neurological disease. The nucleic acid molecules must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of the Cyp4gl polypeptide, or fragment thereof, can be produced.

Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, 1997). For example, a polynucleotide encoding a Cyp4gl polypeptide of the invention, or a fragment thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet

337: 1277-1278, 1991; Cometta et al., Nucleic Acid Research and Molecular Biology 36:311- 322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). In some embodiments, a viral vector is used to administer a polynucleotide encoding a suppressor Cyp4gl polypeptide (or fragment thereof) systemically.

Non-viral approaches can also be employed for the introduction of the therapeutic to a cell of a patient requiring suppression of a neurological disease. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S. A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101 :512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247: 1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.

Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of genes encoding suppressor Cyp4gl polypeptides into the affected tissues of a patient can also be accomplished by transferring a nucleic acid encoding the suppressor Cyp4gl polypeptide into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element.

For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers.

Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above. Delivery of polynucleotides of the invention may also include or be performed in combination with gene or genome editing methods, such as CRISPR-Cas systems, to introduce polynucleotides encoding suppressor Cyp4gl polypeptides in cells. Gene or genome editing methods such as CRISPR-Cas systems are further described in for example, Sander et al. (2014), Nature Biotechnology 32, 347-355; Hsu et al. (2014), Cell 157(6): 1262-1278.

In Drosophila melanogaster, dCyp4gl itself is expressed exclusively in oenocytes, the Drosophila equivalent of the mammalian liver. Gutierrez et al., Nature (2007) 445:275-280. Accordingly, in particular embodiments, suppressor Cyp4gl polynucleotides or polypeptides of the invention are delivered to a liver cell. It is noted that many systems of delivery of therapeutic polypeptides or polynucleotides (particularly nanoparticulate or liposomal delivery systems) result in high accumulation of the therapeutic polypeptides or polynucleotides in the liver. It is also noted that, contrary to other polynucleotide therapies for ALS or neurological disease, the therapeutic Cyp4gl polynucleotides or polypeptides of the invention likely do not need to be delivered to neurons. Thus, currently available systems for delivery of

polynucleotides or polypeptides, such as liposomal delivery, may be particularly suitable for delivery of therapeutic Cyp4gl polypeptides or polynucleotides of the invention.

Screening Assays

The present invention further features methods of identifying modulators of a disease, particularly neurological disease, comprising identifying candidate agents that interact with and/or alter the level or activity of a Cyp4gl polypeptide. The CYP4 class of enzymes plays a unique biochemical role in regulation of body fat mobilization and utilization. The mutants obtained herein define the CYP4 class of enzymes as a potential target in regulating energy metabolism in disease. The CYP4 class of enzymes may, because of their novel active-site chemistry, serve as ideal drug targets to control the pace of lipid metabolism, to achieve some degree of efficacy in treating the whole organism, not just neurons (the current focus of most therapies).

Thus, in some aspects, the invention provides a method of identifying a modulator of a neurological disease, comprising (a) contacting a polypeptide with a candidate agent, wherein the polypeptide is a Cyp4gl polypeptide or fragment thereof, and (b) measuring an activity of the polypeptide contacted with the candidate agent relative to a control activity.

In other aspects, the method comprises (a) contacting a cell or organism with a candidate agent, and (b) measuring a level or activity of Cyp4gl polynucleotide or polypeptide in the cell or organism contacted with the candidate agent relative to a control level or control activity. An alteration in the level or activity of the Cyp4gl polypeptide or polynucleotide indicates the candidate agent is a modulator of neurological disease. In some embodiments, the activity of the Cyp4gl polypeptide is enzymatic activity or a lipid, combinations of lipids, and/or alteration of fatty acid metabolism. The control activity may be the activity of the polypeptide when the polypeptide is not contacted with the candidate agent, or any agent. Alternatively, the control activity may be activity of the polypeptide contacted with a carrier or solvent that does not contain the candidate agent. Likewise, the control activity or control level of the polypeptide may be the activity or level of the polypeptide in a cell when the cell is not contacted with the candidate agent (or any agent) or when the cell is contacted with a carrier that does not contain the candidate agent. Methods of measuring or detecting activity and/or levels of the polypeptide or polynucleotide are known to one skilled in the art. For example, enzymatic activity of the polypeptide may be measured by measuring levels of substrate(s) modified by the polypeptide. Binding activity of the polypeptide may be measured, for example, by immunoassay methods. Polynucleotide levels may be measured by standard methods, such as quantitative PCR, Northern Blot, microarray, mass spectrometry, and in situ hybridization. Standard methods may be used to measure polypeptide levels, the methods including without limitation, immunoassay, ELISA, western blotting using an antibody that binds the polypeptide, and radioimmunoassay.

In still other aspects, the invention provides a method of identifying a modulator of neurological disease, comprising (a) contacting a cell or organism with a candidate agent, and (b) comparing a phenotype of the cell or organism contacted with the candidate agent with a phenotype of a cell or organism comprising a Cyp4gl polynucleotide or polypeptide having a mutation. A similarity in the phenotypes indicates the candidate agent is a modulator of neurological disease. In some embodiments, the cell or organism also comprises a mutation and/or misexpression associated with ALS (e.g., a SOD1 G85R allele). In the method provided, candidate agents that“phenocopy” a phenotype of an organism having a Cyp4gl mutation (in particular, agents that phenocopy suppression of ALS mutations by a suppressor Cyp4gl mutation in an organism) are identified as modulators of ALS or neurological disease. The phenotype may be any observable or measurable phenotype, including without limitation, suppression of ALS associated phenotypes (e.g., weakness of muscles, motor neuron death, shortened lifespan), level and/or activity of Cyp4gl polypeptides or polynucleotides, or levels of lipid and/or fatty acid substrates and/or metabolic products.

In particular embodiments, the candidate agent or modulator of neurological activity inhibits an activity of Cyp4gl. In certain embodiments, the agent or modulator suppresses an ALS phenotype. For example, in some embodiments, the agent or modulator is HET0016. HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)Formamidine (CAS 339068-25-6)) is a potent inhibitor of CYP4V2 (human homolog of Cyp4gl). In particular, HET0016 was shown to be a potent inhibitor of CYP4V2 in the 30 nM range. Nakano et al., Drug Metab. Dispos. (2009) 37(11): 2119-2122. It is expected that HET0016 will phenocopy the effects of the suppressor mutations in a G85R genetic background when supplied in standard Drosophila media.

If the current strategy to treat dying motor neurons (MNs) specifically are misguided, due to the true nature of these diseases in defective metabolism, there are even more tantalizing potential avenues to pursue based on the current invention. Without wishing to be bound by theory, it is believed the Cyp4gl mutations could identify a class of lipids which, when increased, or decreased, would themselves provide an ideal therapeutic index. Some research has been directed towards dietary interventions in ALS, but specific metabolites have not been forthcoming. Perhaps, these studies in Drosophila, extended further, could reveal a specifically balanced dietary intervention (particular mixtures of complex natural lipids and fats) that could prolong the pathologic wasting seen in human neurological disease, especially ALS and dementia. It has been documented that hypercaloric dietary intervention has efficacy, even when begun after symptom onset. Likewise, it has been suggested that intervening during the presymptomatic phase may be more effective. However, these studies are not specific as to the source of calories. Thus, it could be that the current invention could identify tailored mixtures of specific metabolic intermediates (lipid), or a pharmacological method to alter lipid profiles that would optimize the energy balance of motor neurons, providing protection against a“deadly loop” of energy depletion of motor neurons.

Accordingly, the invention also provides methods of identifying lipid substrates or products of Cyp4gl -catalyzed reactions, as modulators of ALS or neurological disease. The methods comprise (a) contacting a polypeptide with a substrate, wherein the polypeptide is a Cyp4gl polypeptide or fragment thereof having enzymatic activity, and (b) detecting a reaction product of the polypeptide contacted with the substrate, wherein detection of a reaction product indicates the substrate and/or reaction product is a modulator of neurological disease. In particular embodiments, the activity or enzymatic activity is omega-hydroxylation of a fatty acid. Detection of the substrate and/or reaction product may be performed according to any standard method known in the art (e.g., NMR or mass spectrometry).

Combination Therapies

In some aspects, the invention features methods of treating a neurode generative disease in a subject, the methods comprising administering to the subject an effective amount of a lipid or fatty acid described herein (e.g., any one or more of the lipids in Table 1). In some embodiments, any combination (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the lipids in Table 1 is administered to the subject. In some other embodiments, the lipid or fatty acid is administered in combination with an effective amount of a composition comprising a Cyp4gl polypeptide or a polynucleotide having a mutation and/or an agent that decreases a level or activity of Cyp4gl polypeptide in the subject. An agent that decrease a level or activity of Cyp4gl polypeptide may be, for example, an inhibitory nucleic acid that reduced expression of Cyp4gl polypeptide (e.g., siRNA) or a small molecule compound that inhibits Cyp4gl polypeptide activity (e.g., HET0016).

Optionally, an anti- neurological disease therapeutic of the invention (e.g., a Cyp4gl polynucleotide or polypeptide comprising a suppressor mutation as described herein, or a lipid or fatty acid described herein) may be administered in combination with any other standard anti- neurological disease (e.g., anti-ALS) therapy; such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin.

Kits

The invention provides kits for the treatment, suppression, or prevention of a neurological disease, particularly amyotrophic lateral sclerosis (ALS) associated with SOD1 mutations (e.g., SOD1 G85R), C9orf72 repeat expansion, or TDP-43 overexpression. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of a suppressor Cyp4gl polypeptide, or fragment thereof (or a polynucleotide encoding such) in unit dosage form. In another embodiment, the kit includes a therapeutic or prophylactic pharmaceutical or nutraceutical composition containing an effective amount of one or more lipids (e.g., any one or more of the lipids in Table 1). In some embodiments, the lipid is a fatty acid. In other embodiments, the lipid or fatty acid is a substrate or product of Cyp4gl polypeptide (e.g., human Cyp4V2) catalyzed omega-hydroxylation of a fatty acid. In some other embodiments, the fatty acid is saturated, unsaturated, or branched. In still other embodiments, the fatty acid comprises a C-10, C-12, C-13, C-14, C-16, C-18, C-20, C-22, or C-24 carbon chain. In various embodiments, the fatty acid is an omega-3, omega-6, or omega-9 fatty acid. In some embodiments, the fatty acid is eicosapentanoic acid (EPA) or docosahexanoic acid DHA.

