BIOMARKERS AND TREATMENT OF NEURONAL INJURY AND NEURODEGENERATION

Abstract

The invention is directed to methods for identifying and treating neuronal injury or neurodegeneration.

Claims

1. A composition comprising a therapeutically effective amount of an FZD5 receptor blocker, a therapeutically effective amount of a long chain fatty acid, and a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein the long chain fatty acid comprises a docosanoid, an elovanoid, or a combination thereof.

3. The composition of claim 1, wherein the FZD5 receptor blocker comprises a peptide comprising SEQ ID NO: 1 (NH-Met-Asp-Gly-Cys-Glu-Leu-CO2H).

4. The composition of claim 1, wherein the FZD5 receptor blocker interacts with an extracellular domain of the FZD5 receptor.

5. The composition of claim 4, wherein the extracellular domain comprises amino acids 28 to 150 and/or amino acids 28 to 238.

6. The composition of claim 3, wherein the peptide is N-terminally butyloxycarbonyl (Boc) protected.

7. The method of claim 1, wherein the fatty acid derived from DHA, EPA, omega-3, or a combination thereof.

8. The composition of claim 2, wherein the docosanoid comprises DHA, neuroprotectins; lipoxin A4; DHA-derived Resolvins; Maresin 1; 10R, 17R diHDHA and its methyl ester derivatives; 10S, 17S diHDHA and its methyl ester derivatives; or any combination thereof.

9. The composition of claim 8, wherein the neuroprotectin comprises NPD1.

10. The composition of claim 2, wherein the elovanoid comprises mono-hydroxylated elovanoid, a di-hydroxylated elovanoid, an alkynyl mono-hydroxylated elovanoid, and an alkynyl di-hydroxylated elovanoid, or any combination thereof.

11. The composition of claim 1, wherein the composition further comprises a MicroRNA.

12. The composition of claim 11, wherein the microRNA is miRNA-224.

13. (canceled)

14. A method for treating a patient afflicted with a condition characterized by neuronal damage and/or neuronal injury, the method comprising administering to the patient a therapeutically effective amount of an FZD5 receptor blocker/antagonist and a therapeutically effective amount of a fatty acid.

15. The method of claim 14, wherein the FZD5 receptor blocker and the fatty acid are administered simultaneously.

16. The method of claim 14, wherein the FZD5 receptor blocker and the fatty acid are administered as a single-dose pharmaceutical composition/formulation.

17. The method of claim 14, wherein the FZD5 receptor blocker and/or fatty acid are administered enterally or parenterally.

18. The method of claim 17, wherein the enteral administration is oral or rectal.

19. The method of claim 17, wherein the parenteral administration is selected from the group consisting of intravascular administration; subcutaneous injection, subcutaneous deposition intramuscular injection, intraperitoneal injection, transdermal, nasal and inhalational.

20. The method of claim 14, wherein the condition comprises ischemic stroke.

21. The method of claim 14, wherein the neuronal damage or neuronal injury is the result of or exacerbated by uncompensated oxidative stress.

22. The method of claim 14, further comprising the steps of: obtaining a sample from the patient; measuring the protein level of Wnt5a protein in the sample and comparing the protein level of Wnt5a protein in the sample to a control sample; wherein the patient is treated if the protein level of Wnt5a protein is changed compared to the control.

23. The method of claim 22, further comprising diagnosing the patient as having a condition characterized by neuronal injury or neuronal damage if the protein level of Wnt5a protein in the sample is higher than that of the control sample.

24. A method for determining the presence of neuronal injury or neuronal damage in a patient, comprising: obtaining a sample from the patient; measuring the protein level of Wnt5a protein in the sample and comparing the protein level of Wnt5a protein in the sample to a control sample; wherein the patient is treated if the protein level of Wnt5a protein is changed compared to the control.

25. The method of claim 24, wherein a higher protein level of Wnt5a protein in the sample relative to the control sample is indicative of a neurodegenerative disease.

26. A method for determining the prognosis of a patient suffering from a condition characterized by neuronal injury or neuronal damage, comprising: obtaining a sample from a patient; measuring the expression level of Wnt5a protein in the sample and comparing the expression level of Wnt5a protein in the sample to a control sample; and determining the prognosis of the patient.

27. The method of claim 1, further comprising repeating the measuring step at one or more intervals.

28. The method of any one of claims 22, 24, or 26, wherein measuring comprises Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay analysis (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunohistochemistry, immunoprecipitation assays, complement fixation assays, flow cytometry, and protein chip (protein microarray), capillary western blot, protein MS, Protein sequencing, HPLC, or gas chromatography.

29. The method of any one of claims 22, 24, or 26, wherein the sample comprises blood, cerebrospinal fluid, tissue biopsies or a combination thereof.

30. The method of claim 29, wherein the blood sample is separated into plasma before measuring.

31. The method of any one of claims 22, 24, or 26, wherein the control sample comprises a sample from a normal subject.

32. The method of any one of claims 22, 24, or 26, wherein the control sample comprises a sample isolated from the patient prior to the onset of the neuronal injury or neuronal damage.

33. The method of any one of claims 22, 24, or 26, wherein the condition is stroke.

34. The method of claim 33, wherein the protein level of Wnt5a protein is measured within seven days, five days, or three days of the stroke event.

35. The method of any one of claims 22, 24, or 26, wherein the neuronal injury or neuronal damage comprises uncompensated oxidative stress.

36. A diagnostic kit for determining brain injury status in a patient comprising: a substrate for collecting a sample from a patient; and means for measuring the protein level of Wnt5a protein.

37. The diagnostic kit of claim 36, wherein the means for measuring comprises Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay analysis (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunohistochemistry, immunoprecipitation assays, complement fixation assays, flow cytometry, and protein chip (protein microarray), capillary western blot, protein MS, Protein sequence, HPLC, or gas chromatography.

Description

FIGURE LEGENDS

[0226] FIG. 1: Docosanoids counteract UOS-dependent NF-κB activation and apoptosis via Wnt5a/FZD5/ROR2.

[0227] A, Experimental design and morphology criteria to determine apoptotic cells. B, Wnt5a enhanced cell death triggered by H2O2. Apoptotic cell percentage was measured using Hoechst staining and quantified by ImageJ. C-E Docosanoids prevented an increase in Wnt5a transcription in cells undergoing UOS. C, DHA, and its derivatives NPD1, 10R, 17R diHDHA, Maresin-1, RvD1 and RvD2. D-F, SYBR green real time PCR was used to determine semi quantitatively the expression of Wnt5a D; FZD5 E in human primary RPE cells and Receptors linked to Wnt signaling; F, in ARPE-19 cells induce a decrease of UOS-triggered Wnt5a transcription. Standardization was performed using p-actin and GAPDH as housekeeping genes. G, Schematic representation of luciferase assay performed with TOPFlash/FOPFlash, and NF-κB/p65 reporter constructs. H, Wnt5a does not affect activation of β-catenin. TOP-Flash (wild type) and FOP-flash (mutated) β-catenin binding sites activity H, and NF-κB binding activity I and J, were measured by the means of luciferase reporter system assay and standardization was made using a plasmid expressing GFP. I, NPD1 does not prevent activation of NF-κB triggered by UOS but it affects its activation when Wnt5a is added. J, ROR2 and FZD5 is involved in the activation of NF-κB by Wnt5a. Human primary hRPE cells were transfected with siRNA targeting FZD5 and ROR2 separately and together or control non-specific siRNA. UOS was induced in the presence or absence of 100 nM NPD1 and Wnt5a. The bars represent the mean of three measurements and the standard error of the mean. *p<0.05.

[0228] FIG. 2: NPD1-dependent cRel binding to promoter A decreases Wnt5a expression.

[0229] A, Representation of siRNA resistant cREL ORF strategy to control off target effects of siRNA. cREL ORF mutations were designed to interfere with siRNA binding while preserving the protein sequence by introducing silent mutations. Mutations were performed by exchanging the third base of some codons without altering the amino acid that they code for B, cRel quantification (top) and Wnt5a (lower) mRNA by means of SYBR green-based real-time PCR in hRPE cells undergoing UOS, +/−NPD1. Cells were transfected with a mixture of 3 siRNAs targeting cRel (left), cREL wild type ORF (middle) and mutated siRNA resistant cREL ORF plus the tried of siRNAs altogether (right). C, Wnt5a mRNA quantification of non-transfected cells (Control for B). D, Model of regulation of NF-κB sites by cRel: in silica analysis of Wnt5a promoter (Katula et al., 2012) showing that the two binding sites for NF-κB have high affinity for p65, p50 and cRel. The cartoon shows the possible direction in which transcription factors elicit their action. NF-κB site prediction is in Table 4. D and E, Other NF-κB binding sites detected by TRED. Region 2 corresponds to the upstream NF-κB binding site and Region 6 to the downstream binding site depicted in D. Regions 1, 3, 4, 5, and 7 showed up in the general TRED search with high score (Table 4) for the three NF-κB. Four amplicons were designed close or sitting on these regions to assess each site. In purple CpG islands that encompass the putative binding sites were depicted (Table 5). E, SYBR green-based real-time PCR using as template the proteinase digested genomic DNA fragments resulting from micrococcal DNAs digestion and cRel pull down. UOS=1600 μM H.sub.2O.sub.2 plus 10 ng/ml TNFα. NPD1: 100 nM and Wnt5a: 50 ng/ml unless stated otherwise. Bars represent mean of three measurements and standard error of the mean. *p<0.05.

[0230] FIG. 3: Secreted Wnt5a is reduced by NPD1 in human RPE cells undergoing UOS.

[0231] Wnt5a protein is released from ARPE-19 (A,B) and hRPE cells (C) in UOS. A, ARPE-19 cells were treated with 600 μM H.sub.2O.sub.2 and 10 ng/ml TNFα for 6 hours in the presence or absence of 100 nM NPD1. Wnt5a was measured in cellular lysate (cWnt5a) and in medium (sWnt5a) by the means of Western blot. B, Cellular content of Wnt5a in 15-LOX-1d and control cells. C, Time course release of sWnt5a in human RPE cells. D, Selective effect of NPD1 on sWnt5a. E and F, Content of sWnt5a in medium of human RPE cells in the presence of 1600 μM H.sub.2O.sub.2 and 10 ng/ml TNFα. E, Exosome enrichment protocol using ultracentrifugation. F, Content of sWnt5a in the different fractions of the medium. The bars represent the mean of three measurements and the standard error of the mean. *p<0.05.

[0232] FIG. 4: NPD1 enhances Wnt5a internalization.

[0233] A, Representative images of colocalized signal of FZD5 and Wnt5a in hRPE cells. RPE cells were incubated for 2 hours with 1600 μM H.sub.2O.sub.2+/−100 nM NPD1 and 50 ng/ml Wnt5a. Immunostaining of Wnt5a (Red) and FZD5 (Green) and z-stack analysis of images using BioImageXD. Pictures taken at 20×. Lasers were set up for each experiment using Control+Wnt5a and used without modification to take the remaining pictures. Colocalization of the two signals are shown in left column (white). B, Total signal intensity of three channels (DAPI=blue, Alexa 488=green and Alexa 594=red) in three random fields per well/condition. On the right of each row, the histogram of intensity vs frequency depicts the number pixels showing the intensity value on X-axis. The upper limit intensity is set at 4095. The black vertical lines for each channel indicates the mode (most frequent observation) to designate the intensity at which each curve reaches its maximum. The signal points or clusters of pixels showing colocalization (A-left column) were quantified using ImageJ of three to six random fields encompassing one or two wells, in up to 3 independent experiments. C, Blow up of a single cell showing vesicles positives to Wnt5a (red), FZD5 (green) or both yellow. The fourth panel shows a drawing of the nucleus (blue) and the position of the vesicles showing colocalization of Fzd5 and Wnt5a. D, Quantification of colocalized spots in human RPE cells undergoing UOS+/−eicosanoids in FIG. 1C. Pearson colocalization coefficient was plotted in FIGS. 13-15. E, Blow up of a cell showing large cluster of Wnt5a signal present most frequently in certain treatments. F, Frequency vs Area histogram for representative fields showing different sizes of clusters of Wnt5a positive signal. Bars represent mean of three measurements and standard error of the mean.*p<0.05.

