Differential Cerebral Cortex Transcriptomes of Baboon Neonates Consuming Moderate and High Docosahexaenoic Acid Formulas

Background Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) are the major long chain polyunsaturated fatty acids (LCPUFA) of the central nervous system (CNS). These nutrients are present in most infant formulas at modest levels, intended to support visual and neural development. There are no investigations in primates of the biological consequences of dietary DHA at levels above those present in formulas but within normal breastmilk levels. Methods and Findings Twelve baboons were divided into three formula groups: Control, with no DHA-ARA; “L”, LCPUFA, with 0.33%DHA-0.67%ARA; “L3”, LCPUFA, with 1.00%DHA-0.67%ARA. All the samples are from the precentral gyrus of cerebral cortex brain regions. At 12 weeks of age, changes in gene expression were detected in 1,108 of 54,000 probe sets (2.05%), with most showing <2-fold change. Gene ontology analysis assigns them to diverse biological functions, notably lipid metabolism and transport, G-protein and signal transduction, development, visual perception, cytoskeleton, peptidases, stress response, transcription regulation, and 400 transcripts having no defined function. PLA2G6, a phospholipase recently associated with infantile neuroaxonal dystrophy, was downregulated in both LCPUFA groups. ELOVL5, a PUFA elongase, was the only LCPUFA biosynthetic enzyme that was differentially expressed. Mitochondrial fatty acid carrier, CPT2, was among several genes associated with mitochondrial fatty acid oxidation to be downregulated by high DHA, while the mitochondrial proton carrier, UCP2, was upregulated. TIMM8A, also known as deafness/dystonia peptide 1, was among several differentially expressed neural development genes. LUM and TIMP3, associated with corneal structure and age-related macular degeneration, respectively, were among visual perception genes influenced by LCPUFA. TIA1, a silencer of COX2 gene translation, is upregulated by high DHA. Ingenuity pathway analysis identified a highly significant nervous system network, with epidermal growth factor receptor (EGFR) as the outstanding interaction partner. Conclusions These data indicate that LCPUFA concentrations within the normal range of human breastmilk induce global changes in gene expression across a wide array of processes, in addition to changes in visual and neural function normally associated with formula LCPUFA.


INTRODUCTION
The vertebrate central nervous system (CNS) is rich in the long chain polyunsaturated fatty acids (LCPUFA) docosahexaenoic acid (DHA) and arachidonic acid (ARA), and this composition is highly conserved across species [1]. Within the CNS, DHA and ARA are found at highest concentration in gray matter [2], and DHA is particularly concentrated in retinal photoreceptor membranes where it has long been known to play a key role in visual excitation [3]. In humans, DHA and ARA accumulate perinatally [4] and many studies of DHA/ARA supplemented formula show improvements in visual acuity [5] and cognitive function [6].
Despite the high demand for LCPUFA during perinatal CNS development, the best current evidence indicates that ARA and DHA can be synthesized only very inefficiently from dietary precursors and must be obtained from the diet [7]. DHA and ARA are present in all human milks studied to date [8], however their concentration is variable. For DHA it is closely linked to the mother's intake of preformed DHA, which is in turn reflective of the mother's intake of fatty fish or fish/marine oil supplements [9,10,11,12]. Dietary factors associated with ARA are less well understood [13]. High levels of precursor fatty acids LA and ALA in formulas yield negligible or at most moderate increases in plasma ARA and DHA concentrations [14,15]. However, in randomized controlled studies where preterm and term infants are fed preformed DHA and ARA supplemented formula, improve-ments in LCPUFA status as well as cognitive development and visual functions are observed [16,17,18,19,20].
While the importance of LCPUFA in infant nutrition has been established, the underlying mechanisms are only beginning to be understood. Brain accretion of LCPUFA is most intense during the brain growth spurt in the third trimester of pregnancy and during early childhood [21,22,23,24]. Selective incorporation and functional properties of LCPUFA, especially DHA, in retinal and neural membranes suggests a specific role in the modulation of protein-lipid interactions, membrane bound receptor function, membrane permeability, cell signaling, regulation of gene expression and neuronal growth [25,26,27,28,29,30]. Additionally, LCPUFA mediate metacrine regulation and changes in gene expression by interacting with nutrient sensitive transcription factors [18,31]. Accordingly, poor nutrition during prenatal life and early infancy may have a lasting influence on neural function, as well as adult risk for chronic diseases [32,33,34]. Studies suggest that infant diets low in LCPUFA can lead to health complications such as insulin resistance, obesity, or blood pressure changes later in life [35,36].
DHA and ARA were introduced in 2002 to infant formulas in the United States, but initial concentrations varied over more than a factor of two (range of DHA 8-19 mg/kcal; ARA 21-34 mg/ kcal), [37] and there are no dose response studies in humans or non-human primates available as a guide to optimal levels. A previous study in our laboratory on 4-week-old baboon neonates with preformed DHA and ARA (0.33%,w/w DHA and 0.67% ARA) in formulas showed DHA concentrations in various regions of the brain similar to breastfed controls, with the important exception of the cerebral cortex; ARA concentrations were not much altered by inclusion of dietary preformed ARA [2]. These results inspired our present study on 12 week old baboon neonates with the higher level of 1.00% DHA, along with 0.67% ARA. We report elsewhere [38] that DHA in the precentral gyrus of cerebral cortex increased beyond that achieved for 0.33% DHA, while regions such as the basal ganglia that reached DHA concentrations similar to breastfed animals at 0.33% DHA did not show further increases with 1.00% DHA. These data demonstrate that formula DHA in the high normal range of breastmilk DHA supports enhanced cortex DHA, but do not reveal how this compositional change may influence metabolic function.
To gather mechanistic information on the role of DHA and ARA in the primate cerebral cortex, we investigated global gene expression for cerebral cortex of animals in this study, consuming two different levels of formula DHA both within the range found in human breastmilk [8]. We report here changes in expression of thousands of genes in 12-week-old baboons in response to two different levels of LCPUFA: 0.33%DHA and 0.67% ARA; 1.00% DHA and 0.67% ARA. We have reported in detail on consequences for tissue fatty acid composition [38] and other factors elsewhere (Hsieh et al., 2007, submitted).

