GM-CSF protects rat photoreceptors from death by activating the SRC-dependent signalling and elevating anti-apoptotic factors and neurotrophins.
BACKGROUND
The term retinitis pigmentosa (RP) comprises a heterogeneous group of hereditary and sporadic human retinal degenerative diseases. The molecular and cellular events still remain obscure, thus hiding effective therapies. Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a hematopoietic factor which plays a crucial role in protecting neuronal cells. Binding of GM-CSF to its receptor induces several intracellular signaling pathways and kinases. Here we examined whether GM-CSF has a neuroprotective effect on photoreceptor degeneration in Royal College of Surgeons (RCS) rats.


METHODS
GM-CSF was injected into the vitreous body of RCS rats either once at the onset of photoreceptor degeneration at day 21, or twice at day 21 and day 42. At day 84, when photoreceptor degeneration is completed, the rats were sacrificed, their eyes enucleated and processed for histological staining and counting the surviving photoreceptor nuclei. The expression of apoptosis-related factors, such as BAD, APAF1 and BCL-2 was examined by Western blot analysis. The expression of neurotrophins such as ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic actor (GDNF), as well as glial fibrillary acidic protein (GFAP) was analysed by Western blots and immunohistochemistry. The expression of JAK/STAT, ERK1/2 and SRC pathway proteins was assessed by Western blot analysis.


RESULTS
GM-CSF protects significantly against photoreceptor degeneration in comparison to control group. After a single injection of GM-CSF at P21, a 4-fold increase of photoreceptors was observed, whereas eyes which received a repeated injection of GM-CSF at P42 showed a 10-fold increase of photoreceptors. Western blot analysis revealed a decreased BAD and an increased pBAD and BCL-2 expression, indicating changed expression profiles of apoptosis-related proteins. Neurotrophic factors examined are up-regulated, whereas GFAP was also modulated. At cell signalling levels, GM-CSF activates SRC-dependent STAT3 which is independent of JAK2, while proteins of the ERK1/2 pathway are not affected.


CONCLUSIONS
The data suggest that GM-CSF is a potent therapeutic agent in photoreceptor degeneration caused by mutation of the receptor tyrosine kinase gene (Mertk), and may be also effective in other photoreceptor degeneration.
Department of Experimental Ophthalmology, School of Medicine, University Eye Hospital Münster and Interdisciplinary Centre for Clinical Research (IZKF), Domagkstrasse 15, 48149 Münster, Germany. maurice.schallenberg@uk-essen.de
Maurice
M
Schallenberg
Petar
P
Charalambous
Solon
S
Thanos
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D053477:Apoptotic Protease-Activating Factor 1 / Q000378:metabolism; D015153:Blotting, Western; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000008:administration & dosage / Q000494:pharmacology*; D007150:Immunohistochemistry; D058449:Intravitreal Injections; D009414:Nerve Growth Factors / Q000378:metabolism*; D020419:Photoreceptor Cells, Vertebrate / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D011922:Rats, Mutant Strains; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D012162:Retinal Degeneration / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D012243:Rhodopsin / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D051025:bcl-Associated Death Protein / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
C506594:Apaf1 protein, rat; D051017:Apoptosis Regulatory Proteins; D053477:Apoptotic Protease-Activating Factor 1; C494821:Bad protein, rat; D005904:Glial Fibrillary Acidic Protein; D009414:Nerve Growth Factors; D019253:Proto-Oncogene Proteins c-bcl-2; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D050796:STAT3 Transcription Factor; C494088:Stat3 protein, rat; D051025:bcl-Associated Death Protein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D012243:Rhodopsin; D019061:src-Family Kinases