RELATED APPLICATIONS

{80011 This application c aims the eneft of U,S. Provisional Patent

Application No, 62 175,871 _s filed on June 15, 2:015 _S which Is Incorporated herein y reference,

SEQUENCE LISTING

{80021 The instant application contains a Se uence Listing which has been submitted electronically in ASCII format and is hereby incorporated by eference in its entirety. The ASCII copy, created on June 13, 2016, is named 14904-15? Sequence UstingjST2§<txf; and is approximately 2 KB in size.,

BACKGROUND

{0003J Peroxisome proHferaior-aciivated receptor o or PPARa belongs to class of nuclear hormone receptors ¹ that participates In a diverse range of biological functions Including control of fatty acid transport and cafaboiism ant- inflammation ⁵ , immuno-moduiaSo i ⁴ , and anti-oxidation* ^' ... However, in a recent tud ⁸ , it has been shown that PPARa also plays an Important role; in the modulation of synaptic function In hippocampus via transcriptional upregulatson of CREB. It: has also been delineated that activation of PPARa In ppocampal neurons leads to the increase in ADAM 10 ^' transcription and subsequent norv amyloldogenic proteolysis of APP These reports highlight a li fd-in ependent rote of PPARa in controlling brain function. Otherwise, If was believed that the presence of peroxisomes in abundance could be important for the compensation of mitochondrial instability m the adult brain hippocampus''..

{0O0 | Like many other nuclear hormone receptors, It is not k own: if all the biological activity of PPAR-a also depends on its binding with the gand and subsequent translocation to the nucleus. Since interaction with figand plays a instrumental role in modulating the biological effect of most nuclear hormone r ce tors^ an investigation into the existenc of endogenous Hgands of PPARa in tie hippocampus was prompted, Successful identification of endogenous modulators of PPARa would aid in understanding t e endogenous regulation hippoeampai function and memory iby PPAR However, Ittle Is known about Ilia presence of endogenous !igands of PPARa n the hippocampus and inert role in regelating the synaptic activity, Although endocannabfnotd-iike molecules including oleoylethanotamide''^ ⁴ and palmitoytetsarK amide^ the fatty acid derivative 20«cart oxy»afachidof fc aoid^, and feufcotrlene B4* ^? have been

considered as endogenous PPARa Uganda, these compounds are ubiquitously present in different issues including live * _* kidne ²⁶ nd brain ¹⁵⁰ , Furthermore, these compounds display a ide range of biological activities starting form antioxidant anti-inflammation to attempt to ind an endogenous ligand of PPAR-a, a recent tud Identifies that 1«paimitoyl*2« oleoyl-sn-glyoef -3- hosp ocholine (16:0/18:1 -GPC) could serve as a potent ligand of In fiver. However, until now, nothing Is known about the presence of endogenous ligand(s In the hippocampus that are capable of modulating the PPARa activity in hippoeampai neurone,

fjMiSSJ In order to identify physiologically available iigands, affinity purification w s performed followed oy gas hase mese spectrometry (GCMS) anaiys.es in the nuclear extracts of !enti*ppara-ov©fexpress©d neurons. Then, the existence of these molecules was confirmed by - affinity purification of hippocampal xtracts collected from i id-type nd Ppam-nui! animals against GST-PPAR- recombinant protein followed by GCMS analyses. These analyses identified three unique llgands 3-hydroxy- 2, 2Hirnethyi butyrate (HM8), hexadecanan ide

(HEX), and 9-Oc!adeceoaroide (OCT) In brain hippocampus. Farther structural analyses revealed that two key amino acid residues Tyrosine 314 and 484 irt the ilgand binding pocket of PPARa ar Important for the binding with tttese igands, which was confirmed by .making site÷directed mutated constructs of PPARtt, subsequent expression of these constructs in neuronal cells using tentlvital strategy, and GCMS analyses of the affinity-purified nuclear fraction. The role of these ligands in c ntroliri¾ the e ression of synaptic proteins nd regulating fhe synaptic function of hippocampal neurons has also been analyzed,

{0 061 The HUB, HEX and OCT ligands induce in® activation of PPARo: m brain eels a d increase syna tic functions a uprcguiation of different synaptic molecules and calcium entry,. What is needed in the art are PPA.R-8 ligands for modulating PPARo activity and: for treatment of disorders such as dementia, neurological disorders, lysosomal storage diaorders and body weight disorders,

BRIEF SUMMARY

{00071 Methods of modulating peroxisome proiiferaic -activated receptor a PPARa) activity in a cell in a subject in need thereof are provided. The methods include administering an effective amount of a PPARa l¾and to the subject, the PPARo g nd being selected from the group consisting of ^' 3~hydroxy-2 _s 2-dsmet^yl butyraie (H B), hexadecananarnid (HEX) sod 9*octadecenamtde (OCT),

[08083 Methods of treating dementia, neurodegenerative disorders, lysosomal storage diseases and body weight disorders in a su jec in need thereof are provided,. The methods include administering an effective amount of a PPA o ligand to the subject. The PPARo ligand is selected from the grou consisting of 3~ hydroxy ^» 2,2^imelhyl butyrate ( MB\ hexadeeananarnide (HEX) and 9 ^»

octadeeenamide (OCT),

BRIEF DESCRIPTION OP THE DRAWINGS

p SSJ PIGS. 1 A*1 L identification of Endogenous Uganda of PP R-a in the mouse br i hippocampus<

[0010] (FIG _* 1A) A flow chart represents the procedur for the affinity purification of nuclear tlgands of PPAR-CL (FIG, IB) GC S analyses of the aoetonllrHe~ and

(FIG,. 10) ohlorofomvreoDnstnjcled nuclear extracts of WT and K0 hippocampus

(«~3 per group) after pulling down with GST-PPARa. (Simitar GCMS analyses were performed in {FIG. ID) acetcnirie- and (FfG, IE) nuclear extracts of Pparaheta-rsul hippocampal tissue {«-3 ^' er grwp)< Chemical structure of

3-hydPO.x , 2, 2-dimethyl btsiyrat® (HMB) (FIG. 1F), hexadecanamlde (HEX) (FIG...

1 G), and 9-Odadeoanamlde or oteanitde (OCT) {FIG, 1H .. (FIG, 1l)Tha immunobloi analyses of etuate collected from glutathione column probed with anti-GST, aotf- GST*PPAR ^« a. and antl GST-PPAHp anybodies (upper panel), and with an(M?PAR*a or ant!-PPARfJ antibody {lower nel), Hlstone 3 (H3) Immunobiot was erformed in the nuclear lys&i© (input)- to show the u it of the nuclear extract (middle anel , (FIG, 1J) Flow-chart of the QC S analysis fer identifying endogenous !gands afte novo synthesized PPARa, GC S analyses

fraction of (FIG. IK) ieni-veetor and (FIG. 1L) leni-PPARa-tBD- transduced hlppocan pai neurons. Results are confirme after three Independent expe iments. 0011 J FIGS. 2A-2G, Analys s of the- interaction of OCT,- HEX, and HU with PPARa by h gh-throughpy!: analyses,

{0012J (Fie, 2A) A sc ematic resentatio of TR-F ET an lysis to an l ze the interaction between endogenous llgarsds and PPARa÷PGC ^« 1 A complex, TR-FRET analyses of (FIG. 28) OCT, {FIG, 2C) HEX, and (FIG. 20} H 8 as plotted

fluorescenc vs. logafthmic scale of Ifgan cot cenirat n. Thermal-shift assay of (FtG. 2E) OCT, (FIG, 2F) HEX, and (FiG. 2G) HUB as described under materials and method section.. Results re confirmed after three indepen e t experiments. {0013] FIGS, 3A-3L A sroieomic approach to study the Inieradton between ilgands and PPARa in a molecular level.

{00 4| (FIG, 3A) Ribbon representations of superposed structures of PPAR-et Slgand binding pocket along with its ligands OCT (FIG, 3A)« HEX (FIG, 38), and HUB (FIG,. 30). grou s of amino acids positioned at a distance of 4 * ound the ligands were also shown In green colour, {FIG. 3D) A plasmid map of PPA a gene cloned in pLenti§ v ctor and the restriction sit© to c one the entire PPARogsne as shown in the middle panel The detailed map of PL. Ppara, Y314D P ara, Y4643DPpara, and Y314D Y464D Pp&m ge e cloned n the vector u i g ientivirel packaging kit as supplied ^' by Life technologies, Thermal shift assay of (FIG.. 3E.) Ft PPARa and (FiG.. 3F) Y314D Y464D PPARa protein. Tfn represents the melting temperature. (FIG, 3G) A flow-chart e resents the strategy of GC S analyses for the defection of endogenous ligands In Ppara-ntili neurone! extracts Infected with !enfi virus particles of different PPARa constructs, (FIGS, 3H-L GCMS analyses in the GFP-afF!niiy purified extracts of Fpara-nyil rilppDcampal neu o s transduced with (FIG, 3H) GFP only, (FIG. 31} GFP-FlPpma, (FIG. 3J) FP~ Υ3Ί 4QPpa _s (FIG, 3 ) GFP- Y464DF am, and (FiG, 3L) GFP-Y 140ίΎ4840Ρ $.?¾ viruses , (001 ^ FIGS. 4A-4S. Tile role of endogenous tigan e of FPARo In FPRE-dnVen. luciferBse acivtty in mouse primary astrocytes and neurons.

(0016] PPRI-tyclferasa sdWty in th© mouse primary astrocytes alter 4 hrs of mcu atton ith (FIG. A) OCT, (FIG, 48) HEX, and (FIG, 4C) HP at their wide range of concentrations. Results am mean ± SO of three Independent experiments *p< O. f vs. mni L (FIG, 40) a cartoon represents the detai s of PPRE ludferas© assay in primary cells Infected with lentivlrus particles of different PPARo constructs, PPRE tacfferase activity was assayed in mouse rimary astrocytes transduced with (E) only vector, {FIG. 4F) FLPpara, (FIG. 4Gj YS14D, (FIG. 4H) Y464D, and (FiG. 4i) Y314D Y464D PFARo genes after the treatment of ifferent closes of HEX, OCT, and HMB, PPRE tuciferase activity in mouse primary astrocytes pre nfected with tent viruses after the treatment of increasing doses of Wy14643 _* fenofibrate, and clctftbrat© ¾FIG, 4J) onl vedor, (FiG, 4K) FLPpara, (FIG, 4L) Y314D _t (FIG, 4M) ¥4640, and (FIG, 4N) Y3140 T464D], PPRE luciferase aaiiviiy was assayed in mouse primary hippooam af neurons transduced with (FIG, 40) only vector, (FiG, 4P) f LPpa , (FiG. 4Q) Y314D, (FIG. 4 ) Y464D _¾ and (FIG.. 4S> Y3140/Y464D

PPARct genes after the treatment of different dos of HEX, OCT, and HUB, Results are mean ± SO of three Independent experiments,

fiil 7| FIGS. SA-SL The role of endog nous igands of PPARo on the

morphological plasticity of hippoeanipal neurons.

(0018| Double immtinostalolng of AP-2 end phalloldin to measure the spine density in hippoeampal neurons transduced w th vector, FLF ara, and 8 £¾¾3m viruses after the treatment wit (PIG, SA) solvent (only DMSO), (F!G> SB) OCT, (FiG, 5C) HEX, and (FIG. 50) H 8.., ΆΜΡΑ-dhven eald m intux was measured In OCT (red), HE (green) and HUB ( &r fe^fcneaW Ppara-nuli hippocarnpal neurons transduced with (FIG., 5E) FLPpara, (FiG, 5F) Y3140 _t (FIG _* SG) ¥4840, and (FiG, 5H) Y314DfY464D PPA a genes. At! neurons ens treated with SO μ of NMDA receptor antagonist 20C to inhibit passive ca dum tow through NMOA receptor , (FIGS. 514.) Similarly H A-driven calcium influx as meas red in the ientiv us» infected Nppoearnpal neurons in trie presence of different endogenous Hgands _* in these cases, Naspm HCf was treated to stop the passive flow of calcium: currents through AMPA receptor, {001 §3 FIGS. 0Ά-8Β. The subcellular localisation of PPA a, $ and y isotypea In mouse brain hippocampus,

{0 20J {FIG. ©A) The i traocula distribution of PPARa _s and y w@r® shown by immunofluorescence {Neu -green _f PPARs ~ red) analyses in the CA1 regions of hippocampus. {FIG.66) Nuclear {ME} and cytoplasmic extracts (CE of ^' hippocampai tissue were irnrnynobiotted for FPAR~o ₍ β, and y, immunobtot analyses were performed in 6-8 weeks οϊ WT and Ppara-nul! mice (n-3 per group). The purSy of the cytoplasmic fraction was validated byGAFDH immunobiot analysts whereas hlstone 3 (H3) immunefclot was performed to evaluate the parity of nuclear fraction. p021J FIGS, 7A-7I. Identification of endogenous ilgands of PPARa in the mouse brain hippocampus,

10 22] GC÷ s ana ses of chloroform* (FIG ^' S. 7A & 7B) and acetonftfsie* (FIB, 7C) reconstituted nuclear extracts of WT hippocampus after puling down with GST÷PPAR«*LBD, Similar GONIS analyses were performed in chloroform {FIG, 7D) and acetonitrie (FIG, 7E) reconstituted nuclear extracts after polling down with GST-PPARp-LBD. FIG, 7F) The tmmunoblot analyses of ©fuale collected from glutathione column probed wi8t ants-GST antibody (tipper panel), and anti- PPARa or antkPPA p antibodies (lower panel), Hisione 3 (H3) tmmunoblot was erformed fn the nuclear lysate (input) to show the purit of the nuclear extract {m ddle panel). GO-MS analyses of the chlomfemvextracted nuclear fraction of i nti-veC'tor- (FIG. 7G) and le«ti-P A o> ( IG, 7H transduced Pp ra-nul hippocampai neurons, FIG. 71) Neuronal extracts infected with teiii-veetof and ie t!-PPARs were analyzed for PFARa and then normalized with actiri. Results were confirmed by three independent experiments,

{00233 FIGS. 8A-8L Analyses of the interaction of OCT, HEX and HUB with PPARa by TR.-F ET,

[00241 TR-FRET analyses of OCT (FIG, SA), HEX (FIG, 8B) and HUB (FIG. 8C) plotted as fluorescence vs. logarithmic scale of Ogand coiicontraHcin,

Thermal-shift assay of OCT (FIG, 8D), HEX (FIG. BE) and HMB (FIG. 8F) was performed as described under the Materials and Method section.