RESULTS AND DISCUSSION
Significance analysis (P,0.05) identified changes in expression levels of 1108 probe sets (ps) for comparisons of L3/C and/or L/ C, representing 2.05% of the total.54,000 ps on the oligoarray. Most ps showed ,2-fold change. For the L/C comparisons, 534 ps were upregulated, and 574 ps were downregulated, while for the L3/C comparisons, 666 ps were upregulated and 442 ps were downregulated, showing that more genes were upregulated in the cerebral cortex in response to increasing formula ARA and DHA. Functional characterization by gene ontology of these differentially regulated genes assigns them to diverse biological processes including lipid and other metabolism, ion channel and transport, development, visual perception, G-protein and signal transduction, regulation of transcription, cell cycle, cell proliferation, apoptosis etc. Known functions were assigned to 702 differentially expressed probe sets, whereas 406 ps had no known functions as shown in Table S1A, S1B, S1C, S1D. Probe sets with $1.4 fold expression change are presented in Table S2. Experimental details for nine genes used for confirmatory RT-PCR analysis are presented in Table S3. We note that in our L/C and L3/C comparisons, expression patterns fall into four groups, L/C and L3/C both upregulated and both downregulated, or one upregulated and one downregulated. Because the L and L3 groups have the same amount of ARA but different amounts of DHA, our treatments do not strictly represent a DHA dose response. The L/C comparison corresponds to inclusion of DHA and ARA at current levels near the worldwide breastmilk means, while the L3 group corresponds to DHA near the worldwide high [8].
Nine genes were tested by quantitative real time PCR to confirm the array results, as shown in Table S4. All were qualitatively consistent with the gene array results.
We highlight results in several categories of gene ontogeny as follows.
Lipid (fatty acid and cholesterol) Metabolism Table 1 presents results from genes related to lipid metabolism that are regulated by dietary LCPUFA.
Genes related to phospholipids biosynthesis (PLA2G6 and DGKE) were differentially expressed. PLA2G6 was downregulated in both groups. This gene codes for the Ca-independent cytosolic phospholipase A2 Group VI. Alterations in this gene have very recently been implicated as a common feature of neurodegener- ative disorders involving iron accumulation [39], as well as the underlying factor in infantile neuroaxonal dystrophy, a neurodegenerative disorder caused by accumulation of iron in the globus pallidus and resulting in death by age 10 [40]. In a previous study of four week old breastfed baboons, the globus pallidus was found to have 15.860.5% DHA (w/w of total fatty acids) and was the richest in DHA of 26 CNS regions examined [2]. The globus pallidus is also rich in ARA, with 10.3% (w/w) in four week old baboons. PLA2 are a superfamily of enzymes that liberate fatty acids from the sn-2 position of phospholipids; in the globus pallidus DHA and ARA are the most abundant acyl groups at this site.
Remarkably, among the elongation and desaturation enzymes associated with LCPUFA synthesis, only a single elongation enzyme was differentially expressed. The human ELOVL5 transcript was downregulated slightly in the L/C group and upregulated in the L3/C group. This enzyme, also called HELO1, catalyzes the two carbon elongation of polyunsaturated 18 and 20 carbon fatty acids [41,42].
We also found that DGKE was upregulated in the L3/C comparison. Genes involved in ceramide metabolism (NSMAF, LASS5), glycosphingolipid metabolism (SPTLC2) and steroid metabolism (OSBP2, UGT2B15) showed increased expression in L3/C group, whereas NSMAF and OSBP2 were downregulated in L/C group.
The best studied role of ARA is as a precursor for eicosanoids including prostaglandins, leukotrienes, and thromboxanes. One of the genes derived from membrane-bound ARA, which catalyze the first step in the biosynthesis of cysteinyl leukotrienes, Leukotriene C4 synthase (LTC4S), is downregulated in both DHA-ARA groups. LTC4S is a potent proinflammatory and anaphylactic mediator [43]. An elevated level of mRNA for PGES3 (prostaglandin E synthase 3) was observed in both the groups. PGES3 is also known as TEBP (telomerase-binding protein p23) or inactive progesterone receptor, 23-KD (p23). p23, a ubiquitous highly conserved protein which functions as a co-chaperone for the heat shock protein, HSP90, participates in the folding of a number of cell regulatory proteins [44,45]. p23 has been demonstrated to bind to human telomerase reverse transcriptase (hTERT) and contribute to telomerase activity [46]. Decreased levels of Annexin A3 (ANXA3) also known as Lipocortin III was observed with increasing DHA.
Genes involved in fatty acid oxidation (ACADSB, ACAD10 and GLYAT) were upregulated, and carnitine palmitoyltransferase II (CPT2) downregulated, in the L3/C group. ACADs (acyl-CoA dehydrogenases) are a family of mitochondrial matrix flavoproteins that catalyze the dehydrogenation of acyl-CoA derivatives and are involved in the b-oxidation and branched chain aminoacid metabolism [47,48]. Both the ACADs family members ACADSB and ACAD10 were upregulated in L3/C group, consistent with greater energy production in the high DHA group. Mitochondrial-specific GLYAT (glycine-N-acyltransferase) also known as acyl CoA:glycine N-acyl transferase (ACGNAT), conjugates glycine with acyl-CoA and participates in detoxification of various drugs and xenobiotics [49,50]. Mawal et al [50] suggested that delayed development of GLYAT might impair detoxification process in children.
SOAT1 (sterol O-acyl transferase) or Acyl-coenzyme A: cholesterol acyl transferase (ACAT) is an intracellular protein which catalyzes the formation of cholesterol esters in endoplasmic reticulum and is involved in lipid droplets that are characteristic of foam cells of atherosclerotic plaques [57,58,59].
Increased expression was detected for ATP8B1, PDE3A in both groups, comparatively more in L3/C, while transcripts involving HNF4A (Hepatic nuclear factor-4a), CLPS and ALDH3B2 showed decreased expression with increasing DHA. Intrahepatic cholestasis, or impairment of bile flow, is an important manifestation of inherited and acquired liver disease resulting in hepatic accumulation of the toxic bile acids and progressive liver damage. Bile acids enhance efficient digestion and absorption of dietary fats and fat-soluble vitamins, and are the main route for excretion of sterols. Expression of ATP8B1 is high in the small intestine, and mutations in ATP8B1 gene have been linked to intrahepatic cholestasis [60,61]. ATP8B1 expression was confirmed by real time PCR (Table S4). PDE3A (phosphodiesterase 3A, cGMP-inhibited) is a 120 kDa protein found in myocardium and platelets [62]. Ding et al [63] showed significantly decreased expression of PDE3A in the left ventricles of failing human hearts. PDE3A expression is required for the regulation of penile erection in humans [64].
Leptin (LEP), which has a role in energy metabolism, was upregulated in L3/C group. Leptin is a secreted adipocyte hormone that plays a pivotal role in the regulation of food intake and energy homeostasis [65,66]. Leptin suppresses feeding and decreases adiposity in part by inhibiting hypothalamic Neuropeptide Y synthesis and secretion [67,68].

