A Hematopoietic Growth Factor, Thrombopoietin, Has a Proapoptotic Role in the Brain

A hematopoietic growth factor, thrombopoietin, has a proapoptotic role in the brain

Hannelore Ehrenreich*†, Martin Hasselblatt*, Friederike Knerlich*, Nico von Ahsen‡, Sonja Jacob*, Swetlana Sperling*, Helge Woldt*, Katalin Vehmeyer§, Klaus-Armin Nave*, and Anna-Leena Sire´ n*

*Max Planck Institute of Experimental Medicine and ‡Departments of Clinical Chemistry and §Hematology and Oncology, Georg-August University, 37075 Goettingen, Germany

Edited by Anthony Cerami, The Kenneth S. Warren Institute, Kitchawan, NY, and approved December 2, 2004 (received for review August 15, 2004) Central nervous and hematopoietic systems share developmental Ͼ95% neurons). Neuronal cell number and viability was assessed features. We report that thrombopoietin (TPO), a stimulator of by trypan blue dye exclusion method. Spontaneous cell death rate platelet formation, acts in the brain as a counterpart of erythro- in neuronal cultures at the time of experiments (5 days plus 15 h) poietin (EPO), a hematopoietic growth factor with neuroprotective was 17 Ϯ 9% (mean Ϯ SD, n ϭ 40). Neuronal survival on properties. TPO is most prominent in postnatal brain, whereas EPO experimental conditions is expressed as percent of spontaneous is abundant in embryonic brain and decreases postnatally. Upon death rate in each particular experiment. Effects of growth factors hypoxia, EPO and its receptor are rapidly reexpressed, whereas on extracellular signal-regulated kinase (ERK)1͞2 phosphorylation neuronal TPO and its receptor are down-regulated. Unexpectedly, were tested on day 5 by incubation at 37°C for 10 min. For TPO is strongly proapoptotic in the brain, causing death of newly measuring long-term effects on neuronal growth, TPO (10 pM) was generated neurons through the Ras-extracellular signal-regulated added to the culture medium at the time of plating and supple- ͞ kinase 1 2 pathway. This effect is not only inhibited by EPO but mented again on day 3. also by neurotrophins. We suggest that the proapoptotic function Primary astrocyte cultures were prepared from the cortices of of TPO helps to select for neurons that have acquired target- 1-day-old Wistar-Imamichi rats as described in refs. 25 and 26, derived neurotrophic support. yielding 98% positive staining for glial fibrillary acidic protein at 2–3 weeks, i.e., the time of experiments. astrocytes ͉ erythropoietin ͉ neurons ͉ differentiation ͉ development Hypoxic conditions were induced by purging an incubator with a mixture of 95% N2͞5% CO2 (neurons) or 90% N2͞10% CO2 n the hematopoietic system, survival, proliferation, and dif- (astrocytes) as described in refs. 25 and 26 and maintained for Iferentiation of cells are regulated by a plethora of growth 15 h. Control experiments were simultaneously performed on factors (1–4). The effect of erythropoietin (EPO) on the gen- the same cell batch under normoxic conditions. For drug treat- eration of red blood cells is well known. The hematopoietic ments, see Supporting Materials and Methods. growth factor thrombopoietin (TPO) stimulates megakaryopoi- esis and thrombocyte formation (1–3, 5, 6). During hematopoi- Experiments in Vivo. For induction of hypoxic͞ischemic brain esis, EPO and TPO can interact in a synergistic and an antagonistic fashion (1–3, 7). injury, a standard method for immature rats (P14 Wistar- EPO and TPO exhibit significant homology in their receptor- Imamichi) was used (27), combining common carotid artery binding domain (20% identity and 25% similarity). Likewise, ligation with hypoxia for 1 h (‘‘moderate’’) or 2 h (‘‘severe’’) in they bind to receptors, erythropoietin receptor (EPOR) and a standardized airtight chamber, flushed with 8% oxygen. ͞ thrombopoietin receptor (TPOR), respectively, that belong to RhTPO (R & D Systems) (1 nmol kg), rhEPO (Janssen-Cilag, the same cytokine receptor superfamily (1–3, 8–10). Previous Neuss, Germany) (1.4 nmol͞kg), or vehicle were injected i.p. studies reported a neurotrophin-like motif in the N-terminal immediately before, 24 h after, and 48 h after hypoxia exposure. receptor binding region of the TPO molecule, with conflicting Brains were removed 72 h after hypoxia. Hematoxylin-eosin data about the presence of TPO in the brain (5, 6, 11–13). stained sections were scored for structural damage in cortex and For EPO, it is well established that the gene is expressed in the hippocampus ipsilateral to the ligation (two to three coronal embryonic CNS. EPO has a marked effect as a survival factor for sections per brain; 20 250ϫ fields per section) from 0–3 (0, Յ1 neurons and their progenitors (14, 15), presumably to overcome apoptotic cell per field; 1, 1–3 apoptotic or dark shrunken phases of physiological hypoxia (16, 17). The widespread but neurons per field; 2, 4–10 apoptotic, dark shrunken, or eosino- rather ‘‘unspecific’’ neuroprotective potential of EPO is regained philic neurons per field; 3, Ͼ10 apoptotic or eosinophilic, in the adult CNS upon distress or injury. This finding has been necrotic cells per field, cortical infarcts). confirmed in rodent models of cerebral ischemia (18–23), brain trauma (18), and neurodegenerative disease (18), as well as in a Expression of TPO and EPO During Brain Development. Forebrain and clinical study with stroke patients (24). hindbrain from C57B6 mice fetuses [embryonic days (E)11, 13, Here we show that TPO plays a previously unrecognized 15, and 18], newborn [postnatal day (P) 0], 14-day-old (P14), and proapoptotic role in the brain. adult mice were used for mRNA or protein extraction. For densitometric analysis of TPO and EPO protein, Western blots Materials and Methods were analyzed by National Institutes of Health image densitom- All experiments were approved by and conducted in accordance etry with ␣-tubulin III as an internal standard. with the regulations of the local Animal Care and Use Com- mittee. For detailed information on all methods see Supporting Materials and Methods, which is published as supporting infor- This paper was submitted directly (Track II) to the PNAS ofﬁce. mation on the PNAS web site. Abbreviations: En, embryonic day n; EPO, erythropoietin; EPOR, EPO receptor; ERK, extracellular signal-regulated kinase; ISOL, in situ oligo ligation; PI3K, phosphatidylinositol Cell Culture. Primary hippocampal neuronal cultures were prepared 3-kinase; Pn, postnatal day n; TPO, thrombopoietin; TPOR, TPO receptor. from newborn Wistar-Imamichi rats, cultured under serum-free †To whom correspondence should be addressed. E-mail: [email protected]. conditions (25, 26), and used for experiments after five days (purity: © 2005 by The National Academy of Sciences of the USA

862–867 ͉ PNAS ͉ January 18, 2005 ͉ vol. 102 ͉ no. 3 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406008102 Downloaded by guest on September 28, 2021 Fig. 1. Antagonistic gene expression of brain EPO and TPO systems. (a) RT-PCR illustrating presence of mRNA of TPO, EPO, and their receptors in fetal and adult rat tissues. HC, hippocampus; CX, cortex. (b) Quantitative PCR of the developing mouse forebrain demonstrates for TPO and EPO mRNA signiﬁcant changes over time (P Ͻ 0.001) and an inverse relationship (similar with hindbrain, data not shown). E11–P0, n ϭ 4; P14 and adult, n ϭ 3; *, P Ͻ 0.01 compared with E11. (c) Quantitative PCR demonstrating an inverse response of TPO, EPO, TPOR, and EPOR mRNA to hypoxia (15 h) in primary hippocampal neurons and cortical astrocytes. n ϭ 5; *, P Ͻ 0.05; **, P Ͻ 0.01 compared with normoxia. (a–c) Elongation factor was used as reference gene.

