A yeast model of optineurin proteinopathy reveals a unique aggregation pattern associated with cellular toxicity

Abstract

SummaryMany neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) are linked to the accumulation of specific protein aggregates in affected regions of the nervous system. SOD1, TDP-43, FUS and optineurin (OPTN) proteins were identified to form intraneuronal inclusions in ALS patients. In addition, mutations in OPTN are associated with both ALS and glaucoma. As the pathological role of OPTN in neuronal degeneration remains unresolved, we created a yeast model to study its potential for aggregation and toxicity. We observed that both wild type and diseaseassociated mutants of OPTN form toxic non-amyloid aggregates in yeast. Similar to reported cell culture and mouse models, the OPTN E50K mutant shows enhanced toxicity in yeast, implying a conserved gainof-function mechanism. Furthermore, OPTN shows a unique aggregation pattern compared to other disease-related proteins in yeast. OPTN aggregates colocalize only partially with the insoluble protein deposit (IPOD) site markers, but coincide perfectly with the prion seed-reducing protein Btn2 and several other aggregation-prone proteins, suggesting that protein aggregates are not limited to a single IPOD site. Importantly, changes in the Btn2p level modify OPTN toxicity and aggregation. This study generates a mechanistic framework for investigating how OPTN may trigger pathological changes in ALS and other OPTN-linked neurodegenerative disorders.

Normal levels of the antiprion proteins Btn2 and Cur1 cure most newly formed [URE3] prion variants

“…In a btn2Δ cur1Δ strain, the copy number of [URE3-1] was higher than in an isogenic BTN2 CUR1 strain (17), again suggesting that these proteins lower prion copy number. The finding that Btn2p colocalizes with Ure2p amyloid aggregates in [URE3] strains undergoing curing (17)(18)(19) suggests a direct mechanism involving sequestration of prion seeds as previously proposed (17). As expected for this model, normal levels of Btn2p/Cur1p are enough to sequester a small number of seeds; higher seed [URE3] variants are only cured by overproduction.…”

“…These proteins are paralogs, members of the Hook family, consistent with their similar effects, but Btn2 localized to a site next to the nucleus, whereas Cur1 was localized largely within the nucleus (17). During the curing process, those [URE3] cells having both Ure2p-GFP aggregates and Btn2-RFP dots show striking colocalization (17)(18)(19). Partial colocalization of Btn2-RFP with Sup35NM-GFP in a [PSI+] strain or with the huntingtin-like Q103-GFP were also observed (17), but overexpression of Btn2p or Cur1p did not cure [PSI+].…”

“…The IPOD site is reported to accumulate nonprion aggregates of Ure2p (20), and such nonprion aggregates were found to not coincide with Btn2p (25). However, prion aggregates of Ure2p do associate with Btn2p (17)(18)(19). Thus, Btn2p (or associated proteins) recognizes the prion form of Ure2p as distinct from nonprion aggregates of the same protein.…”

“…As discussed above, only a tiny minority of cells overexpressing these prion domains form prions, although most cells show aggregates of the overproduced protein, so this inference is not justified. Indeed, Btn2p does colocalize with amyloid aggregates of Ure2p in a [URE3] strain early in the curing process (17)(18)(19). Btn2p is also partially colocalized with amyloid aggregates of Sup35p in a [PSI+] strain (17).…”

“…Btn2p is also partially colocalized with amyloid aggregates of Sup35p in a [PSI+] strain (17). Btn2p also colocalizes with nonamyloid aggregates of optineurin, PrP, Rnq1p, and Q103P (19). Malinovska et al further showed that Btn2p and Cur1p each interact physically with the Hsp40 family member Sis1p, and that both Btn2p and Cur1p are drawn into the nucleus as a result of this association (25).…”