Super-resolution microscopy reveals majorly mono- and dimeric presenilin1/γ-secretase at the cell surface

Abstract

<jats:p>γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer’s disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting ‘hotspots’ or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.</jats:p>

Alteration in synaptic nanoscale organization dictates amyloidogenic processing in Alzheimer's disease

“…Recent evidences indicate that APP and γ-secretase molecules are transiently immobilized on the plasma membrane with the existence of an equilibrium between the nanodomain and extra-nanodomain APP and secretase molecules ( Escamilla-Ayala et al., 2020 ; Kedia et al., 2020 ). The existence of APP nanodomains in neuronal and non-neuronal cells have been confirmed ( de Coninck et al., 2018 ; Kedia et al., 2020 ).…”

“…The existence of APP nanodomains in neuronal and non-neuronal cells have been confirmed ( de Coninck et al., 2018 ; Kedia et al., 2020 ). However, the segregation of γ-secretase into nanodomains in non-neuronal cells is still a matter of debate ( Escamilla-Ayala et al., 2020 ; Liu et al., 2019a ). The results presented here are consistent with previous reports of γ-secretase segregated into nanodomains in the synaptic compartments of neurons ( Schedin-Weiss et al., 2016 ).…”

“…The selective interactions with these molecules could alter the localization and availability of APP and secretases involved in both amyloidogenic and non-amyloidogenic pathways. It is also known that association of APP among themselves and its family of molecules can influence the generation of Ab (Eggert et al, 2018;Gorman et al, 2008;Khalifa et al, 2010). In addition to the foresaid variables, post-translational modifications of APP and secretase as well as its association with lipid rafts is known to interfere with the rate of formation of different proteoforms (Grimm et al, 2017;Hicks et al, 2012;Rajendran and Annaert, 2012).…”

“…Although AD is considered to begin as a synaptopathy, it is not yet understood how the segregation of APP and secretases at nanoscale contributes toward the progression of AD ( De Strooper and Karran, 2016 ; Haass et al., 2012 ; Selkoe et al., 2012 ; Selkoe, 2002 ). This is largely due to the lack of information on (1) the heterogeneity of localization of the amyloidogenic machinery within/outside functional zones of individual synapses ( Harris and Stevens, 1989 ; Harris and Weinberg, 2012 ) and (2) a lack of intuitive understanding of the mechanisms that control the heterogeneity of diffusional collisions between APP and secretases resulting in different proteoforms ( Ben Halima et al., 2016 ; Escamilla-Ayala et al., 2020 ; Kedia et al., 2020 ). Here, we have employed super-resolution imaging and analysis to reveal the subsynaptic organization of these molecules.…”

Tissue Clearing and Expansion Methods for Imaging Brain Pathology in Neurodegeneration: From Circuits to Synapses and Beyond

“…STORM and STED SRM have also been used to visualize the presenilin-1/γ-secretase complex, which is responsible for the proteolytic cleavage of the amyloid precursor protein (APP) that releases Aβ, in both the pre- and post-synaptic compartments of cultured mouse neurons ( Schedin-Weiss et al, 2016 ). More recently, SIM and PALM were used to study the subunit composition and activity of the γ-secretase complex at the plasma membrane in living cells, revealing complex dynamics involving association with specific substrates ( Escamilla-Ayala et al, 2020 ).…”