In some other embodiments, the lipid product of omega-hydroxylation of any one of the fatty acids described or delineated herein (e.g., 12-hydroxy laurate, 14-hydroxy myristate, 16-hydroxy palmitate, 18-hydroxy stearate, 19-hydroxy EPA, 20-hydroxy -EPA, 21 hydroxy DHA, 22- hydroxy DHA). In some embodiments, the lipid is selected from the group of lipids in Table 1. In still other embodiments, the lipid or fatty acid is a signaling molecule. In other embodiments, the lipid or fatty acid is a substrate or product of a reaction or metabolic pathway downstream of omega-hydroxylation of a fatty acid by Cyp4gl (e.g., human Cyp4V2 or other Cyp4 enzyme). For example, the lipid or fatty acid may be a product or substrate in a reaction synthesizing or breaking down fatty acids. In particular embodiments, modulating (e.g., increasing or decreasing) the level of anyone or more of a lipid or fatty acid delineated herein ameliorates an imbalance in lipids and/or reduces or eliminates an energy deficit in a cell or organism.

In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. In some other embodiments, the kit further includes reagents detecting a mutation associated with or causing a neurological disease (e.g., ALS) in a subject. For example, the reagents may be primers or hybridization probes for detection of mutation in SOD1 (e.g., SOD1 G85R), a C9orf72 repeat expansion, or TDP-43 overexpression.

If desired a composition comprising a therapeutic agent of the invention (e.g., Cyp4gl polypeptide or polynucleotide comprising suppressor mutations described herein) is provided together with instructions for administering the agent to a subject having or at risk of developing a neurological disease. The instructions will generally include information about the use of the composition for the treatment or prevention of neurological disease. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof;

precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989);“Oligonucleotide Synthesis” (Gait, 1984);“Animal Cell Culture” (Freshney, 1987);“Methods in Enzymology”“Handbook of Experimental Immunology” (Weir, 1996);“Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);“Current Protocols in Molecular Biology” (Ausubel, 1987);“PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the therapeutic methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1: Forward genetic screen in fly model of amyotrophic lateral sclerosis (ALS) revealed mutations suppressing lethal effects of SOD1 G85R.

Genetic models of human neurological disease were developed, where human disease- causing mutations are introduced into the cognate genes of the facile genetic organism, Drosophila melanogaster. Initial studies revealed that introduced human mutations could very effectively and accurately unravel important details about hereditary disease, such as pediatric epilepsy. This led to modeling ALS in the fly. First, mutations were introduced into one of the first and most common fALS genes, superoxide dismutase (SOD1). Flies carrying human disease mutations (H71Y and G85R) in SOD1 have a range of phenotypes from adult onset loss of motor coordination and early death (H71Y), to an even earlier death during metamorphosis (G85R). Significant efforts were spent in characterizing these mutants as to their expression of mutant SOD proteins, the dosage effects as they relate to disease progression, and genome-wide transcriptomics. All of these studies validated that the Drosophila ALS model recapitulated important aspects of the human disease, including motor neurons’ death.

To make a leap forward in the understanding of motor neuron diseases, the ALS model described above was used, coupled with the power of forward genetics (FIG. 1). In short, forward genetics associates phenotypes with genes in large-scale unbiased screens usually involving mutagenesis of the entire genome of a model system. In this study, the genomes of flies were mutagenized is such a way as to obtain, if possible, suppressor genes that reversed the lethal effects of the SOD1-G85R mutation, an aggressive and fast progressing mutation in human ALS, and likewise completely lethal in flies (FIG. 2A). About 500,000 mutagenized flies or fly strains that would otherwise die of ALS were screened, and five (5) lines of Drosophila carrying mutations which almost completely suppressed, or cured, the deleterious effects of the SOD1 mutation were obtained (FIG. 3A). The mutations are shown in FIG. 4 and FIG. 5.

The results described in the Examples herein were obtained with the following materials and methods.

Materials and Methods

Drosophila strains, EMS mutagenesis and survival screen

Two independently derived homologous recombinant lines of dSodlG85R/Tm3, hshid, sb (Balancer chromosome, Bloomington Drosophila Stock Center number: 1558)

derived from independent targeting transgene stocks were used: dSodlG85R/Tm3,hs- 185 hid,sb, line A and line B. Newly eclosed dSodlG85R/Tm3,hs-hid,sb (line A) males were aged for 1-7 days and were then mutagenized. The stock of dSodlG85R/Tm3,hs-hid,sb (line B) was used for the crosses with the possible suppressor mutation bearing

dSodlG85R/Tm3,hs-hid,sb (line A) mutagenized males to minimize the possibility of homozygosing background mutations. Flies were kept at constant 25oC, on standard molasses food, and under 12-h day /night cycles. 6028 dSodlG85R/Tm3,hs-hid (line A) males were starved overnight (12hr) in vials supplied with only water. Surviving males were fed with 25mM EMS (Sigma M-0880) in 5% sucrose solution for 10 hours. The water and sucrose solution containing EMS was administered with Kimwipe wrapped ceaprene stoppers as described by Christian Bokel (Bokel, 2008). Surviving males were allowed to recover on regular food for 14 hours to allow for the clearance of any excess EMS from the digestive system. Then, the surviving 4800 males were mated with 6400 virgin females dSodlG85R/Tm3,hs-hid (line B). The crosses were set up in bottles (Genesee 32-130) with each bottle containing 15 males and 20 virgin females. (FIGS. 2B-2C) The parents in the bottle stock were passed after 5 days. The progeny in the bottles were heat shocked on days 5 and 6 for 2 hours at 37oC in order to induce hs-hid expression, thus killing any animals containing the Tm3 chromosome. Development was allowed to continue for the remaining cohort of flies, which should be exclusively

dSodlG855RG85R homozygotes and were subsequently screened regularly for survivors over the next two weeks. The surviving progeny were named as EMS1 through EMS145. Flies that survived more than 3 days were crossed with 3 flies (dSodlG85R/Tm3-hsHid) of the opposite sex. The dSodl locus of all the surviving lines were PCR amplified and sequenced with forward primer: 5 '-GC AT GT ATTT CT AAGCT GCTCT GCT ACGGT C AC-3 ' and, reverse primer: 5'- GTCCACTGCTAAGAGCAGCTGCCCTC-3'.

Example 2: Characterization of suppressor mutations from genetic screen

From the forward genetic screen described above, mutations in Cyp4gl of Drosophila, a member of the CYP4 family that has novel catalytic and biological roles, was obtained. In all, four (4) novel missense mutations causing amino acid substitutions in Cyp4gl were found (FIGS. 4 and 5). It is worth emphasizing, all genes of the genome were mutagenized without bias and at random, and four independent changes in only the Cyp4gl that all suppressed ALS- mutant lethality were obtained. The mutations in Cyp4gl were G92E, D112N, S143N, and M333I. The Drosophila line EMS130/35 was found to carry mutation G92E; line EMS102 carried D112N, EMS81 carried S143N, and EMS94 carried mutation M333I. The suppressor alleles Cyp4gl G92E, Cyp4gl D112N, Cyp4gl S143N, and Cyp4gl M333I may be interchangeably referred to herein as“EMS130/35,”“EMS102,”“EMS81,” and“EMS94,” respectively. Likewise, the suppressor mutations G92E, D112N, S143N, and M333I are interchangeably referred to herein as“EMS130/35 mutation,”“EMS102 mutation,”“EMS81 mutation,” and“EMS94 mutation,” respectively.

Genetic and molecular mapping of mutations EMS 81 and EMS 102 revealed that they are dominant gain-of-function alleles of Cyp4gl.

The EMS 81/102 mutations were first balanced over the Fm7 balancer, as described above, and then heterozygote females were crossed to males of a mapping chromosome stock carrying five recessive visible markers. Non-Fm7 heterozyogous females of the FI (where recombination occurs) were then crossed to wild type males, and phenotypes were scored in the F2. The lethal phenotype of the EMS 81/102 mutations was used to infer the location of these mutations by assessing the lack of particular classes of recombinants. In fact, it was clear that both the EMS 81/102 mutations mapped between two tightly linked (1 cM) visible markers (call them p and q). Thus, recombinants on this interval alone could be used to generate mapping data (FIG. 3B). Briefly, the lethal mutation on a wild type background in heterozygous state with a doubly mutant mapping chromosome (p + q, where p, +, and q are the gene order of the p locus, the wild type EMSX locus, and the q locus respectively) will generate males in the FI that are either parental non-recombinant (NR) or recombinant (R) chromosomes (FIG. 3C). In any case where the resultant chromosome retains the lethal (1) EMSX allele, no male progeny will be recovered. Recombination can occur on two intervals that result in viable recombinant offspring (I and II, FIG. 3C). By measuring the ratios of recombinants on this interval (I/(I+II)), one obtains the distance (after multiplying this fraction by lcM) from the p locus. This allowed the mapping of the EMSX mutations to within a O.lcM of one another, strongly suggesting that these were mutations in the same gene.

While the genetic mapping was highly informative, the region of the X chromosome is densely populated with genes, and represents a region where the genetic map is contracted compared to other regions of the X chromosome. In fact, the lcM region that the EMSX mutations mapped to covers 2 Mb of physical distance on the X chromosome containing hundreds of genes. In order to increase mapping accuracy, an interesting and equally surprising observation was exploited. For the EMS 81 allele, it was noticed that when animals were reared in fresh food, complete lethality was always seen with 100% penetrance. However, if the parental animals (and some FI) were carried over in vials to a second generation, and the food was more than two weeks old, and "conditioned" by overcrowding, death, and waste material, EMS 81 hemizygote males could be obtained as viable adults. These males will be referred to herein as pseudo-rescued (EMS 81-PR) males.

