Role of Drosophila Orb2 (CPEB) in Synaptic Protein Synthesis

How a transient experience creates a persistent memory remains a fundamental unresolved issue in neuroscience. One of the molecular processes that is believed to be critical for long- lasting memory is synthesis of new protein at the synapse. However, how synaptic protein synthesis is regulated and how these new proteins confers persistence of memory is largely unknown. Previous studies in Drosophila and Aplysia have implicated that a family of mRNA binding proteins known as Cytoplasmic Polyadenylation Element (CPE) binding proteins is essential for persistent change in experience-dependent synaptic efficacy and persistence of memory. Moreover specific CPEB family members demonstrate biophysical properties that are associated with prion-like proteins. They exist in two distinct physical states: a monomeric and a dominant self-sustaining amyloidogenic aggregated state. This suggested a model in which a transient experience creates persistence molecular alteration in the nervous system via recruiting a stable and self-sustaining amyloidogenic aggregates of neuronal CPEB. The primary objective of this thesis is to determine how Drosophila neuronal CPEB, Orb2, regulates protein synthesis and how conversion to the aggregated state effects its function. Combining in vitro and in vivo studies we find that the monomeric Orb2 represses, while the amyloidogenic oligomeric Orb2 enhances translation and imparts its translational state onto the monomer. The monomer removes, whereas the oligomer stabilizes and elongates the polyA tail of mRNA. In support of these findings, we have identified a two novel proteins: CG13928, which binds only to monomeric Orb2, promotes deadenylation, and CG4612, a putative polyA binding protein, promotes oligomeric Orb2-dependent translation. We posit that monomeric Orb2 keeps target mRNA in a translationally dormant state and experience-dependent conversion of Orb2 to the stable amyloidogenic state activates translation, resulting in persistent alteration of synaptic activity and stabilization of memory. This study also provides an example of an amyloid-based protein switch that turns a repressor into an activator

Vulnerability to HIV infection among sex worker and non-sex worker female injecting drug users in Dhaka, Bangladesh: evidence from the baseline survey of a cohort study

BACKGROUND: Very little is known about female injecting drug users (IDU) in Bangladesh but anecdotal evidence suggests that they are hidden and very vulnerable to HIV through both their injection sharing and sexual risk behaviors. In order to better understand the risks and vulnerability to HIV of female IDU, a cohort study was initiated through which HIV prevalence and risk behaviors was determined. METHODS: All female IDU (those who had injected in the last six months and were 15 years or older) who could be identified from three cities in the Dhaka region were enrolled at the baseline of a cohort study. The study was designed to determine risk behaviors through interviews using a semi-structured questionnaire and measure prevalence of HIV, hepatitis C and syphilis semiannually. At the baseline of the cohort study 130 female IDU were recruited and female IDU selling sex in the last year (sex workers) versus those not selling sex (non-sex workers) were compared using descriptive statistics and logistic regression. RESULTS: Of the 130 female IDU enrolled 82 were sex workers and 48 were non-sex workers. None had HIV but more sex workers (60%) had lifetime syphilis than non-sex workers (37%). Fewer sex worker than non-sex worker IDU lived with families (54.9% and 81.3% respectively), but more reported lending needles/syringes (29.3% and 14.6% respectively) and sharing other injection paraphernalia (74.4% and 56.3% respectively) in the past six months. Although more sex workers used condoms during last sex than non-sex workers (74.4% and 43.3% respectively), more reported anal sex (15.9% and 2.1% respectively) and serial sex with multiple partners (70.7% and 0% respectively). Lifetime sexual violence and being jailed in the last year was more common in sex workers. CONCLUSION: Female IDU are vulnerable to HIV through their injection and sexual risk behaviors and sex worker IDU appear especially vulnerable. Services such as needle exchange programs should become more comprehensive to address the needs of female IDU

Protein Translation in the Pathogenesis of Parkinson's Disease.

Peer reviewed: TruePublication status: PublishedFunder: JPB FoundationIn recent years, research into Parkinson's disease and similar neurodegenerative disorders has increasingly suggested that these conditions are synonymous with failures in proteostasis. However, the spotlight of this research has remained firmly focused on the tail end of proteostasis, primarily aggregation, misfolding, and degradation, with protein translation being comparatively overlooked. Now, there is an increasing body of evidence supporting a potential role for translation in the pathogenesis of PD, and its dysregulation is already established in other similar neurodegenerative conditions. In this paper, we consider how altered protein translation fits into the broader picture of PD pathogenesis, working hand in hand to compound the stress placed on neurons, until this becomes irrecoverable. We will also consider molecular players of interest, recent evidence that suggests that aggregates may directly influence translation in PD progression, and the implications for the role of protein translation in our development of clinically useful diagnostics and therapeutics

Protein Translation in the Pathogenesis of Parkinson’s Disease

In recent years, research into Parkinson’s disease and similar neurodegenerative disorders has increasingly suggested that these conditions are synonymous with failures in proteostasis. However, the spotlight of this research has remained firmly focused on the tail end of proteostasis, primarily aggregation, misfolding, and degradation, with protein translation being comparatively overlooked. Now, there is an increasing body of evidence supporting a potential role for translation in the pathogenesis of PD, and its dysregulation is already established in other similar neurodegenerative conditions. In this paper, we consider how altered protein translation fits into the broader picture of PD pathogenesis, working hand in hand to compound the stress placed on neurons, until this becomes irrecoverable. We will also consider molecular players of interest, recent evidence that suggests that aggregates may directly influence translation in PD progression, and the implications for the role of protein translation in our development of clinically useful diagnostics and therapeutics

Contribution of Orb2A Stability in Regulated Amyloid-Like Oligomerization of <i>Drosophila</i> Orb2

<div><p>How learned experiences persist as memory for a long time is an important question. In <i>Drosophila</i> the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. Here, we report the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.</p></div

Tob stabilizes Orb2A and induces Orb2 oligomerization.

<p>(A) Tob enhances Orb2A stability. Orb2 stability was examined in S2 cells expressing either Orb2A by itself (top panel, Orb2A) or in conjunction with Tob (bottom panel, +Tob). Following the addition of cycloheximide (Chx), samples were taken at the given time points and then analyzed for expression levels of Orb2A by Western blot (left panels). Right panels compare protein half-lives. Half-lives were determined by plotting the percent of protein remaining after time zero and assuming first-order kinetics. The <i>n</i> indicates number of independent experiments performed to determine half-lives. Statistical significance was determined using an unpaired, two-tailed <i>t</i> test. The data are plotted as mean ± SEM. (B) Tob has little effect on Orb2B stability. (C) Both Orb2A and Orb2B significantly enhance Tob stability. (D) Overexpression of Tob increases Orb2 oligomerization. Orb2 was immunoprecipitated from adult head extracts expressing only TdTomato (Elav-Gal4: UAS-TdTom), Tob tagged to TdTomato (Elav-Gal4: UAS-TobTdTom), or Tob lacking a 28 amino acid domain critical for binding to Orb2 (Elav-Gal4: UAS-TobΔ28TdTom). (E) Overexpression of Tob increases the number and size of Orb2AEGFP puncta. (Left panel) Both proteins were expressed in the ellipsoid body of the central complex using a c547Gal4 driver. Each row represents a fly genotype: c547-Gal4:UAS-Orb2AEGFP (Orb2A only), c547-Gal4:UAS-Orb2AEGFP/UAS-TobTdtomato (Orb2A Tob), and c547-Gal4:UAS-Orb2AEGFP/UAS-TobΔ28Tdtomato (Orb2A TobΔ28). Scale bar, 25 µm. (Right panel) Higher magnification image of the boxed region in the left. Puncta were counted in the central portion of the ellipsoid body. Axiovision software was programmed to identify a continuous central region and define aggregates, indicated as white dots. Scale bar, 20 µm. Please see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001786#pbio.1001786.s003" target="_blank">Figure S3</a> for additional images. (F) Puncta number (/100 µm²) and (G) size (average area of aggregates in µm²) were quantified. Statistical analysis was performed using an unpaired two-tailed <i>t</i> test (*) <i>p</i>≤0.05, (**) <i>p</i>≤0.01, and (***) <i>p</i>≤0.001. <i>n</i>, the number of flies examined for each genotype. The data are plotted as mean ± SEM. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001786#pbio.1001786.s002" target="_blank">Figures S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001786#pbio.1001786.s003" target="_blank">S3</a>.</p

Model of activity-dependent Orb2 oligomerization.

