Synaptic Activity Regulated mRNA-Silencing Foci for the Fine Tuning of Local Protein Synthesis at the Synapse

The regulated synthesis of specific proteins at the synapse is important for neuron plasticity, and several localized mRNAs are translated upon specific stimulus. Repression of mRNA translation is linked to the formation of mRNA-silencing foci, including Processing Bodies (PBs) and Stress Granules (SGs), which are macromolecular aggregates that harbor silenced messengers and associated proteins. In a recent work, we identified a kind of mRNA-silencing foci unique to neurons, termed S-foci, that contain the post-transcriptional regulator Smaug1/SAMD4. Upon specific synaptic stimulation, the S-foci dissolve and release mRNAs to allow their translation, paralleling the cycling of mRNAs between PBs and polysomes in other cellular contexts. Smaug 1 and other proteins involved in mRNA regulation in neurons contain aggregation domains distinct from their RNA binding motifs, and we speculate that self-aggregation helps silencing and transport. In addition to S-foci and PBs, other foci formed by distinct RNA binding proteins, such as TDP-43 and FMRP among others, respond dynamically to specific synaptic stimuli. We propose the collective name of synaptic activity-regulated mRNA silencing (SyAS) foci for these RNP aggregates that selectively respond to distinct stimulation patterns and contribute to the fine-tuning of local protein synthesis at the synapse.Fil: Pascual, Malena Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Luchelli, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Habif, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Boccaccio, Graciela Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin

Cytoplasmic Expression of the ALS/FTD-Related Protein TDP-43 Decreases Global Translation Both in vitro and in vivo

TDP-43 is a major component of cytoplasmic inclusions observed in neurodegenerative diseases like frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To further understand the role of TDP-43 in mRNA/protein metabolism and proteostasis, we used a combined approach with cellular and animal models overexpressing a cytoplasmic form of human TDP-43 (TDP-43-ΔNLS), recapitulating ALS/FTD features. We applied in HEK293 cells a method for labeling de novo translation, surface sensing of translation (SUnSET), based on puromycin (PURO) incorporation. While control cells displayed robust puromycilation, TDP-43-ΔNLS transfected cells exhibited reduced ongoing protein synthesis. Next, by using a transgenic mouse overexpressing cytoplasmic TDP-43 in the forebrain (TDP-43-ΔNLS mice) we assessed whether cytoplasmic TDP-43 regulates global translation in vivo. Polysome profiling of brain cortices from transgenic mice showed a shift toward non-polysomal fractions as compared to wild-type littermates, indicating a decrease in global translation. Lastly, cellular level translational assessment by SUNSET was performed in TDP-43-ΔNLS mice brain slices. Control mice slices incubated with PURO exhibited robust cytoplasmic PURO signal in layer 5 neurons from motor cortex, and normal nuclear TDP-43 staining. Neurons in TDP-43-ΔNLS mice slices incubated with PURO exhibited high cytoplasmic expression of TDP-43 and reduced puromycilation respect to control mice. These in vitro and in vivo results indicate that cytoplasmic TDP-43 decreases global translation and potentially cause functional/cytotoxic effects as observed in ALS/FTD. Our study provide in vivo evidence (by two independent and complementary methods) for a role of mislocalized TDP-43 in the regulation of global mRNA translation, with implications for TDP-43 proteinopathies.Fil: Charif, Santiago Elías. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Luchelli, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Vila, Antonella. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; ArgentinaFil: Blaustein, Matías. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; ArgentinaFil: Müller Igaz, Lionel Ivan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentin

A Monoclonal Antibody against p53 Cross-Reacts with Processing Bodies

The p53 tumor suppressor protein is an important regulator of cell proliferation and apoptosis. p53 can be found in the nucleus and in the cytosol, and the subcellular location is key to control p53 function. In this work, we found that a widely used monoclonal antibody against p53, termed Pab 1801 (Pan antibody 1801) yields a remarkable punctate signal in the cytoplasm of several cell lines of human origin. Surprisingly, these puncta were also observed in two independent p53-null cell lines. Moreover, the foci stained with the Pab 1801 were present in rat cells, although Pab 1801 recognizes an epitope that is not conserved in rodent p53. In contrast, the Pab 1801 nuclear staining corresponded to genuine p53, as it was upregulated by p53-stimulating drugs and absent in p53-null cells. We identified the Pab 1801 cytoplasmic puncta as P Bodies (PBs), which are involved in mRNA regulation. We found that, in several cell lines, including U2OS, WI38, SK-N-SH and HCT116, the Pab 1801 puncta strictly colocalize with PBs identified with specific antibodies against the PB components Hedls, Dcp1a, Xrn1 or Rck/p54. PBs are highly dynamic and accordingly, the Pab 1801 puncta vanished when PBs dissolved upon treatment with cycloheximide, a drug that causes polysome stabilization and PB disruption. In addition, the knockdown of specific PB components that affect PB integrity simultaneously caused PB dissolution and the disappearance of the Pab 1801 puncta. Our results reveal a strong cross-reactivity of the Pab 1801 with unknown PB component(s). This was observed upon distinct immunostaining protocols, thus meaning a major limitation on the use of this antibody for p53 imaging in the cytoplasm of most cell types of human or rodent origin

Smaug1 mRNA-silencing foci respond to NMDA and modulate synapse formation

S-foci, the first reported mRNA-silencing foci specific to neurons, may control local mRNA translation in response to NMDA receptor stimulation and synaptic plasticity

Synaptic control of local translation: the plot thickens with new characters

The production of proteins from mRNAs localized at the synapse ultimately controls the strength of synaptic transmission, thereby affecting behavior and cognitive functions. The regulated transcription, processing, and transport of mRNAs provide dynamic control of the dendritic transcriptome, which includes thousands of messengers encoding multiple cellular functions. Translation is locally modulated by synaptic activity through a complex network of RNA-binding proteins (RBPs) and various types of non-coding RNAs (ncRNAs) including BC-RNAs, microRNAs, piwi-interacting RNAs, and small interference RNAs. The RBPs FMRP and CPEB play a well-established role in synaptic translation, and additional regulatory factors are emerging. The mRNA repressors Smaug, Nanos, and Pumilio define a novel pathway for local translational control that affects dendritic branching and spines in both flies and mammals. Recent findings support a role for processing bodies and related synaptic mRNA-silencing foci (SyAS-foci) in the modulation of synaptic plasticity and memory formation. The SyAS-foci respond to different stimuli with changes in their integrity thus enabling regulated mRNA release followed by translation. CPEB, Pumilio, TDP-43, and FUS/TLS form multimers through low-complexity regions related to prion domains or polyQ expansions. The oligomerization of these repressor RBPs is mechanistically linked to the aggregation of abnormal proteins commonly associated with neurodegeneration. Here, we summarize the current knowledge on how specificity in mRNA translation is achieved through the concerted action of multiple pathways that involve regulatory ncRNAs and RBPs, the modification of translation factors, and mRNA-silencing foci dynamics.Fil: Thomas, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires(i); Argentina. Fundación Instituto Leloir; ArgentinaFil: Pascual, Malena Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires(i); Argentina. Fundación Instituto Leloir; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Maschi, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires(i); Argentina. Fundación Instituto Leloir; ArgentinaFil: Luchelli, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires(i); Argentina. Fundación Instituto Leloir; ArgentinaFil: Boccaccio, Graciela Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires(i); Argentina. Fundación Instituto Leloir; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin

RNase treatment or knockdown of specific PB components provokes the simultaneous dissolution of PBs and Pab 1801 puncta.