S §| Equation for fufMengtb protein only; y * 50 « -Q.,0€52x ³ ♦ §,053x ^¾ - Q8Mx + 6012,7; * 45.96321 Equation for full length p tem wfth OCT:

y = 50 ^« ~&0002x ^s ♦ 0.0S2x - S.1349x ^s + 25G,52x ^s - 604L9x + 67653; x ^» 59,6128

Equation for full length protein with HEX:

y « <L0074x ³ - 0.6526x ³ * SI ,967x - 38SJ4; x * 59.2835

Equation forful lengt pro ein with HUB:

y ~ ÷©.08529χ ⁵ * &0S3X ² - 408.09x * 6012,7; x ~ S8,4S47S8

Ribbon representations of syper osed structures of PPA a legend binding pocket along with its Iigands OCT (FIG, 8G), HEX (FIG. SH) and HMS (FIG, 81) are shown, Resylts are confirmed by Ihree independent experiments..

(00281 FIGS, 9A-9L Interaction bet een iigands and PPAR at th© molecular level

(0027] Atom-specific representations of superimposed structures of Y464D-

PPARa iigartd binding pocket along with OCT (F G, ®A), HEX (FIG. 98) and HMB

(FIG. 9C). Amino adds positioned at a distance of 4A ^* around the Iigands were also shown In yellow color. FIG, 9P) Detailed! maps of FL-Ppa , Y3l4D~Pp&ra _>

Y464EHPpa , and Y314B/Y464Q~Ppara are shown. Thermal shift assays of

{FIG- 9E) FL~PPARo and (FIG, SF) Y314D/Y4-64P~PPARq proteins _* Tm

represents the rnellng temperature, (FIG. 9G) Thermal shift assay for ¥4840-

PPARa alone and together with three iigands, GC-MS analyses in GFP-afflnity puriied extracts of P am^ull hip ocarnpal neurons transduced with lentivirions containing GFP (FIG, SH), GFP«FL*Ppam. (FIG, 91), GFP~Y314D»Pp& (FIG, 9 J),

GFP-Y4$4B~Ppa (FIG. 9K), and GfP~¥$14D/Y4$4D~Ppam (FIG, 91).

immj FIGS. 10A-10U Htppocampai Iigands of PPA a induce PPRE-drwen tuciferase activity in primary mouse astrocytes and ^' neurons,

ffj§2i] Astrocytes plated ¹ at 60-70% confluence- were tr nsfected with tk ^«

PPREx3«Ltic, a PPRE-d spend ent luciferase reporter construct After 24 h of transfectJon, cells were treated with different concentrations of HEX (FIG, IDA),

OCT (FIG, 10B) and HUB (FIG, 0G) for 4 h followed by monitoring l clferase activity. Under similar experimental condition, T was performed to understand

? the effect of these Hgartis on eel viability (FIG. 10D, HEX; FIG.. 10E, OCT; FIG. 10F, H B). Results are mean ± SO of three inde endent e eriments. ^: o< 0.001 vs. GOfitmL Ppam-mM astrocytes were transduced with tentivlrions containing empty vector (FIG. 10G), FL-Ppam (FIG... 10H). Y314D*Ppa (FIG. 101), ¥^6 0- Fper¾ (FIG,. 1QJ), and Y3 f 4D Y4B4D~Pp& (FIG, 10 ) for 48 h fbilowe fey transfeetion with tk~PPR£x3~Luc, After 24 h of iransfection, _: ceils were treated with different doses of HEX, OCT nd H 8 for 4 h followed by monitoring iuclferase activity, PPRE !y esterase activity was assayed in Ppar i ll astrocytes transduced with feoiiwraos containing empty vector (FiG. 10L) _t FL-Pp® (FIG, 10M), ¥314D-Ppam (FiG. ION), Y4§4D-Ppam (FIG, 1CK¾ and Y314D/Y464D- Ppara {FIG, 10P) after treatment with different doses of WV14643, feriofibrate, and elGflbrate, PPRE iodferaae activity was assayed in primary ippocampa! neurons transduced with lenivlrions containing empty vector {FIG, 10Q), t* Ppa {FIB, 10R) _f Y314D-Ppam (FIG, 108), Y4mO~Ppam (FIG. 10T), and Y314Q/Y464D*Ppa (FIG.. 1 U) after treatment with different doses of HEX, OCT and HMB. Results are mean ± SD of three independent experiments. *p< 0 Θ1 vs.€ontr L

{00303 FIGS _^ * A-1 The role of endogenous ligertds of PPARe on the morphological piastioity of ^' hippocempa! neurons.

iSI] ar^nuil hlppocaropaf neurons were- tr nsduced with lenfivirlons containing GPP (vector),. FL-Ppa _t and Y464D~Pp&m for 48 h followed by treatment with vehicle (D SO) (FIG. 11A). OCT (FiG, 1 18), HEX (FiG, TIC),

HMB (FIG, 11 D), and WY14643 (FiG, 11E) for 24 h. Then neurons were stained fo phalloidin to measure spine density. FJ A representative picture of dendrite with spines (Cyan color) used for counting area of spine heads. Area of spine beads (FIG, 11 ) and number of spines (FIG. 11 H) In 10 μη of dendrites,.

Resyils are mean, SEM of § neurons per group, p<Q _t Q5 vs ν&≠ύτ ony; ^M p 0. _: G5 vs FL«Ppam. AMPMmen calcium influx was measured i OCT (red),. HEX

(green) and HMB (pufpieHrested Ppa -mii nippocampal neurons transduced with !entivirions containing FL«Fpa (FiG. Hi), Y314D»Ppar® ^' (FIG. 11J) _;t Y464D*

Ppam (FIG, 1 1 ) _s and Y$14D/Y464D-Ppa {HQ, 11L). Ail neurons were treated with 50 ^' pM of MD A receptor antagonist N20C to inhibit passive calcium flow- through N pA receptor. (FIG. 11M-P) Similarly NMDA-driven calcium influx was measured in t e fer*t viys ^« irif®ct©d F a/^-nu!i ppocampaf neurons in the resence of different endogenous ligands _* In fhes® cases,. Naspm-HCl was treated to stop the passive flow of catdum corrupts through AMPA receptor. Results are mean of three inde nd nt experiments,.

FIGS. 12A-12E. The syhoe!ular iocafeatten of PPAR β and y isotypes in mouse br i hippocampus.

{00333 ( iO> I2A) The Intracellular distribute of PPARo, β and y were shown by irjirnurjofluomscenee (NeuR green; PPARs, red) analyses of the CA1 regions of hippocampus. (FIG, 128) Nuctear-enriched (HE) and cytoplasmic -enriched (CE) fractions of h pocarnpai tissues were immuno lottod for PPARo, p\ and y, Histone 3 (M3) and GAPDH were snekided for monitoring purity of nuclear extract and cytoplasmic extract respectkieiy, i unobiot an lyses were performed in 6-8 e k® old male WT and Ppw»*w& n ce |n~3 @r group). Bands were scanned and protein/HS va ues are presented as relative to CE (FIG, 12C, PPAJ o; RG> 12P, PPARP; FIG. 1.2E _S PPARy . Results are mean ± SEM of three mica per group.. VO-0001 vs CE ^,

{003 | FIGS. 13A-1 SC. TR-F ET and extracSon of EC50 values. Curve-fit far OCT (F G. 13A), HEX (FIG, 138) and MB (FIG. 13C).

{0O35| FIGS- 1 -4A-14C. Transduction of Ppa -miii astroc tes with !enivfrions containing different Ppara constructs.

{00363 FIG, 14A) Ppa -nuR astrocytes cultured on coversiips wore transduced wth fentivinons containing FL»Ppara, V3140«Ppara _t Y46 D« para ₍ and

Y 14D Y4S D-Ppara. Forty-eight after tramducte, ieve! of GFP was monitored in an Olympus 1X81 fluorescence microscope.. PAPi was used to vlsualfie nucleus, FIG, 148) Similarly. 48 h after transduction:, the ievel of PPARo PPARo (S3¾Da) + GFP (27 kDa 3 was monitored by Western blot. FIG, 14C) Bands were scanned and values (PPARo/Acttn) p s ted as relative to control. Results are mean * SD of three inde ende t experiments, ⁸ p Ci0O01 vs control,

{0037J Pies. 1 SA*1-SF< Peak integration statistics of (BOMS.. S3i| S _s ^Sis jfi-difne byttoeft^ Henol was used as interna! standard

(arrowhead! fn GO- S analyses {FIG. 1§A„ vector only; FIG. 15 S, Ft Ppara; FIG. 15 C, Y31 D Ppara; FIG, 15 0, Y S4D Ppara; FIG; 15 E _t Y3140/Y464D Ppara). FIG, 15 F) Chemical structure, moleeuir weight and CAS- number of 2 _» 4-Bls £a _s a dtaethylbenytJph-enoL

FIGS. 16A-16H. OCT, HEX and HMS induce PPRS-driven luciferase activity in Pparb-mM astrocytes fn the presence- of PFARy antagonist

tO. WA) Ρμ&:ώ~ηίήΙ primary astrocytes- plated ¾t 80-70% conflyenoa in 12*well plates were transfected with 0,25 g of tkPP 8*34-.uc (a PPRE-dependent ludferas© reporter construct). Twenty-four hours after trarisfeciion, ceils were treated with different concentrations of GW9f¾2 for 30 mfn followed by stimulation with rosiglilazone. After 4 h, iyciferasa activities- we e assa ed Data are mean ± 80 of three different ©xpenYnents. *p < 0,001 versos control; ¾ 0.05 & ^c p < 0,01 versus rosigiiazone, After t ansiti n, cells were afeo treated with GW9662 followed by- stimulation with OCT (FIG, 168), HEX (FIG _* I SC) and H 8 (FIG, 18D).. After 4 % iuciferase activities were assayed, ^s < 0.00 versus control; ns, not significant (FIG, 16E, SEQ ID HO: 9) Promoter map of CREB shows the presence of a consensus PPR.E, ChiP analyses (FIG, 1§F) followed by reaMiro© (FIG, 16C3-H) validation of CREB promoter after puling down with PARcs and PG-CICL Dat are mean ± SO of three different experiments. ^& p < 0,001 versus control.

(0 413 P GS. 17A»1?L Effect, of HEX, OCT and HMB o the expression of synaptic molecules in ^ a-nu!f h ppocampai neurons and neurons transduced with di ferent Ppara co struct,

{0042J {FIG, 1 ?A) Immunafclot analyses foMo ed by em!omeiric analyses of MRM (FIG, 178), G uRI (FIG,. 17C) nd Gf¾B6 (FIG. 1 P) were performad in

Ppam~m and WT hippoearnpaf nearons treated with 5 μ HEX, 5 μΜ OCT and 50 yM HUB. Data are mean ± SO of three different experiments. ^¾ p < 0,05 versus WT- control Immunoc^ocrte kaal anal ses of Nf¾2A (FIG, 1 ?E) and GluR1 (FIG, 17F) in WT and Ppsm-nuii hippocarfipai neurons treated with HEX, OCT and HMB.

Hippocampai neurons were transduced with ientMSFF for 48 h followed by treatment with different Itgands, Inimunobtot analyses followed y relative dansltomethc analyses of CREB in F^ar -pull fitppoeanipai oeuroris trans uced with lentivirions cont ining different P ara construct followed by†re-atnrrent with HEX (F G. 176-H OCT (FIG, !7kJ) and HUB (F G, i? -L), Sands were scanned and pr ssed as relative to control (FIG. 17H, HEX; IG. 17J, OCT; FIG, 17L, H 8 , Data are mean ± SD of three different ex eriments, ^a < 0.05 ¾¾ FLPpara control,

P043J FIG, 18, nalysis of the interaction of GW7647 with PPARo by T -FRIT, TR*FR.ET ana sis of GVY7647 was plotted as fluorescence vs, logarithmic sc e of Mgand concentration,

DETAILED DESCRIPTION

{00 3 he em odiments disclosed below are net in e ded to be exhaustive or to limit the sco e of fee disclosure to th precise form in the followin description. Rather, the em odiments are chosen and described as examples so that others skilled ¹ m the art may utilize Its teachings,

0 §| Methods of modulating peroxis me proisfera or-acflvated receptor n (PPARa) activity m a cell n a subject are provided,. An effective amount of a FPARe ilgand may be administered to the subject.. The PARQ ligand is selected from the group consisting of 3~hydro y-2 _t 2~dimethyi butyrat© (HfylB , hexadecananamkj© (HEX) and 9-oetadecenamld® (OCT),

¾ δ| The PPA © activity may he modulated lo different cells Including

hippocampal neurons and othe brain ceils.

