Pleiotrophin is a neurotrophic factor for spinal motor neurons Ruifa Mi, Weiran Chen, and Ahmet Ho¨ ke* Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21287 Edited by Thomas M. Jessell, Columbia University Medical Center, New York, NY, and approved January 18, 2007 (received for review April 21, 2006) Regeneration in the peripheral nervous system is poor after chronic facial motor neurons against cell death induced by deprivation from denervation. Denervated Schwann cells act as a ‘‘transient target’’ target-derived neurotrophic support. by secreting growth factors to promote regeneration of axons but lose this ability with chronic denervation. We discovered that the Results mRNA for pleiotrophin (PTN) was highly up-regulated in acutely PTN Is Up-Regulated in Denervated Schwann Cells and Muscle After denervated distal sciatic nerves, but high levels of PTN mRNA were Axotomy. To identify candidate neurotrophic factors underlying not maintained in chronically denervated nerves. PTN protected adaptive responses to chronic nerve degeneration, we used focused spinal motor neurons against chronic excitotoxic injury and caused cDNA microarrays to investigate the gene expression of neurotro- increased outgrowth of motor axons out of the spinal cord ex- phic factors in denervated Schwann cells. In microarray experi- plants and formation of ‘‘miniventral rootlets.’’ In neonatal mice, ments, 2 and 7 days after the sciatic nerve transection, PTN mRNA PTN protected the facial motor neurons against cell death induced was up-regulated in the distal denervated segments compared with by deprivation from target-derived growth factors. Similarly, PTN the contralateral side (data not shown). To confirm the up- significantly enhanced regeneration of myelinated axons across a regulation of the PTN mRNA observed in the microarray analysis graft in the transected sciatic nerve of adult rats. Our findings and further explore the pattern of expression, we performed PTN suggest a neurotrophic role for PTN that may lead to previously mRNA measurements in denervated nerves from 2 days to 6 unrecognized treatment options for motor neuron disease and months of denervation. As seen in Fig. 1A, PTN levels were motor axonal regeneration. up-regulated in denervated distal sciatic nerves as soon as 2 days after nerve transection and peaked by 7 days. However, the PTN anaplastic lymphoma kinase ͉ neurotrophism ͉ Schwann cell ͉ mRNA levels returned back to baseline levels by 3 months. This nerve regeneration ͉ denervation observation shows that PTN mRNA is up-regulated in acutely denervated Schwann cells, but this up-regulation is not maintained eripheral nerve injury leads to Wallerian degeneration of over time, and chronically denervated Schwann cells lose their Paxons and denervation of Schwann cells distal to the site of ability to make PTN. This pattern of mRNA expression mirrors injury. Denervated Schwann cells secrete a variety of growth what has been observed with GDNF expression in denervated factors and assume the role of ‘‘transient target’’ for regenerating nerves (21). axons (1, 2). Among these neurotrophic molecules are well- As a potential target derived neurotrophic factor, we examined known ones such as nerve growth factor and glial cell line- the expression pattern of PTN in muscle during development and derived neurotrophic factor (GDNF). Up-regulation of neuro- after denervation. As seen in Fig. 1B, PTN mRNA is expressed at trophic factors allows regeneration of axons when a repair is very high levels in embryonic muscle but is down-regulated to nearly undetectable levels in adult muscle. However, PTN mRNA made promptly or the gap between the transected ends of the is rapidly up-regulated in the adult muscle on denervation within axon is not too long. However, if the duration of denervation is days; this mirrors the expression pattern in denervated Schwann prolonged or when the distances that need to regenerate are very cells. In contrast to muscle, there was no significant up-regulation long, as is frequently the case in humans, success of regeneration of PTN mRNA in denervated footpad skin (data not shown). and functional recovery are suboptimal (3–10). To identify candidate growth factors underlying neurotrophic PTN Causes Increased Outgrowth of Motor Axons Out of Spinal Cord support by Schwann cells, we used cDNA microarrays to investigate Explants. Changes in the pattern of expression of PTN suggested the gene expression of neurotrophic factors in denervated distal that it might have a neurotrophic role in motor neurons. To examine nerve stumps. Pleiotrophin (PTN) gene expression is up-regulated this potential role for PTN, we used the spinal cord explant culture in the distal nerve stump immediately after sciatic nerve transec- system. This system has been used extensively to study neuropro- tion. PTN (also termed heparin binding growth-associated mole- tective and trophic properties of growth factors (22–24). In this cule or heparin binding neurotrophic factor) is a 168-aa, heparin system, spinal cord slice cultures are prepared on culture inserts binding secreted protein. PTN, isolated initially from rat uterus as with semipermeable membranes and allowed to acclimatize to the a mitogen for NIH 3T3 cells (11), is a member of the midkine family that has cysteine- and basic amino acid-rich residues distinct from other heparin binding growth factor families (12–14). Author contributions: R.M., W.C., and A.H. designed research; R.M., W.C., and A.H. per- In addition to the mitogenic effect on fibroblasts, PTN has formed research; R.M., W.C., and A.H. analyzed data; and R.M. and A.H. wrote the paper. activity in a variety of tissues and cell types (14–16). In the nervous This article is a PNAS direct submission. system, it has been shown to induce neurite outgrowth from PC-12 The authors declare no conflict of interest. rat pheochromocytoma cells (17), and cortical (11) and dopami- Abbreviations: ALK, anaplastic lymphoma kinase; DRG, dorsal root ganglion; GDNF, glial nergic neurons (18, 19). PTN is also expressed in the developing cell line-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; PTN, pleiotrophin; THA, threohydroxyaspartate. nervous system and muscle, and plays a role in postsynaptic *To whom correspondence should be addressed at: Department of Neurology, Johns clustering of acetylcholine receptors (20). Here, we show that PTN Hopkins University, 600 North Wolfe Street, Path 509, Baltimore, MD 21287. E-mail: acts as a neurotrophic factor for spinal motor neurons and protects [email protected]. them against chronic excitotoxic cell death in vitro. Furthermore, we This article contains supporting information online at www.pnas.org/cgi/content/full/ show that PTN promotes enhanced regeneration of peripheral 0603243104/DC1. nerve axons after sciatic nerve transection and protects neonatal © 2007 by The National Academy of Sciences of the USA 4664–4669 ͉ PNAS ͉ March 13, 2007 ͉ vol. 104 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603243104 Downloaded by guest on September 23, 2021 A 700 * A 600 500 * Without PTN 400 * 300 200 100 0 Percent change in PTN mRNA -100 2d 7d 1mo 3mo 6mo B100000 * 10000 1000 With PTN * 100 * B C Vehicle PTN 30 10 * 25 Fold difference in PTN mRNA * 20 1 e14 p6 p14 p30 Adult 7d 2mo 15 (3 mo) post- post- dener dener 10 NEUROSCIENCE vation vation Number of Neurites/Slice 5 Fig. 1. Changes in PTN expression. (A) PTN mRNA expression was measured 0 by using quantitative RT-PCR in rat sciatic nerves after transection at the Vehicle PTN midthigh level. PTN mRNA was up-regulated in denervated distal nerves, but ϭ Ͻ this up-regulation was not maintained (n 4–6 animals per time point; *, P Fig. 2. PTN is neurotrophic for spinal motor neurons. (A) Neurotrophism of 0.005 compared with contralateral intact side). (B) Expression of PTN mRNA in PTN was examined in spinal cord explants prepared from postnatal-day-8 rats. rat tibialis anterior muscle during development and after denervation by The explants were cultured on semipermeable membrane inserts with a point ϭ transection of the sciatic nerve at the midthigh level (n 4 animals per time source of PTN in a gelfoam away from the explants, and after 1 week the Ͻ point; *, P 0.005 compared with levels of expression in normal adult muscle). cultures were stained with antineurofilament antibody SMI-32. The motor neurons were identified by their size and location within the explant. PTN induced axonal outgrowth from spinal cord explants and formation of mini- culture conditions for a week. After 1 week, there is a stable ventral rootlets toward the source of PTN; a representative explant is shown. population of surviving motor neurons per slice that remain alive In cultures without PTN, axons remained at the gray–white matter junction in the culture for months. During chronic culture, motor neurons (arrow) and never exited the explants. The images are representative of n ϭ extend axons that can be labeled with anti-neurofilament antibod- 12–16 per group. (Scale bar, 150 ␮m.) (B) In cultures treated with a diffuse ies, but these axons usually remain in the gray matter and rarely source of PTN in the culture medium, PTN increased the number of spinal cross the gray–white matter junction and extend out of the explants. motor axons that crossed the gray–white matter junction and entered the ␮ In spinal cord explants where PTN was applied away from the white matter tracts. (Scale bar, 30 m.) (C) Quantitation was done by counting the number of axons crossing the gray–white matter junction in the ventral explant in the form of gelfoams soaked with recombinant human half of the spinal cord explants (n ϭ 12 explants per group; , P Ͻ 0.005). PTN (100 ng/ml), there was extensive outgrowth of motor axons out * of the explants (Fig.