Using Single-Molecule Fluorescence Microscopy to Uncover Neuronal Vulnerability to Protein Damage

Area of Science:

• Neurobiology
• Molecular Biology
• Cellular Biology

Background:

• Neurodegenerative disorders (NDs) involve toxic protein aggregate accumulation, leading to neuronal death.
• Loss of protein homeostasis (proteostasis) is a key feature of NDs.
• Activating the heat shock response (HSR) via heat shock proteins (HSPs) is a potential therapeutic strategy for NDs.

Purpose of the Study:

• To describe a method for investigating heat shock protein 70 (HSP70) induction in primary hippocampal neurons.
• To analyze neuron-to-neuron variability in HSR activation.
• To study HSP70 mRNA transcriptional induction and localization in primary neurons.

Main Methods:

• Utilized single-molecule fluorescence in situ hybridization (smFISH) to quantify HSP70 induction.
• Applied smFISH in primary hippocampal neurons.
• Quantified HSP70 mRNA levels and localization.

Main Results:

• Developed a method to analyze HSR activation variability in individual neurons.
• Enabled the study of HSP70 mRNA dynamics in response to cellular stress.
• Provided insights into neuron-specific HSR regulation.

Conclusions:

• The described smFISH method allows detailed analysis of HSR activation in primary neurons.
• Understanding neuron-specific HSR regulation is crucial for developing therapies for age-related neurodegenerative disorders.
• This approach may identify druggable targets for treating NDs by modulating proteostasis.