The prion-like RNA-binding protein Orb2, the Drosophila homolog of cytoplasmic polyadenylation element-binding protein (CPEB), switches to self-sustaining aggregates in an experience-dependent manner, and aggregated Orb2 in mushroom body γ-lobe neurons predicts long-term memory strength, prompting the proposal that Orb2 aggregates constitute a putative biochemical engram. Whether the yeast J-domain protein JJJ2 that facilitates Orb2 aggregation has endogenous Drosophila counterparts is already settled: the endogenous Hsp40 chaperone Mrj promotes Orb2A oligomerization, and the J-domain protein Funes promotes Orb2's oligomer-to-amyloid conversion and enhances memory. The open question is a causal, neuron-resolved account of how self-sustaining Orb2 aggregates maintain a specific memory: (i) whether completely eliminating pre-existing Orb2 aggregates after memory consolidation permanently abolishes memory, or whether residual aggregates or N-terminal fragments suffice to seed its recovery; (ii) how many and which neurons must acquire aggregated Orb2 to encode a given memory, and whether behaviorally relevant aggregation occurs outside the mushroom body γ lobe; and (iii) the precise biochemical chain from aggregated Orb2 to sustained synapse-localized translation — which mRNAs are captured and translated at activated synapses, how they are targeted, and how long this translation must continue to maintain a memory.

Background

How a transient experience leaves an enduring physical trace is a longstanding problem in memory research. Long-term memory (LTM), unlike short-term memory, requires new protein synthesis, yet the synaptic proteins that maintain a memory turnover in days — the 'maintenance paradox'. One proposed solution is a self-perpetuating molecular switch. Cytoplasmic polyadenylation element-binding protein (CPEB) is a family of RNA-binding proteins that regulate the polyadenylation and translation of specific mRNAs; in neurons they are implicated in synapse-localized protein synthesis. The Drosophila member, Orb2, contains a prion-like domain — a low-complexity sequence segment able to adopt a self-templating amyloid-like conformation — and can switch from a soluble translation-repressing state to an oligomeric/aggregated state that activates translation of its target mRNAs (Khan et al. 2015; Majumdar et al. 2012). Orb2 encodes two protein isoforms, a rare Orb2A and an abundant Orb2B, with the N-terminal domain of Orb2A nucleating aggregate formation — which is why Li et al. (2016) ask whether residual N-terminal fragments could re-seed aggregates after elimination. Orb2 is required for LTM in courtship and appetitive olfactory conditioning assays (Keleman et al. 2007). The Drosophila mushroom body, a structure of roughly 2000 intrinsic Kenyon cells divided into α/β, α′/β′, and γ lobes, is the main olfactory learning center; the γ lobe is particularly important for memory persisting beyond a day. Li et al. (2016) provided three key observations supporting Orb2 aggregates as a physical substrate of memory ('biochemical engram'): acute reversible inactivation of Orb2 in adult neurons showed Orb2 is needed for encoding, persistent storage, and recall; ectopic expression of JJJ2, a yeast J-domain protein chaperone (DNAJ/Hsp40 family), which facilitates Orb2 aggregation, enhanced LTM formation, including under suboptimal training; and new imaging tools revealed that training-dependent aggregated Orb2 in a subset of mushroom body γ-lobe neurons correlates with and predicts memory strength. A J-domain protein is a co-chaperone that typically stimulates Hsp70 ATPase activity to determine client specificity. The same study left open whether chaperone-guided, self-sustaining Orb2 aggregation is causally sufficient and necessary in the relevant neurons, and what its translational output is. Considerable later work has clarified parts of this: the amyloid core structure of Orb2A fibrils was solved by cryo-electron microscopy (Hervas et al. 2020), Orb2 assembly passes through droplet and fibril intermediates (Ashami et al. 2021; Oroz et al. 2020), Orb2A protein stability is regulated through the Orb2-interacting protein Tob (White-Grindley et al. 2014) and Orb2 mRNA is rendered translation-competent by experience-dependent intron removal (Gill et al. 2017), and two endogenous Drosophila J-domain proteins were identified that regulate Orb2 assembly — Mrj, which promotes Orb2A conversion into prion-like oligomers and is required in mushroom body neurons for LTM (Desai et al. 2024), and Funes (CG10375), which promotes the oligomer-to-amyloid step, enhances memory in γ-lobe Kenyon cells and reward-signaling PAM (protocerebral anterior medial cluster) dopaminergic neurons, and whose J-domain function is required for both amyloid assembly and memory enhancement (Patton et al. 2026). In mice, genomic removal of the Orb2 homolog CPEB3 after memory formation causes established memory to decay without rescue by later CPEB3 re-expression (Fioriti et al. 2015), which is consistent with, but does not directly test, the aggregate-elimination question in Drosophila. What remains unresolved is the causal closure of the engram hypothesis at three levels: the irreversibility test (complete elimination of aggregates after consolidation), the cellular specificity of aggregation, and the identity and required duration of the local translational output that maintains a memory.

