(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



Homeostatic scaling is driven by a translation-dependent degradation axis that recruits miRISC remodeling

['Balakumar Srinivasan', 'National Brain Research Centre', 'Manesar', 'Sarbani Samaddar', 'Sivaram V. S. Mylavarapu', 'Regional Centre For Biotechnology', 'Faridabad', 'James P. Clement', 'Neuroscience Unit', 'Jawaharlal Nehru Centre For Advanced Scientific Research']

Date: 2021-11

Homeostatic scaling in neurons has been attributed to the individual contribution of either translation or degradation; however, there remains limited insight toward understanding how the interplay between the two processes effectuates synaptic homeostasis. Here, we report that a codependence between protein synthesis and degradation mechanisms drives synaptic homeostasis, whereas abrogation of either prevents it. Coordination between the two processes is achieved through the formation of a tripartite complex between translation regulators, the 26S proteasome, and the miRNA-induced silencing complex (miRISC) components such as Argonaute, MOV10, and Trim32 on actively translating transcripts or polysomes. The components of this ternary complex directly interact with each other in an RNA-dependent manner. Disruption of polysomes abolishes this ternary interaction, suggesting that translating RNAs facilitate the combinatorial action of the proteasome and the translational apparatus. We identify that synaptic downscaling involves miRISC remodeling, which entails the mTORC1-dependent translation of Trim32, an E3 ligase, and the subsequent degradation of its target, MOV10 via the phosphorylation of p70 S6 kinase. We find that the E3 ligase Trim32 specifically polyubiquitinates MOV10 for its degradation during synaptic downscaling. MOV10 degradation alone is sufficient to invoke downscaling by enhancing Arc translation through its 3′ UTR and causing the subsequent removal of postsynaptic AMPA receptors. Synaptic scaling was occluded when we depleted Trim32 and overexpressed MOV10 in neurons, suggesting that the Trim32-MOV10 axis is necessary for synaptic downscaling. We propose a mechanism that exploits a translation-driven protein degradation paradigm to invoke miRISC remodeling and induce homeostatic scaling during chronic network activity.

Funding: This work was supported by Ramalingaswami Fellowship (BT/RLF/Re-entry/32/2011) from the Department of Biotechnology, Government of India ( www.dbtindia.gov.in ) and core funding by the National Brain Research Centre ( www.nbrc.ac.in ) to S.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Srinivasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we demonstrate a defined mechanism of synaptic scaling accomplished through an RNA-dependent coordination between translation and proteasome-mediated degradation. We observe that isolated inhibition of either translation or proteasomal activity offsets synaptic homeostasis. Restoration of homeostasis necessitates the combination of both processes. We provide empirical evidence demonstrating that the interaction between translation and protein degradation machineries is direct and RNA dependent. This coordination is achieved when the two apparatuses are tethered to actively translating transcripts linked to miRISC. Synaptic hyperactivity causes an increased abundance of Trim32 and depletion of MOV10 in polysomes; both Trim32 and MOV10 are members of the miRISC. We find that in contexts of chronic hyperactivity, mTORC1-dependent translation of the E3 ligase Trim32 promotes the polyubiquitination and subsequent degradation of MOV10 by proteasome. This is triggered by the mTORC1-mediated phosphorylation of its downstream effector, p70 S6 kinase (p70 S6K). We observe that MOV10 degradation leads to enhanced translation of Arc and results in the reduced distribution of sAMPARs. Loss of MOV10 alone is sufficient to decrease the synaptic strength by reducing sAMPARs and mimic events similar to hyperactivity-driven downscaling. Notably, the observed increase in Arc expression in the context of synaptic downscaling happens via translation and not by transcriptional mechanisms.

