Mammalian Embryonic Cerebrospinal Fluid Proteome Has Greater

Oct 20, 2005 - During early stages of embryo development, the brain cavity is filled with Embryonic Cerebro-Spinal Fluid, which has an essential role ...
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Mammalian Embryonic Cerebrospinal Fluid Proteome Has Greater Apolipoprotein and Enzyme Pattern Complexity than the Avian Proteome Carolina Parada,† AÄ ngel Gato,‡,§ and David Bueno*,†,§ Departament de Gene`tica, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, 08028 Barcelona, Catalonia, Spain, and Departamento de Anatomı´a, Facultad de Medicina, Universidad de Valladolid, C/Ramo´n y Cajal 8, Valladolid, Spain & Laboratorio de Desarrollo y Teratologı´a del Sistema Nervioso, Instituto de Neurociencias de Castilla y Leo´n, Universidad de Valladolid, Valladolid, Spain Received July 14, 2005

Abstract: During early stages of embryo development, the brain cavity is filled with Embryonic Cerebro-Spinal Fluid, which has an essential role in the survival, proliferation and neurogenesis of the neuroectodermal stem cells. We identified and analyzed the proteome of Embryonic Cerebro-Spinal Fluid from rat embryos (Rattus norvegicus), which includes proteins involved in the regulation of Central Nervous System development. The comparison between mammalian and avian Embryonic CerebroSpinal Fluid proteomes reveals great similarity, but also greater complexity in some protein groups. The pattern of apolipoproteins and enzymes in CSF is more complex in the mammals than in birds. This difference may underlie the greater neural complexity and synaptic plasticity found in mammals. Fourteen Embryonic Cerebro-Spinal Fluid gene products were previously identified in adult human Cerebro-Spinal Fluid proteome, and interestingly they are altered in patients with neurodegenerative diseases and/or neurological disorders. Understanding these molecules and the mechanisms they control during embryonic neurogenesis may contribute to our understanding of Central Nervous System development and evolution, and these human diseases. Keywords: embryonic cerebrospinal fluid proteome • rat • chick • human • central nervous system development • neurodegeneration • neuronal complexity

Introduction In vertebrates, at early stages of Central Nervous System (CNS) development, the architecture of the brain primordium reveals the presence of the cavity of brain vesicles, which is filled by Embryonic Cerebro-Spinal Fluid (E-CSF). The E-CSF, which contacts the apical surface of CNS neuroectodermal cells, has several crucial functions during brain development, which * To whom correspondence should be addressed. Tel: 34-934037070. Fax: 34-934034420. E-mail: [email protected]. † Universitat de Barcelona. ‡ Universidad de Valladolid. § These authors contributed equally to this work.

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Journal of Proteome Research 2005, 4, 2420-2428

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have been analyzed in chick and rat. It exerts positive pressure against the neuroepithelial walls and generates an expansive force;1-6 it contributes to the regulation of the survival, proliferation, and neurogenesis of the neuroectodermal stem cells,2,7,8 and it collaborates with the isthmic organizer to regulate neuroectodermal gene expression.9 Until now, few papers have focused on the protein composition of E-CSF.10 Recently, we reported the analysis of avian (Gallus gallus) E-CSF proteome.11 At the stage of maximum proliferation and differentiation of neuroectodermal stem cells, which in chick embryos occurs at developmental stage HH24,12 corresponding to embryonic day 4 (E4), chick E-CSF contains molecules involved in the functions mentioned above. It contains gene products related to the extra-cellular matrix and to the regulation of osmotic pressure. These products are thought to be involved in the growth of cephalic anlagen and the generation of the expansive force exerted by E-CSF.11 Chick E-CSF proteome also includes molecules such as regulators of lipid metabolism, i.e., apolipoprotein AI and AIV, molecules involved in vitamin transport (e.g., retinol-binding protein and transthyretin), and proteins related to cell quiescence and death, which are thought to be responsible, at least partially, for E-CSF functions in regulation of cell proliferation, neural differentiation, and morphogenesis of the cephalic vesicles. Some proteins with antioxidant and antimicrobial properties have also been identified, which may contribute to the generation of a proper environment for the action of the rest of the proteins.11 Apart from this report on chick E-CSF, no other vertebrate species, e.g., mammals, have been analyzed. Unlike mammal E-CSF, adult human CSF has been deeply analyzed. Proteome studies of adult human CSF have shown the presence of 249 known proteins, although most of them are unique to individual subjects, and fewer than 35 are common to all analyzed subjects.13-15 Interestingly, in patients affected by neurodegenerative disorders, the concentration and/or the electrophoretic pattern of some CSF proteins is altered, e.g., apolipoproteins, transthyretin and plasma retinol-binding protein, among others.16,17 Chick E-CSF proteome also includes proteins that in adult humans have been reported to be altered in neurodegenerative diseases and/or neurological disorders such as Alzheimer’s and Parkinson’s Disease. Some of these molecules, which are involved in neuroectodermal cell survival, prolifera10.1021/pr050213t CCC: $30.25