<p>A, live U2OS cells were permeabilized with 0.1% TX100 for five minutes and then exposed to CSKB without (Control) or with 100 µg/ml of RNase (RNase) for additional 5 minutes. The percentage of cells with PBs simultaneous stained with Dcp1a and Pab 1801 was evaluated in 100 cells from duplicate experiments for each treatment and diminished from 75% in control cells to 40% in RNAse-treated cells. The remaining Pab 1801 foci have less than half of the size of control cells. B, U2OS and SK-N-SH cells were treated with specific siRNAs against the PB components Hedls, Rck/p54 or 4ET, and stained with the indicated antibodies. The Pab 1801 puncta vanished when PB are disrupted. The proportion of cells with <i>foci</i> is indicated, as evaluated in 100 cells from duplicate experiments for each treatment. As before (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036447#pone-0036447-g004" target="_blank">Figures 4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036447#pone-0036447-g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036447#pone-0036447-g006" target="_blank">6</a>), all puncta were double stained for each pair of antibodies and single-stained <i>foci</i> were not present in any of the treatments. Bar, 10 µm.</p

The Pab 1801-positive puncta contain PB components.

<p>The indicated p53+/+ or −/− cell lines were simultaneously immunostained with Pab 1801 and antibodies specific for the PB components Dcp1a, Rck/p54, Hedls or Xrn1. In all cases, all the cytoplasmic <i>foci</i> recognized by the Pab 1801 were also recognized by the antibodies against PB markers. Bars, whole cells, 10 µm; magnifications, 1 µm.</p

Pab 1801 puncta are present in p53-null cells.

<p>A, B, p53+/+ or p53−/− HCT116 cells were treated with daunorubicin and immunostained with the Pab 1801 (A) or the Pab DO1 (B). With both antibodies, a nuclear signal is observed upon stimulation of p53+/+ cells but not p53−/− cells. Pab 1801-positive puncta are always present in p53+/+ and p53−/− cells independently of p53 levels. C, p53-null H1299 cells were treated with the indicated drugs and stained with the Pab 1801. The Pab 1801 puncta disappeared upon exposure to cycloheximide and remained unaltered after puromycin treatment. The percentage of cells with punctate Pab 1801 signal was evaluated in 100 cells from duplicate stainings for each treatment. Bar, 10 µm. D, alignment of human and rat p53 N-terminus including the Pab 1801 epitope (grey box). The 10-aa epitope is absent in the rat sequence. E, Hippocampal rat neurons were prepared as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036447#pone.0036447-Baez2" target="_blank">[27]</a> and stained with the Pab 1801 or the Pab DO1. A merge of the immunofluorescence and the phase contrast images is shown. Pab 1801-positive puncta are detected in cell soma and dendrites. Bars, whole cells, 10 µm; magnifications, 1 µm.</p

Pab 1801 puncta and PBs are simultaneously induced by different strategies.

<p>A, B, U2OS cells were treated with two different combinations of arsenite and puromycin: 250 µM arsenite +100 µg/ml puromycin (1× induction) and 500 µM arsenite +250 µg/ml puromycin (2× induction). A, The average number of PBs per cell increased from 4 in control cells to 14 in treated cells, as evaluated in 100 cells from duplicate experiments for each treatment. Induced PBs were stained with the Pab 1801 in all cases. An example of the 2× induction is shown in Ars+Puro panel. B, the size of Dcp1a-foci and Pab 1801 foci increased at the same rate and in a dose-dependent manner. Median values of more than 100 foci size for each treatment were plotted. C, overexpression of the translational repressor Smaug 1 also provoked 1801 foci induction. U2OS cells were transfected with Smaug 1-ECFP and number of foci per cell and foci size were analized in transfected (T) and neighbouring non-transfected cells (NT). The average number of PBs per cell increased from 5 in non-transfected cells to 55 in transfected cells, as evaluated in 100 hundred cells of each condition. In all cases PBs were stained with the 1801 antibody. The foci size increased more than twice, as evaluated for both 1801 and Rck/p54 in 200 non-transfected foci and 750 Smaug1-ECFP transfected foci. Bars, 10 µm.</p