Ion Channel and Transport
Expression levels of transcripts involved in ion channel and transporter activity were altered by dietary LCPUFA (Table S5). Uncoupling protein 2, LOC131873 (hypothetical protein) and ATP11C, which have ion channel activity, are upregulated in both the groups but moreso in L3/C. Other transcripts with ion channel activity, including VDAC3, FTH1, KCNK3, KCNH7 and TRPM1 were upregulated in L3/C group and downregulated in L/C. GLRA2, TRPV2 and HFE are upregulated in L/C and repressed in L3/C. P2RX2, GRIA1 and CACNA1S are repressed in both the groups.
One of our significant observations is the increased expression of uncoupling protein 2 (UCP2), a mitochondrial, proton carrier. Our data shows, for the first time, increased expression of UCP2 in neonatal cerebral cortex associated with dietary LCPUFA; increased expression is observed in both the groups but more in L3/C. QRT-PCR confirmed the array results (Table S4). Nutritional regulation and induction of mitochondrial uncoupling proteins resulting from dietary n3-PUFA in skeletal muscle and white adipose tissue have been observed [69,70]. Increased UCP2 expression is beneficial in diseases associated with neurodegeneration, cardiovascular and type 2 diabetes [71]. Dietary fats in milk increased the expression and function of UCP2 in neonatal brain and protected neurons from excitotoxicity [72].
VDAC3 (voltage-dependent anion channel 3) belongs to a group of pore forming proteins found in the outer mitochondrial membrane and in brain synaptic membranes [73,74]. Massa et al [75] observed a significant reduction of VDAC3 mRNA levels in the skeletal muscle and brains of dystrophin-deficient mdx mice during postnatal development. Mice lacking VDAC3 exhibit infertility [76]. All the transcripts (VDAC3, KCNK3 and KCNH7) having voltage-gated anion channel porin activity were upregulated with increasing DHA. FTH1 (Ferritin heavy chain 1) is required for iron homeostasis and it has been previously shown to be expressed in human brain [77].
Genes encoding small molecule transporters were differentially expressed, including carriers of glucose (SLC2A1, SLC5A4), chloride (SLC12A6), sodium (SLC13A3), monoamine (SLC18A2) and others (SLC26A4, SLC17A6). These transporters might help in exchange of nutrients and metabolites. Members of the cytochrome P and B family of proteins were also differentially expressed. Transcripts encoding VDP, RSAFD1, C1QG and OXA1L were significantly repressed by increasing DHA.

