PA-058
IN VIVO MICRODIALYSIS EXPERIMENTS UNVEILED TAUOPATHYASSOCIATED PROTEOMIC CHANGES IN BRAIN EXTRACELLULAR FLUIDS
Itaru Nishida1 , Kaoru Yamada1 , Asami Sakamoto1, Sari Hirata2, Takeshi Iwatsubo11)
Department of Neuropathology, Graduate school of Medicine, the University of Tokyo, Tokyo, JAPAN2)
College of Arts and Sciences (Junior Division), the University of Tokyo, Tokyo, JAPAN【Introduction】
The brain is supported by two types of extracellular fluids: interstitial fluid (ISF) and cerebrospinal fluid (CSF). Throughout the progression of neurodegenerative
diseases such as tauopathies, various disease-associated alterations manifest in these brain extracellular fluids. ISF serves as the primary compartment for receiving
secretions from brain cells. Therefore, ISF is expected to reflect brain changes such as tau aggregation or other cellular responses most accurately.
To gain comprehensive understanding of the specific proteomic alterations that occur in ISF, we utilized in vivo microdialysis and analyzed how tau
seeding activity and the proteome in ISF alter with age or during the progression of tau pathology.

【Methods & Results】
Figure1. Microdialysis with 3MDa Cut-Off Probes Enhanced Recovery of High Molecular Weight Proteins and Seed-Competent Tau in vitro.
0.5% BSA/aCSF
Push pump

Roller pump

ADialysate

BCSize Exclusion Chromatography
1MDa probe

Dialysate

3MDa probe

Tau Seeding Assay
1MDa probe

3MDa probe

0.1 µL/min
microdialysis probe

3000 kDa (3MDa) cut-off
or1000 kDa (1MDa) cut-off
blue-dextran (2000 kDa) & conalbumin (75 kDa) (A, B)
100 µg/mL Recombinant tauRD (P301S) fibrils (C)1MDaprobein vitro microdialysis

Green: FRET
Blue: DAPI

3MDaprobe

A. A representative picture of dialysate collected through 1MDa and 3MDa probes.
B. Size-exclusion chromatography profile of in vitro dialysates.
C. In vitro dialysates were applied with Lipofectamine 3000 to tau biosensor cells and images were obtained at 72 hours from transduction.

Figure 2. High Molecular Weight Proteins (>1MDa) and Seed-Competent Tau in ISF Were Only Recovered with 3MDa Cut-Off Probes.

A Tau Seeding AssayB

LC-MS/MS Analysis

microdialysis probe
PS19 mouse (3, 9, 12 m.o.)in vivo microdialysis
A. ISF samples from PS19 at different ages were applied with Lipofectamine 3000 to tau biosensor cells.
B. ISF samples from PS19 at different ages were digested by trypsin and analyzed by LC-MS/MS for label free quantification. The raw data were searched against in silico predicted spectral
library using DIA-NN. (version:1.8.1, https://github.com/vdemichev/DiaNN). The peptide abundance of representative proteins in each molecular weight range was quantified and compared(n=3).

Figure 3. Age- or Tau Pathology-Dependent Proteomic Alterations in ISF by LC-MS/MS AnalysisA189 proteinsin 1891 proteinsdecrease in 12 m.o.

41 proteinsin 436 proteins
increase in 12 m.o.

B

Gene Ontology Analysis (Cellular Process)downregulate from 3 m.o. to 12 m.o.
(PS19)

synapse organization
cell junction organization
synaptic signalingchemical synaptic transmission
anterograde trans-synaptic signalingtrans-synaptic signaling
modulation of chemical synaptic transmission
regulation of trans-synaptic signaling
regulation of cellular component organization
postsynapse organization

PS19 ISF
3 m.o. vs 12 m.o.

171 proteinsin 2081 proteinsdecrease in 12 m.o.

Wildtype ISF
3 m.o. vs 12 m.o.

46 proteinsin 816 proteins
increase in 12 m.o.

antimicrobial humoral immune response
mediated by antimicrobial peptide
defense response to Gram-positive bacterium
antibacterial humoral response
innate immune response in mucosa
antimicrobial humoral response
mucosal immune responseorgan or tissue specific immune response
response to bacterium
defense response to bacterium
response to other organism

downregulate from 3 m.o. to 12 m.o.
(Wildtype)mRNA metabolic process
RNA processing
mRNA processing
RNA splicingregulation of mRNA metabolic process
cellular nitrogen compound metabolic process
cellular metabolic processregulation of mRNA processing
nitrogen compound metabolic process
heterocycle metabolic process

upregulate from 3 m.o. to 12 m.o.
(PS19)

upregulate from 3 m.o. to 12 m.o.
(Wildtype)positive regulation of receptor internalization
immune response
cell surface receptor signaling pathway
immune system process
positive regulation of receptor-mediated endocytosis
vesicle-mediated transport
zymogen activation
regulation of receptor internalization
receptor clustering
establishment of localizationA. Volcano plots showing increased (red) and decreased (blue) proteins in ISF of 3-momnth-old vs 12-month-old PS19 or wildtype mice. (n=3)
B. Gene Ontology enrichment analysis were performed using the geneXplain platform on the proteins with significant differences.

【Summary & Conclusions】
・We established a new microdialysis method that enhances the recovery of high molecular weight proteins and successfully identified more than 2,000 proteins in ISF.
・Tau seeding activity was increased with age while total tau levels were decreased in ISF of PS19 mice.
・Proteins related to synaptic signaling were downregulated, while those related to immune responses were upregulated with age in PS19 mice.
・Proteins related to RNA metabolism were downregulated, while those related to receptor internalization were upregulated with age in wildtype mice.

This study revealed dynamic proteomic changes in ISF and suggests that ISF reflect distinct protein profiles that alter with age and tau pathology inbrain.