£0 473 he PPA 0 iigands may be administered to treat dementia,

neurodegenerative disorders, lysosomal storage disorders or obesity,

[00483 *Treating ^Si , *treaf, o Ireatmenf within the context of the instant invention, means an alleviation of symptoms associated with a disorder or disease, or hail of further progression or worsening of thos symptoms, or prevention or prophylaxis of the disease or disorder. For example, within the context of this invention, successful treatment may include an alleviation of symptoms related to dementia,

neurodegenerative disorders, lysosomal storage disorders, and body weight disorders.. The treatment may Include administering an effective amount of a

PPARcr !gand to the subject that results In an alleviate* of symptoms associated with a disorder or disease, or halt of further progression or worsening of those symptoms:, or prevention or prophylaxis of the disease or disorder, |0S4S| 8y way of nw-flmltf g example, oeyrodegenerafve disorders ma be selected from neuronal ceroid lipofuscinosis, A zheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis (ALSJ, Parkinson diseas , incfctdlng

Parkinson's plus diseases such as multiple ystem atrophy ( sA), progressive supranuclear palsy (PSF), corticobasal egene t o (CBD) and dementia w h Lewy bodies (018).

[00SQ] By way of non-limiting example, ly osomal sto age disorders may be selected from Tay-Sa h's disease, Fabry disease, Nismann-Pick disease, Gaucher disease, Hunter Syndrome, Alp a-mannosidGsis, AspartylgiycosaminuHa.,

Cfiolesteryi ester storage disease, Chronic Hexosaminidase A Peflclency,

Cyst nosis, Danon disease. Farter disease, Fycosidosis, and GaiaciosiafldQsis. JfiiSI] The term ^{^} ubject ©r "patient as used herein, refers to a mammal, preferably a human,

P$S2J n some em odiments, practice of the present invention will em loy, unless otherwise indicated, conventional techniques of molecular biology,

immunology _* microbiology, cell biology and recombinant DN , which are within the skit of the art. See g„ Samfcrook, Fritseh and aniatte, ^' MOLECULAR CLONING; A LABORATORY MANUAL, (Cmr t Edition): CURRENT PROTOCOLS IN

MOLECULAR BIOLOGY {F, M. Ausubei ©tat eels.., {Current Edition)); the series METHODS'm ENZYMOLOGY (Academic Press, Inc.); PGR 2; A PRACTICAL APPROACH ICurre t Edition) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (Ft I. Freshney, ed. (198?)), D A Cloning: A Practical Approach, voL I HI! (D. Olcver, ed»); Oligonucleotide Synthesis (N. Gait, ed,, Current Edition); Nucleic Acid Hybrfdfeaien (S„ Hemes & S, Bigglins, ads,. Current Edition); Transcription and Translation (8. Hamas Higglns, ads. _* Current Edition);

Fundamental Virology, 2nd Edition, vol I & II B, N, Fields and 0, M. Knt e, eds, pt353| Pharmaceiiticai co ositi ns.

|0054J he ilgands described herein may be used stone or in compositions together with a pharmaceutically acceptabl carrier or ©xc pient Pharmaceutical compositions of t e present invention comprise a ftefsp¾uticaiiy effective amount of a llgand of PPARo, including 3- ydfoxy-2,2--dim thy| butyfate, hexadscananam ® or

Shootadecenamide, together with one or more pharmaceutically acceptable carders. As «sed erein, the ierrt "pharmiK^uticall acce table carrier* mean a nonto ic, inert, solid, semisolid or liquid filler, diluen encapsulating material or formulation auxiliary of any type. Some exam les of materials which can serve as

pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches «*ch as com starch and potato starch; cellulose and Its derivatives such as sodium carboxymetbyi cellulose, ethyl cellulose, and cellulose acetate;

powdered tragaeaolrr; malt; gelatin; talc; axdpleots such as cocoa butter and suppository waxes: oils such as peanut oil, cottonseed o% saf!to ar oil; sesame oil; olive oil; com oil and soybe n oil; gl cols such a propylene glycol; esters such as ethyl oieate and ethyl Saurate; agar; buffering agents such as mag esium hydroxide and aluminum hydroxide; alglnio aeld; pyrt¾e«-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compaltbte lubricants s tih as sodium lauryl sulfate a d magnesium stearate, as well as coloring agents,, releasing agents, coating agents, sweete ing, flavoring and perfuming agents, preservatives and antioxidants can also be present in the

composition, according to the Judgment of the fcrrnuiaiQr, Other suitable

pharmaceutically acceptable exclpients are described in " emington's

Pharmaceutical Sciences * Mads Pub, Co., ew Jersey, 901, incorporated ^' herein by reference,

DSS] The ilgands described herein may be administered to humans and animals In dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, .adjuvants _* and vehicles as desired,

£0 S$»J Methods of formulation are well known in the art end a e disclosed, for example, in Remington: Ttw $ci c& etfidPrnati of Pfrmn y, U k Publishing Company., Easton, Pa„ 19th Edition {1995), Pharmaceutical compositions for use in the present invention cm be in the form of stents, non-pyr gerslc liquid solutions or suspensions, coated capsules or lipid particles, lyophilzed powder®, or other forms known in the art,

OST] Compositions of the Invention may be formulated for delivery as a liq id aerosol or inhalabte dry powder. Liquid aerosol formulations may be nebulized predominantl i to panicle size that can be delivered to the terminal and respiratory bronchioles, iSSSJ Li s dosage forms for oral adminis ration include phar aceyiealiy acceptable emulsions _* microemutsiorss, ^■ solutions, suspensions, yru s and elixirs. In addition to the cti e llgands, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubizlng agents and emy tellers such as ethyl alcohol, isopropyf alcohol, ©thy! carbonate,. EtOAc, feensyl alcohol, benz l benioate, propylene glycol, 1:,3~&ufylene glycol di ethylformamide, ©lis (in particular, cottonseed, groundnut, corn, germ, olive, castor, sod sesame oils), gl ce ol, t tT¾hydr >foffuFyi alcohol,, polyethylene glycols nd fatty add esters of soroltan, and mixtures thereof, Bess s Inert diluents, the oral compositions can also i clude adjuvants such as ^ ti g agents, emulsifying and suspending agents, sweetening, flavoring, and perfumi ig agents.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and gr nu es.; in such solid dosage forms, the active ii nd is mixed i h at least one inert, pharmaceutically acceptable exc eni or carrier sych as sodium citrate or dlcaldom phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, rnannifol, and silicic acid, o) binders such es, fo example, cartoQXymethylcel ulose, alginates, gelatin, poiyvlnyipyrmldlnooe, sucrose, and acacia, c) humactants such as glycerol, d disintegrating agents such: as agar-agar, calcium carbonate, potato or tapioca starch, alglnic acid, certain silicates, and sodium: carbonate, e) solution retarding agents such as paraffin, I) absorption accelerators such as quaternary ammonium compounds, g) meti g agents such as, for example, acetyl alcohol and glycerol monostearate, h) absorbents such as kaolin nd bentonlts clay _* and I) lubricants such as taio, calcium stearafo, magnesium siearete, solid polyethylene glycols, dium: lauryl sulfate, and mixtures thereof, In the case of capsules, tablets and pills, the dosage form may als comprise buffering agents,

[W§8rj Solid composition of a similar type may also be employed as filters in soft and hardened gelatin capsules using such exdplertts as lactose or milk sugar as well as high molecular weight polyethylene glycols and the Ike.

[01361] The sold dosage forms of tablets, dragees, capsules, pills, and granules can he prepared with coatings and shells suc as enteric coa ings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also fee of a (iom osWon that they release the active ingredients) onl , or preferentially _* in a certain part of t e intestinal tract, optionally, In a de yed m n er. x mples of embedding compositions that can be used include pofyroerie substances and waxes,

[3062] The active Igands can also be in micfo-encaps ^y ialed form with o e or more exclpients as noted above. The solid dosage forms, of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, releas controlling coalings anil other coatings -well known In the pharmaceutical formulating art. in such so id dosage forms t e active ligand may be admixed with at least one Inert diluent such as sucrose,, lactase- or starch.. Such dosage forms may aim comprise,, as is normal p actice additional substances other than inert diluents, e.g., tabtetlng lubricants end othe iabietkig aids such a magnesium staarate and mlcrocrystaine cellulose _* in the case of capsules, tablets and p s, the dosage forms- may also comprise buffering agents. They m

optionally contain opacifying agents and can also be of a composition that th-ey release the active ingredients) only, or preferentially, In a certain part of the intestinal tract, optionally, in a delayed manner.. Examples of embedding compositions thai can be used include polymeric substances and warns,

{(H363J Dosage forms for topics! or transdermal ad inistration of a ligand of this invention include ointments, pastes, creams, lotions, gels, powders,, solutions, sprays. Inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and arty needed preservatives or buffers as may be required. Ophthalmic formulations, ear drops, and ^' t e like are also contemplated as being within the scope of this- invention.

P§64] The ointments, pastes, creams and gels may contain. In addition to an active ligand of this invention, e dplents such as animal and vegetable fats, oils, waxes, paraffins, starch, iragacanth, cellulose derivatives., polyethylene glycols,, silicones,, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof ,

$05iJ Ugands- of the invention may also be formulated for use as topical powders and sprays that can contain. addition to the ilgands of this Invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and p lyamlde powder, or mixtures of these substances. Sprays can addition l y

{OiiiJ Transdermal patches have Its© added advantage of provid ng controlled delivery of a l!garid to the bod . Such dosage terms can foe mad© y dissolving or dispensing the l!garsd In t e proper medium. Absorption enhancers can also be used to i ncrease ite flux of the ligand across the skin. The rale can be controlled by either providing a rate controlling membrane or by dispersing the iigand in a polymer matrix or gel The ligands of the present inve ion can also be administe ed in the form of liposomes. s is k own In the art, li osomes are generally derive from phospholipids or other lipid substances. Liposomes are formed fay memo- or muffHarneiiar hydrated li uid crystals that are dispersed in an aqueou medium. An non-toxic, physiologically acceptable and metabolzabie lipid capable of forming liposomes can be used. The present compositions In li osome form can contain. In addition to a Iigand of the present invention, stabilizers, preservatives, exc leofs, and the Ike, The preferred lipids are he phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic, Methods to form liposomes am known in the art.. See, for example, Frascotf (ed,) _s "Methods In Cell Biology ^* Vol me XIV,

Academic Press, Me York, 1976, p. 33 i eg.

$6?J Aerosolized formulations of the invention may be delivered using m aerosol forming device, such as a Jet, vibrating porous plate or ultrasonic nefcuter, preferably selected to allow the formation of an aerosol particles having with a mass medium average diameter predominantly between 1 to 5 pm, Further, the formyiatan preferably has balanced osmolality ionic strength and chloride ^'

concentration, and the smallest aerosofeaole volume able to deliver effective dos of the iigands of the invention to the site of the Infection, Additionally, the aer solized formulation preferably does not impair negatively tie functionality of the airways and does not cause undesirable side effects,

{0S6SJ Aerosoilzation devices suitable for administration of aerosol formulations of the invention Include, for example, fat, vibrating porous plate, ultrasonic nebulizers and energized dry powder inhalers, that are able to nebulize the formulation of the Inventio into aerosol particle size predominantly in the size range from 1-5 pro. Predominantly in this application means that at least 70% but preferably more tha 90% of all generated aerosol particles are within 1-5 prn range, A fef nebulizer works by air pressure to break a liquid solution Into aeros l d oplets. Vibrating porous plate nebulizers work: by using a sortie vacuum produced by a rapidly vibrating porous plate to extrude a solvent dropfet through a porous p!ate. An ultrasonic nebular wo:r¾s by a piezoelectric crystal that .shears a. l uid Into small aerosol droplets, A ariety of suitable devices are available, including., tor example, AERONEB and AEROOGSE vibrating porous plat® nebulizers (AeroGen. I c., Sunnyvale, California), SIDESTREAfcf nebulizers (Mtedie-Aid Ltd,, West Sussex, England), PARI LC and PARI LC STAR jet nebulizers (Pari Respiratory Equipment,. Inc;, Rich mood , Virginia), and AESOSONIC e¥ilbiss ediziftisc e Produkte (Deu schland GmbH, Heldeo ₍ Germany) and ULT AA1RE (Omr n Healthcare, Inc., Vernon Hills, Illinois) ultrasonic nebulizers.

¾6i] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions m y be formulated according to the known art using suitable dispersing or watting ag nts and suspending agents. The sterile Injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic paf&nterally accepta le diluent or solvent, for example, as a solution i 1,3-propanedlol or Among the acceptable vehicles and solvents that may be employed are water, Rlnge s. solution, U,3,P, and isotonic sodium chloride solution. In addition., sterile, fixed oils are conventionally em loyed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed

Including synthetic mono- or d!g!ycerfcles. In addition, fatty acids such as oleic -acid find use in the preparation of injectables. The injectable formulations can be sterilised, for example, by filtration through a bacterial-retaining flier, or by

incorporating sterilizing agents in. the form of sterile solid compositions which can. be dissolved or dispersed in steril water or otter sterile injectable medium prior to use _*

[6370] in order to prolong the ©feci of a drug, It i often desirable to stow the absorption of the drug from subcutaneous or intramuscu ar injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material wi poor water solubility. The rat© of absorption of the drug then depends upon its rate of dissolute w ich, In turn, may depend upon crystal size and crystalline form. Afernativefy, delayed absorption of a pgrentersliy admi istered dryg form may he ccom lshed toy disso ving or suspending the drug in an oil vehicle, Injectable depot forms are made by: forming mteraencapsule matrices of t e drug in

biodegradable poly me rs such as Ol !aetlde-poiyglyCJo!ide. Depending upon the ratio of drug to polymer and the nature of the part cular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers Include oly^orthoesters and poiy{arthydrides>. Depot Injectabfe formulations may also be re a ed by entra in the drug In liposomes or mlcroemolsions, which are

compatible with body tissues.

