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Alzheimer′s disease (AD) is characterized by deposition of amyloid plaques, neurofibrillary tangles, and neuroinflammation. In order to study microglial contribution to amyloid plaque phagocytosis, we developed a novel ex vivo model by co‐culturing organotypic brain slices from up to 20‐month‐old, amyloid‐bearing AD mouse model (APPPS1) and young, neonatal wild‐type (WT) mice. Surprisingly, co‐culturing resulted in proliferation, recruitment, and clustering of old microglial cells around amyloid plaques and clearance of the plaque halo. Depletion of either old or young microglial cells prevented amyloid plaque clearance, indicating a synergistic effect of both populations. Exposing old microglial cells to conditioned media of young microglia or addition of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was sufficient to induce microglial proliferation and reduce amyloid plaque size. Our data suggest that microglial dysfunction in AD may be reversible and their phagocytic ability can be modulated to limit amyloid accumulation. This novel ex vivo model provides a valuable system for identification, screening, and testing of compounds aimed to therapeutically reinforce microglial phagocytosis. ![][1] Phagocytic function of aged microglial cells in amyloid plaque‐bearing tissue is not irreversibly impaired, but can be restored through factors secreted by young microglia. Microglia function in Aβ clearance and reducing the amyloid burden highlights the need for development of therapeutic approaches modulating microglial activity. [1]: /embed/graphic-1.gif

Numerous membrane-bound proteins undergo regulated intramembrane proteolysis. Regulated intramembrane proteolysis is initiated by shedding, and the remaining stubs are further processed by intramembrane-cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of β-secretase (β-site amyloid precursor protein-cleaving enzyme 1 (BACE1)). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Because of the hairpin nature of NRG1 type III, two membrane-bound stubs with a type 1 and a type 2 orientation are generated by proteolytic processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1-oriented stub is further cleaved by γ-secretase at an ϵ-like site five amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ϵ-cleavage site is only one amino acid N-terminal to a Val/Leu substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III β-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer disease-associated mutations within the amyloid precursor protein. The type 2-oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with 2,2'-(2-oxo-1,3-propanediyl)bis[(phenylmethoxy)carbonyl]-l-leucyl-l-leucinamide ketone inhibits formation of N-terminal intracellular domains and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate that is not only cleaved by multiple sheddases but is also processed by three different I-CLiPs.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid β-peptide (Aβ)1. Two principal physiological pathways either prevent or promote Aβ generation from its precursor (amyloid precursor protein; APP) in a competitive manner1. Modulation of the amyloidogenic pathway is currently exploited by anti-Aβ therapeutic strategies2. Although APP processing has been studied in great detail, unknown proteolytic events appear to hinder stoichiometric analyses of APP metabolism in vivo3. We now identified higher molecular weight C-terminal fragments of APP (CTF-η) in addition to the long-known α- and β-secretase (a disintegrin and metalloproteinase; ADAM10 and β-site APP cleaving enzyme 1; BACE1) generated CTF-α and CTF-β. Generation of CTF-η is mediated in part by membrane bound matrix-metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504/505 of APP(695) releasing a truncated, soluble APP ectodomain (sAPP-η). Upon shedding of sAPP-η CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (Aη-α and Aη-β). Aη peptides are therefore distinct from N-terminally extended Aβ variants4,5, since they do not extend to the γ-secretase cleavage sites. η-Secretase produced CTFs are enriched in dystrophic neurites in an AD mouse model and human AD brains6. Genetic and pharmacological inhibition of BACE1 activity results in a robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor hippocampal long-term potentiation (LTP) was reduced. Strikingly, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo, LTP was lowered. Furthermore, in vivo single cell two-photon calcium imaging revealed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major physiologically relevant APP processing pathway but may also suggest potential translational relevance for therapeutic strategies targeting APP processing.