InteractiveFly: GeneBrief

orb2: Biological Overview | References

Gene name - orb2

Synonyms -

Cytological map position - 66E4-66E5

Function - RNA-binding protein

Keywords - amyloid-like oligomers, prion, stabilized memory, synapse, brain

Symbol - orb2

FlyBase ID: FBgn0264307

Genetic map position - chr3L:8935401-8947582

Classification - RRM_6: RNA recognition motif

Cellular location - cytoplasmic

NCBI link: EntrezGene

Orb2 orthologs: Biolitmine

Recent literature
Kruttner, S., Traunmuller, L., Dag, U., Jandrasits, K., Stepien, B., Iyer, N., Fradkin, L. G., Noordermeer, J. N., Mensh, B. D. and Keleman, K. (2015). Synaptic Orb2A bridges memory acquisition and late memory consolidation in Drosophila. Cell Rep 11: 1953-1965. PubMed ID: 26095367
To adapt to an ever-changing environment, animals consolidate some, but not all, learning experiences to long-term memory. In mammals, long-term memory consolidation often involves neural pathway reactivation hours after memory acquisition. It is not known whether this delayed-reactivation schema is common across the animal kingdom or how information is stored during the delay period. This study shows that, during courtship suppression learning, Drosophila exhibits delayed long-term memory consolidation. It is also shown that the same class of dopaminergic neurons engaged earlier in memory acquisition is also both necessary and sufficient for delayed long-term memory consolidation. Furthermore, evidence is presented that, during learning, the translational regulator Orb2A tags specific synapses of mushroom body neurons for later consolidation. Consolidation involves the subsequent recruitment of Orb2B and the activity-dependent synthesis of CaMKII. Thus, these results provide evidence for the role of a neuromodulated, synapse-restricted molecule bridging memory acquisition and long-term memory consolidation in a learning animal.

Burguete, A. S., Almeida, S., Gao, F. B., Kalb, R., Akins, M. R. and Bonini, N. M. (2015). GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function. Elife 4. PubMed ID: 26650351
Microsatellite expansions are the leading cause of numerous neurodegenerative disorders. This study demonstrates that GGGGCC and CAG microsatellite repeat RNAs associated with C9orf72 in ALS/FTD and with polyglutamine diseases, respectively, localize to neuritic granules that undergo active transport into distal neuritic segments. In cultured mammalian spinal cord neurons, the presence of neuritic GGGGCC repeat RNA correlates with neuronal branching defects and the repeat RNA localizes to granules that label with FMRP, a transport granule component. Using a Drosophila GGGGCC expansion disease model, this study characterized dendritic branching defects that are modulated by FMRP and Orb2. The human orthologues of these modifiers are misregulated in induced pluripotent stem cell-differentiated neurons from GGGGCC expansion carriers. These data suggest that expanded repeat RNAs interact with the mRNA transport and translation machinery, causing transport granule dysfunction. This could be a novel mechanism contributing to the neuronal defects associated with C9orf72 and other microsatellite expansion diseases.

Hervas, R., Li, L., Majumdar, A., Fernandez-Ramirez Mdel, C., Unruh, J. R., Slaughter, B. D., Galera-Prat, A., Santana, E., Suzuki, M., Nagai, Y., Bruix, M., Casas-Tinto, S., Menendez, M., Laurents, D. V., Si, K. and Carrion-Vazquez, M. (2016). Molecular basis of Orb2 amyloidogenesis and blockade of memory consolidation. PLoS Biol 14: e1002361. PubMed ID: 26812143
Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Using an array of biophysical, cell biological and behavioural assays this study has characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, it was found that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. It was also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.
Li, L., Sanchez, C. P., Slaughter, B. D., Zhao, Y., Khan, M. R., Unruh, J. R., Rubinstein, B. and Si, K. (2016). A putative biochemical engram of long-term memory. Curr Biol 26(23): 3143-3156. PubMed ID: 27818176
How a transient experience creates an enduring yet dynamic memory remains an unresolved issue in studies of memory. Experience-dependent aggregation of the RNA-binding protein CPEB/Orb2 is one of the candidate mechanisms of memory maintenance. Using tools that allow rapid and reversible inactivation of Orb2 protein in neurons, this study found that Orb2 activity is required for encoding and recall of memory. From a screen, a DNA-J family chaperone, JJJ2, was identified that facilitates Orb2 aggregation, and ectopic expression of JJJ2 enhances the animal's capacity to form long-term memory. Tools were developed to visualize training-dependent aggregation of Orb2. Aggregated Orb2 in a subset of mushroom body neurons can serve as a "molecular signature" of memory and predict memory strength. These data indicate that self-sustaining aggregates of Orb2 may serve as a physical substrate of memory and provide a molecular basis for the perduring yet malleable nature of memory.
Cervantes, S. A., Bajakian, T. H., Soria, M. A., Falk, A. S., Service, R. J., Langen, R. and Siemer, A. B. (2016). Identification and structural characterization of the N-terminal amyloid core of Orb2 isoform A. Sci Rep 6: 38265. PubMed ID: 27922050
Orb2 is a functional amyloid that plays a key role in Drosophila long-term memory formation. Orb2 has two isoforms that differ in their N-termini. The N-terminus of the A isoform (Orb2A) that precedes its Q-rich prion-like domain has been shown to be important for Orb2 aggregation and long-term memory. However, besides the fact that it forms fibrillar aggregates, structural information of Orb2 is largely absent. To understand the importance of the N-terminus of Orb2A and its relation to the fibril core, solid-state NMR and EPR data were recorded on fibrils formed by the first 88 residues of Orb2A (Orb2A88). These data show that the N-terminus of Orb2A not only promotes the formation of fibrils, but also forms the fibril core of Orb2A88. This fibril core has an in-register parallel beta-sheet structure and does not include the Q-rich, prion-like domain of Orb2. The Q-rich domain is part of the unstructured region, which becomes increasingly dynamic towards the C-terminus.
Gill, J., Park, Y., McGinnis, J. P., Perez-Sanchez, C., Blanchette, M. and Si, K. (2017). Regulated intron removal integrates motivational state and experience. Cell 169(5): 836-848. PubMed ID: 28525754
Myriad experiences produce transient memory, yet, contingent on the internal state of the organism and the saliency of the experience, only some memories persist over time. How experience and internal state influence the duration of memory at the molecular level remains unknown. A self-assembled aggregated state of Drosophila Orb2A protein is required specifically for long-lasting memory. In the adult fly brain the mRNA encoding Orb2A protein exists in an unspliced non-protein-coding form. The convergence of experience and internal drive transiently increases the spliced protein-coding Orb2A mRNA. A screen identified pasilla, the fly ortholog of mammalian Nova-1/2, as a mediator of Orb2A mRNA processing. A single-nucleotide substitution in the intronic region that reduces Pasilla binding and intron removal selectively impairs long-term memory. It is posited that pasilla-mediated processing of unspliced Orb2A mRNA integrates experience and internal state to control Orb2A protein abundance and long-term memory formation.
Soria, M. A., Cervantes, S. A., Bajakian, T. H. and Siemer, A. B. (2017). The functional amyloid Orb2A binds to lipid membranes. Biophys J 113(1): 37-47. PubMed ID: 28700922
Lipid membranes interact with and influence the aggregation of many amyloid-forming proteins. Orb2 is a cytoplasmic polyadenylation element-binding protein homolog in Drosophila melanogaster that forms functional amyloids necessary for long-term memory. One isoform, Orb2A, has a unique N-terminus that has been shown to be important for the formation of amyloid-like aggregates and long-term memory in vivo. Orb2A is also found enriched in the synaptic membrane fraction. Sequence and hydropathy analysis suggests that it can form an amphipathic helix, which is ideal for lipid membrane interaction. Circular dichroism and site-directed spin labeling coupled with electron paramagnetic resonance was used to test the first 88 amino acids of Orb2A for lipid interaction. Orb2A1-88 was shown to interact with anionic lipid membranes using an amphipathic helix at its unique N-terminus. This interaction depends on the charge of the lipid membrane and the degree of membrane curvature. Transmission electron microscopy and electron paramagnetic resonance were used to show that the presence of anionic small unilamellar vesicles inhibits amyloid fibril formation by Orb2A. This inhibition by anionic membranes could be a potential mechanism regulating Orb2A amyloid formation in vivo.
Sanguanini, M. and Cattaneo, A. (2018). A continuous model of physiological prion aggregation suggests a role for Orb2 in gating long-term synaptic information. R Soc Open Sci 5(12): 180336. PubMed ID: 30662713
The regulation of mRNA translation at the level of the synapse is believed to be fundamental in memory and learning at the cellular level. The family of cytoplasmic polyadenylation element binding (CPEB) proteins emerged as an important RNA-binding protein family during development and in adult neurons. Drosophila Orb2 (homologue of mouse CPEB3 protein and of the neural isoform of Aplysia CPEB) has been found to be involved in the translation of plasticity-dependent mRNAs and has been associated with long-term memory. Orb2 protein presents two main isoforms, Orb2A and Orb2B, which form an activity-induced amyloid-like functional aggregate, thought to be the translation-inducing state of the RNA-binding protein. This study presents a first two-states continuous differential model for Orb2A-Orb2B aggregation. This model provides new working hypotheses for studying the role of prion-like CPEB proteins in long-term synaptic plasticity. Moreover, this model can be used as a first step to integrate translation- and protein aggregation-dependent phenomena in synaptic facilitation rules.
Oroz, J., Felix, S. S., Cabrita, E. J. and Laurents, D. V. (2020). Structural transitions in Orb2 prion-like domain relevant for functional aggregation in memory consolidation. J Biol Chem. PubMed ID: 33093173
The recent structural elucidation of ex vivo Drosophila Orb2 fibrils revealed a novel amyloid formed by interdigitated Gln and His residue side chains belonging to the prion-like domain.  However, atomic-level details on the conformational transitions associated with memory consolidation remain unknown.  This study has characterized the nascent conformation and dynamics of the prion-like domain (PLD) of Orb2A using a nonconventional liquid-state NMR spectroscopy strategy based on (13)C detection to afford an essentially complete set of (13)Cα, (13)Cβ, (1)Hα and backbone (13)CO and (15)N assignments.  At pH 4, where His residues are protonated, the PLD is disordered and flexible, except for a partially populated α-helix spanning residues 55-60, and binds RNA oligos, but not divalent cations.  At pH 7, in contrast, His residues are predominantly neutral and the Q/H segments adopt minor populations of helical structure, show decreased mobility and start to self-associate.  At pH 7, the His residues do not bind RNA or Ca(++), but do bind Zn(++), which promotes further association. These findings represent a remarkable case of structural plasticity, based on which an updated model for Orb2A functional amyloidogenesis is suggested.
Gilmutdinov, R. A., Kozlov, E. N., Yakovlev, K. V., Olenina, L. V., Kotov, A. A., Barr, J., Zhukova, M. V., Schedl, P. and Shidlovskii, Y. V. (2021). The 3'UTR of the orb2 gene encoding the Drosophila CPEB translation factor plays a critical role in spermatogenesis. Development. PubMed ID: 34473243
CPEB proteins are conserved translation regulators involved in multiple biological processes. One of these proteins in Drosophila, Orb2, is a principal player in spermatogenesis. It is required for meiosis and spermatid differentiation. During the later process orb2 mRNAs and proteins are localized within the developing spermatid. To evaluate the role of orb2 mRNA 3'UTR in spermatogenesis, the CRISPR/Cas9 system was used to generate a deletion of the orb2 3'UTR, orb2R. This deletion disrupts the process of spermatid differentiation but has no apparent effect on meiosis. Differentiation abnormalities include defects in the initial polarization of the 64-cell spermatid cysts, mislocalization of mRNAs and proteins in the elongating spermatid tails, altered morphology of the elongating spermatid tails, and defects in the assembly of the individualization complex. These disruptions in differentiation appear to arise because orb2 mRNAs and proteins are not properly localized within the 64-cell spermatid cyst.


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, it was 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. This study 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 (Majundar, 2012).

Learning changes the efficacy and number of synaptic connections. Memory is the maintenance of that altered state over time. Synaptic modification is likely to include both quantitative and qualitative changes in local protein composition. However, this model of memory raises a fundamental question that remains unanswered: how does the altered protein composition of a synapse persist for years when the molecules that initiated the process should disappear within days (Majundar, 2012)?

The protein composition of a synapse can be altered in several ways including synthesis of new proteins. Local synthesis of new proteins at the synapse has been shown to be essential for stabilizing the functional changes and physical growth of the activated synapse. Previously, a family of RNA-binding proteins, known as cytoplasmic polyadenylation element-binding proteins (CPEBs), were identified as regulators of activity-dependent protein synthesis at the synapse (Alarcon, 2004; Atkins, 2004; Huang, 2002, Huang, 2003, Huang, 2006; Kundel, 2009; Si, 2003a; Wells, 2001; Wu, 1998). In the sea snail Aplysia, a neuron-specific variant of CPEB, ApCPEB, is required not for the initial changes in synaptic efficacy or growth following serotonin stimulation but for the maintenance of these changes beyond 24 hr (Miniaci, 2008; Si et al., 2003a, 2010). In Drosophila, reduction in Orb2, a member of the CPEB protein family, does not affect short-term memory (< 3 hr) but does prevent the memories from persisting beyond 12 hr (Keleman, 2007). In mice, deletion of the CPEB-1 gene reduces long-term potentiation evoked by theta-burst stimulation (Alarcon, 2004) and growth-hormone application (Zearfoss, 2008). Together, these observations suggest that CPEBs play a role in stabilizing activity-dependent changes in synaptic efficacy. However, how CPEB-dependent changes in molecular composition of the synapse persist over time is unknown (Majundar, 2012).

Previously, based on the self-sustaining amyloidogenic (prion-like) properties of Aplysia CPEB, it was hypothesized that the activity-dependent conversion of CPEB to the amyloidogenic state provides a self-sustaining mechanism for the persistent change in molecular composition of the synapse and thereby persistence of memory (Heinrich, 2011; Si, 2003a; Si, 2010). Consistent with this idea, in Aplysia sensory neurons ApCPEB forms amyloidogenic aggregates when overexpressed, and the number of aggregates increases following stimulation with serotonin (Si, 2010). Moreover, an antibody that recognizes oligomeric ApCPEB selectively blocks the persistence of long-term facilitation of the sensory-motor neuron synapse beyond 24 hr (Si, 2010). However, the central question of whether such conversion of neuronal CPEB to the amyloid-like state is necessary for the persistence of memory remains unanswered. To address the behavioral significance of the amyloid-like state of CPEB, Drosophila Orb2 was studied. Drosophila Orb2 protein carry a prion-like domain (Si, 2003b) and target synaptic growthrelated proteins, suggesting that Orb2 is not only structurally but also functionally similar to ApCPEB (Mastushita-Sakai, 2010; Majundar, 2012 and references therein).

This paper has asked two specific questions. First, does the Orb2 protein form amyloid-like oligomers in the adult Drosophila brain in an activity-dependent manner? Second, is this oligomerization necessary for long-term memory? It was found that Drosophila Orb2 forms stable SDS-resistant, amyloid-like oligomers upon neuronal stimulation, and Orb2 mutant defective in activity-dependent oligomerization is specifically impaired in forming stable long-term memories. These observations support the hypothesis that self-sustaining amyloid-like conversion of neuronal CPEB is involved in the persistence of memory (Majundar, 2012).

Previously, based on studies primarily with Aplysia CPEB, it was postulated that self-sustaining amyloidogenic oligomers, similar to yeast prion-like proteins, might be the basis of the persistence of activity-dependent increase in synaptic efficacy and the persistence of memory (Heinrich, 2011; Si, 2003b, Si, 2010). However some of the earlier analysis was performed under overexpression or in heterologous conditions. This study found that like other amyloids, in physiological concentration, in the adult Drosophila brain, Orb2 forms tetramers or hexamers that are resistant to heat, SDS, and chaotropic reagents. Stimulation of behaviorally relevant neurons increases the level of oligomeric Orb2, which is enriched in the synaptic membrane fraction. These observations suggest that the unusual amyloidogenic oligomerization of Orb2/CPEB is conserved across species, and the oligomer may indeed act to stabilize activity-dependent increase in synaptic efficacy (Majundar, 2012).

A mutation in the rare Orb2A isoform that results in reduced oligomerization, without lowering the amount of Orb2B protein, produces a selective deficit in the stabilization of memory beyond 24 hr. This is different than loss of both isoforms, which leads to earlier memory deficit. One interpretation of these data is that Orb2B activity is required for the formation of long-term memory, whereas Orb2A activity is required for the persistence of memory beyond 24 hr. However, both Orb2A and Orb2B form amyloidlike oligomers and when overexpressed in Dorb2/+ background can rescue the male courtship suppression memory (Keleman, 2007) as well as olfactory-reward memory at 24 hr, suggesting functional similarity. How can these observations be reconciled (Majundar, 2012)?

When expressed in S2 cells, both Orb2A and Orb2B act as translation repressor (Mastushita-Sakai, 2010). In the adult brain, the Orb2B protein is constitutively expressed in a large number of neurons, but the Orb2A protein is 100-fold less abundant, expressed in fewer neurons, and deletion of Orb2A reduces overall Orb2 oligomerization in vivo. Together, these results suggest the following model. Orb2B-mediated translation repression is critical for the formation and consolidation of memory up to 24 hr, and when ectopically expressed, Orb2A rescues this repressive function of Orb2B. However, regulated conversion of Orb2 proteins to the oligomeric state is necessary for long-term stabilization of memory beyond 24 hr, and the Orb2A protein regulates this conversion. This model implies that Orb2A and Orb2B have nonredundant functions in long-term memory, and neurons in which Orb2 oligomerizes are the site for long-term memory storage in Drosophila (Majundar, 2012).

The recent modENCODE project has reported four new protein isoforms in the Orb2 locus that would be affected in the Orb2 deletion mutants. However, the Orb2 oligomers or the behavioral phenotypes observed in this study are not dependent on these isoforms. The Orb2 antibodies used in this study do not recognize the common region between Orb2B and the new isoforms. Moreover, Orb2A and Orb2B cDNA as well as a genomic construct that does not code for any of these new proteins isoforms can rescue behavioral deficit. The function of these new isoforms has yet to be determined (Majundar, 2012).

Although the amyloidogenic forms of PrP (Prusiner, 1998) and other proteins are pathogenic, it is now evident that amyloids can underlie epigenetic heritable phenotypes in yeast (Alberti, 2009; Wickner, 2008) and can serve normal physiological functions in other organisms. However, in most cases it is unclear how amyloid formation is regulated, if at all. The low level of Orb2 oligomers in the adult brain and their virtual absence from the body raise the possibility that although Orb2B protein is widely expressed, Orb2 oligomerization per se is limited, perhaps only in neurons in which Orb2A is expressed. The Orb2A protein, due to its propensity to oligomerize, may form the seed that recruits Orb2B protein, resulting in the temporally and spatially restricted conversion of Orb2A/Orb2B into self-sustaining oligomers. In this regard, Orb2 oligomerization may resemble seeded formation of curli amyloid on the surface of bacteria, in which oligomerization of the major curli subunit CsgA is seeded by the membrane-bound minor subunit CsgB (Hammer, 2007; Majundar, 2012 and references therein).

Curiously, this study found that Orb2A mRNA in the adult brain retains an intronic sequence with stop codons. Among age-matched pCasper-Orb2AEGFP flies, heterogeneity in the Orb2A protein level was observed, particularly in the synaptic-neuropil region. The low abundance, presence of unprocessed mRNA, and immense propensity to oligomerize imply that in the adult head, expression of Orb2A is regulated and may constitute the rate-limiting step in Orb2 oligomerization and thereby long-lasting memory formation. Moreover, although Orb2A mRNA is present in the body (Si, 2003a), the Orb2A protein was undetectable, suggesting that it is present either in very low levels or only in certain cell types. What function Orb2A serves outside the nervous system is not known (Majundar, 2012).

It is now evident that a number of proteins with very different primary amino acid sequences can form self-templating amyloids (Toyama, 2011). What sequence and structural features distinguish a regulated functional amyloid from unregulated inactive or pathogenic amyloids? Although these studies were initiated with the observation that Aplysia CPEB contains a Q-rich unstructured domain, this study found that the Q-rich region of Orb2 is important for the stability but not formation of oligomer. Similarly, a coiled-coil domain outside the amyloid-forming domain of Aplysia CPEB regulates its oligomerization (Fiumara, 2010). These observations suggest that identification of functional amyloid based on primary amino acid sequence is challenging. Highlighting this point, it was found that a single nonpolar to polar amino acid change in the N-terminal 8 amino acids of Orb2A affects not only the efficiency of oligomerization but also the nature of the amyloid oligomer. Structural analysis of wild-type and mutant Orb2 proteins may help us to understand what features distinguish functional amyloid from nonfunctional amyloid (Majundar, 2012).

The Drosophila Lingerer protein cooperates with Orb2 in long-term memory formation

Recently mated Drosophila females were shown to be reluctant to copulate and to exhibit rejecting behavior when courted by a male. Males that experience mate refusal by a mated female subsequently attenuate their courtship effort toward not only mated females but also virgin females. This courtship suppression persists for more than a day, and thus represents long-term memory. The courtship long-term memory has been shown to be impaired in heterozygotes as well as homozygotes of mutants in orb2, a locus encoding a set of CPEB RNA-binding proteins. This study shows that the impaired courtship long-term memory in orb2-mutant heterozygotes is restored by reducing the activity of lingerer (lig), another putative RNA-binding protein gene, yet on its own the loss-of-function lig mutation is without effect. It was further shown that Lig forms a complex with Orb2. It is inferred that a reduction in the Lig levels compensates the Orb2 deficiency by mitigating the negative feedback for Orb2 expression and thereby alleviating defects in long-term memory (Kimura, 2014).