 2005 American Chemical Society

technical notes

Parada et al.

Figure 1. 2-DE analysis of rat E-CSF proteins. The proteins were separated on a pH 3-11 IPG strip, followed by a 12.5% SDS-gel, as described in Materials and Methods. The gel was stained with silver nitrate for MS. The identities of proteins were deduced from their similarity to rat (Rattus norvegicus) proteins, or to mouse (Mus musculus) for nidogen-2. The identities assigned are listed in Table 1. On the right-hand side of the 2-DE separation, a MW marker (BioRad, Broad Range Standard) was included. The single bands correspond to apparent MW of 209 kDa (myosin); 124 kDa (β-galactosidase); 80 kDa (BSA); 49.1 (ovalbumin); 34.8 kDa (carbonic anhydrase); 28.9 kDa (soybean trypsin inhibitor); 20.6 kDa (lysozyme); and 7.1 kDa (aprotinin), from top to bottom.

tion and differentiation in vertebrate embryos, might take part to some degree in similar functions in the adult brain.2,9,11 Nevertheless, the specific functions of these molecules in both E-CSF and adult CSF are unclear. Using proteomic approaches we identify the E-CSF proteome in the rat, at a developmental stage equivalent to that of the chick, which also corresponds to the period of maximum neuroectodermal stem cell proliferation and neurogenesis. Rat and chick E-CSF proteomes are similar, although rat is more complex in certain groups of proteins, e.g., apolipoproteins, which may be involved in the control of neural diversity, and has soluble enzymes present, just like adult human CSF, but unlike chick E-CSF, revealing phylogenetic brain differences between these groups of vertebrates. Knowledge of a mammalian E-CSF proteome will help us understand the processes that convert the neuroectoderm into a functional brain, and open a new window onto understanding of the function of adult CSF on neural stem cells.

Materials and Methods Obtaining Embryonic Cerebrospinal Fluid. Rats (Rattus norvegicus) were naturally mated, with the morning of appearance of a vaginal plug designated as embryonic day 0.5 (E0.5). Embryos at stage E12.7 were dissected from extra-embryonic membranes and washed in sterile saline solution, and their E-CSF was aspired under the dissecting microscope with a glass microneedle (30 µm inner diameter at the tip), carefully placed in the middle of the mesencephalic cavity and connected to a micro-injector (Nanoject II). E-CSF was slowly aspired so as to avoid contact with the neuroepithelial wall, thus avoiding the sample being contaminated by neuroepithelial cells. All experiments were approved by the Committee for animal research and welfare of the Universitat de Barcelona (Commite` EÅ tic d′Experimentacio´ Animal).

To minimize protein degradation, E-CSF samples were kept at 4 °C during this procedure. E-CSF from 70 embryos (0.2 mg of total protein content) was pooled and used in all procedures. Although the presence of cells was not detected in the preparation by microscopic inspection, pooled E-CSF samples were centrifuged at 2000 × g at 4 °C for 10 min to avoid the presence of any insoluble particles that might interfere with 2D-electrophoresis. Pellet was not detected in any of the samples. After centrifugation, samples were immediately precipitated in icecold acetone (1:8 v/v). Precipitated samples were kept at -20 °C until analysis, which was performed within 24 h of withdrawal. 2D-Electrophoresis. One mg of total protein was resuspended in 450 µL of rehydration buffer (7 M urea, 2 M thiourea, 80 mM DTT, 2% CHAPS, 0.5% IPG buffer, bromophenol blue) and loaded onto 24 cm, pH 3-11 NL IPG strip.18 IEF separation was performed in an IPGphor system (Amersham Bioscience) according to manufacturer’s instructions. Focused strips were equilibrated with equilibration buffer (65 mM DTT, 50 mM TrisHCl, 6 M urea, 30% glycerol, 2% SDS, bromophenol blue) for 15 min followed by (135 mM IAA, 50 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS, bromophenol blue) for 15 min.19 The equilibrated strips were directly applied to 12.5% acrylamide gel20 and separated at 3 V/gel for 30 min and at 18 V/gel for 6 h in a Ettan DALT II system (Amersham Bioscience). Gels were fixed overnight, subjected to silver mass spectrometry compatible staining21 and then scanned. In-Gel Digestion. Proteins were in-gel digested with trypsin (Sequencing grade modified, Promega) in the automatic Investigator ProGest robot of Genomic Solutions. Briefly, excised gel spots were washed sequentially with ammonium bicarbonate buffer and acetonitrile. Proteins were reduced and alkylated by treatment with 10 mM DTT solution for 30 min and with a 100 mM solution of iodoacetamide, respectively. After sequential washings with buffer and acetronitrile, proteins were Journal of Proteome Research • Vol. 4, No. 6, 2005 2421

technical notes

Rat Embryonic Cerebro-Spinal Fluid Proteome Table 1. E-CSF Proteins Identified in the Tryptic Digest, as Described in Material and Methodsa

protein ID

species

accession no. (Mascot database)

agrin albumin alpha fetoprotein angiotensinogen apolipoprotein A-I apolipoprotein A-IV apolipoprotein B apolipoprotein E apolipoprotein M calreticulin collagen alpha 1 (III) collagen alpha 1 (V) collagen alpha1 (XI) corticosteroid-binding globulin (CBG) (Transcortin) DJ-1 protein Eef1 g protein heparan sulfate proteoglycan core protein (HSPG) (Perlecan) (PLC) laminin receptor 1 lumican malate dehydrogenase 1, NAD (soluble) nidogen-2 peroxiredoxin 2 (peroxidase) phosphatase 2, alpha isoform of regulatory subunit A phosphatidylethanolamine binding protein plasma retinol-binding protein serine (or cysteine) proteinase inhibitor, clade F, member 1 set beta isoform transferrin transthyretin, chain D tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide

Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus

gi|202799 gi|55391508 gi|228784 gi|19705570 gi|6978515 gi|8392909 gi|34863099 gi|37805241 gi|46237618 gi|11693172 gi|3171998 gi|19745166 gi|34859865 gi|399194

scoreb

no. of peptide matched

% sequence coverage

MWckDa

pId

429>47 from 196>47 to 987> 46 from 203>45 to 1424>46 from 109>45 to 175>46 from 425>46 to 655>47 190>49 174>46 104>45 from 109>45 to 175>46 61>44 67>48 from 248>45 to 276>47 95>45 256>47

10 from 4 to 22 from 9 to 56 from 3 to 4 from 27 to 34 4 3 2 from 8 to 11 2 2 from 5 to 7 2 9

5 from 6 to 35 from 10 to 60 from 11 to 13 from 45 to 52 10 47 Rattus norvegicus gi|51261278 76>48 Rattus norvegicus gi|34872189 340>47

5 2 6

33 2 1

20190 50371 488524

6.32 6.31 6.09

Rattus norvegicus gi|8393693 316>48 Rattus norvegicus gi|13591983 185>47 Rattus norvegicus gi|15100179 287>46

4 4 7

18 11 24

32917 38654 36631

4.80 6.00 6.16

Mus musculus gi|23592218 178>47 Rattus norvegicus gi|8394432 191>46 Rattus norvegicus gi|55926139 203>47

5 5 3

2 18 8

156609 21963 66079

5.20 5.34 5.00

Rattus norvegicus gi|8393910

2

14

20902

5.48

131>46

Rattus norvegicus gi|206589 486>47 Rattus norvegicus gi|29293811 from 821>45 to 827>46

