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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 6, 2004 as doi:10.1096/fj.04-1789fje. |
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,1
* Institute of Physiology, Otto-von-Guericke University, Magdeburg, Germany;
AG Developmental Biology, Faculty of Biology, Ruhr-University, Bochum, Germany;
Department Anatomy, Fac. Medicine, University of La Laguna, Tenerife, Spain; and
AG Molecular Mechanisms of Plasticity, Department of Neurochemistry/Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany
1Correspondence: AG Molecular Mechanisms of Plasticity, Department of Neurochemistry/Molecular Biology, Leibniz Institute for Neurobiology, Brenneckestr. 6, 39118 Magdeburg, Germany. E-mail: kreutz{at}ifn-magdeburg.de
SPECIFIC AIMS
Our aims were to 1) clarify the molecular identity of a biological activity present in cortex-conditioned medium that promotes the survival of thalamic explant cultures and 2) investigate its cellular origin and regulation of expression.
PRINCIPAL FINDINGS
1. Identification of Y-P30 as the biological activity present in cortical conditioned media supporting the survival of thalamic explant monocultures
Findings from our laboratory show that maintenance of organotypic monocultures of the dorsal thalamus can be improved when arranged as a coculture with an age-matched cortical explant or a conditioned cortex medium, suggesting the existence of trophic factors in the cortex-conditioned medium. However, supplementing thalamus monocultures with known growth factors of the neurotrophin family failed to mimic the cortex-conditioned activity. We therefore hypothesized that an essential yet unidentified biological activity is secreted from cortical tissue that promotes survival of thalamic cells. We isolated from the cortex-conditioned medium a single polypeptide factor responsible for the survival-promoting effects of thalamic monocultures (Fig. 1
) using a 2-step experimental strategy. First, we used biochemical purification steps and subsequent sequence analysis by mass spectroscopy and Edman degradation. Second, since cerebellar microexplant cultures can be prepared in a standardized manner and much higher numbers than thalamus-cortex cocultures, we used this culture system to control and measure the biological activity after each purification step by quantifying the length of neurite extension and explant size.
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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 identical to the first 16 amino acids of survival-promoting peptide (Y-P30; Fig. 1h
). 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, mimicking the effects of CM (Fig. 1f
). Moreover, incubation of Y-P30 or CM in the presence of an antiserum generated against the synthetic peptide abolished the sprouting response (Fig. 1g
), indicating that neutralization of Y-P30 reduces survival and sprouting-promoting activity released from the cortex. Next, the effects of the isolated factor was tested on organotypic thalamic monocultures and compared with those of CM and cocultured cortices. Addition of 2 µM of Y-P30 to essential culture medium was sufficient to improve survival of organotypic thalamus monocultures to a level indistinguishable from CM. Quantitative measurements of the surface area and the length of sprouts extending from cocultures revealed a significant growth and sprouting-promoting effect of Y-P30 identical to those of CM. We conclude that the biological activity present in CM is identical to Y-P30.
2. Identification of the cellular origin of the Y-P30 peptide
Y-P30 was originally purified from oxidatively stressed neural cell lines and has been shown to promote the survival of neurons in vitro and in vivo, but its origin and mechanism of action are unknown. Y-P30 is derived from an mRNA encoding a larger polypeptide in human (Fig. 1h
). Cloning of the full-length rat cDNA revealed that the open reading frame contained 110 amino acids identical to the human protein, confirming that the corresponding mRNA encodes a precursor protein that gives rise to at least two peptides with biological activity (Fig. 1h
): dermcidin and Y-P30.
Northern blot analysis of rat tissues revealed the presence of a transcript in total blood cells during early postnatal development, but it was absent from other postnatal tissues, including brain (Fig. 2
a). Further RT-PCR analysis demonstrated the presence of Y-P30/dermcidin mRNA in peripheral blood mononuclear cells (PBMCs) and juvenile rat sweat glands from the foot pad, but not in juvenile rat blood neutrophils (Fig. 2c
). 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. We therefore tested whether the Y-P30 mRNA is expressed in PBMCs of pregnant rats. Indeed, RT-PCR analysis of PBMC mRNA from pregnant rats showed an abundant transcript at postconceptional day 18 (Fig. 2d
). Transcripts were detected from E6 until postnatal day 3 (P3) and were absent in PBMCs collected from age-matched nonpregnant rats (Fig. 2e
). Likewise, the transcript was present in the blood of pregnant but not nonpregnant humans (Fig. 2f
). Expression of Y-P30 was restricted to the first 29 wk of pregnancy (Fig. 2f
).
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3. Y-P30 synthesized by PBMCs accumulates in neurons
Immunostaining revealed the presence of Y-P30 immunoreactivity mainly in the neocortex and hippocampus (Fig. 2g
) and its absence in subcortical areas, including the thalamus (Fig. 2g
). Y-P30 immunoreactivity was prominently found in pyramidal cells, with accumulation in cell somata and dendrites suggestive of an intracellular localization (Fig. 2h
). Y-P30 immunoreactivity was present until P28 (Fig. 2h
). Y-P30 immunoreactivity was detected in the human fetal brain with a localization similar to that in the rat. Double immunofluorescence staining on cortical primary cultures showed that Y-P30 is present exclusively in neurons.
In the next set of experiments we sought 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. Western blot analysis and immunostaining of neonatal rat brain revealed the presence of a peptide that harbored a myc fusion tag within the Y-P30 peptide. The presence of the recombinant Y-P30 peptide in cortical cells could be revealed with immunostainings using an anti-myc antibody.
4. Y-P30 transcription can be induced by CNS lesions in PBMCs of the adult rat
To address the question of whether expression of the factor can be induced in the adult CNS, we used a white matter injury model, a controlled crush of the optic nerve. We demonstrated the presence of Y-P30 transcript in the optic nerve and blood of adult male rats receiving lesions, but not in their sham control counterparts. A systemic inflammation induced by injection of lipopolysaccharide did not result in the transcription of Y-P30 mRNA in blood cells, indicating that the induction of transcription is not governed by a general inflammatory response.
CONCLUSIONS AND SIGNIFICANCE
Here 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 affects neuronal survival and differentiation of the infant brain. We propose a unique interaction between the maternal immune system and the presence of Y-P30 in the pre- and early postnatal brain; although the nature of its role is yet to be elucidated, it is tempting to speculate that Y-P30 might be an epigenetic factor, especially for establishing the thalamo-cortical circuitry. So far, Y-P30 is the only factor isolated that is sufficient to improve survival of organotypic thalamus explanted at postnatal ages and cultured in isolation from the cortex.
An unresolved issue is by which mechanisms the peptide actually accumulates in neurons. We showed that a recombinant Y-P30 containing peptide deriving from the blood of the mother accumulates in cortical neurons of the offspring. 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. We found that transcription of the polypeptide factor can be induced after white matter lesions of the adult CNS in CNS tissue as well as in the blood. It is conceivable that Y-P30 has a specific pathophysiological role within the interaction of the immune system and the CNS after CNS injury.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1789fje;
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