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First published online 23 January 2007
doi: 10.1242/jcs.03354

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Anti-NR1 N-terminal-domain vaccination unmasks the crucial action of tPA on NMDA-receptor-mediated toxicity and spatial memory

Karim Benchenane^1,*, Hervé Castel¹, Michel Boulouard², Rosemarie Bluthé³, Monica Fernandez-Monreal^1,, Benoit D. Roussel¹, José P. Lopez-Atalaya¹, Sabrina Butt-Gueulle², Véronique Agin¹, Eric Maubert¹, Robert Dantzer³, Omar Touzani⁴, François Dauphin², Denis Vivien^1, and Carine Ali¹

¹ INSERM, INSERM-Avenir `tPA in the working brain'; Université de Caen Basse-Normandie; Cyceron; 14074 Caen Cedex, France
² Université de Caen Basse-Normandie, EA 3915, CERMN, Pharmacologie-Physiologie, UFR des Sciences Pharmaceutiques, 14032 Caen, France
³ INRA, U1244; Université de Bordeaux 2; CNRS, FRE2723, 33077 Bordeaux, France
⁴ CNRS, UMR-6185; Université de Caen Basse-Normandie; Cyceron; 14074 Caen Cedex, France

View larger version (39K): [in this window] [in a new window]   Fig. 1. Immunisation against the NTD of the NMDA receptor NR1 subunit prevents both the interaction of tPA with NR1 and subsequent cleavage of NR1 in vivo. (A) Schematic representation of the hypothesis: antibodies raised against the NTD-NR1 should interact with endogenous NR1 and thus prevent the potentiating effect of tPA on NMDA receptor signalling. (B) 1, 5 or 10 µg of recombinant N-terminal domain of NR1 (NTD-NR1, amino acids 19-371) was separated by SDS-PAGE prior to detection with sera collected from NTD-NR1- or CFA-immunised mice. Immunoblot is representative of three independent experiments performed with sera from three different animals for each group. (C) tPA activity in cortical and striatal extracts measured by zymography assay in CFA-treated mice and vaccinated mice (n=3). (D) Evidence of IgG immunoreactivities in the cerebral parenchyma of NTD-NR1- or CFA-immunised mice (representative photomicrographs taken from the same anatomical level of the cortex) (E) Protein extracts from cerebral cortices of control and immunised mice were subjected to immunoprecipitation against tPA or NR1 before immunoblotting against NR1 as described in the Materials and Methods section. Immunoblot is representative of three independent experiments. IB, immunoblot; IP, immunoprecipitation; CFA, CFA-injected; NTD, NTD-NR1-injected.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Immunisation against the NTD of the NMDA receptor NR1 subunit prevents the neurotoxic effect of endogenous and exogenous tPA. (A) Excitotoxic lesions were performed, 4-5 days after the last inoculation, by injecting NMDA (10 nmol) into the striatum. After 30 minutes, tPA (1 mg/kg) or vehicle was injected over 15 minutes. Brains were harvested 24 hours later, and cerebral lesions were measured by analysing thionine-stained cryostat sections (see representative section for each group). Lesion volume was analysed by a two-way ANOVA and Bonferroni/Dunn (n=9-11 mice per group, mean ± s.e.m.; **P<0.01). (B,C) Focal permanent ischemia was induced by MCA occlusion and cerebral lesions were measured after 24 hours of injury. (B) Representative staining and (C) lesion volumes analysed by Student's t-test (n=7-8 mice per group, mean ± s.e.m.; *P<0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 3. Immunisation has no effect on tPA-independent behaviour tasks. (A,B) Spontaneous locomotor activity in an activity cage. No difference was seen in either group. (C,D) Mice were subjected to an elevated plus-maze, which allows the measurement of the anxiety level. (C) The activity of tPA-deficient mice in the closed or open arms of the maze did not differ from that of WT mice. (D) Accordingly no behaviour difference was seen in the plus-maze between control, CFA-treated and NTD-NR1-vaccinated mice.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Immunisation leads to an impaired spatial memory comparable to that observed in tPA-deficient mice. (A) In the Y-maze task, spatial memory was clearly impaired in tPA-deficient mice when compared with WT littermates as assessed by the time spent in the new arm compared with the two others. (B) A similar deficit was observed in NTD-NR1-immunised mice when compared with control or CFA-injected mice. (C) Plot of all the parameters used to quantify the performance in this task (time spent and number of entries in each arm). There is a positive correlation between the effect of tPA deletion and that of vaccination (R2=0.67, P<0.001).

View larger version (16K): [in this window] [in a new window]   Fig. 5. Impairment of performance on object recognition in tPA-deficient mice is not mediated by the tPA/NR1 interaction. (A) In the novel-object-recognition test, WT mice exhibit good recognition performances as indicated by a strong preference towards the novel objects. By contrast, recognition memory is impaired in tPA-deficient mice, as indicated by the absence of preference towards the novel object. (B) In the same test, vaccination does not affect recognition memory. No differences were found among control, CFA-treated and NTD-NR1-vaccinated mice. *P<0.05.

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