[0234] FIG. 5: Pitstop2 halts the internalization of Wnt5a and its subsequent activation of NF-κB.

[0235] A, Experimental design. B and C, quantification of clusters showing colocalization in the absence B, or presence c, of 25 μM Pitstop2. 100 μg/ml Box5, an inhibitor of Wnt5a biding to FZD5 was used to interrupt Wnt5a effect. ImageJ Quantification of the objects resulting from the 3D colocalization analysis. Pearson's colocalization coefficient for these experiments was plotted in Figure X+1. D, Western blot analysis of Wnt5a in response to UOS in the presence or absence of NPD1 and Pitstop2. E, Pitstop2 interferes with the activation of NF-κB/p65. Reporter assay of three NF-κB/p65 binding sites in tandem driving the expression of luciferase ORF, the construct was depicted in FIG. 2A. NPD1=100 nM and UOS=1600 μM H.sub.2O.sub.2. F, Perinuclear distribution of Wnt5a G, vesicle like signal in the Z axe of the Z-stack. Whole arrow shows a fusion between a FZD5 and Wnt5a positive to a large Wnt5a positive cluster. Arrowhead shows already fused colocalized cluster. H, Model of internalization and recycle of Wnt5a and FZD5 to activate NF-κB/p65. The bars represent the mean of three measurements and the standard error of the mean. *p<0.05.

[0236] FIG. 6: DHA prevents Wnt5a overexpression and secretion in response of ischemia reperfusion.

[0237] A, Timeline of MCAo. DHA, Box5 or saline were administered at 1 h after 2 h of MCAo and rats were sacrificed on days 1, 2, 3 or 7. B, Effect of DHA on neurological recovery. Total score (normal score=0, maximal deficit=12), tactile placing (dorsal, lateral, proprioceptive reactions; normal score=0, maximal deficit=2) after MCAo. DHA or saline was administered at 1 h after 2 h of MCAo and rats sampled on days 1, 2, 3 or 7. Values are mean±SD; n=4 rats/group. *Significantly different from corresponding saline group (p<0.05, repeated measures ANOVA followed by Bonferroni tests). C-E, 400 μg Box5 IV administration, 1 h after MCAo showed effects on neurological recovery and infarct size resembling DHA treatment. C, Total neurological score at days 1, 3 and 7; D, MRI quantification at day 7 of the lesion volume depicting total, core and penumbra and; E, representative coronal sections showing T2 weighted image (T2WI), the defined core and penumbra region (red and blue, respectively) in the second column; and a 3D reconstruction of the lesion. F, Wnt5a mRNA assessment by means of SYBR-green real-time PCR in rat cortex Ipsilateral (Ipsi) or contralateral (Contra) of MCAo, treated with saline (vehicle) or DHA. G,H Wnt5a protein in plasma 2 h after MCAo with DHA (N=4); G, or Box5 (N=4); H, at 1, 3 and 7 days post-surgery. I, Western blot of tissue A1 and A2 (Ipsi and Contra) in Saline and DHA treated animals 1 day after MCAo or Sham-MCAo. J, Quantification of Wnt5a and NF-κB linked gene expression in MCAo. MCAo and saline treatments (vehicle) and DHA for 3 days (N=3) and each reaction was run in triplicate. The mRNA were measured using SYBR green RT-PCR. The cartoon show the color-coded region to test gene expression. Bars represent t mean of three measurements and standard error of the mean. *p<0.05. K, Wnt5a inflammatory signaling after increased abundance due to ischemia/reperfusion.

[0238] FIG. 7. Wnt5a was up-regulated in ARPE-19 cells deficient in 15-LOX-1 undergoing UOS and downregulated by NPD1.

[0239] Design for experiments leading to the identification of Wnt5a (left). UOS-triggered increase in Wnt5a expression was reversed by NPD1 in 15-LOX-1d. 15-LOX-1d cells, which shows depletion in NPD1 synthesis (Calandria et al., 2009), were used to determine genes regulated by the lipid messenger in a microarray assay. UOS=600 μM H.sub.2O.sub.2 plus 10 ng/ml TNFα Representative values of three independent experiments. ANOVA and test for false positives was applied to select regulated genes on microarray output.

[0240] FIG. 8. Quantification of mRNA of Wnt5a in primary human RPE cells.

[0241] Confirmation of the NPD1-regulation of Wnt5a transcription. UOS was carry out using 1600 μM H.sub.2O.sub.2 for hRPE cells, plus 10 ng/ml TNFα to confirm microarray output using SYBR green-based real-time PCR in human primary cells. Representative values of three independent experiments. Bars represent the mean+standard error of the mean of 3 different experimental subjects.

[0242] FIG. 9. Wnt5a enhances the percentage of cell death induced by UOS in ARPE-19 cells.

[0243] Hoechst-positive ARPE-19 cells were beyond H2O2-induced levels. The criteria used to determine Hoechst positive cells and the experimental design is depicted in FIG. 1A. The bars represent the mean of three measurements and the standard error of the mean. *p<0.05.

[0244] FIG. 10. Quantification of mRNA of FZD5 and ROR2 in silenced cells.

[0245] A, Design of luciferase reporter assay performed with COX-2 promoter construct. UOS was induced by the addition of 10 ng/ml TNFα and 600 μM H.sub.2O.sub.2 in ARPE-19 cells. The concentrations of Wnt5a used was 50 ng/ml. Cells were co-transfected with the constructs driving the expression of luciferase and a plasmid that constitutively expressed green fluorescent protein for standardization. Luciferase activity is denoted in luciferase activity units (LUC) and standardized using GFP fluorescent (GFP). B, Wnt5a and IL-1β effect time course on COX-2 promoter activity. COX-2 promoter fragment from −830 bp to the site of transcription start contains one NF-κB binding site at −448 bp. Luciferase activity (LUC) was measured in ARPE-19 cells in the presence of 50 ng/ml Wnt5a or 20 ng/ml IL1-β. C, NPD1 prevents Wnt5a-induced activation of COX-2 promoter in hRPE cells. Luciferase activity measured in cells incubated with 50 ng/ml Wnt5a in the presence or absence of 100 nM (+) and 200 nM (++) NPD1. The bars represent the mean of three measurements and the standard error of the mean. *p<0.05.

[0246] FIG. 11. Quantification of mRNA of FZD5 and ROR2 in silenced cells (Related to FIG. 1J).

[0247] A, FZD5 and B, ROR2 mRNA quantification on Negative control, FZD5 and FZD5 plus ROR2 siRNA transfected human RPE cells. Controls corresponding to experiment. The bars represent the mean of three measurements and the standard error of the mean. *p<0.05.

[0248] FIG. 12. Validation of FZD5 antibody for Immunocytochemistry (Related to FIG. 4, A-F and FIGS. 5, B, C, F and G).

[0249] Histograms of Intensity vs frequency for FZD5 signal (red) and siRNA tracer signal (black). (B and D) representative pictures of human RPE cells transfected with B, FZD5 siRNA and D, Negative control siRNA. To allow the comparison, confocal lasers were set to the negative control parameters and pictures were taken without changing them. White=tracer siRNA; blue=DAPI and red=FZD5.

[0250] FIG. 13. Pearson's colocalization coefficient (PCC) for first experiment in the series of colocalization by immunocytochemistry, Related to FIGS. 4 and 5.

[0251] The PCC values obtained for all the slices of z-stacks of three fields were averaged and plotted. The bars represent the mean of three measurements and the standard error of the mean. *p<0.05.

[0252] FIG. 14. Pearson's colocalization coefficient (PCC) for experiment in FIG. 4C showing colocalization of by immunocytochemistry (Related to FIGS. 4 and 5).

[0253] The PCC values obtained for all the slices of z-stacks of three to six fields were averaged and plotted. The bars represent the mean of three measurements and the standard error of the mean. *p<0.05.

[0254] FIG. 15. Pearson's colocalization coefficient (PCC) for experiment in FIGS. 5, B and C showing colocalization of by immunocytochemistry (Related to FIGS. 4 and 5).

[0255] The PCC values obtained for all the slices of z-stacks of three fields were averaged and plotted. The bars represent the mean of three measurements and the standard error of the mean. *p<0.05.

[0256] FIG. 16. Mechanisms by which DHA and BOX5 alleviate Wnt5a-mediated cell damage.

[0257] Mechanism of action of combinatorial therapy the Box5 peptide and Docosanoids at strategic upstream and downstream different points of the signaling pathway that contribute to an enhancement of the blockage of the inflammatory/cell damaging signaling triggered by Wnt5a. This results in neuroprotection.

[0258] FIG. 17. Western blot expression of vesicular and secreted Wnt5a in the post mortem brains of two Alzheimer Disease (AD) patients as compared to a human control.

[0259] Wnt5a intracellular life is spent is a vesicular form. The supernatant fraction obtained by centrifuging a tissue lysate at 3000 rpm contains cytoplasm, membranes and mitochondria. In this fraction, vesicles are present. In the 100000 rpm, only soluble cytosolic molecules and small structures are present. Western blot performed from two postmortem cortical Alzheimer Disease patient samples (cases #195 and 158) showed Wnt5a was elevated in both fractions while in a normal control the soluble Wnt5a is almost negligible and is very low abundant in vesicular form. These results indicate a relationship between an increase in Wnt5a synthesis (intracellular vesicular form 3000 g) and secreted (soluble 100000 g) pro-inflammatory Wnt5a and Alzheimer's disease. Thus, without wishing to be bound by theory, patients of AD can present increased release of Wnt5a and thus can display the ligand in blood or cerebrospinal fluid.

