Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsa knock-in (KI) mice expressing solely the secreted ectodomain APPsa. Here, we generated double mutants (APPsa-DM) by crossing APPsa-KI mice onto an APLP2-deficient background and show that APPsa rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsa-DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsa-DM muscle showed fragmented postsynaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsa-DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABA A receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.

Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsa knock-in (KI) mice expressing solely the secreted ectodomain APPsa. Here, we generated double mutants (APPsa-DM) by crossing APPsa-KI mice onto an APLP2-deficient background and show that APPsa rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsa-DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsa-DM muscle showed fragmented postsynaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsa-DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABA A receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.

Understanding the nano-architecture of protein machines in diverse subcellular compartments remains a challenge despite rapid progress in super-resolution microscopy. While single-molecule localization microscopy techniques allow the visualization and identification of cellular structures with near-molecular resolution, multiplex-labeling of tens of target proteins within the same sample has not yet been achieved routinely. However, single sample multiplexing is essential to detect patterns that threaten to get lost in multi-sample averaging. Here, we report maS 3 TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach capable of fully automated 3D direct STORM (dSTORM) imaging and solution exchange employing a re-staining protocol to achieve highly multiplexed protein localization within individual biological samples. We demonstrate 3D super-resolution images of 15 targets in single cultured cells and 16 targets in individual neuronal tissue samples with <10 nm localization precision, allowing us to define distinct nano-architectural features of protein distribution within the presynaptic nerve terminal.

The amyloid precursor protein family (APP/APLPs) has essential roles for neuromuscular synapse development and for the formation and plasticity of synapses within the CNS. Despite this, it has remained unclear whether APP mediates its functions primarily as a cell surface adhesion and signaling molecule or via its numerous proteolytic cleavage products. To address these questions, we followed a genetic approach and used APP⌬CT15 knockin mice lacking the last 15 amino acids of APP, including the highly conserved YENPTY protein interaction motif. To circumvent functional compensation by the closely related APLP2, these mice were bred to an APLP2-KO background to generate APP⌬CT15-DM double mutants. These APP⌬CT15-DM mice were partially viable and displayed defects in neuromuscular synapse morphology and function with impairments in the ability to sustain transmitter release that resulted in muscular weakness. In the CNS, we demonstrate pronounced synaptic deficits including impairments in LTP that were associated with deficits in spatial learning and memory. Thus, the APP-CT15 domain provides essential physiological functions, likely via recruitment of specific interactors. Together with the well-established role of APPs␣ for synaptic plasticity, this shows that multiple domains of APP, including the conserved C-terminus, mediate signals required for normal PNS and CNS physiology. In addition, we demonstrate that lack of the APP-CT15 domain strongly impairs A␤ generation in vivo, establishing the APP C-terminus as a target for A␤-lowering strategies.

No abstract