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Mrj is a chaperone of the Hsp40 family that regulates Orb2 oligomerization and long-term memory in Drosophila [1]

['Meghal Desai', 'National Centre For Cell Science', 'Savitribai Phule Pune University Campus', 'Pune', 'Ankita Deo', 'Institute Of Bioinformatics', 'Biotechnology', 'Ibb', 'Savitribai Phule Pune University', 'Jagyanseni Naik']

Date: 2024-05

Orb2 the Drosophila homolog of cytoplasmic polyadenylation element binding (CPEB) protein forms prion-like oligomers. These oligomers consist of Orb2A and Orb2B isoforms and their formation is dependent on the oligomerization of the Orb2A isoform. Drosophila with a mutation diminishing Orb2A’s prion-like oligomerization forms long-term memory but fails to maintain it over time. Since this prion-like oligomerization of Orb2A plays a crucial role in the maintenance of memory, here, we aim to find what regulates this oligomerization. In an immunoprecipitation-based screen, we identify interactors of Orb2A in the Hsp40 and Hsp70 families of proteins. Among these, we find an Hsp40 family protein Mrj as a regulator of the conversion of Orb2A to its prion-like form. Mrj interacts with Hsp70 proteins and acts as a chaperone by interfering with the aggregation of pathogenic Huntingtin. Unlike its mammalian homolog, we find Drosophila Mrj is neither an essential gene nor causes any gross neurodevelopmental defect. We observe a loss of Mrj results in a reduction in Orb2 oligomers. Further, Mrj knockout exhibits a deficit in long-term memory and our observations suggest Mrj is needed in mushroom body neurons for the regulation of long-term memory. Our work implicates a chaperone Mrj in mechanisms of memory regulation through controlling the oligomerization of Orb2A and its association with the translating ribosomes.

Taking a cue from the yeast prion literature, where the protein folding machinery/chaperones act as key regulators of prions, we hypothesize, chaperones may also play a role in the regulation of Orb2A oligomerization. In yeast, the Hsp70, Hsp40, and Hsp104 chaperones have been found to regulate the oligomerization and propagation of prions [ 29 , 31 , 44 – 46 ]. Using an immunoprecipitation-based screen and a yeast-based prion conversion assay, here we identify Drosophila Mrj as a regulator of Orb2A’s prion-like conformational conversion. Mrj stands for the mammalian relative of DnaJ [ 47 ] and functions as a chaperone in mammals. Mammalian Mrj is now referred to as DNAJB6 according to the HUGO gene nomenclature [ 48 ]. We find Drosophila Mrj to behave similarly to mammalian DNAJB6 as a chaperone and interfere with the aggregation of pathogenic Huntingtin. While knockout of Mrj in mice is embryonic lethal [ 49 ], in Drosophila we find it not to be an essential gene and observe this knockout to have a reduced amount of Orb2 oligomers. We further find that Mrj knockout exhibits a deficit in long-term memory and Mrj is needed in specific mushroom body neurons for the regulation of long-term memory. Our observations suggest Mrj interacts with translating ribosomes and might play a role in regulating Orb2A’s association with the translating ribosomes.

Orb2 has 2 isoforms, Orb2A and Orb2B, both containing the common prion-like domain and a C terminal RNA-binding domain. The 2 isoforms differ in the position of the prion-like domain, enrichment in the brain, and propensity to aggregate. Orb2A has 8 amino acids while Orb2B has 162 amino acids, in front of the prion-like domain. In comparison to Orb2B, Orb2A is less abundant in the brain. Orb2A has a higher propensity to aggregate compared to Orb2B. Though both the Orb2A and Orb2B isoforms interact among themselves and are present in the oligomers, it is Orb2A that acts as a seed for Orb2 oligomerization. This is supported by several lines of evidence. Firstly, a deletion of Orb2A causes a drastic reduction in the formation of endogenous Orb2 oligomers in the brain. Secondly, a point mutation of the fifth Phenylalanine to Tyrosine (F5Y) in Orb2A reduces its ability to oligomerize and interferes with the maintenance of long-term memory [ 40 ]. This residue is specific to only Orb2A and is not present in Orb2B. Thirdly, genetic deletion experiments suggested Orb2A does not need its RNA-binding domain and Orb2B does not need its prion-like domain for the maintenance of memory [ 43 ]. Finally, the addition of the prion-like domain of Orb2A can seed monomeric Orb2B to oligomerize and result in its change to a translational activator [ 41 ]. All this evidence suggests oligomerization of Orb2A is crucial for the formation of Orb2 oligomers and the maintenance of memory. Hence in this study, we asked, what regulates the oligomerization of Orb2A, how this regulator affects the overall Orb2 oligomers in the brain, and if this regulator plays any role in long-term memory.