G-Proteins and Signaling
Numerous genes encoding G-protein activity were differentially regulated (Table S5), and the majority were induced by high DHA. GNA13, GNA14, PTHR2, RCP9 and FZD3 showed increased expression in both DHA groups. EDG7, SH3TC2, GNRHR, ADRA1A, BLR1, GPR101, GPR20 and OR8G2 were downregulated in L/C and upregulated in L3/C. NPY1R is downregulated in both the groups.
DHA regulates G-protein signaling in the brain and retina [78]. G-proteins are membrane-associated proteins which promote exchange of GTP for GDP and regulate signal transduction and membrane traffic [79]. GNA13 deficiency impairs angiogenesis in mice [80] while GNA14 activates the NF-kB signaling cascade [81]. Parathyroid hormone receptor 2 (PTHR2) is activated by parathyroid hormone and is relatively abundant in the CNS [82,83]. RCP9, also known as calcitonin gene-related peptidereceptor component protein, may have a role during hematopoiesis [84]. Tissir and Goffinet [85] showed expression of FZD3 during postnatal CNS development in mice. FZD3 array results were confirmed by SYBR green real time PCR assay (Table S4).
Neuropeptide Y is a 36-amino acid peptide with strong orexigenic effects in vivo [86]. Two major subtypes of NPY (Y1 and Y2) have been defined by pharmacologic criteria. NPY1R was suggested to be unique for the control of feeding [87]. Pedrazzini et al [88] observed a moderate but significant decrease in food intake in mice lacking the NPY1R gene.
NF1 is a tumor-suppressor gene; mutations in this gene cause neurocutaneous defects [91]. NF1 gene expression and function are needed for normal fracture healing [92]. NF1 expression levels were confirmed by QRT-PCR (Table S4). WSB1 is a SOCS-boxcontaining WD-40 protein expressed during embryonic development in chicken [93]. RAS and RAS related gene families of small GTPases (RIT1, KRAS, RERG and RAPGEF6) were upregulated by increasing DHA.
Diets deficient in n-3 PUFA induce substitution of n-6 DPA (22:5n-6) in neural membranes, and impairment of functions mediated by G protein mediated signaling, such as visual perception, learning and memory, and olfactory discrimination. Abun-dant evidence indicates that this results in reduced rhodopsin activation, and signaling in rod outer segments compared to DHAreplete animals [78,94,95,96,97]. Table 2 shows differential expression of 24 genes related to development. The products of 11 transcripts play a role in nervous system development. The expression of TIMM8A, NRG1, SEMA3D and NUMB genes were upregulated in both L/C and L3/C groups. HES1 and SIM1 were downregulated in both the groups. GDF11, SMA3/SMA5, SH3GL3 were downregulated in L/C and upregulated in L3/C. The mRNA levels of growth factors FGF5 and FGF14 displayed increased abundance in L/C and decreased abundance in L3/C.

Development
TIMM8A also known as Deafness/Dystonia Peptide 1 (DDP1) is a well conserved protein organized in mitochondrial intermembrane space. Loss-of-Function mutations in the TIMM8A gene cause Mohr-Tranebjaerg syndrome (a progressive neurodegenerative disorder with deafness, blindness, dystonia and mental deficiency) and Jensen syndrome (opticoacoustic nerve atrophy with dementia) [98,99,100]. TaqMan assay confirmed the array results (Table S4). NRG1 is essential for the development and function of the CNS facilitating the neuronal migration and axon guidance [101,102]. NUMB negatively regulates notch signaling and plays a role in retinal neurogenesis, influencing the proliferation and differentiation of retinal progenitors and maturation of postmitotic

Visual Perception
Nine transcripts having a role in visual perception were differentially expressed (Table 3). Genes coding for LUM, EML2, TIMP3 and TTC8 were upregulated in both the supplement groups. IMPG1 was upregulated in L3/C and downregulated in L/C. RGS16 and TULP2 were upregulated in L/C and downregulated in L3/C. RAX and IMPDH1 were downregulated in both the supplement groups. Lumican (LUM), is an extracellular matrix glycoprotein and a member of the small-leucine-rich-proteoglycan (SLRP) family [105]. It is widely distributed in the corneal stoma and connective tissues [106]. Lumican helps in the establishment of corneal stromal matrix organization during neonatal development in mice. Those lacking lumican exhibit several corneal related defects [107]. It is important for corneal transparency in mice [108]. TaqMan assay showed 5-fold more upregulation of LUM more than the microarray data (Table S4). Mutations in TIMP3 gene result in autosomal dominant disorder Sorsby's fundus dystrophy an age-related macular degeneration of retina [109]. Clarke et al [110] suggested that a possible mechanism for retinal degeneration in Sorsby's fundus dystrophy was traceable to nutrition.
IMPG1 is a proteoglycan which participates in retinal adhesion and photoreceptor survival [111]. Higher amounts of DHA in the infant formula increased the expression of IMPG1. Expression of RAX transcript is decreased in both the supplement groups. Increased RAX expression is seen in the retinal progenitor cells during the vertebrate eye development and is downregulated in the differentiated neurons [112,113]. DHA is well known to promote neurite growth in the brain [30]; this could be the possible reason for RAX downregulation in our study.

Integral to Membrane/Membrane Fraction
Transcripts that are integral part of biological membranes or within the membrane fractions were differentially expressed (Table   S5). EVER1, PERP, Cep192, SSFA2, LPAL2, TMEM20, TM6SF1 were upregulated in both the groups. ORMDL3, SEZ6L, HYDIN, TA-LRRP, PKD1L1 were upregulated in L3/C and downregulated in L/C. MFAP3L was upregulated in L/C and downregulated in L3/C. Transcripts of GP2 and SYNGR2 were downregulated in both the groups.
Numbers of transcripts were upregulated by increased DHA in the formulas. LCPUFA can affect biological membrane functions by influencing membrane composition and permeability, interaction with membrane proteins, membrane-bound receptor function, photoreceptor signal transduction and transport [114,115,116]. Mutations in EVER1 or transmembrane channel-like 6 (TMC6) gene cause epidermodysplasia verruciformis, a type of skin disorder [117]. HYDIN is a novel gene and nearly-complete loss of its function due to mutations causes congenital hydrocephalus in mice [118]. The exact function of GP2 is unknown, but it has been associated with the secretory granules in the pancreas [119].