EMS 81-PR males were found to be fertile and mobile for at least a week. This allowed us to cross these males to molecularly -defined deficiency bearing balanced stocks carrying a series of five deficiencies spanning the 2Mb regions implicated in genetic mapping studies (Cook et al, 2012) (FIG. 3D). One of these deficiencies (BSCdf2) failed to complement EMS 81, giving rise to female progeny that died in exactly the same manner as hemizygous EMS 81 males, namely, as pharate adults during eclosion. All other deficiencies tested complemented the lethal phenotype of EMS 81. Thus, we were able to localize the EMS 81 mutation to a ~260kb deficiency, however, this region still contained around 35 annotated transcription units.

To further refine the mapping, a series of molecularly defined duplication stocks carrying ~80kb duplications inserted into a common site on the third chromosome, and covering almost completely the entire X chromosome (Venken et al, 2010) (FIG. 3E) was utilized. Females balanced for EMS 81 or 102 (EMSX/Fm7) were crossed to seven duplication-bearing lines. Duplications could be followed by the presence of eye color, as the EMS stocks were in a white-eyed background. Three duplications (DP2, DP3, DP4) rescued the lethality of EMS 81 and 102. The combination of rescue by these deletions allowed the delimitation of a much smaller genetic interval containing only two genes (here labeled A and B). A lethal mutation (molecularly uncharacterized) happened to have been attributed to the gene B, which is referred to herein as Su(G85R). This mutation (l(l)x) was obtained as a balanced stock and EMS 81-PR males were crossed to this stock, resulting in failure of complementation of the l(l)x mutation.

The Su(G85R) gene was sequenced for EMS 81, EMS 102 and the l(l)x mutations. In the case of the l(l)x stock, a 13nt deletion was found in the open reading frame of Su(G85R), predicted to truncate the protein at about 2/3 its normal length. Thus, l(l)x is almost certainly a null mutation for Su(G85R). Mutations were also found in the EMS 81 and 102 mutant stocks, G-to-A mutations, which is consistent with EMS as the mutagen. Both mutations cause missense change in different and highly conserved positions within the Su(G85R) protein, which is known to play a role in metabolism. The Su(G85R) gene is Cyp4gl.

Mapping suppressor lines EMS 35, EMS 94 and EMS 130

The mutations EMS 35, 94 and 130 were backcrossed in the balanced dSodlG85R background through females for >10 generations and it was observed that suppressed males were obtained in the expected ratios. In order to test initial linkage, suppressed G85R/G85R dSodl homozygote males were crossed to the balanced G85R stock. In this case, only suppressed G85R homozygous females were obtained. Such females, when crossed to balanced G85R males gave rise to suppressed males and females in the expected ratios. Thus, these suppressor mutations, whose only phenotype appeared to be suppression of G85R homozygosity (in contrast to EMS 81 and 102) also appeared to map to the X chromosome.

Suppressor Lines Also Suppresses Eclosion Defect and Short Life Span of

dSodlH71 Y/H71 Y

EMS 81 and 102 were introgressed into a balanced dSodlH71Y genetic background, using alleles of EMS 81 and 102 that had been recombined with the tightly linked w+ gene, allowing the suppressor alleles to be monitored via eye color. Homozygosity for

dSodlH71 Y/H71 Y normally confers a high level of unviability in the pupal stage, and animals only live for about 16 days, with a profound loss of motor function within the first week.

Females homozygous for dSodlH71Y/H71Y but also containing either EMS 81 or EMS 102 appeared in much greater numbers from the doubly balanced stock, and initial experiments showed that they live for at least one month with no apparent loss of locomotor function, compared to controls lacking the suppressor alleles which behaved as previously described. These experiments strongly support the notion that EMS 81 and 102, despite being selected for suppression of G85R homozygous lethality, also suppress unviability and locomotor defects in a less severe ALS model.

Nature of Cyp4gl and CYP4 class of proteins

Mapping and molecular studies have revealed the nature of the Cyp4gl gene in flies, a member of the ubiquitous cytochrome P450 (CYP) class of proteins found in all life forms, including bacteria. FIG. 6 depicts the structure of the dCyp4gl (Drosophila melanogaster Cyp4gl) ALS-suppressor gene. The dCyp4gl (CG3972) gene in Drosophila is an intronless gene. CYP proteins play two major roles in biology; detoxifying (biodefense) against many xenobiotics and antibiotics, and biosynthesis of endogenous molecules such as steroids and lipids. There are as many as 37 families of CYP genes. The CYP4 family has a role in the metabolism of fatty acids mobilized from fat stores in animals. The novel chemistry at its active site (found only in the CYP4 family), allows it to perform omega-hydroxylation of fatty acids, a necessary function to utilize excess free fatty acids. The dCyp4gl gene is known to play a key role in lipid metabolism, in particular, in regulation of triacylglyceride (TAG) content of Drosophila fat storage cells. Interestingly, dCyp4gl itself is expressed exclusively in oenocytes, the Drosophila equivalent of the mammalian liver. Gutierrez et al., Nature (2007) 445:275-280.

FIG. 7 shows an alignment of dCyp4gl and hCYP4V2, the closest human homologue to dCyp4gl. The amino acid in the position mutated in EMS81 (position 143) is conserved between dCyp4gl and hCYP4V2. The hCYP4V2 gene is mutated in Bietti’s Crystalline Dystrophy (BCD) a retinal degeneration syndrome. hCYP4V2 protein has a fairly broad expression pattern including liver. Patients with BCD display a systemic dyslipidemia. A mouse model of BCD recapitulates clinical features of the human disease including systemic dyslipidemia.

FIG. 8 shows an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in other invertebrates. Suppressor alleles (EMS35/130, EMS102, and EMS81) isolated as dominant suppressors of SOD1-G85R all mapped to the dCyp4gl gene on the X chromosome. EMS81 and EMS102 alleles were homozygous lethal in Drosophila and were also invariant positions in invertebrate lineages. EMS35/130 and EMS94 were mutations in less conserved positions and were viable in males. EMS35/130 result from the same

nucleotide/amino acid change.

FIG. 9 shows an alignment of dCyp4gl (Drosophila melanogaster Cyp4gl) with Cyp4gl homologs in vertebrates. Suppressor alleles occurred in regions of high conservation, but only EMS81 serine (S) was conserved from flies through mammals.

Example 3: Modeling of Cyp4gl structure revealed a suppressor mutation located in substrate binding pocket.

Modeling of the active site of the CYP4A11 protein (related to CYP4V2) revealed residues lining the substrate binding pocket and active site involved in omega-hydroxy lation. Chang et al., Proteins (1999) 34(3):403-415. FIG. 10 shows an alignment of dCyp4gl and hCYP4V2 showing conserved residues lining a substrate binding pocket. The EMS81 mutation lies very near residues deep in the substrate binding pocket near the active site.

Without intending to be bound by theory, it is believed at least some of the suppressor mutations obtained in the genetic screen described herein alter the activity of Cyp4gl, particularly enzymatic activity or fatty acid / lipid metabolism activities of Cyp4gl. HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)Formamidine (CAS 339068-25-6)) is a known potent inhibitor of CYP4V2 (FIG. 11). In particular, HET0016 was shown to be a potent inhibitor of CYP4V2 in the 30 nM range. Nakano et al., Drug Metab. Dispos. (2009) 37(11): 2119-2122.

To further investigate the relationship between Cyp4gl activity and a neurode generative disease (e.g., AES) phenotype, the ability of HET0016 to phenocopy the effects of the suppressor mutations in a G85R genetic background when supplied in standard Drosophila media is assessed.

Example 4: Mutations in Cyp4gl suppressed additional ALS causing mutations.

There are other genetic models of ALS in Drosophila based on very different causative gene mutations, or misexpression models. One of these is the C9orf72 hexanucleotide repeat expansion model that is the most common form of familial ALS (fALS) and sporadic ALS (sALS). It is proposed that a hexanucleotide which is expanded (GGGGCC)_n in the disease causes ALS or dementia when the repeat number expands (n= 1000s) through a toxic RNA- based mechanism. Overexpression of the C9orf72 toxic repeat globally in flies was found to lead to a phenotype indistinguishable from the lethality seen with SOD1-G85R expression. Preliminary data revealed that the suppressor mutations (G92E, D112N, S143N, and M333I in Cyp4gl) obtained from the forward genetic described above also suppressed the lethal phenotype of C9orf72 repeat overexpression in Drosophila.

In addition, it has been reported for another ALS-causing gene, TDP-43, that motor neuron overexpression of TDP-43 leads to a lethal phenotype that appears very similar to SOD1- G85R. The TDP-43 gene’s normal biological role is currently hotly debated, but it is has been shown to regulate body fat composition. Given the commonalities of phenotype of these other ALS models, whether the suppressor mutations in Cyp4gl also suppress these other seemingly unrelated genetic forms of ALS will be tested. If so, then the work in Drosophila may have revealed an important underlying cause of ALS, and possibly, many age-related cognitive disorders. Suppressors will be tested with the TDP43 model of neurodegeneration in

Drosophila.

Example 5: Modulation of lipid metabolism

The relationship between Cyp4gl activity and disease phenotype underlying neurodegenerative disease (e.g., ALS, Parksinon’s disease, dementia, or Huntington’s disease), is further determined by measuring the ability of various lipids described herein (e.g., lipids listed in Table 1) to phenocopy the effects of the suppressor mutations in a G85R genetic background when supplied in standard Drosophila media. The ability of the various lipids listed in Table 1 to modulate fatty acid or lipid metabolism in subject (particularly a subject having a neurodegenerative disease characterized by an energy deficit, such as ALS), is also assessed. Further, the ability of the lipids in Table 1 to prevent, ameliorate, or treat a neurodegenerative disease (e.g., ALS) in a subject is assessed. In addition, the ability of omega- hydroxylated products of the fatty acids in Table 1 to modulate fatty acid or lipid metabolism in a subject and to prevent, ameliorate, or treat a neurodegenerative disease (e.g., ALS) in a subject is assessed.