TABLES

[0260]

TABLE-US-00001 TABLE 1 [DNA constructs and siRNAs, Related to constructs and siRNAs], Related to FIGURES 2A-D, 2G, 211, 3A, 3B, and 6E. Reference/catalog number and Constructs Type Gene/protein company COX2 830 bp upstream PTGS2 Calandria promoter transcription (NM_000963.1, et al., 2012. reporter initiation site NP_000954) vector Wild Type REL (untagged)- REL (NM_002908) Origene cREL UNIQUE VARIANT True ORF expression 1 of Human v-rel Cat #SC126639 vector reticuloendotheliosis viral oncogene homolog (avian) (REL) siRNA Human REL ORF REL (NM_002908) GeneART gene resistant designed silent mutant Human cDNA Clone synthesis of JMC cREL affecting binding of designed cREL expression Trilencer 3 siRNAs mutant. Calandria vector et al., 2015 NFkB 3 tandem copies of p65 Qiagen, Cignal reporter binding sequence NFκB Reporter vector driving the expression (luc) Kit: of luciferase. Cat #CCS-013L TOP Super8XTOPflash 7 TCF/LEF Addgene repository Flash construct M50, Beta- binding sites: Plasmid #12456. catenin reporter. AGATCAAAGGgggta (Veeman et al, 2003) TCF/LEF sites (SEQ ID NO: 2), with upstream of a TCF/LEF binding site luciferase reporter. in CAP letters, and a spacer in lower case, separating each copy of the TCF/LEF site. FOP M51 Super 6 mutated TCF/LEF Addgene repository flash 8 × FOPFlash binding sites that were Plasmid #12457. (TOPFlash mutant) cloned into the pGL3 (Veeman et al, 2003) vector (Promega), cREL REL (Human) 3 unique Human cREL Origene Trilencer siRNA 27mer siRNA duplexes (NM_002908) Cat #SR304027 FZD5 Human FZD5 21-mer Human FZD5 Silencer select siRNA siRNA duplexes (NM_003468) Validated Ambion, Life Technologies- Thermo Cat #4390824. ID: s15416 ROR2 Human ROR2 21-mer Human ROR2 Silencer select siRNA siRNA duplexes (NM_004560) Ambion, Life Technologies- Thermo Cat #4390824. ID: s9758 Negative Non-specific binding Allstars. Qiagen control siRNA sequence Cat #1027292 siRNA Alexa Fluor 488 conjugated Negative Non-specific binding Allstars. Qiagen control siRNA sequence Cat #1027287 siRNA Alexa Fluor 488 conjugated

TABLE-US-00002 TABLE 2 [Primers information], Related to FIGS. 1B, 1D, 2E, 2F, 3B, 3C, 7D, and 7G. Target Sequence SEQ ID NO: Source Rat Wnt5a Forward primer  3 RealTimePrimers.com 5′-TTACCCAAACCGGACTGTTA-3′ Reverse primer  4 5′-AGCCTTTTCGGTTCATCTCT-3′ Human Wnt5a Forward primer  5 Campioni et al., 2008. 5′-CAAAGCAACTCCTGGGCTTA-3′ Reverse primer  6 5′-CCTGCTCCTGACCGTCC-3′ Rat Cxcl1 Forward primer  7 RealTimePrimers.com 5′-GCGGAGAGATGAGAGTCTGG-3′ Reverse primer  8 5′-TCCAAGGGAAGCTTCAACAC-3′ Rat ACTB Forward primer  9 RealTimePrimers.com 5′-CACACTGTGCCCATCTATGA-3′ Reverse primer 10 5′-CCGATAGTGATGACCTGACC-3′ Rat TNFa Forward primer 11 Ohtomo et al., 2010 5′-AACTCGAGACAAGCCCGTAG-3′ Reverse primer 12 5′-GTACCACCAGTTGGTTGTCTTTGA-3′ Rat IL6 Forward primer 13 RealTimePrimers.com 5′-CTTCCTACCCCAACTTCCAA-3′ Reverse primer 14 5′-ACCACAGTGAGGAATGTCCA-3′ Rat B2m Forward primer 15 RealTimePrimers.com 5′-TGCTACGTGTCTCAGTTCCA-3′ Reverse primer 16 5′-GCTCCTTCAGAGTGACGTGT-3′ Rat MMP13 Forward primer 17 RealTimePrimers.com 5′-CCTCTTCTTCTCAGGGAACC-3′ Reverse primer 18 5′-GGAATTTGTTGGCATGACTC-3′ Rat MMP9 Forward primer 19 RealTimePrimers.com 5′-ACTTCTGGCGTGTGAGTTTC-3′ Reverse primer 20 5′-TGTATCCGGCAAACTAGCTC-3′ Rat MMP2 Forward primer 21 RealTimePrimers.com 5′-CTTCAGGTTCTCCAGCATGA-3′ Reverse primer 22 5′-CCGTAAGGGAGACACCAGAT-3′ Rat IL-1b Forward primer 23 Nakazawa et al., 2011. 5′-TCAGGAAGGCAGTGTCACTCATTG-3′ Reverse primer 24 Rat ICAM1 Forward primer 25 Ammirante et al., 2010. 5′-CTGTCAAACGGGAGATGAATGGT-3′ Reverse primer 26 5′-TCTGGCGGTAATAGGTGTAAATGG-3′ Rat MCP1 Forward primer 27 Nakazawa et al., 2006. 5′-ATGCAGGTCTCTGTCACGCTTCTG-3′ Reverse primer 28 5′-GACACCTGCTGCTGGTGATTCTCTT-3′ Rat E-Selectin Forward primer 29 Hannawa et al., 2005. 5′-TGCGATGCTGCCTACTTGTG-3′ Reverse primer 30 5′-AGAGAGTGCCACTACCAAGGGA-3′ Rat Ywhaz Forward primer 31 Gubern et al., 2009. 5′-GATGAAGCCATTGCTGAACTTG-3′ Reverse primer 32 5′-GTCTCCTTGGGTATCCGATGTC-3′ Rat Sdha Forward primer 33 Gubern et al., 2009. 5′-TCCTTCCCACTGTGCATTACAA-3′ Reverse primer 34 5′-CGTACAGACCAGGCACAATCTG-3′

TABLE-US-00003 TABLE 3 [ChIP assay primers for SYBR green based real-time PCR], Related to FIGS. 3E, 3F, and 3G. Promoter Primers Forward SEQ ID NO: Reverse SEQ ID NO: Wnt5a A1 5′-GCATCCCACTACCC 35 5′-GCTGCCTTGACATGGA 39 Promoter A AAGTCC-3′ ACCTCA-3′ A2 5′-CAGCAATAAGTTCC 36 5′-GCTTTGGGGCCACAGA 40 GGGGCG-3′ ACAATC-3′ A3 5′-GCCTCTCCGTGGAA 37 5′-GATGCGCCCAGGAATG 41 CAGTTGC-3′ G-3′ A4 5′-CGCCAGTGCCCGCT 38 5′-CAGCCGAGGAATCCGA 42 TCAG-3′ GC-3′

TABLE-US-00004 TABLE 4 [TRED and TF bind analysis on the Promoter sequence (Katula et al., 2012)], Related to FIGS. 3D-3G. Position/Sequence TRED Score TFBind Score cREL Region 1 [192 . . . 201] TAGAAATTCC 3.92 [214 . . . 223] CCGGTTTTGC 2.21 [215 . . . 224] CGGTTTTGCC 3.3 193 (+) SGGRNWTTCC TAGAAATTCC  0.819522 194 (−) SGGRNWTTCC AGAAATTCCG  0.844549 Region 2 [357 . . . 366] GGGACTTTGC 5.08 358 (+) SGGRNWTTCC GGGACTTTGC  0.854004 Region 3 [1445 . . . 1454] GCGACTTTCA 4.12 Region 4 [1550 . . . 1559] CGGCATCTCC 3.3 1565 (−) SGGRNWTTCC GAAAAAGCCA  0.850945 Region 5 [1945 . . . 1954] CCTAATTACC 1.99 1939 (−) SGGRNWTTCC GGAAAGCCCT  0.887097 Region 6 [2103 . . . 2112] GGGCGCATCC 2.6 Region 7 [2284 . . . 2293] GGCGACTTCC 3.71 2285 (+) SGGRNWTTCC GGCGACTTCC  0.814516 p65 Region 1 [192 . . . 201] TAGAAATTCC 4.17 194 (−) GGGRATTTCC AGAAATTCCG  0.868024 Region 2 [357 . . . 366] GGGACTTTGC 6.19 358 (+) GGGRATTTCC GGGACTTTGC  0.861557 Region 3 [1445 . . . 1454] GCGACTTTCA 1.95 Region 4 [1550 . . . 1559] CGGCATCTCC 2.56 1551 (+) GGGRATTTCC CGGCATCTCC  0.769102 1552 (−) GGAMTTYCC GGCATCTCCC  0.803246 Region 5 [1937 . . . 1946] TGGAAAGCCC 2.14 1938 (+) GGGRATTTCC TGGAAAGCCC  0.782754 1939 (−) GGGRATTTCC GGAAAGCCCT 0.86491 Region 6 [2104 . . . 2113] GGCGCATCCC 1.79 2105 (+) GGGRATTTCC GGCGCATCCC  0.765749 Region 7 [2284 . . . 2293] GGCGACTTCC 2.72 2285 (+) GGGRATTTCC GGCGACTTCC  0.771018 NFkB/p50 Region 1 193 (−) NGGGACTTTCCA TAGAAATTCCGG  0.760042 Region 2 [357 . . . 366] GGGACTTTGC 5.92 358 (+) GGGGATYCCC GGGACTTTGC  0.750555 Region 3 [1445 . . . 1454] GCGACTTTCA 0.86 Region 4 [1551 . . . 1560] GGCATCTCCC 1.74 1551 (−) GGGGATYCCC CGGCATCTCC  0.790315 1552 (−) GGGAMTTYCC GGCATCTCCC 0.803246 Region 5 [1937 . . . 1946] TGGAAAGCCC 2.08 1939 (−) GGGGATYCCC GGAAAGCCCT 0.79498 Region 6 [2104 . . . 2113] GGCGCATCCC 3.78 2105 (−) GGGGATYCCC GGCGCATCCC 0.75522 Region 7 [2285 . . . 2294] GCGACTTCCT 2.95 2285 (+) GGGGATYCCC GGCGACTTCC  0.754331

TABLE-US-00005 TABLE 6 Antibodies Antibody Company Cat. No WNT5A Thermo Scientific MA5-15511 FZD5 EMD Millipore 06-756 Human Albumin ABCAM AB28405 rat Albumin ABCAM AB53435 cREL CST 12659S GAPDH EMD Millipore MAB374 b-actin Abcam ab8229 ECL Plex goat-a-rabbit IgG Cy5 GE-Amersham PA45011 ECL Plex goat-a-mouse IgG Cy3 GE-Amersham PA43010V

TABLE-US-00006 TABLE 5 [CpG islands detected by MethPrimer (Li and Dahiya, 2002). Criteria: Island size >100, GC Percent >50.0, Obs/Exp >0.6): 5 CpG island(s) were found in the sequence], Related to FIGURES 3E, 3F, and 3G. Size (Start-End) Island 1 175 bp (137-311) Island 2 173 bp (483-655) Island 3 144 bp (664-807) Island 4 793 bp  (948-1740) Island 5 338 bp (1929-2266)

TABLE-US-00007 TABLE 7 Recombinant proteins Protein Company Cat. No h/mWNT5A R&D systems 645-WN hWNT3A R&D systems 5036-WN PEDF EMD Millipore GF134 TNFα Cell sciences CSI15659A Basic FGF Stemgent 03-0002

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Example 3

[0313] cRel and Wnt5a Frizzled 5 Receptor-Mediated Inflammatory Regulation Reveals Dual Targets for Neuroprotection

[0314] Wnt5a is engaged in a multitude of cell signaling processes. Here, we demonstrate that Wnt5a triggers inflammatory responses via NFkB/p65 and damage in retinal pigment epithelial (RPE) cells undergoing uncompensated oxidative stress (UOS) and in ischemic stroke. We found that Wnt5a Clathrin-mediated uptake leads to NFkB/p65 activation. Wnt5a is secreted in an exosome-independent fashion. Docosahexaenoic acid (DHA) and its derivative, Neuroprotectin D1 (NPD1), upregulate c-Rel expression that blunts Wnt5a abundance by competing with NFkB/p65 in the Wnt5a promoter A. Wnt5a increases in ischemic stroke penumbra and blood, while DHA reduces Wnt5a abundance with concomitant neuroprotection. Peptide inhibitor of Wnt5a binding, Box5, is also neuroprotective. DHA-decreased Wnt5a expression is concurrent with a drop in NFkB-driven inflammatory cytokines expression, uncovering mechanisms after stroke, as in RPE cells exposed to UOS. Limiting the Wnt5a activity via Box5 reduces stroke size, indicating neuroprotection sites pertinent to onset and progression of retinal degenerations and stroke consequences.