Programmed Cell Death/Apoptosis
Transcripts with apoptotic activity were differentially expressed (Table S5). Seven out of nine transcripts in our study were upregulated with increasing DHA, including CARD6, TIA1, BNIP1, FAF1, GULP1, CASP9 and FLJ13491. Programmed cell death (PCD) plays an important role during the development of immune and nervous systems [120]. Jacobson et al [121] proposed PCD as an important event in eliminating unwanted cells during development. Mice with targeted deletion of CASP3 die perinatally due to vast excesses of cells deposition in their CNS as a result of decreased apoptotic activity [120]. CARD6 (caspase recruitment domain protein 6) is upregulated in both the groups. It is a microtubule-interacting protein that activates NF-B and takes part in the signaling events leading to apoptosis [122]. TIA1 is upregulated in L3/C and downregulated in L/C. TIA1 is a member of RNA-binding protein family with pro-apoptotic activity, and it silences the translation of cyclooxygenase-2 (COX2). Narayanan et al, [123] suggested that DHA indirectly increases the expression of genes which downregulate COX2 expression. The COX2 enzyme catalyzes the rate-limiting step for prostaglandin production, which influence many processes including inflammation [124]. Downregulation of TIA1 in L/C could be due to the influence of ARA, the major COX2 substrate, rather than that of DHA which is a competitive inhibitor. GULP1 assists in efficient removal of the apoptotic cells by phagocytosis [125]. CASP9 activates caspase activation cascade and is an important component of mitochondrial apoptotic pathway [126].

Cytoskeleton and Cell adhesion
Dietary LCPUFA regulated expression of several transcripts involved in cytoskeleton and cell adhesion (Table S5). The expression of 27 ps involved in cytoskeleton was altered. MYO1A and MYO5A were upregulated with increasing amounts of DHA whereas MYO1E showed decreased expression. Myosin-1 isoforms are membrane associated molecular motors which play essential roles in membrane dynamics, cytoskeletal structure and signal transduction [127]. COL4A6 and COL9A3 showed increased expression whereas COL4A2 and COL9A2 showed decreased expression with increasing DHA. Type IV collagen is the major component of the basement membrane. Mild forms of Alport nephropathy is associated with deletion in COL4A6 gene [128] and eye abnormalities are common in people afflicted with Alport syndrome [129]. WASL, also known as neural WASP (WASP), was upregulated in both the groups. Actin cytoskeleton regulation is vital for brain development and function. WASL is an actin- regulating protein and mediates filopodium formation [130,131,132]. HIP1 (huntingtin interacting protein 1) and HOOK2 (hook homolog 2) were downregulated in both the groups. The expression levels of 15 transcripts involved in cell adhesion changed as a result of dietary LCPUFA (Table S5). BTBD9, CD44, ARMC4, CD58, LOC389722 and PCDHB13 showed increased expression in both the groups. Glycoprotein CD44 is a cell-surface adhesion molecule that is involved in cell-cell and cell-matrix interactions [133] while PCDHB13 is a member of protocadherin beta family of transmembrane glycoproteins [134]. NLGN3 and CYR61 were downregulated in both groups.

Peptidases
Several transcripts having peptidase activity were differentially expressed (Table S5). SERPINB6 is significantly upregulated in L3/C and downregulated in L/C. Of note, the ADAM families of proteins (ADAM17, ADAM33, and ADAMTS16) were upregulated and ADAMTS15 was downregulated in both the supplement groups. ADAM proteins are membrane-anchored glycoproteins named for two of the motifs they carry: an adhesive domain (disintegrin) and a degradative domain (metalloprotease) [135]. These proteins are involved in several biological processes including cell-cell interactions, heart development, neurogenesis and muscle development [136,137,138,139]. ADAM17 is required for proteolytic processing of other proteins and have been reported to participate in cleaving of the amyloid precursor protein [140,141]. Loss of ADAM17 is reported in abnormalities associated with heart, skin, lung and intestines [142,143,144]. Real time PCR confirmed array results of ADAM17 (Table S4). ADAM33 has been recently implicated as an asthma and bronchial hyperresponsiveness gene [145]. It is required for smooth muscle development in the lungs helps in airway wall ''modeling'', and proper functioning of lungs throughout life [146,147].
CTSB (Cathepsin B) also known as amyloid precursor protein secretase (APPS) was upregulated. It is involved in the proteolytic processing of amyloid precursor protein [148]. Felbor et al [149] reported deficiency of CTSB results in brain atrophy and loss of nerve cells in mice. CTSC (Cathepsin C) was downregulated in the L/C group and upregulated in the L3/C group. Loss of function mutations in CTSC gene are associated with tooth and skin abnormalities [150].

Cell Cycle, Cell Growth and Cell Proliferation
Fifteen transcripts having a role in cell cycle regulation, growth and proliferation were differentially expressed (Table S5). Four of the transcripts SESN3, RAD1, GAS1 and PARD6B involved in cell cycle regulation were upregulated in both the groups. Cell growth factors, INHBC and OGN were induced in both the groups. FGFR1OP is a positive regulator of cell proliferation and showed increased expression. KAZALD1, CDC20 and CDKN2C were downregulated.
Growth arrest specific gene 1 (GAS1) expression is positively required for postnatal cerebellum development. Mice lacking GAS1 had significantly reduced cerebellar size compared to wild type mice [152]. Liu et al [152] proposed that GAS1 perform dual roles in cell cycle arrest and in proliferation in a cell autonomous manner. PARD6B has a role in axonogenesis [153].
INHBC is a member of transforming growth factor-beta superfamily (TGF-beta) and is involved cell growth and differentiation [154,155]. Osteoglycin (OGN) is also known as Mimecan and Osteoinductive factor (OIF). Mimecan is a member of smallleucine rich proteoglycan gene family and is a major component of cornea and other connective tissues [156,157]. It has a role in bone formation , cornea development and regulation of collagen fibrillogenesis in corneal stroma [157,158,159]. CDC20 regulates anaphase-promoting complex [160].

