Annual Report of Activities CNC 2008 - Center for Neuroscience and ...

Neuronal Cell Death and Neuroprotection | Head: Carlos DuarteObjectivesNumerous disorders of the CNS are characterizedby neuronal cell death, which may arise from thederegulation of the activity of neurotransmittersystems or insufficient neurotrophic support. Inbrain ischemia there is an excessive accumulationof the neurotransmitter glutamate, and theresulting overactivation of glutamate receptorscauses neuronal death (excitotoxicity). The activityof glutamatergic synapses in the hippocampus isnormally regulated by the neurotrophin BDNF(e.g. J Biol Chem 282: 12619‐12628 [2007]), which isalso an endogenous neuroprotectant, counteractingto some extent the effects of glutamate as a toxin.This group studies molecular mechanismscontributing to excitotoxic cell damage, particularlyin the hippocampus, a brain region particularlyvulnerable to glutamate toxicity, andneuroprotection by BDNF (brain‐derivedneurotrophic factor). Furthermore, this groupinvestigates the mechanisms controlling theexpression of neuroprotective factors uponneuronal injury, both in cell cultures and in a ratmodel of Parkinson’s disease.Main AchievementsThe contribution of Ca2+‐permeable AMPAreceptors overactivation to dell death wasinvestigated in HEK293 cells constitutivelyexpressing GluR4‐containing AMPARs.Excitotoxicity mediated by these receptors wasfound to involve the calcium‐dependent activationof the JNK pathway. The GluR4 subunit wasshown to bind the JIP‐1 protein, a scaffold thatparticipates in the JNK cascade induced by deathstimuli in neurons, and current studies will allowmapping the JIP‐1 domain interacting with GluR4.Moreover, JNK‐specific GluR4 phosphorylationwas increased upon excitotoxic stimulation.Studies with the GluR4 subunit mutated at thephosphorylation site will allow understanding thefunctional role of this phosphorylation.Fig. 1. Excitotoxic stimulation of Ca 2+ ‐permeable AMPA andNMDA receptors for the neurotransmitter glutamate leads tocell death due to activation of the JNK signaling pathway andinhibition of the ubiquitin‐proteasome system, respectively.Cell death under excitotoxic conditions is mediatedby deregulation of the major proteolytic systems inneurons. Excitotoxic stimulation of hippocampalneurons down‐regulated proteasome activity by amechanism sensitive to cathepsin‐L inhibitors. Thedecrease in proteasome activity was moresignificant in the nuclear fraction than in thecytoplasmic fraction, and was mediated byactivation of extrasynaptic NMDA receptors.Excitotoxic stimulation with glutamate alsodecreased the deubiquitinase activity, pointing to ageneral deregulation of the ubiquitin‐proteasomesystem.Excitotoxic stimulation was found to downregulatethe full‐length TrkB (TrkB‐FL) receptorsfor the neurotrophin BDNF in hippocampal andstriatal neurons, by a calpain‐dependentmechanism, and upregulated the truncated form ofthe receptors (TrkB‐T). Under the same conditionsthere was a decrease in the signaling activity ofTrkB‐FL, and current studies will allowdetermining the role of a putative dominantnegative effect of TrkB‐T. The effect on TrkB‐Toccurred at the transcription level, and wascoupled to the down‐regulation of RhoA signalingactivity. Future studies will address how thechange in TrkB protein levels affectneuroprotection under excitotoxic conditions.24