[0315] Wnt signaling pathways are associated with normal functions and pathology, including development and cancer (Nusse and Clevers, 2017). From the wingless family of ligands, Wnt5a is a secretory lipid-modified glycoprotein that, in certain cases, activates calcium-dependent signaling via interaction with Frizzled proteins, Ror1/2, RYK, and RTK (De, 2011). However, Wnt5a activity is driven by cellular context and is also able to activate beta-catenin via promiscuous interaction with LRP5/6 (Ring et al., 2014). During tissue morphogenesis and differentiation, Wnt5a is involved in synaptogenesis (Varela-Nallar et al., 2010) via Ca++ dependent signaling. Wnt5a has also been associated with inflammatory diseases like rheumatoid arthritis (Rauner et al., 2012; Sen et al., 2001) and atherosclerosis (Ackers et al., 2015). Moreover, Wnt5a is released by macrophages (Naskar et al., 2014; Pereira et al., 2008) to activate NFkB (Zhao et al., 2014). Because NFkB/p65 is a Wnt5a transcriptional activator (Katula et al., 2012), it heightens its own expression. Thus, we asked if the modulators of uncompensated oxidative stress (UOS) and cell survival (Calandria et al., 2012; Mukherjee et al., 2004), DHA/NPD1, regulate Wnt5a expression and its availability. Using human primary retinal pigment epithelial (hpRPE) cells, we have addressed events relevant to retinal degenerations by activating NPD1 synthesis from DHA (Bazan, 2006, 2007; Calandria et al., 2009) since these cells support photoreceptor integrity. In these cells, we show that cREL mediates Wnt5a transcriptional regulation by NPD1. In brain ischemia reperfusion, DHA fosters neuronal survival via NPD1 synthesis that in turn activates NFkB/cRel (Calandria et al., 2015). We provide evidence that Wnt5a is upregulated in stroke penumbra and augmented in the bloodstream, favoring activation of immune cells and their recruitment into damaged brain. In addition, DHA decreased bloodstream and penumbra Wnt5a abundance, leading to neuroprotection. Altogether, these results indicate inflammatory modulatory signaling mediated by DHA/NPD1 engages Wnt5a in responses to neural cell injury.

[0316] Docosanoids Inhibit UOS-Triggered WNT5a and Fzd5 Transcription with Concomitant Reduction in Apoptosis

[0317] Observations showed that UOS induced by H.sub.2O.sub.2 plus TNFα triggers NPD1 synthesis via 15-lipoxygenase-1 (15-LOX-1) in RPE cells and the silencing of this enzyme results in NPD1 depletion (Calandria et al., 2009). Six hours after the initiation of UOS, 15-LOX-1 deficient cells display a 2-fold increase in Wnt5a expression (FIG. 24A) that was brought down to below controls by NPD1. Also, DHA plus pigment epithelium-derived factor (PEDF), a neurotrophin agonist of NPD1 synthesis (Mukherjee et al., 2007), prevented Wnt5a upregulation in hpRPE cells (FIG. 24B). To test the idea that other docosanoids (FIG. 18Bi-vi) downregulated Wnt5a transcription, we confronted hpRPE cells for 6 hours using 1,600 μM H.sub.2O.sub.2 and 10 ng/ml TNFα (FIG. 18A) in the presence or absence of DHA (FIG. 18Bi), NPD1 (FIG. 18Cii), 10R, 17R diHDHA (FIG. 18Biii), Maresin-1 (FIG. 18Biv), RvD1 (FIG. 18Bv) or RvD2 (FIG. 18Biv). All docosanoids decreased the expression of Wnt5a to control levels (FIG. 18C). Recombinant Wnt5a potentiated cell death by UOS in ARPE-19 cells (FIG. 24C) and in hpRPE (FIG. 18D). A hexapeptide that corresponds to the amino acid portion 332 to 337 of Wnt5a with the t-Boc substitution in the N-terminal (t-Boc-NH-Met-Asp-Gly-Cys-Glu-Leu-CO.sub.2H (SEQ ID NO: 1)), Box5 is a Wnt ligand analog that blocks binding to receptors (Jenei et al., 2009). Box5 or NPD1 hindered apoptosis by UOS in the presence of the Wnt ligand. Wnt5a alone had no effect on RPE cells (FIG. 24C), indicating that the Wnt ligand enhanced apoptosis in susceptible cells undergoing UOS but not in resting cells.

[0318] To assess the co-regulation of Wnt5a receptors/co-receptors (FZD4 and 5, LRP5/6, RYK, and ROR1/2) (Mikels and Nusse, 2006), we assayed their expression by SYBR green-based real-time PCR. hpRPE were exposed to UOS, FZD5 mRNA rose 2-fold and then dropped to control levels when docosanoids were added (FIG. 18E). DHA leads to the synthesis of 10R, 17R diHDHA, Maresin 1, RVD1, RVD2, and NPD1 (FIG. 18B i-vi), however, this fatty acid alone did not affect FZD5 expression, indicating that its conversion to lipid mediators is required to counteract the effect of UOS on FZD5. Unlike FZD5, other Wnt signaling receptors: FZD1, FZD4, LRP5, LRP6, ROR2, and RYK, remained unchanged, although ROR1 expression was reduced under UOS and re-established by NPD1 (FIG. 18F). These results also advocate that FZD5 is linked to Wnt5a signaling in RPE cells undergoing UOS.

[0319] WNT5a-Dependent Activation of NFkB Requires FZD5 and ROR2

[0320] Wnt5a elicits positive and negative actions on β-catenin activity, depending on the receptor context and the presence of other Wnt ligands (Mikels and Nusse, 2006). We tested whether or not Wnt5a by itself activates β-catenin using TOP Flash/FOP flash reporter system in hpRPE cells undergoing UOS+/−NPD1 (FIG. 18G). In the absence of Wnt5a or Wnt3a, β-catenin activity was not significantly altered by UOS or NPD1 (FIG. 18H). When Wnt3a was added, luciferase rose almost twice, which is consistent with the β-catenin co-activation of TCF/LEF reporter system. Wnt5a alone did not affect β-catenin (FIG. 18H), indicating that, in this case, Wnt5a signaling in the absence of activated β-catenin does not involve TCF/LEF-related gene expression.

[0321] Without wishing to be bound by theory, Wnt5a interacts with FZD5 and ROR2 to trigger inflammatory gene expression via NFkB activation (Naskar et al., 2014; Sato et al., 2015). To determine if Wnt5a activates NFkB, hpRPE were transfected with a construct that encompassed 3 p65 high-affinity binding sites in tandem, driving the expression of the luciferase reporter gene (FIG. 18G). hpRPE cells exposed to UOS showed increased luciferase activity, and NPD1 did not affect NFkB/p65 as shown previously (FIG. 18I) (Calandria et al., 2015). Exposure of 2 hours of Wnt5a heightened NFkB activation in the presence of UOS, and NPD1 reduced luciferase activity (FIG. 18I). These results indicate that the activation of NFkB/p65 via Wnt5a is exerted by a different signaling pathway than the one triggered by UOS/TNFα, and in this case, the effect is responsive to NPD1 (FIG. 18K). To assess whether or not FDZ5 and ROR2 were involved in NFkB activation by Wnt5a, cells were co-transfected with siRNAs targeting the 2 receptors (FIG. 25). hpRPE cells co-transfected with control siRNA showed the same pattern of NFkB activity seen in FIG. 1I. ROR2 or FZD5 siRNAs separately abolished the difference between UOS and UOS+NPD1 (FIG. 18J). The co-transfection of both siRNAs, against ROR2 and FZD5 together, induced a higher NFkB activation that was not affected by NPD1. When Wnt5a was added to the double knockdown cells, the NFkB activity did not differ from controls, but it did show a slight yet significant difference with the UOS-treated cells, indicating both receptor and co-receptor are required to activate NFkB/p65.

[0322] NPD1 Stimulates cRel Binding to Wnt5a Promoter a that in Turn Prevents Transcriptional Activation

[0323] The Wnt5a gene is under the regulation of 2 promoters located in exon1a and exon2 (Vaidya et al., 2016). Promoter A drives the expression of the largest form, variant-1, and contains at least 2 binding sites for NFkB (Katula et al., 2012). In-silico analysis using TRED software showed that both sites have a high affinity for 3 NFkB members: p50, cRel, and p65. Downstream, p50 and cRel binding are opposed to p65 site, indicating that p65 and cRel activity compete to oppose each other (FIG. 19A and Table S1). To confirm a direct link between cRel expression and Wnt5a transcription, we over-expressed the transcription factor in hpRPE (FIG. 19B). cRel overexpression decreased Wnt5a mRNA (FIG. 19B). The increase in cRel availability dominantly shut off Wnt5a expression regardless of the treatment. Altogether, the data indicate that cRel blocks Wnt5a expression triggered by UOS, which may be a key modulatory NPD1 function.

[0324] Based on the regions found to bind NFkB members (Table S1), we designed 4 sets of primers that bound in the proximity or within the regions of interest to perform SYBR green-based real-time PCR detection (FIG. 19D-F and Table S3). FIGS. 19D and F and Table S2 also show regions of high probability for methylation, a mechanism to block Wnt5a transcription (Vaidya et al., 2016). Micrococcal DNase digested genomic DNA fragments from UOS or UOS-plus-NPD1-treated RPE cells, +/−rWnt5a, were pulled down by a cRel antibody and used to test the 4 sets of primers. Amplicon A1, localized close to Region 1 and overlap to Region 2 cRel/p65 binding sites (FIG. 19D), showed no differences between treatments, indicating no differential binding of cRel to the genomic DNA in the presence of Wnt5a and NPD1 (FIG. 19E). Methylation of Region 1 may occur since it was predicted by the Methprimer software (Table S5). The amplicon 2 encompassing Region 3 and 4 displayed twice the amount of cRel bound to genomic DNA under NPD1 treatment. Within the proximity of Region 5 and overlaps Region 6 (FIG. 19F), the amplicon 3 displayed the largest differences between treatments, reaching more than 20-fold when NPD1 was added to RPE cells undergoing UOS in the absence of Wnt5a and 10-fold when Wnt5a was present. The results obtained for amplicon 3 indicate competition between the Wnt5a-activated p65 and NPD1-activated cRel (FIG. 19E). Finally, Amplicon 4, which is located upstream of Region 7, showed no differences in the absence of Wnt5a but did show a decrease in RPE cells undergoing UOS in the presence of rWnt5a. NPD1 restored the cRel binding to control levels, indicating that in the presence of NPD1, cRel displaces the initially bound p65 (FIGS. 19E and F). These data indicate that there is a binding interactive competition between p65 and cRel that depends on availability and other factors, such as methylation for NPD1-mediated cell survival.

[0325] Extracellular Availability of Wnt5a is Mainly Endosome-Free and is Controlled by NPD1

[0326] Western blot assays using cellular lysates and medium (precipitated by Methanol/Chloroform to bring down secreted Wnt5a) from hpRPE undergoing UOS+/−NPD1 to assess Wnt5a release were used. We found that UOS induced an increase in secreted Wnt5a in ARPE-19 cells (FIG. 20A). NPD1 addition decreases band intensity close to control levels (FIG. 20A). Wnt5a cellular abundance remained constant for all the treatments in cells including 15-LOX-1d cells, indicating a tight regulation of the balance between secreted (sWnt5a) and cellular Wnt5a (cWnt5a) (FIGS. 20A and B).

