Mummy‘s Little Helpers: How a Maternally Derived Blood-Borne Factor Promotes Brain Development

Recently, the enigmatic survival-promoting peptide Y-P30 has entered the scene of developmental neuroscience. It has been identified as a survival factor for young neurons of the mammalian thalamus, a structure representing the gate for sensory information en route to the processing at the cortical level. Like all neurons in the brain, thalamic neurons depend on target-derived or paracrine trophic support, and Y-P30 seems to do exactly this job [1,2].

Desperately wanted: trophic support for thalamic neurons

The story began with the observation that a slice of dorsolateral thalamus explanted at the day of birth thrives if a slice of neocortex is cocultured in the same roller culture tube, but not when maintained alone [1]. We also noted that the thalamic slices easily sprout axons towards the cocultured cortex slice to re-establish the thalamocortical connection in vitro[3]. Physical contact and reciprocal axonal connections were not important. Intriguingly, medium sampled from monocultured cortex was as protective as a cortex slice in direct neighborhood, arguing for a diffusible agent. Interestingly, none of the four neurotrophins was able to keep the solitary thalamus alive. It rather seemed as if a novel target-derived factor was secreted from the cortical neurons.

We then set out to purify the mysterious diffusible agent from cortex-conditioned medium. Microexplant cultures of neonatal cerebellum were employed as an easy-to-standardize read-out system to monitor the biological activity during purification. The active fractions caused the neuritic processes radiating from the microexplants to become longer, and these growth-promoting fractions were analyzed further. Eventually a peptide was purified and after N-terminal Edman degradation it was identified as Y-P30. A synthetic peptide was made and mimicked the effects of the conditioned medium. In the presence of the Y-P30 peptide, a thalamic slice was now able to survive solitarily without the cortex [1].

The biology of Y-P30 is mind boggling

Y-P30 is a derivative of the dermcidin (DCD) gene, which by proteolytic processing gives rise to different bioactive peptides [4–6]. One product from the precursor peptide is the C-terminal DCD, which is secreted as part of the innate immune system by the skin sweat gland. It has antimicrobial actions and comprises a mix of at least six major bioactive fragments [4,5]. Following cleavage of the signal peptide from the N-terminus, the glycosylated proteolysis-inducing factor (PIF) is generated.

Proteolysis-inducing factor was the first identified peptide of this precursor and is known as a cachexia factor that in rodent models is involved in the hypermetabolic state causing the loss of muscle tissue in cancer [6–8]. The murine PIF and its human counterpart for human cachexia associated protein (HCAP), and DCD are sporadically overexpressed by a variety of tumors and tumor cell lines. For instance, a majority or pancreatic and prostate cancers produce PIF [6–8] and breast cancers express DCD [9], and the higher the expression levels the greater is the weight loss in the patients and the worse seems the prognosis. PIF signaling is proinflammatory since it elicits an interleukin response and activates NFκB-mediated gene expression [10]. Y-P30, formerly also termed survival-promoting peptide or diffusible survival/evasion peptide (DSEP) [6,11–13], contains a similar sequence of 30 amino acids but is not glycosylated.

Y-P30 was originally identified from media conditioned by oxidatively stressed human neuroblastoma cells, which happened to be DCD gene expressing. Y-P30 and DSEP have been shown to be neuroprotective on injured neurons in the adult brain [11–13]. Intriguingly, DCD was found to bind, in particular, to dopaminergic and noradrenergic neurons in the human brain, suggesting that it helps protect these neuron types from reactive oxygen species that occur during transmitter biosynthesis [9]. Yet, attempts to identify the mRNA of the Y-P30/DCD precursor in brain tissue failed. The explanation was simple: Y-P30 is produced by peripheral blood mononuclear cells of the maternal immune system. Brain, heart, lung, liver, spleen and kidney from adult rats are negative for the transcript [1]. Interestingly, reverse transcription-PCR analysis of mRNA extracted from pregnant rats showed abundant Y-P30 transcript levels, but it was absent in the blood of nonpregnant female and also male rats. Immunostaining using an affinity-purified polyclonal antibody directed against Y-P30 showed the immunoreactivity in pyramidal neurons of the early postnatal neocortex and the hippocampus, but not in most subcortical areas. The peptide was also found in the fetal human brain cortex and hippocampus of the 16th and 25th gestational week, and pregnant human females had the transcript in the blood from week 5 until the third trimester [1].