[0071 J A Igand described herein can be administered alone or In combination wit otter llgartds, for a possible combination therapy being staggered or given in epen ently of one another. Long-farm therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, m described above. Other possible treatments are therapy to maintain the patient's status after the initial treatment, or even preventive therapy, for example in patents at rsk.

pO?2J Effective amounts of the iigands of the invention generally include an amount sufficient to detectahiy an In ibition or alleviation of symptoms. The amount of active ingredient that: may e combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and th particular mode of administration, it will be understood _* however, that the specific dose level for any particular patient w ll depend upon a variety of factors Including the activity of the specific ligand employed, the age, body weight, general eal h, sex, diet, time of administration, routs of administration, rate of excretion, drug combination, and th seve ty of the particular dise se un going therapy. The thera eutically effectiv amount for a gi en situation can be readily determined by routine experimentation a d is within the skill and Judgment of the ordinary clinician.

{βδ?3| If the !gan is administered in combination, with another compound, the term ^* a mount that is effective to modulate PPA o activity" is understood to mean that, amount of a ligand In combination wit the additional compound to achieve the desired effect:. In other words, a suitable combination thera y according to ^' the current invention encompasses an amount of the Igand and an amount, of the additional compound, either of which when given alon at that particular dose would not constitute an effective am tf, but administered^ so combination would e m

"amount that Is effective to modulate FPARa activity ^* ,

{00743 ft l be understood, however, that the total dally usage of the Itgands nd compositions of the resent invention will be decided by the attending physician within the scope of sound m dical Judgment, The specific t era eutically effective dose level for any particular patient will de end upon a variet of factors including the disorder being treated and the -severity of the disorder; the activity of the specifi ilgand employed; the specific composition em loyed; the age, body weight, general health, sex an diet of the patient; the time of administration, route of ^' administrati , and rate of excretion of the specific tigand e mployed; the d uration of it*e treatment; drugs used in combination or coincidental with the specific Ilgand employed; and like factors well kno n In th medical arts,

{ Q75| The dose of a Igaod to o administered to warm-blooded a imals _* fo example humans- of approximately 70 kg tody eig t, is preferably from

approximately 3 mg to approximately 5 g. _f more preferably from proximately 10 mg to approximately 1.5 g, most preferably from about 100 rng to about 1000 mg per person per day, divided preferably into 1 to 3 single doses w lch ay, fo exam le, be of the same size. Usually, children receive half of the adult dose,

P§?g] Results

{00773 Role of PRARs in the ex ression of piastlcity-related gene In

hippocampus

{0078| PARa Is strongly e p essed ¹ in hlppocampal. neurons* ** , Since

bippocampai neurons are equipped wi h: a wide-spectrum of synaptic proteins related to Long term potentiation {L ^' TP}- and long term depression (LTP ibe role of PPARa In regulating the expression of different L?P~ and LTD-assoe ated synaptic molecules was examined, LTP causes a persistent Increase in synaptic strength between pre- and postsynaptic neurons, whereas LTD causes a persistent

reduction of synaptic strength:. An mR A-based microarray followed by heat map analyses dearly indicated that hippocampus of Ppam-mttt (KG) mica, but not wild type (WT) mice, downreg laiori of 28 ge es* ypreguiatlon of 34 genes and no alteration in 22 genes, (Data not shown,) !test of the downregwl&ted mRNAs are involved in LTP, including the ionotropic & P receptors Gria 1 and Gri S mRNAs; Sonotro c HMD receptors Gr 1< Gm2® and Gf#?2¾»m As Immediate early genes !EGs) mRNAs Including Am, Hmmrl and as; and different synaptic membrane e coded ^; mRMAs AdsmfO, Dig4, S npo, and Adcyl. On the contrary, most of the unregulated m NAs are associated wit h LTD including different protein phosphatase mRNAs such as.; Ngfr, Pickl, No8l _t -m$ Nfkbl, The dswnmgulat n of some cruda! tTP-assoeSated m ^' MAs in O hippocampus including Am, Grssl, Grf« a, Gr#i2& _;! a d Cmh was se arately confirmed by r al~ime PGR analyses,

S munohistoche ical analyses of PSD*95(enceded by e Dlg4 gene) in the presynaptic fibers of CA1 hippocampus and immunobiot assay of N 2A (encoded by Grin2 GISJ I (encoded by G 1 PSD-95, Arc, and CRE further indicated t at hippocampus of KO brain e re sed; less LTP~associated molecuies than the hippocampus of WT mice.

|§ §] Identification of novel nuclear ligands of PPARp m the hippocam us PPARs am nuclear receptors that require the binding of ligands for activation of gene ex ress n. Immunosta ng of hlppocampsl section (Fig, 12A) and immunobiot analyses of nudear-enrched fraction of hippoeampal extracts (Figs. 128--E) clearly demonstrated that PPARa, hot na ther PPARfS nor PPARy, was present I the nuclei, hese results suggest that the hippocampus as endogenous e p essio of PPARct aganoist and the such ligands shouid he present within the nucleus, i order to identify these ligands, a gas Chromatography mass specrtromeiric (GC&IS) technology w s adopted. Briefly, nuclear extracts were prepared form mouse, hippocampus _f incubated with a QST agged P ARo iigand b nding domain (LBD),. purified with affinity chromatography, reconstituted with chloroform or acefcwftrle, and SC S analyses preformed, {Fig. 1A ₈ Figs, 7A-C). Analysis chloroform extracts displayed two distinct peaks matching 9-octadecenamfd (OCT) with an mfe at .23.03 minute (Fig, ?A) and he adeeanarrilde (HEX) with an o¾¾ at 21 M minute {Fig JB). On the other hand, GC-MS analyses of the aceton&riie fraction of affinity purified hippocampai nuclea extract resulted in a distinct peak of m& 160.0 at 14,48 minutes that mated the NtST fibrary for 3-hydrox (2, 2)-dimeth t butyric add ethyl ester (H S) (Fig. 7C interestingly, SC S analyses of hippocampai nuclear extracts after pulling down with PPA p-iBD d d: not exhibit arty peak(Fig> 7D-E) _S suggesting that these three hippocampai isgands could he specific for PPARs, The fraction ©f hfopocampal nuclear extracts ©luted through Ilia glutathione column was further immune-blotted to validate the accuracy of ®m fSn¾ puriftestet procedures, which dearly showed thai a! parameters including the amount of hippocampej tissue, amount of recombinant protein, and the olume of eiuate were kept constant in ail cases t rough ut ®m assay (Fig, 11,. Fig, ?F)÷ However, t e afcove«menfi©ned assay was unable to demonstrate If these figands could display similar interacto with de «QV0-synthes¾ed PPARo, Therefore., n-ext cyltyred Ppara-snu! h ppocampai neurons were infected with lentsvlrai particles of fuiMength PPARo and then immunopredpiiaSon performed followed by GC S analyses {Fig. 1J _S Fig. 7G»H), Similar to the previous observations, both OCT (Fig, I ) and HEX (Fig, 1L) war© found to be bound to de «ο ο-synt esfeecf PPARo m ieot LPa^-tmn dyeed (fig. 7H) _f but not with empty tenti-^otor-transduced Ppars- ull neurons (Fig. 7Gj„ The efficiency of gene transduction was measured by irnmunobiot analyses of cell extract with PPARo antibody (Fig. 71, In addition _s †hese analyses succassfyliy i entified a group of biological iigands of PPARa, which ere endogenously produced In the hippocampus, Some of these detected compounds are suifur^ontsihing unknow compounds such as thtoles ihW 220-240), thiosemicarbe ooes 0JW 190-200) are! Wazolldln esters M 250-270) (Table 2), However, these compounds were excluded from this study because of their unk o n blosynthetle pathway, relatively poor match-factor {<§§)« and commercial unavailability, Trarig-O-di!hiaoe-4, 5 dsol is the oxidize product of DTI used in the buffer whereas O-Gaiaotono 1 _F 4-laefone ¾ 6-ocjylidene is excluded because of the commercial unavailability of this compound required to confirm its association with PPARCL Taken togethe _* our GC-MS analyses identified OCT, HEX and U m three putative, eudogenously produced _* but also commercially available, PPARo Igands,

{QlSSrj Next, Time Resolved-Fluorescence Resonance Energy Transfer f?R~ FRET) analysis was performed to confirm the interaction oeiween these lig&nds and PPARo- The optimized TR-FRET analysis (f¾ 2A, Fig, 8A-C)) Indicated that P6C- let -PPARo LBD complex displayed a strong Interaction with ail these three ligands (Fig, 2B-D, Fig. BA-G). Fig, 13 shows how these d tes wer fit end SCS0 values extracted for OCT (A), HEX {SJ and H B (C). fn all cases, TR-FRET signals (Fig. 2D, Figs, 8A»C» released by PPARQ LSD showed a steady Increase, Although the • 04 ί slgm Intensity was observed higher In HMB compared to OCT (Fig. 2B, Fig. 8A and HEX (Pig. 2C _t F¾, SB), both OCT and HEX gene ated FRET signals at much lower∞ncentrations than that of HMB,. On the other hand, a large thermal shift was obser ed a evidenced fay a change In mgitlpg temperature of py tiled PFARa-LSD protein ¾hen ncubated with t ese llgands (F lg. 2B G,„ F g, 8D-F) suggesting that these iigan s truly interact wtth the ligaod inding domain of PPARo wih high efficiency.

|0SS1| oiaeylar c racte iz tio of the intaraclon betwee th e llgand binding domain of P ARo and lis novel llgands.

;[0 S2] The nexi aim was to characterize the moecu ar interaction of these llgands. with t e PPARG-L6D, The in si∞ computer-aided chemlnformatlG analyses gener ted a reasonable docked pose of these lig&nds in th ΡΡΛΚό -LSD (Fig. 3A« C, Rg.8G-i}, The docked pose of ail th ee llgands stowed two potential hydrogen bonds betwee the llgand and two active-site residues, Tyr 31 (Y314) and Tyr 464 (Y484) {Fig.. 3B), of the PPA a-LBD. The Igand- binding surface is a phip&thic, as it shared boti a negatively charged electrostatic surface and a law patches of a partial positively charged surface with mo tly lipophilic, and so e hydroph!fe p tches. Imposing the most stringent docking protocols, a reasonable docked poses of OCT (a total score of 10, 15, a polar score of 1.05, and a crash score of™1.49; total binding energy ~2BM k i noi), HEX (a total score of 10,01 , a polar score of 1.81 _s arid a cr sh seore of -1.04; total binding energy -28.3 kcal moJ), and HMB (a total score of 5.63, a polar score of 1,9¾ and a crash score of -1.55; total binding energy -10,5 kcal mo ) were obtained for PPA ©\< Interestingly, in the case of both PPARp and PPARy, by applying similar docking protocols, we -failed to ob ain any docked pose for these llgands, suggesting that the nteraction of all three llgands with PPARo-LSD Is specific and not possible In other PPA Isofomts. To further confirm th s observation, In li matatfcm analysis was performed, In which OCT, HEX, and HMB were placed In the ligand-blndihg pocket of Y464D~PPARa After energy minimization {total binding energy is -15.6 koai/mot for OCT* -14.3

kcal mol for HEX and -5,04 k alftnol for HUB), all th oe llgands wore obs ved to be located far (>4A*) from aspartate (O) residue to establish any hydrogen bond (Fig:, 9A-C), suggesing that the mutation of tyrosine 484 to aspaitate ^■ significantly impairs H e interaction of these ligands wfth PPARo. However, in si?co modeling ©f pratelrt-ligand in er c ion is hypothetical and requires rigorous experimental analysis for further validation.

entivsrys-f iecisatec cf no expression studies were performed, where wild type fulhiength {G P~FLPpa ) was overexpos and three different USD* mutated PPARef {GPP~Y31 0 _! GFP-Y464D and GFP~Y.31:4P Y484D Pp&m)

recombinant proteins (Fig. 9D) m ® ex ressed in neurons followed by binding analyses with three endogenous ligamfs. Briefly, site directed mutagenesis was performed in the mouse PPARa with Y314 and Y464 residues replaced separately or together w¾ aspartate D), After that,, the entire ffiouse GFP-Ppm gene (GFP-- FLPpar ) and three different mutated §en s were cloned in the pLeni V5*TOPO leniSviraS expression vector (Fig, 3D), packaged in !entivras partfcie with HEK293FT c l s, purified full length a d mutated PPARa oteins In a QFP-affin!ty column, anil finally thermal shift assays were performed in order to analyze thei conformational stability.. Bath MS le g (Fig. 3E, Fig, 9E) and mut ted {Fig. 3F _:i . Fig. 9F) proteins displayed a similar pattern of thermal shift with equivalent melting temperature Cirri! suggesting that the mu ations In Y314 and Y464 residues did not alter the

conformational stability of P AR Moreover, OCT, HEX and HUB did not alter the Tm in Y464B-PPARa _:i emomfmti g that mutation of tyrosine 464 to aspartate sig ificantl Impacted the binding of these ligands to the LBO of PPARo (Figure 9G},. In another experiment Pp®m*m hippocampai neurons were transduced with different la&tMrai P Rd constructs and transduction efficiencies were basically the same in all casts (Fig. 14A and the level of PPARo was comparable in coils transduced with different constructs (Figs,. 14B-G). After 48 h of transduction, the cells- were homogenized, passed: through GFP-affinity colymn, ¾lu:ied _f fractionated with ehloroform-met anoi, artd finally analyzed by GC-IMS for the detectors of ligands. Interestingly, we observed that lie .aflniy-punfied nttciaar extract of

GPP ^« FtP ara (Fig, 31, Fig., 91), out not fe *GFP «trahs ti«ed (Fig, 3H, Fig, §H)

F ara-nuil neurons contained these ligands. Interestingly, the mutation of Y3 4- was found to partiall impact the !kpnd binding affinity of PPARa as we detec ed low amount of both OCT and HEX m the nuclear extract of ,fe«lf»GFF*Y134 »Ppara transduced Ppara-raiit neurons (Rg< 3 J, FfO, i J), On the o er h nd , mutation of the ¥464 completely knocked dmm the Igand binding affinity as we o ser ed: profound loss of ligand banding in both fe«iM3FP*Y4S D-Ppara (Fig, 3K, ig. 9 ) and f - OP P ^« Y314D ¥464D÷Ppa.fa (Fig:, 31, R§, §L)4ransduGed Ppam-nuii neurons.

