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

Neurodegenerative disorders (NDs) are diverse age-related conditions also described as "conformational diseases." The hallmark of NDs is the accumulation of disease-specific proteins as toxic misfolded aggregates in some areas of the brain. They lead to the loss of protein homeostasis (proteostasis) that causes neuronal dysfunction and death. A potential therapeutic strategy for NDs is to prevent the accumulation of misfolded proteins by activating the heat shock response (HSR). The HSR maintains proteostasis through the upregulation of heat shock proteins (HSPs), molecular chaperones that recognize misfolded proteins, and either refold them to their functional conformations and/or target them for degradation. However, how to manipulate the expression of HSPs to obtain a therapeutic effect in neurons remains unclear. Furthermore, the regulation of the HSR in neurons is more complex than what we have learned from culturing somatic nonneuronal cells. This chapter describes a method to investigate the induction of HSP70 in primary hippocampal neurons using single-molecule fluorescence in situ hybridization (smFISH). Quantification of smFISH provides the means to analyze neuron-to-neuron variability in the activation of the HSR and enables us to study the transcriptional induction and localization of HSP70 mRNA in primary neurons. This information might be critical to find the druggable steps for developing effective therapies to treat age-related NDs.