Problem Statement

Provide a causal, neuron-resolved account of how self-sustaining aggregates of the prion-like RNA-binding protein Orb2 maintain a specific long-term memory in Drosophila, by answering three linked questions that Li et al. (2016) left open: (1) Elimination and irreversibility — if pre-existing Orb2 aggregates are completely eliminated after a memory has been consolidated (rather than partially reduced), that is, reduced below the detection limit of biochemical aggregate assays, is the memory permanently abolished, or can residual aggregates or N-terminal Orb2 fragments remaining below that detection limit re-seed aggregates and allow the memory to recover once monomeric Orb2 is restored? (2) Cellular specificity — how many, and which, individual neurons within the mushroom body γ lobe (or elsewhere) must acquire aggregated Orb2 for a given long-term memory to be encoded and maintained, and does training-dependent Orb2 aggregation outside the γ lobe contribute to memory in a behaviorally relevant way? (3) Translational output — what is the precise biochemical sequence linking aggregated Orb2 to sustained synapse-localized translation: which specific mRNAs are captured and translated at activated synapses, by what targeting mechanism, and for how long must this Orb2-dependent translation continue in order to maintain a memory? All three parts together form the complete account, and each is independently resolvable: a part is resolved only when every query listed within it is addressed by a defensible experimental design or mechanistic model that is consistent with the cited literature, and an account that resolves only some of the parts — or only some of the queries within a part — leaves the remainder open. The endogenous-chaperone part of this question (whether functional equivalents of the yeast J-domain protein JJJ2 that facilitate Orb2 aggregation in vivo exist in Drosophila) has been answered affirmatively by the identification of Mrj and Funes and is therefore out of scope; speculation about JJJ2 equivalents in other species lies outside this Drosophila-specific question. What remains is the elimination, cellular-specificity, and translational-output components of the biochemical-engram hypothesis.