This study has successfully demonstrated that lig phenotypically interacts with orb2 by genetic experiments and that the Lig protein is physically associated with Orb2 as well as Orb, Rin and dFMR1 proteins by immunoprecipitation experiments. Orb and Orb2 belong to the CPEBs, a family of sequence-specific RNA binding proteins. Rin is a member of the G3BPs, which were originally characterized by their binding to the RasGAP SH3 domain. Recent experiments showed that G3BPs also contribute to RNA metabolism. FMR1 has been shown to bind to various mRNA elements, including a G-rich RNA structure, G-quartet, and U-rich stretches, to repress translation. In contrast, the biochemical functions of Lig remain unknown. The presence of an RGG box-like region in the Lig protein implies its RNA binding activity, although this activity has yet to be demonstrated experimentally. However, the genetic interaction of lig with three genes encoding RNA-binding proteins as well as the molecular association of Lig with these proteins strongly suggest that Lig contributes to the protein complex regulating RNA functions. In particular, it is of interest to note that Orb, G3BPs (Rin relatives) and dFMR1 are all contained in a messenger ribonucleoprotein (mRNP) complex by which mRNAs are transported to the correct destination and then translated. The mRNP complex also contains protein kinase C (PKC) and the receptor for activated PKC (RACK1). Interestingly, the Lig protein has multiple consensus target sites for protein kinase C-mediated phosphorylation, as does G3BP2 (Kimura, 2014).

Phosphorylation of these proteins might modulate the activity of mRNPs in regulating the transport and translation of mRNAs. We demonstrated that Lig, Orb, Rin and dFMR1 all colocalize in a dot-like structure in cellular processes of S2 cells. The association of dFMR1 with this structure has been reported by Ling et al. (2004), who have also shown, by time lapse imaging, that this 'granule' actually travels along the process in a kinesin-1-dependent manner. They conclude that the granule represents mRNP (Kimura, 2014).

In the nervous system, de novo synthesis of certain proteins takes place at synapses in an activity-dependent manner, leading to long-term potentiation or depression of neurotransmission specifically at the active synapses. For such local protein synthesis to occur, translationally dormant mRNPs need to travel from the soma to synaptic sites on dendrites. In the rat hippocampal slices, the amount of PKCβ2/RACK1 complexes bound to polyA-mRNAs is increased after mGluR1/5 (metabotropic glutamate receptor 1/5) stimulation, suggesting that synaptic activity alters the mRNP composition in dendrites (Kimura, 2014).

Such changes in mRNP may change the stability of mRNAs or translational states in the dendrites, ultimately affecting the synaptic structure and function and thus the behavioral outcome. In fact, loss of FMR1 alters the dendritic structure, which has been implicated in cognitive impairments in fragile X patients. A recent study showed that FMR1 increases the stability of mRNA by binding to the 3'UTR of the PSD-95 (postsynaptic density-95) mRNA. In contrast to FMR1, G3BP1 (a Rin homolog) has a phosphorylation-dependent endoribonuclease activity, which mediates c-myc mRNA cleavage. Thus FMR1-binding stabilizes mRNAs, whereas G3BP1 (Rin)-binding promotes degradation of mRNAs (Kimura, 2014).

This observation raises the intriguing possibility that the Lig-containing protein complex would bi-directionally control the synaptic efficacy by regulating the stability of mRNAs through the interactions with FMR1 and G3BP1 (Rin). It is noteworthy in this context that Orb2 binds to the 3'UTR of numerous mRNAs, resulting in the translational suppression of many of these targets (Matsushita-Sakai, 2010). orb2 mRNA is one such target (Matsushita-Sakai, 2010). It is envisaged that a reduction in Orb2 induced by a mutant copy of orb2 could be compensated by a simultaneous reduction in the amount of Lig by a mutant copy of lig. In fact, a recent study has shown that Lig upregulates rin transcription (not translation) in S2 cells (Baumgartner, 2013). It is therefore plausible that a reduction in lig activity could mitigate the orb2 mRNA degradation by decreasing the level of Rin, thereby restoring the memory deficits in orb2 heterozygotes. The effect of Lig on mRNA degradation might not manifest under the condition that Orb2 expression is stabilized by the mutually opposing actions of dFMR1 and Rin. Thus Lig could function as a homeostatic regulator of localized translation that confers robustness on the memory traces stored in a specific synapse. This hypothesis awaits a rigorous experimental test using a combinatorial approach aided by molecular imaging of synaptic activities and a sophisticated learning paradigm aided by use of a tethered fly (Kimura, 2014).

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. This study reports 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 (White-Grindley, 2014).

Previous work suggested that conversion of neuronal CPEB to an amyloid-like oligomeric state provides a molecular mechanism for the persistence of memory. However, it is not known how Orb2 oligomerization is regulated so that it will occur in a neuron/synapse-specific and activity-dependent manner. This study reports that factors that influence Orb2A stability and thereby abundance regulate Orb2 oligomerization (White-Grindley, 2014)

Tob, a previously known regulator of SMAD-dependent transcription and CPEB-mediated translation, associates with both forms of Orb2, but increases the half-life of only Orb2A. Stimulation with tyramine or activation of mushroom body neurons enhances the association of Tob with Orb2, and overexpression of Tob enhances Orb2 oligomerization. Both Orb2 and Tob are phosphoproteins. Phosphorylation destabilizes Orb2-associated Tob, whereas it stabilizes Orb2A. Tob promotes Orb2 phosphorylation by recruiting LimK, and PP2A controls the phosphorylation status of Orb2A and Orb2B (White-Grindley, 2014).

PP2A, an autocatalytic phosphatase, is known to act as a bidirectional switch in activity-dependent changes in synaptic activity. PP2A activity is down-regulated upon induction of long-term potentiation of hippocampal CA1 synapses (LTP) and up-regulated during long-term depression (LTD). Similarly, Lim Kinase, which is synthesized locally at the synapse in response to synaptic activation, is also critical for long-term changes in synaptic activity and synaptic growth (White-Grindley, 2014).

Based on these observations a model is proposed for activity-dependent and synapse-specific regulation of amyloid-like oligomerization of Orb2. It is postulated that in the basal state synaptic PP2A keeps the available Orb2A in an unphosphorylated and thereby unstable state. Neuronal stimulation results in synthesis of Orb2A by a yet unknown mechanism. The Tob protein that is constitutively present at the synapse binds to and stabilizes the unphosphorylated Orb2A and recruits the activated LimK to the Tob-Orb2 complex, allowing Orb2 phosphorylation. Concomitant decreases in PP2A activity and phosphorylation by other kinases enhances and increases Orb2A half-life. The increase in Orb2A level as well as phosphorylation may induce conformational change in Orb2A, which allows Orb2A to act as a seed. Alternatively, accumulation and oligomerization of Orb2A may create an environment that is conducive to overall Orb2 oligomerization. In the absence of an in vitro Orb2A-Orb2B oligomerization assay, it is not possible to distinguish between these two possibilities (White-Grindley, 2014).

For Tob, initial Orb2 association stabilizes Tob. However, association with Orb2 as well as suppression of PP2A activity leads to additional phosphorylation, which results in dissociation of Tob from the Orb2-Tob complex and destabilization. The destabilization of Orb2-associated Tob provides a temporal restriction to the Orb2 oligomerization process. The coincident inactivation of PP2A and activation of LimK may also provide a mechanism for stimulus specificity and synaptic restriction (White-Grindley, 2014).

Orb2A and Orb2B are phosphorylated at multiple sites, including serine/threonine and presumably tyrosine residues. These phosphorylation events are likely mediated by multiple kinases because overexpression of LimK did not affect Orb2 phosphorylation to the extent observed with the inhibition or activation of PP2A, raising several interesting questions. In what order do these phosphorylations occur? What function do they serve individually and in combination? What kinases are involved? Moreover, similar to mammalian CPEB family members, in addition to changing stability, phosphorylation may also influence the function of Orb2A and Orb2B (White-Grindley, 2014).

Does Tob regulate Orb2 function? In mammals Tob has been shown to recruit Caf1 to CPEB3 target mRNA, resulting in deadenylation, and CPEB3 is known to act as a translation repressor when ectopically expressed. This study found Drosophila Tob also interacts with Pop2/Caf1 and Orb2A and Orb2B can repress translation of some mRNA. Orb2 has also been identified as a modifier of Fragile-X Mental Retardation Protein (FMRP)-dependent translation, and Fragile-X is believed to act in translation repression (Cziko, 2009). Therefore, the Tob-Orb2 association may contribute to Orb2-dependent translation repression, and the degradation of Orb2-associated Tob may relieve translation repression. Additionally, if the oligomeric Orb2 has an altered affinity for either mRNA or other translation regulators, Tob can affect Orb2 function by inducing oligomerization. However, the relationship between Tob phosphorylation and its function is unclear at this point (White-Grindley, 2014).

Does involvement of Tob both in transcription and translation serve a specific purpose in the nervous system? Tob inhibits BMP-mediated activation of the Smad-family transcription activators (Smad 1/5/8) by promoting association of inhibitory Smads (Smad 6/7) with the activated receptor. In Drosophila BMP induces synaptic growth via activation of the Smad-family of transcriptional activators, and subsequent stabilization of these newly formed synapses via activation of LimK. These studies suggest Tob and LimK also regulate Orb2-dependent translation, raising the possibility Tob may coordinate transcriptional activation in the cell body to translational regulation in the synapse (White-Grindley, 2014).

Amyloidogenic oligomerization transforms Drosophila Orb2 from a translation repressor to an activator

Memories are thought to be formed in response to transient experiences, in part through changes in local protein synthesis at synapses. In Drosophila, the amyloidogenic (prion-like) state of the RNA binding protein Orb2 has been implicated in long-term memory, but how conformational conversion of Orb2 promotes memory formation is unclear. Combining in vitro and in vivo studies, this study finds that the monomeric form of Orb2 represses translation and removes mRNA poly(A) tails, while the oligomeric form enhances translation and elongates the poly(A) tails and imparts its translational state to the monomer. The CG13928 protein, which binds only to monomeric Orb2, promotes deadenylation, whereas the putative poly(A) binding protein CG4612 promotes oligomeric Orb2-dependent translation. These data support a model in which monomeric Orb2 keeps target mRNA in a translationally dormant state and experience-dependent conversion to the amyloidogenic state activates translation, resulting in persistent alteration of synaptic activity and stabilization of memory (Khan, 2015).

Previous studies have suggested that the self-sustaining amyloidogenic state of specific isoforms of neuronal CPEB produces an enduring mark in the activated synapse. However, the biochemical consequence of this enduring marking of the synapse remains largely unknown. This study reports that, when Drosophila Orb2 adopts the amyloid-like state, it gains a new function, and the translational-repressive function of the monomeric form of the protein changes to a translation-activating function. Therefore, Drosophila Orb2 provides an example of an amyloid-based protein switch that turns a repressor into an activator. Recently, regulated aggregation-dependent transcriptional or translational control has been reported by others (Berchowitz, 2015; Cai, 2014 and Kwo, 2013). Therefore, it is conceivable that, in multicellular eukaryotes, amyloid-like protein aggregations are used to create altered activity states and, in some cases, to create persistent change in cellular physiology from transient stimuli (Khan, 2015).

