Copper and zinc ions govern the trans-directed dimerization of APP family members in multiple ways.

The amyloid precursor protein (APP) and its homologs amyloid precursor-like protein 1 (APLP1) and APLP2 have central physiological functions in transcellular adhesion that depend on copper and zinc mediated trans-directed dimerization of the extracellular domains E1 and E2. Copper binds to three distinct sites in APP, one in the copper binding (CuBD) and growth factor-like (GFLD) domains each within E1, and one in the E2 domain. For APLP1 and APLP2, metal binding has so far only been shown for the E2 domain. Zinc binding has been reported for all APP family members to a unique site in the E2 domain and an additional site essential for APLP1 E2 domain trans-dimerization. Using isothermal titration calorimetry, co-immunoprecipitation, and in vitro bead aggregation assays, we show that copper promotes cis- as well as trans-directed dimerization of APLP1 and APLP2, similar as reported previously for APP. Furthermore, we report a APP-specific zinc binding site with nanomolar affinity located in the E1 domain, whereas no binding of zinc to the individual subdomains GFLD or CuBD was detected. Zinc binding did not affect the cis- but trans-dimerization of APP and APLP1. Furthermore, zinc binding inhibited copper-induced trans-directed dimerization of APP. Together, we identified a high-affinity APP-specific zinc binding site in the E1 domain and revealed contrasting cis- and trans-directed dimerization properties of APP, APLP1, and APLP2 in dependence on zinc and copper ions. Consequently, changes in metal ion homeostasis, as reported in the context of synaptic activity and neurodegenerative diseases, appear as key modulators of homo- and heterotypic trans-cellular APP/APLPs complexes.

Modulation of BAG3 Expression and Proteasomal Activity by sAPPα Does Not Require Membrane-Tethered Holo-APP.

Maintenance of intracellular proteostasis is essential for neuronal function, and emerging data support the view that disturbed proteostasis plays an important role in brain aging and the pathogenesis of age-related neurodegenerative disorders such as Alzheimer's disease (AD). sAPPalpha (sAPPα), the extracellularly secreted N-terminal alpha secretase cleavage product of the amyloid precursor protein (APP), has an established function in neuroprotection. Recently, we provided evidence that membrane-bound holo-APP functionally cooperates with sAPPα to mediate neuroprotection via activation of the Akt survival signaling pathway and sAPPα directly affects proteostasis. Here, we demonstrate that in addition to its anti-apoptotic function, sAPPα has effects on neuronal proteostasis under conditions of proteasomal stress. In particular, recombinant sAPPα significantly suppressed MG132-triggered expression of the co-chaperone BAG3 and aggresome formation, and it partially rescued proteasomal activity in a dose-dependent manner in SH-SY5Y neuroblastoma cells. In analogy, sAPPα was able to inhibit MG132-induced BAG3 expression in primary hippocampal neurons. Strikingly, these sAPPα-induced changes were unaltered in APP-depleted SH-SY5Y cells and APP-deficient neurons, demonstrating that holo-APP is not required for this particular function of sAPPα. Importantly, recombinant sAPPbeta (sAPPß) failed to modulate BAG3 expression and proteostasis in APP-proficient wild-type (wt) cells, indicating that these biological effects are highly selective for sAPPα. In conclusion, we demonstrate that modulation of proteostasis is a distinct biological function of sAPPα and does not require surface-bound holo-APP. Our data shed new light on the physiological functions of APP and the interplay between APP processing and proteostasis during brain aging.

Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain.

Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function.

Differential role of APP and APLPs for neuromuscular synaptic morphology and function.

The analysis of mouse models indicated that APP and the related APLPs are important for synapse formation and function. The synaptic role of APP is, however, complex due to partially overlapping functions within the gene family. APP/APLPs are proteolytically cleaved and have both adhesive and signaling properties. Mice lacking individual APP family members are viable, whereas APP/APLP2 and APLP1/APLP2 double knockout (DKO) mice die shortly after birth. Here, we analyzed the morphology of the neuromuscular junction (NMJ) of lethal APLP1/APLP2-DKO mice in comparison to lethal APP/APLP2-DKO mutants and viable single KO mice. We report that, surprisingly, the NMJ phenotype of APLP1/APLP2-DKO mice shows striking differences as compared to APP/APLP2-DKO mice. Unexpectedly, APLP1/APLP2-DKO mice exhibit normal endplate patterning and lack presynaptic nerve terminal sprouting. However, at the level of individual synapses we show that APLP1/APLP2-DKO mice exhibit reduced size of pre- and postsynaptic compartments and reduced colocalization. As APP/APLP2-DKO and APLP1/APLP2-DKO mice show similar penetrance of early postnatal lethality, this suggests that deficits at the level of individual synapses due to impaired synaptic apposition and/or deficits in transmitter release may cause lethality. Using an in vitro cell-adhesion assay, we observed that APP trans-dimerization is considerably less efficient than APLP2 trans-interaction. Thus, differences between APP/APLP2 and APP/APLP1 NMJ formation may be in part explained by differences in APP/APLP2 trans-dimerization properties. Collectively, our study further highlights the distinct and essential role of APLP2 at NMJ synapses that cannot be compensated by APP.

Structural aspects and physiological consequences of APP/APLP trans-dimerization.

The amyloid precursor protein (APP) is one of the key proteins in Alzheimer's disease (AD), as it is the precursor of amyloid ß (Aß) peptides accumulating in amyloid plaques. The processing of APP and the pathogenic features of especially Aß oligomers have been analyzed in detail. Remarkably, there is accumulating evidence from cell biological and structural studies suggesting that APP and its mammalian homologs, the amyloid precursor-like proteins (APLP1 and APLP2), participate under physiological conditions via trans-cellular dimerization in synaptogenesis. This offers the possibility that loss of synapses in AD might be partially explained by dysfunction of APP/APLPs cell adhesion properties. In this review, structural characteristics of APP trans-cellular interaction will be placed critically in context with its putative physiological functions focusing on cell adhesion and synaptogenesis.