<p>The model depicts the sequence of events that leads to oligomerization of Orb2A and destabilization of Tob. PP2A keeps both Orb2 and Tob in an unphosphorylated form, which particularly destabilizes Orb2A. Synaptic activation leads to Orb2A synthesis, and the unphosphorylated Orb2A is bound and stabilized by unphosphorylated Tob. Tob recruits activated LimK to the complex, which phosphorylates Orb2A and Tob. The initial phosphorylation of Tob and Orb2 can trigger additional phosphorylation by other kinases, which leads to the dissociation and destabilization of Tob. On the other hand, the phosphorylation of Orb2A leads to further stabilization and/or changes in conformation to induce oligomerization. The oligomeric Orb2A may directly or indirectly induce oligomerization of Orb2B. The phosphorylation of Orb2A and Orb2B may also regulate the function of both proteins.</p

Tob and Orb2 are phosphorylated, and phosphorylation regulates Tob-Orb2 association.

<p>(A) Orb2 is phosphorylated in the adult fly head. Blotting of Orb2 immunoprecipitate with phospho-tag™ detects phosphorylated Orb2B. Treatment with calf-intestinal phosphatase (PPase) that removes phosphate groups from proteins shows specificity of phospho-tag™. (B) Both Orb2A and Orb2B are phosphorylated at a low level in S2cells. Orb2 immunoprecipitate from the S2 cell is probed with phospho-tag™. (C) Tob is phosphorylated in the adult fly brain. (Left panel) Approximately 2 mg of total head extracts were immunoprecipitated with preimmune (pre) or immune Tob serum. The phospho-tag™ detects a band at the position of Tob only in the immune but not in preimmune lane. Treatment with λ-phosphatase (PPase) reduces phospho- tag™ signal. (Right panel, top) Total adult head extracts were treated with calf-intestinal phosphatase (PPase). Change in phosphorylation status of Tob was assessed as a change in mobility by Western blot analysis. (Right panel, bottom) Exogenously expressed Flag-tagged Tob protein is also phosphorylated in the adult brain. (D) The addition of the phosphatase inhibitor, calyculin (CY), dissociates the Orb2-Tob complex. S2 cells were transfected with Orb2A with and without Tob and treated with 10 µm CY for 1 h prior to Tob immunoprecipitation. CY treatment almost completely abolished Tob association with Orb2A oligomers and reduced association with the monomers. (E) Unphosphorylated Tob has a greater affinity for Orb2A. S2 cell lysates were first treated with the indicated units of phosphatase (uPPase), and subsequently the Orb2A-Tob complex was immunoprecipitated. More Orb2A was found to be associated with Tob following phosphatase treatment. Western blots of the lysates show the level of Orb2A and Tob (input). The 4%–12% gradient gels were used in these experiments. (F) Hyperphosphorylated Tob does not associate with Orb2. Untagged Orb2 and FLAG-tagged Tob complex was immunopurified using anti-Orb2 antibodies and probed with phosphor-tag™ (top panel). Phosphorylated proteins correspond to the size of Orb2 (bottom panel) but not Tob (middle panel). The hyperphosphorylated Tob proteins are visible in the total extract (input), however they are absent in the Orb2-Tob complex. The 8% gel in Tris-Glycine buffer was used in this experiment.</p

PP2A regulates Tob-Orb2 phosphorylation and stability.

<p>(A–C) Tob is destabilized upon PP2A inhibition when in complex with Orb2. (A) The phosphatase inhibitor calyculinA (CY) does not significantly affect the half-life of Tob alone. (B and C) In the presence of CY, the increase in Tob stability normally observed in the presence of Orb2A (B) or Orb2B (C) was significantly reduced. (D) In S2 cells Tob is phosphorylated when coexpressed with Orb2A or Orb2B. Changes in Tob phosphorylation was confirmed by λ-phosphatase (PPase) treatment. (E) PP2A inhibitor CY, okadaic acid, but not PP1 inhibitor tautomycin increases Orb2A and Orb2B phosphorylation, as evident in shift electrophoretic mobility. (F) The Orb2 proteins are phosphorylated in multiple sites. (G) PP2A inhibitor CY increases the half-life of Orb2A and Orb2B. (H) Overexpression of PP2A catalytic subunit Mts destabilizes Orb2A and Orb2B. The RNA binding protein Hrp36 serves as loading control. Statistical significance was measured with two-tailed <i>t</i> test (*) <i>p</i>≤0.05, (***) <i>p</i>≤0.001. <i>n</i>, the number of independent experiments for each experimental group. The shift in molecular weight associated with phosphorylation was assayed in 8% SDS-PAGE. The half-life determination experiments were assayed in 4%–12% SDS-PAGE. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001786#pbio.1001786.s005" target="_blank">Figure S5</a>.</p