Response to Stress
MSRA, SOD2, GSTA3 and GSR genes were differentially expressed (Table S5). MSRA was upregulated in both the supplement groups. SOD2 is downregulated in L/C and upregulated in L3/C. GSR is upregulated in the L/C and downregulated in the L3/C. GSTA3 is downregulated in both the groups.
Oxidative damage to proteins by reactive oxygen species is associated with oxidative stress, aging, and age-related diseases [161,162,163]. MSRA is expressed in the retina, neurons and the nervous system [162]. Knock-outs of the MSRA gene in mice result in shortened life-spans both under normoxia and hyperoxia conditions [164]. MSRA also participates in the regulation of proteins [165]. MSRA plays an important role in neurodegenerative diseases like Alzheimer's and Parkinson's by reducing the effects of reactive oxygen species [163]. Overexpression of MSRA protects human fibroblasts against H2O2-mediated oxidative stress [166]. SOD2 belongs to the iron/manganese superoxide dismutase family. It encodes a mitochondrial protein and helps in the elimination of reactive oxygen species generated within mitochondria [167]. In our study increased amount of DHA reduced the expression of glutathione-related proteins GSR and GSTA3.

Kinases and Phosphatases
Phosphorylation and dephosphorylation of proteins control a multitude of cellular processes. Several proteins having kinase activity were altered (Table S5). Of note, transcripts involving STK3, STK6, HINT3, TLK1, DRF1, GUCY2C and NEK1 were significantly upregulated with increasing DHA. A number of MAP kinases were downregulated in L3/C group, including MAP4K1, MAPK12, MAP3K2 and MAP3K3. Other transcripts which showed significantly decreased expression were CKM, LMTK2, NEK11, TNK1, BRD4 and MGC4796.

Ubiquitin Cycle
Twenty-five probe sets having a role in the ubiquitination process were differentially expressed (Table S5). Interestingly, five members of F-box protein family (FBXL7, FBXL4, FBXL17, FBXW4 and FBXW8) showed increased expression in L3/C group. F-Box proteins participate in varied cellular processes such as signal transduction, development, regulation of transcription and transition of cell cycle. They contain protein-protein interaction domains and participate in phosphorylation-dependent ubiquitination [169,170]. Proteins associated with anaphasepromoting complex (CDC23 and ANAPC1) were downregulated in L3/C group.

Ingenuity Network Analysis
We explored relationships among sets of genes using Ingenuity Systems network analysis. Out of 1108 differentially expressed probe sets in our data, 387 probe sets (34.93%) were found in the Ingenuity Pathway Analysis (IPA) knowledge database, and are labeled ''focus'' genes. Based on these focus genes, IPA generated 41 biological networks (Table S6). Among these 41 networks, 24 had scores of .8 and the top 2 networks with 35 genes had scores of 49. We focus here on the most significant network.
The top network identified by IPA is associated with nervous system development and function, cellular growth and proliferation (Figure 1). Epidermal growth factor receptor (EGFR) is the most outstanding interaction partner found within the network. EGFR interacts with TIMP3, NRG1, ADAM17, EDG7 and FGF7; all are upregulated, and involved in neural or visual perception development. EGFR signaling is implicated in early events of epidermal, neural and eye development. Loss of EGFR signaling results in reduced brain size and loss of larval eye and optic lobe in drosophila [171]. EGFR expression is required for postnatal forebrain and astrocytes development in mice [172]. Functional pathway analysis conducted on this network using the IPA tool set identified three genes, ADAM17, NUMB and HES1, involved in the Notch signaling pathway which regulates nervous system and eye development [173,174]. ADAM17 and NUMB were upregulated while HES1 was repressed in both the groups. This analysis suggests that LCPUFA influence many processes with influences that converge on EGFR.
LCPUFA are known to directly interact with nutrient sensitive transcription factors such as peroxisome proliferator-activated receptors (PPARs), liver6receptors, hepatic nuclear factor-4a, sterol regulatory binding proteins, retinoid6receptors and NF-B. Upon ingestion, LCPUFA can elicit a transcriptional response within minutes [31,175,176,177]. Microarray studies on LCPUFAsupplementing animals have identified several tissue-specific pathways regulated by LCPUFA, particularly involving the liver, adipose, and brain tissue transcriptome [26,178,179]. Using murine 11K Affymetrix oligoarrays, Berger et al [178] [180] showed increased hepatic expression of lipolytic and decreased expression of lipogenic genes. However, in the hippocampus brain region, increased expression of HTR4 and decreased expression of TTR and SIAT8E, genes involved in the regulation of cognition and learning, as well as POMC, a gene associated with appetite control, was identified. The first paper published on the brain gene transcriptome with respect to LCPUFA supplementation by Kitajka et al. in 2002 [181] demonstrated that feeding fish oil (DHA 26.9%) to rats increased expression of genes involved in lipid metabolism (SPTLC2, FPS), energy metabolism (ATP synthase subunit d, ATP synthase H + , cytochromes, IDH3G), cytoskeleton (Actin related protein 2, TUBA1), signal transduction (Calmodulins, SH3P4, RAB6B small GTPase), receptors, ion channels and neurotransmission (Vasopressin V1b receptor, Somatostatin), synaptic plasticity (Synucleins) and regulatory proteins (protein phosphatases). In the same study, fish oil supplementation also significantly reduced the expression of phospholipase D and Transthyretin. In related work, Kitajka et al [26] , using rat cDNA microarrays with 3,200 spots, found results similar to those previously reported. Barcelo-Coblijn et al. [182] were the first to report moderation of age-induced changes in gene expression in rat brain as a result of diets rich in fish oil (DHA 11.2%). In this study, 2 month old rats showed increased expression of SNCA and TTR, however, 2-year old rats exhibited no significant changes. In addition, Puskas et al. [183] demonstrated that administration of omega-3 fatty acids from fish oil (5% EPA and 27% DHA; total fat content: 8%) for 4 weeks in 2 year old rats induced expression of transthyretin and mitochondrial creatine kinase and decreased expression of HSP86, ApoC-I and Makorin RING zinc-finger protein 2, genes in hippocampus brain region. Finally, Flachs et al [179] showed increased expression of genes for mitochondrial proteins in adipose tissue.
In comparison with previous brain transcriptome analyses, the present study employing the use of high-density Affymetrix oligoarrays (.54,000 ps) revealed genes differentially regulated by LCPUFA at ranges mimicking breastmilk. With the exception of SPTLC2, which we also found to be upregulated in the L/C and L3/C comparisons, none of the remaining, previously identified genes, were differentially expressed in our dataset. Many factors are likely to contribute to the observed differences in differentially expressed genes between our study and previous work. One likely source is the difference in dietary DHA/ARA, which is within the range of human and baboon breastmilk; previous studies used much higher amounts of DHA, from 11.2% to 27% [182,183]. Also, interactions between levels of ARA and DHA supplied in our study add some complexity to the interpretation since the three treatments do not represent a strict dose response to DHA. However, our DHA and ARA come from sources that are routinely consumed by human infants in commercial infant formulas, and thus are directly relevant to that group. Despite lower levels of DHA/ARA, genes in our data set show subtle changes in expression. Moreover, the magnitude of these results is not surprising given the nutritional focus of the study, in which subtle, widespread shifts in transcription may have profound biological effects. Our data indicate that LCPUFA supplementation within the ranges of breastmilk will induce global changes in gene expression across numerous biological processes.