Aplysia CPEB behaves like functional prion-like proteins [ 25 – 27 ]. Prions were discovered as protein-based infectious particles associated with neurodegenerative diseases like Creutzfeldt–Jakob disease, Scrapie, and Bovine spongiform encephalopathy [ 28 ]. For the disease-causing prions, they can exist in 2 distinct conformational variants: one monomeric and another oligomeric amyloid-like form. The oligomeric form is toxic, dominant, and self-perpetuating as it can convert the monomeric to the amyloid form. Many proteins sharing similar dominant and self-perpetuating properties were discovered in yeast and were classified as yeast prions [ 29 – 33 ]. In recent times, several proteins which are nontoxic and similar in characteristics to prions are discovered. In these, since the conversion to the oligomeric amyloid-like form is mediated by a signal and the amyloid form can have a beneficial physiological function, they are described as functional prions [ 34 – 38 ]. For Aplysia CPEB, it is suggested that synaptic stimulation causes CPEB to convert to its prion-like state and this state can self-sustain as long as monomers are getting synthesized. The prion-like oligomers can further regulate the protein synthesis of the target mRNAs needed for the maintenance of long-term memory. This model gets support from studies with Drosophila Orb2, where a point mutation in Orb2 that disrupted its prion-like oligomerization caused an impairment in the persistence of long-term memory [ 39 , 40 ]. Biochemically separated monomeric and oligomeric forms of Orb2 exhibit functional differences from each other. In in vitro translation assays with its target mRNAs, the monomer was found to act as a translational repressor by decreasing its poly-A tail length while the oligomer acted as a translation activator by elongating its poly-A tail [ 41 ]. This observation is similar to the idea that prions based on their conformational difference and oligomeric status can have different biochemical functions. These translation-enhancing oligomers when visualized using cryo-electron microscopy, appear as amyloids and like prions can seed the conversion of the monomers to the amyloid form [ 42 ].

Memory is the experience-dependent ability to preserve and recover information from the past. The molecular mechanism behind long-term memory is long-term potentiation (LTP). LTP is the persistent change observed in synaptic strength accompanied by structural changes like new synaptic growth and stabilization, caused due to repeated patterns of electrical stimulation [ 1 – 3 ]. Several studies ranging across species suggest that protein synthesis plays a crucial role in regulating long-term memory and LTP [ 4 – 15 ]. The translation regulator in Aplysia, cytoplasmic polyadenylation element binding (CPEB) protein is crucial for the maintenance phase of long-term facilitation and stabilization of learning-induced new synaptic growth [ 16 , 17 ]. Its Drosophila homolog Orb2 is necessary for the persistence of long-term memory and has among its mRNA targets, genes regulating protein turnover, synapse formation, and neuronal growth [ 18 , 19 ]. The mouse homologs CPEB1, CPEB2, and CPEB3 are also implicated in the regulation of memory processes [ 20 – 24 ].