21 58 from 18 to 21 from 38 to 42

20331 46493

5.67 6.04

Rattus norvegicus Rattus norvegicus Rattus norvegicus Rattus norvegicus

2 from 47 to 50 9 from 6 to 12

32086 76908 13122 29274

4.1 6.35 6.04 4.55

gi|545265 gi|15825992 gi|3212535 gi|13928824

from 63>47 to 108>45 from 985>45 to 1480>46 253>46 from 255>46 to 488>47

from 3 to 10 from 43 to 45 63 from 17 to 39

a Species similarity used to deduce identity, accession no., score, no. of peptides matched, percentage sequence coverage, MW and pI corresponding to available genomic information are given. Score, number of peptides matched, and percentage sequence coverage ranges for proteins with more than 1 spot analyzed are given. All proteins were identified by ESI-MS/MS mass spectrometry. b Score X > Y indicates identity or extensive homology (p < 0.05). c theoretical molecular mass calculated from the amino acid sequence. d theoretical pI calculated from the predicted mature protein.

digested overnight, at 37 °C with 0.27 nmol of trypsin. Tryptic peptides were extracted from the gel matrix with 10% formic acid and acetonitrile; the extracts were pooled and dried in a vacuum centrifuge. Acquisition of MS/MS Spectra. Proteins excised from the two-dimensional gels were analyzed by ESI-MS/MS mass spectrometry22 (Q-TOF Global, Micromass-Waters). Q-TOF was calibrated using 200 fmol Glu1-fibrinopeptideB peptide fragmentation by direct injection. Samples were analyzed by online liquid chromatography tandem mass spectrometry (CapLC-nano-ESI-Q-TOF) (CapLC, Micromass-Waters; Actual flow: 200 nl/min). The HPLC gradient was 5-65%B in 40 min linear gradient (A: 0.1% formic acid/2% ACN/H2O; B: 0.1% formic acid/10% H2O/ACN). Samples were resuspended in 15 µL of 10% formic acid solution and 4 µL were injected to chromatographic separation in a reversed-phase capillary C18 column (75 µm internal diameter and 15 cm long, PepMap column, LC Packings). The eluted peptides were ionized via 2422

Journal of Proteome Research • Vol. 4, No. 6, 2005

coated nano-ES needles (PicoTipTM, New Objective). A capillary voltage of 1800-2200 V was applied together with a cone voltage of 80 V. The collision in the CID (collision-induced dissociation) was 25-35 eV and argon was employed as collision gas. Data were generated in PKL file format, which was submitted for database searching (NCBInr) in MASCOT server. Mass tolerance used for the search was 100-200 ppm.

Results and Discussion General Overview of Rat E-CSF Proteome. Using proteomic approaches we identified and analyzed the proteome of E-CSF from rat embryos at developmental stage E12.7. The results are shown in Figure 1 and summarized in Table 1. Thirty-one proteins contained within E-CSF were identified. The number of peptide matches and the percentage of sequence coverage for each identified gene product are shown in Table 1. Proteins with individual ions scores indicating identity or extensive homology (p < 0.05) were included. Proteins matching only

technical notes

Parada et al.

Table 2. Classification of the Proteins Identified According to Their Functional or Structural Characteristics, as Described in the Literature on Systems Other than E-CSF

group 1: extracellular matrix proteins

group 2: proteins of osmotic pressure and ion carriers

group 3: regulators of lipid metabolism

group 4: retinol and corticosteroid carriers

Agrin

group 5: enzymes or enzyme regulators

group 6: antioxidant and antimicrobial proteins

Angiotensinogen Apolipoprotein AI Retinol binding Malate Albumin protein dehydrogenase 1 Collagen alpha 1 Calreticulin Apolipoprotein Trasnthyretin Tyrosine 3Alpha-fetoprotein (III) AIV monooxygenase/ tryptophan 5monooxygenase activation protein Collagen alpha 1 Transferrin Apolipoprotein B Corticosteroid Peroxiredoxin 2 (V) binding globulin Collagen alpha 1 Apolipoprotein E Phosphatase 2 (XI) Heparan sulfate Apolipoprotein M Serine (or cystein) protein proteinase inhibitor Laminin receptor 1 Lumican Nidogen-2