Conclusions
The impact of DHA and ARA on infant baboons was both significant and widespread. We identified several novel differentiallyexpressed transcripts in 12-week old baboon cerebral cortexes modulated by dietary LCPUFA. The majority of probe sets showed subtle changes in gene transcription. In the cerebral cortex, we observed increased expression of mitochondrial proton carrier, UCP2 (uncoupling protein 2) in both groups, but more in L3/C. PLA2G6, implicated in childhood neurodegeneration, was differentially expressed. TIA1, a silencer of the COX2 gene translation is upregulated in L3/C. Increased expression was observed for TIMM8A, NRG1, SEMA3D and NUMB, genes involved in neural development. LUM, EML2, TIMP3 and TTC8 genes with roles in visual perception were upregulated. Hepatic nuclear factor-4a (HNF4A) showed decreased expression with increasing DHA. RARA was repressed in both the groups. A network involving 35 genes attributed to neural development and function was identified using Ingenuity pathway analysis, emphasizing EGFR as the most outstanding interaction partner in the network. In this network EGFR interacts with genes involved in neural or visual perception, TIMP3, NRG1, ADAM17, EDG7 and FGF7. Although subtle, the upregulation of NUMB and downregulation of HES1 in the Notch signaling pathway, not previously shown to interact with fatty acids, supports the involvement of LCPUFA, particularly DHA, in neural development. Interestingly, no known desaturases and only one elongase, LCPUFA biosynthetic enzymes, were differentially expressed in cerebral cortex. In a study of liver gene expression in preparation, fatty acid desaturases SCD and FADS1 were significantly downregulated in liver, where we identified a multifunctional protein TOB1 which is significantly upregulated.
These data represent the first comprehensive transcriptome analysis in primates and have identified widespread changes in cerebral cortex genes that are modulated by increases in DHA, induced by dietary means. Importantly, the range of DHA used here is within limits of human and primate breastmilks, the natural food for infants, and indicate that CNS gene expression responds to LCPUFA concentrations.

MATERIALS AND METHODS
Details of experimental design, animal characteristics, and tissue sampling are available elsewhere [38] and will be outlined briefly here.

Animals and Diets
The animal phase took place at the Southwest Foundation for Biomedical Research (SFBR), San Antonio, TX, and was approved for animal care and research protocols from SFBR and Cornell University Institutional Animal Care and Use Committee (IACUC). Twelve baboon neonates born spontane-ously around 182 days gestation were randomized into 3 groups (n = 4 per group). They were fed for 12 weeks on one of three formulas: C: Control (no DHA-ARA); L: 16LCPUFA (0.33%DHA-0.67%ARA); L3: 36LCPUFA (1.00%DHA-0.67%ARA). Formulas in color-coded cans were kindly provided by Mead-Johnson Nutritionals (Evansville, IN) in ready-to-feed form, 2 colors per treatment, so that investigators were masked to the treatments.

Sampling and Array Hybridization
Twelve week old baboon neonates were anesthetized and euthanized at 84.461.1 days. Tissue collected from the precentral gyrus of the cerebral cortex was placed in RNALater according to vendor instructions and was used for the microarray analysis and validation of microarray results.
Microarray studies utilizing baboon samples with human oligonucleotide arrays have been successfully carried out previously [184,185]. Cerebral cortex global messenger RNA in the three groups was analyzed using Affymetrix Genechip TM HG-U133 Plus 2.0 arrays ,http://www.affymetrix.com/products/arrays/ specific/hgu133plus.affx.. The HG-U133 Plus 2.0 has .54,000 probe sets representing 47,000 transcripts and variants, including 38,500 well-characterized human genes. One hybridization was performed for each animal (12 chips total). RNA preparations and array hybridizations were processed at Genome Explorations, Memphis, TN ,http://www.genome-explorations.com.. The completed raw data sets were downloaded from the Genome Explorations secure ftp servers.