Expression Analysis by Quantitative Real-Time RT-PCR. First-strand expression of EPOR (increased) and TPOR (decreased) in cDNA was generated from total RNA by random priming cultured neurons. In astrocytes, however, both TPOR and (GIBCO͞Pharmacia, Freiburg, Germany). Detailed informa- EPOR mRNA were augmented by hypoxia (Fig. 1c). tion on rat and mouse primer pairs is available in Supporting Materials and Methods. PCR reactions were carried out on a Antagonistic TPO͞TPOR and EPO͞EPOR Protein Expression in Neurons LightCycler real-time PCR machine (Roche Molecular Bio- and Astrocytes During Brain Development and After Hypoxia. At the chemicals). RACE of rat TPOR͞Mpl mRNA was performed to protein level, TPO and TPOR expression was weak in the fetal allow for the design of rat-specific primers in the 3Ј UTR that are brain and prominent in the juvenile hippocampus, with no not sensitive toward differentially spliced isoforms (28). obvious difference (in whole-tissue lysates) 6 h and 24 h after moderate hypoxia͞ischemia (data not shown). Quantification of Western Blotting. Protein extracts transferred to nitrocellulose protein levels between E11 and adult stages indicated again a membranes were incubated with rabbit͞goat anti-TPO (Sigma), regulatory dissociation of the two growth factors: whereas TPO EPO, TPOR͞Mpl, or EPOR antibodies (Santa Cruz Biotechnol- peaked in the adult brain, EPO decreased to a nearly undetect- ogy), mouse anti-phospho-p44͞42-ERK (Thr-202͞Tyr-204) (New able level (Fig. 2a). For both TPO and TPOR, cultured neurons England Biolabs) or rabbit pan-ERK polyclonal antibody (New revealed weak but distinct specific labeling of cell bodies and England Biolabs). Immunoreactive bands were visualized by using processes (Fig. 2a, and Fig. 5 a–d, which is published as sup- secondary antibodies coupled to horseradish peroxidase by en- porting information on the PNAS web site). In agreement with hanced chemoluminescence (Amersham Pharmacia). the mRNA data, immunostaining was reduced in neurons after 15 h of hypoxia, whereas that of EPO and EPOR was enhanced. Immunohistochemistry and in Situ Oligo Ligation (ISOL). For infor- In hypoxic astrocytes, staining of TPO was weaker and staining mation on this subject, see Supporting Materials and Methods. of TPOR was unchanged as compared with normoxia (Fig. 2b). Taken together, neuronal EPO and TPO systems are regulated

Statistical Analysis. Data, expressed as mean Ϯ SEM in figures NEUROSCIENCE and text, were compared by ANOVA with post hoc planned comparisons or Duncan test, Kruskal-Wallis ANOVA with Mann–Whitney U test, or the Fisher exact probability test.

Results Inverted Pattern of TPO͞TPOR and EPO͞EPOR mRNA Expression in Brain Cells During Development and upon Hypoxia. In studies aimed originally at understanding the role of EPO in neuroprotection (21, 24, 25, 29), we noticed that TPO and TPOR also are widely expressed in the rodent CNS (Fig. 1a). We quantified and compared TPO and EPO mRNA in the mouse brain at various developmental stages by real time RT-PCR. Although TPO mRNA steady-state levels increased between E11 and adult, those of EPO decreased between E11 and E15 and stayed low thereafter (Fig. 1b). Unexpectedly, when neuronal or astrocytic cultures were Fig. 2. Antagonistic TPO͞TPOR and EPO͞EPOR protein expression in brain challenged with hypoxia (Ͻ1% O2) for 15 h, the steady-state tissue and cultured neurons and astrocytes. (a) Densitometric analysis of levels of EPO mRNA and TPO mRNA became inversely related. 70-kDa (TPO) and 38-kDa (EPO) bands corresponding to known sizes of TPO (3) In hippocampal neurons, EPO mRNA increased by Ϸ400%, and EPO (22) in Western blots of developing mouse forebrain demonstrate Ͻ whereas TPO mRNA decreased by Ϸ40% (Fig. 1c). Such a loss significant changes over time (P 0.01) and an inverse relationship of TPO and EPO (hindbrain similar, data not shown). E11–P0, n ϭ 4; P14 and adult: n ϭ 3–4; is remarkably high because a 40% decrease of TPO mRNA *, P Ͻ 0.05 compared with E11. (b) Confocal images illustrating presence of would be expected for the complete shutoff of TPO transcription TPO, EPO, and their receptors in neurons and astrocytes in culture (green in combination with a calculated mRNA half-life of 18.75 h. A fluorescence). Red fluorescence shows nuclear counterstaining with pro- similar dissociating response after hypoxia was observed for the pidium iodide. N, normoxia; H, hypoxia. (Scale bar, 50 ␮m.)

