Unveiling epigenetic-driven synaptic remodeling by parallel analysis of nuclei and synapses using NucleuSynapse-Tag.

Area of Science:

• Neuroscience
• Molecular Biology
• Cell Biology

Background:

• Neuronal function depends on coordinated molecular mechanisms between cellular compartments, particularly nucleus-synapse communication.
• Simultaneous quantitative analysis of nuclear and synaptic molecular content presents a significant technical challenge.

Purpose of the Study:

• To develop a novel method for parallel quantitative analysis of nuclear and synaptic molecular content from a single brain sample.
• To investigate the role of epigenetic mechanisms in modulating synaptic protein levels.

Main Methods:

• Neurons were genetically tagged with EGFP (Enhanced Green Fluorescent Protein) via AAV injection into the mouse dentate gyrus.
• Nuclear (P1) and synaptosomal (P2) fractions were isolated from the same hippocampal homogenate using differential centrifugation.
• Immunolabeling and flow cytometry were employed for quantitative analysis of tagged nuclei and synaptosomes.

Main Results:

• The NucleuSynapse-Tag protocol successfully isolated and identified EGFP-tagged neuronal nuclei (NeuN+, PROX1+) and synaptosomes (FM4-64+, VGluT1+).
• Parallel analysis demonstrated that epigenetic mechanisms modulate brain-derived neurotrophic factor (BDNF) protein levels at the synapse.
• This provides the first direct evidence linking epigenetic regulation to synaptic BDNF levels.

Conclusions:

• The NucleuSynapse-Tag protocol enables quantitative, parallel analysis of molecular markers in both nuclei and synapses from a single sample.
• This approach facilitates comprehensive studies of molecular remodeling in interdependent subcellular compartments crucial for neuronal function and plasticity.
• Integration with tools like AAV and Cre mice enhances the scope of molecular remodeling analysis.