Throughout ties©, analyses, we used 2, 4 ^« fo!s (o, o-dinelEiyi oen l) phenol as an internal standard (Supplementary Figure 5A-F), We normalized peak area of different ligands with that of Internal standard and then quantised the binding affinity of t ese ligands with d if ©rent construct of FPA s by peak integration statistics (Table 1 Taken together, the detailed GC~MS analyses c!easly Indicated that boil Y314 and Y464 esidues of the PPA a-LBD were crucial for its Interaction with endogenous ligands.

$84] Next, the role of thes Sgs ds In controlling the transcriptional activity of PPARo was monitored. First, a PPRE~ddven Suoifer se activity assay was performed in cultured astrocytes treated with different concentrations of OCT (F¾, 4A _« Fig. 108), HEX (Fig, 4B _t .Fig, 10A), and HUB (Fig, 40, Fig. 10C), At! three iigandsligands increased the PP E teiferase activity In a dose dependant ma ner (Fig. 10A-C). However, PPRE-iuciferaae .gerie-transfecied astrocytes displayed significant level of cytotoxicity with higher concentrations of HEX (F g, 10D), OCT (Fig, IDE) and H 8 (Fig, 10F), justifying the decrease of PPRE-lucferase activity with higher doses of ligands (Figs _* 10A-C), Consistent with the TR-FRET assay, both OCT and HEX Incieased P ^' PRE-lucHemse activity a much ^' lower concentration as compared to HUB (Fig, 10 -C,), Similarly, these ligands were also able to induce PPRE iuciferas© activity In Ppara null astrocytes transduced with fenti-FLPPara (Fig, 4E _t Fig. 10£ ₍ but not ienti-vector (Fig, 10D,}.

OSSJ To further confirm the specificity of these ligands to PPARo, we performed PPRE-ludferase assay in PPAR0 O (Pp&rb~niM) astrocytes.. These astrocytes were pre-treafed with PPARy-sntagorast G 9662 to nullify the involvement of PPAR In reporter assay. Inhibition of roslgiifazone-mediated increase In PPRE-luclferas.e activity by 6W86i2 (Fig,. ISA) suggests that this inhibitor is capable of suppressing the function of PPARy in Pparfe-nyfi astrocytes. OCT (Fig. 10B), HEX (Fig. 16C) nd HMB (Fig. 18D) markedly increased FP .E iuciferase activity sn Pparfy-mill astrocytes. Interesting fy _< QW9S6-2: remained unable to inhibit OCT-, HEX- and HfclB-medlated increase in P RE-!udferase activity in PpartMiull astrocytes (Fig, 8B-D), indicating the - specificity of these ligands to ards PPARcs, To further confirm this finding, we performed ChiP analyses of the C EB promoter (Pig, 16E) as- described recently ⁶ and observed that all three ligands stimulated the m mnt of PPARo and its coactlvaior PGC1« to fie CRES promoter (Figs, 16F ^« H). Since Y314 and Y464 esidues of PPARa-LBD were crucial for the interaction with ip ocampa! Uganda, we examined whether these residues ware also Involved In hippoearnpat iltpn - mediated activatio of PPARo, As expected, HEX, OCT and HMB remained nab e to induce PPRE~tinv©n lueiferase activity in Pp&m~nuil astrocytes {Fig, 110), However; all three ligands markedly induced PPRE reporter activity in Pparsf-hull astrocytes that were transduced with ientlvirtons containing PL-Ppm® (Fig;, 10 ^' H). On the other hand, Y314D mutation in PPAR ^» UBD displayed partial induction of PPRE-iyelferase activity (Fig, 40, Fig. ICS J as we observed in our GG- MS analysis that the lnterac¾cfi of ell three ligands was partially compromised with Y31 D PPARo. Consistent with the GC-MS results, all three ligands were unable to stimulate PPRE-teifwase activity in Ppara-rmH astrocytes infected with tentiviruses containing either Y464 ^» P sra (Fig, 4H _f Fig., 1GJ) or ¥S14D/Y4S4DPpara {Fig, 41, Fig. 10 ) viruses, suggesting that the Y4640 mutation I suf icient to knockdown PPARo aeii:yation by its endogenous hippoc m ai iigands, ComnwdaS ilgands of PPARo ( Y1464-3, fenoflbrate and ciofihrate) were also unable to induce- PPRE* iyciferase activity in pem-nul! astrocytes (Ft§> 10L), However, hese commercial ligand® markedly induced PPRE4ydfera.se activity in f^are-ftuM astrocytes tha were transduced with fan8~FL ^» Pp8m {Figure 1Q ), On the other hand, commercial llgsnds of PPARo displayed ludferase activity when Ppam-oull astrocytes were transduced ig, ION), tenS*Y484 * Pp& (Fig. 4 _S fig. 10O), a 4N, Fig, 10P) suggesting that both Y314 and Y464 residues of PPARa are important: for the binding with commerciall available ilgands,. Simitar to astroc tes* the transduction of either }mt) ^» Y4$ 0 ^» Pp®m {Fig, 4R, Fig, 1.0T) or &n# ^» 31 W 6 D*Pp r (Fig.4S _f Fig, J% hui neither tenU~Pl«Pfmm (Fig, 4P _f Fig, 1QGMR) nor i ii Y iit^ psm (Fig, 4Q, Fig. IDS), completely abrogated he PPRE4uofera$© ae&vity in OCT-, HEX-, and HMB reated Ppars-nul ti poc mpai neurons. Ga!ieeiivety, these results suggest a mandatory ¹ role far the Y464 residue and a partial roie for Il a Y314 residue in the binding and acivatfon of PPAR by endog oiis hippocampal Hgands,

¾S6] The rate of fie endogenous !igands of FFARa in regulating trie synapt c function of hf ocampsi nsiirons

po 7] Next we Investigated whether these lppoqampai ilgands- were capable of im ro i g synaptic function of hippoeampai neurons., immynabiot (Fig.. 1.7A) followed by refaiv dens teatric analyses (Fig. 178-D) and immunofluorescence anal ses of MR2A. {Fig. 17E) and GluR (Fig. 7F) dearly demonstrated that HEX, OCT and H S upr gutetet!, NR2A _* GiuRI and CREB in T, but not Ppara-nul, Nppocampai neurons, suggesting that these ligands inomased the express on of synaptic moiaeuies v a FFARa,

08S] Dendritic spines a e the crucial mediators* of synaptic transmission among central neurons and often serve as a primary candidate for the. tong term

morphological su bstrates of neuronal plasticity ^ϊ¾?3 . Therefore, the ©fleet of these ilgandson the Increase of spine density _, in cultured hippocampal neurons w s studied. Briefly, mouse Ppam-nufl ftiopocampal neurons were transduced with lentivims containing empty vector, FL-Ppam- _> or Y464D- Ppars for a week followed by the treatment wit OCT, HEX, and H S for four more days. After that, neurons ware labeled wit phatoldsn to monitor the spin© density. Interestingly, the

transduction, of Ppsra-nui neurons wf ill fen -Y48 D-Ppara, but not tenii-FL-Ppara, significantly attenuated the density of de d ite spines (Fig _* SA _t Fig, 11A). oreover, the treatment with OCT (Fig. SB, Fig. 11 B% HEX (Fig, 5C, Fig, 1.1C), and HMS (Fig, 5D) and the synthetic agonist Y14643 (Fig- 110) simulated the density of spines only when P ara iull neurons were transduced with tefiti* .»Ppa , but not with tenf^Y46 D- pam further suggesting that: the ^■ PPA & Y48 residue is crucial for the induction of morphological plas-tldty by its endogenous Ugan a, This observation was further validated by measuring in© area of spine head Fig, 11 F-G) and number of spines (Fig, 11 H) In HEX, OCT, and HfolEMmated F^am-nuil neurons, HEX (Fig., 17G-H OCT (F g, 17M) and H B (Fig, 1 TK«L) simulated the expression of CRE8 in Ppa ^« mM bippocampal neurosis that were transduced with fent tens containing the FL-Ppara gene. On the other h nd, HEX, OCT and HM8 rema ned unable to increase the ex essi n of CREB to Ppam-nuli h pocampal neurons that were transduced with tm *Y4§4D~Pp®m and l &Y484i 314B*Pf®m{ Fig, 17G-L), Moreover, Y314P mutation only partially restored the ex ression of CREB In response to OCT, HEX, and HM8 In Ppam-mll neurons (Fig. 17G-L).

sst] Calcium oscillation throug meta&otroplc rece to s has been implicated in synaptic plasticity and recently we have demonstrated thai both AMPA and N DA ©tidied .much weaker calcium iniux nd a smaller am litude oseillaflon in P ar iull than WT h ppocampat neurons' ⁸ . Consistently, we have seen that HEX.,. OCT and HUB stimulated AMPA* and DA-w dicaied calcium influx In ten&FL* Ppst&4mndueed Ppara- ull hippoeampal neurons (Fig. 11 t-PjL However, HEX, OC and H B remained unable to Increase AMPA- |Rg. IE-H, Fig, 11i~L} and N DA- (Fig. 51-L, Fig, 1 ΪΜ-Ρ) mediated calcium Influx in Ppsra^ ti ihippoeampaf neurons that wore transduced with either i ti~Y4§4D~Ppa or ienl Y314D/Y464D-Pfiam. On the other hand, t ~Y314D~Ppam was only able to partially restore HEX-, OCT- and R S-elidted calcium Influx in ArVIPA- or M OA-treated parshroAi hippocampai neurons (Figure 6J & M These results suggest pivotal role of ¥464 residue and limited role of Y314 residue of PPARo: in O T HEX- _¥ and HMiS- stimulated calcium influx through HMO A. and AMFA-sensiliv© receptors,

t Dj Discussion

SIJ Since PPARa has been reported to e localised in the different parts of the brain ³² and might play crucial role In controlling different brain function ^6,14 , ther is a growing interest m identifying the endogenous agonist for PAR- In this tissue. Although different studies speculated anandamides or 9-Gly!ethanoiarnide could serve as central ligands of PPARa ^" "* , them is no experimental evidence that shows the molecular interactio between 9-oieoyiethano!amicie and PPA , _: however 9~ oleoyletharsol mide was shown to display PPAR ^« independent effects ^' - ⁶ , Moreover, there are many structurally ^' .similar fatly aeyf amides available In the CNS that have not been evaluated a potential endoge ous Hgan s of PPARa. The isolation and characterization of three novel Ilgands of PPAR have been delineated

(octadeeenamide (OCT), hex-adeeananiide (HEX), and 3-hydro.5ty-2 _S 2^l ethyi butyraie CHIMB}) from the hippocampus. First, GC- S analyses of PPARa LBD- p lle down fraction of hsppocarn si nu-clear extract revealed th existence of these com ound . lnt©f©s§«gSy, these three compounds were detected only In PFA os L8D-, but not PPARji LBD-puJted down fraction of hippocampal nuofear extract suggesting that these Ilgands are specific for PPARos. In addition to these three major !igands, we also detected some thionated compounds including thtazofes {mw 220-240) _S thiQsernicarbazon.es (mw 180*200), and thia¾o idlne esters {mw 2B0- 270) white performing GC-MS anal ses. S& nd, da novo est blishment of PPARa by fentivhal transduction of the Ppam gsm Ppa -null hsppocarrtpa! neurons followed by s milar GOMS analysis also resulted n the detection of these three ilgands. Third, further characterisation of these molecules by TR-PRET and thermal shift assay revealed that HEX, OCT and HMS strongiy i teract d. ^' With ttii® L.BD of PPARa, The h¾ t ropgtiput studies indicated that ell t ree Ilgands served as full ilgands of PPARa as we observed the slope of the curve derived from both FRET and thermal-shift assay shifted along the positive direction of X axis. White

measu ing their affinity, ECS0 values- of these Ilgands (ECSOoe ~ 4>31 Μζ ECSOHSX ²² 4,36 μ _» ECSOHMS - 31,8 μΜ) were observed higher than the same for GW?847(E0¾5 - 6.62 n!VI), a pharmacological agonist of PPARa (Fig, 18). These results suggest that these newly discovered ppocaropai Ilgands have less affinity coniparad to commercially available iigands.