The requirement for discrete sets of sensors and effectors is fulfilled within neurons through varied mechanisms including translation and ubiquitin proteasome system (UPS) degradation. An enhanced degradation of postsynaptic density (PSD) proteins including GluA1 and GluA2 has been observed in contexts of altered network excitability [ 26 ], whereas complete inhibition of UPS activity was shown to occlude synaptic compensation [ 27 ]. The integral role of de novo translation in synaptic homeostasis was recently highlighted when proteomic analysis of neurons undergoing upscaling and downscaling revealed a remarkable diversity of newly synthesized proteins. Of particular interest was the significant enrichment in the expression of the proteasome core complex during downscaling [ 28 , 29 ]. The demand for the translation of proteasome complexes implies that proteasomes work alongside translation mechanisms during downscaling. Reports documenting the colocalization of ribosomes and the proteasome in neuronal dendrites [ 30 , 31 ] further emphasize the possibility that these two opposing machineries physically interact within the postsynaptic compartment. The remodeling of the proteome through the dynamic regulation of protein biogenesis and degradation has been termed as cellular “proteostasis” [ 32 ]. However, several questions remain unexplored in the context of cellular proteostasis during homeostatic scaling, such as (a) What factor establishes the link between translation and protein degradation machineries to shape the proteome during scaling? (b) Which process among translation and degradation takes precedence? (c) What are the signaling mechanisms that connect events of “sensing” the bicuculline-mediated hyperactivity and the final down-regulation of sAMPARs?

Till date, not much is known about the repertoire of molecular “sensor” cascades that serve to link events where neurons sense deviations in the network firing rate and subsequently initiate the scaling process. Few molecular sensors have been identified; the eukaryotic elongation factor eEF2 and its dedicated kinase, eEF2 kinase or CamKIII, are the two reported thus far [ 10 ]. One cascade discovered in this context is the mammalian Target Of Rapamycin Complex-1 (mTORC1) signaling pathway that regulates presynaptic compensation in neurons by promoting BDNF synthesis in the postsynaptic compartment [ 11 , 12 ]. In contrast, AMPA receptors (AMPARs) have been identified, by overwhelming consensus, to be the predominant “end-point-effectors” in all paradigms of synaptic scaling [ 13 – 16 ]. Unlike NMDARs, AMPARs undergo de novo translation during network destabilizations [ 17 ], and chronic changes in the postsynaptic response during scaling have been attributed to the abundance of surface AMPARs (sAMPARs) (GluA1 and GluA2 subunits) [ 18 ]. Among the key modifiers of AMPAR expression, miRNAs are known to play pivotal roles in synaptic scaling [ 19 – 22 ]. Relief from translational repression by miRNAs necessitates that mRNAs exit the functional microRNA-induced silencing complex (miRISC). This requires miRISC to undergo dynamic changes in its composition [ 23 , 24 ], a cellular phenomenon previously termed as miRISC remodeling [ 25 ]. However, what remains surprising is our lack of knowledge about how compositional changes within the miRISC are achieved during scaling.

Neurons employ a unique strategy, known as synaptic scaling, to counter the runaway excitation and subsequent loss of input specificity that arise due to Hebbian changes; they rely on a compensatory remodeling of synapses throughout the network while maintaining differences in their synaptic weightage [ 1 – 6 ]. Synaptic scaling is achieved by a complex interplay of sensors and effectors within neurons that serve to oppose global fluctuations in a network and establish synaptic homeostasis by modifying postsynaptic glutamatergic currents in a cell-autonomous manner [ 7 – 9 ]. In the context of homeostatic scaling, “sensors” are classified as molecules that sense deviations in the overall network activity, and “effectors” scale the neuronal output commensurately.