Lipid substrates or products of the Cyp4gl polypeptide (or Cyp4 enzyme) catalyzed omega-hydroxy lation of fatty acids, and lipid substrates and products of reactions downstream of the fatty omega-hydroxylation reaction are assessed for their ability to modulate lipid metabolism, modulate or reduce an imbalance in lipids, and reduce an energy deficit in a cell or organism. The lipid substrates or products are further assessed for their ability to ameliorate or treat a neurodegenerative disease (e.g., ALS) in a subject. These lipid substrates or products include fatty acids (e.g. saturated, unsaturated, or branched fatty acids). The lipids also include fatty acids having a C-10, C-12, C-13, C-14, C-16, C-18, C-20, C-22, or C-24 carbon chain. The lipids further include omega-3, omega-6, or omega-9 fatty acids. The lipids also include eicosapentanoic acid (EPA), docosahexanoic acid DHA, 2-hydroxy laurate, 14-hydroxy myristate, 16-hydroxy palmitate, 18-hydroxy stearate, 19-hydroxy EPA, 20-hydroxy-EPA, 21 hydroxy DHA, and 22-hydroxy DHA.

In some embodiments, the lipid is lauric acid (or dodecanoic acid), mystiric acid (tetradecanoic acid), oleic acid, linoleic acid,□ -linoleic acid, arachidonic acid, eicosapentanoic acid, docosahexanoic acid, or an omega-hydroxylated product of any one of the lipids (e.g., lipids in Table 1) described herein. Table 1: List of lipids

LIPID TYPE LIPID SPECIES LM ID

Fatty acid 10Z-heptadecenoic acid LMFA01030283 Fatty acid 11,14,17-eicosatrienoic acid LMFA01030159 Fatty acid 11,14-eicosadienoic acid LMFA01030130 Fatty acid 13,16,19-docosatrienoic acid LMFA01030408 Fatty acid 13,16-docosadienoic acid LMFA01030132 Fatty acid 5,8,11, 14, 17-eicosapentaenoic acid LMFA01030759 Fatty acid 5,8,11-eicosatrienoic acid LMFA01030157 Fatty acid 7, 10, 13 , 16, 19-docosapentaenoic acid LMFA01030184 Fatty acid 7Z, 10Z, 13Z, 16Z-docosatetraenoic acid LMFA01030178 Fatty acid 9Z-palmitoleic acid LMFA01030056 Fatty acid alpha-linolenic acid LMFA01030152 Fatty acid Arachidic acid LMFA01010020 Fatty acid Arachidonic acid LMFA01030001 Fatty acid Behenic acid LMFA01010022 Fatty acid bishomo-gamma-linolenic acid LMFA01030158 Fatty acid Cerotic acid LMFA01010026 Fatty acid cis-erucic acid LMFA01030089 Fatty acid cis-selacholeic acid LMFA01030092 Fatty acid DHA (docosahexaenoic acid) LMFA01030185 Fatty acid gamma-linolenic acid LMFA01030141 Fatty acid Laurie acid LMFA01010012 Fatty acid Lignoceric acid LMFA01010024 Fatty acid Linoleic acid LMFA01030120 Fatty acid Margaric acid LMFA01010017 Fatty acid Myristic acid LMFA01010014 Fatty acid Oleic acid LMFAO 1030002 Fatty acid Palmitic acid LMFA01010001 Fatty acid Pentadecanoic acid LMFA01010015 Fatty acid Stearic acid LMFA01010018 Fatty acid Stearidonic acid LMFA01030357 Fatty acid Tricosanoic acid LMFA01010023 Fatty acid EPA (eicosapentanoic acid)

Hydroxy lated fatty acid 12-hydroxy laurate

Hydroxy lated fatty acid 14-hydroxy mystirate

Hydroxy lated fatty acid 16-hydroxy palmitate

Hydroxy lated fatty acid 19-hydroxy EPA

Hydroxy lated fatty acid 20-hydroxy EPA

Hydroxylated fatty acid 21 -hydroxy DHA

Hydroxylated fatty acid 22 -hydroxy DHA

Eicosanoid 10-HDoHE LMFA04000027 Eicosanoid 11,12-DiHETrE LMFA03050008 Eicosanoid 1 lbeta-PGE2 LMFA03010060 Eicosanoid 1 lbeta-PGF2alpha LMFA03010036

Eicosanoid 11-HDoHE LMFA04000028

Eicosanoid 11-HETE LMFA03060085

Eicosanoid l l-trans-LTC4 LMFA03020020

Eicosanoid 12,13-DiHOME LMFA01050351

Eicosanoid 12-epi-LTB4 LMFA03020015

Eicosanoid 12-HETE LMFA03060088

Eicosanoid 12S-HEPE LMFA03070008

Eicosanoid 12S-HHTrE LMFA03050002

Eicosanoid 13.14-dihydro-15-keto-PGD2 LMFA03010022

Eicosanoid 13.14-dihydro- 15 -keto-PGE2 LMFA03010031

Eicosanoid 13,14-dihydro- 15-keto-PGF2alpha LMFA03010027

Eicosanoid 13-HDoHE LMFA04000029

Eicosanoid 13-OxoODE LMFA02000016

Eicosanoid 13(S)-HODE LMFA01050349

Eicosanoid 13(S)-HOTrE LMFA02000051

Eicosanoid 13(S)-HOTrE(gamma) LMFA01050145

Eicosanoid 14.15-DiHETrE LMFA03050010

Eicosanoid 14.15-EpETrE LMFA03080005

Eicosanoid 14-HDoHE LMFA04000030

Eicosanoid 15 -deoxy -delta- 12, 14-PGD2 LMFA03010051

Eicosanoid 15 -deoxy -delta- 12, 14-PGJ2 LMFA03010021

Eicosanoid 15-HEPE LMFA03070032

Eicosanoid 15-HETE LMFA03060087

Eicosanoid 15-keto-PGF 1 alpha LMFA03010150

Eicosanoid 15-keto-PGF2alpha LMFA03010026

Eicosanoid 15-Oxo-ETE LMFA03060051

Eicosanoid 15(S)-HETrE LMFA03050007

Eicosanoid 16(17)-EpDPE LMFA04000037

Eicosanoid 16-HDoHE LMFA04000031

Eicosanoid 16R-HETE LMFA03060069

Eicosanoid 17-HDoHE LMFA04000032

Eicosanoid 17-HETE LMFA03060091

Eicosanoid 18-HEPE LMFA03070033

Eicosanoid 18-HETE LMFA03060092

Eicosanoid 19,20-DiHDPE LMFA04000043

Eicosanoid 20-HDoHE LMFA04000033

Eicosanoid 20-HETE LMFA03060009

Eicosanoid 2,3-dinor-l lb-PGF2alpha LMFA03010011

Eicosanoid 4-HDoHE LMFA04000024

Eicosanoid 5.6-DiHETrE LMFA03050004

Eicosanoid 5.6-EpETrE LMFA03080002

Eicosanoid 5-HEPE LMFA03070027

Eicosanoid 5-HETE LMFA03060084

Eicosanoid 5-iPF2alpha-VI LMFA03110011

Eicosanoid 5S,15S-DiHETE LMFA03060010 Eicosanoid 5S,6S-Lipoxin A4 LMFA03040003

Eicosanoid 5(S)-HETrE LMFA03050005

Eicosanoid 6-trans- 12-epi-LTB4 LMFA03020014

Eicosanoid 6-trans-LTB4 LMFA03020013

Eicosanoid 7-HDoHE LMFA04000025

Eicosanoid 8,9-DiHETrE LMFA03050006

Eicosanoid 8-HDoHE LMFA04000026

Eicosanoid 8-HETE LMFA03060086

Eicosanoid 8-iso-PGF2alpha LMFA03110001

Eicosanoid 8(S)-HETrE LMFA03050011

Eicosanoid 9-HEPE LMFA03070029

Eicosanoid 9-HETE LMFA03060089

Eicosanoid 9(S)-HODE LMFAO 1050278

Eicosanoid 9(S)-HOTrE LMFA02000024

Eicosanoid bicyclo-PGE2 LMFA03010034

Eicosanoid LTB4 LMFA03020001

Eicosanoid LTC4 LMFA03020003

Eicosanoid LTE4 LMFA03020002

Eicosanoid PGD1 LMFA03010049

Eicosanoid PGD2 LMFA03010004

Eicosanoid PGD3 LMFA03010142

Eicosanoid PGE2 LMFA03010003

Eicosanoid PGF2alpha LMFA03010002

Eicosanoid PGJ2 LMFA03010019

Eicosanoid T etranor- 12(R)-HETE LMFAO 1050143

Eicosanoid TXB2 LMFA03030002

Eicosanoid TXB3 LMFA03030006

Triacylglycerol TG(48: l)

Triacylglycerol TG(48:2)

Triacylglycerol TG(50:0)

Triacylglycerol TG(50: l)

Triacylglycerol TG(50:2)

Triacylglycerol TG(50:3)

Triacylglycerol TG(50:4)

Triacylglycerol TG(52: 1)

Triacylglycerol TG(52:2)

Triacylglycerol TG(52:3)

Triacylglycerol TG(52:3)

Triacylglycerol TG(52:4)

Triacylglycerol TG(52:5)

Triacylglycerol TG(54:2)

Triacylglycerol TG(54:3)

Triacylglycerol TG(54:4)

Triacylglycerol TG(54:5)

Triacylglycerol TG(54:6)

Triacylglycerol TG(56:6) Diacylglycerol l,2-DG( 30:0)