P§9¾] The in i o naly is, slie-dlrec ed mutation οί Y314 and Y464 residues of P ARa followed ^; by fenivirai manipulation of these hjppocampai neurons revealed minimal binding of PPARa with these Ilgands as evident torn the GC-MS analyses:. The results also found that boil ¥314 end Y ^' 484 residues of

PPARs are Involved in the interaction wth these ilgands, with the PPARa Y464 residue being mors crlleaf than the Y31 residue in terms of its Interaction with the endogenous igands. This observations was further validated |j> analysis of the transcriptional activity of PPARa via PPRE iuelferase assays where Y4S4D mutation of PPARadid not restore PPRE*luciferase activity In OCT*, HEX*, and HMB-tieai d f¾5#/¾ ^« iiull hippocampa! neurons. The mutatio of tyrosine to aspartate might .gener te a conformational instability to PPARa protest. However, the therms! melti g curve of FL-PPA a and mutated PPARa did not show much difference in terms of the melt ng temperature of the protein suggesting that this mutation does not affect the conformational .stability of PR AROL Previous studies have reported the 9- oteyjeihanolemlns could serve as a ¾and for PPARa in the brain; however we could not detect 9*oieytete,ftoiamlne in hippocampus by GO-MS after pypng dow the bJppocampal extracts with- recombinant FPARe LBD _* One possibility is that PPARa LBOhes been ulled down aniy from the nuclear extracts and that - olevle hanolamlne is not present in the nucleus. The nyclear fraction of PPARa was targets for its llgand detection as PPARa is constRuiSveiy present i nuclei of hippocam al neurons

!$0S3J Recently, it has been s own that PPARa regulates the transcription of CREB and controls the expression of CREB-associateo ^* synaptic genee^ in another study, we have shown t at statin-mediated nuclear ^■ activation of PPARa is also important to regulate the expression of neumtrophins i different brain cells ³ . Our detailed molecular interaction analyses reveal that statins interact with 1331 and ¥334 residues of PPARa LSD i the presence of P SC a and controls the transcription of CRES, However, commercially available ilgands and the endogenous Ngands described in this study, do not interact with these two residues of PPARa, Instead, these molecules interact wit Y314 and Y4S4 residues of t e PPARa LBD _* Ou site-directed mutatageoesi studies followed by GOMS analyses eoiffrroed that these residues of PPARa controlled I s association with endogenou ¾and¾ however PPARa Y4S4 residue appeared to be more crucial ten Y314, Moreover, the PPRE-drlyen reporter assay indicated that the mutation of Y4S4 of PPARa completely abolished the activation of PPARa, whereas the mutation of Y31 only partially compromised the transcriptional efficiency of PPARa suggesting the importance of Y464 in the PPARa-tBD Is ie most crucial amino acid residue for its interaction with endogenous llgands.

Characterizing drugs fo improving synaptic plasticity is an Important area of research. Interestingly, these bippocarrtpaS Uganda Increased synaptic properties of hippoea npal ne rons. However, these com ounds siimyiated the expression f different synaptic molecules In T _f but not In Fpar *nuii neurons. Stimulation of dendritic spine formation and increase in HMD A- and AMPA-drlven calcium influx by hippocampal Uganda in Ppara-nutl hippocampal neurons upon establishment of FLPp& , but not Y464DPfmm _> indicates the importance of Y464 residue of PPARo in synaptic propertie of h p oca pi ligands. While Y464 res due of PPARa was folly responsible for the functioning of these i!gands, Y314 residue was also partly involved In this process. Earlier studies suggest that OCT could bo beneficial n control Sing sleep as it has boon found in the

cerebrospinal fluid during sleep deprivation ^' ^. Since OCT and two other compounds HEX and H B are constitutively present In the hippocampus as

PPARo. Uganda, it would bo interesting; to see if these compounda Increase sleep via PPARo.

|0894j Materials and Methods;

i§S| Animals; Animal maintain ng and experiments wer In accordance with National Institute of Health guidelines sod were approved by the institutional Antmai Care and Us committee of the Rush University of Medical Center, Chicago, IL Ppam~n uli and their wild-type (WT) controls were purchased from Jackson

Laboratory.:. Mice were housed n ventilated micro-Isolator cages in a

environmentally controlled vivarium (7:00 AM, iJiQQPM. light cycle: temperature maintained at 21~23* ¼ humidity 35-55%), Animals were provided standard mouse chow and water ad libitum and closely monitored for health and overall welt-being daily by veterinary staff and the investigator,

$SS] Reagents: Rabbit polyclonal anthPPA o antibod Abeam; Cat #

3b18S159; WB and IHC), mouse nti-MeoN antibody (MiHipore Cat# AB377), rabbit polyclonal anti-PPARp antibody (Abeam; Cat abS§37; Wi and IHC), pnti- PPARy antibody (Abeam; Cat #«£66343; WB and IHC), antl-N DA 2A antibody (Cell Signaling for W8 at a dilution of 1 :1000, Cat #4205: Abeam for IHC, Cat# ab169873), a«iM3iu 1 antibody (Ceil Signaling for WB at a dilution of 1:1000, Cat #13185; Abeam for IHC, Cat# ab131507), anti-CRES antibody ( Cell Signaling for WB at a dilution of 1 :1000 a d IC at a dilution of :20u\ Cat # 9104}, and antl*Arc antibody {A e m: for B a! a dilution of 1 : 1000, Cat # ab11893) were used in this stud . Different pharmacological compounds including §^tadeeen mlde ^'

<Cai#02136), hexed£t½namide (Caf S350435} _s 2 _! ~b5SCa _! ¾-Q meth i benzyl) pheno (Cat #372129), gemfibrozil (Cat #09518), cWbrale (Cst#C66 3) _i fenofihrate ic moioi, GWS662 ccai#M6iai), wr ms fCat#c?¾si) _P and rr- ased toxicity assay kit (Stock No. T0X ) were purchased from Slgma-Al neh, GST- PPARo-LiP sod ST-PPA p-LBD were purchased from Prote n: One.,. On the other hand, 3*hydroxy 2, 2-dsmethyl butyric acid e fyl ester iC&t #sc»216 §2) was purchased from Santa Cruz.

[0097] Isolation of Mouse Hippocampal Neurons; Hippocampal neurons were isolated from fetuses E18) of pregnant female Pp&m-mW and strain-matched WT iltarniat© mice as described by us ^δ · ^,3<3a -* with some mo ificatio s Briefly, dissection and Isolation procedures were parformed In n ice*co!d, sucrose buffer solution {sucrose 0.32 M„ Ids 0,025 M; pH 7.4} ^Jf , The sk n and the skull we e carefully removed from the brain by scissors followed by peeling off the meninges by a pair of f ne tweessrs. Next, a fine incision was made In the middle line around the circle of Willis a d medial temporal lobe was opened op. Hi pocampus was isolated as a thin slice of tissue located near the cortical ed e of medial temporal lob®, Hippocampal tissues isolated from all fetal pups (n >1 Q) were combined together and homogenized with 1 ml of trypsin for 5- minutes at 3? ^* C followed by neutralization of trypsin, The single ceil suspension of hippocampal tissue was plated in the po!y-D- lysine pr^coafed 75 mm flask.: Five mirt after plating, th supernatant® were carefully removed and replaced with complete neurobasai media. The next day, 10 A was added to remove glial contamination In the neuronal culture. The pur cultures of hippocampal neurons were allowed to differentiate f Lilly for 9-10 da s before treatment ^{38>¾ s} ,

[0038] Isolation of Mouse Astrocytes: Astrocytes were isolated Irom mixed glial cultures of 7 d old mouse pups according to the procedure of Guilars and Baker as described earlier ^{i si<} ,

OfiiJ Lenfivifal cloning of FLRpam and mutated Ppaia {&&bdPp m}:

J00180] SO* di med mutation: Mouse PPARo ORF ctoned in pC V&AC-GFP vector (cat # MG 227841} was purchased from Qrigene, G227§41 was mutated at Tyr3l4 with aspartate (¥3140) nd Tyr 64 with aspartate {¥4840} by site^freeted muiatkm kit CStratagene) ¹ ', Two primers lo opposite orientate, were used to amplify the mutated plasmid in a s ngle FCf? re ction. The PCR product was precipitated with sthanot and then phosphoryiated by T4 kinase.. The phosphoryleted fragment was selMlgated by T4 DMA Igase and digested w th restriction enz me Dpnl to eliminate the non-mutated template. The mutated piasmid was cloned and amplified ire Escherichia coll (PH5~a strain) competent cells.,

Ρ#101| G nerating pLentl6 J ^S-TQPG® constructs of FtPpara and AsbdPpara p£H 82| Briefly, each construct was amplified by PGR. using primer pair

(sequence) and every product had a single adenosine (A) to the 3 ^* end.. Then the TOFQ cloning reactio was erformed using Inv!trogen kit {K531S-20) with pLentl6<3 5~TOFO vector. For transformation One-Shot StolS competent cells were used. Sequencing of the clones was performed at AOGT inc;

p$183| Pr iii M. L§:n(i¥ir . m.293FT C&'is

{Oil 04] 293FT cells were cultured with 95% eonfiuency In OpI-ME media without antibiotics. Next day, ViraPo er ^:rM Packaging Mix (9 pg/reactfon) and pLenti expression piasmld ON A containing either FLPpsra or ksMPfrn (3 pgfreaeta} {12 pg total) were mixed In 1,5 ml of serum-free Ορ!Ι- ΕΙ¾ϋ> f Medium,. In another tube, 36 μΐ of Upofectamine® 2000 was added in 1 ,5 ml of serum-free Ο !Ι ^» ΜΕΜ# I edium with gentle- mix, .After § minutes of incubation at room em e ure, both the reactions were combined and Incubated for 20 n ns, .After that, the mixtu e was applied to H.E -293FT calls and imttbaied overnight at 37°C in a humidified 5% COg incubator. The ext day, the media was replaced with serum-free Opti* E media and further incubated for 48-72 hrs at 37*0 in a humidified S%€€ incubator and then supernatant containing viral particles was collected. ^' Viral partietes were concentrated wit lehti- oncenf ator soiulon and MQI was calculated,

0105J Isol tion of Nuclear Extracts and Gas Chromatography M ss Spectra {GG ^» MS) analysis of PPAf¾HI§and interaction

P010i| Sample preparation

Either El 8 cultured ^' mouse hlppocarnpal neurons -or .hi pocampal tissue of 6-8 week old male CS7/SL6 mice were homogenized in lee-ook nondetergent hypotonic buffe

£10 m HEPES (pH 7,9), IS mM gCfe, 10 mM CI, 100 mM DTT, protease and phosphatase nhi itor cocktail]., After 10 rnin of additional incubation in the hypotonic buffer, the homogersate was eentrfuged at 8,000 g ai 4*C for 10 min> Next, the pellet wa homogenized in ce-co d extraction buffer [10 M HSPES ( H 7,9), 1,5 mM MgCI _f c 0.21 M UaCl Q.,2 mi EDTA, 25% (v v) glycerol, 100 mM DTT, protease and: phosphatase inhibitor cocktail! placed on a rotating shaker at 4X for 1 h _f and then centrifyge at 18,000 g for 10 min. The supernatant (nuclear fraction) was incubated w! 1..5 pg of GS PPA a LBD {Protein One) at 4X for § h In a. .rotating shaker,. The reaction mixture was passed through glutathione column (Pierce ⁵ * GST Spin Purification K t), was ed four imes [SO mi Tris HC (pH 7A 100 m NaCt, oteas and phosphatase inhibitor eocktaiij and trse« elated with frs glutathione:. The eluate was transferred to methanol: chloroform; water (4:3:1) mixture and then eentrtf ged at. 14,000 rpm for 90 sec, The organic phase was collecte , evaporated Irs the SpeedVac fieconstaftifed with 30 pi chloroform or aeotorsitrile, and then analyzed by SO-MS, in: another case, S18 matured hippocampa! saymns were tcartsduced wit lentivirat particles conjugated with PPARa-LBD or rJiiferen GFP- tagged mutated constructs followed by pulling down with anti-PFARo! antibody or passing the extract through GFP-coiumn of Vector Fusion-Aid GPP Kit (Cat # 8- 0732), After that, the e!uate was collected from the column with S ^: NaCt solution, concentrated with FD-10 desalting column and analyzed for GC-- S,

{001071 GG-MS analyses

601081 A JIOL GCMale II {MOL USA, Paabody MA) mass spectrometer s used in these ex e iments. The gas chromatograph was an Agilent 6890 tus

(Wilmington DE) e ui ed with -a G1513A auto nJecior with 100 vial sample tray connected: to a G1 §12A controller, Tt gas chromatography column was a fused sice, capillary column with a nonpo!ar 5% henyl 8S% dimeihylpolysiloxam phase (Agilent HP~5ms), 30 meters lo g, 0,25 mm Internal diameter, 0.25 pm film

thickness. The carrier gas was ^' Helium |¾9.9&S5% Research Grade) run through a SsTG triple filter Restefc Corp,) at a constant Sow rate 11 mUmin, The injector w s held at 275 ^* 0 nd was fitted with an Agilent 4mm IO single tajper split liner containing deactivated glass wool. One pL of solution was injected at a split ratio of 20:1 ₁ The initial oven temperature was 4QX held at 2 min _s raised to 300X at a rate of 10X (Figure 2A*E) or 20T (Figure 2K & 21) er rrm then held for 17 min {Figure 2A-E) or 30 mif* (Figure 2K & 21). This exp a ns the variable retention times of the identified compou ds. otal fun time was 45 min.

