1. Scribble Scaffolds a Signalosome for Active Forgetting
Isaac Cervantes-Sandoval, Molee Chakraborty, Courtney MacMullen, Ronald L. Davis 
Neuron 
Volume 90, Issue 6, Pages (June 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1 Knocking Down scrb Impairs Memory Loss
(A) Knocking down scrb expression with the pan-neuronal nsyb-gal4 enhanced 3 hr memory performance relative to the gal4- and uas-only control groups. One-way ANOVA, ∗p < n = 6. Error bars indicate SEM.
(B) Memory retention is enhanced in scrbRNAi flies. Flies expressing scrbRNAi performed at levels similar to the control group shortly after acquisition but exhibited reduced memory decay when tested at multiple time points after training. Two-way ANOVA, ∗p < n = 6. Error bars indicate SEM.
(C) Knocking down scrb expression only during adulthood using the TARGET system increased memory performance. The gal80ts transgene inhibits Gal4 activity at 18°C but not at 30°C. Two-way ANOVA, ∗p < n = 6–12. Error bars indicate SEM.
(D) Knocking down scrb expression partially impaired acquisition when trained with 1 or 2 shock pulses but not when trained with 3 to 12 shock pulses. Two-way ANOVA, ∗p < n = 6. Error bars indicate SEM.
(E) Knocking down scrb expression did not alter memory acquisition when flies were trained with variable shock intensity. Two-way ANOVA. Error bars indicate SEM.
(F) Knockdown of scrb expression did not lead to increased protein synthesis-dependent LTM after only one cycle of training. Two-way ANOVA,∗p < n = 6. Error bars indicate SEM.
(G) Downregulation of scrb increased both anesthesia-sensitive and anesthesia-resistant memory. Two-way ANOVA, ∗p < n = 6. Error bars indicate SEM.
Neuron  , DOI: ( /j.neuron )
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3. Figure 2
Knocking Down scrb Expression Impairs Interference-Based Forgetting
(A) Control flies trained with a reversed contingency expressed considerable memory to the more recent learning event, but scrbRNAi flies exhibited impaired forgetting demonstrated by poor performance to the reversed contingency. Two-way ANOVA, ∗p < n = 6. Error bars indicate SEM.
(B) When trained to a reversed contingency but using a third odor for the test to measure the memory performance exclusively to odor A, scrbRNAi flies performed significantly better than control flies. Two-way ANOVA, ∗p < n = 6. Error bars indicate SEM.
(C) The scrbRNAi flies were impaired in retroactive interference introduced by new learning occurring 1.5 hr after the first learning episode. Two-way ANOVA, ∗p < n = 9. Error bars indicate SEM.
(D) Proactive interference decreased memory performance in control but not scrbRNAi flies. Two-way ANOVA, ∗p < n = 6. Error bars indicate SEM.
Neuron  , DOI: ( /j.neuron )
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4. Figure 3 Scrb Is Required in MBn and DAn for Normal Memory Loss
(A) The scrbRNAi line was crossed to the gal4 lines listed. Knocking down scrb expression in MBn (c772-gal4, 238y-gal4, R13F02-gal4) or DAn (th-gal4) enhanced memory retention. Pairwise t test, ∗p < n = 6. Error bars indicate SEM.
(B) Knocking down scrb expression in α/β (c739-gal4) and/or γ (1471-gal4; 247-gal4) MBn enhanced memory retention. Pairwise t test, ∗p < n = 6. Error bars indicate SEM.
(C) The scrb1 heterozygous mutants exhibited enhanced 3 hr memory. Pairwise t test, ∗p < n = 6. Error bars indicate SEM.
(D and E) Knocking down scrb expression in MBn and DAn using a second and third RNAi line enhanced memory performance. Pairwise t test, ∗p < n = 6. Error bars indicate SEM.
(F) Endogenous expression of scrb::GFPCA07683 in the Drosophila brain, immunostained with anti-GFP and anti-nc82 antibodies.
(G) Endogenous expression of Scrb in Drosophila brain, immunostained with anti-Scrb and anti-nc82.
Neuron  , DOI: ( /j.neuron )
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5. Figure 4
DAn Activity Is Upstream of Rac1 and Scrb in the Regulation of Forgetting
(A) Overexpressing the uas-damb transgene in MBn of adult flies impaired 3 hr memory but left learning intact. One-way ANOVA, ∗p < Error bars indicate SEM.
