Decay accelerating factor (CD55) protects neuronal cells from chemical hypoxia-induced injury.
BACKGROUND
Activated complement system is known to mediate neuroinflammation and neurodegeneration following exposure to hypoxic-ischemic insults. Therefore, inhibition of the complement activation cascade may represent a potential therapeutic strategy for the management of ischemic brain injury. Decay-accelerating factor (DAF, also known as CD55) inhibits complement activation by suppressing the function of C3/C5 convertases, thereby limiting local generation or deposition of C3a/C5a and membrane attack complex (MAC or C5b-9) production. The present study investigates the ability of DAF to protect primary cultured neuronal cells subjected to sodium cyanide (NaCN)-induced hypoxia from degeneration and apoptosis.


METHODS
Cultured primary cortical neurons from embryonic Sprague-Dawley rats were assigned one of four groups: control, DAF treatment alone, hypoxic, or hypoxic treated with DAF. Hypoxic cultures were exposed to NaCN for 1 hour, rinsed, followed by 24 hour exposure to 200 ng/ml of recombinant human DAF in normal medium. Human DAF was used in the present study and it has been shown to effectively regulate complement activation in rats. Neuronal cell function, morphology and viability were investigated by measuring plateau depolarization potential, counting the number dendritic spines, and observing TUNEL and MTT assays. Complement C3, C3a, C3a receptor (R) production, C3a-C3aR interaction and MAC formation were assessed along with the generation of activated caspase-9, activated caspase-3, and activated Src.


RESULTS
When compared to controls, hypoxic cells had fewer dendritic spines, reduced plateau depolarization accompanied by increased apoptotic activity and accumulation of MAC, as well as up-regulation of C3, C3a and C3aR, enhancement of C3a-C3aR engagement, and elevated caspase and Src activity. Treatment of hypoxic cells with 200 ng/ml of recombinant human DAF resulted in attenuation of neuronal apoptosis and exerted significant protection against neuronal dendritic spine loss and plateau depolarization reduction. Furthermore, treatment with DAF resulted in decreased accumulation of C3a, MAC, C3a-C3aR interaction, caspase-9, activated caspase-3, and pTyr416-Src (activated Src) tyrosine kinase.


CONCLUSIONS
DAF was found to reduce neuronal cell death and apoptosis in NaCN induced hypoxia. This effect is attributed to the ability of DAF to limit complement activation and inhibit the activity of Src and caspases 9 and 3. This study supports the inhibiting of complement as a neuroprotective strategy against CNS ischemia/reperfusion injury.
Wang
Ying
Y
Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
Li
Yansong
Y
Dalle Lucca
Shawn L
SL
Simovic
Milomir
M
Tsokos
George C
GC
Dalle Lucca
Jurandir J
JJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D018958:CD55 Antigens / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects; D015926:Complement C3a / Q000378:metabolism; D003165:Complement System Proteins / Q000502:physiology; D003712:Dendrites / Q000502:physiology; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D009474:Neurons / Q000187:drug effects*; D018696:Neuroprotective Agents; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D011951:Receptors, Complement / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D018958:CD55 Antigens; D018696:Neuroprotective Agents; D011951:Receptors, Complement; C085567:complement C3a receptor; D015926:Complement C3a; D003165:Complement System Proteins; D019061:src-Family Kinases; D053148:Caspase 3; D020169:Caspases