# Synthetic Reconstruction of Human Chaperone Networks in Yeast Models of Neurodegeneration

> **NIH NIH K99** · UNIVERSITY OF PENNSYLVANIA · 2024 · $115,290

## Abstract

Project Summary
In aging neurons, the accumulation of key misfolded proteins into aggregates is a hallmark of many
neurodegenerative diseases. For example, pathological forms of TDP43 become mislocalized to the cytoplasm
and accumulate in aggregates in frontotemporal dementia (FTD) and other Alzheimer Disease-related
Dementias (ADRD). Highly intricate networks of enzymes called molecular chaperones combat these processes.
In ADRD, it is unknown how the chaperone networks fail against pathological forms of ADRD proteins such as
TDP43, FUS, and TAU. A major challenge to studying chaperone networks is the combinatorial complexity. The
canonical Hsp70 network consists of 54 Hsp40, 12 Hsp70, and 16 Hsp110 gene variants, creating a landscape
of 12,155 possible protein expression combinations. Unique combinations of the Hsp40-Hsp70-Hsp110 proteins
are hypothesized to confer specificity for different misfolded proteins in the complex human proteome. This
hypothesis is widely accepted but it has never been directly tested due to technical limitations. This NIH K99/R00
proposal outlines a plan to directly test this hypothesis by building the first exhaustive map of a chaperone
network against the ADRD-associated proteins TDP43, FUS, and TAU. To achieve this goal, aim 1 will leverage
a new genetic technique developed by Dr. Edward Barbieri to express and study all 12,155 possible
combinations of the human Hsp40-Hsp70-Hsp110 network in yeast models of ADRD. The chaperones identified
as having activity against TDP43 in yeast will be further studied in aim 2 using human cells and in vitro assays.
With human cell models, Dr. Barbieri will study the effect of the TDP43-active chaperones on cytoplasmic TDP43
aggregation and assess if the chaperones restore native TDP43 function in mRNA splicing in both HEK-293T
cells and neurons. Using in vitro biochemistry, he will measure chaperone activity for prevention and reversal of
TDP43 aggregation. During the R00 phase in aim 3 Dr. Barbieri will apply the chaperone network screen to study
the TAU aggregation and he will expand the chaperone networks studied in yeast by including Hsp40 pairs and
small HSPs. Lastly, Dr. Barbieri will combine the skills he learns during the K99 phase to develop a screen for
combinatorially overexpressing all 194 human chaperones directly in human cell models of ADRD to study
proteostasis networks in the native context. Together, the experiments outlined in this proposal will identify key
chaperones as therapeutic targets for ADRD. Dr. Barbieri will perform the K99 phase mentored in the Shorter
lab at the University of Pennsylvania, a world class biochemistry lab with expertise in the study of chaperones
and ADRD. This is an ideal training setting for Dr. Barbieri to acquire new skills in biochemistry. Furthermore,
Dr. Barbieri assembled an advisory committee to provide expertise in ADRD and formal training in the human
cell assays. The new skills will complement his current expertise in mo...

## Key facts

- **NIH application ID:** 10800710
- **Project number:** 5K99AG075242-02
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Edward Matthew Barbieri
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $115,290
- **Award type:** 5
- **Project period:** 2023-03-15 → 2026-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10800710

## Citation

> US National Institutes of Health, RePORTER application 10800710, Synthetic Reconstruction of Human Chaperone Networks in Yeast Models of Neurodegeneration (5K99AG075242-02). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10800710. Licensed CC0.

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