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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 19, 2004 as doi:10.1096/fj.04-2493fje. |
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* Department of Neurology, University Hospital Zurich (USZ), Zurich, Switzerland; and
Institute of Veterinary Physiology, Vet Suisse Faculty, University of Zurich, Zurich, Switzerland
1Correspondence: Department of Neurology, University Hospital Zurich, Frauenklinikstr. 26, Zurich CH-8091 Switzerland. Email: uelkan.kilic{at}usz.ch
SPECIFIC AIMS
The hematopoietic growth factor erythropoietin (Epo) exhibits neuroprotective activity under experimental conditions of brain, retinal, and spinal cord ischemia and, according to a recent proof-of-concept trial, in human patients with ischemic stroke. Epo might raise new hope for treatment of many neurological disorders in which neurons are lost, including more delayed conditions such as Parkinson’s and Alzheimer’s diseases. The factor might be particularly suitable as a neuroprotectant, since it is considered safe and has few side effects. The aims of this study were to 1) demonstrate whether human Epo, when expressed in a transgenic mouse (termed tg21 line) exhibiting elevated Epo levels in the brain but not the blood, protects against subacute degeneration of axotomized retinal ganglion cells (RGCs), and 2) elucidate the underlying signaling mechanisms involved in Epo’s neuroprotective activity. RGC are central nervous system (CNS) -derived neurons that, due to their accessibility outside the cranial cavity, present an almost exemplary opportunity to study cell death mechanisms and neuroprotection strategies in vivo.
PRINCIPAL FINDINGS
1. Retinae of tg21 mice express human Epo
To find out whether human Epo is expressed in retinae of tg 21 mice, radioimmunoassays (RIA) were performed. RIA revealed significantly increased Epo levels in retinae of tg21 animals, when compared with nontransgenic mice (94.4±6.6 vs. 59.5±2.1 mU Epo/mg).
2. Epo receptor is expressed on RGCs of wild-type and tg21 mice
To elucidate whether RGCs carry the Epo receptor, immunohistochemistries were prepared. Staining showed a robust expression of Epo receptor on murine RGCs. Expression levels did not differ between wild-type and tg21 mice.
3. Epo protects against RGC degeneration in vivo
To study whether human Epo protects against experimentally induced degeneration of RGCs, RGCs were retrogradely prelabeled with fluorogold, which was injected into both superior colliculi. RGCs were axotomized 3 days later. RGC survival was assessed 14 days after optic nerve transection by stereological counting of viable ganglion cells. Transgenic Epo markedly increased the density of surviving RGCs at 14 days after axotomy (P< 0.01, Fig. 1
A–C). Neuroprotection was seen in all retinal eccentricities, both close to the posterior pole of the eye and distant to it (Fig. 1B
). Ganglion cell densities in nonlesioned retinae did not differ between mouse lines (Fig. 1C, D
). Hence human Epo protects against the subacute degeneration of RGCs in vivo.
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4. Constitutive ERK-1/-2, Akt and STAT-5 activities are elevated in nonlesioned retinae of tg21 mice
To elucidate signaling mechanisms mediating Epo’s neuroprotection, Western blot analyses were performed using retinal homogenates. Nonlesioned retinae showed that phosphorylation levels of ERK-1/-2, Akt, and STAT-5 were higher in tg21 than in wild-type mice (Fig. 2
). Constitutive JNK, JAK-2, Bcl-XL, and caspase-3 levels did not differ between mouse lines (Fig. 2)
.
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5. ERK-1/-2 and Akt signaling are increased whereas JNK and caspase-3 signaling are decreased after axotomy in tg21 mice
In tg21 mice, ERK-1/-2 phosphorylation increased after RGC axotomy (Fig. 2)
. Akt phosphorylation remained high after lesioning whereas JNK activity mildly decreased (Fig. 2)
. In wild-type mice, optic nerve transection did not induce any signaling changes, neither of phosphorylated Akt nor of MAP kinases (Fig. 2)
. Our data suggested a role of ERK-1/-2 and Akt signaling in Epo’s neuroprotection. Caspase-3 activity was inhibited in axotomized retinae of Epo expressing, but not of control mice (Fig. 2)
, indicating that Epo signaling converged in the inhibition of executioner caspase-3.