Similar to mammalian CPEB1, Orb2 acts as a repressor as well as an activator of translation through regulating poly(A) tail length (Udagawa, 2012). A U-rich sequence in the 3' UTR is important for the recruitment of Orb2 monomer and oligomer. The recruitment of the monomer results in shortening of the poly(A) tail and reduction in translation, while the oligomer protects and increases poly(A) tail length and translation. The following in vitro observations suggest that the oligomerization results in not just loss of monomeric function, but gain of a new function. (1) Addition of Orb2 oligomer in extract lacking any monomeric protein (orb2-/-) increases translation, suggesting that oligomeric Orb2 has activities of its own in addition to inhibiting the monomeric Orb2 function. (2) The oligomeric Orb2 binds mRNA, and it not only lacks the deadenylating activity of monomeric Orb2, but it also can add to the poly(A) tail and stabilize mRNA. And finally, (3) oligomeric Orb2 is the primary form of Orb2 that associates with polyribosomes in the adult neuron. Therefore, the net outcome of Orb2 oligomerization is reduction in deadenylation, increase in poly(A) tail length, stabilization of mRNA, and enhanced translation. However, the relative contributions of inhibition of deadenylation and elongation of the poly(A) tail in Orb2-oligomer-mediated translation are unclear (Khan, 2015).

Mechanistically, the zinc-finger domain containing protein CG13928 that only interacts with monomeric Orb2 enhances the repressive function. Since embryo extract lacking CG13928 can still support Orb2-dependent translation repression, CG13928 likely facilitates the recruitment of the deadenylation complex to the mRNA. The mechanistic basis of Orb2 oligomerization-mediated translation enhancement is less clear. Unlike monomeric Orb2, no oligomer-specific interacting protein has been identified, and although CG4612 is required for oligomerization-mediated translation, it binds to both forms of the Orb2 protein. However, the oligomer may enhance translation not only by recruitment of a specific protein, but also by increasing the affinity for existing interactors. For example, an mRNA bound by oligomeric Orb2 will have more CG4612 compared to a monomer-bound mRNA and, therefore, more of the CG4612-associated proteins. Such a mechanism would be reminiscent of CPEB1-mediated polyadenylation; unphosphorylated CPEB1 has weak affinity for the CPSF160 complex, and phosphorylation increases the affinity of CPEB1 for CPSF160, leading to polyadenylation and translation activation. In mammals, PABPs are multifunctional proteins and, in some cases, are known to activate translation by interacting with the translation initiation complex. Curiously, the other PABP, Pabp2, interacts with both forms of Orb2 yet does not have a significant effect on Orb2 function except for translation inhibition at a high concentration. Further studies would be required to dissect the detailed mechanism of translational activation and the role of PABPs and other proteins in Orb2-mediated translation activation (Khan, 2015).

Among the two isoforms of Orb2, Orb2A and Orb2B, no significant difference in mRNA binding, translation activity, or the kind of protein complexes that they form were found. However, unlike Orb2B, which is widely distributed, Orb2A is extremely low in abundance, expressed only in a subset of neurons, and is primarily synaptic. Therefore, based on their abundance and distribution, it is postulated that Orb2B regulates Orb2 target mRNA distribution, stability, and translation, while Orb2A controls where and when amyloid-like oligomerization and translational activation will ensue. Indeed, just the N-terminal prion-like domain of Orb2A is sufficient to support long-term memory, and it was found that the N-terminal domain of Orb2A is sufficient to induce Orb2 oligomerization and change the translational state of Orb2B (Khan, 2015).

Although direct evidence that the amyloidogenic oligomer regulates translation only in the activated synapse is still lacking, based on these observations, a plausible model is proposed for synapse-specific persistent translation. It is posited that Orb2B binds to the target mRNA and that the bound mRNA is transported to the synapses and kept in a repressed state via association with the deadenylation complex. Synaptic activation increases the local concentration of the low abundant Orb2A protein via phosphorylation (White-Grindley, 2014) and/or other yet unknown mechanisms. Increase in the Orb2A protein level triggers self-sustaining amyloidogenic oligomerization of Orb2A-Orb2B. Binding of the oligomer to the 3' UTR prevents deadenylation and recruits polyadenylation complex, and both of these events result in enhanced translation. Because of the self-sustaining and stable nature of the amyloid state, a local and self-sustaining translation activation of Orb2 target mRNA is created, maintaining the changed state of synaptic activity over time (Khan, 2015).

In mammals, there are four CPEB proteins, CPEB1-4, and all of them are expressed in the adult nervous system. Among these four isoforms, recent studies suggest a functional role of aggregated mammalian CPEB3 similar to Aplysia and Drosophila (Fioriti, 2015 and Stephan, 2015). CPEB3 forms amyloid-like oligomers in the adult hippocampus, and removal of CPEB3 from the hippocampus affects both the consolidation and expression of long-term memory (Fioriti, 2015). Functionally, CPEB3 regulates neuronal protein synthesis and ubiquitination, and SUMOylation regulates translation inhibitory function and aggregation of CPEB3 (Drisaldi, 2015 and Pavlopoulos), 2011). Intriguingly, constitutive removal of CPEB3 improved some forms of memory (Huang, 2014), although the mechanistic basis of the consequences of constitutive removal of CPEB3 remains unclear. Among the other CPEB family members, variants of CPEB2 have putative prion-like domains, although it remains to be seen whether they confer prion-like properties and whether CPEB2 is required for long-term memory (Khan, 2015).

Models for the biochemical basis of long-lasting memory anticipated an experience-dependent molecular switch and included autolytic kinases and even prions. It has been proposed that such a biochemical switch of long-lasting memory would possess certain properties: (1) be engaged by a temporally defined physiological stimulus, (2) form in response to some, but not all, experiences, (3) produce a change in the neuronal properties that elicit appropriate behavioral responses, and (4) deal with the natural turnover of individual proteins to enable a persistent change in behavioral output (Khan, 2015).

The emerging evidence from Aplysia, Drosophila, and Mouse suggests that the self-sustaining aggregates of CPEB may be a biochemical switch involved in at least some forms of long-term memory for the following reasons. (1) Activity-dependent conversion to the amyloidogenic state suggests that it can be engaged by behavioral training. (2) Phosphorylation, SUMOylation, and other mechanisms can confer the specificity and selectivity to amyloidogenic conversion. (3) Orb2-dependent translation of mRNAs can alter the protein composition of the synapse, thereby altering synaptic properties and neuronal output. Finally, once triggered, the stable and self-sustaining capacity of the amyloidogenic state would outlast turnover of individual molecules to sustain memory over the long term. However, some key questions remain unanswered. How and what ensures selective engagement of the self-sustaining state of neuronal CPEB only in response to long-term memory inducing stimuli? Once engaged, how long does it persist, and is the continued presence of the amyloidogenic state necessary for the persistence of memory? Is amyloid formation correlated with or predictive of long-lasting memory? Can a transient memory be stabilized by artificial recruitment of the amyloidogenic state (Khan, 2015)?

Orb2 as modulator of Brat and their role at the neuromuscular junction

How synapses are built and dismantled is a central question in neurobiology. A wide range of proteins and processes from gene transcription to protein degradation are involved. Orb2 regulates mRNA translation depending on its monomeric or oligomeric state to modulate nervous system development and memory. Orb2 is expressed in Drosophila larval brain and neuromuscular junction (NMJ), Orb2 knockdown causes a reduction of synapse number and defects in neuronal morphology. Brain tumor (Brat) is an Orb2 target; it is expressed in larval brain related with cell growth and proliferation. Brat downregulation induces an increase in synapse number and abnormal growth of buttons and branches in neurons. In absence of Orb2, Brat is overexpressed suggesting that Orb2 is negatively regulating Brat mRNA translation. Orb2 or Brat control the expression of specific genes related to neuronal function. Orb2 is required for Liprin and Synaptobrevin transcription meanwhile Brat is required for Synaptobrevin and Synaptotagmin transcription. This study presents evidences of a novel genetic mechanism to regulate synapse fine tuning during development and propose an equilibrium between Orb2 conformational state and nervous system formation (Santana, 2017).

How synapses are built and the genetic relationship between partners involved in synapse formation is poorly understood. The current data propose a molecular mechanism where Brat and Orb2 genetically interact and modulate the expression of synaptic proteins for the correct development of the NMJ. Orb2 is a regulator of translation of specific mRNAs after synaptic stimulation. Its role in the adult brain as a key player controlling learning and memory has been widely studied. However, little is known about Orb2 role during nervous system development. This study has explored the role of Orb2 in NMJ and brain development through the regulation of Brat. It is demonstrate that Orb2 plays a central role during NMJ development. Although Orb2 downregulation reduces synapse number, Orb2 upregulation is not sufficient to generate new active zones. Orb2 protein can adopt different conformational states. Upon synaptic stimulation Orb2 changes its conformational state and interacts with target mRNAs. It is possible that synapse stimulation and Orb2 conformational changes are necessary to induce synaptogenesis. Therefore, Orb2 upregulation would require additional events to yield synaptic changes (Santana, 2017).

Orb2 directly activates translation of other synaptic proteins as neuroligin, branchless or synaptobrevin. These data are compatible with real-time qPCR results that show no significant changes in Syb or Brp transcription after Orb2 reduction. Moreover, a reduction was observed in the number of synaptic spots labeled with Brp. It is suggested that Orb2 is activating or repressing mRNA translation of a set of synaptic proteins involved in synaptic formation and stabilization. Orb2 down-regulation might be affecting some of these proteins and as a consequence induces a reduction in the NMJ active zones represented by a decrease in Brp spots. Further experiments and deeper analysis of mRNA partners of Orb2 and its relation with Brp might be done to elucidate this question (Santana, 2017).

Besides, Orb2 interacts with Brat mRNA. Orb2 can activate or repress translation depending on the conformational state. This study has provided evidences that Orb2 repress Brat translation, possibly through its monomeric form. In conclusion, this study has shown that Brat downregulation increases the number of active zones. However, Brat upregulation does not affect synapse number. Given that Brat (as Orb2) is a repressor of translation in combination with other factors, Nanos and Pumilio, it is plausible that Brat alone may not suffice to have a significant effect on synaptic genes. Volume is not affected by Brat or Orb2 modifications, suggesting at least two different signaling pathways, one related with growth and other with synapse formation and stabilization. This hypothesis correlates with qPCR results where transcription levels of genes related with active zones formation (Brp) are altered but not with genes related with vesicle trafficking (Syb) (Santana, 2017).

These evidences suggest that the increase in synapse number is not sufficient to properly build an NMJ. Moreover, Brat or Orb2 regulation is not enough to induce an effect in NMJ formation. Proteins related with synaptic transmission, trafficking and other factors are involved and NMJs showing an increase in synapse number, branches and boutons can show otherwise defects in synapse transmission and vesicle trafficking. It is proposed that both proteins regulate a set of transcription factors which, in turn, modulate the expression of other genes. In conclusion, the architecture of an NMJ includes a collection of interrelated players which are in equilibrium to maintain motor neuron integrity (Santana, 2017).

