A maternal blood-borne factor promotes survival ... - The FASEB Journal

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The FASEB Journal express article 10.1096/fj.04-1789fje. Published online December 6, 2004.

A maternal blood-borne factor promotes survival of the developing thalamus Peter Landgraf,*,† Frank Sieg,* Petra Wahle,‡ Gundela Meyer,§ Michael R. Kreutz,† and Hans-Christian Pape* *Institute of Physiology, Otto-von-Guericke University, Magdeburg, Germany; ‡AG Developmental Biology, Faculty of Biology, Ruhr-University, Bochum, Germany; §Department of Anatomy, Faculty of Medicine, University of La Laguna, Tenerife, Spain; and †AG ‘Molecular Mechanisms of Plasticity’, Department of Neurochemistry/Molecular Biology, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany Corresponding author: Michael R. Kreutz, AG ‘Molecular Mechanisms of Plasticity’, Department of Neurochemistry/Molecular Biology, Leibniz Institute for Neurobiology, Brenneckestr. 6, 39118 Magdeburg, Germany. E-mail: [email protected] ABSTRACT In this report, we describe the identification of a polypeptide survival-promoting factor that is produced by maternal and early postnatal peripheral blood mononuclear cells (PBMCs) of the immune system in Long-Evans rats and humans. The factor, termed Y-P30, most likely arises from proteolytic processing of a larger precursor protein and accumulates mainly in pyramidal neurons of the developing cortex and hippocampus but not in astrocytes. It was released from neurons grown in culture and substantially promotes survival of cells in explant monocultures of perinatal thalamus from the offspring. Y-P30 mRNA was not detectable in infant or adult brain and was present only in blood cells of pregnant rats and humans but not in nonpregnant controls. However, Y-P30 transcription could be induced in PBMCs of adult animals by a central nervous system lesion (i.e., optic nerve crush), which points to a potential role of the factor not only in neuronal development but also in neuroinflammation after white matter injury. Keywords: Y-P30 • survival-promoting peptide • sprouting • optic nerve crush • inflammation

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revious findings from our laboratory have shown that the maintenance of organotypic monocultures of dorsal thalamus can be improved by use of coculture with an agematched cortical explant or cortex-conditioned medium (CM), which suggests the existence of trophic factors in the CM (1, 2). However, supplementing thalamic monocultures with known growth factors of the neurotrophin family failed to mimic the cortex-conditioned activity. We therefore hypothesized that an essential and yet unidentified biological activity is secreted from cortical tissue, which promotes survival of thalamic cells. In fact, by using a twostep experimental strategy, we isolated from the CM (1, 2) a single polypeptide factor responsible for the survival-promoting effects of thalamic monocultures. First, we used biochemical purification steps and subsequent sequence analysis by mass spectroscopy and Edman degradation analysis. Second, because cerebellar microexplant cultures can be prepared