Microarray Data Analysis
Raw data (.CEL files) were uploaded into Iobion's Gene Traffic MULTI 3.2 (Iobion Informatics, La Jolla, CA, USA) and analyzed by using the robust multi-array analysis (RMA) method. In general, RMA performs three operations specific to Affymetrix GeneChip arrays: global background normalization, normalization across all of the selected hybridizations, and log2 transformation of perfect match oligonucleotide probe values [186]. Statistical analysis using the significance analysis tool set in Gene Traffic was utilized to perform Multiclass ANOVA on all probe level normalized data. Pairwise comparisons were made between C vs L and C vs L3 and all probe set comparisons reaching P ,0.05 were included in the analysis. Gene lists of differentially expressed probe sets were generated from this output for functional analysis.

RNA Isolation and RT PCR
RT PCR was conducted on nine genes to confirm the results of the array analysis. Total RNA from 30 mg samples of baboon cerebral cortex brain tissue homogenates was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA). Each RNA preparation was treated with DNase I according to the manufacturer's instructions.
The yield of total RNA was assessed by 260 nm UV absorption. The quality of RNA was analyzed by 260/280 nm ratios of the samples and by agarose gel electrophoresis to verify RNA integrity.
One microgram total RNA from each group (C, L, L3) was reverse-transcribed into first strand cDNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). The iScript reverse transcriptase is a modified MMLV-derived reverse transcriptase and the iScript reaction mix contains both oligo(dT) and random primers. The generated first strand cDNA is stored at 220uC until used.
Quantitative real-time PCR using SYBR green and TaqMan assay methods was used to verify the differential expression of selected genes that were upregulated in L3/C comparison. All the primers were gene-specific and generated from human sequences ,www.ensembl.org.. PCR primers were designed with Primer-Quest software (IDT, Coralville, IA) and ordered from Integrated DNA Technologies (IDT, Coralville, IA). Initially primers were tested by polymerase chain reactions with baboon cerebral cortex brain cDNA as template in a 30 ml reaction volume using Eppendorf gradient thermal cycler (Eppendorf), with 1 mm of each primer, 0.25 mM each of dNTPs, 3 ml of 106PCR buffer (Perkin-Elmer Life Sciences, Foster City, CA, USA), 1.5 mM MgCl 2 and 1.5 U Taq polymerase (Ampli Taq II; Perkin-Elmer Life Sciences). Thermal cycling conditions were: initial denaturation at 95uC for 5 min followed by 25-35 cycles of denaturation at 95uC for 30 s, annealing at 60uC for 1 min and extension at 72uC for 1 min, with a final extension at 72uC for 2 min. PCR products were separated by electrophoresis on 2% agarose gel stained with ethidium bromide and bands of appropriate sizes were obtained. The PCR products of LUM, TIMM8A, UCP2, ß-ACTIN, ADAM17 and ATP8B1 were sequenced and deposited with GenBank (Acc Numbers: DQ779570, DQ779571, DQ779572, DQ779573, DQ779574 and DQ779575, respectively).
Initially standardized primers for genes (ATP8B1, ADAM17, NF1, FZD3, ZNF611, UCP2, EGFR and control ß-ACTIN) were used for SYBR green real time PCR assay (Power SYBR Green PCR Master Mix, Applied Biosystems, Foster City, CA). We used the baboon LUM, TIMM8A and ß-ACTIN sequences to design TaqMan Assay (Assay by Design; ,www.appliedbiosystems. com.). The selected gene symbols, primer pairs and probe details are depicted in Table S3. Quantitative real time PCR reactions were done with the Applied Biosystems Prism 7300/7500 real time PCR system (Applied Biosystems, Foster City, CA). After 2 minutes of UNG activation at 50uC, initial denaturation at 95uC was carried out for 10 minutes, the cycling conditions of 40 cycles consisted of denaturation at 95uC for 15 seconds, annealing at 60uC for 30 seconds, and elongation at 72uC for 1 minute. For SYBR green method UNG activation step is eliminated. All reactions were done in triplicate and ß-ACTIN was used as the reference gene. Relative quantification was performed by using comparative CT method (ABI Relative Quantification Chemistry guide # 4347824).

Network Analysis
We used a new web-delivered bioinformatics tool set, Ingenuity pathway analysis (IPA 3.0) ,http://www.ingenuity.com., to identify functional networks influenced by our dietary treatments. IPA is a knowledge database generated from the peer-reviewed scientific publications that enables discovery, visualization and exploration of functional biological networks in gene expression data and delineates the functions most significant to those networks. The 1108 differentially expressed probe sets identified by microarray data, as discussed below, were used for network analyses. Affymetrix probe set ID's were uploaded into IPA and queried against all other genes stored in the IPA knowledge database to generate a set of networks having up to 35 genes. Each Affymetrix probe set ID was mapped to its corresponding gene identifier in the IPA knowledge database. Probe sets representing genes having direct interactions with genes in the IPA knowledge database are called ''focus'' genes, which were then used as a starting point for generating functional networks. Each generated network is assigned a score according to the number of differentially regulated focus genes in our dataset. These scores are derived from negative logarithm of the P indicative of the likelihood that focus genes found together in a network due to random chance. Scores of 4 or higher have 99.9% confidence level of significance as defined in detail elsewhere [188].