Ehrenreich et al. PNAS ͉ January 18, 2005 ͉ vol. 102 ͉ no. 3 ͉ 863 Downloaded by guest on September 28, 2021 Fig. 3. TPO and EPO exert opposite actions on neuronal survival. (a) Dose–response curves of TPO (filled circles) and EPO (open circles) effects on cell death rate in primary hippocampal neurons under normoxic conditions. n ϭ 4; *, P Ͻ 0.05 compared with control. (b) EPO (100 pM) abolishes the effect of TPO on neuronal death. Black circles, TPO alone; gray circles, TPOϩEPO; n ϭ 4. Experiments independent from a. *, P Ͻ 0.05 compared with control, #, P Ͻ 0.05 compared with TPO. (c) Granulocyte colony-stimulating factor (GCSF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and brain-derived neurotrophic factor (BDNF) (each at 1 nM) abolish the death-promoting effect of TPO (100 pM). n ϭ 6–17; *, P Ͻ 0.001 compared with control; #, P Ͻ 0.001 compared with TPO alone. (d) Antagonism of TPO-induced (100 pM) neuronal death with a Janus kinase (JAK) 2-transphosphorylation inhibitor (AG490), an inhibitor of ERK1͞2 (PD98059) or a caspase inhibitor (Ac-VAD-CHO) but not a PI3K inhibitor (LY294002). The latter abolishes the protection against TPO-induced cell death by EPO (100 pM). n ϭ 4–5; *, P Ͻ 0.05 compared with control; #, P Ͻ 0.05 compared with TPO. (e) TPO (100 pM) and EPO (100 pM) induce ERK1͞2 phosphorylation in hippocampal neurons (representative of five separate experiments). (f) Proposed EPOR-TPOR signaling cross talk. TPO dimerizes TPOR and transphosphorylates receptor- associated JAK-2. Activation of Ras-mitogen-activated protein kinase pathway leads to caspase activation and apoptosis. EPO opposes TPO effects through activation of PI3K-Akt͞protein kinase B pathway. (g–i) Primary hippocampal cultures on day 4. Representative confocal images of nuclear staining with propidium iodide (Left; red fluorescence). Arrows mark apoptotic cells. Identical fields in Right demonstrate fluorescent labeling (green) with markers for neural precursors (nestin), early postmitotic neurons (␤-tubulin III), or mature neurons (MAP2). (Scale bar, 25 ␮m.)

in opposite ways, both during brain development and after on neuronal and hematopoietic cell survival, we performed addi- hypoxia. tional experiments on murine bone marrow hematopoietic cell cultures by using the same preparation and concentrations of TPO TPO and EPO Exert Opposite Actions on Neuronal Survival. We as in the neuron cultures (Supporting Materials and Methods and Fig. anticipated that TPO, like EPO, would enhance neuronal survival. 6, which is published as supporting information on the PNAS web Surprisingly, TPO, added to cultured hippocampal neurons for 15 h site). In this preparation, TPO was clearly proproliferative and, at concentrations as low as 10 pM, increased the spontaneous cell interestingly, had a dose–response curve almost identical to that of death rate by Ͼ60% (determined under normoxia) (Fig. 3a). the proapoptotic effect of TPO on neurons. Higher concentrations diminished this effect, and TPO lost its At all concentrations tested, the cell-killing effect of TPO was death promoting activity at 10 nM (i.e., the ‘‘toxic’’ concentration completely antagonized by 100 pM EPO (Fig. 3b). Interestingly, range of EPO, see below), resulting in a bell-shaped dose–response granulocyte colony-stimulating factor (1 nM), another hemato- curve (Fig. 3a). To have a direct comparison of the effect of TPO poietic growth factor with neuroprotective potential (and a