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in a standardized manner and much higher numbers than can thalamus-cortex cocultures, we used the cerebellar culture system to control and measure biological activity after each purification step by quantifying the length of neurite extension and explant size. MATERIALS AND METHODS Cell cultures Long-Evans rats at different postnatal (P) days were killed by decapitation and used for preparation of organotypic co- and monocultures (P0) and cultures of cerebellar microexplants (P5). Dissociated cell cultures from cortex were prepared from embryos at embryonic stage E19, as described previously for the hippocampus (3). For explant cultures, the occipital cortex was dissected as described previously (4) and coronally sliced with a tissue chopper (McIlwain, Mickle Industries, Surrey, England) into 350-µm-thick slices, transferred immediately into Gey's balanced salt solution (GBSS; Gibco Invitrogen, Karlsruhe, Germany) supplemented with 0.65% D-glucose (final concentration; Merck, Darmstadt, Germany), and allowed to recover at 7°C for 1 h. Slices with perpendicular orientation were placed as monocultures on coverslips with 10 µl of chicken plasma (Cocalico, Reamstown, PA), which was coagulated with 10 µl of thrombin (final concentration 25 U/ml; ICN, Meckenheim, Germany). Coverslips were placed in roller tubes (Nunc, Wiesbaden, Germany) and supplied with semiartificial culture medium [two fourths Basal Medium Eagle, one fourth HBSS, one fourth inactivated horse serum, 2 mM L-glutamine (Gibco), and 0.65% D-glucose (Merck)]. At 2 days in vitro (DIV), 10 µl of a solution containing equal volumes of uridine, cytosine-β-D-arabinofuranoside, and 5-fluorodeoxyuridine (4.4 µM final concentration; Sigma, Deisenhofen, Germany) was applied for 24 h to retard glial growth. Cultures were maintained for up to 20 DIV. CM from monocultures was harvested every fourth day, pooled, lyophilized to complete dryness, and redissolved in 1/10 volume of 125 mM NaCl and 5 mM HEPES (pH 7.3), followed by extensive desalting against the same buffer in dialysis tubes (Roth, Karlsruhe, Germany). The dorsal thalamus was dissected, as described previously (4, 5), into 350-µm-thick frontal slices, which were immediately transferred to GBSS supplemented with antagonists of excitatory amino acid receptors [50 µM 2-amino-5-phosphonovaleric acid and 3 µM kynurenic acid (Sigma), 0.65% glucose, 2.5 mM Na2HPO4, pH 7.5]. Slices were placed as monocultures on defatted, baked coverslips as described above or were grown as cocultures, arranged with cortical tissue at a distance of ≤5 mm, on the coverslips. In this case, the thalamus was orientated with the habenula nucleus facing cortical layer VI, and medium was supplemented with 25 mM KCl. Cultivation conditions were as described above. Cerebelli were prepared at P5, transferred into ice-cold GBSS supplemented with 0.65% glucose, separated from remaining blood vessels, and chopped with a razor blade. The resulting material was pressed through a 0.5-mm cannula with a 125-µm gauze tissue and then centrifuged for 3 min at 100g and 4°C. The supernatant was removed, and the remaining pellet was resuspended in 1 ml of Start V medium (Biochrom, Berlin, Germany), followed by a second centrifugation under the same conditions. This supernatant was discarded, and the microexplants were resuspended in 0.5 ml of Start V medium. Then, 40 µl of this suspension was placed on defatted, baked, and poly-D-lysine-coated (PDL, MW 150,000–300,000; Roche Diagnostics GmbH, Mannheim, Germany) coverslips, which were incubated for 3 h at 36.5°C and 5% CO2, to allow


the cells to adhere to the PDL surface. Coverslips were then placed in small dishes, and after addition of 1 ml of Start V medium they were cultivated as described above. Purification of survival-promoting activity from CM CM (70–80 ml) from 5–20 DIV was harvested and dialyzed against 50 mM potassium phosphate, pH 7.0; loaded onto a Blue Sepharose matrix; and eluted with a salt gradient leading up to 1.5 M KCl. Fractions were desalted into 10 mM sodium phosphate, pH 7.0; tested for biological activity; and subsequently bound to a hydroxyapatite matrix. After the binding, proteins were eluted by a stepwise gradient, with the use of 0.4 M sodium phosphate, pH 6.8. The remaining fractions were again dialyzed in 10 mM potassium phosphate, pH 7.0; tested; and separated in the same buffer on a heparin-polystyrene matrix. With a stepwise gradient up to 2 M KCl, bound proteins were eluted from this matrix. Cation exchange chromatography was performed on an SO3-residue matrix after extensive desalting against 10 mM citrate, pH 4.5. Bound proteins were eluted with a stepwise gradient up to 1 M NaCl in the same buffer. Active fractions were dialyzed in 10 mM NaCl, pH 7.0, and separated via high-performance liquid chromatography (HPLC), by using a Superdex G-75 matrix and C18 reverse-phase column. The purification procedure was repeated seven times, and the remaining active fractions were independently sequenced by conventional Edman degradation and HPLC analysis (done at ChromaTec GmbH, Greifswald, Germany). In all cases, a sequence between 14 and 16 amino acids identical to the N-terminal part of Y-P30 was obtained. Generation of antibodies and a Y-P30 synthetic peptide Polyclonal antibodies raised against Y-P30 were generated by a commercial supplier (Pineda Antikörper-Service, Berlin, Germany) by using the provided synthetic peptide as the antigen. A glutathione S-transferase (GST)-tagged fusion protein was used for affinity purification of highly specific antibodies. Specificity of the antisera was checked by binding to different recombinant Y-P30 fusion proteins on Western blots. Synthetic Y-P30 was obtained from Biotrend Chemikalien GmbH (Köln, Germany) or Dr. Thorsten Nürnberger (Leibniz-Institute of Plant Biochemistry, Halle, Germany). After synthesis, both peptides were analyzed by mass spectroscopy. Purity was greater than 85%. For both peptides, identical biological activity was confirmed in all assays. Viability test of organotypic thalamic monocultures and thalamocortical cocultures Vital dyes (Syto 21, 0.25 µg/ml, or propidium iodide, 10 µg/ml; Molecular Probes, Eugene, OR) were diluted in HBSS and added to organotypic cultures 4 h before estimation of viability. Viability was photographically documented under epifluorescence illumination. Immunocytochemical labeling of organotypic cultures and cerebellar microexplants Organotypic cultures were fixed in 4% paraformaldehyde for 3 h. Cerebellar microexplants were fixed in increasing paraformaldehyde concentrations (1.2, 2, 3, and 4% for 2 min each). Fixed cultures were preincubated at 4°C in blocking solution (0.1 M PBS, 0.3% Triton X-100, 10% goat normal serum, 2% bovine serum albumin; Sigma). After 1 h, primary antibodies (rabbit anti-glial fibrillary acidic protein (GFAP), used at a dilution of 1:1000, Dako, Glostrup, Denmark; mouse anti-microtubule-associated protein 2 (MAP2), 1/200; Boehringer Mannheim,


Mannheim, Germany) were added for overnight incubation. Then, cultures were washed with 0.1 M PBS and incubated with secondary antibodies (Cy3-conjugated goat anti-rabbit IgG, used at a dilution of 1:500, Dianova, Hamburg, Germany; Alexa Fluor 488-conjugated goat anti-mouse, used at a dilution of 1:300, Molecular Probes; in 0.1 M PBS, 0.2% Triton X-100) for 3 h, washed, and covered with coverslips. Immunolocalization of Y-P30 in rat and human brain Immunostaining of rat brain was performed with free-floating sections according to published procedures (6). Y-P30 antibody was diluted 1:750 in 10% goat normal serum and 0.03% Triton X-100 and was incubated for 36 h at 4°C. Paraffin sections 7 µm thick from Bouin- and Carnoyfixed fetal human brains aged 16, 17, 18, 23, and 25 gestational weeks were rehydrated, incubated with 0.3% Triton X-100 in 50 mM Tris buffer pH 7.4 for 15 min, blocked with 3% bovine serum albumin and 1% normal goat serum in Tris-buffered saline (TBS), and incubated overnight with the Y-P30 antibody at a dilution of 1:150, followed by biotinylated goat antirabbit and avidin-biotin-horseradish peroxidase complex (Dakopatts, Hamburg, Germany). The reaction product was developed with 0.02% diaminobenzidine and 0.001% H2O2 in Tris buffer. Controls included omitting the primary antibody or staining with rabbit-anti-Y-P30 antibody preabsorbed with an excess of synthetic Y-P30 peptide. Separation of blood cells Blood from rats and humans was harvested in the presence of anticoagulant (10 U of heparin/ml) and was then 10-fold diluted with PBS. Cells were pelleted by centrifugation (800g, 20 min), the supernatant was removed, and the remaining pellet was dissolved to the original volume with PBS. The cell suspension was layered onto Lympholyte-M (Cedarlane Laboratories Limited, Hornby, Canada; at a 1:1 ratio) and centrifuged again (800g, 30 min). Peripheral blood mononuclear cells (PBMCs) were collected by aspiration, centrifuged (800g, 15 min), washed with PBS, and finally resuspended in mRNA isolation buffer. Neutrophil granulocytes were harvested from the remaining pellet, resuspended in PBS, and layered onto a 0.96% NaCl solution containing 1% polyvinyl alcohol 72,000 Da (1:1 ratio). After the erythrocytes settled at 37°C, granulocytes were harvested by aspiration of the upper phase, centrifugation (300g, 10 min), and resuspension in 0.87% ammonium chloride at 4°C. After a final centrifugation, cells were washed with PBS and resuspended in mRNA isolation buffer. RNA extraction, Northern blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) For RNA preparations, organs were harvested from P2 rats. Organ-specific mRNA (1 µg) was blotted after electrophoretic separation onto nitrocellulose membrane and hybridized at high stringency with a 32P-labeled probe representing the open reading frame (ORF) of the human expressed sequence tag cDNA clone DKFZp313H1523 (AL598959). mRNA was isolated by using the Oligotex mRNA purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RT was performed with the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) or Sensiscript-RT kit (Qiagen). The mRNA from each tissue was divided and