[0327] Frizzled 5 mRNA expression was found to be upregulated by UOS in hpRPE cells and downregulated by NPD1. To determine the FZD5 protein availability, a western blot assay was performed in cell extracts from hpRPE cells exposed to UOS 1+/−NPD1 or Box5 when Wnt5a was added. Contrary to the levels of mRNA (FIG. 18E), cells exposed to UOS displayed a similar FZD5 content than controls, indicating receptor regulation by degradation. The presence of Box5 decreased FZD5 abundance by half of the level, even in the presence of Wnt5a, showing that the inhibition of the binding of the true ligand enhances receptor degradation as well. Intriguingly, when NPD1 was added in the absence of Wnt5a, no differences in the FZD5 receptor were observed although, when the ligand was present, a steep decrease in the receptor content was evident (FIG. 20C), indicating that NPD1 enhances FZD5 degradation via internalization of the FZD5/Wnt5a complex.

[0328] Wnt5a display in western blots, besides the light band that ran between 52 and 38 KDa, a higher molecular mass band with all the antibodies used in this study. Since Wnt5a is glycosylated and palmitoylated (Kurayoshi et al., 2007), we tested if the band above 52 KDa was a highly glycosylated form of the ligand by total deglycosylation (Degly) of the precipitated medium in comparison non-deglycosylated samples (Gly). Deglycosylation followed the patterned of intensity observed when the cells were exposed to UOS+/−NPD1 or Box5 (FIG. 20D), however, the samples that were not digested showed a different pattern due to different affinity of the antibodies to the modified Wnt5a. Overexpression of Wnt5a showed that Wnt5a increases after deglycosylation, but not noticeable change in the hyper glycosylated state by the antibody (FIG. 26), indicating that hyper glycosylated secreted protein may be a mechanism by which the cell modulates ligand activity.

[0329] Active Wnt5a can be released in exosomes (Gross et al., 2012). To address whether Wnt5a was released by hpRPE cells in exosomes we performed medium ultracentrifugation from cells undergoing UOS for 10 hours (FIG. 20E). Western blot of the first pellet after spinning at 300 rpm (dead cells pulled-down), showed a 35 KDa band as that of mature Wnt5a. The bands pattern closely resembles the one in whole cells (FIG. 20F). Pellets of 2,000 and 10,000 rpm, containing cell debris (FIG. 20E), displayed no bands and the pellet from ultracentrifugation at 100,000 rpm (exosomes) lack a 42 KDa band. The supernatant was then precipitated using Methanol/Chloroform and a 42 KDa emerged, indicating that most of Wnt5a is not contained in exosomes. However, a band above 52 KDa appeared in the pellet obtained in the 100K×g centrifugation and a very intense band in the supernatant that was further precipitated, indicating that the glycosylated forms were present in exosomes, but mainly in soluble manner. These results indicated that Wnt5a is mainly release in an exosome-free manner by hpRPE cells undergoing UOS.

[0330] Wnt5a/FZD5 Clathrin-Mediated Endocytosis is Required for NFkB Activation.

[0331] Wnt5a is processed to maturity in the ER/Golgi where it binds to its receptor and join the recycled protein pool (Willert and Nusse, 2012). To ascertain localization and interaction between FZD5 and Wnt5a, we incubated hpRPE undergoing UOS with Wnt5a+/−NPD1 or other lipid mediators for 2 hours. Immunocytochemistry in detergent-permeabilized cells was analyzed by BioImageXD to assess colocalization of FZD5/Wnt5a signal and by ImageJ or IMARIS software to count colocalized objects in each field. The mean of vesicles/field that showed colocalization of FZD5/Wnt5a was increased with the addition of the ligand in the control and UOS cells (FIG. 21A-C), indicating that the Wnt ligand promotes its own internalization. NPD1 and its bioactive stereoisomer 10R, 17R diHDHA decreased the amount of vesicles per field in the presence or absence of Wnt5a, but not the other docosanoids and related bioactive lipids tested (FIG. 21i).

[0332] The population of the FZD5/Wnt5a vesicles showed various amplitudes of sizes (FIG. 21A, C, D and FIG. 27C). The addition of Wnt5a induced an increase in the size of vesicles containing FZD5 receptor/Clathrin and, Wnt5a/Clathrin in resting cells reflecting an increase in FZD5 and Wnt5a internalization. However, the vesicles that showed colocalization between the FZD5 and Wnt5a remained unchanged in control cells, indicating that at least part of the Wnt5a uptake may be mediated by other receptors in resting cells (FIG. 21 D). Conversely, under UOS conditions, the size of the vesicles colocalizing the signals FZD5/Wnt5a, FZD5/Clathrin and Wnt5a/Clathrin decreased with the addition of Wnt5a to the medium, showing the same trend and thus indicating that under oxidative stress conditions Wnt5a and FZD5 may be internalized together via clathrin. Neither NPD1 nor Box5 affected the magnitude of the Wnt5a effect on the size of FZD5/Wnt5a vesicles under UOS conditions. FZD5/Clathrin and Wnt5a/Clathrin vesicle sizes remained unchanged with the addition of Wnt5a to UOS cells+/−NPD1 or Box5 (FIG. 21 D). Together, the variation in vesicle size points to a complex mechanism of uptake of Wnt5a that involves clathrin, FZD5 and/or other receptors.

[0333] To determine vesicles load, we used the mean of the intensity as an indicator for each vesicle. Furthermore, to assess the role of enhanced expression Wnt5a on the load of the vesicles, we overexpressed variant 1 of the human Wnt5a and subjected them, along with control hpRPE cells, to UOS. The load of the FZD5/clathrin, Wnt5a/clathrin and Wnt5a/FZD5 vesicles in control cells overexpressing and exposed to external Wnt5a vs control showed the same pattern; the control vesicles did not differ in the load between overexpressing the ligand and cells exposed to external Wnt5a. However, cells that overexpressed Wnt5a depicted more intensity than those exposed to external Wnt5a for the 3 types of colocalization observed, indicating that increased Wnt5a expression triggers enhanced upload of FZD5 and Wnt5a in the vesicle. Moreover, Wnt5a overexpression mimicked the intensity observed in the UOS-treated cells for the 3 types of colocalization in agreement with the consequences of oxidative stress. The addition of Wnt5a raised the intensity of the vesicles as well. External Wnt5a did not affected vesicle loads that showed FZD5/clathrin or Wnt5a/clathrin signal in the presence of Box5 under UOS but did affected those in which Wnt5a and FZD5 colocalized (FIG. 21 E). In addition, cells exposed to UOS and NPD1 displayed higher Wnt5a/FZD5 and FZD5/Clathrin vesicle intensity when Wnt5a was added, but Wnt5a/Clathrin remained unaffected, indicating that the internalization of Wnt5a have proceed via FZD5/Clathrin (FIG. 21 E).

[0334] To assess the vesicles that were positive for Wnt5a, FZD5 or both, we analyzed the confocal z-stacked pictures for control and UOS alone or treated with Wnt5a Box5 and NPD1 (FIG. 21 F upper panels depicts the IMARIS rendering of the actual flattened Z-stack picture in lower panels). UOS increased the number of Wnt5a- and FZD5-positive vesicles compared to control cells. While Box5 and NPD1 decrease the number of FZD5-positive vesicles, the addition of Wnt5a raised the number and the mean intensity of vesicles showing Wnt5a signal in agreement with FIG. 21 B. Box5 did not modify the increase in intensity and number induced by UOS, however, Wnt5a-positive vesicles displayed a difference in mean intensity distribution when NPD1 was added.

[0335] Wnt5a signaling required FZD5 and ROR2 to activate p65/NFkB under UOS conditions (FIG. 18J). The pattern of colocalization between FZD5/Clathrin, Wnt5a/Clathrin and Wnt5a/FZD5 (FIGS. 21 D and E) are compatible with Wnt5a/FZD5 internalization mediated by Clathrin- or Clathrin-mediated endocytosis (CME) (Feng and Gao, 2015). Thus, we asked if sWnt5a plus FZD5 internalization is linked to p65/NFkB activation when hpRPE cells were transfected with the NFkB/p65 reporter construct with UOS+/−200 nM NPD1 plus 50 ng/ml rWnt5a (FIG. 22A). We found that the addition of Pitstop2 in cells undergoing UOS in the presence of Wnt5a decreases the activation of p65/NFkB, indicating that the internalized Wnt5a is responsible for NFkB activation (FIGS. 22B and C).

[0336] Wnt5a overexpression disclosed a high number of vesicles that colocalize with FZD5/Wnt5a signal as well as with Wnt5a- and FZD5-positive vesicles, even in the presence of NPD1. FZD5 overexpression display low number of FZD5 vesicles while the overexpression of ROR-2 induced a steep increase in the Wnt5a/FZD5- and Wnt5a-positive vesicles in controls and NPD1-treated cells, but more so in cells undergoing UOS (FIG. 22D). The co-expression of ROR-2 and FZD5 increased the number and intensity of the expression of the 2 genes separately, supporting the observation in FIG. 18G, which shows FZD5, ROR2 and Wnt5a signal together to activate NFkB.

[0337] Without wishing to be bound by theory, the heterogeneity of the signal within the vesicles, the size and the intensity observed by immunocytochemistry (FIG. 22F) indicate that NPD1 enhances internalized Wnt5a degradation by favoring the conversion of early endosome to lysosome pathway. Large vesicles that were labeled positive for Wnt5a are shown (FIG. 22G). By increasing Wnt5a degradation, NPD1 can induce a decrease in Wnt5a-triggered NFkB induction independently of cRel activation. Altogether, the results agree with the mechanism indicated in FIG. 22H.

[0338] Ischemic Stroke Activates Wnt5a Expression: DHA and Box5 Elicit Neuroprotection

[0339] Intravenously (IV) DHA reduces ischemia-reperfusion (I-R) brain damage. To test whether IR increases Wnt5a secretion and signaling, we induced stroke in rats by middle cerebral artery occlusion (MCAo) for 2 hours and, 1 hour later, injected IV saline (vehicle) or DHA. Neurological scores, tactile and proprioceptive tests 1, 2, 3, and 7 days after MCAo showed severe neurological impairments in saline-treated rats (FIGS. 23A and B). DHA treatment improved neurologic scores, including tactile (dorsal and lateral) and proprioceptive forelimb placing reaction (FIG. 23B). To test whether Wnt5a is involved in post ischemia-reperfusion damage, we injected the receptor blocker Box5 after MCAo (FIG. 23A) and found neurological protection remarkable similar to that obtained by DHA treatment (FIG. 23D). Moreover, MRI illustrated decreased volume of brain damage (FIGS. 23 D and E) resembling those observed by DHA injection (Belayev et al., 2017). Furthermore, we assessed Wnt5a mRNA in A1 penumbra, A3 stroke core, and as control A2 and A4 that correspond to contralateral parts of A1 and A3 (FIG. 23K). The penumbra, an area surrounding the ischemic core, is subject to moderate damage and may survive the ischemic reperfusion when treatment is applied. We found that the Wnt5a mRNA was increased in the ipsilateral hemisphere 1-3 days post-surgery and that DHA blocked this surge (FIG. 23F). After stroke, both hemispheres work synergistically to overcome damage (Buga et al., 2008). In this case, the increase in Wnt5a mRNA level was detected only in the ipsilateral hemisphere; the contralateral showed no surge in Wnt5a expression 1-3 days after surgery, indicating a local induction of mRNA expression. However, Wnt5a protein abundance showed that the levels of ipsilateral and contralateral hemispheres A1 and A2 did not differ from one another, and they were both high in saline and low in DHA-treated animals (FIG. 23I). In addition, Wnt5a was enhanced in blood after MCAo 1 day post-stroke and was decreased at day 3 (FIG. 23G). Impairment of the interaction Wnt5a/receptor with Box5 that induced changes in the size of the infarct observed in the MRI (FIGS. 23D and E) and an improvement in neurological score (FIG. 23C) failed to reduce the plasma Wnt5a content, indicating a difference in the action between DHA and Box5. In agreement with the activation of NFkB/p65, the expression of NFkB-activated inflammation mediators IL6, TNFα, CCL1, MCP1, and IL1(3 follows the same trend as Wnt5a after DHA (FIG. 23J). MMP13, MMP2 and MMP9 expression is enhanced when Wnt5a-ROR2 is activated (Yamagata et al., 2012). The 3 mRNAs showed the same trend of expression as Wnt5a (FIG. 24J), indicating the activation of ROR2 by Wnt5a. Other genes, such as E-selectin and ICAM-1, that are involved in inflammatory signaling and are known to be activated by Wnt5a (Kim et al., 2010), were found to follow the same pattern of Wnt5a expression. These results indicate that the effect of Wnt5a on those genes is restricted specifically to the penumbra, not the contralateral side. As Wnt5a is available in both hemispheres, stress is required for Wnt5a to act as an inflammatory mediator. These results altogether point to Wnt5a as a non-conventional inflammation mediator and DHA/NPD1 signaling as a regulatory mechanism that specifically switches off Wnt5a-triggered gene expression and Wnt5a extracellular availability.

