Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory.

A long-standing question in the study of long-term memory is how a memory trace persists for years when the proteins that initiated the process turn over and disappear within days. Previously, we postulated that self-sustaining amyloidogenic oligomers of cytoplasmic polyadenylation element-binding protein (CPEB) provide a mechanism for the maintenance of activity-dependent synaptic changes and, thus, the persistence of memory. Here, we found that the Drosophila CPEB Orb2 forms amyloid-like oligomers, and oligomers are enriched in the synaptic membrane fraction. Of the two protein isoforms of Orb2, the amyloid-like oligomer formation is dependent on the Orb2A form. A point mutation in the prion-like domain of Orb2A, which reduced amyloid-like oligomerization of Orb2, did not interfere with learning or memory persisting up to 24 hr. However the mutant flies failed to stabilize memory beyond 48 hr. These results support the idea that amyloid-like oligomers of neuronal CPEB are critical for the persistence of long-term memory.

Aplysia CPEB can form prion-like multimers in sensory neurons that contribute to long-term facilitation.

Prions are proteins that can assume at least two distinct conformational states, one of which is dominant and self-perpetuating. Previously we found that a translation regulator CPEB from Aplysia, ApCPEB, that stabilizes activity-dependent changes in synaptic efficacy can display prion-like properties in yeast. Here we find that, when exogenously expressed in sensory neurons, ApCPEB can form an amyloidogenic self-sustaining multimer, consistent with it being a prion-like protein. In addition, we find that conversion of both the exogenous and the endogenous ApCPEB to the multimeric state is enhanced by the neurotransmitter serotonin and that an antibody that recognizes preferentially the multimeric ApCPEB blocks persistence of synaptic facilitation. These results are consistent with the idea that ApCPEB can act as a self-sustaining prion-like protein in the nervous system and thereby might allow the activity-dependent change in synaptic efficacy to persist for long periods of time.

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.

Drosophila Orb2 targets genes involved in neuronal growth, synapse formation, and protein turnover.

In the study of long-term memory, how memory persists is a fundamental and unresolved question. What are the molecular components of the long-lasting memory trace? Previous studies in Aplysia and Drosophila have found that a neuronal variant of a RNA-binding protein with a self-perpetuating prion-like property, cytoplasmic polyadenylation element binding protein, is required for the persistence of long-term synaptic facilitation in the snail and long-term memory in the fly. In this study, we have identified the mRNA targets of the Drosophila neuronal cytoplasmic polyadenylation element binding protein, Orb2. These Orb2 targets include genes involved in neuronal growth, synapse formation, and intriguingly, protein turnover. These targets suggest that the persistent form of the memory trace might be comprised of molecules that maintain a sustained, permissive environment for synaptic growth in an activated synapse.

Activation of cryptic 3' splice sites within introns of cellular genes following gene entrapment.

Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3'-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3' splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3' splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3' splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3' splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3' splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3' splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3' processing and polyadenylation of cellular transcripts.

RISC-y Memories.

Local protein synthesis in the synapse is required for synaptic plasticity and has been implicated in learning and memory. However, direct evidence that behavioral training induces local protein synthesis has been lacking. In this issue of Cell, Ashraf et al. (2006) observe persistent local protein synthesis in the antennal lobe synapses of the fruit fly following olfactory-avoidance learning. This protein synthesis is regulated by the RNA-induced silencing complex (RISC).

P2P-R expression is genetically coregulated with components of the translation machinery and with PUM2, a translational repressor that associates with the P2P-R mRNA.

P2P-R is a nuclear protein with potential functional roles in the control of gene expression and mitosis. The P2P-R protein also interacts with the p53 and Rb1 tumor suppressor proteins. To search for additional functional associations of P2P-R, we employed the WebQTL database that contains the results of cDNA microarray analysis on forebrain, cerebellum, and hematopoietic stem cell (HSC) specimens of multiple BXD recombinant inbred strains of mice. Using WebQTL, gene products were identified that show genetically based coexpression with P2P-R. Initial studies identified general groups of mRNAs that share common functional roles and high covariation in expression with P2P-R. These functional groups involved the regulation of transcription, nucleotide binding, translation control, and ion transport. The findings related to translational mechanisms were further evaluated. In HSCs, expression of P2P-R mRNA demonstrates an impressive expression correlation with a group of gene products associated with translation; high expression of P2P-R specifically was associated with decreased expression of 29 ribosomal protein mRNAs. In all three tissues that were screened using the WebQTL database, a strong positive expression covariance between P2P-R and the Pum2 gene product also was observed. PUM2 is a member of the highly conserved Puf family of RNA binding proteins that often function as gene-specific translation regulators. The ability of Puf proteins to repress translation is mediated by their binding to specific elements located in the 3' untranslated region (UTR) of their target mRNAs. To assess the functional significance of the strong genetic correlation in expression of P2P-R and PUM2, the 3' UTR of the P2P-R mRNA was analyzed and found to contain one perfect consensus and two near-perfect consensus PUM2 binding sequences. PUM2 pull-down methods combined with reverse transcription and RT-PCR confirmed that PUM2 does indeed bind P2P-R mRNA. These results suggest that P2P-R expression may be translationally regulated by PUM2 and that P2P-R may modulate translation by influencing ribosomal protein gene expression. This study represents the first description of a RNA target for mammalian Puf proteins and the first molecular confirmation of information obtained using the WebQTL database.