Quantifying inter-organelle membrane contact sites using proximity ligation assay in fixed optic nerve sections

Area of Science:

• Cell Biology
• Neuroscience
• Biochemistry

Background:

• Membrane contact sites (MCS) are vital for cellular functions.
• Dysfunction of MCS proteins is linked to neurological and optic nerve diseases.
• Existing imaging techniques struggle to quantify MCS in mammalian optic nerve tissues.

Purpose of the Study:

• To establish a novel method for quantifying inter-organelle MCS in mammalian optic nerve tissues.
• To investigate the effect of protrudin gene therapy on MCS in the optic nerve.

Main Methods:

• Proximity Ligation Assay (PLA) was adapted for use in mammalian optic nerve tissues.
• Adeno-associated virus (AAV) gene therapy was used to deliver wild-type or active protrudin.
• Quantification of mitochondria-endoplasmic reticulum (ER) and mitochondria-late endosome contacts.

Main Results:

• Proximity Ligation Assay (PLA) proved effective for quantifying MCS in optic nerve tissue.
• Gene therapy with protrudin significantly increased mitochondria-ER contact sites.
• Upregulation of protrudin also significantly increased mitochondria-late endosome contact sites.

Conclusions:

• PLA is a viable and efficient technique for quantifying MCS in the optic nerve.
• Protrudin upregulation via gene therapy enhances mitochondrial-ER and mitochondrial-late endosome interactions in the optic nerve.
• This provides a new tool for studying optic nerve diseases linked to MCS dysfunction.