CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and L-type Ca2+channels upstream of p38 MAPK.
BACKGROUND
The chemokine receptor CXCR4 (CD184) and its natural ligand CXCL12 contribute to many physiological processes, including decisions about cell death and survival in the central nervous system. In addition, CXCR4 is a co-receptor for human immunodeficiency virus (HIV)-1 and mediates the neurotoxicity of the viral envelope protein gp120. However, we previously observed that CXCL12 also causes toxicity in cerebrocortical neurons but the cellular mechanism remained incompletely defined.


METHODS
Primary neuronal-glial cerebrocortical cell cultures from rat were exposed to a neurotoxicity-inducing CXCL12 concentration for different times and the activity of the stress-associated mitogen-activated protein kinase p38 (p38 MAPK) was assessed using an in vitro kinase assay. Neurotoxicity of CXCL12 and cellular localization of p38 MAPK was analyzed by immunofluorescence microscopy. Pharmacological inhibition of NMDA-type glutamate receptor-gated ion channels (NMDAR) of L-type Ca2+channels was employed during 12- and 24-h exposure to neurotoxic amounts of CXCL12 to study the effects on active p38 MAPK and neuronal survival by Western blotting and microscopy, respectively. Neurotoxicity of CXCL12 was also assessed during pharmacological inhibition of p38 MAPK.


RESULTS
Here, we show that a neurotoxic amount of CXCL12 triggers a significant increase of endogenous p38 MAPK activity in cerebrocortical cells. Immunofluorescence and Western blotting experiments with mixed neuronal-glial and neuron-depleted glial cerebrocortical cells revealed that the majority of active/phosphorylated p38 MAPK was located in neurons. Blockade of NMDAR-gated ion channels or L-type Ca2+channels both abrogated an increase of active p38 MAPK and toxicity of CXCL12 in cerebrocortical neurons. Inhibition of L-type Ca2+channels with nimodipine kept the active kinase at levels not significantly different from baseline while blocking NMDAR with MK-801 strongly reduced phosphorylated p38 MAPK below baseline. Finally, we confirmed that directly blocking p38 MAPK also abrogated neurotoxicity of CXCL12.


CONCLUSIONS
Our findings link CXCL12-induced neuronal death to the regulation of NMDAR-gated ion channels and L-type Ca2+channels upstream of p38 MAPK activation.
Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, Bldg. 10, La Jolla, CA, 92037, USA.
Ana B
AB
Sanchez
Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, Bldg. 10, La Jolla, CA, 92037, USA.|Present address: UC San Diego Health, 200 W. Arbor Drive #8765, San Diego, CA, 92103, USA.
Kathryn E
KE
Medders
Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, Bldg. 10, La Jolla, CA, 92037, USA.
Ricky
R
Maung
Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, Bldg. 10, La Jolla, CA, 92037, USA.
Paloma
P
Sánchez-Pavón
Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, Bldg. 10, La Jolla, CA, 92037, USA.
Daniel
D
Ojeda-Juárez
Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, Bldg. 10, La Jolla, CA, 92037, USA. mkaul@SBPdiscovery.org.|Department of Psychiatry, University of California, San Diego, 9500 Gilman Drive, San Diego, CA, 92093, USA. mkaul@SBPdiscovery.org.
Marcus
http://orcid.org/0000-0003-3202-670X
M
Kaul
CXCL12; CXCR4; Calcium channel; Cell death; Immunofluorescence microscopy; Inhibitors; Kinase activity; Neurotoxicity; p38 MAPK
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