one peptide or not matching any known secreted protein were not included. The identities of proteins were deduced from their similarity to available Rattus norvegicus sequences. The genetic products not matching any sequence from this species were identified by their similarity to other vertebrate species sequences (i.e., nidogen-2, from Mus musculus). Unidentified protein spots correspond to either low concentrated proteins, hydrophobic proteins or proteins with too few trypsin targets. The pI and MW listed in Table 1 correspond to available genomic information (see also the accession no.). A few proteins with different MW were observed, probably due to the presence of truncated forms as a result of physiological or artifactual proteolysis, and/or modified forms as a result of post-translational modifications, e.g., glycosylation. Similarly, some proteins showed spots focusing at different pI, probably corresponding to differences in phosphorylation. On the other hand, the proteins were present in the E-CSF in very different amounts, being alpha-fetoprotein the most represented protein (Figure 1), There was a wide range of protein amounts in the E-CSF, alpha-fetoprotein being the most represented (Figure 1) probably due to the particular function of each gene product in the embryonic development. The proteins identified were classified in seven groups established according to their functional or structural characteristics, as described in the literature for systems other than E-CSF (Table 2). The first group includes proteins related to extra-cellular matrix, i.e., agrin, collagen alpha 1 (III), (V), and (XI), heparan sulfate protein, laminin receptor 1, lumican, and nidogen-2. The second group includes gene products associated with the regulation of osmotic pressure and ion transport, i.e., angiotensinogen, calreticulin and transferrin. The third group includes apolipoproteins AI, AIV, B, E, and M, which are regulators of lipid metabolism. The fourth group includes retinol and corticosteroid carriers, i.e., retinol binding protein, transthyretin, and corticosteroid-binding globulin. The fifth group includes several types of enzymes and enzyme regulators, i.e., malate dehydrogenase 1, tyrosine 3-monooxygenase/

group 7: proteins with unknown function during embryonic development

DJ-1 protein Eef1 g protein

Phosphatidylethanolamine binding protein Set beta isoform

tryptophan 5-monooxygenase activation protein, peroxiredoxin 2, phosphatase 2, and serine (or cystein) proteinase inhibitor. The sixth group includes antioxidant and antimicrobial proteins, i.e., albumin and alpha-fetoprotein. The last group includes proteins whose structure and/or function has not been described in detail, i.e., DJ-1 protein, Eef1 g protein, phosphatidylethanolamine binding protein, and set beta isoform. Implications of Rat E-CSF Proteome for CNS Development and Neurodegenerative Diseases. It is known that the development of the CNS from relatively simple anlagen involves the simultaneous and interdependent action of various developmental mechanisms, including the establishment of positional identities, morphogenesis and histogenesis. It has been demonstrated that diffusible molecules, such as growth factors and morphogens secreted locally by organizing centers, regulate these processes by controlling neighbor cells in an autocrine/ paracrine manner.23,24 In addition, the architecture of the brain primordium reveals another component, the cavity of brain vesicles, which is filled by E-CSF, with which a physiologically sealed system is established. It was recently reported that during CNS development E-CSF also has important functions in CNS development, such as favoring brain expansion and growth, regulating cell behavioral parameters, i.e., neuroectodermal stem cell survival, proliferation, and primary neurogenesis, and collaborating with the isthmic organizer in the establishment of the proper pattern of gene expression at early stages of CNS development.2,5,6,7 It has also recently been shown that chick E-CSF contains molecules that are thought to be responsible for the reported functions of this embryonic fluid in this species.11 At a developmentally equivalent stage to that of chick, rat E-CSF proteome exhibits a complex protein pattern that is very similar to chick E-CSF. The presence of extracellular matrix molecules (Group 1) and of proteins related to control of osmotic pressure and ion carriers (Group 2) confirms that in mammals E-CSF is also involved in the regulation of brain expansion and growth through generation of positive pressure against neuroepithelial Journal of Proteome Research • Vol. 4, No. 6, 2005 2423

technical notes

Rat Embryonic Cerebro-Spinal Fluid Proteome

Table 3. Summary of the Available Information on the Proteins Identified, as Described in the Literature on Systems Other than E-CSFa

protein ID

Homo Gallus sapiens gallus (CSF)b,c (E-CSF)