Diacylglycerol 1.2-DG( 30: 1)

Diacylglycerol 1.2-DG( 30:2)

Diacylglycerol 1,2-DG( 32:0)

Diacylglycerol 1,2-DG( 32: 1)

Diacylglycerol 1,2-DG( 32:2)

Diacylglycerol 1,2-DG( 32:3)

Diacylglycerol 1,2-DG( 34:0)

Diacylglycerol 1,2-DG( 34: 1)

Diacylglycerol 1,2-DG( 34:2)

Diacylglycerol 1,2-DG( 34:3)

Diacylglycerol 1,2-DG( 34:4)

Diacylglycerol 1,2-DG( 36:0)

Diacylglycerol 1,2-DG( 36: 1)

Diacylglycerol 1,2-DG( 36:2)

Diacylglycerol 1,2-DG( 36:3)

Diacylglycerol 1,2-DG( 36:4)

Diacylglycerol 1,2-DG( 36:5)

Diacylglycerol 1,2-DG( 38:0)

Diacylglycerol 1,2-DG( 38: 1)

Diacylglycerol 1,2-DG( 38:2)

Diacylglycerol 1,2-DG( 38:3)

Diacylglycerol 1,2-DG( 38:4)

Diacylglycerol 1,2-DG( 38:5)

Diacylglycerol 1,2-DG( 38:6)

Diacylglycerol 1,2-DG( 40:4)

Diacylglycerol 1,2-DG( 40:6)

Diacylglycerol 1.2-DG( 40:7)

Diacylglycerol 1.3-DG( 30:0)

Diacylglycerol 1,3-DG( 30: 1)

Diacylglycerol 1,3-DG( 30:2)

Diacylglycerol 1,3-DG( 32:0)

Diacylglycerol 1,3-DG( 32: 1)

Diacylglycerol 1,3-DG( 32:2)

Diacylglycerol 1,3-DG( 32:3)

Diacylglycerol 1,3-DG( 34:0)

Diacylglycerol 1,3-DG( 34: 1)

Diacylglycerol 1,3-DG( 34:2)

Diacylglycerol 1,3-DG( 34:3)

Diacylglycerol 1,3-DG( 34:4)

Diacylglycerol 1,3-DG( 36:0)

Diacylglycerol 1,3-DG( 36: 1)

Diacylglycerol 1,3-DG( 36:2)

Diacylglycerol 1,3-DG( 36:3)

Diacylglycerol 1,3-DG( 36:4)

Diacylglycerol 1,3-DG( 36:5) Diacylglycerol l,3-DG( 38:0)

Diacylglycerol 1.3-DG( 38: 1)

Diacylglycerol 1.3-DG( 38:2)

Diacylglycerol 1,3-DG( 38:3)

Diacylglycerol 1,3-DG( 38:4)

Diacylglycerol 1,3-DG( 38:5)

Diacylglycerol 1,3-DG( 38:6)

Diacylglycerol 1,3-DG( 40:4)

Diacylglycerol 1,3-DG( 40:6)

Diacylglycerol 1,3-DG( 40:7)

Glycerophospholipid LPC(16:0)

Glycerophospholipid LPC(16:0e)

Glycerophospholipid LPC(16:0p)

Glycerophospholipid LPC(16: 1)

Glycerophospholipid LPC(18:0)

Glycerophospholipid LPC(18:0e)

Glycerophospholipid LPC(18: 1)

Glycerophospholipid LPC(18:2)

Glycerophospholipid LPC(20:3)

Glycerophospholipid LPC(20:4)

Glycerophospholipid LPC(22:5)

Glycerophospholipid LPC(22:6)

Glycerophospholipid LPE(16:0)

Glycerophospholipid LPE(18:0)

Glycerophospholipid LPE(18: 1)

Glycerophospholipid LPE(18:2)

Glycerophospholipid LPE(20:4)

Glycerophospholipid LPE(22: 1)

Glycerophospholipid LPE(22:6)

Glycerophospholipid N-Acyl-PS(52: l)

Glycerophospholipid N-Acyl-PS(54:2)

Glycerophospholipid PA(32:0)

Glycerophospholipid PA(32: 1)

Glycerophospholipid PA(34:0)

Glycerophospholipid PA(34: 1)

Glycerophospholipid PA(34:2)

Glycerophospholipid PA(36:0)

Glycerophospholipid PA(36: 1)

Glycerophospholipid PA(36:2)

Glycerophospholipid PA(36:3)

Glycerophospholipid PA(36:4)

Glycerophospholipid PA(38:2)

Glycerophospholipid PA(38:3)

Glycerophospholipid PA(38:4)

Glycerophospholipid PA(38:5)

Glycerophospholipid PA(38:6) Glycerophospholipid PC(30: l)

Glycerophospholipid PC(32:0)

Glycerophospholipid PC(32: l)

Glycerophospholipid PC(32:2)

Glycerophospholipid PC(34:0)

Glycerophospholipid PC(34: l)

Glycerophospholipid PC(34: le)

Glycerophospholipid PC(34:2)

Glycerophospholipid PC(34:2e)

Glycerophospholipid PC(34:3)

Glycerophospholipid PC(36:0)

Glycerophospholipid PC(36: l)

Glycerophospholipid PC(36: le)/PC(36:0p)

Glycerophospholipid PC(36:2)

Glycerophospholipid PC(36:2e)/PC(36: lp)

Glycerophospholipid PC(36:3)

Glycerophospholipid PC(36:4)

Glycerophospholipid PC(36:4e)/PC(36:3p)

Glycerophospholipid PC(36:5)

Glycerophospholipid PC(38:2)

Glycerophospholipid PC(38:3e)/PC(38:2p)

Glycerophospholipid PC(38:4)

Glycerophospholipid PC(38:5)

Glycerophospholipid PC(38:5e)/PC(38:4p)

Glycerophospholipid PC(38:6)

Glycerophospholipid PC(40:2)

Glycerophospholipid PC(40:4)

Glycerophospholipid PC(40:5)

Glycerophospholipid PC(40:6)

Glycerophospholipid PC(40:7)

Glycerophospholipid PC(40:8)

Glycerophospholipid PE(32: l)

Glycerophospholipid PE(34:0)

Glycerophospholipid PE(34: l)

Glycerophospholipid PE(34: lp)

Glycerophospholipid PE(34:2)

Glycerophospholipid PE(34:2p)

Glycerophospholipid PE(36:0)

Glycerophospholipid PE(36: l)

Glycerophospholipid PE(36:2)

Glycerophospholipid PE(36:2e)/PE(36: lp)

Glycerophospholipid PE(36:3)

Glycerophospholipid PE(36:3e)/PE(36:2p)

Glycerophospholipid PE(36:4)

Glycerophospholipid PE(36 : 4e)/PE(36 : 3p)

Glycerophospholipid PE(36:5) Glycerophospholipid PE(36:5e)/PE(36:4p)

Glycerophospholipid PE(38: l)

Glycerophospholipid PE(38:2)

Glycerophospholipid PE(38:3)

Glycerophospholipid PE(38:4)

Glycerophospholipid PE(38:5)

Glycerophospholipid PE(38:5e)/PE(38:4p)

Glycerophospholipid PE(38:6)

Glycerophospholipid PE(38 : 6e)/PE(38 : 5p)

Glycerophospholipid PE(40: l)

Glycerophospholipid PE(40:4)

Glycerophospholipid PE(40:5)

Glycerophospholipid PE(40:5e)

Glycerophospholipid PE(40:6)

Glycerophospholipid PE(40:6e)

Glycerophospholipid PE(40:7)

Glycerophospholipid PE(40:7e)

Glycerophospholipid PE(42: l)

Glycerophospholipid PE(42:5)

Glycerophospholipid PE(42:5p)

Glycerophospholipid PE(42:6)

Glycerophospholipid PE(42:6p)

Glycerophospholipid PE(42:7)

Glycerophospholipid PG(34: l)

Glycerophospholipid PG(34:2)

Glycerophospholipid PG(36: l)

Glycerophospholipid PG(36:2)

Glycerophospholipid PG(36:3)

Glycerophospholipid PG(36:4)

Glycerophospholipid PG(36:5)

Glycerophospholipid PG(38:4)

Glycerophospholipid PG(38:5)

Glycerophospholipid PG(38:6)

Glycerophospholipid PG(40:4)

Glycerophospholipid PG(40:5)

Glycerophospholipid PG(40:6)

Glycerophospholipid PG(40:7)

Glycerophospholipid PG(40:8)

Glycerophospholipid PG(40:9)

Glycerophospholipid PI(32: l)

Glycerophospholipid PI(34:0)

Glycerophospholipid PI(34: l)

Glycerophospholipid PI(34:2)

Glycerophospholipid PI(36:0)

Glycerophospholipid PI(36: l)

Glycerophospholipid PI(36:2) Glycerophospholipid PI(36:3)

Glycerophospholipid PI(36:4)

Glycerophospholipid PI(36:5)

Glycerophospholipid PI(38:2)

Glycerophospholipid PI(38:3)

Glycerophospholipid PI(38:4)

Glycerophospholipid PI(38:5)

Glycerophospholipid PI(38:6)

Glycerophospholipid PI(40:3)

Glycerophospholipid PI(40:4)

Glycerophospholipid PI(40:5)

Glycerophospholipid PI(40:6)

Glycerophospholipid PS(32: 1)

Glycerophospholipid PS(34:0)

Glycerophospholipid PS(34: 1)

Glycerophospholipid PS(34:2)

Glycerophospholipid PS(36:0)

Glycerophospholipid PS(36: 1)

Glycerophospholipid PS(36:2)

Glycerophospholipid PS(36:3)

Glycerophospholipid PS(36:4)

Glycerophospholipid PS(38: 1)

Glycerophospholipid PS(38:2)

Glycerophospholipid PS(38:3)

Glycerophospholipid PS(38:4)

Glycerophospholipid PS(38:5)