Results

Identification of chaperone interactors of Orb2 We started with identifying homologs of the yeast Hsp70, Hsp40, and Hsp104 families of proteins. The Drosophila genome lacks any Hsp104 homolog but it has members of the Hsp40 and Hsp70 classes of proteins [50–52]. So, for our screen to identify Orb2A regulators, we decided to focus on these 2 groups. The Hsp40 group of proteins is classified as proteins containing DnaJ domains. The Drosophila genome contains 39 such genes (S1 Fig). For the Hsp70 class, Drosophila has both the heat shock inducible Hsp70 (Hsp68, Hsp70Aa, Hsp70Ab, Hsp70Ba, Hsp70Bb, Hsp70Bc, and Hsp70Bbb) and the constitutively expressing Hsc70 (Hsc70-1, Hsc70-2, Hsc70-3, Hsc70-4, Hsc70-5, and Hsc70Cb) (S2 Fig). We made a library of 37 of the Hsp40 genes tagged with HA epitope and 4 of the Hsp70 genes tagged with Flag epitope. The 4 Hsp70 genes were selected based on their sequence variability from each other. We next transfected each of these constructs with Orb2A in Drosophila S2 cells and used these cells to perform immunoprecipitation with an anti-Orb2 antibody (Fig 1A and 1C). The immunoprecipitate was probed in a western blot for the presence of HA-tagged Hsp40 or Flag-tagged Hsp70 protein. From this immunoprecipitation-based screen, we observed the proteins CG4164, CG9828, DroJ2, CG7130, Tpr2, Mrj, Hsc70-1, Hsc70-4, Hsc70Cb, and Hsp70Aa as interactors of Orb2A (Fig 1B and 1D). For 31 Hsp40 proteins, we could not detect interaction with Orb2A in our immunoprecipitation screen (S3 Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Identification of Mrj as a regulator of Orb2A’s prion-like oligomerization. (A) Schematic of immunoprecipitation assay to identify Hsp40 interactors of Orb2A. Individual constructs coding for members of the Hsp40 family with HA tag were cotransfected in S2 cells with Orb2A construct and immunoprecipitation was performed with anti Orb2 antibody. The immunoprecipitate was probed with an anti-HA antibody to detect if the Hsp40 proteins were interacting with Orb2A. (B) From 37 proteins of the Hsp40 family, the immunoprecipitation (IP) screen identified CG9828, CG4164, Mrj, Tpr2, DroJ2, and CG7130 as interactors. Lys is the input lysate, Ctl IP is the control IP with beads only and Orb2 IP is the IP with anti-Orb2 antibody. (C) Schematic of immunoprecipitation assay to identify Hsp70 interactors of Orb2A. Individual constructs coding for members of the Hsp70 family with Flag tag were cotransfected in S2 cells with Orb2A construct and immunoprecipitation was performed with anti Orb2 antibody. The immunoprecipitate was probed with an anti-Flag antibody to detect if the Hsp70 proteins were interacting with Orb2A. (D) All 4 proteins of the Hsp70 family screened in the immunoprecipitation experiment, Hsp70Aa, Hsc70-1, Hsc70Cb, and Hsc70-4 were found to be interacting with Orb2A. (E) Schematic of the yeast based screen. A Sup35 knockout strain rescued by a chimeric construct expressing Orb2A’s prion-like domain tagged with the C-terminal domain of Sup35 is transformed with galactose inducible Hsp40 and Hsp70 constructs. A premature stop codon in the Ade 1–4 gene is used as a reporter. The screen consists of inducing the chaperones with galactose in the prion negative strain and screening for their ability to change the color to white and grow in adenine-deficient media. (F) Galactose induction of individual chaperones of both Hsp40 and Hsp70 family of proteins in prion negative Orb2APrD-Sup35C strain, caused only Mrj to convert the prion negative state of the cells to prion positive state, as evidenced by the change in the colony color to white in YPD media and causing it to now grow in adenine. (G) Schematic of SDD-AGE assay to quantitate the change in Orb2A-GFP oligomerization in presence of Mrj. Sf9 cells were infected with viruses for Orb2A alone and Orb2A with Mrj. The lysate from these cells was centrifuged and the resulting pellet was resuspended in an SDS-containing buffer, subjected to SDD-AGE, and further probed with anti-Orb2 antibody. (H) Representative SDD-AGE blots showing increased levels of Orb2A oligomers in presence of Mrj. (I) Quantitation of Orb2A oligomers in presence and absence of Mrj. Data is represented as a relative fold change for Orb2A in presence of Mrj as compared to without Mrj. Data is represented as mean ± SEM and significance is tested using two-tailed Student’s unpaired t test. (J) Recombinant GST-Mrj bound to Glutathione beads on incubation with purified recombinant Orb2A-His from E. coli could pulldown Orb2A. The same blot is probed first with an anti-Orb2 antibody followed by probing with an anti-Mrj antibody. The band marked with * is the GST-Mrj protein and the band below it is possibly a breakdown product from the former. GST protein bound to Glutathione beads was used as a negative control. (K) Recombinant GST-Mrj bound to Glutathione beads on incubation with purified recombinant Orb2A-GFP-His from Sf9 cells could pulldown Orb2A-GFP. The same blot is probed first with an anti-Orb2 antibody followed by probing with an anti-Mrj antibody. The band marked with * is the GST-Mrj protein and the band below it is possibly a breakdown product from the former. GST protein bound to Glutathione beads was used as a negative control. The data underlying this figure are available at: https://figshare.com/s/f5d913a0a289339ee16b. https://doi.org/10.1371/journal.pbio.3002585.g001

Drosophila Mrj converts Orb2A from non-prion to a prion-like state We next asked if any or all of these interactors can regulate the oligomerization of Orb2. Toward this, we tested these genes in a heterologous yeast chimeric Sup35-based system. Sup35 is a translation terminator that can exist in both non-prion and prion forms [29,53–55]. Replacement of Sup35’s prion-like NM domain with putative prion-like domains of other proteins was previously used to identify several new prions [56,57]. In this assay, when the NM domain is replaced with the N terminal 162 amino acids of Orb2A (Orb2A-PrD), the prion-like behavior could be visualized as a red (non-prion) or white (prion) colony color. While the white colonies grow in adenine-deficient media, the red colonies are unable to grow in the same [39]. To check what effect the Orb2 interactors will have on the non-prion state of Orb2A, we made Galactose inducible constructs for expressing the 10 Orb2 interactors and transformed these into red-colored, adenine negative, Orb2A-PrD-C-Sup35 strain. The transformed colonies were independently grown and then induced with Galactose (Fig 1E) and plated with serial dilutions on complete media (YPD) and adenine-deficient media. Out of all the 10 genes from the Hsp40 and Hsp70 groups and controls, we observed only Mrj coexpression could change the red color of the colony into white. These cells could also now grow in adenine-deficient media (Fig 1F). This yeast-based screen of interactors of Orb2 suggested Mrj could convert the non-prion form of Orb2A to its prion-like state. A conversion of Orb2A from its non-prion to prion-like form suggests an increase in its oligomeric state. We tested this in Sf9 cells by coexpressing Orb2A with and without Mrj. The cells were lysed and centrifuged and the pellet was further resuspended in an SDS-containing buffer and then resolved on a semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) (Fig 1G) [56,58]. SDD-AGE has been previously reported to detect SDS-resistant Orb2 oligomers as a smear in agarose gels [40,41,59]. On probing the SDD-AGE blot with an anti-Orb2 antibody and quantitating the intensity of the smear, we observed a significant increase in Orb2 oligomers in the presence of Mrj (Fig 1H and 1I). This suggests that Drosophila Mrj can increase the oligomerization of Orb2A. We further asked if Mrj can interact directly with Orb2A and to address this used recombinant protein-based pulldown assays using GST-Mrj bound to Glutathione beads. We added these beads to either recombinant Orb2A (6X Histidine tagged from E. coli) or recombinant Orb2A-GFP (6X His tagged from Sf9 cells) and on performing western blots with the pulldown beads noticed the presence of Orb2A in both these cases (Fig 1J and 1K). Overall, these pulldown experiments suggest Drosophila Mrj can directly interact with Orb2A.

Drosophila Mrj knockout is viable and does not show any gross developmental defect We next used the CRISPR-Cas9 system to generate a knockout of Drosophila Mrj by introducing a Gal4 cassette in the Mrj locus (Fig 3A). We confirmed the knockout by using both PCR and western blotting with an anti-Mrj antibody (Fig 3B and 3C). Here, we found the Drosophila Mrj knockout line to be homozygous viable. Developmentally, there was no defect in any organization of any body structure, and there was no sterility associated with the homozygous line. Using the Mrj KO Gal4 line with reporter CD8GFP, we could detect strong expression in the brain including areas of optic lobes, olfactory lobes, and mushroom body (S5D Fig). In terms of overall cellular organization, we observed no defect in the actin cytoskeleton and the gross organization of the mushroom body as evidenced by staining with Phalloidin and anti-FasII antibody (Fig 3D). We next checked if the absence of Mrj caused endogenous proteins to aggregate in the brain. Towards this, we looked for the Drosophila homolog of p62, Ref(2)P which is a regulator of protein aggregates and is present in ubiquitinated protein aggregates associated with aging and neurodegenerative disorders [75]. On Ref(2)P immunostaining to label ubiquitinated protein aggregates, we could not detect any difference in the labeling between the wild-type and Mrj knockout brains (Fig 3E). We also stained the wild-type and Mrj knockout brains with anti-ubiquitin antibody and again saw no difference in ubiquitin labeling in the brains (Fig 3F). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Drosophila Mrj is not an essential gene unlike its mammalian homolog. (A) Schematic of the genomic organization of the Mrj gene and the knockin of the Gal4-loxP-3XP3-RFP-loxP cassette in the locus to make Mrj knockout. The black arrows represent the PCR primers for confirming the Mrj knockout, and the green and purple arrows represent the PCR primers for up and down PCR to check the knockin of the cassette in the Mrj locus. (B) Confirmation of Mrj knockout using genomic DNA PCR with the knockout, up and down PCR primers. The red * marks the amplified band for Mrj in the wild type (+/+) and its absence in the Mrj knockout (-/-). GAPDH PCR amplification (blue **) was used as the loading control in the PCR. (C) Confirmation of Mrj knockout using western blot using an anti-Mrj antibody. Anti-α-Tubulin antibody was used as the loading control. (D) Immunostaining of wild type (+/+) and Mrj knockout (-/-) with anti-FasII antibody and Phalloidin shows no gross difference in the overall morphology of the mushroom body and the brain. Scale bar is 20 micron. (E) Immunostaining of wild type (+/+) and Mrj knockout (-/-) with anti-Ref(2)P antibody and Phalloidin and (F) with anti-Ubiquitin antibody and Phalloidin shows no gross difference between the two sets. (G) Phalloidin staining of muscles from third instar larvae of wild type (+/+) and Mrj knockout (-/-) shows no gross difference. (H) Quantitation of the average speed of wild type (+/+) and Mrj knockout (-/-) shows no significant difference between the two. Data is represented as mean ± SEM and Mann–Whitney U test is used to check significance. (I) Kaplan–Meier survivor curve shows no significant difference in the life span of wild type (Red) and Mrj knockout (-/-) (Blue). Significance was tested with log-rank Mantel–Cox test. (J) Representative image of silver-stained gel of soluble and insoluble protein fractions, from wild type (+/+) and Mrj knockout (-/-) fly heads. (K) Quantitation of soluble to insoluble protein ratio from wild-type (+/+) and Mrj knockout (-/-) flies showed no significant difference. Data is represented as mean ± SEM and a two-tailed Student’s unpaired t test is used to check significance. The data underlying this figure are available at: https://figshare.com/s/f5d913a0a289339ee16b. https://doi.org/10.1371/journal.pbio.3002585.g003 As mutations in human DNAJB6 are associated with limb-girdle muscular dystrophy (LGMD), we checked if the muscles in Mrj knockout fly show any defect in terms of organization or degeneration. Using the Mrj KO Gal4 line with CD8GFP as a reporter, we could see the expression of the reporter GFP in the muscles and NMJ synapses (S5E–S5G Fig) suggesting the similarity of Drosophila Mrj with human DnaJB6 expression. On staining the larval muscles with phalloidin, we observed no evidence of degeneration or disorganization in the Mrj knockout compared to the wild type (Fig 3G). We next checked the adult Mrj knockout fly for any defect in locomotion and found them to show no significant difference in their walking speed in comparison to the wild type (Fig 3H). This ruled out the possibility of any drastic muscle defect later in development. In terms of life span, we observed no difference between the knockout and wild-type animals (Fig 3I). Since Mrj is known to prevent the formation of pathogenic Htt aggregates, we next asked if Mrj knockout animals show any difference in the total protein distribution in the brain between the soluble and insoluble fractions in comparison to the wild type. We lysed fly heads in Triton X-100 containing lysis buffer and separated the supernatant as the soluble fraction. The remaining pellet was solubilized using an SDS containing lysis buffer and separated as an insoluble fraction. We resolved these fractions in SDS-PAGE, performed silver staining (Fig 3J), and quantitated the ratio of soluble/insoluble protein. Here, we also observed no significant difference in terms of the amount of soluble/insoluble protein ratio between the wild-type and Mrj knockout animals (Fig 3K). These results together suggested that under normal growing conditions, Mrj knockout animals were indistinguishable from wild-type ones in terms of their development, brain and muscle organization, and locomotion abilities. Also, for ubiquitinated aggregates and insoluble protein amounts no difference could be seen between the two sets.