separately reverse primed with oligo d(T)12-18, pd(N)6, and the 3'-primer of the putative ORF. The cDNAs obtained were then pooled and analyzed by PCR. Each PCR amplification mixture contained 0.5 µl of template; 1× Taq PCR reaction buffer; 2.5 U of Taq DNA polymerase (Qiagen) or PfuTurbo DNA Polymerase (Stratagene, La Jolla, CA); 0.2 mM dNTP; and each primer at 0.5 µM (sequences: 5'-GGG AAT TCA TGA GGT TCA TGA CTC TCC TCT TC3'/5'-ACG CGT CGA CTC ACT ATA GTA CTG AGT CAA GGA CGT-3'). This primer pair amplifies the entire ORF of the Y-P30 precursor protein, beginning with its start codon and ending with the stop codon. To subclone the PCR product, specific restriction sites (EcoRI/SalI) were added at the 5'-ends of the primers. PCR conditions were as follows: denaturation at 95°C, followed by 40 cycles at 95°C for 45 s, 58°C for 45 s, and 72°C for 1 min and a final elongation step at 72°C for 7 min. Each PCR was done twice with two independent mRNA preparations. The PCR products were separated on 1.5% agarose gels, and the identity of the PCR fragment was verified by cDNA sequencing after shotgun cloning of the amplicon. Nested primed PCR To identify transcript levels of even low abundance in blood samples, PCR products were also amplified in a second step via nested primed PCR. Amplicons from the first reaction were used as a template with the following PCR conditions: 2 min denaturation at 95°C, 30 cycles at 95°C for 45 s, 56°C for 45 s, and 72°C for 1 min and a final elongation step at 72°C for 7 min (sequence of nested primers: 5'-AGC ATG AGG TTC ATG ACT CTC-3'/5'-CAC GCT TTC TAG ATC TTC GAC-3'). The primers for nested primed PCR are located at positions 57– 78/321–342 of the Y-P30 cDNA. Analysis of morphological data Morphometric data from organotypic thalamic explants were analyzed by using Neurolucida, version 2.1 (MicroBrightField, Colchester, CT). A total of 10–25 explant cultures from three independent experiments were analyzed. Generation and purification of Y-P30 recombinant proteins and analysis of transport to the embryonic brain On the basis of a two-step PCR strategy, an myc epitope tag was introduced into the ORF of the Y-P30 precursor protein (between amino acids 48 and 49), cloned into the pTrcHis2 vector (Invitrogen, Carlsbad, CA), and subsequently transformed into Escherichia coli, strain TOP10F'. Overexpression of the recombinant protein was performed according to the manufacturer’s protocol. Cells were harvested, washed, and resuspended in a native purification buffer (50 mM NaPO4, 0.5 M NaCl, pH 8.0), including an EDTA-free protease inhibitor cocktail (Complete Mini, EDTA-free; Roche Diagnostics GmbH), and finally frozen at –80°C. The cells were then thawed and broken by a French pressure cell press (Spectronic Instruments, Rochester, NY). The remaining homogenate was supplemented with 1% Triton X-100, gently shaken for 1 h at 4°C, and subsequently centrifuged to pellet the cell debris. The ProBond purification system (Invitrogen) was used to purify the soluble polyhistidine-containing recombinant protein from


