Introduction
Acute neurological diseases such as stroke and head trauma lead to neuronal death. To a large measure, neuronal death is triggered by excitotoxicity, a term that denotes the killing of neurons by glutamate-triggered influx of CaP2+P into neurons, mainly by NMDA receptors, prominent molecular machines in excitatory synapses, which are designed for high CaP2+P permeability and are activated by the prevalent transmitter L-glutamate (1). To prevent NMDA receptor-mediated cell death after stroke, numerous efforts by the pharmaceutical industry were directed towards blocking NMDA receptors. Unfortunately, these efforts failed, in large part due to harmful side effects caused by NMDA receptor blockers.Remarkably, the same NMDA receptors that are involved in mediating cell death after stroke, are crucial to the brain’s efforts to restore function after stroke-induced brain damage. Indeed, NMDA receptor-mediated CaP2+P influx under physiological as opposed to pathophysiological conditions is required for the plasticity of the brain (2), to alter network function, synaptic strength and synaptic connectivity, thereby forming the basis for functional recovery from stroke.
Results/Project Status
We therefore set out to test the role in functional recovery following stroke of the two main NMDA receptor subtypes in the forebrain. We use mice for our studies, as we have generated mouse lines in which these two receptor subtypes are functionally impaired.
The two main NMDA receptor subtypes are characterized by the subunit composition NR1/NR2A and NR1/NR2B, and differ by the modulatory NR2 subunit while incorporating the same principal NR1 subunit. Interestingly, the NR2B subtype is highly expressed in the central nervous system throughout embryonic development and postnatal life, whereas the NR2A subtype commences expression after birth and reaches peak by approximately 3 weeks of age in the rodent (3). Thus the NR2A receptor subtype is a late form of the receptor whereas the NR2B subtype represents the early form. The early form mediates much of the developmentally important circuit formation, whereas the late form is thought to participate in the modification of synaptic communication essential for learning by the adult brain. However, the distinct roles of the two main subtypes in the adult brain need elucidating. We are in process of adapting for the mouse a stroke paradigm which was first developed for the rat. This paradigm involves setting a photothrombotic, laser-assisted unilateral lesion into the motor cortex, leading to the impaired use of the contralateral forelimb (4). Appropriate behavioural tests (Fig. 1) are then employed to monitor the functional recovery of the impaired forelimb during the weeks following the insult. Although the photothrombotic lesion is not directly a relevant model for human stroke, which usually occurs by blood vessel occlusion, it has the distinct advantage that the lesions can be produced at a high level of reproducibility in different mice, This ensures comparable levels of impairment among the lesioned mice, essential for an analysis of molecular mechanisms underlying functional recovery from stroke. Returning to the relevance of the two main NMDA receptors for functional recovery from stroke, we will compare the recovery processs in mice of different genetic make-up. We will compare wild-type mice with mice having either the NR1/NR2A subtype or the NR1/NR2B subtype functionally impaired (5, 6). We predict that the recovery process is protracted in mice lacking the subtype most important for functional recovery. Functional recovery from photothrombotic lesions will require the formation of novel synaptic connections between neuronal populations not damaged by the lesion, including connections across the hemispheres. New wiring between cells rests on transcriptional programs set in motion in the active neurons, programs triggered, in part, by the CaP2+P influx through synaptically activated NMDA receptor channels. We plan to determine the genes most relevant for restructuring the synaptic connections for functional recovery of the lesioned brain, and further determine which of the two NMDA receptor subtypes is involved in triggering their transcriptional activity.
Genes for functional recovery
In view of the considerations outlined above, we plan to monitor cohorts of mice with altered NMDA receptor subtype function in their progress of functional recovery from photothrombotic lesion. We anticipate that we will be able to detect genotype-specific differences in the speed and extent of recovery, which will depend on the extent of reproducibility concerning the location and size of the lesion. Once this goal is reached, we will ask important questions as to the activity of key genes underlying the recovery process. Such recovery promoting genes will be revealed by transcriptional profiling of several small excised brain regions in the vicinity of the lesion and the corresponding regions on the contralateral side. Transcriptional profiling will be performed with the help of genome-wide Affymetrix chips. It will also be important to determine the transcriptional profiles during the recovery process to reveal the temporal aspects of different signaling cascades relevant to recovery. Moreover, the dependence of these cascades on the NMDA receptor subtypes will be relevant to understanding mechanistic aspects of circuit rewiring.
Outlook
The program outlined above will extend over several years. Numerous technical aspects need testing, the behavioural paradigms require setting up, and mice of the different genotypes will have to be bred in sufficient numbers. At the end of this endeavour, we hope to learn about select genes which by up- or downregulation will contribute to the rewiring of a damaged brain and hence, promote functional recovery from stroke. Moreover, we hope to determine to what extent the regulation of these recovery promoting genes is controlled by the two main NMDA receptor subtypes. Finally, and most importantly, the data generated from this program may reveal new avenues to therapeutic intervention in stroke.
Lit.: 1. Lee JM, Zipfel GJ, Choi DW. The changing landscape of ischaemic brain injury mechanisms. Nature. 1999; 399:A7-14. 2. Klintsova AY and Greenough WT. Synaptic plasticity in cortical systems. Curr Opin Neurobiol. 1999; 9:203-208. 3. Monyer H et al. Developmental and regional expression in the rat brain and functional properties of four NMDA receptor subtypes. Neuron. 1994; 12:529-540. 4. Witte OW and Stoll G. Delayed and remote effects of focal cortical infarctions: secondary damage and reactive plasticity. Adv Neurol. 1997; 73:207-227. 5. Sprengel R et al. Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo. Cell. 1998; 92:279-289. 6. Köhr et al. Intracellular domains of the NMDA receptor subtypes are determinants for long-term potentiation induction. J Neurosci. 2003; 23: 10791-10799.