-d

Agrin

+

Albumin

+d

+

Angiotensinogen

+

-e

Alpha fetoprotein

-

-e

Apolipoprotein A-I

+

+

Apolipoprotein A-IV +

+

Apolipoprotein B

2424

+

-e

originc

extracellular matrix heparan sulfate proteoglycan of neuronal origin

function in developmentc

increases the affinity of FGF2 to its receptor.

alteration in human diseasesc

accumulated within cerebrovascular amyloid deposits.

control of formation, maintenance and regeneration of neuromuscular junction. axonal growth. establishment of blood-brain barrier (BBB). placenta proliferation of microglia. raised intracerebral levels associated with Alzheimer’s disease formation of scars after breakdown of BBB. liver cell differentiation. not associated placenta anlagen growth. regulator of electrolyte homeostasis and pressure. liver major serum protein. altered in patients with autosomal recessive spinocerebellar ataxia, cerebellar atrophy and peripheral neuropathy. yolk sac increased in CSF of patients suffering intracranial germ cell tumor. liver lipid transport. accumulated in amyloid plaques in Alzheimer’s disease patients. intestine control of collaborates with embryo growth reelin receptor (candidate through MAP gene for Alzheimer’s kinase activation. disease). liver lipid transport. low level in schizophrenic patients. intestine control of embryo growth through MAP kinase activation. liver lipid transport. increased in serum and amyloid plaques of Alzheimer’s disease patients. yolk sac polymorphisms associated with Alzheimer’s disease, vascular dementia and Parkinson’s disease. polymorphisms increased in CSF of associated with multiple sclerosis and Alzheimer’s disease, neurological disease vascular dementia and patients Parkinson’s disease. increased in CSF of multiple sclerosis and neurological disease patients

Journal of Proteome Research • Vol. 4, No. 6, 2005

refs

39

40, 41

42-49

50-56

57- 59

60

61-66

technical notes

Parada et al.

Table 3. (Continued)

protein ID

Apolipoprotein E

Apolipoprotein M Calreticulin

Homo sapiens (CSF)b,c

Gallus gallus (E-CSF)

+

-e

-

-e

-

-e

originc

liver

liver kidney

function in developmentc

alteration in human diseasesc

lipid transport.

control of embryo growth through MAP kinase activation. lipid transport. Ca(2+)-binding protein

refs

APOE epsilon 4 is associated with Alzheimer’s disease

16, 67

not reported

68

factor contributing to neuronal oxidative stress and neuronal demise in Alzheimer’s disease patients

69-72

Collagen alpha 1(III) Collagen alpha 1 (V) Collagen alpha 1 (XI) Corticosteroid-binding globulin (CBG) (Transcortin)

+

+ + -e -

extracellular matrix extracellular matrix extracellular matrix liver

control of neural synapses through transduction system of NMDA. not reported not reported not reported cortisol transport.

DJ-1 protein

-

-

kidney unknown

fertilization.

Eef1 g protein Heparan sulfate proteoglycan core protein precursor (HSPG) (Perlecan) (PLC) Laminin receptor 1

+

-e

unknown extracellular matrix

not reported cell proliferation.

-

-e

extracellular matrix

not reported

Lumican

-d

-e

extracellular matrix

Malate dehydrogenase 1, NAD (soluble) Nidogen-2

+

-

unknown

control of mossy fiber growth and regeneration. acts in rat hippocampus.

-

-e

extracellular matrix

not reported

Peroxiredoxin 2 (peroxidase)

+

-

unknown

Phosphatase 2, alpha isoform of regulatory subunit A Phosphatidylethanolamine binding protein Retinol binding protein

-d

-

unknown

axonal marker of developing neurons. related to neurotrophins.

-

-

unknown

not reported

not reported

+

+

liver

as retinol carrier, in morphogenesis and neural differentiation.

altered in Alzheimer’s disease patients

not reported not reported not reported not reported

73, 74

altered in Parkinson’s disease patients, as it normally acts as a activator that protects against apoptosis. not reported amyloidosis and Tau aggregation in Alzheimer’s disease

75

amyloidosis in Alzheimer’s disease. not reported

78

brain damage marker.

32, 80

interaction with amyloid precursor proteins in Alzheimer’s disease. altered in substancia nigra pars compacta in Parkinson’s.

