Contribution of Orb2A Stability in Regulated Amyloid- Like Oligomerization of Drosophila Orb2

How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. Here, we report the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories. Author Summary The formation of stable long-term memories involves the synthesis of new protein, however the biochemical basis of this process is unclear. A family of RNA binding proteins, Cytoplasmic Polyadenylation Element Binding (CPEB) proteins, are known to regulate synaptic activity and stabilization of memory. The Drosophila CPEB is called Orb2, and its amyloid-like oligomers are critical for the persistence of long-lasting memories. Amyloid formation is often unregulated and stochastic in nature, and the amyloid state is usually dominant and self-sustaining. However, to serve as a substrate for long-lasting memory, the amyloid-like oligomerization of Orb2 must be regulated in a space-, time-, and stimulus-specific manner. Orb2 has two protein isoforms: Orb2A, which is present only in low abundance, and Orb2B, which is the abundant form. Orb2A is important for oligomerization as well as memory persistence. Previous studies suggested that Orb2A may act as a seed to induce oligomerization of the constitutive Orb2B isoform. Therefore, the availability of Orb2A protein would be an important determinant of Orb2 oligomerization. Here we have analyzed how Orb2 conversion to the oligomeric state is regulated. We find that Orb2A is a very unstable protein and that phosphorylation-dephosphorylation of this isoform via canonical neuronal signaling modules can regulate Orb2A stability, and thereby its abundance. We also show that Tob, a known regulator of CPEB-mediated translation, acts as a stabilizer for Orb2A and triggers Orb2 oligomerization. These observations suggest that amyloid formation can be regulated in a dynamic manner by controlling the availability of the seeding Orb2A protein.