Glycerophospholipid PS(38:6)

Glycerophospholipid PS(40:3)

Glycerophospholipid PS(40:4)

Glycerophospholipid PS(40:5)

Glycerophospholipid PS(40:6)

Glycerophospholipid PS(40:7)

Sphingolipid Cer(dl6: 1/17:0) LMSP02010015

Sphingolipid Cer(dl6: 1/22:0) LMSP02010016

Sphingolipid Cer(dl 6: 1/23:0) LMSP02010017

Sphingolipid Cer(dl 6:2/23:0)

Sphingolipid Cer(dl8:0/13:0) LMSP02010018

Sphingolipid Cer(dl8:0/14:0) LMSP02020016

Sphingolipid Cer(dl8:0/15:0) LMSP02010019

Sphingolipid Cer(dl8:0/16:0) LMSP02020001

Sphingolipid Cer(dl8:0/17:0) LMSP02010031

Sphingolipid Cer(dl8:0/18:0) LMSP02020008

Sphingolipid Cer(dl8:0/18:l) LMSP02020015

Sphingolipid Cer(dl8:0/20:0) LMSP02020009

Sphingolipid Cer(dl 8: 0/22:0) LMSP02020010

Sphingolipid Cer(dl 8: 0/24:0) LMSP02020012 Sphingolipid Cer(dl 8: 0/24:1) LMSP02020011

Sphingolipid Cer(dl8:0/26:0) LMSP02020014

Sphingolipid Cer(dl8:0/26:1) LMSP02020013

Sphingolipid Cer(dl8:0/26:2)

Sphingolipid Cer(dl8: 1/14:0) LMSP02010001

Sphingolipid Cer(dl8: 1/16:0) LMSP02010004

Sphingolipid Cer(dl8: 1/17:0) LMSP02010020

Sphingolipid Cer(dl8: 1/18:0) LMSP02010006

Sphingolipid Cer(dl8: l/18:l) LMSP02010003

Sphingolipid Cer(dl8: 1/19:0) LMSP02010032

Sphingolipid Cer(dl8: 1/20:0) LMSP02010007

Sphingolipid Cer(dl8: 1/22:0) LMSP02010008

Sphingolipid Cer(dl 8: 1/23:0) LMSP02010021

Sphingolipid Cer(dl8: 1/24:0) LMSP02010012

Sphingolipid Cer(dl8: l/24:1) LMSP02010009

Sphingolipid Cer(dl 8: 1/25:0) LMSP02010013

Sphingolipid Cer(dl8: 1/26:0) LMSP02010011

Sphingolipid Cer(dl8: l/26:1) LMSP02010010

Sphingolipid Cer(dl8:2/14:0) LMSP02010022

Sphingolipid Cer(dl 8:2/15:0) LMSP02010023

Sphingolipid Cer(dl8:2/16:0) LMSP02010024

Sphingolipid Cer(dl8:2/18:l) LMSP02010025

Sphingolipid Cer(dl8:2/20:0) LMSP02010026

Sphingolipid Cer(dl8:2/20:1) LMSP02010027

Sphingolipid Cer(dl 8:2/21 :0) LMSP02010028

Sphingolipid Cer(dl 8:2/22:0) LMSP02010029

Sphingolipid Cer(dl 8:2/23:0) LMSP02010030

Sphingolipid Cer(dl 8:2/23:1)

Sphingolipid Cer(dl 8:2/24:2)

Sphingolipid HexCer(dl6: 1/17:0)

Sphingolipid HexCer(dl6: 1/22:0)

Sphingolipid HexCer(dl6: 1/23:0)

Sphingolipid HexCer(d 16:2/23:0)

Sphingolipid HexCer(d 18 : 0/ 13 : 0)

Sphingolipid HexCer(d 18 : 0/ 14 : 0)

Sphingolipid HexCer(d 18 : 0/ 16 : 0)

Sphingolipid HexCer(d 18 : 0/ 17: 0)

Sphingolipid HexCer(d 18 : 0/ 18 : 0)

Sphingolipid HexCer(d 18 : 0/ 18 : 1)

Sphingolipid HexCer(d 18:0/20:0)

Sphingolipid HexCer(d 18:0/22:0)

Sphingolipid HexCer(d 18:0/24:0)

Sphingolipid HexCer(dl8:0/24: 1)

Sphingolipid HexCer(d 18:0/26:0)

Sphingolipid HexCer(dl8:0/26: 1)

Sphingolipid HexCer(d 18:0/26:2) Sphingolipid HexCer(dl8: 1/14:0)

Sphingolipid HexCer(d 18 : 1/ 16 : 0)

Sphingolipid HexCer(dl8: 1/17:0)

Sphingolipid HexCer(dl8: 1/18:0)

Sphingolipid HexCer(dl8: 1/18: 1)

Sphingolipid HexCer(dl8: 1/19:0)

Sphingolipid HexCer(dl8: 1/20:0)

Sphingolipid HexCer(dl8: 1/22:0)

Sphingolipid HexCer(dl8: 1/23:0)

Sphingolipid HexCer(dl8: 1/24:0)

Sphingolipid HexCer(dl8: 1/24: 1)

Sphingolipid HexCer(dl8: 1/25:0)

Sphingolipid HexCer(dl8: 1/26:0)

Sphingolipid HexCer(dl8: 1/26: 1)

Sphingolipid HexCer(d 18 : 2/ 14 : 0)

Sphingolipid HexCer(d 18 : 2/ 15 : 0)

Sphingolipid HexCer(d 18 : 2/ 16 : 0)

Sphingolipid HexCer(d 18:2/18: 1)

Sphingolipid HexCer(d 18:2/20:0)

Sphingolipid HexCer(dl8:2/20: 1)

Sphingolipid HexCer(d 18:2/21:0)

Sphingolipid HexCer(d 18:2/22:0)

Sphingolipid HexCer(d 18:2/23:0)

Sphingolipid HexCer(dl8:2/23: 1)

Sphingolipid HexCer(d 18:2/24: 1)

Sphingolipid HexCer(d 18:2/24:2)

Sphingolipid C31:0 Sphingomyelin

Sphingolipid C32:0 Sphingomyelin

Sphingolipid C32: l Sphingomyelin

Sphingolipid C32:2 Sphingomyelin

Sphingolipid C33:0 Sphingomyelin

Sphingolipid C33: l Sphingomyelin

Sphingolipid C33:2 Sphingomyelin

Sphingolipid C34:0 Sphingomyelin

Sphingolipid C34: l Sphingomyelin

Sphingolipid C34:2 Sphingomyelin

Sphingolipid C35:0 Sphingomyelin

Sphingolipid C35: l Sphingomyelin

Sphingolipid C36:0 Sphingomyelin

Sphingolipid C36: l Sphingomyelin

Sphingolipid C36:2 Sphingomyelin

Sphingolipid C36:3 Sphingomyelin

Sphingolipid C37: l Sphingomyelin

Sphingolipid C38:0 Sphingomyelin

Sphingolipid C38: l Sphingomyelin

Sphingolipid C38:2 Sphingomyelin Sphingolipid C38:3 Sphingomyelin

Sphingolipid C39: l Sphingomyelin

Sphingolipid C39:2 Sphingomyelin

Sphingolipid C40:0 Sphingomyelin

Sphingolipid C40: l Sphingomyelin

Sphingolipid C40:2 Sphingomyelin

Sphingolipid C40:3 Sphingomyelin

Sphingolipid C41: l Sphingomyelin

Sphingolipid C41:2 Sphingomyelin

Sphingolipid C41:3 Sphingomyelin

Sphingolipid C42: l Sphingomyelin

Sphingolipid C42:2 Sphingomyelin

Sphingolipid C42:3 Sphingomyelin

Sphingolipid C42:4 Sphingomyelin

Sphingolipid C43:l Sphingomyelin

Sphingolipid C43:2 Sphingomyelin

Sphingolipid C43:3 Sphingomyelin

Sphingolipid C44: l Sphingomyelin

Sphingolipid C44:2 Sphingomyelin

Sphingolipid C44:3 Sphingomyelin

Sphingolipid C16 Sphinganine LMSP01040001

Sphingolipid Sphinganine LMSP01020001

Sphingolipid Sphingosine LMSP01010001

Sphingolipid C16 Sphinganine-P LMSP01050006

Sphingolipid C16 Sphingosine-P LMSP01050005

Sphingolipid C19 Sphingosine-P LMSP01050004

Sphingolipid Sphinganine-P LMSP01050002

Sphingolipid Sphingosine-P LMSP01050001

Sphingolipid LacCer(d 18:0/14:0) LMSP0501AB13

Sphingolipid LacCer(d 18:0/16:0) LMSP0501AB14

Sphingolipid LacCer(d 18:0/18:0) LMSP0501AB15

Sphingolipid LacCer(dl8:0/18: l) LMSP0501AB22

Sphingolipid LacCer(d 18:0/20:0) LMSP0501AB16

Sphingolipid LacCer(d 18 : 0/22 : 0) LMSP0501AB17

Sphingolipid LacCer(d 18 : 0/24 : 0) LMSP0501AB18

Sphingolipid LacCer(d 18 : 0/24 : 1) LMSP0501AB21

Sphingolipid LacCer(d 18:0/26:0) LMSP0501AB19

Sphingolipid LacCer(d 18:0/26: 1) LMSP0501AB20

Sphingolipid LacCer(dl8: 1/14:0) LMSP0501AB11

Sphingolipid LacCer(dl8: 1/16:0) LMSP0501AB03

Sphingolipid LacCer(dl8: 1/18:0) LMSP0501AB04

Sphingolipid LacCer(dl8: 1/18: 1) LMSP0501AB12

Sphingolipid LacCer(dl8: 1/20:0) LMSP0501AB05

Sphingolipid LacCer(dl8: 1/22:0) LMSP0501AB06

Sphingolipid LacCer(dl8: 1/24:0) LMSP0501AB07

Sphingolipid LacCer(dl8: 1/24: 1) LMSP0501AB09 Sphingolipid LacCer(dl8: 1/26:0) LMSP0501AB08