81

altered in the hippocampus of Alzheimer’s disease patients.

84, 85

76, 77

79

82, 83

28, 86, 87

placenta

Journal of Proteome Research • Vol. 4, No. 6, 2005 2425

technical notes

Rat Embryonic Cerebro-Spinal Fluid Proteome Table 3. (Continued)

protein ID

Serine (or cysteine) proteinase inhibitor, clade F), member 1

Homo sapiens (CSF)b,c

Gallus gallus (E-CSF)

+

-

function in developmentc

alteration in human diseasesc

establishment of DV polarity in the Drosophila embryo. general mechanisms for achieving spatial control in diverse biological processes. maintenance and development of neuronal cells. not reported cell differentiation and proliferation. regulation of anionic concentration. as an RBP carrier, in morphogenesis and neural differentiation

increased in CSF in injured blood brain barrier

originc

Set beta isoform Transferrin

+

+

unknown liver

Transthyretin Chain D (prealbumin)

+

+

liver

Tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein (14-3-3)

+

-

unknown

not reported

refs

88, 89

altered in disorders similar to Alzheimer’s disease.

not reported not reported

90

altered in Alzheimer’s disease patients, as normally it protects against amyloidosis.

91, 92

altered in multiple sclerosis and massive brain destruction.

34

increased in patients suffering neurological diseases. a Presence within adult human CSF and chick E-CSF, embryological origin, function in development in systems other than E-CSF, alterations reported in adult human diseases and specific references are given. b CSF from adult humans (http://au.expasy.org/cgi-bin/map2/def?CSF_HUMAN) c According to literature, at the analyzed development stages unless stated d Present in the brain. e Protein with similar function.

walls. Rat E-CSF also contains several molecules involved in signal transduction pathways, such as apolipoproteins AI and E (Group 3). In adults, apolipoproteins are thought to be responsible for cholesterol transport, synapse formation, modulation of neurite outgrowth and synaptic plasticity, destabilization of microtubules and β-amyloid clearance.25 Apolipoproteins may influence similar processes during embryonic development, although their precise embryonic function is unknown.26 Rat E-CSF proteome also shows the presence of retinol transporters (Group 4). Active retinoids act as morphogens during CNS development, regulating both the establishment of anterior-posterior patterning and primary neurogenesis.27,28 We also identified several enzymes (Group 5) and other proteins (Group 7), whose function during embryonic development has not yet been established. Function for each particular protein reported in the literature in system other than E-CSF are depicted in Table 3. All in all, our results indicate that mammal E-CSF may have the same functions in neuroectodermal stem cells as avian E-CSF. Interestingly, almost half the proteins contained in rat E-CSF are also present in adult human CSF. As the study of neurodegenerative disorders is currently a hot topic in research, generating a huge amount of information, most of those proteins have been associated in some way, either as cause or consequence, with the pathogeny of neurodegenerative diseases, e.g., Alzheimer’s and Parkinson’s diseases (Table 3). Thus, as molecules contained in the E-CSF are known to be involved in neuroectodermal cell survival,2,7 a process that is severely impaired in these neurodegenerative diseases, it is tempting to suggest that they may perform to some degree 2426

Journal of Proteome Research • Vol. 4, No. 6, 2005

similar functions in the adult brain, despite eventually any CSF protein may be potentially related in some way to these neurodegenerative disorders. It has been suggested that adult CSF may act as a fluid pathway for delivering diffusible signals and could thus affect the cell behavior of determined brain parenchyma cells.29 Recently, it has been shown that in adult brain the stem cells located at the subventricular zone have transitory contact with the ventricular brain cavity,30,31 which suggests that adult neurogenesis could be controlled by the molecules contained within CSF. Thus, understanding these molecules and the mechanisms they control during embryonic neurogenesis not only contributes to the general understanding of CNS development, but it may also contribute to a deeper understanding of these human diseases. Comparison between Rat and Chick E-CSF Proteomes. As stated above, at a developmentally equivalent stage corresponding to maximum neuroectodermal stem cell proliferation and differentiation, rat (E12.7) and chick (E4) E-CSF show the presence of proteins belonging to the same protein groups established, although the identity of specific proteins differ in some cases, suggesting that there are inter-specific differences. The main differences rely on enzymes, enzyme regulators and apolipoproteins. Rat E-CSF includes a group of proteins that have no functional homologous in chick E-CSF, i.e., enzymes and enzyme regulators. Interestingly, most of these enzymes are also present in adult human CSF,32-34 in which at least some of them have been linked to the aetiology of neurodegenerative diseases. However, very little is known on their actual role in adults and nothing in embryos, as this is the first report on