Drosophila Mrj knockout shows reduced amounts of Orb2 oligomers We next asked what happens to endogenous Orb2 oligomers in Mrj knockout animals. In western blots from fly heads, the detectable form is Orb2B representing its relatively higher abundance in the brain. We could not detect any significant difference in Orb2B levels between the wild type and Mrj knockout (Fig 5A). Next, we separated the soluble and insoluble fractions from wild-type and Mrj knockout fly heads and probed them with an anti-Orb2 antibody (Fig 5B). In this assay, while we could detect the presence of Orb2B in both the soluble and insoluble fractions in the wild-type fly head lysate, in the Mrj knockout, Orb2B was more enriched in the soluble fraction (Fig 5C and 5D). This indicated that Mrj knockout has less Orb2B in the insoluble fraction, indicating lighter or lesser Orb2 oligomers. We further tested this by immunoprecipitating Orb2 from wild-type and Mrj knockout animals and subjecting the immunoprecipitate to SDD-AGE (Fig 5E). On probing the SDD-AGE blots, we observed the presence of significantly reduced levels of Orb2 oligomers in Mrj knockout brains in comparison to the wild type (Fig 5F and 5G). Since the insoluble fraction here was solubilized using 0.1% SDS containing buffer, we wondered if the reduced amounts of Orb2 oligomers that we detect in the absence of Mrj are actually due to a reduction in Orb2 oligomerization or due to the migration of Orb2 oligomers to even more aggregated state which cannot be solubilized using 0.1% SDS. To address this, we took the wild-type and Mrj knockout fly heads, lysed them first in the TritonX-100 containing solubilizing buffer, the pellet was further solubilized using 0.1% SDS containing buffer. The remaining pellet was again resolubilized using 2% SDS containing buffer (Fig 5H). On running these different fractions in SDD-AGE we observed while there is more Orb2 present in the Triton X-100 soluble fraction from Mrj knockout, the amount present in both the 0.1% and 2% SDS soluble fractions was decreased in comparison to the wild type (Fig 5I and 5J). SDS-PAGE and immunoblotting with anti-Orb2 antibody showed a similar trend for the monomeric Orb2B (Fig 5K and 5L). So, overall these results suggest that the Mrj knockout has decreased amounts of Orb2 oligomers and thus the chaperone Mrj is probably regulating the oligomeric status of Orb2. An alternate possibility is early interactions of Mrj with Orb2 affect the latter’s oligomerization process, by regulating a conformational change in Orb2 which now makes it an amenable unlocked substrate on which other chaperones or other regulatory proteins might act to drive the oligomerization process. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Mrj knockout shows reduced Orb2 oligomers. (A) Western blot of fly head extracts from wild type and Mrj KO shows similar amounts of Orb2B in both of them. α-Tubulin is used as a loading control here. (B) Schematic of the process of preparing the soluble and insoluble fractions from fly head extract to check for differential distribution of Orb2B. (C) Western blot of soluble and insoluble fractions from wild-type and Mrj KO fly heads show a reduced amount of Orb2B in the insoluble fraction of Mrj knockout. α-Tubulin was used as the loading control. (D) Quantitation of relative Orb2B level ratio in soluble to insoluble fractions show a significant increase in Mrj KO flies as compared to control wild-type flies. Data is represented as mean ± SEM and significance is tested using two-tailed Student’s paired t test. (E) Schematic of the Orb2 pulldown experiment followed by SDD-AGE to check for Orb2 oligomers. (F) Representative SDD-AGE blot shows the reduced amount of Orb2 oligomers in Mrj knockout. (G) Quantitation of Orb2 oligomers in Mrj knockout and wild type. Data is represented as mean ± SEM and two-tailed Student’s unpaired t test is used to check significance. (H) Schematic of sequential detergent-based extraction using TritonX-100, 0.1% SDS, and 2% SDS containing buffers to check for differential distribution of Orb2 in wild type and Mrj knockout. (I) Probing the SDD-AGE of different fractions from wild-type and Mrj knockout fly heads show, an increased presence of Orb2 in the TritonX-100 extracted fraction from Mrj knockout in comparison to