“Dominant-Negative Effect” Mechanism in OPTNE478G-Linked Amyotrophic Lateral Sclerosis (S43.004)

Objective: Pathological protein inclusions containing OPTN are a common feature in neurodegenerative diseases, including ALS. OPTN primarily localizes to autophagosomes. Our data suggest that autophagic dysfunction is the key molecular event in OPTN-linked ALS. Background: OPTN mutations cause ALS in both autosomal recessive and autosomal dominant fashion, an uncommon phenomenon in genetic diseases. A convergent mechanism to explain this phenomenon is lacking. The dominant OPTNE478G mutation provides a clue to key mechanistic insight in the pathogenesis in OPTN-linked ALS, and possible in other forms of ALS as well, because OPTN protein aggregates have been shown to be a common pathological hallmark in vast majority of the ALS cases, except for SOD1-linked cases. Design/Methods: Expression constructs. The XhoI/BamHI fragment containing full-length wild-type OPTN, or OPTN-E478G was cloned into the pEGFP-N1 or pmCherry-C1 expression vectors (Clontech). We also co-transfected UBQLN2-GFP and GFP-LC3 (Addgene, 11546) with OPTN constructs. Cell culture, transfection, Western blot and confocal microscopy were performed. Results: In Neuro2A cells, we find that cells expressing OPTNE478G accumulate UbG76V-GFP (a UPS reporter substrate) and LC3 (an autophagosome-associated protein). Furthermore, LC3-positive autophagosomes fail to recruit mutant OPTN (confocal studies). Western blots showed decreased conversion of LC3-I to LC3-II, indicating disrupt autophagic function in OPTNE478G mutation. Wild-type OPTN would aggravate OPTNE478G-related autophasomal dysfunction in double transfected cell lysate. Conclusions: OPTNE478G, a well-established autosomal dominant ALS mutation, acts in a “dominant-negative effect” mechanism in the OPTNE478G-mediated ALS in our study. The OPTN-linked ALS appears to be the first neurodegenerative disorder showing a “dominant-negative effect’ in its pathogenesis. Further studies of the physiological functions of OPTN should provide key insights into the pathogenesis of motor neuron degeneration in ALS due to OPTN deficiency. Disclosure: Dr. shi has nothing to disclose. Dr. Fecto has nothing to disclose. Dr. Esengul has nothing to disclose. Dr. Siddique has nothing to disclose. Dr. Deng has nothing to disclose.