Additional file 1 of Accumulation of saposin in dystrophic neurites is linked to impaired lysosomal functions in Alzheimer’s disease brains
Creators: name: Sharoar, Md Golam
name: Palko, Sarah
name: Ge, Yingying
name: Saido, Takaomi C.
name: Yan, Riqiang
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: 29999 Physical Sciences not elsewhere classified
subject: FOS: Physical sciences
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Developmental Biology
subject: 111714 Mental Health
subject: FOS: Health sciences

description: Additional file 1: Supplemental Fig. 1. Absence of cathepsins and endosomal proteins in DNs. (A) The fixed brain sections from a 6-month-old APPNL-G-F mouse were co-labelled either with LAMP1 and cathepsin B or cathepsin D. (B) The fixed brain sections from a 6-month-old APPNL-G-F mouse were co-labelled with 6E10 and EEA1/ RAB11 or Aβ42 and RAB4A/ RAB6A antibodies. (C) AD postmortem brain sections were either triple stained with Thio-S, SAP-C and RTN3 antibodies. Cathepsins or endosomal proteins were not appeared in DNs in APPNL-G-F mouse brains. Supplemental Fig. 2. LAMP1, SAPs, ATG9A and RTN3 are differentially enriched in different populations of DNs in various ages of AD mouse brains. (A) Fixed brain sections from 1.5-, 3, 6-, 9- and 15-month (m)-old APPNL-G-F mice were co-stained either with LAMP1 and SAPs or LAMP1 and RTN3. SAPs- and RTN3-marked DNs appeared in similar time point, but LAMP1-marked DNs appeared early during plaque growth. (B) The fixed brain sections from 3- and 15-m-old APPNL-G-F, 2.5- and 10-m-old 5xFAD and 6- and 16-m-old PA mice were triple-stained with LAMP1, ATG9A and RTN3. (C) 15-m-old APPNL-G-F mouse brain sections were co-stained with LAMP1 and cathepsin B or cathepsin D. LAMP1+-DNs is reduced in older AD mouse brains and it mostly labeled center of the plaque, which was colocalized with cathepsins. Supplemental Fig. 3. Disruption of plaque surrounding axonal network in older APPNL-G-F mouse brains. The Fixed brain samples from 1.5- and 15-month (m)-old APPNL-G-F mice were co-stained with LAMP1 and SMI31 antibodies. SMI31 labeled axons were interact and LAMP1 labelled lysosomes were visible (indicated by white arrowhead) in axons at younger age when plaque formation just started. The axonal integrity was destroyed, and lysosomes were not visible in plaque surrounding area at older age. Supplemental Fig. 4. LAMP1 is mostly localized in microglia in older AD mouse brains. (A) The brain sections from 2.5-, 4 and 10-month (m)- old 5xFAD mice were co-labelled with LAMP1 and IBA1 or SAMI31. (B) The fixed brain section from PA mice at 6 and 16 months were co-stained with LAMP1 and IBA1 antibodies. (C) The fixed brain samples from a 10-m-old 5xFAD, a 16-m-old PA and a 9-m-old APPNL-G-F mouse were triple-stained with LAMP1, ATG9A and IBA1. (D) Fixed brain section of a 9-m-old APPNL-G-F mouse were triple-stained with cathepsin-D, LAMP1 and IBA1. LAMP1 mostly colocalized with IBA1 and cathepsin D in older AD mouse brains. Supplemental Fig. 5. Changes of proteins levels in AD mouse brains. (A) The protein level of LAMP1, PSAP, SAP-C, SAPs, ATG9A and calnexin were detected in the cortex of each age group (m, month) of APPNL-G-F mice using Western blotting. (B) The band intensity for each protein was measured using Fiji software and the average of each age group was calculated after normalizing with calnexin. (C) The protein level of LAMP1, PSAP, SAP-C, SAPs, ATG9A and calnexin were detected in the cortex of each age group of 5xFAD mice. (D) The band intensity for each protein was measured and normalized with calnexin. * indicate a significant difference at P ≤ 0.05. The level of LAMP1, SAPs and SAP-C were increased during plaque growth in AD mouse brains. Supplemental Fig. 6. Increased level of SAP-C and galectin-3 in 5xFAD lysosomes. Variable sized organelles were enriched in mitochondria (MF)- and lysosomal (LF)- fractions by sequential centrifugation. (A) MF and LF obtained from 5- month-old WT and 5xFAD mouse brains were subjected to Western blot analysis. (B) Band intensity for each protein was measured and standardized to GAPDH. * indicate significant differences at P