the wild type. However, for both the 0.1% and 2% SDS extracted fractions the Orb2 oligomer levels were found to be reduced in the Mrj knockout. (J) Quantitation of the SDD-AGE shows increased levels of Orb2 oligomer in the TritonX-100 soluble fraction in Mrj knockout and decreased levels in the 0.1% and 2% SDS soluble fractions. Data is represented as mean ± SEM and two-tailed Student’s unpaired t test is used to check significance. (K) Probing the SDS-PAGE of the same extracted fractions with anti-Orb2 antibody shows a similar trend of increased amounts of Orb2B monomers in the TritonX-100 extracted fraction and decreased amounts of Orb2B monomers in the 0.1% and 2% SDS extracted fractions from Mrj knockout in comparison to wild type. For the 2% SDS extracted fraction, 25% of the total eluate was run to avoid saturation of the signal. The right panel shows the same blot stained with Ponceau stain. (L) Quantitation of the SDS-PAGE shows increased levels of Orb2B in the TritonX-100 soluble fraction in Mrj knockout and decreased levels in the 0.1% and 2% SDS soluble fractions. Data is represented as mean ± SEM and two-tailed Student’s unpaired t test is used to check significance. The data underlying this figure are available at: https://figshare.com/s/f5d913a0a289339ee16b. https://doi.org/10.1371/journal.pbio.3002585.g005

Mrj is needed in the mushroom body for the regulation of long-term memory As our observations imply Orb2 oligomerization is dependent on Mrj, we next asked if Mrj is needed for long-term memory. We took the wild-type and Mrj knockout animals and subjected them to courtship suppression-based memory assay (Fig 6A). Male flies when subjected to repeated rejections by mated females, suppress their courtship behavior post-training. Compared to wild-type animals, mutants defective in their ability to retain memory show a faster recovery of the courtship behavior suggesting a decrease in their capacity to remember. At post-training time points of 2 h and 12 h, we could not detect any significant difference in memory scores between the wild-type and Mrj knockout groups, suggesting a normal formation of early and intermediate-term memory. However, at the 16-h and 24-h time points, the memory score became significantly reduced for the Mrj knockout in comparison to the wild type (Fig 6B). This suggests Mrj is needed for regulating long-term memory in Drosophila. We also did a control experiment where we crossed the Mrj knockout flies with another P element inserted line (KG04490) near the Mrj locus which does not disrupt the Mrj locus. The progeny which was heterozygous for Mrj knockout were tested for memory at 16- and 24-h time points in comparison with the wild-type flies. Here, we did not notice any significant difference between the memory scores of the 2 groups (S6A Fig), suggesting loss of 1 copy of Mrj is not sufficient to cause a memory deficit. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Mrj regulates long-term memory. (A) Schematic of male courtship suppression-based memory assay. (B) Mrj knockout flies show significant memory deficit in comparison to the wild-type flies from 16 h onwards. Data are represented as mean ± SEM and Mann–Whitney U test is done to test for significance. (C) Representative image of the expression pattern of Mrj KO Gal4 visualized using the reporter CD8GFP (green) and counterstained with anti-FasII antibody (magenta) shows the expression of GFP in mushroom body neurons. Scale bar is 20 micron. (D) Confirmation of knockdown of Mrj by pan-neuronal expression of an Mrj RNAi line using a western blot with anti-Mrj antibody. Anti-α-Tubulin antibody was used as the loading control. (E) Representative image of the expression pattern of 201Y Gal4 visualized using the reporter CD8GFP. (F) Knockdown of Mrj in specific mushroom body neurons using 201Y Gal4 causes a significant memory deficit in comparison to the control from 16 h onwards. Data are represented as mean ± SEM and Mann–Whitney U test is done to test for significance. The data underlying this figure are available at: https://figshare.com/s/f5d913a0a289339ee16b. https://doi.org/10.1371/journal.pbio.3002585.g006 We next asked if Mrj is expressed in brain structures that are relevant for memory. Towards this, we used the Mrj knockout line, where we