Abstract
Abnormally high concentrations of extracellular glutamate in the brain may cause neuronal damage via excitotoxicity. Thus, tight regulation of glutamate release is critical to neuronal function and survival. Excitotoxicity is caused mainly by overactivation of the extrasynaptic NMDA receptor (NMDAR) and results in specific cellular changes, including calcium-induced activation of calpain proteases. Here, we report that presenilin-1 (PS1) null mouse cortical neuronal cultures have increased amounts of calpain-dependent spectrin breakdown products (SBDPs) compared with WT cultures. NMDAR antagonists blocked accumulation of SBDPs, suggesting abnormal activation of this receptor in PS1 null cultures. Importantly, an increase in SBDPs was detected in cultures of at least 7 days in vitro but not in younger cultures. Conditioned medium from PS1 null neuronal cultures at 8 days in vitro contained higher levels of glutamate than medium from WT cultures and stimulated production of SBDPs when added to WT cultures. Use of glutamate reuptake inhibitors indicated that accumulation of this neurotransmitter in the media of PS1 null cultures was due to increased rates of release. PS1 null neurons showed decreased cell surface expression and phosphorylation of the GluN2B subunit of NMDAR, indicating decreased amounts of extrasynaptic NMDAR in the absence of PS1. Inhibition of γ-secretase activity in WT neurons caused changes similar to those observed in PS1 null neurons. Together, these data indicate that the PS1/γ-secretase system regulates release of glutamate, tyrosine phosphorylation, and surface expression of GluN2B-containing NMDARs.
Highlights
Calpain Is Activated in PS1Ϫ/Ϫ Neurons Due to High Levels of Glutamate Release—Mouse cortical neuronal cultures at 7 days in vitro (DIV) prepared from PS1 null (PS1Ϫ/Ϫ) embryonic brains showed increased levels of cleaved spectrin breakdown products (SBDPs) compared with neuronal cultures from WT mice (Fig. 1A, lanes 1 and 4)
These products are known to be derived from the calpain-dependent cleavage of neuronal spectrin and are induced in response to NMDA receptor (NMDAR) activation by its agonists such as glutamate [7]
To examine whether the conditioned media of PS1Ϫ/Ϫ neuronal cultures contain factors that stimulate the NMDAR-dependent increase in SBDPs, the conditioned medium from PS1Ϫ/Ϫ cultures was used to replace the media of WT neuronal cultures
Summary
Results: Inhibition of presenilin-1/␥-secretase increases release of glutamate, stimulating NMDAR and calpain while decreasing phosphorylation and surface expression of neuronal GluN2B. Conclusion: Presenilin-1/␥-secretase regulates glutamate release, NMDAR-dependent calpain activity, and modification of GluN2B. Inhibition of ␥-secretase activity in WT neurons caused changes similar to those observed in PS1 null neurons Together, these data indicate that the PS1/␥-secretase system regulates release of glutamate, tyrosine phosphorylation, and surface expression of GluN2B-containing NMDARs. Presenilins (PSs) are the catalytic subunits of the ␥-secretase complexes that mediate production of amyloid- peptides, the precursors of amyloid depositions of Alzheimer disease brains. We report that PS1 null cortical neuronal cultures contained high amounts of SBDPs, indicating increased calpain activity in the absence of PS1, a defect rescued by NMDAR antagonists. The cell extract was incubated overnight with streptavidin-agarose beads, and bound proteins were detected as we described recently [7]
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