Results

RNA-dependent cosedimentation of the proteasome and translation regulators The colocalization of polyribosomes and proteasomes to sites of synaptic activity [30,31] lead us to examine whether the components of the 26S proteasomal machinery could remain physically associated with actively translating transcripts in order to make the necessary proteomic changes. These components include proteins forming the 19S regulatory subunits and the 20S proteasome core. We analyzed polysomes from the hippocampus of 8- to 10-week-old rats and assessed whether the sedimentation pattern of proteasomes match those of actively translating, polyribosome-associated mRNA fractions. We observed that several components of the proteasomal machinery such as α7 subunit of the 20S proteasome; and Rpt1, Rpt3, and Rpt6 subunits of the 19S proteasome cosedimented with translation initiation factors such as eIF4E and p70 S6K within actively translating polysomes (Figs 3A, 3B, and S2A). We also detected the polysomal distribution of MOV10, a helicase and an RNA-binding protein known to be polyubiquitinated upon synaptic activation, and Trim32, an E3 ligase, both components of the miRISC [23,33] (Fig 3A and 3B). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. RNA-dependent association between active proteasomes and translating polyribosomes. Absorbance profile at 254 nm (A 254 ) and western blot analysis of fractionated cytoplasmic extracts from hippocampal tissue incubated in the absence or presence of MgCl 2 or RNase or EDTA. Monosome (80S), 60S ribosome, and polysome fractions are as indicated. Western blots performed from tri-chloroacetic acid–precipitated fractions to determine the distribution of the translation regulators eIF4E and p70 S6K; α7 subunit of the 20S core of the proteasome, Rpt1, Rpt3, Rpt6 of the 19S cap, and miRISC proteins MOV10 and Trim32 in the presence or absence of MgCl 2 or RNase or EDTA. (A) A 254 profile in the presence of MgCl 2 . (B) Western blots of the fractions obtained in (A). (C) A 254 profile obtained in the presence of RNase. (D) Western blots of the fractions obtained in (C). (E) A 254 profile in the presence of EDTA. (F) Western blots of the fractions obtained in (E). Rpt3 blots with different exposures are distinguished by a vertical black line to denote they represent separate panels within the figure. Two blots with different exposures are shown in the main figure and raw data to visualize the specific band of Rpt3. (G) A 254 profile of fractionated cytoplasmic extract used for determining activity of proteasomes. (H) Quantitation of catalytic activity of proteasomes present in alternate fractions from two polysome preparations. See also S2 Fig. The data underlying this figure are available at https://figshare.com/articles/dataset/Homeostatic_scaling_is_driven_by_a_translation-dependent_degradation_axis_that_recruits_miRISC_remodeling/16768816. miRISC, miRNA-induced silencing complex; p70 S6K, p70 S6 kinase. https://doi.org/10.1371/journal.pbio.3001432.g003 RNase or EDTA treatment of cytoplasmic lysates prior to density gradient fractionation led to a complete collapse of the polysome profile, simultaneously shifting the sedimentation of Rpt6, Rpt1, α7, eIF4E, MOV10, Trim32 to the lighter fractions (Figs 3C–3F, S2B, and S2C). The disruption of association between the translational and proteasomal components on RNase and EDTA treatment suggests that translating transcripts are necessary to recruit the translation and proteasome machineries. These observations ruled out the possibility that the observed cosedimentation was a result of similar densities between the protein complexes and polysomes. Trim32 and MOV10 in specific high-density sucrose fractions (fraction # 8/11/15) were not detected due to loss of proteins during the TCA precipitation step (Fig 3B). Furthermore, we saw that the polysome-associated 26S proteasome is catalytically active as detected by its ability to cleave a fluorogenic proteasome substrate that is blocked by the proteasome inhibitor epoxomicin (Figs 3G, 3H, and S2D).