the supernatant according to the manufacturer’s protocol. Collected fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Samples (500 µg) of the recombinant protein, dissolved in 10 mM phosphate buffer, pH 7.4, were injected into the tail vein of pregnant rats at embryonic stage E12 and E16. The offspring of these animals were killed at P0 to analyze the presence of recombinant protein in the brain. Brains were removed and immediately shock-frozen in liquid nitrogen. For histological investigations, brains of the offspring were prepared at P4. Brains of animals at P0 were homogenized in 5 mM HEPES buffer, pH 7.4, containing 0.32 mM sucrose and protease inhibitor. The homogenate was then centrifuged for 10 min at 1000g and 4°C. The pellet obtained (P I) contained cell debris and nuclei and was solubilized with 4× SDS sample buffer (250 mM Tris-HCl, pH 6.8, 1% SDS, 40% glycerol, 20% β-mercaptoethanol, 0.004% bromphenol blue). The supernatant was centrifuged again for 15 min at 12,000g and 4°C. The resulting pellet (P II) contained the crude membrane fraction and was processed as described for the first pellet. In a final centrifugation step at 100,000g and 4°C, microsomes were pelleted (P III); proteins of the cytosol remained in the supernatant (S III). Both fractions were solubilized as described above. For immunoblotting experiments, 30 µg of each solubilized protein fraction was separated on 5– 20% SDS-PAGE gradient gels and subsequently transferred to nitrocellulose membranes (90 min, 200 mA). The transfer buffer contained 25 mM Tris, 192 mM glycine, 0.02% SDS, and 20% methanol. After the membranes were blotted, they were blocked with 5% dry milk and 0.1% Tween 20 in 1× TBS for 2 h. The membranes were then incubated at 4°C overnight with a 1:1000 dilution of antibodies raised against the c-myc epitope or 6xhis tag, in 1× TBS containing 0.1% Tween 20. After the washing, the blots were incubated with horseradish peroxidaseconjugated antibodies (1:4000) and were finally developed by using ECL Films. Optic nerve crush and footpad inflammation Optic nerve crush was performed with 12-week-old adult male Sprague-Dawley rats as described previously (7). Survival time after the crush for sampling of blood and optic nerves was 4 days (n = 3). With the same rat strain, lipopolysaccharide (LPS) was injected into the footpad to induce systemic inflammation according to published procedures (n = 4) (8). The inflammatory reaction was visually inspected. Animal experiments were performed in accordance with German laws and the regulations of the state of Saxony-Anhalt. RESULTS Identification of Y-P30 as the biological activity present in CM supporting the survival of thalamic explant monocultures Peptide extracts from CM significantly enhanced the average length of extending neurites (Fig. 1A–D). This effect was readily visible from 12 to 72 h after supplementation of the media with the peptide extract (Fig. 1E). Subsequent purification followed by Edman sequence analysis yielded a partial peptide sequence that was identical to the first 16 amino acids of the survivalpromoting peptide Y-P30 (Table 1; Fig. 1H) (9). Incubation of cerebellar microexplants with a synthetic peptide containing the 30 amino acids of Y-P30 indeed increased neurite length at