[0340] Discussion

[0341] Wnt5a fosters neuronal survival by negatively regulating the cell cycle (Zhou et al., 2017), protects against neurodegeneration through glucose metabolism promotion (Cisternas et al., 2016) and displays high abundance that correlate with the aggressiveness of cancer (Binda et al., 2017). We found that several forms of Wnt5a are released by human RPE cells and that deglycosylation allows visualization of Wnt5a. We are currently validating whether glycosylation is a cellular regulatory mechanism of Wnt5a extracellular availability. In addition, the context in which Wnt5a targets a cell also determines its activity. By characterizing the vesicles containing FZD5/Wnt5a, FZD5/clathrin or Wnt5a/clathrin, we defined that the variation does not always follow the same pattern for the 3 types of vesicles (FIGS. 21D and E), indicating that other receptors are involved in the inflammatory signaling of hpRPE cells. Wnt5a also triggers the internalization of FZD4 (Chen et al., 2003), though whether or not this pathway is conducive to the activation of NFkB is unclear. Moreover, the presence of Wnt5a enhanced apoptosis beyond the levels induced by UOS, resembling TNFα action (FIG. 18D and FIG. 24), whereas the sole presence of Wnt5a did not trigger RPE cell death (FIG. 18D and FIG. 24C). Thus, within cells in a susceptible state, Wnt5a affects their fate since they may succumb to initial insults, such as UOS, adding another layer of regulation to the Wnt ligand function. Insults and exposure to Wnt5a enhances susceptibility to containment of cell integrity and survival.

[0342] Wnt ligands are secreted via exosomes (Gross et al., 2012), but Wnt5a was not in the exosomal form in hpRPE cells when we extracted them by ultracentrifugation (FIG. 20F) and by differential solubility. Only by precipitation of 100,000 rpm supernatant did we rescue the sWnt5a (FIG. 20F). Therefore, in RPE cells under UOS, our data show that even Wnt5a is trafficked via vesicles (FIG. 22H), and its release is produced mainly in an exosome-free manner (FIG. 20F). Wnt5a signal was found alone and together with FZD5, signifying that at least 2 distinctive events occur in RPE cells (FIGS. 21A, D, G and H). Wnt5a signal increases with the addition of recombinant Wnt5a (FIG. 21F and FIG. 23B), reflecting the ability of Wnt5a to enhance not only its own expression but also FZD4, 2, and 5 receptor endocytosis (Chen et al., 2003; Kurayoshi et al., 2007; Shojima et al., 2015). Vesicular Wnt5a was detected in RPE cells (FIG. 21 and FIG. 22) as was secretion of mature Wnt5a in vesicles, and Golgi supported maturation of the protein (Kurayoshi et al., 2007). Therefore, different size vesicles carrying Wnt5a detected in RPE cells (FIG. 21F) can harness maturation, degradation and sorting of Wnt5a through a dynamic, steady state with intracellular Wnt5a remaining constant (FIG. 20A-D) while extracellular release is controlled; transcription is variable depending on UOS (FIG. 18D and FIG. 19C; FIG. 25 and FIG. 25). NPD1 decreases soluble Wnt5a during UOS (FIG. 20A-D and FIG. 22D) while CME-inhibitor, Pitstop2, interrupted Wnt5a endocytosis in resting cells and in NPD1 treated cells, ensuing in an increase in sWnt5a under these conditions (FIG. 22D) and pointing to the role of NPD1 in the fate of secreted Wnt5a.

[0343] As a macrophage activator in the non-sterile innate response (Pereira et al., 2008), Wnt5a also mediates non-sterile inflammatory responses in non-immune cells (Zhao et al., 2014). Our current results indicate that non-immune cells evoke the inflammatory response in sterile conditions. The signaling pathways involved in RPE cell damage and in stroke display similarities in the stroke penumbra, Wnt5a synthesis increase (FIGS. 23F and I) in the ipsilateral side (FIG. 23F), Wnt5a protein elevation in both sides (FIG. 23I) and presence in the blood plasma after stroke, providing a potential stroke biomarker candidate. Systemic Wnt5a can be involved in the recruiting and stimulation of innate immune cells since Wnt5a activates microglia, dendritic cells and macrophages (Shimizu et al., 2016), enhancing other pathways such as non-canonical Wnt and TLR-triggered signaling. DHA treatment brought down bloodstream Wnt5a protein after 24 hours of stroke onset (FIG. 23G). This was an early event considering that at 3-7 days after stroke the levels of Wnt5a in blood decreased. Box5 administration did not affect Wnt5a abundance as DHA did, indicating that Box5 and DHA act differently. Without wishing to be bound by theory, DHA conversion into NPD1 activates cRel expression (Calandria et al., 2015), which in turn halts Wnt5a transcription, release and autocrine and paracrine binding to FZD5, while Box5 impedes the latter. Our data show Box5 and DHA protection at the neurological/behavioral level as well as by reduction of stroke damage (FIG. 23C-E); however, DHA seems to produce a sustained improvement. The source of circulating Wnt5a is unknown and currently under investigation. There are at least 2 sources of secreted Wnt5a: blood cells such as monocytes (Sessa et al., 2016) or endothelial cells that produce the Wnt ligand in certain conditions, inducing permeabilization and angiogenesis (Korn et al., 2014; Skaria et al., 2017). The elevation in Wnt5a found in the contralateral hemisphere (FIG. 23F), can be explained by damage or permeabilized brain blood barrier that allows the entrance of the circulating Wnt ligand. Alternatively, without increasing its transcription, the hike in the contralateral Wnt5a protein may be explained by glutamate excitotoxicity in the contralateral area at a lesser degree than in the ipsilesional side (Li et al., 2012). Finally, gene expression linked to Wnt5a, UOS and inflammation via NFkB/p65 or Wnt signaling markedly increased in ipsilateral but not in the contralateral side, and such an increase was counteracted by DHA as indicated by the gene expression profile (FIG. 23J).

[0344] Transcellular inflammation signaling is not well understood. Without wishing to be bound by theory, non-immune cells under UOS conditions can transfer inflammatory signals that may only affect susceptible cells and lead to their damage. NPD1 interferes with the Wnt5a feedback loop at 2 strategic signaling points, promoting cell survival in bystander cells. The ischemic stroke model provided an in vivo test of our observations at the RPE cell level. We have previously found that this lipid mediator synthesis correlated with cRel, a member of NFkB/p65, activity in RPE cells and in post-stroke penumbra (Calandria et al., 2015). We demonstrate that cRel binds to at least 2 regions in the Wnt5a promoter with high affinity (FIG. 19D-F and Table S4). cRel overexpression suppresses the Wnt5a transcription in response to UOS probably displacing those NFkB dimers containing p65 (FIG. 19A-C). Post-stroke, we observed a similar trend seen in the RPE: Wnt5a transcription was elevated at 1 and 3 days after stroke only in penumbra at the ipsilateral hemisphere while the expression in the contralateral side was not affected (FIG. 23F). These results indicate that only susceptible cells affected directly by ischemia reperfusion promote the Wnt5a positive feedback loop at the transcriptional level. DHA enhances NPD1 synthesis and induces translocation of cRel in neurons (Calandria et al., 2015), which prevents p65-driven activation of Wnt5a transcription. Stressors like NMDA glutamate receptor activation trigger transcription-independent Wnt5a translation (Li et al., 2012), which means that Wnt5a transcription and translation may be uncoupled events and may explain some of our observations (FIGS. 23F and I). Therefore, without wishing to be bound by theory, translation alone is linked to secretion and sequestration from the extracellular space via CME in the RPE (FIG. 22H) and may be plausible in ischemic stroke as well.

[0345] These findings uncovered a new participant in the transfer of inflammatory signals occurring in retina and brain under UOS and how endogenous neuroprotection mediators derived from DHA may halt damage to enhance cell survival. The understanding of these new neuroprotective cellular and molecular mechanisms will allow the exploration of therapeutic avenues to target onset and early progression of brain and retina damage that include neurodegenerative diseases.

[0346] Experimental Model and Subject Details

[0347] Transient Middle Cerebral Artery Occlusion (MCAo)

[0348] Animals were housed and treated in compliance of LSU Health Sciences Center Institutional Animal Care and Use Committee (IACUC) protocols. The right MCA was occluded for 2 h by intraluminal filament, as we described previously (Belayev et al., 2011). Briefly, the right common carotid artery (CCA) and external carotid artery (ECA) were exposed through midline neck incision, and then completely isolated from the surrounding nerves. The occipital branches of the ECA and pterygopalatine artery were ligated. A 4-cm of 3-0 nylon filament, coated with poly-L-lysine was advanced to the origin of MCA through the proximal ECA via internal carotid artery. The filament was inserted 20 to 22 mm from the bifurcation of the CCA, according to the animal's body weight. The neck incision was then closed and the rats were returned to their cages. After 2 h of MCAo, the rats were re-anesthetized with the same anesthetic combination and the intraluminal filament was gently removed. The animals were allowed to survive for different times, according to the experimental protocol, with free access to water and food.