Sphingolipid LacCer(dl8: 1/26: 1) LMSP0501AB10

Sphingolipid SM(dl8:0/13:0) LMSP03010033

Sphingolipid SM(dl8:2/14:0) LMSP03010034

Sphingolipid SM(dl6: 1/16:0) LMSP03010035

Sphingolipid SM(dl8: 1/14:0) LMSP03010028

Sphingolipid SM(dl8:0/14:0) LMSP03010030

Sphingolipid SM(dl7:2/16:0)

Sphingolipid SM(dl8:2/15:0) LMSP03010036

Sphingolipid SM(dl6: 1/17:0) LMSP03010037

Sphingolipid SM(dl8: 1/15:0) LMSP03010039

Sphingolipid SM(dl8:0/15:0) LMSP03010091

Sphingolipid SM(dl6: l/18: l) LMSP03010040

Sphingolipid SM(dl8: l/16: l) LMSP03010041

Sphingolipid SM(dl8:2/16:0) LMSP03010090

Sphingolipid SM(dl6: 1/18:0) LMSP03010042

Sphingolipid SM(dl8: 1/16:0) LMSP03010003

Sphingolipid SM(dl8:0/16:0) LMSP03010004

Sphingolipid SM(dl7: 1/17:0) LMSP03010043

Sphingolipid SM(dl8: 1/17:0) LMSP03010044

Sphingolipid SM(dl9: 1/16:0) LMSP03010045

Sphingolipid SM(dl8:0/17:0) LMSP03010046

Sphingolipid SM(dl8:2/18: l) LMSP03010047

Sphingolipid SM(dl6: 1/20: 1) LMSP03010048

Sphingolipid SM(dl8:0/18:2) LMSP03010049

Sphingolipid SM(dl8: l/18: l) LMSP03010029

Sphingolipid SM(dl8:2/18:0) LMSP03010050

Sphingolipid SM(dl9: 1/17: 1) LMSP03010051

Sphingolipid SM(d20:2/16:0)

Sphingolipid SM(dl6: 1/20:0) LMSP03010052

Sphingolipid SM(dl8: 1/18:0) LMSP03010001

Sphingolipid SM(dl6:0/20:0) LMSP03010053

Sphingolipid SM(dl8:0/18:0) LMSP03010020

Sphingolipid SM(dl9:0/17:0) LMSP03010054

Sphingolipid SM(dl8: 1/19:0) LMSP03010055

Sphingolipid SM(dl9: 1/18:0) LMSP03010056

Sphingolipid SM(dl 6: 1/22:2)

Sphingolipid SM(dl 8: 1/20:2)

Sphingolipid SM(dl8:2/20: l) LMSP03010057

Sphingolipid SM(dl 6: 1/22: 1) LMSP03010058

Sphingolipid SM(dl7:2/21:0)

Sphingolipid SM(dl8: 1/20: 1) LMSP03010059

Sphingolipid SM(dl8:2/20:0) LMSP03010060

Sphingolipid SM(dl6: 1/22:0) LMSP03010061

Sphingolipid SM(dl8: 1/20:0) LMSP03010005

Sphingolipid SM(dl6:0/22:0) LMSP03010063 Sphingolipid SM(dl8:0/20:0) LMSP03010021

Sphingolipid SM(dl6:2/23:0)

Sphingolipid SM(dl8:2/21:0) LMSP03010064

Sphingolipid SM(dl6: 1/23:0) LMSP03010065

Sphingolipid SM(dl7: 1/22:0) LMSP03010066

Sphingolipid SM(dl8: 1/21:0) LMSP03010067

Sphingolipid SM(dl9: 1/20:0) LMSP03010068

Sphingolipid SM(d20: 1/19:0) LMSP03010069

Sphingolipid SM(dl 6: 1/24:2)

Sphingolipid SM(dl 8: 1/22:2)

Sphingolipid SM(dl8:2/22: l) LMSP03010070

Sphingolipid SM(dl 6: 1/24: 1) LMSP03010071

Sphingolipid SM(dl 8: 1/22: 1) LMSP03010072

Sphingolipid SM(dl8:2/22:0) LMSP03010092

Sphingolipid SM(dl6: 1/24:0) LMSP03010073

Sphingolipid SM(dl8: 1/22:0) LMSP03010006

Sphingolipid SM(dl8:0/22:0) LMSP03010022

Sphingolipid SM(dl8:2/23: l)

Sphingolipid SM(dl 7: 1/24: 1) LMSP03010074

Sphingolipid SM(dl 8: 1/23: 1)

Sphingolipid SM(dl8:2/23:0) LMSP03010075

Sphingolipid SM(d20: l/21: l)

Sphingolipid SM(dl6: 1/25:0) LMSP03010076

Sphingolipid SM(dl7: 1/24:0) LMSP03010077

Sphingolipid SM(dl8: 1/23:0) LMSP03010078

Sphingolipid SM(dl8: 1/24:3)

Sphingolipid SM(dl8:2/24:2)

Sphingolipid SM(dl 8: 1/24:2)

Sphingolipid SM(dl8:2/24: l) LMSP03010080

Sphingolipid SM(d20: 1/22:2)

Sphingolipid SM(dl 8: 1/24: 1) LMSP03010007

Sphingolipid SM(dl8:2/24:0) LMSP03010081

Sphingolipid SM(dl8: 1/24:0) LMSP03010008

Sphingolipid SM(dl 6: 1/27:2)

Sphingolipid SM(dl8:2/25: l)

Sphingolipid SM(dl 7: 1/26: 1) LMSP03010082

Sphingolipid SM(dl 8: 1/25: 1)

Sphingolipid SM(dl8:2/25:0) LMSP03010083

Sphingolipid SM(dl9: 1/24: 1) LMSP03010084

Sphingolipid SM(d20: 1/23: 1)

Sphingolipid SM(dl8: 1/25:0) LMSP03010027

Sphingolipid SM(dl9:0/24: l) LMSP03010085

Sphingolipid SM(dl9: 1/24:0) LMSP03010086

Sphingolipid SM(d20: 1/23:0) LMSP03010087

Sphingolipid SM(dl 7: 1/27:2)

Sphingolipid SM(dl 8: 0/26:3) Sphingolipid SM(dl 8: 1/26:2)

Sphingolipid SM(d20: 1/24:2)

Sphingolipid SM(dl 8: 0/26:2)

Sphingolipid SM(dl8: 1/26: 1) LMSP03010009

Sphingolipid SM(dl9: 1/25: 1)

Sphingolipid SM(dl 6: 0/28: 1)

Sphingolipid SM(dl 8: 0/26: 1) LMSP03010025

Sphingolipid SM(dl9:0/25: l)

Sphingolipid SM(d20: 0/24: 1) LMSP03010088

Sphingolipid SM(dl7:0/27:0) LMSP03010089

Sphingolipid GalCer(dl6: 1/22:0) LMSP0501AC09

Sphingolipid GalCer(d 16: 1/23:0) LMSP0501AC10

Sphingolipid GalCer(d 18:0/16:0) LMSP0501AC11

Sphingolipid GalCer(d 18:0/20:0) LMSP0501AC12

Sphingolipid GalCer(d 18 : 0/22 : 0) LMSP0501AC13

Sphingolipid GalCer(d 18 : 0/24 : 0) LMSP0501AC14

Sphingolipid GalCer(d 18:0/26:0) LMSP0501AC15

Sphingolipid GalCer(d 18:0/26: 1) LMSP0501AC16

Sphingolipid GalCer(d 18:0/26:2)

Sphingolipid GalCer(dl8: 1/16:0) LMSP0501AC01

Sphingolipid GalCer(dl8: 1/18:0) LMSP0501AC02

Sphingolipid GalCer(dl8: 1/20:0) LMSP0501AC03

Sphingolipid GalCer(dl8: 1/22:0) LMSP0501AC04

Sphingolipid GalCer(d 18: 1/23:0) LMSP0501AC17

Sphingolipid GalCer(dl8: 1/24:0) LMSP0501AC05

Sphingolipid GalCer(dl8: 1/24: 1) LMSP0501AC07

Sphingolipid GalCer(dl8: 1/26:0) LMSP0501AC06

Sphingolipid GalCer(dl8: 1/26: 1) LMSP0501AC08

Sphingolipid GalCer(d 18:2/16:0) LMSP0501AC18

Sphingolipid GalCer(d 18 : 2/20 : 0) LMSP0501AC19

Sphingolipid GalCer(d 18 : 2/20 : 1) LMSP0501AC20

Sphingolipid GalCer(d 18 : 2/21 : 0) LMSP0501AC21

Sphingolipid GalCer(d 18 : 2/22 : 0) LMSP0501AC22

Sphingolipid GalCer(d 18 : 2/23 : 0) LMSP0501AC23

Sphingolipid GalCer(dl8:2/23: 1)

Sphingolipid GlcCer(dl6: 1/22:0) LMSP0501AA30

Sphingolipid GlcCer(dl6: 1/23:0) LMSP0501AA31

Sphingolipid GlcCer(dl8:0/16:0) LMSP0501 AA04

Sphingolipid GlcCer(d 18:0/20:0) LMSP0501AA20

Sphingolipid GlcCer(d 18 : 0/22:0) LMSP0501AA21

Sphingolipid GlcCer(d 18 : 0/24:0) LMSP0501AA23

Sphingolipid GlcCer(d 18:0/26:0) LMSP0501AA25

Sphingolipid GlcCer(dl8:0/26: 1) LMSP0501AA24

Sphingolipid GlcCer(d 18:0/26:2)

