31 March 2007. Excitotoxic cell death, the end for many cells after ischemic brain injury and in some neurodegenerative diseases, is triggered by glutamate-induced calcium influx. Downstream, activation of the p38α map kinase leads to apoptosis, but just how calcium and p38 connect has been an open question. New results from Michael Courtney’s lab at the University of Kuopio in Finland show that the small GTPase Rho is the missing link. Their work, published in the March 18 online edition of Nature Neuroscience, establishes Rho activation by calcium influx as a necessary and sufficient event to turn on p38 and cause cell death in primary neurons.
Their results open up a potential new target for drugs to block excitotoxicity, and may have some additional relevance to Alzheimer disease. Inhibitors of Rho and its downstream kinase Rock can modulate amyloid-β (Aβ) peptide production (Zhou et al., 2003; Leuchtenberger et al., 2006). In addition, Rho is farnesylated, and changes in Rho/Rock pathway activity have been implicated in alterations of amyloid precursor protein processing in response to statins (see ARF related news story).
Finding Rho in the excitotoxicity pathway was unexpected, the authors write, since many previous attempts have failed to detect Rho-mediated activation of p38α. This is because the pathway only functions in primary neurons, and not in cultured cell lines, they found. To look at Rho’s signaling role in response to excitotoxic stimuli, first authors Maria Semenova, Anu Maki-Hokkonen, and Jiong Cao used a strategy to inactivate Rho by expressing the Clostridium botulinum C3 exoenzyme, a toxin which ADP ribosylates and inactivates Rho proteins specifically. When the toxin gene was expressed in primary cerebellar neurons, it blocked activation of p38 by glutamate without affecting calcium influx. They subsequently found that glutamate stimulation activated Rho, but not the related protein Rac, in the same neurons. Rho activation was also seen in mouse brain after ischemia.
Blocking Rho not only inhibited p38α activation, but it also decreased cell death. The same effect was elicited with a dominant negative Rho mutant, suggesting that the protein was necessary for cell death. Finally, they showed that expression of constitutively active Rho was sufficient to cause cell death. They found a similar role for Rho in p38 activation and cell death after glutamate stimulation in cortical and hippocampal neurons in culture.
Using a sensitive florescence resonance energy transfer (FRET) assay for Rho activation in primary cell culture, the investigators showed that glutamate activated Rho in an NMDA receptor-dependent manner. They found a rapid, glutamate concentration-dependent increase in calcium levels, Rho activation, p38 activation, and cell death, further supporting the idea that these events are all linked. However, they did not find out how calcium stimulated Rho, although they ruled out two candidate pathways, the Rho115 guanine exchange factor and the calmodulin-dependent kinase II. Likewise, the downstream targets of Rho are also unknown, but the investigators showed the pathway did not require either RockI or II kinase activity.
The results fill in one more piece of the puzzle of excitotoxic cell death, and open up the possibility of a new target for inhibiting the pathway. “Inhibition of Rho has proven to be beneficial in models of axonal regeneration, and regulation of the release of amyloidogenic Aβ42 peptide from neuroblastoma cells," the authors write. “In light of the present data, Rho inhibition might also help to increase survival subsequent to excitotoxic challenge." The map kinases including p38 and its cousins are involved in many aspects of neuron life and death (see ARF related news story), suggesting that more roles for Rho remain to be discovered.—Pat McCaffrey.
Semenova MM, Maki-Hokkonen AM, Cao J, Komarovski V, Forsberg KM, Koistinaho M, Coffey ET, Courtney MJ. Rho mediates calcium-dependent activation of p38alpha and subsequent excitotoxic cell death. Nat Neurosci. 2007 Mar 18; [Epub ahead of print] Abstract