{001 S9] The mass spectrometer was a benchtop magnetic sector operating at a nominal resolving power of 500 using an accele ating voltage of 2500 volte. The s ec rometer was operated in full scan El mode (*Ve) with the filament operating at 70 eV scanning from m z 10 to miz 850 using a liner magnet scan, Ttjo scan speed was 0.3 sec par scan. The sol ent delay was 4.0 min. Data analysis was performed using the TS8 Pro software {Shrader analytical & Consulting laboratories inc., Detroit MY} provided with trie spectrometer. Reconstructed ion current (RIG) chromatographic peafes us ng ions unique to each compound wore used for quantitation.. Mass calibration was performed using peril orpkerosene (PF ), {0 110| in Slice structural analyses of PPARcr compiexed with OCT,. HEX. nd HUB.

{0 111] Ugand Frepa ration

{00112| Ugamte (OCT, HEX and HMB) were subjected to UgPrep module implemented In Tripo software* ¹ , which converted the 2D to 3D structure. Then using the Ionization engi e, the llgand was prepared at H 7,0 ± 1, The appropriate stereoisomers were gone rated along with the tow energetic conformers.

p0113J Protein Preparation

f¾0114| The crystal structures for FPA.Ro (3V!3.pd% β C3G X.pdrj}, and y |3U9Q.pdb) ware imported froni the pdb databank,. The protein preparation modulo of Tripos was utilised to fix up the hydrogen bonding orientation, bo d orders, charges, missing side chain atoms, missing loop, profanation at physiological pH, and side chain bumps. Finally, staged minimization was performed fo ail three protein structures.

[0011 §J Dockin g of the Llgands

{00 1 i| The Surflex docking module ¹ implemented In Tripos was used to carry out the docking of OCT, HEX and HUB in PPA , p and y crystal structures., After the docking, three major scoring functions such as Total Score fa function of -Log e), Crash Score {penalty score reflecti g the inappropriate penetration of the licpnd Into the active site pocket) nd Pola Soore (depleting ail the favorable polar interactions) were o ained _*

[0011 ?J We also computed the binding free energy of OCT, HEX and HUB In PPARo, using olecular Mec anic Generalized Born Surface Area appre-ach ⁴² . To account for the structural deformation upon binding, we included adaptation expense that ccounts for changes In the Intramolecular energetics (&G¾. For !igarsd strain energy we specified a 5i gi of trie receptor from the centroid of the llgand to be flexible so that tie protein structure was re a ed in the computation of the binding energy of fie bgands.

{00118] To soften the potential for the non-polar part of the ligands, the van der Waafs radii of the atoms were sealed to 0.8 In a regular docking expehment. This allowed! tr e dock pose to show as a successful pose ever* if the distance etween the iigand atoms and the protein atoms are less than 1 A away from each other, V¥e increased the sealing factor to 1.2, in order to eliminate the unreasonable poses,

111] TR-F ET analysis

{0012®] TR-FRET assay was performed using Lanthascreen TR*FRET PPAR» alpha ooaciivatof ass y kit (Cat # PV4684). In t is assay, different do es of statin drugs were Incubated with GST-tagged recombinant PPARo L8D protein, Terbium f ^' b Bagse-d anti GST antibody and Fluorescein {FLHagged PGCIa: as directed in the ma ufacturer's protocol. The entir reaction was set up In coming 384 well plate by an automated robotic injector.. Each plate was centrifuged, incubated in a dark, place for 30 rrsins, and then analyzed "molecular devices analyst" machine equipped with dichrolo mirror. The excitation wavelength and emission wavelength were set at 340 nm and 540 nm wavelengt respectively,

0121] Thermal shift assays

[00122] Thermal shift assays were ^e fofmed In Applied: Blosystems 7SO0

standard real-time thermal cycler machine with commercially available thermal shift dye kit (Ufa technologies _. ; Cat # 4461146).. Fo each reaction, purified protein (Q pg to tpg) was added to 18 pt of thermal shit buffer provided with the kit, and 1÷ 2pl of dye. Reactio was set 98 wet! PGR plate In file dark and then placed in the thermal cycler machine using ^; fee fallowing two-stage program f { 2S°C for .2 mim) 1 cycle; (2? ^S C for 15 see, 2:6 ^* C for 1 mm) 70 eyctes;: auto increment 1*G "for both st ges], The liter was set at ROX with no cjuencher filler d a si e Iter,

1001 3] JVllcroarxay analyses

(00124] RHA samples ere collected from hippocampal tissue of T a d Ppara-nuli (a O) mice using G!agen RNeasy kit (Get# 74104), Q«¾ftt¾yar»d purity of N;A were determined using the NanoDrop LIE {Nanodrop

Technologies. Wilmington, DE. USA). The mRNA of ach sample was converted into cDNA using Superscript III First-Strand synthesis Kit (T ermoflsher; Cat# 18080-051 ). Next,, each dDNA sample was diluted at 1:2 ratio, mixed with SYBR Oreen qPCR Master M (Applied Bim siems _* Ther ofislier; Cat # 4S0©155) _i and then a!quoted o 96 well Mouse Plasticity oPCR-arrays (SASiosdenoes:;: Cat #ΡΑΜΜ-1262 . Teen 96-¾#ell plate was placed In AB Prism 7500 standard qPCR S stem and run with stage 2, ste 2 (δΟ.δ'Ό^Ι :00 mln) "data collectio " module. Once PCR is done, Ct values were Imported from the PGR console and uploaded in SABiosclences we site for further analyses _* As recommended, we used online software modules to proceed wit further calculations. Data normalization was performed by correcting all Ct values with th average Ct values of 12 constantly expressed housekeeping genes HKGs) present on the array. PCR* array results were di layed by olostergram analyses with three colo

presentation from green (low expression) to black to red (high expression).

|001 5J T-PCR Analysis

JW126] Total RNA was digested with DN&se and RT-PCR was carried out as described earlier ¹⁷ - ¹⁸⁶ using a RT-PCR kit from Clanteek GAPDH {glyceraidehyde-3- phosphata dehydrogenase) was used to ascertain that an equivalent amount of cDNA was synthesized from d liferent samples.

p9127 ReaHime PCR Analysis

|0O128J Real time PCR was performed In the ABI~Prlsm?70® s uence detection system (Applied Blosystems, Foster City,. CA) as described eariier ^{: S} using

TaqMan Universal Master mix nd F Wabeled probes and primers (Applied Sroaystems).: Data ware processed b the ABI Sequenc Detection System 1.8 software a nd analyzed by AN O VA, (001 S| immunobtot anai sis

(001 $0| For whole-ceil and tissue !ysates, sanipias were homogenized In RtPA buffer containing protease and phosphatase inhibitors (Sigma , passed 10 tim s through a 26-gaygo needle, rotated end over end for SO rnln at 4*0, and cantrffuged for 10 rnin at 18,000 * g. The supernatant was alquoted and stored at -8D ³ C uritll tise> Protein conc ntratio s wore d e min d using a NanoOrop 2000 (Thermo Fis er), and 15-30 pg sample was heat-denatured and resolved on 10% or 12% pQlyacf !amfde-SDS gels, transferred to 0,45 μηΐ. nit ocel ulose membranes under semidfy conditions (1SV for 12 mln). Membranes were blocked for 1 h with biodking buffer (Li-Oar), incubated with primary ant bodies overnight at 4 ^a C under s king conditions, washed, incubated with IR^ e abe|ed secondary antibodies (1:17,000; U-Cor) for 45 m n at room temperature, washed, and visualized tih the Odyssey Infrared imaging System: (Li-Cor). Blots were converted to grayscale and then binary, analyzed using Fiji, and normalized to appropriate loading controls,

JIMH31J Irrimunohlst chemical analysis

(00132| Hi pocampa! regions were dissected from 18÷day~otd embryonic fetus as described elsewhere (PMID: 25007337% combined and plated in poiy-D-lysine coated plate for anof er 2-3 weeks for branchi g. After that, these neurons w re transduced with QFP-contalning ten¾ torjs for 2 & Neurons were stained with Dyi¾fti~554~e©r gated phaloWtn (Cat # 21834: Tbermoisfter) as per manufactures protocol and visualized fluorescence microscope. For tissue staining, 10 pro paraifn embedded mou e brain blppocarnpai sections wars mad© from 8- to10- week-old male WT and Ppa <m mice and imrnyno-stalned with anti ^' -PPARa arid a tf-NeoN antibodies,

(00133] Siatisfeal analyses

3 J All values are expressed as the mean ± SO, Difference among means were analyzed using one- or two-way ANGVA with dose of ligends or genotype as the independent factors. Differences in behavioral measures were examined by independent one*way or mpe ted÷ easures ANOVAs. using SPSS.. Homogeneity of variance between test groups was exami ed using tevene's test P t- oc analyses of between-sybjecis effects were conducted using Scheffe's, Tukey% or Garo s-

Noweil tests, where appropriate, p < 0.DS was considered statistically significant (0013SJ The following experiments wll: be conducted to test the efficacy of

H S, HEX and OCT for treatment of neurodegenerative disorders, lysosomal storage disorders and body weight control. The examples set forth below

illustrate t e experiments exemplify t e use of HMB. Similar experiments will be conducted with HEX and OCT,

$00136J Adopii iy-t ftsfBrr&d Experiments! A rgfc £ricep a(omy®M$ (EAE), S o el Female SJL/J mice {4-5 weeks old) will be used, Gonor mm will be im unized s,o with 400 bovine Myelin Basic Protein (MSP) end 60 pg M tubmwhsts. in incomplete Freund's Adjuvant (I A). Animals will be sacrificed 10-12 days post-immunization, and the draining lymp nodes- will be harvested and single cell suspensions will be cultured in R i 1640 supplemented with 10% Fetal Bovine Serum (FBS) _:f 50 igfml BP, 50 p 2;-ΜΕ, 2 mU L-gtuiamine, 100 U mt penicillin and 100 pg m! streptomycin. On day 4, cells will be harvested and re-suspended In Hank's b lanced salt solution (HBSS), A total of 2 x 10 viable cells In volume of 2:00 pL will be Injected Into the tall vein of naiv mice. Pertussis toxin (160 ng mouse; Stgma-Aldrfch) wi be infected once via ip, route on Q day post-iranster (dpi) of cells. Mice will be treated wit? OCT (2 and 5 mg/kg body wf d), HEX {2 and S mg/kg bod wt d) and HUB (10 and 20 g kg o y wt d) vis gavage _* During feeatment these drugs will be- mixed In 0-5% methyleslulose. Therefore, control animals will receive only 0.5% memylcellulose as vehicle. Six mice will be used in each group. Female m ce (4«5 week old) will be randomly selected for any group. Experimental a imals will be scored dally tor 30 days by a masted investigator, as follows: 0„ no clinical disease; 0.5, lleemcSon; 1, tail weakness; 1 J, tali paralysis; 2, hind limb weakness; 3, hind limb paralysis; 3<S, ferel b weakness; 4, foreilmb paralysis; S. _f moribund or death.

it 37] i m g EAE in 58§ PLP-TCR Tg mic&. PIP^..^ -specific 586 TC -

T§ mice (provided by Prof. Vijay Kuchroo, Harvard Medical School, Boston, MA) will be used,. Female Tg mice |4~5 weeks old) will be Immunized with 10 μ§ of

PLP13£MS1 in iub&rcuio In I FA. as described above. Mk® will be treated with OCT (2 and S. mi kg body wt d) _s HEX (2 and § mg/kg ody wt d) and HUB (10 and 20 mg kg body t/d) via gavage. Du ing treatment these drugs will be mixed in 0-5% methylceilulose. Therefore, control animals- it receive only 0.5% methyiselylose as vehicle. Six mice- will be used in each group. Female mice (4~ 5 week old) will be randomly selected for any group. Experimental -animals will be scored daily for 30 days tsy a masked inve tig tor.

p0 38J Chro ic EAE, C57SL/6 mice will be immunized wit 106 p§ of OG3S- 55 as described above. Mice will also receive two doses of pertussis ^' t xi (150 ng mouse) on 0 and 2 dpi. Mica wi be treated with OCT (2 and S mg kg body wt d), HEX (2 and 5 mg kg body wt/d) and HU (10 arid 20 mg/kg body wt d) vi gava-ge. During treatment, these drugs will be mixed ^' in 0.5% methyicelluiose. Therefore, control animals will receive only §£% mefhyieelUo-se as vehicle. Six mice will b used in each group. Female mice -5 week old) will be randomly select d for any group. Experimental animals will be scored daily for ^' 30 days by a masked investigator,

P§13§] Hmtotogic&t mi mtopy. On 1 dpi {first chronic phase), five mice from each of the following groups control _* EAE, EAE*HMB, and EAE+vehioie) will be anesthetized. After perfusion with phosphate buffered -saline (PBS) (pH 7,4) and then with 4% (wAr) paraformaldehyde solution in PBS, cerebellum and whole spina! cord will be di sected out from each mouse. The tissues will be further f xed and then divided into halves: one-half will be used lor histological staining and the other half will be used for myelin staining. For histological analysis, routine histology will be performed to obtai perivascular cuffing and

morphological details of CNS tissues of EAE mice. Paraformaid ^' e ytie-fixe tissues will be embedded in paraffin, and serial sections (4 pro) w ll be cut Sections will be stained with conventional H&E staining method. Digital Image wll be collected under bright-field setting using a x40 objective. Slides wll be assessed in a blinded fashion by three examiners for inflammation in different anatomical compartments (meninges and parenchyma). Inflammation will be scored using the following scale as described: for meninges and parenchyma: 0 _> no infiltrating cells; 1 _} few nfiltrating cells; ^' 2, numerous infiltrating cells; and ¾ widespread Infiltration, For vessels: ø« no cuffed vessel: 1 , one or two cuffed vessels per section; 2 _S three to ive cuffed vessels per section and 3, more than five cuffed vessels per section. At feast s x serial sect ons of each spinal cord from each of five mice per group will be scored and statistically analyzed by

ANOVA.