technical notes the occurrence of these molecules during embryonic development. This phylogenetic difference between mammals and avians makes it tempting to speculate that the presence of these gene products is related to the development and/or maintenance of the mammal brain. Moreover, the most remarkable difference between chick and rat E-CSF proteomes is an increase in the number of members of the apolipoprotein family in rat. In chick E-CSF only two different apolipoproteins, AI and AIV, have been identified, whereas rat E-CSF includes at least five different apolipoproteins, those found in the chick fluid as well as apolipoproteins B, E, and M. The best analyzed of these are apoAI and apoE.26,35 Studies of phylogenetic distribution of apolipoproteins have shown that apolipoprotein AI is highly conserved through vertebrate evolution, while apolipoprotein E is absent in the avian species analyzed, i.e., chicken and quail, although it is present in organisms such as zebrafish and turtle. In zebrafish, apoE expression correlates with endogenous lipid nutrition and yolk lipoprotein synthesis during embryonic development, suggesting that in this species its role is restricted to lipid metabolism.26 The functional significance of the absence of the apoE gene in the avian lineage is unclear, although it has been suggested that the absence of apoE allows for more rapid uptake of lipoproteins into oocytes and for rapid oocyte growth, which are characteristic of birds, since in other species apoE encourages sequestration of lipoproteins into other tissues.35 In mammals, apoE is thought to act by encouraging neuronal complexity, as it is involved in multiple signaling pathways in primary neurons to control the activation of neurite outgrowth, neuronal migration, and synaptic plasticity.25 Moreover, this apolipoprotein is involved in the aetiology and progress of Alzheimer’s disease and other neurodegenerative disorders. Since mammal brain is more complex than avian brain in terms of neuronal diversity and synaptic plasticity, especially in the cortex36-38, the presence of apoE in rat E-CSF at E12.7, as well as of apoB and M, whose function has not been so thoroughly analyzed, indicates that E-CSF reflects the phylogenetic differences between avian and mammal brain. It is tempting to speculate that E-CSF also contributes to the establishment of these differences from this very early stage of development, when primary neurogenesis starts.

Acknowledgment. We thank Dr. Eliandre de Oliveria and David Bellido (Proteomic Platform-SCT-PCB, University of Barcelona) for their assistance in protein identification. This research was financed by the Ministerio de Sanidad y Consumo Instituto de Salud Carlos III (Grant No. 02/0915, to D. Bueno; and Grant Number 02/0961, to A. Gato), co-financed by the European Community FEDER; by the Junta de Castilla y Leo´n (Grant Nos. VA049/04 and VA17/03, to A. Gato); and by the Generalitat de Catalunya (Grant No. GRQ93-1044, to Prof. Jaume Bagun ˜ a`). References (1) Miyan, J. A.; Nabiyouni, M.; Zendah, M. Can. J. Physiol. Pharmacol. 2003, 81, 317-328. (2) Gato, A.; Moro, J. A.; Alonso, M. I.; Bueno, D., et al. Anat. Rec. 2005, 284A, 475-484. (3) Desmond, M. E.; Jacobson, A. G. Dev. Biol. 1977, 57, 18-198. (4) Jeliken, R.; Peixeder, T. Folia Morphol. 1970, 18, 102-110. (5) Alonso, M. I.; Gato, A.; Moro, J. A.; Martin, P.; Barbosa, E. Cells Tissues Organs. 1999, 165, 1-9. (6) Alonso, M. I.; Moro, J. A.; Martin, P.; Barbosa, E.; Gato, A. Eur. J. Anat. 2000, 4, 161-167. (7) Owen-Lynch, P. J.; Draper, C. E.; Mashayekhi, F.; Bannister, C. M.; Miyan, J. A. Brain 2003, 126, 623-631.

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