Submission Date

7-18-2025

Document Type

Paper- Restricted to Campus Access

Department

Biology

Second Department

Neuroscience

Faculty Mentor

Erica Gorenberg

Comments

Presented during the 27th Annual Summer Fellows Symposium, July 18, 2025 at Ursinus College.

Project Description

Parkinson’s disease (PD) is marked by the accumulation of misfolded α-synuclein (α-syn) into toxic aggregates, contributing to dopaminergic neuronal degeneration. Molecular chaperones, particularly the Hsp70 system, have been shown to mediate disaggregation of α-syn fibrils in vitro. DnaJB1, a co-chaperone of Hsp70, enhances substrate targeting and disaggregase activity, yet its role in mitigating α-syn toxicity in human cells remains unclear. This study investigates whether expression of DnaJB1 promotes cell survival in the presence of aggregated α-syn in human HEK293T cells. Plasmids encoding α-syn and DnaJB1 fused to fluorescent proteins were constructed using restriction enzyme cloning. PCR amplification incorporated XhoI/XmaI and BamHI-HF/NotI-HF restriction sites for α-syn and DnaJB1 constructs, respectively. Amplified products were verified by agarose gel electrophoresis, digested with restriction enzymes, and purified. Following ligation into expression vectors, plasmids were transformed into competent E. coli and purified by midi prep. Constructs were confirmed by restriction digest and DNA quantification. Transfection into HEK293T cells will allow assessment of DnaJB1’s impact on α-syn-mediated cytotoxicity using cell viability assays. This work aims to translate in vitro disaggregation findings into a human cellular model and explore the therapeutic potential of enhancing proteostasis through chaperone modulation in neurodegenerative diseases.

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