References

  1. Li L, Sanchez CP, Slaughter BD, Zhao Y, Khan MR, Unruh JR, Rubinstein B, Si K (2016) A Putative Biochemical Engram of Long-Term Memory. Current Biology 26(23):3143-3156. doi:10.1016/j.cub.2016.09.054 https://doi.org/10.1016/j.cub.2016.09.054
  2. Majumdar A, Cesario WC, White-Grindley E, Jiang H, Ren F, Khan MR, Li L, Choi EM-L, Kannan K, Guo F, Unruh J, Slaughter B, Si K (2012) Critical Role of Amyloid-like Oligomers of Drosophila Orb2 in the Persistence of Memory. Cell 148(3):515-529. doi:10.1016/j.cell.2012.01.004 https://doi.org/10.1016/j.cell.2012.01.004
  3. Keleman K, Krüttner S, Alenius M, Dickson BJ (2007) Function of the Drosophila CPEB protein Orb2 in long-term courtship memory. Nature Neuroscience 10(12):1587-1593. doi:10.1038/nn1996 https://doi.org/10.1038/nn1996
  4. Khan MR, Li L, Pérez-Sánchez C, Saraf A, Florens L, Slaughter BD, Unruh JR, Si K (2015) Amyloidogenic Oligomerization Transforms Drosophila Orb2 from a Translation Repressor to an Activator. Cell 163(6):1468-1483. doi:10.1016/j.cell.2015.11.020 https://doi.org/10.1016/j.cell.2015.11.020
  5. Mastushita-Sakai T, White-Grindley E, Samuelson J, Seidel C, Si K (2010) Drosophila Orb2 targets genes involved in neuronal growth, synapse formation, and protein turnover. Proceedings of the National Academy of Sciences USA 107(26):11987-11992. doi:10.1073/pnas.1004433107 https://doi.org/10.1073/pnas.1004433107
  6. White-Grindley E, Li L, Mohammad Khan R, Ren F, Saraf A, Florens L, Si K (2014) Contribution of Orb2A Stability in Regulated Amyloid-Like Oligomerization of Drosophila Orb2. PLoS Biology 12(2):e1001786. doi:10.1371/journal.pbio.1001786 https://doi.org/10.1371/journal.pbio.1001786
  7. Gill J, Park Y, McGinnis JP, Perez-Sanchez C, Blanchette M, Si K (2017) Regulated Intron Removal Integrates Motivational State and Experience. Cell 169(5):836-848.e15. doi:10.1016/j.cell.2017.05.006 https://doi.org/10.1016/j.cell.2017.05.006
  8. Stepien BK, Oppitz C, Gerlach D, Dag U, Novatchkova M, Krüttner S, Stark A, Keleman K (2016) RNA-binding profiles of Drosophila CPEB proteins Orb and Orb2. Proceedings of the National Academy of Sciences USA 113(45):E7030-E7038. doi:10.1073/pnas.1603715113 https://doi.org/10.1073/pnas.1603715113
  9. Fioriti L, Myers C, Huang Y-Y, Li X, Stephan JS, Trifilieff P, Colnaghi L, Kosmidis S, Drisaldi B, Pavlopoulos E, Kandel ER (2015) The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3. Neuron 86(6):1433-1448. doi:10.1016/j.neuron.2015.05.021 https://doi.org/10.1016/j.neuron.2015.05.021
  10. Hervas R, Rau MJ, Park Y, Zhang W, Murzin AG, Fitzpatrick JAP, Scheres SHW, Si K (2020) Cryo-EM structure of a neuronal functional amyloid implicated in memory persistence in Drosophila. Science 367(6483):1230-1234. doi:10.1126/science.aba3526 https://doi.org/10.1126/science.aba3526
  11. Oroz J, Félix SS, Cabrita EJ, Laurents DV (2020) Structural transitions in Orb2 prion-like domain relevant for functional aggregation in memory consolidation. Journal of Biological Chemistry 295(52):18122-18133. doi:10.1074/jbc.RA120.015211 https://doi.org/10.1074/jbc.RA120.015211
  12. Ashami K, Falk AS, Hurd C, Garg S, Cervantes SA, Rawat A, Siemer AB (2021) Droplet and fibril formation of the functional amyloid Orb2. Journal of Biological Chemistry 297(1):100804. doi:10.1016/j.jbc.2021.100804 https://doi.org/10.1016/j.jbc.2021.100804
  13. Chatterjee S, Bahl E, Mukherjee U, Walsh EN, Shetty MS, Yan AL, Vanrobaeys Y, Lederman JD, Giese KP, Michaelson J, Abel T (2022) Endoplasmic reticulum chaperone genes encode effectors of long-term memory. Science Advances 8(12):eabm6063. doi:10.1126/sciadv.abm6063 https://doi.org/10.1126/sciadv.abm6063
  14. Desai M, Hemant, Deo A, Naik J, Dhamale P, Kshirsagar A, Bose T, Majumdar A (2024) Mrj is a chaperone of the Hsp40 family that regulates Orb2 oligomerization and long-term memory in Drosophila. PLoS Biology 22(4):e3002585. doi:10.1371/journal.pbio.3002585 https://doi.org/10.1371/journal.pbio.3002585
  15. Patton K, Yi Y, Burt R, Ng KK, Mukhi M, Khaki PSS, Hervas R, Si K (2026) A J-domain protein enhances memory by promoting physiological amyloid formation in Drosophila. Proceedings of the National Academy of Sciences USA 123(5):e2516310123. doi:10.1073/pnas.2516310123 https://doi.org/10.1073/pnas.2516310123
  16. Hicks DA, Aubrey LD, Kwok JCF, Radford SE (2025) Role of CPEBs in Learning and Memory. Journal of Neurochemistry 169(9):e70226. doi:10.1111/jnc.70226 https://doi.org/10.1111/jnc.70226
  17. Garcia-Pardo J, Ventura S (2024) Cryo-EM structures of functional and pathological amyloid ribonucleoprotein assemblies. Trends in Biochemical Sciences 49(2):119-133. doi:10.1016/j.tibs.2023.10.005 https://doi.org/10.1016/j.tibs.2023.10.005