Although activation of calcium-activated neutral protease (calpain) by the NMDA receptor has been suggested to play critical roles in synaptic modulation and neurologic disease, the nature of its substrates has not been completely defined. In this study, we examined the ability of calpain to cleave the NMDA receptor in cultured hippocampal neurons. Activation of the NMDA receptor by agonist application led to rapid calpain-specific proteolysis of spectrin and decreased levels of NR2A/2B subunits. Cleavage of the NR2A/2B subunit created a 115 kDa product that retained the ability to bind 125I-MK-801 and is predicted to be active. Increases in levels of this product appeared within 5 min of NMDA receptor activation and were stable for periods of >30 min. Subtype-specific antibodies demonstrated that the NR2B subunit was cleaved in these primary cultures, but the NR2A subunit was not. An inhibitor of calpain blocked both the decrease of intact NR2B and the increase of the low molecular weight form, whereas neither caspase nor cathepsin inhibitors had an effect on these events. Cell surface biotinylation experiments demonstrated that the 115 kDa fragment remained on the cell surface. This NR2B fragment was also found in the rat hippocampus after transient forebrain ischemia, showing that this process also occurs in vivo. This suggests that calpain-mediated cleavage of the NR2B subunit occurs in neurons and gives rise to active NMDA receptor forms present on the cell surface after excitotoxic glutamatergic stimulation. Such forms could contribute to excitotoxicity and synaptic remodeling.

The perforant pathway projection from layer II of the entorhinal cortex to the hippocampal dentate gyrus is especially important for long-term memory formation, and is preferentially vulnerable to developing a degenerative tauopathy early in Alzheimer’s disease (AD) that may spread over time trans-synaptically. Despite the importance of the perforant pathway to the clinical onset and progression of AD, a therapeutic has not been identified yet that protects it from tau-mediated toxicity. Here, we used an adeno-associated viral vector-based mouse model of early-stage AD-type tauopathy to investigate effects of the mTOR inhibitor and autophagy stimulator rapamycin on the tau-driven loss of perforant pathway neurons and synapses. Focal expression of human tau carrying a P301L mutation but not eGFP as a control in layer II of the lateral entorhinal cortex triggered rapid degeneration of these neurons, loss of lateral perforant pathway synapses in the dentate gyrus outer molecular layer, and activation of neuroinflammatory microglia and astroglia in the two locations. Chronic systemic rapamycin treatment partially inhibited phosphorylation of a mechanistic target of rapamycin substrate in brain and stimulated LC3 cleavage, a marker of autophagic flux. Compared with vehicle-treated controls, rapamycin protected against the tau-induced neuronal loss, synaptotoxicity, reactive microgliosis and astrogliosis, and activation of innate neuroimmunity. It did not alter human tau mRNA or total protein levels. Finally, rapamycin inhibited trans-synaptic transfer of human tau expression to the dentate granule neuron targets for the perforant pathway, likely by preventing the synaptic spread of the AAV vector in response to pathway degeneration. These results identify systemic rapamycin as a treatment that protects the entorhinal cortex and perforant pathway projection from tau-mediated neurodegeneration, axonal and synapse loss, and neuroinflammatory reactive gliosis. The findings support the potential for slowing the progression of AD by abrogating tau-mediated neurotoxicity at its earliest neuropathological stages.

N-Methyl-D-aspartate (NMDA) receptors are calcium-permeable glutamate receptors that play putative roles in learning, memory, and excitotoxicity. NMDA receptor-mediated calcium entry can activate the calcium-dependent protease calpain, leading to substrate degradation. The major NMDA receptor 2 (NR2) subunits of the receptor are in vitro substrates for calpain at selected sites in the C-terminal region. In the present study, we assessed the ability of calpain-mediated proteolysis to modulate the NR1a/2A subtype in a heterologous expression system. Human embryonic kidney (HEK293t) cells, which endogenously express calpain, were cotransfected with NR1a/2A in addition to the calpain inhibitor calpastatin or empty vector as control. Receptor activation by glutamate and glycine as co-agonists led to calpain activation as measured by succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-aminomethyl coumarin (Suc-LLVY-AMC). Calpain activation also resulted in the degradation of NR2A and decreased binding of 125 I-MK-801 ( 125 I-dizocilpine) to NR1a/2A receptors. No stable N-terminal fragment of the NMDA receptor was formed after calpain activation, suggesting calpain regulation of NMDA receptor levels in ways distinct from that previously observed with in vitro cleavage. NR2 subunit constructs lacking the final 420 amino acids were not degraded by calpain. Agonist-stimulated NR1a/2A-transfected cells also had decreased calcium uptake and produced lower changes in agonist-stimulated intracellular calcium compared with cells cotransfected with calpastatin. Calpastatin had no effect on either calcium uptake or intracellular calcium levels when the NR2A subunit lacked the final 420 amino acids. These studies demonstrate that NR2A is a substrate for calpain in situ and that this proteolytic event can modulate NMDA receptor levels.