Endocytosis and autophagy are known to be enhanced in some cases of neuronal death, and there is evidence that they can be directly involved in the death mechanism. We here report that both these trafficking events are enhanced by cerebral ischemia in neonatal (P12) rats, in which the main (cortical) branch of the middle cerebral artery was occluded by cauterization in association with clamping of the common carotid artery on the same side for 1.5 h. We also analyze the endocytic phenomenon pharmacologically in dissociated neuronal cultures exposed to NMDA. The enhanced endocytosis was shown in rats that received an intracerebroventricular injection of 4.4 kD FITC-dextran at the time of carotid unclamping, and were sacrificed 24 h later. There was strong neuronal endocytosis restricted to the ischemic zone, which was specifically labelled by immunocytochemistry for early endosomal antigen 1 (EEA1). In dissociated neuronal cultures a similar NMDA-induced endocytosis of FITC-dextran was observed; it increased with the dose of NMDA and the duration of NMDA treatment, and with a toxic dose the strongest endocytosis was observed microscopically to be in neurons stained with propidium iodide (a marker for dying or dead cells). However, to study specifically the endocytotic mechanism without the complication of cell death, we determined a combination of NMDA concentration and timing that enhanced endocytosis but with minimal cell death, as judged by LDH release, and measured the endocytosis in cellular extracts by fluorescence spectrometry. This revealed two components of dextran internalisation: they were both confirmed to be endocytic by the fact that they did not occur at 4°C, but they had completely different pharmacological profiles. One occurred constitutively (even in the absence of NMDA) and was blocked by inhibitors of classical fluid-phase dynamin-independent endocytosis (e.g. by wortmannin). The other component was induced by NMDA, insensitive to fluid-phase inhibitors, but sensitive to inhibitors of dynamin-mediated endocytosis such as 0.4 M sucrose and also to the JNK pathway inhibitor D-JNKI1, a powerful neuroprotectant. The latter inhibitors had little effect on the constitutive component of endocytosis. Although most dynamin-mediated endocytosis is receptor-mediated, competition experiments showed that the NMDA-induced endocytosis did not depend on a receptor specific for dextran. Enhanced lysosomal activity, presumably autophagy, was shown in neurones within the ischemic region (again at 24 h after carotid unclamping) by acid phosphatase histochemistry and by immunohistochemistry for lysosome-associated membrane protein (LAMP)-1. Cathepsin D was also increased in the ischemic region, but appeared to be in glia, not in neurons. The importance of these results is twofold.
They suggest that a specific JNK-dependent endocytic pathway and an autophagic pathway may be targets for neuroprotection in cerebral ischemia and neonatal asphyxia. The induced endocytosis may provide a means for delivering neuroprotective agents specifically into the cells that need them.
