Identification of a splice variant of optineurin which is defective in autophagy and phosphorylation

Area of Science:

• Cell Biology
• Molecular Biology
• Autophagy Research

Background:

• Optineurin (Optn) is a crucial autophagy receptor involved in both selective and non-selective autophagy.
• Dysfunctional autophagy is implicated in various neurodegenerative diseases.

Purpose of the Study:

• To identify and characterize novel splice variants of mouse optineurin.
• To investigate the functional consequences of a newly identified truncated optineurin isoform on autophagy and cargo clearance.

Main Methods:

• Identification and characterization of mouse optineurin splice variants using molecular biology techniques.
• Expression analysis of the truncated variant (d157mOptn) in various tissues and cells.
• Assessment of d157mOptn phosphorylation status and interaction with TBK1.
• Functional assays in Optn-deficient cells to evaluate autophagosome and autolysosome formation.
• Analysis of d157mOptn's role in clearing mutant huntingtin aggregates in neuronal cells.

Main Results:

• A novel splice variant, d157mOptn, was identified, producing a truncated protein lacking the N-terminal 157 amino acids.
• d157mOptn exhibits reduced interaction with TBK1 and is not phosphorylated at S187, unlike wild-type Optn.
• The truncated variant impairs autophagosome and autolysosome formation in Optn-deficient cells.
• d157mOptn fails to clear mutant huntingtin aggregates in neuronal cells, unlike wild-type Optn.
• Phosphorylation of Optn at S187 requires the intact N-terminal sequence and a functional C-terminal ubiquitin-binding domain.

Conclusions:

• A novel, functionally impaired optineurin isoform (d157mOptn) has been identified in mice.
• This isoform is defective in promoting autophagy, likely due to impaired TBK1 interaction and lack of phosphorylation.
• d157mOptn may act as a dominant-negative inhibitor of wild-type Optn function, potentially contributing to disease pathogenesis.
• The N-terminal domain of Optn is critical for its autophagic functions and interaction with TBK1.