[0349] Cell Culture, Treatments, and Transfection

[0350] Primary human RPE cultures were obtained from human eyecups, provided by the National Disease Research Interchange (NDRI), as described previously (Calandria et al., 2012). hRPE cells were grown and maintained in high-glucose MEM (Life Technologies Corporation) supplemented with 10% FBS (Tissue Culture Biologicals, Inc.), 5% NCS, non-essential amino acids, Penicillin-Streptomycin (100 U/mL), human fibroblast growth factor (FGF) 10 ng/mL and incubated at 37° C. with a constant supply of 5% CO2. ARPE-19 cells, were plated and grown in DMEM/F-12 containing 10% FBS and 1× penicillin/streptomycin at 37° C., 5% CO2, 99% relative humidity for 24 h. Silenced 15-LOX-1 cells, described in detail elsewhere (Calandria et al., 2009), are derived from ARPE-19 by stably silencing 15-LOX-1. 15-LOX-1 deficient cells were maintained in the same medium as ARPE-19 with the addition of 500 μg/mL Geneticin (Life Technologies Corporation). Transfection was performed in all cases using Lipofectamine 2000 (Life Technologies Corporation) following the manufacturer's recommendations. Transfection efficiency was assessed using an expression plasmid-carrying GFP ORF or by including a negative control siRNA conjugated with Alexa Fluor® 488 or 546 (QIAGEN). siRNA efficiency was assessed using SYBR green-based real-time PCR using the primers of Table S4 and the percentage of silencing achieved (FIG. 24). Experiments with transfection efficiency that yielded less than 80% were discarded. For siRNA, we used 6 μl of Lipofectamine 2000 per 50 pmol of siRNA per mL of incubation medium, and, for plasmids, we used 6 μl of Lipofectamine 2000 per 2 μg of plasmid per mL of incubation medium. Cells were incubated for 5 hours at 37° C., 5% CO2 and 99% relative humidity. After the medium was changed, the cells were left to recover for 24 hours before starting treatments. When siRNA/plasmid co-transfection was required, siRNA and plasmid-carrying transfection mixes were prepared separately and added one after the other in the same well. Oxidative stress treatments were performed using 600 (ARPE-19) or 1600 (hRPE) μM H2O2, along with 10 ng/mL TNFα, for the required time indicated in each experiment (these concentrations were previously tested to achieve over 50% apoptosis on ARPE-19 and hpRPE cells). The treatment we used employed the low serum (0.5% serum) without hFGF. Treatment to induce uncompensated oxidative stress (UOS) was performed after 8 hours of low-serum medium incubation unless specifically noted. Wnt5a, Wnt3a or NPD1 was added immediately before induction of UOS. In the secretion experiments, Pitstop 2 was added to the medium 2 hours before harvesting.

[0351] Immunocytochemistry and Hoechst Staining

[0352] Immunostaining was performed in 8-well slide chambers and the Hoechst staining in 24 well plates. To carryout immunocytochemistry, a previously described protocol was followed (Calandria et al., 2012). Briefly, cells were fixed using 4% paraformaldehyde in PBS 1× for 20 min at room temperature or overnight at 4° C. After three washes with PBS, cells were permeabilized using 0.1% Triton™ X-100 for five minutes and blocked using 10% normal serum and 1% BSA for one hour. Primary antibody incubation took place overnight at 4° C. and secondary antibody coupled to Alexa Fluor® 488 and 594 were used to detect Wnt5a and FZD5 respectively. Hoechst staining was performed using same protocol of fixation, but permeabilization was done using methanol for 20 min at room temperature. Immediately after 20 ng/mL Hoechst 33342 (Life Technologies) in PBS 1× was added. Images for Hoechst staining were obtained using Nikon Ti-U inverted fluorescence microscopes with NIS-Elements BR 3.00 software (NIKON Inc, Melville, N.Y., USA) and ICC images from Olympus FV1200 confocal with Fluoview software FV10-ASW Version 04.02.02.09 (Olympus Corp Center Valley, Pa., USA). The analysis was performed using ImageJ 1.48 (National Institutes of Health). Dying cells were identified by size (0 to 50 pixels), intensity threshold and circularity. The ratio of hyperpyknotic cells over the total was calculated for nine randomly chosen fields per sample obtained from three independent wells per experiment. Each experiment was repeated at least three times to confirm the findings. In the experiments involving early stages of apoptosis, we assessed the total number of cells per field and counted them to ensure unbiased observations. Colocalization of Wnt5a and FZD5 was performed using BioImageXD (Kankaanpaa et al., 2012) and IMARIS 9.3 (Bitplane, Oxford instruments, Belfast, UK) on z-stack images obtained at 20× magnification in an Olympus FluoView1200 confocal laser-scanning microscope. Settings were adjusted in the conditions that showed higher intensity and were used throughout the remaining samples. Pearson's colocalization coefficient (PCC) was obtained using FV1200 analysis software for every field in each experiment, and the mean and SEM are depicted in Figures S1-S5. Validation of the FZD5 primary antibody used in the study is depicted in Figure S2. Colocalized objects and plots of pixels vs intensity were obtained using ImageJ (Schneider et al., 2012). IMARIS software analysis was performed building a colocalization channel using the Costes thresholding, and the colocalized elements were analyzed using the spots function using the settings different spots sites, background subtraction and local contrast. The output obtained consisted on a collection of sizes (one per element in each picture) or the mean of the intensity in each spot. The data was plotted as boxplot and analyzed using BioVinCi 1.1.5 (Bioturing, San Diego, Calif., USA).

[0353] Protein Precipitation and Western Blot

[0354] When secretion of Wnt5a was assessed, 1 mL of medium was collected, centrifuged at 13,000 rpm/5 min at 4° C. to remove cell debris and precipitated using methanol/chloroform (Wessel and Flügge, 1984). The pellet was re-suspended and denatured in 100 μl of 2× Laemmli sample buffer (Bio-Rad Laboratories) at 95° C. for 5 min. MCAo brain tissue and cell samples were homogenized using RIPA (Thermo Fisher Scientific) buffer supplemented with protease and phosphatase inhibitor cocktails (Sigma). The amount of 30 μg of total protein was loaded in NuPAGE® Novex® 4-12% Bis-Tris precast gels (Life Technologies Corporation) or Mini-PROTEAN TGX Stain free gels 4-15% and ran at constant 120V for 1 hour and 20 min or 250 V for 40 min respectively. Proteins were transferred to μm Nitrocellulose or LF-PVDF membrane using Bio-Rad Trans-Blot® Turbo™ System (Bio-Rad Laboratories). ECL™ Plex Fluorescent Rainbow Markers (GE Healthcare) were used as the ladder for protein's molecular weight. Membranes were blocked using 5% non-fat dry milk (Bio-Rad Laboratories) in TBS with 1% Tween® 20 (TBST-10X) for 1 hour and incubated overnight with primary antibodies. Anti-mouse or anti-rabbit secondary antibodies conjugated with Cy3 or Cy5 were used to visualize the protein of interest. Immunoblots were documented using LAS 4000 imaging system (GE Healthcare Life Sciences) or ChemiDoc MP Imaging System (BioRad Laboratories). A time course exposition was produced in each case to prevent quantification of saturated images. Densitometry data was obtained using ImageQuant™ TL software and XRS Blot Chemi Software (BioRad Laboratories).

[0355] SYBR Green-Based Real-Time PCR

[0356] Brain samples were homogenized on ice by Dounce homogenizer and total RNA was extracted by TRIzol Reagent (Life Technologies Corporation). Cell samples, total RNA was extracted by RNeasy Mini Kit (QIAGEN) following manufacturer's protocol. The purity and concentration of RNA were determined by NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). cDNA first strand was obtained from one microgram of total RNA using iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, CA, USA). The resulting cDNA was used as template SYBR-green or Eva-green based real-time PCR quantification using SsoAdvance Universal Supermix (Bio-Rad Laboratories). Data was collected and analyzed using CFX Manager 3.0 software, the ΔΔCt method. Melting curve was produced for every run to assure a unique amplified product per primer set. Primers are depicted in Table S4.

[0357] Luciferase Assay

[0358] For activation of canonical NF-κB, cells were co-transfected with plasmid p65/p50 promoter consensus sequence Cignal NF-κB Reporter Kit, (QIAGEN) along with both positive (constitutively-active promoter) or negative controls and GFP by using Lipofectamine 2000 (Life Technologies) following company protocols. β-catenin activity was measured using TOP Flash/FOP Flash constructs obtained from Addgene (Cambridge, Mass., USA) (Veeman et al., 2003). Except TOP/FOP flash, the Luciferase activity was standardized using a construct expressing GFP constitutively under CMV virus promoter. To assess luciferase activity, cell lysates were obtained using Passive Lysis Buffer (Promega) and mixed with a luciferase assay reagent (Promega). Chemiluminescence produced by luciferase and fluorescence from GFP was detected using Appliscan 2.3. Data were analyzed using SkanIt 2.3 (Thermo Fisher Scientific).

[0359] Detection of DNA Binding Motifs and Methylation

[0360] To analyze the consensus binding sequences of the Wnt5a promoter A (Katula, et al., 2012), two searching engines were used: TRED (http://rulai.cshl.edu/TRED), which uses the JASPAR database, and TFBind (http://tfbind.hgc.jp/) that uses TRASFAC database (Jiang et al., 2007; Tsunoda and Takagi, 1999). Table S1 contains scores and positions obtained for the 7 regions identified that cREL potentially binds (FIG. 19). Analysis of the methylation was performed using MethPrimer (http://www.urogene.org/methprimer2/) (Li and Dahiya, 2002).

[0361] ChIP Assay

[0362] The chromatin immunoprecipitation assay was performed using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, Boston, Mass., USA) following the manufacturer's recommendations including ChIP validated cREL antibody. Positive control Histone H3 antibody and normal serum was provided by the kit, as well as control primers for human RPL30 Exon 3. Primers A1 to 4 were designed using Primer-Blast at NCBI platform (Ye et al., 2012) and are depicted in Table S5. The immunoprecipitated samples real-time PCR values were standardized using 10% of the input chromatin preparation using primers A1, A2, and A3 pooled values and RPL30 Exon 3 values.

[0363] Behavioral Tests

[0364] Behavioral tests were conducted before, during MCAo (at 60 min), and then at 24 h, 48 h, 72 h, or 7 days after MCAo by an investigator who was blinded to the experimental groups. The battery consisted of two tests, (1) postural reflex to examine the upper body posture when the rat was suspended by tail, and (2) forelimb placing test to assess the forelimb placing responses to visual, tactile and proprioceptive stimuli (Belayev et al., 2011). Neurologic function was graded on a scale of 0 to 12 (normal=0, maximal deficits=12), as we described previously (Belayev et al., 2011). The severity of stroke injury was assessed by behavioral examination of each rat at 60 min after onset of MCAo. Rats that did not demonstrate high-grade contralateral deficit (score, 10-11) were excluded from further study. Two animals were excluded for this reason.

[0365] Treatment Groups

[0366] Docosahexaenoic acid (DHA; 5 mg/kg, Cayman, Ann Arbor, Mich., USA) or vehicle (0.9% saline) was administered intravenously into the femoral vein at a constant rate over 3 min using an infusion pump at 3 h after onset of MCAo. For western-blot study rats were sacrificed on days 1, 3, or 7; for real-time PCR study rats were sacrificed on days 1, 2, or 3.

[0367] Quantification and Statistical Analysis

[0368] Data is presented as histograms with mean values±SD or Boxplot, which depict median quartiles 1, 3 and maximum and minimum observations. Repeated measures analysis of variance (ANOVA) followed by Bonferroni procedures to correct for multiple comparisons was used for intergroup comparisons. Multiple comparisons was performed using Tukey's Honest Significant Difference. Plotting and statistical analysis was performed using Excel 2016 or BioVinCi 1.1.5 (developed by BioTuring Inc., San Diego, Calif., USA, www.bioturing.com). Differences at P<0.05 were considered statistically significant.

[0369] Data and Software Availability

[0370] NIS-Elements BR 3.00, FV10-ASW Version 04.02.02.09, ImageQuant™ TL, LAS 4000 imaging system, LAS 4000 imaging system, CFX Manager 3.0, MiniTab 18, SkanIt 2.3, IMARIS 9.3.1, BioVinci 1.1.5, XRS Blot Chemi Software and Excel 2016, are licensed software and the licenses were own by NG Bazan Lab. ImageJ 1.48, BioImageXD, TRED, and TFBind are free resources available online at the websites mentioned in the key resources table

[0371] Supplemental Table Titles

[0372] Table S1. TRED and TF bind analysis on the Promoter sequence (Katula et al. 2012), Related to FIG. 20, D-G. Table S1 discloses SEQ ID NOS 43-57, 43, 58, 48, 59, 50-51, 60-66, 56, 67-68, 48, 69, 50, 70-71, 61-62, 72, 65 and 73-75, respectively, in order of appearance.

