Journal: bioRxiv

Article Title: Spectrum of γ-Secretase dysfunction as a unifying predictor of ADAD age at onset across PSEN1 , PSEN2 and APP causal genes

doi: 10.1101/2024.11.11.622936

Figure Lengend Snippet: A. Schematic representation of PSEN1 and PSEN2 structural heterogeneity. The colour gradient displays local homology between PSEN1 and PSEN2 (based on NCBI’s Basic Local Alignment Search Tool (BLAST) using BLOSUM 62 matrix). B. GSEC-APPC83 co-structure (PDB: 6IYC). PSEN forms the catalytic subunit, while Nicastrin, PEN2, and APH1A/B are essential subunits of the GSEC complex. C. Schematic representation of PSEN1- and PSEN2-type GSEC complex subcellular localizations. PSEN1-type GSEC complexes (green) are broadly distributed, while PSEN2-type GSECs are restricted to late endosomes. Image created with BioRender. D. APP cleavage by BACE1 generates APPC99, the direct GSEC substrate. The initial GSEC-mediated cut (endopeptidase activity) releases AICD50-99 or AICD 49-99 and generates longer Aβ fragments (Aβ48 or Aβ49). These fragments undergo sequential γ-cleavages to produce Aβ peptides of various lengths. Pathogenic mutations destabilize the GSEC-APP/Aβ complex, reducing sequential cleavage efficiency (processivity) and increasing release of longer, more toxic Aβ peptides. E. Age at symptom onset (AAO) associated with mutations in PSEN1, PSEN2, and APP genes. PSEN1 harbours most ADAD mutations with broadly distributed AAOs (23-75y). PSEN2 and APP mutations are associated with later onsets (33-64y and 39-87y, respectively). Box plots show the median (centre line) and 25-75 percentiles. Dots represent individual mutations plotted as averaged mean ± SD. Data sourced from Alzforum database ( www.alzforum.org/mutations ) and literature (see Supplementary Tables S1 and S2 ).

Article Snippet: The following antibodies were used in western blot analyses: mouse anti-NCT (9C3) kindly provided by Prof. Wim Annaert; anti-human PSEN1-CTF (MAB5232) purchased from Merck Millipore; anti-human PSEN2-CTF (ab51249) purchased from Abcam and anti-human PEN2 (DGG8) purchased from Cell Signaling.

Techniques: Activity Assay

Journal: G3: Genes|Genomes|Genetics

Article Title: The human RAP1 and GFAPɛ proteins increase γ-secretase activity in a yeast model system

doi: 10.1093/g3journal/jkad057

Figure Lengend Snippet: RAP1 and GFAPɛ colocalize with subunits of the γ-secretase complex. SH-SY5Y cells were grown, fixed, and probed with antibodies as indicated in each panel. Representative images are shown that include images of each fluorophore individually with a larger, merged image. The box in the merged images indicates the region that is increased in size for the inset at the top right corner. Arrows in the inset indicate examples of colocalization. a) RAP1 and GFAPɛ colocalize. Cells were probed with DAPI, antibodies to RAP1 and antibodies to GFAPɛ. b) RAP1 colocalizes with the γ-secretase subunit PSENEN. Cells were probed with DAPI, antibodies to RAP1, and antibodies to PSENEN. c) RAP1 and GFAPɛ colocalize with the γ-secretase subunit PS1. DAPI stain was not used in this image. Cells were probed with antibodies to PS1, antibodies to RAP1 and antibodies to GFAPɛ.

Article Snippet: The samples were first incubated with primary antibodies to GFAPɛ (described above) or PSENEN (rabbit polyclonal anti-PEN2 antibodies, Invitrogen, PA5-20302) in blocking solution.

Techniques: Staining