(B) Overexpression of the Damb receptor in MBn during adulthood accelerated forgetting. Co-expression of the Damb receptor and dominant-negative Rac1N17 phenocopied the expression of Rac1N17 alone (right). Flies of the various genotypes maintained at 18°C during adulthood performed with no significant differences (left). One-way ANOVA, ∗p < n = 6. Error bars indicate SEM.
(C) Flies were stimulated at 32°C for 5 min at 1.5 hr after training and memory was tested at 3 hr. The control flies, maintained at 23°C (left), showed a significant increase in performance when scrb was knocked down in MBn (gray and red bars) compared to their respective control groups (black and white bars). TrpA1-based stimulation of DAn neurons induced forgetting (white bar; middle), but this effect was partially suppressed when scrb was knocked down in the MBn (red bar). The right panel illustrates the magnitude of the suppression due to scrb inhibition when the performance of the two relevant genotypes at 32°C (middle) was normalized to their performance at 23°C (left). One-way ANOVA, ∗p < n = 6. Error bars indicate SEM.
Neuron  , DOI: ( /j.neuron )
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6. Figure 5
Scribble Physically and Genetically Interacts with Rac1, Pak3, and Cofilin, Scaffolding a Signalosome for Active Forgetting
(A) Expressing constitutively active Rac1 (Rac1V12) in adult MBn accelerated forgetting even when scrb was knocked down in same neurons (right). Flies of the various genotypes maintained at 18°C during adulthood performed with no significant differences (left). One-way ANOVA, ∗p < n = 6–10. Error bars indicate SEM.
(B) Expressing constitutively active Cofilin (tsrS3A) or scrbRNAi in adult MBn impaired memory loss. Co-expression of tsrS3A and scrbRNAi did not further reduce forgetting (right). Flies of the various genotypes maintained at 18°C during adulthood performed with no significant differences (left). One-way ANOVA, ∗p < n = 6. Error bars indicate SEM.
(C) Scrb was immunoprecipitated using scrb::GFPCA07683 flies and anti-GFP antibodies. The Scrb immunoprecipitate co-immunoprecipitated Rac1, Pak3, and Cofilin (top). Immunoprecipitates of Rac1 co-immunoprecipitated Scrb (bottom).
(D) Pan-neuronal knockdown of scrb significantly reduced the levels of inactive p-Cofilin but not to total Cofilin compared to control groups. One-way ANOVA, ∗p < n = 4. Error bars indicate SEM.
Neuron  , DOI: ( /j.neuron )
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7. Figure 6 Scrb Is Required in DAn to Maintain Normal Ongoing Activity
(A) Schematic diagram illustrating the regions of interest recorded for DAn projections to the MB lobes.
(B) Knocking down scrb using th-gal4 decreased DAn ongoing activity in the α/α’ and MV1 regions but not V1 or aimpr. Pairwise Mann-Whitney test, ∗p < n = 12–23. Error bars indicate SEM.
(C) Knocking down scrb using R13F02-gal4 increased the DAn ongoing activity in the MV1 region but not in α/α’, V1 and aimpr neuropil regions. Pairwise Mann-Whitney test, ∗p < n = 20–37. Error bars indicate SEM.
(D) Representative recordings of DAn spontaneous activity in flies where scrb expression was reduced using the th-gal4 driver (red) compared to control flies (gray). Ten minute recordings of the DAn processes in the α/α’, V1, MV1, and aimpr regions.
(E) Representative recordings of DAn spontaneous activity in flies where scrb expression was reduced using the R13F02-gal4 driver (red) compared to control flies (gray). Ten minute recordings of the DAn processes in the α/α’, V1, MV1, and aimpr regions.
Neuron  , DOI: ( /j.neuron )
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8. Figure 7 Model for the Role of Scrb in Forgetting
Ongoing activity of DAn through the Damb receptor modulates the strength of the “forgetting signal” (red arrow) in the MBn. The Scrib protein scaffolds a signaling complex consisting of Rac1, Pak3, and Cofilin. Knocking down scrb in MBn impairs postsynaptic signaling of forgetting (middle). Knocking down scrb also increases ongoing DAn activity through a retroactive signal (blue arrow). Scrb is also required in the presynaptic compartment, e.g., in DAn for normal memory loss by maintaining the ongoing activity in these neurons (right).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions