A VAPB mutant linked to amyotrophic lateral sclerosis generates a novel form of organized smooth endoplasmic...

Area of Science:

• Cell Biology
• Neuroscience
• Protein Biochemistry

Background:

• Vesicle-associated membrane protein-associated protein B (VAPB) is an ER-resident protein crucial for lipid transport.
• Mutations in VAPB, such as P56S-VAPB, are associated with familial amyotrophic lateral sclerosis (ALS) and lead to inclusion body formation.
• The precise biogenesis and structural characteristics of these mutant VAPB inclusions remain poorly understood.

Purpose of the Study:

• To elucidate the cell biological basis of P56S-VAPB pathogenicity by investigating its biogenesis and the nature of the inclusions it forms.
• To characterize the membrane insertion mechanism of P56S-VAPB compared to wild-type VAPB.
• To analyze the ultrastructure of the inclusion bodies generated by the P56S-VAPB mutant.

Main Methods:

• Post-translational translocation assays were performed in cell-free systems and cultured mammalian cells.
• Confocal imaging and electron microscopy were utilized to characterize the inclusion bodies.
• Expression levels of mutant VAPB were compared to endogenous wild-type VAPB.

Main Results:

• Mutant P56S-VAPB inserts into endoplasmic reticulum (ER) membranes post-translationally, similar to wild-type VAPB.
• P56S-VAPB rapidly clusters to form inclusion bodies that remain continuous with the ER.
• These inclusions represent a novel form of organized smooth ER (OSER), characterized by parallel cisternae and a dense cytosolic layer.
• Inclusion formation was observed even at endogenous-like expression levels of the mutant protein.

Conclusions:

• The ALS-linked P56S-VAPB mutant induces significant restructuring of the ER.
• The formation of novel OSER structures by mutant VAPB may be a key factor in its pathogenicity in motoneurons.
• Understanding mutant VAPB biogenesis and inclusion formation provides insights into the molecular mechanisms of familial ALS.