concentrations as low as 2 nM, thus mimicking the effects of CM (Fig. 1F). Moreover, incubation of either Y-P30 or CM in the presence of an antiserum generated against the synthetic peptide abolished the sprouting response (Fig. 1G), which indicated that neutralization of Y-P30 reduced survival and the sprouting-promoting activity released from the cortex. Next, the effects of the isolated factor on organotypic thalamic monocultures were evaluated and compared with those of CM and cocultured cortices. Addition of 2 µM Y-P30 to essential culture medium was sufficient to improve survival of organotypic thalamic monocultures to a level indistinguishable from that achieved with CM (Fig. 2). Moreover, quantitative measurements of the surface area and length of sprouts extending from cocultures revealed significant growth and sprouting-promoting effects of Y-P30 identical to those of CM (Fig. 2). From these data, we conclude that the biological activity present in CM is identical to that of Y-P30 or a larger peptide containing this peptide sequence. Identification of the cellular origin of the Y-P30 peptide Y-P30 was originally purified from oxidatively stressed neural cell lines (9) and has been shown to promote the survival of neurons in vitro and in vivo (9, 10). Its origin and mechanism of action, however, remain unknown. Interestingly, Y-P30 is derived from mRNA encoding a larger polypeptide in humans (Fig. 1H), and it was proposed to represents a proteolytic fragment of a larger polypeptide precursor (9–12). Cloning of the full-length rat Y-P30 cDNA (GenBank accession number: AF531422) revealed that the ORF contained 110 amino acids identical to the human protein. Initial attempts to clone Y-P30 from rodent brain tissue failed. Moreover, a more sensitive RTPCR analysis also failed to detect the transcript in cortex and total brain of neonatal rats as well as in organotypic cortex monocultures delivering Y-P30 peptide into the CM (Fig. 3). The next question thus concerned the origin of the Y-P30 peptide. Northern blot analysis of rat tissues revealed the presence of the transcript in total blood cells during early postnatal development, but it was absent from other postnatal tissues including brain (Fig. 3A). While this work was in progress, dermcidin was identified as an antimicrobial peptide from human sweat glands (12). Dermcidin was also found to be part of the same precursor protein as Y-P30 and therefore derived from the same mRNA. In this previous study, the corresponding mRNA was exclusively detected in sweat glands but not in fetal and adult brain tissue (12). Thus, further RT-PCR analysis demonstrated the presence of the Y-P30 mRNA in PBMCs, and, in agreement with data for humans, in juvenile rat sweat glands from the footpad, but not in juvenile rat blood neutrophils (Fig. 3C). Although PBMCs in fetal blood mainly derive from bone marrow, the vast majority of secreted peptides circulating in the fetal bloodstream derive from maternal PBMCs. These peptides readily pass the umbilical cord and are thought to control the protein expression of immune cells in the developing fetus (13). We therefore tested whether the Y-P30 mRNA is expressed by PBMCs of pregnant rats. Indeed, RT-PCR analysis of PBMC mRNA from pregnant rats showed abundant transcript at postconceptional day 18 (E18; Fig. 3D). Transcripts were detected from E6 until postnatal day 3 (P3) and were absent in PBMCs collected from agematched nonpregnant rats (Fig. 3E). Likewise, the transcript was also present in the blood of pregnant but not of nonpregnant women (Fig. 3F). Expression of Y-P30 was restricted to the first 29 weeks of pregnancy and could not be detected thereafter (Fig. 3F). Thus, the temporal


expression profile overlaps with the time course of the initial formation of thalamocortical connections in human brain (14, 15). Y-P30 synthesized by PBMCs accumulated in neurons We next determined the localization of Y-P30 in neonatal brain tissue by using an affinity purified polyclonal antiserum directed against Y-P30. Immunostaining revealed the presence of Y-P30 immunoreactivity mainly in the neocortex and hippocampus; it was absent in subcortical areas including the thalamus (Fig. 3G). Y-P30 immunoreactivity was prominent in pyramidal cells, with accumulation in cell somata and dendrites, which suggested an intracellular localization (Fig. 3H). Y-P30 immunoreactivity was present until P28 and not thereafter (Fig. 3H). No immunolabel was found in adult rat brain tissue sections and after preabsorption of the antiserum with Y-P30 peptide (not shown). Y-P30 immunoreactivity was also detected in human fetal brain, with a localization similar to that in the rat (Fig. 4). Y-P30 immunoreactivity was observed in somata and dendrites of neurons in the cortical plate and hippocampus, with strong labeling of vertical processes (Fig. 4). Thus, this accumulation of Y-P30 immunoreactivity, together with the presence of Y-P30 transcript in human blood cells at early stages of gestation, suggests a similar survival-promoting role in the human brain. Y-P30 immunoreactivity was present exclusively in neurons of primary cortical cultures To further analyze the cellular localization of Y-P30, we performed double-immunofluorescence staining with primary cortical cultures starting from 1 DIV until 35 DIV. In these experiments, we showed that Y-P30 was exclusively present in neurons, as evidenced by its absence in GFAPimmunolabeled astrocytes and its prominent localization in MAP2-labeled neurons at early culture stages (Fig. 5). At later stages, however, the labeling signal was much weaker, which supports the idea that the neuronal protein levels of the factor decline with increasing maturation (Fig. 5). Recombinant Y-P30 was transported from maternal blood to the infant’s brain In the next set of experiments, we sought out to determine whether a recombinant fusion-tagged Y-P30 precursor protein injected into the tail vein of the mother passes the umbilical cord and accumulates in cortical neurons of the infant brain. The fusion-tagged construct is shown schematically in Fig. 6A. Western blot analysis and immunostaining of neonatal rat brain showed the presence of a peptide that harbored an myc fusion tag within the Y-P30 peptide (Fig. 6). The presence of the recombinant Y-P30 peptide in cortical cells was also be revealed by immunostaining with an anti-myc antibody (Fig. 6). Thus, recombinant Y-P30 injected into the blood circulation of the mother accumulated in the brain of the offspring. Y-P30 transcription was induced by central nervous system lesions in PBMCs of the adult rat Because the peptide was originally purified from oxidatively stressed neural cell lines (9) and has potent survival-promoting effects on neurons in vitro and the adult brain in vivo (9, 10), a major obstacle at this stage of investigation was how these findings relate to absence of Y-P30 in the


mature brain. We decided to address the question of whether expression of the factor can be induced in the adult central nervous system (CNS) by using a well-established CNS white matter injury model: controlled crush of the optic nerve (7, 8). Using this paradigm, we demonstrated the presence of the Y-P30 transcript in the optic nerve and blood of adult male rats with optic nerve crush lesions but not in their sham control counterparts (Fig. 7). Importantly, a systemic inflammation induced by injection of LPS into rat footpads did not result in the transcription of Y-P30 mRNA in blood cells, which indicated that induction of transcription is not governed by a general inflammatory response (Fig. 7). Thus, Y-P30 expression was induced in the adult male CNS after trauma, which suggests an endogenous neuroprotective role. DISCUSSION In this paper, we describe a hitherto unknown interaction between cells of the maternal immune system and the developing brain of the offspring. Our findings suggest that a protein synthesized in maternal mononuclear cells has an impact on neuronal survival and differentiation of the infant brain. Thus, most of the Y-P30 peptide in fetal as well as early postnatal blood has been generated by PBMCs of the maternal organism and will accumulate in neurons of the pre- and postnatal brain, particularly in the cortex. In consequence, we propose a unique interaction between the maternal immune system and the presence of Y-P30 in prenatal and early postnatal brain. Although the nature of the role of Y-P30 is yet to be elucidated, it is tempting to speculate that Y-P30 may be an epigenetic factor, especially for the establishment of thalamocortical circuitry. In favor of this notion is the time course of its accumulation in and release from cortical neurons, which fits nicely to the time course of axonal termination and its decrease with the beginning of synaptogenesis in these brain areas (14, 15). Most important, however, Y-P30 is so far the only isolated factor that can improve survival of organotypic thalamus explanted at postnatal ages and cultured in isolation from the cortex (16– 20). The peptide also significantly promoted extension of thalamic neural sprouts and had similar effects in cerebellar microexplant cultures. These unique properties make it even more a likely that Y-P30 has a major trophic role in the developing brain, even though it lacks major properties of classical neurotrophins. It will therefore be of major interest to identify regulatory factors that contribute to transcriptional regulation of Y-P30 mRNA in maternal and fetal PBMCs and, in particular, to uncover the details of how the expression is correlated with CNS development. Another unresolved issue is the nature of the mechanisms by which the peptide actually accumulates in neurons. To this end, we showed that a recombinant Y-P30 containing peptide derived from the blood of the mother accumulated in cortical neurons of the offspring. Accordingly, although PBMCs in fetal blood derive mainly from bone marrow, the vast majority of secreted peptides circulating in the fetal bloodstream derive from maternal PBMCs. These peptides readily pass through the umbilical cord and are thought to control the protein expression of immune cells in the developing fetus (13). Moreover, the only explanation for our findings is that a specific neuronal uptake system exists for the peptide. In this regard, Cunningham et al. (9) reported that Y-P30 is an IgG binding peptide. IgGs are known to accumulate in early generated cortical neurons (21, 22), which suggests IgG as a possible carrier for the peptide. If a transcytotic mechanism underlies Y-P30 uptake, the question of how Y-P30 is stored and released from maturing neurons should be addressed, because the peptide is present in primary cortical neurons but not in dissociated glial cells, and Y-P30 is released from neurons in vitro in


an activity-dependent manner (P. Landgraf, A. Thiele, and M. R. Kreutz, unpublished observations). A major question at this stage of investigation is how the original discovery of the peptide by purification from oxidatively stressed neural cell lines (9) and its potent survival-promoting effects on neurons in vitro in this study and the adult brain in vivo (9, 10) relate to absence of YP30 in the mature brain. We found that transcription of the polypeptide factor was induced after white matter lesions of the adult CNS in CNS tissue as well as in blood. The presence of Y-P30 transcript in the crushed optic nerve under such conditions can be most likely explained by infiltration of macrophages into the lesion site (23). In contrast, activation of macrophages as induced by application of LPS in footpads did not lead to detectable levels of Y-P30 mRNA in blood cells, which excludes the possibility that any type of inflammation can induce Y-P30 gene transcription. Moreover, this finding points to the presence of a yet unknown signaling process deriving from damaged neural tissue that regulates transcription of the Y-P30 gene in PBMCs of the adult. It is therefore conceivable that Y-P30 has a rather specific pathophysiological role within the interaction of the immune system and the CNS after CNS injury. Finally, using cocultures of thalamus and cortex, we frequently observed that addition of Y-P30 or CM to the culture medium led to establishment of connections between both explants, which contained glial and neuronal elements and always originated from the thalamus. This result was never observed when the medium was not supplemented with either Y-P30 or CM (P. Landgraf, H. C. Pape, F. Sieg, and M. R. Kreutz, unpublished observations). Thus, because Y-P30 also enhanced sprouting of the explanted perinatal thalamus and thus regeneration of the thalamocortical projection in vitro, a deeper understanding of this role will most likely also lead to new therapeutic avenues in the treatment of neurodegenerative disease states affecting the thalamus and other CNS regions. ACKNOWLEDGMENTS Supported by the BMFT (Förderschwerpunkt Neurotraumatologie, TP A11 to H. C. P. and P. W.), DFG (Leibniz-Program to H. C. P., Kr1879/1-2 to M. R. K., SFB 509 TP C2 to P. W.), LSA (FKZ: 2508A to M. R. K.), and the Fritz Thyssen Stiftung (to M. R. K.). We would like to thank Drs. Schenk and Mathies for kind supply of human blood samples and Dr. T. Nürnberger for the gift of synthetic Y-P30 peptide. The support and critical comments of Drs. E. D. Gundelfinger and K.-H. Smalla and the professional technical assistance of M. Marunde, A. Reupsch, K. Schumacher, and, in particular, S. Mücke are also gratefully acknowledged. REFERENCES 1.

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Table 1 Biochemical purification of Y-P30 from cortex-conditioned media Chromatography Blue Sepharose

Hydroxyapatite

Heparin-polystyrene Gel filtration Cation exchange

Running conditions Active fractions Buffer A: 50 mM KH2PO4, pH 7.0 Buffer B: 50 mM KH2PO4/1.5 M KCl, pH 7.0 22–38 stepwise gradient, 2.0 ml/min Buffer A: 10 mM NaPO4, pH 7.0 Buffer B: 400 mM NaPO4, pH 6.8 9–14 stepwise gradient, 0.8 ml/min Buffer A: 10 mM NaPO4, pH 7.0 Buffer B: 10 mM NaPO4/2 M KCl, pH 7.0 36–38 stepwise gradient, 1.0 ml/min 20 mM NaPO4, 200 mM NaCl, 0.05% Tween 80, pH 7.0, 0.1 17–21 ml/min Buffer A: 10 mM citrate, pH 4.5 Buffer B: 10 mM citrate, 1 M NaCl, pH 4.5 22–25 stepwise gradient, 1.0 ml/min

The basic material for each biochemical purification procedure was 70–80 ml of CM; each procedure was repeated seven times under identical conditions. The mean of the corresponding start activity of CM is shown in Fig. 1F. Those and the activity of all of the obtained fractions were tested by bioassay of cerebellar microexplants. Remaining fractions with activity comparable to that of CM were pooled and dialyzed according to the required chromatographic conditions. The final analysis using an HPLC C18 reverse-phase column and sequencing was performed by ChromaTec GmbH.


Fig. 1

Figure 1. Effects of CM and peptide extracts on rat cerebellar microexplant cultures labeled with immunofluorescent MAP2 (green) and GFAP (red). Examples of microexplants are shown immediately (2 h) after preparation (A), after 2 DIV (B), and after 2 DIV and supplementation with CM (C) or 2 µM Y-P30 (D). E) Average neurite length in microexplants at different times in culture under control conditions (open bars) and after supplementation with CM (filled bars). The sprouting response is depicted as the quotient between the length of the neurites and the diameter of the respective microexplant. Data are the averages ± SD from 25–55 independent experiments; significant differences from control are indicated (***P