864 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406008102 Ehrenreich et al. Downloaded by guest on September 28, 2021 receptor of the same cytokine receptor superfamily) (30), pre- day 4 or day 6), nuclear condensation and expression of apo- vented TPO-induced neuronal death. In addition, neurotrophins ptosis markers were most frequently seen in early postmitotic known to play a role in hippocampus (31), i.e., nerve growth neurons (␤-tubulin III-positive) but never in precursors (nestin- factor, neurotrophin-3, and BDNF (each at 1 nM), salvaged positive) and rarely in mature neurons (microtubule-associated- neurons from TPO-mediated cell death (Fig. 3c). protein 2-positive) (Fig. 3 g–i). In fact, TPO administration The spontaneous death rate of hippocampal neurons is higher resulted in a significant decrease of ␤-tubulin III immunoreac- under hypoxic compared with normoxic conditions (21, 25). tive cells relative to the total number of cells (39 Ϯ 6% control Remarkably, adding TPO to neuronal cultures that were kept versus 27 Ϯ 6% TPO, P Ͻ 0.05, n ϭ 4). under hypoxia (Ͻ1% O2; 15 h) did not further increase cell death (100% under normoxia, 148 Ϯ 6% under hypoxia alone, 153 Ϯ In Vivo Effects of TPO in the Pathological Brain: ‘‘Gain-of-Function’’ 7% under hypoxia plus 10 pM TPO; n ϭ 5). This result may be Experiments. To investigate the in vivo role of TPO in the brain, explained by the demonstrated loss of neuronal TPOR under we exploited our observation that TPO in neurons is reduced hypoxia (compare Figs. 1c and 2b). Growing neurons in the after hypoxia (compare Figs. 1c and 2b). We experimentally continuous presence of 10 pM TPO (‘‘chronic condition’’ of 6 applied TPO to juvenile rats in combination with a standard days instead of only 15 h) enhanced apoptotic cell death. Under model of hypoxic͞ischemic brain damage (27). In this setting, TPO, the percentage of ISOL-positive apoptotic cells increased unphysiologically high levels of TPO should result in an infor- to 58 Ϯ 1% compared with 31 Ϯ 3% under continuous placebo mative gain-of-function phenotype. The entire procedure in- treatment (P Ͻ 0.01, n ϭ 4), suggesting that the phenomenon is volved a one-sided permanent carotid artery ligation, followed not subject to any tolerance. 2 h later by exposure to a moderate or severe hypoxia (i.e., 1 or When applied to primary hippocampal neurons under nor- 2 h of 8% oxygen). Immediately before hypoxia (and 24 h and moxic conditions, EPO did not promote cell survival (25), 48 h thereafter), we gave i.p. injections of TPO (1 nmol͞kg, or consistent with low neuronal EPOR expression under normoxia vehicle placebo). After 3 days, the tissue damage of the ipsilat- (compare Figs. 1c and 2b). At higher concentrations (10 nM), eral cortex and hippocampus was scored as described under however, EPO also had a cell death promoting effect (Fig. 3a). Materials and Methods. Thus, both EPO and TPO have bell-shaped dose–response As expected, carotid artery ligation by itself (i.e., without hyp- curves. oxia) did not cause obvious brain pathology. However, 50% of artery-ligated animals that additionally received TPO exhibited a Signaling Pathways Involved in TPO and EPO Action on Neuronal damage score of Ն1 (compared with only 8% of placebo-treated Survival. The deleterious effect of TPO on neuronal survival was rats; n ϭ 10–12 per group; P Ͻ 0.05). The number of cleaved unexpected, and the antagonistic response of EPO was unex- caspase-3-positive neurons in TPO versus placebo-treated rats was plained. To determine the underlying mechanisms, we studied 114 Ϯ 62 versus 1 Ϯ 1 cells͞square unit, respectively (n ϭ 9 per candidate second-messenger systems, previously associated with group; P ϭ 0.02). At that time point, there was not yet any EPOR signaling (21, 23, 32), in primary hippocampal neurons. difference in the number of apoptotic (ISOL-positive) cells in Both AG-490 (20 ␮M), an inhibitor of JAK2-transphosphory- adjacent sections (9 Ϯ 4 cells͞square unit in TPO-treated versus lation, and PD-98059 (50 ␮M), an inhibitor of ERK1͞2, com- 11 Ϯ 7 cells͞square unit in placebo-treated rats; n ϭ 8–10 per pletely eliminated TPO-induced neuronal death, as did the group). These data suggest that TPO triggers a cell death program caspase-3 inhibitor Ac-VAD-CHO (50 ␮M) (Fig. 3d). In con- in distressed neurons in vivo. To detect potential systemic effects of trast, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY- TPO on the hematopoietic system or on thrombosis͞fibrinolysis, 294002 (100 ␮M) did not