{00140! Staining for my&lirt. Sections will be sta ed with Loxol fast blue for myelin. Slides wit! be ssessed: in a blinded fashion for demyelnation by three examiners using the following scale; 0 ₍ normal white matter; 1 rare foci; 2 _f a few areas of e yeiinatlan; and 3, large areas of denomination. At least six serial sections of each spinal cord from each of five mice per group will be scored and statistically analyzed by ANOVA,

{00 ] S&mi-quantaMh/e RT-PCR analysis. Total RNA will be isolated from splenic T cells and spina! cord by using tie R leasy mini kit fQiagen _f Valencia, CA and from spleen and cerebellum by using the Ultraspec-il RNA reagent (Bkrtecx laboratories, Inc, Houston, TX) following manufacturer' protocol. To remove any contaminating genomic DNA _f total RNA will be digested with D ase, Semi- uantitative RT-FCR will be carried out using a RT-PCR kit from Clonetech (Mountain View _* CA), Briefly, 1 pg of total RNA will be reverse transcribed, using oligoidX) as primer and M&ifLV reverse transcriptase (G rttech) in a 20 μΐ reaction mixture.. The resting cDNA will be appropriately-diluted, and diluted cDNA will be amplified using Titanium Taq DNA polymerase and the following primers. Amplified products will be electrophoresed oh a 1 % agarose gels and visualized by et dium bromide staining,

fii 42] (SEQ ID NO: 1 ) | OS: Sense: S ^! - CCGTTOiGAAGTTTCTGGCAGCAGC^

1001 3] {SEQ ID NO:2) Aniisense: S'-GGCTGTCAGAGCCTCGTGGCTTTGG-

{00144! (SEQ ID NO:3) it-I B: Sense: S'-CTCCATGAGGTTTGTACAA.GG-S ^*

100145] (SEQ ID NO:4) Anilsense; S'-TGCTGATGTACCAGTTGGGG-S'

{001403 (SEQ ID NO:5)MI& Sense: S ~TGeAGAGA.TTCACCGAGQAGA~3 ^?

{001471 {SEQ ID NG;6) Antisense; 6' ^« TGAAGGTCGTCGGACTCTGAG*3'

{001481 (SEQ ID NO:?} GAPPH: Sense: S'-GGTGAAGGTCGGTGTGAACG-S ^* PS1 S] S£G ID NO:8)

I §0] The relative expression of each gene with respect: to GA.FDH wl b measured after scanning the bands with a Floor Chem 8800 Imaging System {Alpha Irinotech, San Leandro, CA|,

l 51 Real-time PGR ana ysis will fc© performed ysing the A.8l-Pr§sm?70Q se uence detection system (Applied Biosystems, Foster Cit ,. CA Briefly, reactions will be performed in a 06-wefi optical reaction plates on cONA

equivalent to SO ng DNasenf g sted MA In a volume of 25 pL, containing 12,5 pL Taq an Universal Master mix and optimized concentrations of FAM-lapeled probe, forward and reverse primers following i © manufacturer's protocol. All primers and FA -!shefe probes for mouse genes and GAPOH wili oe obtained from Applied Biosystems, The rn A expressions of respective genes will be normalized to tie level of GAPOH mRNA. Data will be processed by the ABI Sequence Detection System M software and analyzed by ANOVA,

£00152] Flow cytometry. Two-color flow cytometry wilt be performed,. Briefly, 1 x 10 ^s lymph node ceils (L C) or splenoeyies suspended in flow staining buffer will be incubated at 4 ^* C with appropriately diluted FITC- abeled Ab to C0 far 30 rotn, washed, and re-suspended in fixation and pormeabi ization solution. Following; incubation in dark for 30 min, ceils will be washed, blocked with test Fc block (anti-mouse GDI 8/32) in permeablliiatlon buffer, and subsequently inculcated with appropriately diluted PE-la eled Abs to Pox S at 4 ^* C I tie dark. After incubation, the cell suspension will be eentrifuged, washed thrice, and re- suspended in flow staining: buffer, The cells then will be analyzed through FAGS (BD Biosciences, San do e, 0.A), Ceils will be gated based on morphologic l characteristics. Apoptotic and necrotic cells will not fee accepted for FAC3 analysts.

£80153J OCT, HEX and HMB wiif fee tested ^' for in ibition of infiifr&ikm of momnuciear ceiis _t inflammation and demyaim&tmn in the spina! cord ofEAE.

Infiltration of autoreactive T cells and associated mononuclear cells is a hallmark

OfEAE as well as M . OCT, HEX and HMS treatment: will be tested for attenuation of infiltration and inflammation In adoptively-transferred EAE mice, ice receiving OCT, HEX and HUB from S dpi (onset of the acute phase) mil be saedffced on 16 dpi H i E staining will be examined for widespread infltratlon of inflammatory eel Into the spinal cord of EAE mice verses HUB treated mic . The rela i e level of inflammatory cells will be quaoilated,

{8015 1 OCT, HEX and: HUB treatment: wi h© examined to determine het er these drugs are capable of inhibitrtg the expression of proinflammatory molecu es in the spinal cord of EAE mice. Expression of ^' pro-Inflammatory molecules like INOS and IL ^» 1p wilt be observed In the spinal cord of untreated EAE mice compared to control mice and Hy B-treated mice,

{001 SSJ Demyalinatfon is the most important pathological feature in MS, which is also modeled in EAE animals. Therefore. It will be e amined whether OCT, HEX and HM8. treatment rotects EAE mice from demyalnation. Spinal cord sections will be stained by luxe! fast: blue (LFB) for myelin and observed for widespread demyelination■ zones in the white matter or restoration of myeli . To confirm these fndings, the expression of three myelin genes, myelin basic protein (M:BP) and proteofipid protein (PIP), will be examined.

{001 §§J Experiments will also be carried out testing H!¾f8, HEX and/or OCT treatment in connection with Parkinson's disease (PDJ, Examples are describ d using H S,

JW1 i?l Animate and MPTP intoxication. Six- to eight-week old: CS7BL/8 mice will be used. For acute ΜΡΎΡ intoxication, n ce will receive four intraperitoneal (ip>) injections of fV!PTP-HCI (W mg/kg of free base; Sigma Chemical Co, _s St Louts, MO) in saline at 2-h intervals, Control..animals will receive only saline., |001 SSI HAI8 fre&imBBt M ice will be tr ated with HUB (10 and 20 mg/kg: body wt/rJ) via gavage. HUB will he mixed In G.5% methytee!!ufose (MC)and from 3 h after the last injection of M TP, mice will be gavaged with 100 pL H B-mlxed MC once dally using gavage needle,. Therefore,, control MPTP mice received only MC as vehicle.

[0 533 Western bht A y s _* Jmmunobioi analysis for DJ-1 and TH will b carried out Briefly, cell homogenates will b electropboresed, proteins will be transferred onto a nitrocellulose membrane, and bands were visualized with an Odyssey sreteed scanner afte immurieJabelng, with respective primary antibodies followed by infra-red fluofoprtore-tagge secondary antibody

{Invitrogen},

i16i] HPLG. afys&s _* Striatal level of dopamine will be quantified in

Complete Stand-Alone HPLC-ECD System EICOMHTEC-500 (JM Science lm., Grand Island, NY), Briefly, mice will be sacrificed by cervical dislocation, alter ? days of PTP intoxication and their striata will bo collected and immediately frozen rs dry ice and stored at ^« 80C until analysis. On the day of the analysts, tissues will be sonicated in perchloric acid containing isoproterenol and resulting homogeniates were centrluged at 2:0,000 x g for I S mm at 4C, After pH adjustment and filtration, 10μΙ of supernatant will be injected onto an Eico pak 8C-30DS column (Complete Standalone HPLC-ECD System EiCOMHTEOSQO from JM Science Inc., Grand Island, NY) and analyzed fallowing tie

manufacturer's protocol

P§181] UpregySatlon and/or maintenance of PD elated: beneficial protein such as DJ-1 Irs the nigra during neurodegenerative Insults- may have therapeutic efficacy PD. PTF intoxication should decrease the level of Dj-1 in wo in the nigra. Oral treatment of MPTP-info^caied mice with HMB will be tested for protection of In the nigra- Protectio of nigral tyrosine hydroxylase (TH) levels and dopamine (DA) levels In the striata will also be investigated after MPTP treatment in the presence: and: absence of HMB.

[00102| Futur experiments will evaluate whether HWB treatment improves motor toetions in PTP-lntoxicated mice, JMale C57/BL8 mice wi: be Intoxi ate with fctPTP and from 6 hours after the last injection of MPTP, the mice will: receive HUB (10 and 20 mg kg body weigbtfday) via gavage. The mice wiil be tested for motor functions (A, rotorod; B _s movement time; C, number of movements; O, rest: time; E, horizontal activity; F, total distance; G _s rearing:: and H, stereotypy) 7 days after the last injection of MPTP, The data will be means ± SEM of si mice per group,

{O l S3J Additional experiments will be conducted to etermine if HMB, HEX and/or OCT treatment protects htppooarnpai neurons and improves memory and learning in 5XF AD mice, an animal model for Alzheimer's disease. Briefly, six-month GM mate 5X AD mice will be treated with OCT (2 and 5 mg/kg bc y i/d), HEX (2 and 5 mg/kg body wt d) and H¾iB (10 and 20 mg/kg body wt d) via gavaga. During treatment, these drugs will be mixed in 0.5% methytcelfulose. Therefore, control animate wilt receive only 0.5% mefJ yfoeiu ase as vehicle. After 30 d of treatment mice will be tested for Barnes m ze, T maze and Novel Object Recognition,

Conclusion w ll be drawn from: analysts of at least six mica per group. Hippocampai sections will be also double-labele for Ne«N (marker of neuron) and TUNEL

(marker of apoptosis).. Results will represent an lys s of t o hippocampat sections of each of six mice per grou ,

(091 S4] Experiments will be also carded out In fibroblasts of patients of Batten disease, one of the lysosomal storage disorders _* Fibroblasts of patients with late infantile Ba ten disease will be treated with OCT (1 a d 2 pM) _t HEX (1 and 2≠&) and HUB (10 and 20 μΜ) for 2:4 h followed by measuring the activity of tripeptidyi peptidase 1 {JPPi } activity as described by us ^!S , After drug treatment celts w i be also monitored for lysosomal biogenesis using Lysotracker as described by us , p016SJ In order to examine whether OCT _s HEX and HUB can induce weight toss, obese (ob/ob) mice will be treated with OCT (2 and 5 mg/kg body wi d}, HEX (2 and S mg kg body wt d) and HUB (10 and 20 mg/kg body wt d) via gavage.

During treatment, these drugs will be mixed in 0.5% rnetbyfeslWose. Therefore.,, on© group of animals will receive only 0.5% fi ethyteellutase a vehicle. After a month of treatment, body weight will be monitored. Six. mice writ be used i each group,

(091S6| The above Figures and disclosure are intended to be Illustrative and not exhaustive. This description will suggest many variations and alternatives to one of ordinary skill in the art. All such variations and alternatives are Intended to be encompassed within the scope of the attached claims. Those familiar win the art may recognize other equivalents to the specific embodiments described herein which equivalents are also intended to be encompassed by the attached claims, (001673 Refere ces (001681 1 - Issemann, I- & Green, 8, Activation of a member of the steroid hormone receptor superfarnsiy b peroxisome prolifer tes. Matum 347 _? 645-50 (1990),

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[002053 36, Saha, RJ , & Pahan, K, Differential regulation of n-sup roxid© dlsmutase in neurons and astroglia by HIV-1 120: Implications tor H ^associated dementia. Free R ic Biol Atetf 42 _;i . 1866-78 (2007).

[062061 39. Glutei, P.,. & Baker, T,J« Char cterizaSon of ameboid microglia isolated from developing mammalian brain, J NmftO&ci .2163-78 (1986),

[002071 4Q,Dasgypta, S„ Jana, M., Liu, X, & Pahan, , Rote of very-late aniigen-4 (VLA-4) in myelin basic protein-primed T cel contact-Induced expression of proinflammatory cytokines in microglial cells, J Biot dwrn 278» .22424-31 (2003), {002081 1. Tripos International, 2011-2014 Certara, L ,210 NL Tucker Bivd, Suite 350, St Loute MO 63101 {2011}.

[00209] 42, im, . _s Feig, M> & Brooks, C,L _S 3rd. An impllet membran generalized bom t eory for the study of structure, stability, and Interactions, of membrane proteins, &mp y$j > 2900-18 (2003), Table 1, Peak integration statistics of mass spactrometric analyses for HEX arjd OCT in Pf r3*tmll hJppoeampal neurons infected Witt* tentivirions

containing different Ppara constructs.

Ppam'n il hippocampal reeurorss ere transduced with Sentivirions containing different Ppara constructs for 48 ft followed by the affinity purification through C3FP column. After that, tn© ©Med fraction was fractionated wUh cnlorofornv methanol extraction procedure arid tfse organic pitas© was analyzed by SC$iS> Peaks for Hexadecanarrnde fHEX) and S-ocfadece artticte |OCT) ere analyzed and their detaile peak Integration statistics were displayed a&ove. Peak area was adjusted with baseline and then normalized! with the peak area of the internal standard I2 _i 4*Bis « _s iSHd methyli½n2yi}p ¾ o!l _i The normalized value was shown as trie relative abu dance and finaliy presented Sn a percent scale.

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