Immediate upregulation of proteins belonging to different branches of the apoptotic cascade in the retina after optic nerve transection and optic nerve crush.
OBJECTIVE
To further investigate the molecular signals underlying optic nerve (ON) injury, the authors analyzed in adult control, ON-transected, and ON-crushed retinas the expression pattern and time-course regulation of the following proteins, all of which are linked to apoptosis through different pathways: Stat 1, caspase 11 (inflammation and death), cathepsins C and B (lysosomal death pathway), calpain 1 (endoplasmic reticulum stress), calreticulin (apoptosis marker), Jun (early response), and aryl hydrocarbon receptor (cell cycle arrest).


METHODS
Adult female rats were subjected to intraorbital optic nerve transection (IONT) or intraorbital optic nerve crush (IONC). Protein from naive and ON-injured adult rat retinas was extracted at different times postlesion, and Western blotting experiments were performed. For immunohistofluorescence analyses, retinal ganglion cells (RGCs) were retrogradely identified with fluorogold applied to the superior colliculi 1 week before injury.


RESULTS
Western blotting analyses revealed upregulation of all the analyzed proteins as early as 12 hours postlesion (hpl), peaking at 48 hpl, in agreement with our previous RNA study findings. Furthermore, immunohistofluorescence to radial sections showed that all but Stat 1 were expressed by the primarily injured neurons, the RGCs, as seen by colocalization with fluorogold.


CONCLUSIONS
All analyzed proteins were upregulated in the retina after IONT or IONC as early as 12 hpl, indicating that ON injury regulates several branches of the apoptotic cascade and suggesting that commitment to death might be an earlier event than previously anticipated.
Agudo
Marta
M
Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, Murcia, Spain. martabar@um.es
Pérez-Marín
Maria C
MC
Sobrado-Calvo
Paloma
P
Lönngren
Ulrika
U
Salinas-Navarro
Manuel
M
Cánovas
Isabel
I
Nadal-Nicolás
Francisco M
FM
Miralles-Imperial
Jaime
J
Hallböök
Finn
F
Vidal-Sanz
Manuel
M
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D015153:Blotting, Western; D064032:Calbindin 2; D020169:Caspases / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism; D020789:Cathepsin C / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D009409:Nerve Crush; D020221:Optic Nerve Injuries / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000473:pathology; D064030:S100 Calcium Binding Protein G / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation
D051017:Apoptosis Regulatory Proteins; D064032:Calbindin 2; D016755:Proto-Oncogene Proteins c-jun; D018336:Receptors, Aryl Hydrocarbon; D064030:S100 Calcium Binding Protein G; D050794:STAT1 Transcription Factor; C494081:Stat1 protein, rat; D020789:Cathepsin C; C533920:Ctsc protein, rat; C491071:Casp4 protein, rat; D020169:Caspases; D002401:Cathepsin B; C533916:Ctsb protein, rat