affect TPO-induced neuronal death. platelet counts as well as plasma D-dimers were determined in This finding indicates that the PI3K-Akt͞protein kinase B juvenile rats (n ϭ 4 per group) after three injections of TPO or pathway is not involved in death signaling through TPOR. placebo (at time points 0, 24, and 48 h) upon killing at 72 h (see However, this pathway is activated by EPO (21, 23) and is critical Supporting Materials and Methods). In agreement with ref. 35, there for the antagonistic effect of EPO toward TPO. In the presence was no difference among the groups with respect to these param- of LY-294002, EPO was unable to prevent TPO-induced cell eters (TPO, 555 Ϯ 100 ϫ 103͞␮l platelets and 0.04 Ϯ 0.02 ng͞␮l NEUROSCIENCE death (Fig. 3d). D-dimers, n ϭ 4; placebo, 559 Ϯ 93 ϫ 103͞␮l platelets and 0.03 Ϯ Both TPO and EPO administration to hippocampal neurons 0.01 ng͞␮l D-dimers, n ϭ 4). caused an increased phosphorylation of ERK1͞2 (Fig. 3e), which could be blocked by AG-490 (20 ␮M) (data not shown). We note In Vivo Effects of TPO Administration upon Hypoxic͞Ischemic Brain that the Ras-ERK1͞2 pathway has been implicated in both cell Damage. When the carotid ligation protocol was followed by death and survival, whereas the PI3K-Akt͞protein kinase B severe (2 h) hypoxia, the damage was extensive but about the pathway acts only in a protective fashion (21, 23, 33). Therefore, same between TPO-treated and placebo-treated rats (data not the interaction of TPO and EPO on neuronal survival is best shown). However, if exposed to only moderate (1 h) hypoxia explained with a cross talk between two intracellular signaling after ligation, TPO-treated animals had a significantly higher pathways that associate with EPOR and TPOR. TPOR engage- damage score and incidence of apoptosis than placebo-treated ment activates Ras-ERK1͞2, leading to neuronal apoptosis that rats (Fig. 4 a–c). This finding indicates that TPO worsens the can be prevented downstream by EPO through activation of outcome of moderate hypoxic͞ischemic brain injury. In marked PI3K-Akt͞protein kinase B (summarized in Fig. 3f). contrast, administration of EPO (3 ϫ 1.4 nmol͞kg i.p.) was neuroprotective, even under severe hypoxia (damage score 1.0 Ϯ TPO-Induced Apoptotic Cell Death and Neuronal Differentiation 0.3 in hypoxia plus EPO versus 1.8 Ϯ 0.4 in hypoxia alone; n ϭ Stage. TPO is known to inhibit the differentiation of cultured 12 per group; P Ͻ 0.01). Also, apoptotic cells were reduced in embryonic stem cells (34). We wondered whether the death- EPO-treated compared with placebo-treated rats (62 Ϯ 41 promoting effect of TPO could be linked to the differentiation versus 378 Ϯ 172 ISOL-positive cells per square unit in corre- of neuronal progenitors, e.g., during brain development or sponding coronal sections; n ϭ 8 per group; P Ͻ 0.05). repair. We determined in hippocampal neuronal cultures the Interestingly, the areas of cortical and hippocampal damage in developmental stage of cells undergoing TPO-induced apoptosis TPO-treated rats showed a higher density of cells positive for by using a series of well known differentiation markers (nestin, TPOR than in placebo-treated rats and a significantly more ␤-tubulin III, and microtubule-associated-protein 2). When pri- intense specific TPOR immunostaining of individual cells (Figs. mary cultures were continuously exposed to TPO (10 pM until 4d and 5 e and f). In fact, TPOR immunoreactivity was fre-