TABLE-US-00008 TABLE S1 [TRED and TF bind analysis on the Promoter sequence (Katula et al., 2012)], Related to FIGS. 3D-3G. Position/Sequence TRED Score TFBind Score cREL Region 1 [192 . . . 201] TAGAAATTCC 3.92 [214 . . . 223] CCGGTTTTGC 2.21 [215 . . . 224] CGGTTTTGCC 3.3 193 (+) SGGRNWTTCC TAGAAATTCC  0.819522 194 (−) SGGRMWTTCC AGAAATTCCG  0.844549 Region 2 [357 . . . 366] GGGACTTTGC 5.08 358 (+) SGGRNWTTCC GGGACTTTGC  0.854004 Region 3 [1445 . . . 1454] GCGACTTTCA 4.12 Region 4 [1550 . . . 1559] CGGCATCTCC 3.3 1565 (−) SGGRNWTTCC GAAAAAGCCA  0.850945 Region 5 [1945 . . . 1954] CCTAATTACC 1.99 1939 (−) SGGRNWTTCC GGAAAGCCCT  0.887097 Region 6 [2103 . . . 2112] GGGCGCATCC 2.6 Region 7 [2284 . . . 2293] GGCGACTTCC 3.71 2285 (+) SGGRNWTTCC GGCGACTTCC  0.814516 p65 Region 1 [192 . . . 201] TAGAAATTCC 4.17 194 (−) GGGRATTTCC AGAAATTCCG  0.868024 Region 2 [357 . . . 366] GGGACTTTGC 6.19 358 (+) GGGRATTTCC GGGACTTTGC  0.861557 Region 3 [1445 . . . 1454] GCGACTTTCA 1.95 Region 4 [1550 . . . 1559] CGGCATCTCC 2.56 1551 (+) GGGRATTTCC CGGCATCTCC  0.769102 1552 (−) GGGAMTTYCC GGCATCTCCC  0.803246 Region 5 [1937 . . . 1946] TGGAAAGCCC 2.14 1938 (+) GGGRATTTCC TGGAAAGCCC  0.782754 1939 (−) GGGRATTTCC GGAAAGCCCT 0.86491 Region 6 [2104 . . . 2113] GGCGCATCCC 1.79 2105 (+) GGGRATTTCC GGCGCATCCC  0.765749 Region 7 [2284 . . . 2293] GGCGACTTCC 2.72 2285 (+) GGGRATTTCC GGCGACTTCC  0.771018 NFkB/p50 Region 1 193 (−) NGGGACTTTCCA TAGAAATTCCGG  0.760042 Region 2 [357 . . . 366] GGGACTTTGC 5.92 358 (+) GGGGATYCCC GGGACTTTGC  0.750555 Region 3 [1445 . . . 1454] GCGACTTTCA 0.86 Region 4 [1551 . . . 1560] GGCATCTCCC 1.74 1551 (−) GGGGATYCCC CGGCATCTCC   0.790315 1552 (−) GGGAMTTYCC GGCATCTCCC  0.803246 Region 5 [1937 . . . 1946] TGGAAAGCCC 2.08 1939 (−) GGGGATYCCC GGAAAGCCCT 0.79498 Region 6 [2104 . . . 2113] GGCGCATCCC 3.78 2105 (−) GGGGATYCCC GGCGCATCCC 0.75522 Region 7 [2285 . . . 2294] GCGACTTCCT 2.95 2285 (+) GGGGATYCCC GGCGACTTCC  0.754331

[0373] Table S2. CpG islands detected by MethPrimer (Li and Dahiya, 2002). Criteria: Island size >100, GC Percent >50.0, Obs/Exp >0.6): 5 CpG island(s) were found in the sequence, Related to FIG. 20, E-G.

TABLE-US-00009 TABLE S2 CpG islands detected by MethPrimer (Li and Dahiya, 2002). Criteria: Island size >100, GC Percent >50.0, Obs/Exp >0.6): 5 CpG island(s) were found in the sequence], Related to FIGURES 3E, 3F, and 3G. Size (Start-End) Island 1 175 bp (137-311) Island 2 173 bp (483-655) Island 3 144 bp (664-807) Island 4 793 bp  (948-1740) Island 5 338 bp (1929-2266)

[0374] Table 3. DNA constructs and siRNAs, Related to FIG. 18, G-J, and FIG. 19, B-C. Table S3 discloses “7 TCF/LEF binding sites: AGATCAAAGGgggta” as SEQ ID NO: 2.

TABLE-US-00010 TABLE S3 DNA constructs and siRNAs, Related to FIGURES 1G-1J and 2B-2C. Reference/catalog number and Constructs Type Gene/protein company Wild Type REL (untagged)- REL (NM_002908) Origene True ORF cREL UNIQUE VARIANT Cat #SC126639 expression 1 of Human v-rel vector reticuloendotheliosis viral oncogene homolog (avian) (REL) NFkB 3 tandem copies of p65 Qiagen, Cignal reporter binding sequence driving NFκB Reporter vector the expression of (luc) Kit: luciferase. Cat #CCS-013L Human Open Reading frame Origene True ORF Wnt5a Cat #SC126838 variant 1 Human Open Reading frame Origene True ORF FZD.sub.5 Cat #SC117952 Human Open reading Frame Origene True ORF ROR2 Cat #SC117279 TOP Super8XTOPflash 7 TCF/LEF Addgene repository Flash construct M50, Beta- binding sites: Plasmid #12456. catenin reporter. AGATCAAAGGgggta Zebrafish prickle, a TCF/LEF sites with TCF/LEF modulator of upstream of a binding site noncanonical luciferase reporter. in CAP letters, and a Wnt/Fz signaling, spacer in lower case, regulates gastrulation separating each copy movements. of the TCF/LEF site). (Veeman et al, 2003) FOP M51 Super 6 mutated TCF/LEF Addgene repository flash 8 × TOPFlash binding sites that were Plasmid #12457. (TOPFlash mutant) cloned into the pGL3 (Veeman et al, 2003) vector (Promega) FZD5 Human FZD5 21-mer Human FZD5 Silencer select siRNA siRNA duplexes (NM_003468) Validated Ambion, Life Technologies- Thermo Cat #4390824. ID: s15416 ROR2 Human ROR2 21-mer Human ROR2 Silencer select siRNA siRNA duplexes (NM_004560) Ambion, Life Technologies- Thermo Cat #4390824. ID: s9758 Negative Non-specific binding Proprietary Allstars Qiagen control siRNA sequence Cat #1027292 siRNA Alexa Fluor 488 conjugated

[0375] Table S4. Primers information, Related to FIG. 18, C, E, F and FIGS. 23, F and J. Table S4. disclose SEQ ID NOS 3-10, 82, 76, 13-34 and 77-78, respectively, in order of appearance.

TABLE-US-00011 TABLE S4 Primers information, Related to FIGS. 1C, 1E, 1F, 6F and 6J. Target Sequence Source Rat Wnt5a Forward primer RealTimePrimers.com 5′-TTACCCAAACCGGACTGTTA-3′ Reverse primer 5′-AGCCTTTTCGGTTCATCTCT-3′ Human Wnt5a Forward primer Campioni et al., 2008. 5′-CAAAGCAACTCCTGGGCTTA-3′ Reverse primer 5′-CCTGCTCCTGACCGTCC-3′ Rat Chemokine Forward primer RealTimePrimers.com C-X-X motif 5′-GCGGAGAGATGAGAGTCTGG-3′ ligand 1 (Cxcl1) Reverse primer NM_030845 5′-TCCAAGGGAAGCTTCAACAC-3′ Rat ACTB Forward primer RealTimePrimers.com 5′-CACACTGTGCCCATCTATGA-3′ Reverse primer 5′-CCGATAGTGATGACCTGACC-3′ Rat TNFa Forward primer RealTimePrimers.com 5′-AACTCGAGACAAGCCCGTAG-3′ Reverse primer 5′-GTACCACCAGTTGGTTGTCTTTGA-3′ Rat IL6 Forward primer RealTimePrimers.com 5′-CTTCCTACCCCAACTTCCAA-3′ Reverse primer 5′-ACCACAGTGAGGAATGTCCA-3′ Rat B2m Forward primer RealTimePrimers.com 5′-TGCTACGTGTCTCAGTTCCA-3′ Reverse primer 5′-GCTCCTTCAGAGTGACGTGT-3′ Rat MMP13 Forward primer RealTimePrimers.com 5′-CCTCTTCTTCTCAGGGAACC-3′ Reverse primer 5′-GGAATTTGTTGGCATGACTC-3′ Rat MMP9 Forward primer RealTimePrimers.com 5′-ACTTCTGGCGTGTGAGTTTC-3′ Reverse primer 5′-TGTATCCGGCAAACTAGCTC-3′ Rat MMP2 Forward primer RealTimePrimers.com 5′-CTTCAGGTTCTCCAGCATGA-3′ Reverse primer 5′-CCGTAAGGGAGACACCAGAT-3′ Rat IL-1β Forward primer Nakazawa et al., 2001. 5′-TCAGGAAGGCAGTGTCACTCATTG-3′ Reverse primer 5′-ACACACTAGCAGGTCGTCATCATC-3′ Rat ICAM1 Forward primer Ammirante et al., 2010. 5′-CTGTCAAACGGGAGATGAATGGT-3′ Reverse primer 5′-TCTGGCGGTAATAGGTGTAAATGG-3′ Rat MCP1 Forward primer Nakazawa et al., 2006. 5′-ATGCAGGTCTCTGTCACGCTTCTG-3′ Reverse primer 5′-GACACCTGCTGCTGGTGATTCTCTT-3′ Rat E-Selectin Forward primer Hannawa et al., 2005. 5′-TGCGATGCTGCCTACTTGTG-3′ Reverse primer 5′-AGAGAGTGCCACTACCAAGGGA-3′ Rat Ywhaz Forward primer Gubern et al., 2009. 5′-GATGAAGCCATTGCTGAACTTG-3′ Reverse primer 5′-GTCTCCTTGGGTATCCGATGTC-3′ Rat Sdha Forward primer Gubern et al., 2009. 5′-TCCTTCCCACTGTGCATTACAA-3′ Reverse primer 5′-CGTACAGACCAGGCACAATCTG-3′ Human cREL Forward primer Calandria et al., 2015 5′-CAGGAGGAAGAGCAGTCGTC-3′ Reverse primer 5′-GCAGGAATCAATCCATTCAA-3′

[0376] Table S5. ChIP assay primers for SYBR green based real-time PCR, Related to FIG. 20, E-G. Table S5 discloses the “Forward” sequences as SEQ ID NOS 35-38 and the “Reverse” sequences as SEQ ID NOS 39-42, all respectively, in order of appearance.

TABLE-US-00012 TABLE S5 ChIP assay primers for SYBR green based real-time PCR, Related to FIGS. 3E-3G. Sequence Promoter Primers Forward Reverse Wnt5a A1 5′-GCATCCCACTACCCAAGTCC-3′ 5′-GCTGCCTTGACATGGAACCTCA-3′ Promoter A A2 5′-CAGCAATAAGTTCCGGGGCG-3′ 5′-GCTTTGGGGCCACAGAACAATC-3′ A3 5′-GCCTCTCCGTGGAACAGTTGC-3′ 5′-GATGCGCCCAGGAATGG-3′ A4 5′-CGCCAGTGCCCGCTTCAG-3′ 5′-CAGCCGAGGAATCCGAGC-3′

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EQUIVALENTS

[0417] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.