introduced a Gal4 cassette in the locus. On using this line to express CD8GFP, we observed labeling in the mushroom body lobes (Fig 6C). As, in the Drosophila brain, the mushroom body is the center for the formation and storage of memory, we enquired if Mrj is needed in the mushroom body to regulate memory. Toward this, we used an Mrj RNAi line. We first checked for the ability of the Mrj RNAi line to knock down Mrj in the brain. On inducing Mrj RNAi using a pan-neuronal Elav Gal4 driver, we could not detect Mrj in the RNAi fly head extract in comparison to the control (Fig 6D). This indicated the effective knockdown of endogenous Mrj to non-detectable levels. Driving the same Mrj RNAi line using a glial cell-specific driver Repo Gal4, did not show any decrease in Mrj levels (S5H Fig). We also took advantage of the Mrj KO Gal4 line by using it to drive NLS-GFP and immunostained these brains with a glia-specific anti-Repo antibody. Here, we could see cells that were positive for both Repo and Mrj Gal4 driven GFP (S5I Fig), suggesting Mrj apart from neurons is most likely also expressed in glial cells. On our observation of not detecting any Mrj on driving the Mrj RNAi line using Elav Gal4, one explanation can be the majority of the endogenous Mrj is probably contributed from the neurons. Another possibility is, it is reported that Elav Gal4 expresses in neuroblasts and glial cells at earlier developmental stages [76], and so the Elav Gal4 is also knocking down Mrj in these cells along with the neurons causing a drastic decrease of Mrj levels. Using this Mrj RNAi line we now asked, if Mrj is specifically required in the mushroom body neurons for memory. We used a mushroom body-specific 201Y Gal4 line which extensively expresses in the γ lobes along with some neurons of the α/β lobes (Fig 6E) to knock down Mrj in mushroom body neurons. Previously, the expression of Orb2 transgenes using this 201Y Gal4 line was found to be sufficient to rescue memory deficits in Orb2 mutants [18]. Hence, this Gal4 line provides us with the advantage of knocking down Mrj only in the Orb2-relevant neurons. Though earlier we could not detect any gross developmental defects in Mrj knockout flies, we wondered if there might be fine undetected developmental defects. To rule out memory deficiency caused due to such defects, we decided to perform the Mrj knockdown in the adult stage after all development has taken place. Towards this, we coupled the 201Y Gal4 line with a temperature-sensitive Gal80 ts [77,78] and used this to perform the Mrj knockdown with the inducible Mrj RNAi line. These flies were allowed to lay eggs, grow, and eclose at 22°C temperature. At this temperature, the Gal80 would suppress the transcriptional activity of Gal4 and the Mrj RNAi would not be induced. Post eclosion, these flies were shifted to 30°C, where the Gal80 becomes inactive resulting in restoration of the transcriptional activity of Gal4 causing the Mrj RNAi to knockdown Mrj in the mushroom body neurons. We next subjected these mushroom body-specific Mrj knockdown animals to courtship suppression-based memory assay as described earlier (Fig 6A). At post-training time points of 2 h and 12 h, we could not detect any significant difference in memory scores between the control and Mrj knockdown groups, suggesting a normal formation of early and intermediate-term memory. However, at the 16-h time point, the memory score became significantly reduced for the Mrj knockdown animals in comparison to the control animals (Fig 6F). This difference became even greater at the 24-h time point where the memory score for Mrj knockdown decreased drastically compared to the control (Fig 6F). As a control for the same experiment, we left the crosses of the 201YGal4; Tub Gal80 ts with the control and Mrj RNAi lines to grow and eclose at 18°C, where the Gal80 ts will not be inactivated and hence the RNAi will not be induced. These flies when tested in the memory assays showed no significant difference in the memory scores at any of the time points including 16 and 24 h (S6B Fig). Together these experiments suggest Mrj is needed in mushroom body 201Y-specific neurons for regulating long-term memory in Drosophila.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002585

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