This leads to the intriguing possibility that Y-P30 transfers from maternal blood to fetal blood to then become accumulated by brain neurons. Indeed, recombinant Y-P30 with a fusion tag injected into the tail vein of a pregnant dam accumulated in the postnatal brain of the pups as determined by protein blot and immunohistochemistry detecting the tag [1]. This argued for a transplacental import, possibly via the immunoglobulin-binding properties of Y-P30 [11]. Maternal immunoglobulins are present in pre-and postnatal neurons, for example, in the transient neurons of the cortical subplate [14–16]. Also their role was enigmatic; but in the new context it may well be that maternal immunoglobulins serve as bulk carriers for Y-P30 and after unloading their cargo become dumped in neurons destined to undergo cell death.

It was of central importance to characterize the Y-P30 signaling. A cDNA library from human fetal brain was screened for interaction partners using the yeast two-hybrid system. As one candidate, a neurotrophic protein called pleiotrophin was identified and the interaction was subsequently confirmed using pull-down assays. Pleiotrophin, also termed heparin-binding growth-associated molecule, is a cytokine of the midkine family [17]. For instance, it induces neurite growth, and this has been shown for thalamic neurons and other developing axonal tracts where pleiotrophin and the heparan sulfate proteoglycan syndecan-3 are spatiotemporally coexpressed [17–19]. Indeed, the action of pleiotrophin is partly mediated by syndecan, and syndecan signaling via cortactin and src-family kinases is known to regulate neurite growth [20,21]. Intriguingly, similar to DCD/Y-P30, pleiotrophin and syndecan, in addition to the other pleiotrophin receptors anaplastic lymphoma kinase and receptor protein typrosine phosphatase β/ζ, are implicated in cancer, invasion and tumor angiogenesis [22–24]. We now found that Y-P30 can also bind directly to syndecan-2 and -3 and it strongly enhances the interaction of pleiotrophin and the syndecans in a noncompetitive manner [2].

A maternal peptide is imported into the infant‘s brain to enhance survival, migration & axonal growth

Taken together this leads to a model in which maternal Y-P30 is transported to the developing infants brain, taken up and stored in maturing neurons. It becomes released, and indeed, cortical neurons in a freshly prepared dissociated culture display high Y-P30 immunoreactivity, which declines with ongoing differentiation in vitro[1]. In the extracellular space, Y-P30 forms large aggregates with itself and with pleiotrophin and these complexes are captured by the heparan sulfate side chains of the membrane-bound syndecans. One cellular effect is an enhancement of neurite growth in short-term dissociated cultures of embryonic rat thalamus. The heparan sulfate chains are important because removing them with heparitinase compromises the Y-P30/pleiotrophin-induced neurite outgrowth [2].

Currently, ongoing experiments strongly suggest that axons are a prime target for Y-P30. We found that axons of neurons differentiated from mouse embryonic stem cells and axons of postnatal rat retinal neurons grow faster than controls when exposed to Y-P30 [25,26]. Moreover, axons from retinal neurons show a rapid appearance of β-actin immunoreactivity to the growth cones in response to Y-P30 [27]. By contrast, dendrites of cortical pyramidal cells and GABAergic interneurons and of the stem cell-derived neurons failed to grow in response to Y-P30 [25–27]. Axonal growth cone migration involves mechanisms similar to cell migration.

Analysis of the cerebellar microexplants already led us to speculate on a role in migration, because in the presence of Y-P30 more cell bodies seemed to move out of the explants [1]. Ongoing studies now reveal that Y-P30 strongly enhances the migration [26]. It is highly likely that the broad spectrum of different actions of Y-P30 during neuronal development might also be of use after neuronal injury. The mechanisms might be similar since brain injury increases syndecan and pleiotrophin expression [28] and evokes a reappearance of Y-P30 mRNA, for example, after a crush lesion of the optic nerve [1]. Thus, the documented neuroprotective role of the peptide provides another largely unexplored area of investigation.

The concept of neurotrophic factors originating from the periphery of the developing body instead of cells from within the developing brain is well accepted. Less is known on the neurotrophic roles of factors delivered along the maternofetal route. Y-P30 has now been characterized as such a factor. Although the exact mechanisms remain to be unravelled, we suggest that it acts by promoting pleiotrophin–syndecan signaling for migration and axonal growth. Together this leads to the view that a little helper from the mother coaches the wandering and the wiring of neurons in the infant‘s brain.

Financial & competing interests disclosure

The work has been funded by the Land Saxony-Anhalt (N2/TP5), the Leibniz Society and Deutsche Forschungsgemeinschaft GRK 736, SFB779. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

The work has been funded by the Land Saxony-Anhalt (N2/TP5), the Leibniz Society and Deutsche Forschungsgemeinschaft GRK 736, SFB779. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.