Ehrenreich et al. PNAS ͉ January 18, 2005 ͉ vol. 102 ͉ no. 3 ͉ 865 Downloaded by guest on September 28, 2021 widespread cellular TPOR immunoreactivity, TPOR-associated apoptosis was more cell type-specific; most frequently we found signs of apoptosis in ␤-tubulin III-positive neurons. In fact, TPO administration resulted in a significant decrease in ␤-tubulin III immunoreactivity, determined as staining density in the cortex (3 Ϯ 1 versus 6 Ϯ 2 arbitrary units͞mm2, TPO versus placebo, respectively, P Ͻ 0.05, n ϭ 11 per group). In contrast, NF-200- positive (mature) neurons were rarely labeled, and glial fibrillary acidic protein-positive astrocytes and nestin-positive precursors were never labeled with apoptotic markers. Discussion Here we show that TPO͞TPOR are expressed in the brain in astrocytes and neurons of various differentiation stages. TPO͞ TPOR and EPO͞EPOR display an inverse pattern, the latter decreasing with increasing brain maturation but up-regulated and protective upon distress, and the former following the opposite rule. Whereas the protective role of the EPO͞EPOR system in the brain has extensively been documented (18–22, 24, 29) the ‘‘detrimental’’ role of TPO͞TPOR is entirely novel. In fact, the most surprising finding of this work is that a prominent member of the cytokine type 1 receptor superfamily, TPOR͞TPO, induces powerful proapoptotic signaling in cells of the nervous system. In contrast, related growth factors, EPO, granulocyte colony-stimulating factor, and growth hormones have all been shown to act in an antiapoptotic fashion on neurons (4, 21, 23, 25, 30, 37). Similarly, TPO has antiapoptotic effects in the hematopoietic system (1–3, 34). To the best of our knowledge, the previously uncharacterized proapoptotic function of TPO in the nervous system has not yet been described for any other cell type. In the brain, TPO-induced apoptosis appears to be restricted predomi- nantly to maturating neuronal cells, suggesting a role for TPO in the selection of differentiated neurons. The necessity of developmental apoptosis in the brain has long been recognized (38, 39). Among others, type ␤ TGF has been shown to play a pivotal proapoptotic part in the developing nervous system of the chicken embryo (40). Now, TPO is another factor to be considered in the regulation of neuronal apoptosis. Remarkably, it acts at very low concentrations. We note that in human cerebro- spinal fluid the level of TPO (Ϸ1 pM by ELISA) (13) is Ϸ10% of the maximally effective proapoptotic concentration in vitro (10 pM TPO), and serum levels are up to 10-fold higher (7), emphasizing the physiological relevance of our findings. Fig. 4. TPO increases tissue damage upon cerebral hypoxia͞ischemia in Another somewhat puzzling finding is the bell-shaped dose– juvenile rats. (a) Representative low-magnification photomicrograph depict- response curve for both EPO and TPO effects on cultured ing cortical (CX) and hippocampal (HC) areas of a placebo-treated (Left) and neurons. Considering the structural similarity of the receptor a TPO-treated (Center) rat 72 h after right carotid artery ligation and exposure binding domain in EPO and TPO (3), we cannot exclude that ϫ to hypoxia (1 h, 8% O2). Underneath corresponding high (250 ) magnifica- EPO (when present at high concentrations) binds to the neu- tion images. Damage scores (cortex plus hippocampus) of placebo- versus ronal TPOR, and vice versa. This interaction could explain the TPO-treated rats are shown in Right (n ϭ 10). (b–d) Representative cortical cell death promoting effect of EPO at very high concentrations, ͞ sections and quantification of labeled cells in hypoxic ischemic hemisphere i.e., 1,000 times the antiapoptotically effective dose, or the loss (cortex plus hippocampus) of placebo- versus TPO-treated rats. (b) Cleaved caspase-3 (n ϭ 8–9). (c) ISOL-positive apoptotic cells (n ϭ 9). (d) TPOR immu- of the cell-killing effect of TPO at a similarly high concentration range. In experiments performed with transfected cell lines, noreactivity (n ϭ 9). (a–d) *, P Ͻ 0.05, **, P Ͻ 0.01. however, such nonspecific binding of TPO to EPOR and EPO to TPOR has not been demonstrated (41, 42). Interestingly, a bell-shaped dose–response curve of EPO, with a gradual loss of quently colocalized with two apoptosis markers, cleaved its protective effect upon increasing concentrations, has been caspase-3 and ISOL (Fig. 7 a and b, which is published as found in vivo by others (20) and ourselves (43). supporting information on the PNAS web site). Our in vivo