Cryo-EM structure of a neuronal functional amyloid implicated in memory persistence in Drosophila

How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element-binding (CPEB) protein, is a putative substrate of long-lasting memories. This study isolated aggregated Drosophila CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo-electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and 'seed' for further translationally active aggregation. The 31-amino acid protofilament core adopted a cross-beta unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory (Hervas, 2020).

A memory, once formed, is maintained in a physical state that does not require continuous presence of the original experience. The substrate of memory has been extensively studied at the neuronal network level. However, how the underlying biochemical changes, induced by initial experience, withstand degradation to maintain altered network properties-and hence, memory-remains unclear. One proposed solution is a self-renewing protein system that can recruit and modify newly made constituents as older ones are degraded. A prionlike assembly has the ability to promote the conformational conversion of other existing or newly formed monomers, creating a selfsustaining protein 'conformational memory' that outlives its individual constituents. A prion-like protein with a defined role in memory in different species is the mRNA binding cytoplasmic polyadenylation element-binding (CPEB) family of proteins. In Drosophila melanogaster, inhibition of Orb2 aggregation interferes with memory consolidation, facilitation of Orb2 aggregation lowers the threshold for long-term memory formation, and inactivation of Orb2 after memory formation interferes with memory recall. These observations suggest that the aggregated state of Orb2 creates a protein conformational memory (Hervas, 2020).

Many prion-like proteins, prevalent among RNA-binding protein, form amyloids, which are filamentous protein aggregates composed of cross-β cores. However, in the nervous system, amyloids are commonly associated with disease. Recently, new classes of protein assemblies formed by prion-like proteins have been reported, such as the nonamyloid filaments or the gel-like state, physical states that are believed to be more amenable to regulation. These observations raise a fundamental question: What is the physical nature of the Orb2 aggregate in the brain that acts as a biochemical substrate of memory? To characterize the function and structure of endogenous Orb2 aggregates, this study purified Orb2 protein from ~3 million 3- to 7-day-old adult D. melanogaster heads. There was no qualitative difference between Orb2 purified from synaptosomes or total head extracts, and whole-head Orb2 was used in subsequent experiments. Purified Orb2 contained monomers as well as heat- and SDS-resistant aggregates. Quantitative mass spectrometry [liquid chromatography / tandem mass spectrometry (LC-MS)/MS] revealed that 97.5% of the purified protein is Orb2. Unlike in the adult nervous system, Orb2 protein is monomeric in the early embryo. Orb2 purified from 0- to 2-hour-old embryos remained monomeric, suggesting that the purification does not induce Orb2 aggregation (Hervas, 2020).

Size-exclusion chromatography revealed monomeric (75 to 160 kDa), oligomeric (160 to 670 kDa), and aggregated (>1 MDa) Orb2 in the adult head. Monomers were not visible, and oligomers appeared as heterogeneous globules, which is reminiscent of oligomers of other amyloid-forming proteins. The aggregates appeared as unbranched helical filaments with an average length of ~750 Å, width of ~100 Å, and helical crossover distance of ~550 Å. JJJ2, a yeast DnaJ-domain protein, enhances Orb2 aggregation and facilitates long-term memory formation in Drosophila. JJJ2, but not its close relative JJJ3, converted Orb2 oligomers into filaments, suggesting that oligomers are on-pathway precursors for filaments. Incubation of Orb2 monomers with traces of Orb2 filaments induced monomer aggregation in a time-dependent manner, and seeded-aggregates themselves acted as a seed. Orb2 monomer alone did not aggregate in the same time period, and Orb2 filaments did not cause aggregation of monomeric heterologous prion-like RNA-binding protein RBM3. Both endogenous and seeded Orb2 filaments bound Thioflavin T, were recognized by the amyloid-specific antibody OC, and were resistant to protease treatment. Orb2 binds to the 3' untranslated regions (UTRs) of mRNAs; monomeric Orb2, which interacts with CG13928, decreases translation, whereas aggregated Orb2, which interacts with CG4612, enhances translation. Tequila, a protease, is one of the mRNA targets of Orb2 and required for long-term memory. All purified Orb2 species bound to the 3'UTR of Tequila mRNA, and mutations in the Orb2-binding site UUUUGU to GCUUGU reduced binding of all species. Gold-labeled wild type, but not mutant (M2P) mRNA, colocalized with the oligomers and filaments. In addition to mRNA, when incubated with recombinant binding partners, Orb2 filaments bound only to GC4612. N-terminal-deleted CG4612, CG4612D, and monomer binding-partner CG13928 did not bind to Orb2 filaments. When incubated with wild-type Tequila 3'UTR mRNA and CG4612 protein, the Orb2 filaments were decorated with both mRNA and CG4612 protein. In an Orb2- dependent translation assay, the addition of the Orb2 monomers reduced translation, whereas oligomers and filaments, both endogenous and seeded, enhanced translation. Thus, Orb2 filament purified from fly retains aspects of its physical and functional properties (Hervas, 2020).

Cryo-electron microscopy (cryoEM) was used to determine the atomic structure of Orb2 filaments, which were manually picked and used for helical reconstruction in RELION. The final reconstruction, at an overall resolution of 2.6 Å, showed that the Orb2 filament is composed of three identical protofilaments related by C3 symmetry with separated β-strands along the helical axis. The filament core has a triangular cross section, with a side of ~80 Å and a 15- Å diameter channel along the helical axis. Successive rungs of three hairpin-shaped protofilaments form extended b-sheets along the helical axis, which is typical of the cross-b amyloid structure, with a twist of -1.55° and a rise of 4.75 Å. Surrounding the stable fibril core, lower-resolution densities are visible that likely correspond to the RNA-recognition motif and protein interaction domain of Orb2. We could identify densities that correspond to peptide-group oxygen atoms, ordered solvent molecules in each interface, and alternative conformations of histidine side chains. Consequently, it was possible to build and stereochemically refine an atomic model de novo. The resulting model of the amyloid core comprised 31 amino acid residues from the glutamine-rich prion-like domain of Orb2. Orb2 protein has two isoforms in the fly brain that share the prion-like domain but differ in their N-terminal extension. The low abundant Orb2A seeds aggregation of prevalent Orb2B, which can subsequently seed Orb2B protein. Of the protein in the sample, 97.5% is Orb2B, and the protofilament core structure extends from residues 176 to 206 of Orb2B (Hervas, 2020).

The protofilament core adopts a simple hairpin-like fold, composed of two β-strands, β1 (residues 176 to 186) and β2 (residues 197 to 206), with a wide turn (residues 187 to 196) in between. Perpendicular to the helical axis, a difference in height of 6 Å between the highest point in the turn and the lowest point in the tip of the β2 allows an ordered hydrogen bonding network of the β-stranded regions. The cross-b packing of the two β-strands is made of a tight interdigitation of glutamine residues, Q179, Q181, Q183, and Q185 from β1 and Q200, Q202, and Q204 from β2. A single leucine, L198, separates this block of seven glutamines from a cluster of four more interior glutamine residues in the hairpin turn: Q187, Q190, Q193, and Q196. Next to H189, there is an additional nonproteinaceous density that runs contiguously across the rungs. This density is compatible with linear chains of aliphatic hydrocarbons and polyamines, which may have copurified with Orb2. The three equivalent protofilament packing interfaces are made of eight polar residues: five glutamines and three histidines. These polar residues form an extensive network of hydrogen bonds on each side and across the interface. Orb2 mutations, known to disrupt Orb2 aggregation, were mapped on the polar interface between protofilaments and the cross-β packing that stabilizes the protofilament (Hervas, 2020).

Moreover, removal of 54 amino acids from the glutamine-rich region specifically interfered with long-term, but not short-term, memory. Several structures of functional amyloids, produced in vitro from truncated protein, have been solved. By contrast, Orb2 filament represents a biochemically active full-length functional amyloid extracted from the endogenous source. The threefold symmetry of the ordered core of Orb2 resembles that of b-amyloid 1-40 filaments seeded from Alzheimer's disease brain tissue or assembled in vitro. However, unlike the hydrophobic β-amyloid 1-40 core, Orb2 forms a hydrophilic core stabilized by interdigitated glutamines. When Orb2 filaments were assembled from recombinant protein, filaments were longer, morphologically distinct, and less biochemically active and had negligible seeding capacity compared with endogenous ones under the experimental conditions, suggesting that the same prion-like protein can adopt distinct structures in vitro and in vivo. Other proteins with glutamine-rich sequences are known to produce amyloids. The interdigitated cross-β structure observed in Orb2 filaments could be extended on both sides of a parallel β-sheet made of only glutamine residues, which would allow for the formation of stable, multilayered cross-β structures from long polyglutamine sequences, such as in pathological glutamine expansions (Hervas, 2020).

There are a number of ways that 'molecular memories' can be created: increase in the amount of a protein, where the kinetics of decay to the basal state would be the duration of memory; a stable protein or protein state, the half-life of which would inform the duration of memory; or a feed-forward molecular circuit, whose activity could be reciprocally perpetuated across time. A self-sustaining amyloid formed by a single polypeptide uses all three mechanisms. The amyloid fold itself is not 'memory,' but the amyloid fold reorganizes the rest of the Orb2 protein to create a persistent alteration in the synthesis of specific synaptic proteins. Memory is the altered synaptic state that results from the change in functional state of the Orb2. However, amyloid formation is generally assumed to be irreversible in physiological conditions; then, how can memory be dynamic? Or is it possible that some memories are irreversible and appear lost merely because of an inability to retrieve them? First, amyloids are not necessarily irreversible and could exist in a dynamic equilibrium with the available monomer. Second, the Orb2 amyloid core is based on a hydrophilic, glutamine- and histidine-rich fold, and the protonation state of histidine residues could influence Orb2 amyloid stability. Lowering pH destabilized Orb2 filaments, suggesting that functional amyloid could be amenable to modification or even dissolution. The current findings question the assumption that amyloid formation in the brain is always an unintended consequence that leads to dysfunction. It is postulated that the brain fosters a cellular environment that is permissive to the formation of an amyloid-like state of certain proteins in order to meet the diversity of functional requirements imposed on it (Hervas, 2020).

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. This study has 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 (Mastushita-Sakai, 2010).

This study used a candidate gene approach combined with a genome-wide screen to identify potential Drosophila Orb2 targets. Surprisingly, the use of orb2 null flies, despite inherent limitations, turned out to be quite useful in identifying Orb2 targets. Clearly, all of the Orb2 targets have yet to be determined, and it is yet to be determined if the protein levels of these targets are indeed altered in response to a learning-related stimulus in an Orb2-dependent manner. However, the identification of these targets provides clues to Orb2 function and a plausible molecular makeup of the long-lasting memory trace (Mastushita-Sakai, 2010).