6. JAK-2, STAT-5, and Bcl-XL signaling are attenuated after optic nerve transection in tg21 mice
Optic nerve transection reduced retinal JAK-2, Bcl-XL, and STAT-5 phosphorylation levels in tg21 mice (Fig. 2)
. In nontransgenic animals, JAK-2 and Bcl-XL levels remained unchanged after lesioning whereas STAT-5 activity increased, even above levels of transgenic mice (Fig. 2)
. Reduced STAT-5 and Bcl-XL signaling argues against an involvement of these pathways in Epo’s neuroprotective activity, at least in the experimental paradigm and mouse line used.
7. Epo-induced neuroprotection in vivo is mediated by ERK-1/-2 signaling
To determine whether ERK-1/-2 or Akt signaling pathways are responsible for EPO’s neuroprotection, we locally applied the solvent DMSO with and without signal transduction inhibitors into the eye’s vitreous space at 0, 4, 7, and 10 days after axotomy. Whereas phosphatidyl inositol-3' kinase (PI3K)/ Akt inhibitor Wortmannin did not influence RGC survival to major extent, inhibition of ERK-1/-2 using PD98059 abolished the protection effects of Epo (Fig. 1D
). Our data show that Epo-induced neuroprotection critically depends on activation of the ERK-1/-2, but not of Akt (Fig. 3
).
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CONCLUSIONS
Using the RGC axotomy model, we demonstrated in the transgenic mouse line tg21, which expresses human Epo preferentially in the CNS, that hematopoietic growth factor Epo protects CNS-derived neurons in vivo against delayed neuronal degeneration via ERK-1/-2 signaling. Our data were obtained using 1) stereological assessments of retrogradely prelabeled RGCs in axotomized retinae, 2) Western blot analyses for various signal transduction factors and apoptosis-related proteins previously implicated in Epo’s neuroprotective activity as well as 3) additional in vivo studies, in which signal transduction inhibitors were applied into the vitreous space and in which RGC survival was assessed. Our study reveals a marked neuroprotective action of Epo, which was even above that of other growth factors previously assessed in the same model.
Among the large number of signaling factors previously involved in Epo’s neuroprotective activity in vitro, only ERK-1/-2 and Akt signaling were activated after optic nerve transection in our tg21 animals; the JAK-2, STAT-5, JNK, and Bcl-XL pathways were inhibited at the same time. Signal transduction inhibition studies subsequently showed that the neuroprotective action of Epo was fully reversible after inhibition of ERK-1/-2 with PD98059, but not of PI3K/ Akt with Wortmannin. The inhibition of JAK-2, STAT-5, and Bcl-XL pathways in axotomized RGCs of tg21 mice argues against a role of these signaling factors in Epo’s neuroprotection. Our observation is noteworthy since 1) STAT-5 is a major signaling pathway activated by the Epo receptor, and 2) Bcl-XL is a crucial death-inhibitory protein localized in mitochondria, preventing caspase-3-dependent apoptosis both in vitro and in vivo. According to our data, inhibition of caspase-3 induced by Epo occurs most likely not via a mitochondrial loop, but directly via cytosolic pathways (i.e., ERK-1/-2).
The neuroprotective action of Epo shown in this study may open new perspectives for treatment of subacute and chronic neurodegenerative diseases that might also profit from Epo under clinical conditions. Neurodegenerative illnesses, such as Parkinson’s and Alzheimer’s diseases, are frequent disorders that are major causes of long-term disability and impose tremendous burdens to public health care. There are no efficacious treatments available at present that allow prevention of neuronal degeneration in a causative manner. Establishing new strategies in this context would not only create entirely new perspectives for neurological therapy and open hopes for millions of patients concerned, but also have favorable socio-economical consequences. Development of clinically applicable protection strategies deserve concerted efforts as a priority issue in clinical neurosciences. Epo is a highly attractive candidate and chances appear favorable that it will thus be possible to establish new treatments.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2493fje;
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