The γ-secretase has come a long way since its original discovery as the protease responsible for the production of amyloid-β peptides during sequential cleavage of the amyloid precursor protein. The unusual intramembrane cleavage catalyzed by the secretase turns out to be a popular regulatory maneuver for many cells, and the list of γ-secretase substrates continues to expand, encompassing a myriad of functions both in and outside of the nervous system. A report in the May 4 Journal of Cell Biology adds a new substrate to the tally, one with a critical role in synapse formation and plasticity. In the paper, Eiji Inoue and colleagues at the KAN Research Institute in Kobe, Japan (part of the Eisai pharmaceutical company), propose that γ-secretase processing of the receptor tyrosine kinase EphrinA4 (EphA4) in synapses, and release of its intracellular domain fragment, promotes dendritic spine formation. Two γ-secretase mutants that cause familial early onset AD show decreased activity for Eph4A processing. The results add support to the idea that γ-secretase mutations induce disease via a loss-of-function mechanism, and suggest that multiple substrates could mediate the mutants’ effects. In the case of EphA4, the FAD mutations might lead to lower dendritic spine density, and synapse loss.
Using mouse hippocampal neurons as a model system, Inoue and colleagues first determined by immunofluorescent staining that the catalytic subunit of γ-secretase, presenilin1 (PS1), resides in synapses. The enzyme seemed to have a hand in the formation and maintenance of synapses, since treatment of cells with a γ-secretase inhibitor reduced the clustering of AMPA-type glutamate receptors, a step in synapse maturation, and also reduced the density of dendritic spines, the structures that harbor synapses, by 20-25 percent. Similar results were seen when both presenilin genes (PS1 and PS2) were silenced by RNAi knockdown. The results are consistent with previous work showing a loss of synapses in PS1/PS2 conditional knockout mice (see ARF related news story and Saura et al., 2004).
To identify the γ-secretase substrate(s) responsible for the synaptic effects, the investigators purified the cholesterol-rich lipid raft membrane fraction from crude synaptic membranes, where PS1 has been found to be colocalized with other substrates. They used mass spec to identify 324 proteins in the fraction. The batch included EphA4, a transmembrane receptor and a likely substrate candidate. EphA4 bears a consensus cleavage site for γ-secretase, and it is a relative of the known γ-secretase substrate EphB (Georgakopoulos et al., 2006). Moreover, EphA4 signaling has been implicated in dendrite retraction (see Murai et al., 2003 and ARF related news story on Fu et al., 2007).
Like other secretase substrates, EphA4 has an extracellular domain that needs to be cleaved before γ-secretase can get at the transmembrane region. That cleavage is accomplished in the neurons by matrix metalloprotease, the authors show. After that, processing of the remaining C-terminal fragment depended on γ-secretase, as it was blocked by the secretase inhibitor or by PS1/2 knockdown. In vitro experiments showed that γ-secretase could release an intracellular domain fragment (ICD) of EphA4 from membrane preparations of cells overexpressing the receptor. In contrast to other γ-secretase substrates including Notch and EphB, EphA4 did not require ligand binding to trigger processing. Instead, processing appeared to be regulated by synaptic activity and glutamate receptor activation.
To look for a functional effect of the ICD in cells, the researchers overexpressed that fragment only. They found the ICD was sufficient to increase spine number, and that the γ-secretase inhibitor no longer affected spine density in the ICD-expressing cells. “These results suggest that the processing of EphA4 by γ-secretase has a critical role in the formation of dendritic spines, and that EphA4 is one of the critical substrates of γ-secretase that regulates the morphogenesis of dendritic spines,” the authors conclude. In further support of this idea, they show that Eph4A knockdown in cultured rat hippocampal primary neurons decreased spine number, with a strong effect on the mushroom spines containing mature synapses. The γ-secretase inhibitor had no effect on spine density in these cells.
How does Eph4A cleavage regulate spines? In NIH3T3 fibroblasts, overexpressing the EphA4 ICD activated the Rac signaling pathway and led to actin cytoskeleton reorganization and the formation of lamellipodia. In neurons, Rac regulates dendritic spine morphogenesis (Tashiro and Yuste, 2004), and the authors showed that expression of a dominant-negative Rac inhibited increased dendrite number in response to the EphA4 ICD in cultured neurons. The ICDs of several γ-secretase substrates have been shown to move to the nucleus and regulate gene expression, but neither nuclear localization nor the kinase activity of EphA4 was necessary for Rac activation or the increase in lamellipodia in NIH3T3 cells. The results suggest a complex dual role for EphA4 in synapse formation. Where previous work showed that binding of Ephrin protein ligands to EphA4 results in dendrite retraction, the current study suggests that ligand-independent processing can promote dendrite formation.
To look at the effect of AD PS1 mutants, the researchers measured EphA4 processing by γ-secretase in membrane preparations from PS1/2 knockout cells that were engineered to express normal PS1, a catalytically dead mutant (PS1D385A) or one of two AD mutants (M146L or E280A). The AD mutant proteins made much less ICD than the wild-type PS1, indicating that processing of EphA4 was impaired by FAD-linked mutations. More work is needed to see if ineffective processing as a result of FAD mutations plays a role in synapse loss in AD, or presents another complication to the use of γ-secretase inhibitors as potential treatments.—Pat McCaffrey
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