The targets can be broadly classified into the following groups: (1) genes involved neuronal growth and synapse formation, (2) genes involved in synaptic function, (3) genes involved in proteolysis, and (4) genes with unknown (predicted) functions. Although direct evidence is lacking, it is postulated that, in the adult fly brain, Orb2-dependent persistent regulation of these target genes in the activated synapse might stabilize altered synaptic function and synapse number and thus, memory in the following ways. First, the suppression of ubiquitin-mediated protein degradation machinery, as well as other proteases, might allow for the accumulation of otherwise unstable molecules. In contrast, activation of the proteosomal pathway and proteases might remove inhibitory constraints at the synapse to create and maintain a permissive environment for synaptic growth. The involvement of Ubiquitin carboxy-terminal hydrolase (Uch) and ubiquitin-mediated protein-turnover pathways in synaptic plasticity has previously been observed in Aplysia and in mice. Because ubiquitination can act as a posttranslational modification in addition to a signal for degradation, the ubiquitination pathway can, in principle, modulate stability, function, or membrane distribution of proteins at the activated synapse. Second, the cell polarity and asymmetric growth genes, such as atypical protein kinase C (DaPKC), widerborst, and glaikit, might allow the activated synapse to capture or modify the globally distributed gene product selectively at the activated synapse. In Drosophila, DaPKC is required for the formation of glutamatergic synapses in the NMJ. DaPKC is believed to be a homolog of mouse atypical protein kinase C (PKMζ), and in mice, the continuous activity of PKMζ is necessary for the persistence of memory for months (Shima, 2007). Interestingly, in mice, PKMζ mRNA is localized in the dendrites (Muslimov, 2004). Taken together, these observations in flies and mice raise the possibility that Orb2-dependent regulation of DapKC/PKMζ at the activated synapse contributes to memory-related synaptic growth. Finally, the regulation of the cytoskeleton-remodeling complexes, such as Capulet, Still-life, and Actin 5C, initiators of de novo synapse formation, such as Neuroligin, and growth regulators, such as Brain tumor, Pathetic, and Branchless , might regulate the maintenance of newly grown synapse for a long time. Future studies should be directed to addressing these postulates (Mastushita-Sakai, 2010).

Drosophila CPEB Orb2A mediates memory independent of its RNA-binding domain

Long-term memory and synaptic plasticity are thought to require the synthesis of new proteins at activated synapses. The CPEB family of RNA binding proteins, including Drosophila Orb2, has been implicated in this process. The precise mechanism by which these molecules regulate memory formation is however poorly understood.Gene targeting and site-specific transgenesis was used to specifically modify the endogenous orb2 gene in order to investigate its role in long-term memory formation. The Orb2A and Orb2B isoforms, while both essential, have distinct functions in memory formation. These two isoforms have common glutamine-rich and RNA-binding domains, yet Orb2A uniquely requires the former and Orb2B the latter. It was further shown that Orb2A induces Orb2 complexes in a manner dependent upon both its glutamine-rich region and neuronal activity. It is proposed that Orb2B acts as a conventional CPEB to regulate transport and/or translation of specific mRNAs, whereas Orb2A acts in an unconventional manner to form stable Orb2 complexes that are essential for memory to persist (Kruttner, 2012).

Local translation of mRNAs in both pre- and postsynaptic compartments is thought to be important for the synaptic modifications that underlie long-lasting memories. The CPEB family of proteins regulate local translation, and the Drosophila CPEB protein Orb2 is acutely required for long-term memory. However, the detailed molecular mechanism of CPEB function in synaptic plasticity and memory formation remains elusive (Kruttner, 2012).

This study has shown that the two Orb2 isoforms, Orb2A and Orb2B, both contribute to long-term memory formation, albeit by distinct mechanisms. The two isoforms share the same RNA-binding and Q domains, yet each uniquely requires only one of these domains for its function in long-term memory formation. Specifically, the Q domain is essential in Orb2A but not Orb2B, whereas the RNA-binding domain is required in Orb2B but not Orb2A. Moreover, it was found that Orb2A lacking its RNA-binding domain is able to fully complement Orb2B lacking its Q domain. Such interallelic complementation often reflects the formation of the heteromeric complexes between the encoded proteins, and indeed it was observed that Orb2A and Orb2B are present in the same protein complexes in vitro and in vivo, and that formation of these heteromeric Orb2A:Orb2B complexes acutely depends on Orb2A and its Q domain. Moreover, these complexes are induced within 6 hr after feeding with biogenic amines (thought to provide learning signals relevant for memory formation), corresponding to the time course of memory decay in Orb2ΔQ mutants. It is therefore proposed that Orb2A:Orb2B heteromeric complexes are induced at specific synapses by the relevant learning signals and required for memory persistence beyond 6 hr (Kruttner, 2012).

Dopamine is thought to provide a reinforcement signal in Drosophila courtship learning. For short-term memory this dopamine signal is provided by neurons that innervate the gamma lobe of the mushroom body, and for long-term memory Orb2 is required in intrinsic gamma lobe neurons. Gamma lobe synapses are thus a likely site of Orb2 complex formation and the structural and functional modifications that underlie courtship learning in Drosophila. Orb2 also functions in long-term memory in an appetitive learning paradigm, which likely maps to a distinct class of mushroom body neuron. Indeed, specific long-term memories may be stored at various sites in the fly brain, extending even beyond the mushroom body. The broad distribution of Orb2 throughout the nervous system suggests that it may contribute generally to long-term synaptic plasticity and memory formation, regardless of where these memories are stored (Kruttner, 2012).

Why might Orb2A have such a critical role in Orb2 complex formation and long-term memory, when most of its residues are shared with the evidently more abundant Orb2B, including the Q and RNA-binding domains? The efficacy of complex formation of proteins containing Q domains is thought to be determined by the length of the preceding N-terminal sequences. In this regard it is interesting to note that Orb2A has an N-terminal extension of 9 amino acids, compared to the 162 N-terminal residues of Orb2B. Additionally, a single point mutation in the unique Orb2A N-terminal extension decreases Orb2 multimer formation in the Drosophila brain and impairs long-term memory retention beyond 48 hr (Majumdar, 2012). Thus, both the size and sequence of Orb2A's unique N-terminal extension might endow it with a greater propensity to aggregate than Orb2B, and thereby nucleate heteromeric Orb2 complexes through the Q domain of Orb2A (Kruttner, 2012).

It has been suggested that the activation of Orb2 and other CPEB proteins occurs via the prion-like properties of their Q domains (Heinrich, 2011; Krishnan, 2005; Majumdar, 2012; Si, 2003; Si, 2010). Such Q domains occur however in a wide range of proteins with diverse biochemical functions, in which they are generally thought to mediate homo- and heterotypic interactions. In some of these proteins, for example, the Q domains serve as polar zippers in the assembly of large multimeric complexes. Whatever the means by which the Q domain of Orb2 contributes to complex formation, the current data suggest that this is restricted to Orb2A, as Orb2B does not require its Q domain to interact with Orb2A and function in long-term memory formation. This mechanism is likely to be conserved among CPEB proteins, as the Q domain of Orb2A can be replaced with the analogous domain from CPEBs of Aplysia and mouse, but not with the prion domain of ScUre2 (Kruttner, 2012).

The data support and extend a model (Majumdar, 2012) in which the two Orb2 isoforms form heteromeric complexes that are essential for long-term memory formation. It is further proposed that, upon neuronal stimulation, Orb2A, which may be present in more limiting amounts, restricted locations, or under specific circumstances, provides the spatial and temporal specificity for heteromeric complex formation and synaptic plasticity. Orb2B, in contrast, appears to be more broadly and highly expressed and may mediate a more general function of Orb2 in development (Cziko, 2009; Hafer, 2011; Richter, 2007; Shieh and Bonini, 2011). Orb2 has been reported to be present in the messenger RNPs, as we have also observed here specifically for Orb2B, and is thought to control mRNA transport and translational repression. During learning, Orb2A might interact with Orb2B-containing RNPs at the relevant synapses, releasing the associated mRNAs from translational repression or possibly even converting Orb2B from a translation repressor to an activator (Kruttner, 2012).

CPEB molecules are conserved across a wide range of species and most of them exist in multiple isoforms generated through alternative splicing, often varying only in their N terminus. This is the case for mCPEB3, for example, the Q domain of which is able to substitute for the Q domain of Orb2A both biochemically and behaviorally. It is tempting to speculate that the model proposed in this study is not unique for Drosophila Orb2 but might also extend to other members of the CPEB family. Moreover, because Orb2A functions in long-term memory without its RNA-binding domain, it is possible that proteins lacking an RNA-binding domain, and hence not even recognized as canonical CPEB molecules, might function in a fashion analogous to Orb2A in Drosophila and other species (Kruttner, 2012).

Function of the Drosophila CPEB protein Orb2 in long-term courtship memory

Both long-term behavioral memory and synaptic plasticity require protein synthesis, some of which may occur locally at specific synapses. Cytoplasmic polyadenylation element-binding (CPEB) proteins are thought to contribute to the local protein synthesis that underlies long-term changes in synaptic efficacy, but a role has not been established for them in the formation of long-term behavioral memory. This study found that the Drosophila melanogaster CPEB protein Orb2 is acutely required for long-term conditioning of male courtship behavior. Deletion of the N-terminal glutamine-rich region of Orb2 resulted in flies that were impaired in their ability to form long-term, but not short-term, memory. Memory was restored by expressing Orb2 selectively in fruitless (fru)-positive gamma neurons of the mushroom bodies and by providing Orb2 function in mushroom bodies only during and shortly after training. These data thus demonstrate that a CPEB protein is important in long-term memory and map the molecular, spatial and temporal requirements for its function in memory formation (Keleman, 2007).

The CPEB protein Orb2 has multiple functions during spermatogenesis in Drosophila melanogaster

Cytoplasmic Polyadenylation Element Binding (CPEB) proteins are translational regulators that can either activate or repress translation depending on the target mRNA and the specific biological context. There are two CPEB subfamilies and most animals have one or more genes from each. Drosophila has a single CPEB gene, orb and orb2, from each subfamily. orb expression is only detected at high levels in the germline and has critical functions in oogenesis but not spermatogenesis. By contrast, orb2 is broadly expressed in the soma; and previous studies have revealed important functions in asymmetric cell division, viability, motor function, learning, and memory. This study shows that orb2 is also expressed in the adult male germline and that it has essential functions in programming the progression of spermatogenesis from meiosis through differentiation. Like the translational regulators boule (bol) and off-schedule (ofs), orb2 is required for meiosis and orb2 mutant spermatocytes undergo a prolonged arrest during the meiotic G2-M transition. However, orb2 differs from boule and off-schedule in that this arrest occurs at a later step in meiotic progression after the synthesis of the meiotic regulator twine. orb2 is also required for the orderly differentiation of the spermatids after meiosis is complete. The differentiation defects in orb2 mutants include abnormal elongation of the spermatid flagellar axonemes, a failure in individualization and improper post-meiotic gene expression. Amongst the orb2 differentiation targets are orb and two other mRNAs, which are transcribed post-meiotically and localized to the tip of the flagellar axonemes. Additionally, analysis of a partial loss of function orb2 mutant suggests that the orb2 differentiation phenotypes are independent of the earlier arrest in meiosis (Xu, 2012).