Sphingolipid GlcCer(dl8: 1/16:0) LMSP0501AA03

Sphingolipid GlcCer(dl8: 1/18:0) LMSP0501AA05 Sphingolipid GlcCer(dl8: 1/20:0) LMSP0501AA06 Sphingolipid GlcCer(dl8: 1/22:0) LMSP0501AA07 Sphingolipid GlcCer(dl8: 1/23:0) LMSP0501AA32 Sphingolipid GlcCer(dl8: 1/24:0) LMSP0501 AA09 Sphingolipid GlcCer(dl8: 1/24: 1) LMSP0501AA08 Sphingolipid GlcCer(dl8: 1/26:0) LMSP0501AA11 Sphingolipid GlcCer(dl8: 1/26: 1) LMSP0501AA10 Sphingolipid Glc Cer(d 18:2/16:0) LMSP0501AA33 Sphingolipid GlcCer(d 18 : 2/20 : 0) LMSP0501AA34 Sphingolipid GlcCer(dl8:2/20: 1) LMSP0501AA35 Sphingolipid GlcCer(d 18:2/21 : 0) LMSP0501AA36 Sphingolipid Glc Cer (d 18 : 2/22 : 0) LMSP0501AA37 Sphingolipid GlcCer(d 18:2/23 : 0) LMSP0501AA38 Sphingolipid GlcCer(dl8:2/23: 1)

Sterol lipid 24S-hydroxy -cholesterol LMST01010019 Sterol lipid 25-hydroxy-cholesterol LMST01010018 Sterol lipid 27-hydroxy-cholesterol LMST01010057 Sterol lipid 4beta-hydroxy-cholesterol LMST01010014 Sterol lipid 7alpha-hydroxy-cholesterol LMST01010013 Sterol lipid 7-Dehydrocholesterol LMST01010069 Sterol lipid 7-oxo-cholesterol LMST01010049 Sterol lipid Campe sterol LMST01030097 Sterol lipid Cholestenone LMST01010015 Sterol lipid Cholesterol LMST01010001 Sterol lipid Desmosterol LMST01010016 Sterol lipid Lanosterol LMST01010017 Sterol lipid Lathosterol LMST01010089 Sterol lipid Sitosterol LMST01040129 Sterol lipid CE(14:0) LMST01020004 Sterol lipid CE(14: 1) LMST01020021 Sterol lipid CE(15:0) LMST01020027 Sterol lipid CE(15: 1) LMST01020022 Sterol lipid CE(16:0) LMST01020005 Sterol lipid CE(16: 1) LMST01020006 Sterol lipid CE(16:2) LMST01020024 Sterol lipid CE(17:0) LMST01020026 Sterol lipid CE(17: 1) LMST01020023 Sterol lipid CE(18:0) LMST01020007 Sterol lipid CE(18: 1) LMST01020003 Sterol lipid CE(18:2) LMST01020008 Sterol lipid CE(18:3) LMST01020009 Sterol lipid CE(20:0) LMST01020010 Sterol lipid CE(20: 1) LMST01020011 Sterol lipid CE(20:2) LMST01020012 Sterol lipid CE(20:3) LMST01020013 Sterol lipid CE(20:4) LMST01020014 Sterol lipid CE(22:0) LMST01020016 Sterol lipid CE(22: l) LMST01020025 Sterol lipid CE(22:2) LMST01020017 Sterol lipid CE(22:4) LMST01020018 Sterol lipid CE(22:5) LMST01020031 Sterol lipid CE(22:6) LMST01020019 Prenol lipid Coenzyme Q10 LMPR02010001 Prenol lipid Coenzyme Q9 LMPR02010004 Prenol lipid Dolichol-16 LMPR03070026 Prenol lipid Dolichol-17 LMPR03070002 Prenol lipid Dolichol-18 LMPR03070003 Prenol lipid Dolichol-19 LMPR03070001 Prenol lipid Dolichol-20 LMPR03070004

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A nutraceutical composition comprising a lipid in a nutraceutically acceptable carrier, wherein the lipid is selected from the group consisting of the lipids in Table 1.

2. A dietary supplement comprising a lipid, wherein the lipid is selected from the group consisting of the lipids in Table 1.

3. A therapeutic composition comprising an effective amount of a lipid in a

pharmaceutically acceptable excipient, wherein the lipid is selected from the group consisting of the lipids in Table 1.

4. The therapeutic composition of claim 3, wherein an effective amount is an amount that ameliorates at least one symptom of a neurodegenerative disease characterized by an energy deficit.

5. The therapeutic composition of claim 3, wherein an effective amount is an amount that delays onset or progression of at least one symptom of amyotrophic lateral sclerosis (ALS).

6. The therapeutic composition of claim 3, wherein the lipid comprises at least about 5- 75% of the weight of the composition.

7. A kit comprising the therapeutic composition of claim 3 and instructions for the use of the composition for treating or ameliorating a symptom of or delaying progression of a neurodegenerative disease characterized by an energy deficit.

8. The kit of claim 7, further comprising a capture reagent for detecting a level or sequence of a gene selected from the group consisting of SOD1, C9orf72 repeat, and TDP-43.

9. A method of reducing or ameliorating an effect of a mutation associated with a neurodegenerative disease characterized by an energy deficit, the method comprising administering to the subject a composition comprising a lipid selected from the group consisting of the lipids in Table 1, thereby delaying progression of, or reducing or ameliorating an effect of a mutation associated with the neurodegenerative disease characterized by an energy deficit in the subject.

10. A method of treating a neurodegenerative disease characterized by an energy deficit in a subject, comprising administering to the subject a composition comprising a lipid selected from the group consisting of the lipids in Table 1, thereby beating or delaying progression of the neurodegenerabve disease characterized by an energy deficit in the subject.

12. The method of any one of claims 9-10, wherein the composibon is administered to the subject by oral adminisbation.

13. The method of any one of claims 9-10, wherein the composibon is administered to the subject in a drink.

14. The method of any one of claims 9-13, wherein the subject has a mutation and/or misexpression associated with ALS.

15. The method of claim 14, wherein the mutation is SOD1-G85R, TDP-43 misexpression, or C9orf72 repeat expansion.

16. The method of any one of claims 9-15, wherein the subject is human.

17. The method of any one of claims 9-16, wherein the composition is the composition or supplement of any one of claims 1-6.

18. The method of any one of claims 14-15, wherein the mutation and/or misexpression associated with ALS is measured using the kit of claim 8.

19. The method, composition, or kit of any one of claims 4, 7, or 9-18, wherein the neurodegenerabve disease characterized by an energy deficit is amyobophic lateral sclerosis (ALS).

20. The method of claim 19, wherein the ALS is sporadic ALS.

21. The composition, supplement, or method of claim 21, wherein the lipid is selected from the group comprising of lauric acid, mystiric acid, palmitic acid, oleic acid, linoleic acid, gamma-linoleic acid, arachidonic acid, EPA, and DHA, 12-hydroxy laurate, 14-hydroxy mystirate, 16-hydroxy palmitate, 19-hydroxy EPA, 20-hydroxy EPA, 21-hydroxy DHA, and 22- hydroxy DHA.

22. A nutraceutical composition comprising an omega-hydroxylated fatty acid, wherein the fatty acid is selected from the group consisting of the fatty acids in Table 1.

23. A dietary supplement comprising an omega-hydroxylated fatty acid, wherein the fatty acid is selected from the group consisting of the fatty acids in Table 1, the lipid is selected from the group consisting of the lipids in Table 1.

24. A therapeutic composition comprising an effective amount of an omega-hydroxylated fatty acid in a pharmaceutically acceptable excipient, wherein the fatty acid is selected from the group consisting of the fatty acids in Table 1.

25. The therapeutic composition of claim 24, wherein an effective amount is an amount that ameliorates at least one symptom of a neurodegenerative disease characterized by an energy deficit.

26. The therapeutic composition of claim 24, wherein an effective amount is an amount that delays onset or progression of at least one symptom of amyotrophic lateral sclerosis (ALS).

27. The therapeutic composition of claim 24, wherein the omega-hydroxylated fatty acid comprises at least about 5-75% of the weight of the composition.

28. A kit comprising the therapeutic composition of claim 24 and instructions for the use of the composition for treating or ameliorating a symptom of or delaying progression of a neurodegenerative disease characterized by an energy deficit.

29. The kit of claim 28, further comprising a capture reagent for detecting a level or sequence of a gene selected from the group consisting of SOD1, C9orf72 repeat, and TDP-43.

30. A method of reducing or ameliorating an effect of a mutation associated with a neurodegenerative disease characterized by an energy deficit, the method comprising administering to the subject a composition comprising an omega-hydroxylated fatty acid, wherein the fatty acid is selected from the group consisting of the fatty acids in Table 1, thereby delaying progression of, or reducing or ameliorating an effect of a mutation associated with the neurodegenerative disease characterized by an energy deficit in the subject.

31. A method of treating a neurodegenerative disease characterized by an energy deficit in a subject, comprising administering to the subject a composition comprising an omega- hydroxylated fatty acid, wherein the fatty acid is selected from the group consisting of the fatty acids in Table 1, thereby treating or delaying progression of the neurodegenerative disease characterized by an energy deficit in the subject.

32. The method of any one of claims 30-31, wherein the composition is administered to the subject by oral administration. 33. The method of any one of claims 30-31, wherein the composition is administered to the subject in a drink.

34. The method of any one of claims 30-33, wherein the subject has a mutation and/or misexpression associated with ALS.

35. The method of claim 34, wherein the mutation is SOD1-G85R, TDP-43 misexpression, or C9orf72 repeat expansion.

36. The method of any one of claims 30-35, wherein the subject is human.

37. The method of any one of claims 30-36, wherein the composition is the composition or supplement of any one of claims 21-27.

38. The method of any one of claims 34-35, wherein the mutation and/or misexpression associated with ALS is measured using the kit of claim 29.

39. The method, composition, or kit of any one of claims 25, 28, or 30-38, wherein the neurodegenerative disease characterized by an energy deficit is amyotrophic lateral sclerosis (ALS).

40. The method of claim 39, wherein the ALS is sporadic ALS.