Proteasome and the regulators of translation directly interact with each other within excitatory neurons Whole-cell patch clamp recordings from hippocampal excitatory neurons demonstrated that the coregulation of translation and proteasome-mediated protein degradation is necessary for synaptic homeostasis. Consistent with this observation, cosedimentation of proteasome subunits along with polysomes linked to protein synthesis regulators and members of the miRISC led us to enquire whether components of the ternary complex directly interact with each other in excitatory neurons of the hippocampus. To evaluate this, we immunoprecipitated the 19S proteasomal complex using Rpt6 antibody from hippocampal neurons. We observed the coprecipitation of eEF2, a translation elongation factor that functions as a “sensor” of change in network activity (Fig 4A). We also found that a known regulator of mTORC1-dependent protein synthesis; p70 S6K as well as its phosphorylated form [34] coprecipitated with the 19S proteasome (Fig 4A). We further analyzed the proteins interacting with polysomes within excitatory neurons by expressing haemagglutinin (HA)-tagged ribosomal protein Rpl22 (HA-Rpl22) that gets incorporated into polysomes [35,36] (Figs 4B–4D and S2E). We reasoned that the analysis of HA-Rpl22-affinity purified complexes would confirm whether the polysome-associated translation and degradation machineries directly interact with each other. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Interaction between proteasome and actively translating RNA-associated polyribosomes. (A) Proteasome-associated protein complex was immunoprecipitated from hippocampal lysate using antibody against Rpt6 or mouse IgG. Western blot of purified protein complex performed using antibodies against Rpt6, eEF2, p70 S6K, and phospho-p70 S6K. (B) RiboTag mouse when crossed with CamKIIa promoter-driven Cre recombinase mouse results in the deletion of wild-type Rpl22 ribosomal protein and replacement of HA-tagged Rpl22 in forebrain excitatory neurons. (C) A 254 profile showing indicated fractions of monosome and polysome. (D) Polysome fractions from (C) showing enrichment of HA-Rpl22 as detected by western blot using antibody against HA. (E) HA-tagged Rpl22 containing polyribosome was affinity-purified using antibody against HA. Western blot analysis of affinity-purified complex shows the presence of HA, Rpt6, Trim32, and MOV10. See also S2 Fig. (F) MOV10 immunoprecipitated from hippocampal lysates. Western blot analysis of MOV10-immunoprecipitated protein complex shows the coprecipitation of Trim32 with miRISC components MOV10 and Ago. (G) Detection of HspA2 and Rpt6 in HA affinity-purified protein complex from HA-Rpl22 expressing neurons by western blot using antibody against HspA2, Rpt6 and HA. (H, I) A 254 profile showing indicated fractions of monosome and polysome obtained from cytoplasmic lysates treated with (H) or without (I) RNase prior to density gradient fractionation. See also S2 Fig. (J) HA-tagged Rpl22 containing ribosomes affinity-purified from heavy fractions of sucrose gradient using antibody against HA. Western blot analysis of affinity-purified complex with antibodies against HA, p70 S6K, eIF4E, 20S Core subunits, Rpt6, and Rpt1. The data underlying this figure are available at https://figshare.com/articles/dataset/Homeostatic_scaling_is_driven_by_a_translation-dependent_degradation_axis_that_recruits_miRISC_remodeling/16768816. Ago, Argonaute; HA, haemagglutinin; IgG, immunoglobulin G; IP, immunoprecipitation; miRISC, miRNA-induced silencing complex; p70 S6K, p70 S6 kinase; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001432.g004 Our western blot analysis of HA-Rpl22 affinity-purified protein complex showed that Rpt6 directly interacts with Trim32 and MOV10 (Fig 4E). Immunoprecipitation of MOV10 from hippocampal neurons resulted in the coprecipitation of both Argonaute (Ago) and Trim32, confirming that the latter is an integral component of the Ago-containing miRISC (Fig 4F). We also detected the chaperone protein HspA2 in the HA-affinity purified fraction along with Rpt6 (Fig 4G), suggesting that HspA2 could tether proteasomes to actively translating transcripts. The direct interaction between components of the translation and proteasome machinery could occur without the participation of polysome-associated, translating mRNA. To evaluate whether the observed association was RNA dependent or RNA independent, HA-Rpl22 affinity-purified protein complexes from polysome fractions of hippocampal tissue lysates treated with or without RNase were analyzed (Fig 4H, 4I, S2F and S2G Fig). Our western blot analysis revealed that the 20S proteasome core, Rpt6, and Rpt1 coprecipitated with eIF4E and p70 S6K (Fig 4J). RNase treatment of the cytoplasmic lysate prior to density gradient fractionations prevented this interaction on the actively translating, heavier fractions of the polysome (Fig 4J). This demonstrates that polysome-associated, translating RNA act as scaffolds to facilitate the direct interaction between protein synthesis and degradation modules.

[END]

[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001432

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.