The Drosophila CPEB protein Orb2 has a novel expression pattern and is important for asymmetric cell division and nervous system function

Cytoplasmic polyadenylation element binding (CPEB) proteins bind mRNAs to regulate their localization and translation. While the first CPEBs discovered were germline specific, subsequent studies indicate that CPEBs also function in many somatic tissues including the nervous system. Drosophila has two CPEB family members. One of these, orb, plays a key role in the establishment of polarity axes in the developing egg and early embryo, but has no known somatic functions or expression outside of the germline. This study characterized the other Drosophila CPEB, orb2. Unlike orb, orb2 mRNA and protein are found throughout development in many different somatic tissues. While orb2 mRNA and protein of maternal origin are distributed uniformly in early embryos, this pattern changes as development proceeds and by midembryogenesis the highest levels are found in the CNS and PNS. In the embryonic CNS, Orb2 appears to be concentrated in cell bodies and mostly absent from the longitudinal and commissural axon tracts. In contrast, in the adult brain, the protein is seen in axonal and dendritic terminals. Lethal effects are observed for both RNAi knockdowns and orb2 mutant alleles while surviving adults display locomotion and behavioral defects. It was also shown that orb2 funtions in asymmetric division of stem cells and precursor cells during the development of the embryonic nervous system and mesoderm (Hafer, 2011).

Genes and pathways affected by CAG-repeat RNA-based toxicity in Drosophila

Spinocerebellar ataxia type 3 is one of the polyglutamine (polyQ) diseases, which are caused by a CAG-repeat expansion within the coding region of the associated genes. The CAG repeat specifies glutamine, and the expanded polyQ domain mutation confers dominant toxicity on the protein. Traditionally, studies have focused on protein toxicity in polyQ disease mechanisms. Recent findings, however, demonstrate that the CAG-repeat RNA, which encodes the toxic polyQ protein, also contributes to the disease in Drosophila. To provide insights into the nature of the RNA toxicity, we extracted brain-enriched RNA from flies expressing a toxic CAG-repeat mRNA (CAG100) and a non-toxic interrupted CAA/G mRNA repeat (CAA/G105) for microarray analysis. This approach identified 160 genes that are differentially expressed specifically in CAG100 flies. Functional annotation clustering analysis revealed several broad ontologies enriched in the CAG100 gene list, including iron ion binding and nucleotide binding. Intriguingly, transcripts for the Hsp70 genes, a powerful suppressor of polyQ and other human neurodegenerative diseases, were also upregulated. Therefore it was tested and shown that upregulation of heat shock protein 70 mitigates CAG-repeat RNA toxicity. It was then assessed whether other modifiers of the pathogenic, expanded Ataxin-3 polyQ protein could also modify the CAG-repeat RNA toxicity. This approach identified the co-chaperone Tpr2, the transcriptional regulator Dpld, and the RNA-binding protein Orb2 as modifiers of both polyQ protein toxicity and CAG-repeat RNA-based toxicity. These findings suggest an overlap in the mechanisms of RNA and protein-based toxicity, providing insights into the pathogenicity of the RNA in polyQ disease (Shieh, 2011).

Cytoplasmic polyadenylation element binding protein is a conserved target of tumor suppressor HRPT2/CDC73

Parafibromin, a tumor suppressor protein encoded by HRPT2/CDC73 and implicated in parathyroid cancer and the hyperparathyroidism-jaw tumor (HPT-JT) familial cancer syndrome, is part of the PAF1 transcriptional regulatory complex. Parafibromin has been implicated in apoptosis and growth arrest, but the mechanism by which its loss of function promotes neoplasia is poorly understood. This study reports that a hypomorphic allele of hyrax (hyx), the Drosophila homolog of HRPT2/CDC73, rescues the loss-of-ventral-eye phenotype of lobe (Akt1s1). Such rescue is consistent with previous reports that hyx/parafibromin is required for the nuclear transduction of Wingless (Wg)/Wnt signals and that Wg signaling antagonizes lobe function. A screen using double hyx/lobe heterozygotes identified an additional interaction with orb and orb2, the homologs of mammalian cytoplasmic polyadenylation element binding protein (CPEB), a translational regulatory protein. Hyx and orb2 heterozygotes lived longer and were more resistant to starvation than controls. In mammalian cells, knockdown of parafibromin expression reduced levels of CPEB1. Chromatin immunoprecipitation (ChIP) showed occupancy of CPEB1 by endogenous parafibromin. Bioinformatic analysis revealed a significant overlap between human transcripts potentially regulated by parafibromin and CPEB. These results show that parafibromin may exert both transcriptional and, through CPEB, translational control over a subset of target genes and that loss of parafibromin (and CPEB) function may promote tumorigenesis in part by conferring resistance to nutritional stress (Zhang, 2010).

Genetic modifiers of dFMR1 encode RNA granule components in Drosophila

Mechanisms of neuronal mRNA localization and translation are of considerable biological interest. Spatially regulated mRNA translation contributes to cell-fate decisions and axon guidance during development, as well as to long-term synaptic plasticity in adulthood. The Fragile-X Mental Retardation protein (FMRP/dFMR1) is one of the best-studied neuronal translational control molecules and this study describes the identification and early characterization of proteins likely to function in the dFMR1 pathway. Induction of the dFMR1 in sevenless-expressing cells of the Drosophila eye causes a disorganized (rough) eye through a mechanism that requires residues necessary for dFMR1/FMRP's translational repressor function. Several mutations in dco, orb2, pAbp, rm62, and smD3 genes dominantly suppress the sev-dfmr1 rough-eye phenotype, suggesting that they are required for dFMR1-mediated processes. The encoded proteins localize to dFMR1-containing neuronal mRNPs in neurites of cultured neurons, and/or have an effect on dendritic branching predicted for bona fide neuronal translational repressors. Genetic mosaic analyses indicate that dco, orb2, rm62, smD3, and dfmr1 are dispensable for translational repression of hid, a microRNA target gene, known to be repressed in wing discs by the bantam miRNA. Thus, the encoded proteins may function as miRNA- and/or mRNA-specific translational regulators in vivo (Cziko, 2009).

It is suggested, that as for previously identified sev-dfmr1 suppressors Ago1, Lgl, and Me31b, analysis of PABP, Smd3, Rm62, Orb2, and Dco proteins, encoded by the sev-dfmr1 suppressor genes identified in this study, will help elucidate how dFMR1 works in translational regulation, RNA targeting and localization, and ncRNA pathway function (Cziko, 2009).

Three lines of evidence indicate that the genes identified encode proteins with translational repressor activity. First, with the exception of Dco, all of these proteins have been previously implicated in some aspect of RNA metabolism and are present on dFMR1-containing neuritic granules in which RNA is repressed and transported. Second, the rough-eye phenotype observed in sev-dfmr1 has been linked to the ability of FMRP to repress mRNA translation. Thus, it would be expected that the phenotype would be alleviated by mutations that reduce the efficiency of translational repression. Third, overexpression of Dco, Pabp, Orb2, or Rm62 inhibits the dendritic growth of neurons, a phenotype predicted for neuronal translational repressors. These observations are consistent with the idea that translation of RNAs in neurites, which promotes dendritic branching, is inhibited by overexpression of Dco, Pabp, Orb2, or Rm62. Thus, genetic interaction data, molecular localization, and one functional test in dendrites indicate that Dco/Dbt, PABP, Rm62, or SmD3 function as neuronal translational repressors (Cziko, 2009).

The identification of several canonical translational-factor encoding genes as suppressors of sev-dfmr1 highlights the point that individual translational control molecules work in multicomponent complexes and therefore have several functional interactions. PABP is one example of a protein that is currently believed to perform two opposing functions of translational control. In addition to its well-studied role as a translational activator, PABP can mediate translational repression, e.g., of Vasopressin mRNA although the exact mechanism remains unclear. Dual roles in activation and repression are also suggested by the observation that reduced or elevated levels of PABP have similar effects at the Drosophila neuromuscular junction (NMJ). Additionally, PABP associates with particles containing BC1, a neuron-specific noncoding RNA with translational repressor function, as well as a CYFIP-FMRP complex that may function as a repressor in some contexts but as an activator in others. Similarly, Orb2 homologs (CPEBs) though required for translational activation of CPE-containing mRNAs via poly-A polymerase, also allow translational repression in combination with Maskin or Cup proteins (Cziko, 2009).

It was somewhat surprising that SmD3, a splicing factor, was identified in a screen for translational repressors. However, SmD3 has additional nonsplicing functions: in Caenorhabditis elegans, the Sm proteins are required for germ cell mRNP assembly and RNA localization. Such a role in translational regulation and mRNP assembly is more consistent with functions predicted by the genetic experiments (Cziko, 2009).

Rm62/Dmp68 is a member of the DEAD-box helicase family that has been shown to be associated with a dFMR1-containing RNAi silencing complex. It also has additional roles during transcription and mRNA processing as well as potentially in miRNA processing as part of the Drosha complex. Based on the biochemical evidence for Rm62's presence in FMRP-containing complexes, it is not surprising that rm62 mutations show strong genetic interactions with dfmr1. However, the mechanism of suppression remains unknown (Cziko, 2009).

Finally Dco/Dbt, is by far the most elusive protein in regard to its potential function in the translational regulatory pathway. Dco/Dbt, a casein kinase I (CKI) is best known from circadian biology where it phosphorylates Per and expedites its degradation. dFMR1 protein has several phosphorylation sites, one of which in S2 cells has been demonstrated to be phosphorylated by a CKII protein. While the functional requirement for CKI-dependent dFMR1 phosphorylation is as of yet not understood, there is considerable evidence that the phosphorylation state of FMRP may actually determine its role in translation. Biochemical data demonstrate that most FMRP in granules is in the phosphorylated state while FMRP in the polysome fraction is dephosphorylated, suggesting a mechanism to switch state from an activator to a repressor, and an important regulatory role for kinases that phosphorylate FMRP (Cziko, 2009).

Another interesting potential link between the two proteins is the behavioral observation that patients with Fragile-X Mental Retardation often display circadian disturbances. This altered circadian rhythm is also present in the Drosophila dfmr1 mutants that usefully model fragile-X syndrome (Cziko, 2009).

The identification of these proteins as sev-dfmr1 modifiers illustrates the many possibly regulatory roles of RNA-associated proteins. In addition, the data associating Dco/Dbt with RNA regulation indicates unexplored and novel mechanisms of RNA regulation in neurons (Cziko, 2009).

Given that dFMR1/FMRP is thought to function in miRNA-dependent translational repression, it was of particular interest to asking whether these dFMR1 interactors had any role in this pathway. To address this issue, a sensitive in vivo assay that uses a fluorescent reporter was employed to reveal the strength of translational repression via an endogenous (bantam) miRNA. When combined with genetic mosaic analysis, this assay can be used to study null mutations in candidate genes, as long as the mutations do not cause cell lethality. The assay appears more sensitive than typically used cell-based assays on the evidence of prior analysis of Me31B, whose requirement for miRNA function, clearly seen in the in vivo assay, is only evident in double-knockdown experiments in the more commonly used cell-culture assays (Cziko, 2009).

In vivo experiments revealed no requirement for the sev-dfmr1 interacting proteins Dco, Orb2, Rm62, and SmD3 in miRNA repression. For reasons explained above, it is unlikely that this reflects a weakness in the experimental assay for miRNA function. A bigger surprise was the finding that the dFMR1 itself appeared dispensable for miRNA function in vivo. Because the allele used is a well-characterized null allele, and the absence of dFMR1 in the mutant clones is confirmed by antibody staining, the conclusion that dFMR1 is not a core, essential component of the RISC/miRNA pathway is strong. This conclusion is not inconsistent with any of the existing data showing biochemical association between RISC and FMRP and genetic interactions between Ago1 and FMRP. However, it is also consistent with recent observations indicating the dispensability of FMRP for RISC function in cultured cells. It is suggested that the function of dFMR1 and, by extension, FMRP may be restricted to a subset of transcripts, for instance those with UTRs containing both FMRP binding motifs and miRNA target elements. Indeed similar models that account for the mRNA specificity of FMRP have been previously proposed (Cziko, 2009).

These data provide a foundation on which to design further experiments to understand the specific roles of FMR1 and its interacting proteins in translational control (Cziko, 2009).

The CPEB3 Protein Is a Functional Prion that Interacts with the Actin Cytoskeleton

The mouse cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a translational regulator implicated in long-term memory maintenance. Invertebrate orthologs of CPEB3 in Aplysia and Drosophila are functional prions that are physiologically active in the aggregated state. To determine if this principle applies to the mammalian CPEB3, this study expressed it in yeast and found that it forms heritable aggregates that are the hallmark of known prions. In addition, this study confirmed in the mouse the importance of CPEB3's prion formation for CPEB3 function. Interestingly, deletion analysis of the CPEB3 prion domain uncovered a tripartite organization: two aggregation-promoting domains surround a regulatory module that affects interaction with the actin cytoskeleton. In all, these data provide direct evidence that CPEB3 is a functional prion in the mammalian brain and underline the potential importance of an actin/CPEB3 feedback loop for the synaptic plasticity underlying the persistence of long-term memory (Stephan, 2015).

In this study it was asked whether mouse CPEB3 possessed bona fide prion properties, and it was found that it indeed exhibits three hallmarks of known prions: (1) the ability to form amyloid in vitro that can be visualized as long fibers by TEM and detected by birefringence in CR-stained samples, (2) the ability to form SDS-resistant oligomers in yeast and the mouse, and (3) the ability to form heritable foci in yeast that are modulated by Hsp104. The CPEB3 prion was further characterized by deletion analysis, which revealed a complex prion domain with a tripartite organization. The first and third domains, PRD1 and PRD2, were required for CPEB3's prion properties. PRD1 (aa 1-191/217) is essential for prion seed formation and foci heritability. PRD2 (aa 284-449) is essential for CPEB3 foci formation. Of these two domains, PRD1 has an amino acid composition that is the most similar to that of other prions with Q-rich prion domains: it contains an extremely Q-rich stretch (aa 1-32), two P/Q-rich tracts (aa 89-98 and 167-191), and a region with a relatively high P/Q content (aa 191-217). Importantly, deletion of PRD1 in the mouse interfered with CPEB3's activation of its targets b-actin and GluR2, the AMPA receptor critical for synaptic plasticity. These results, together with the previous finding that the CPEB3 Q-rich N terminus mediates the positive effect of the ubiquitin-ligase Neuralized on learning and memory (Pavlopoulos, 2011), strongly suggest that CPEB3 is a functional prion and that CPEB3 prion formation is necessary for CPEB3 function in the context of long-term memory maintenance. Considering earlier findings on the CPEB prion in invertebrates, this evidence for CPEB3 as a functional prion in the mammalian brain suggests that a CPEB-mediated, prion-based mechanism of regulation of memory persistence is conserved in mammals (Stephan, 2015).

In that context, it is worth mentioning that CPEB3 has been implicated in episodic memory in humans (Vogler, 2009) and that the CPEB3 prion-forming determinants identified here are conserved in the human protein. The middle region of the CPEB3 N terminus identified in the deletion analysis, LMD (aa 191/217-284), is a regulatory domain that influences the distribution of CPEB3 along the cell periphery and, specifically, the formation of characteristic rims of CPEB3 foci. This region contains two unusual sequence features, an extremely A/S-rich tract (aa 226-239) and a run of VG dipeptide repeats (aa 273-283). The LMD is the most intriguing part of the CPEB3 prion domain, as it constitutes a central core located between two prion-promoting domains and modulates a functional interaction with the actin cytoskeleton. This prion domain architecture, in addition to CPEB3 SUMOylation in the basal state (Drisaldi, 2015), could provide a possible explanation for why CPEB3 is a repressor of its targets in the basal state and only switches to an activator upon synaptic stimulation. Given that the two CPEB3 prion determinants PRD1 and PRD2 are spatially separated, it is possible that CPEB3 exists in a "closed" conformation in the basal state, resulting from intramolecular interactions between PRD1 and PRD2. Neuronal-stimulation-induced signaling and the concomitant increase in CPEB3 levels could push these domains to favor intermolecular, prion-based PRD1/PRD1- and PRD2/PRD2-type interactions, which would be stabilized by interactions with actin-binding proteins through the now-exposed CPEB3 LMD domain. This interaction with the actin cytoskeleton could in turn act to maintain CPEB3 in the "open" conformation, locally concentrating the protein and leading to more efficient prion seeding. Such a requirement for cytoskeletal interactions could constitute a general property of functional prion formation, ensuring that it only occurs in a tightly regulated context and safeguarding the cell against toxic, out-of-control aggregation. In support of that idea, interaction with actin has been reported for [PSI+ ] and [LSB2+ ] in yeast and [Het-s] in Podospora anserina. Conversely, escape from this regulatory control might promote the deleterious aggregation at the root of some prion diseases and amyloid-based neurological disorders. In fact, cytoskeletal interactions are known for huntingtin, alpha-synuclein, tau, and the precursor form of the prion protein, Pro-PrP (Stephan, 2015).

Considering that actin is highly conserved between yeast and mammals, it was not surprising that the finding of a CPEB3/actin interaction in yeast was reproduced in the mouse brain and specifically at synapses, reflecting an important physiological function of CPEB3. Mapping of the CPEB3/actin interaction to a region between PRD1 and PRD2 has some fascinating implications in the context of sustained synaptic plasticity and the maintenance of long-term memory. On the one hand, actin has been extensively studied in that context and has been hypothesized to be a candidate for the synaptic tag. Additionally, actin dynamics are known to be modulated by the AMPA receptors, themselves well-established CPEB3 targets. The current results demonstrate that CPEB3 colocalizes and interacts with actin and depends on the actin cytoskeleton for its aggregation. On the other hand, previous work in invertebrates had identified neuronal actin mRNA as a target of Aplysia CPEB, and this study confirms in the mouse that b-actin mRNA is a CPEB3 target and that stimulation-induced CPEB3 aggregation leads to an increase of actin protein levels. Taken together, these observations suggest the possibility of a positive feedback loop at the basis of activity-induced synaptic plasticity, based on interdependence of actin filament dynamics and CPEB3 prion formation. This loop would ultimately result in prionbased stabilization of actin ultrastructure and establishment of the long-lasting synaptic modifications that underlie long-term memory persistence (Stephan, 2015).

Protein-only mechanism induces self-perpetuating changes in the activity of neuronal Aplysia cytoplasmic polyadenylation element binding protein (CPEB)

Neuronal cytoplasmic polyadenylation element binding protein (CPEB) plays a critical role in maintaining the functional and morphological long-lasting synaptic changes that underlie learning and memory. It can undergo a prion switch, but it remains unclear if this self-templating change in protein conformation is alone sufficient to create a stable change in CPEB activity: a robust 'protein-only' biochemical memory. To investigate, advantage was taken of yeast cells wherein the neuronal CPEB of Aplysia is expressed in the absence of any neuronal factors and can stably adopt either an active or an inactive state. Reminiscent of well-characterized yeast prions, this study found that CPEB can adopt several distinct activity states or 'strains.' These states are acquired at a much higher spontaneous rate than is typical of yeast prions, but they are extremely stable -- perpetuating for years -- and have all of the non-Mendelian genetic characteristics of bona fide yeast prions. CPEB levels are too low to allow direct physical characterization, but CPEB strains convert a fusion protein, which shares only the prion-like domain of CPEB, into amyloid in a strain-specific manner. Lysates of CPEB strains seed the purified prion domain to adopt the amyloid conformation with strain-specific efficiencies. Amyloid conformers generated by spontaneous assembly of the purified prion domain (and a more biochemically tractable derivative) transformed cells with inactive CPEB into the full range of distinct CPEB strains. Thus, CPEB employs a prion mechanism to create stable, finely tuned self-perpetuating biochemical memories. These biochemical memories might be used in the local homeostatic maintenance of long-term learning-related changes in synaptic morphology and function (Heinrich, 2011).

Structural insights into a yeast prion illuminate nucleation and strain diversity

Self-perpetuating changes in the conformations of amyloidogenic proteins play vital roles in normal biology and disease. Despite intense research, the architecture and conformational conversion of amyloids remain poorly understood. Amyloid conformers of Sup35 are the molecular embodiment of the yeast prion known as [PSI], which produces heritable changes in phenotype through self-perpetuating changes in protein folding. This study determined the nature of Sup35's cooperatively folded amyloid core, and this information was used to investigate central questions in prion biology. Specific segments of the amyloid core form intermolecular contacts in a 'Head-to-Head', 'Tail-to-Tail' fashion, but the 'Central Core' is sequestered through intramolecular contacts. The Head acquires productive interactions first, and these nucleate assembly. Variations in the length of the amyloid core and the nature of intermolecular interfaces form the structural basis of distinct prion 'strains', which produce variant phenotypes in vivo. These findings resolve several problems in yeast prion biology and have broad implications for other amyloids (Krishnan, 2005).

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 it was found that a translation regulator CPEB from Aplysia, ApCPEB, that stabilizes activity-dependent changes in synaptic efficacy, can display prion-like properties in yeast. This study finds 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, it was found 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 (Si, 2001).


Search PubMed for articles about Drosophila Orb2

Alarcon, J. M., Hodgman, R., Theis, M., Huang, Y. S., Kandel, E. R. and Richter, J. D. (2004). Selective modulation of some forms of schaffer collateral-CA1 synaptic plasticity in mice with a disruption of the CPEB-1 gene. Learn Mem 11: 318-327. PubMed ID: 15169862

Alberti, S., Halfmann, R., King, O., Kapila, A. and Lindquist, S. (2009). A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. Cell 137: 146-158. PubMed ID: 19345193

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Biological Overview

date revised: 17 November 2021

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