gain-of-function model may help to understand why hypoxia physiologically down-regulates the neuronal TPO Cells Preferentially Targeted by TPO in the Hypoxic Brain. Double- system. Under conditions of distress, the presumably regulatory immunolabeling of TPOR-positive cells within the damaged proapoptotic action of TPO would not be desirable. TPO cortical and hippocampal areas identified nestin-positive pro- injected at that time point is detrimental for the damaged brain: genitors, ␤-tubulin III-positive (young postmitotic) neurons, and It leads to enhanced expression of its own receptor before NF-200-positive (mature) neurons, as well as glial fibrillary inducing cell death. However, despite widespread up-regulation acidic protein-positive glial cells (Fig. 7 c–f). Nestin-positive cells of TPOR on immature precursors and mature neurons, induc- at the site of injury could be derived from adult stem cells and tion of apoptosis is restricted to maturating neurons, whereas could be involved in tissue repair (36). In contrast to the progenitors are largely unaffected. A reduction in maturating

866 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406008102 Ehrenreich et al. Downloaded by guest on September 28, 2021 neurons in turn will compromise the brain’s repair capacity, decreasing EPO and rising TPO expression in the brain make it thereby increasing the damage. increasingly difficult for newly generated neurons to survive. The We have no direct evidence yet that TPO, when given shifting ratio of proapoptotic TPO over neuroprotective EPO is peripherally, crosses the intact blood–brain barrier, as has been then likely to make other forms of neuronal support necessary, unequivocally demonstrated for EPO (18, 29, 44). However, for example by target-derived neurotrophins. Thus, brain TPO hypoxia͞ischemia, as applied in the present study, is well known may contribute to the timing of neuronal selection by neurotro- to compromise blood–brain barrier function (45). Also, the phins. We would predict that a complete lack of TPO or TPOR demonstrated effect of TPO on neurons, together with the expression in mutant mice does not perturb gross brain devel- absence of systemic changes affecting the hematopoietic or opment. However, a temporal delay in neurotrophic selection the fibrinolytic system, make it very likely that the effect of TPO may alter the final size of neuronal populations. We note that a is a direct action on the brain. An additional indirect effect on mutation of the human TPOR͞Mpl1 gene, causing congenital thrombotic capacity due to hyperactive platelets, however, can- amegakaryocytic thrombocytopenia, has also been associated not be entirely ruled out. with abnormal brain MRI findings (46). In mice, TPO and At a more general level, we found that two classical hemato- TPOR mutations are viable (1), but neurodevelopmental defects poietic growth factors, EPO and TPO, are differentially regu- have not been analyzed. Studies with conditional mouse mutants lated in the mammalian brain, where they influence neuronal of the EPO and TPO system would help to better understand the survival in an antagonistic way. In contrast to the globally CNS functions of hematopoietic growth factors. neuroprotective effects of EPO during early brain development (14, 15), the action of TPO is selectively proapoptotic and We thank Prof. Hans Thoenen (Max Planck Institute for Neurobiology, Martinsried, Germany) for carefully reading the manuscript; Sandra increasingly prominent during later brain development. The Hartung for technical assistance; and all members of the H.E. laboratory effect of TPO is overcome by neurotrophins such as neurotro- for stimulating scientific discussions and helpful criticism. This work was phin-3, brain-derived neurotrophic factor and nerve growth supported in part by the DFG Research Center for Molecular Physiology factor. Based on these findings, we suggest a model in which of the Brain.

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