The two presenilin‐1 (PS1) and presenilin‐2 (PS2) homologs are the catalytic core of the γ‐secretase complex, which has a major role in cell fate decision and Alzheimer's disease (AD) progression. Understanding the precise contribution of PS1‐ and PS2‐dependent γ‐secretases to the production of β‐amyloid peptide (Aβ) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Aβ release without affecting the processing of other γ‐secretase substrates. To that end, we studied PS1‐ and PS2‐dependent substrate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1‐PS2 double‐KO noted PSdKO) or stably re‐expressing human PS1 or PS2 in an endogenous PS‐null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L‐685,458). We found that murine PS1 γ‐secretase plays a predominant role in APP and Notch processing when compared to murine PS2 γ‐secretase. The inhibitors blocked more efficiently murine PS2‐ than murine PS1‐dependent processing. Human PSs, especially human PS1, expression in a PS‐null background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1‐ than human PS2‐dependent γ‐secretase activity.

The Amyloid Precursor Protein (APP) has been extensively studied as the precursor of the βamyloid peptide (Aβ) peptide, the major component of the senile plaques found in the brain of Alzheimer's disease (AD) patients. However, the function of APP per se in neuronal physiology remains to be fully elucidated. APP is expressed at high levels in the brain. It resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell-cell interaction and/or activation of intracellular signaling pathways. In this respect, the APP intracellular domain (AICD) was reported to act as a transcriptional regulator. Here, we used a transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and upon maturation of primary cortical neurons. Surprisingly, the overall transcriptional changes were subtle, but a more detailed analysis pointed to genes clustered in neuronal-activity dependent pathways. In particular, we observed a decreased transcription of Neuronal PAS domain protein 4 (NPAS4) in APP-/-neurons. NPAS4 is an inducible transcription factor (ITF) regulated by neuronal depolarization. The down-regulation of NPAS4 co-occurs with an increased production of the inhibitory neurotransmitter GABA and a reduced expression of the GABA A receptors alpha1. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Patch-clamp investigation did not reveal any functional decrease of GABA A receptors activity, but LTP measurement supported an increased GABA component in synaptic transmission of APP-/-mice. Together, NPAS4 appears to be a downstream target involved in APP-dependent regulation of inhibitory synaptic transmission.

Summary Amyloid precursor protein (APP) cleavage by the β-secretase produces the C99 transmembrane (TM) protein, which contains three dimerization-inducing Gly-x-x-x-Gly motifs. We demonstrate that dimeric C99 TM orientations regulate the precise cleavage lines by γ-secretase. Of all possible dimeric orientations imposed by a coiled-coil to the C99 TM domain, the dimer containing the 33 Gly-x-x-x-Gly 37 motif in the interface promoted the Aβ 42 processing line and APP intracellular domain-dependent gene transcription, including the induction of BACE1 mRNA, enhancing amyloidogenic processing and signaling. Another orientation exhibiting the 25 Gly-x-x-x-Gly 29 motif in the interface favored processing to Aβ 43/40 . It induced significantly less gene transcription, while promoting formation of SDS-resistant “Aβ-like” oligomers, reminiscent of Aβ peptide oligomers. These required both Val24 of a pro-β motif and the 25 Gly-x-x-x-Gly 29 interface. Thus, crossing angles imposed by precise dimeric orientations control γ-secretase initial cleavage at Aβ 48 or Aβ 49, linking the former to enhanced signaling and Aβ 42 production.

A key hallmark of Alzheimer’s disease is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-β (Aβ) peptide. The Aβ peptide is a product of sequential cleavage of the Amyloid Precursor Protein, the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by γ-secretase activity releases Aβ of several lengths and the Aβ42 isoform in particular has been identified as being neurotoxic. The misfolding of Aβ leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric Aβ42 and show its assembly enhancing properties which are dependent on the Aβ monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers.

A key hallmark of Alzheimer’s disease (AD) is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-β (Aβ) peptide. The Aβ peptide is a product of sequential cleavage of the Amyloid Precursor Protein (APP), the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by γ-secretase activity releases Aβ of several lengths and the Aβ42 isoform in particular has been identified as being neurotoxic. The misfolding of Aβ leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric Aβ42 and show its nucleating properties which are dependent on the Aβ monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers.