Proteostatic regulation in neuronal compartments
Authors: Stefano L. Giandomenico, Beatriz Alvarez-Castelao, Erin M. Schuman
Journal: Trends in Neurosciences (2021)

Dynamic local regulation of protein synthesis and degradation allows neuronal synapses to modify their proteome autonomously during plasticity.While our understanding of how mRNAs are localized to different neuronal compartments has rapidly advanced, a comprehensive and mechanistic understanding of how synaptic activity triggers translation of certain transcripts over others is still lacking.The UPS degrades protein substrates selectively and specifically, but the local role of the UPS in neuronal compartments remains underexplored. Recent efforts in chemical biology and proteomics have established new promising tools and techniques to bridge this knowledge gap.PTMs are a rich regulatory resource in cells. Future efforts should focus on broadening our knowledge of local regulation of PTMs in response to neuronal activity and how specific PTMs contribute to neuronal proteostasis. Neurons continuously adapt to external cues and challenges, including stimulation, plasticity-inducing signals and aging. These adaptations are critical for neuronal physiology and extended survival. Proteostasis is the process by which cells adjust their protein content to achieve the specific protein repertoire necessary for cellular function. Due to their complex morphology and polarized nature, neurons possess unique proteostatic requirements. Proteostatic control in axons and dendrites must be implemented through regulation of protein synthesis and degradation in a decentralized fashion, but at the same time, it requires integration, at least in part, in the soma. Here, we discuss current understanding of neuronal proteostasis, as well as open questions and future directions requiring further exploration. Neurons continuously adapt to external cues and challenges, including stimulation, plasticity-inducing signals and aging. These adaptations are critical for neuronal physiology and extended survival. Proteostasis is the process by which cells adjust their protein content to achieve the specific protein repertoire necessary for cellular function. Due to their complex morphology and polarized nature, neurons possess unique proteostatic requirements. Proteostatic control in axons and dendrites must be implemented through regulation of protein synthesis and degradation in a decentralized fashion, but at the same time, it requires integration, at least in part, in the soma. Here, we discuss current understanding of neuronal proteostasis, as well as open questions and future directions requiring further exploration. Most catalyzed chemical reactions inside cells depend on protein levels, and fine-tuning protein concentrations is key to ensure proper cellular function. Because throughout the cellular lifespan proteins accumulate damage, become dysfunctional and need to be replaced continually, protein synthesis and protein turnover are central to cellular physiology and function. The dynamic regulation of a balanced and functional proteome (i.e., proteostasis) concerns all proteins whose levels need to be adjusted in space and time in response to intracellular and extracellular cues. Thus, the specific parameters of cellular proteostasis vary across cell types and states. However, cellular proteostasis invariably relies on the precise control of protein synthesis, folding and conformational maintenance, post-translational modifications (PTMs), degradation, and secretion [1.Hipp M.S. et al.The proteostasis network and its decline in ageing.Nat. Rev. Mol. Cell Biol. 2019; 20: 421-435Google Scholar]. Precise control of these parameters is already challenging in cells that have little or no polarity, but becomes a particularly impressive feat for highly polarized cells, such as neurons. Neurons stand out from all other cell types in their unique morphology and high degree of compartmentalization; characteristics that are central to neuronal computation. Often, extrinsic signals are spatially localized so that only a confined portion of the neuron receives a certain signal, with the neuronal portion being, for instance, a cluster of dendrites (influenced by a neuromodulator), a single dendritic branch, a synaptic neighborhood, or even an individual synapse. How these local signals are transmitted to the somata remains largely unknown. Work over the last two decades has shown that neurons have the capacity to tune their proteome locally through regulation of local protein synthesis, degradation, and PTMs to regulate multiple aspects of dendritic and axonal biology [2.Holt C.E. Schuman E.M. The central dogma decentralized: New perspectives on RNA function and local translation in neurons.Neuron. 2013; 80: 648-657Google Scholar]. Nevertheless, to date, we still lack a comprehensive understanding of how different cellular degradation pathways interact with the protein synthesis machinery to shape and maintain the local proteome. In this review, we discuss current understanding of neuronal local proteostatic regulation, and key knowledge gaps in the field. We also comment on present technological limitations and promising recent advances to address some of the outstanding questions. Studies in primary cultured neurons and mathematical modeling have estimated that ~0.7% of the synaptic protein content of a neuron is turned over every hour at glutamatergic postsynaptic densities [3.Cohen L.D. et al.Metabolic turnover of synaptic proteins: kinetics, interdependencies and implications for synaptic maintenance.PLoS One. 2013; 8e63191Google Scholar]. Although this figure is likely to change depending on parameters such as neuron type, number of synapses, and activity, it suggests that maintaining the protein content of synapses places a considerable demand on the cellular protein synthesis machinery. Recent proteomic studies have shown that, at a population level, protein turnover rates change depending on the activity state of neurons [4.Dörrbaum A.R. et al.Local and global influences on protein turnover in neurons and glia.Elife. 2018; 7e34202Google Scholar]. At the level of an individual neuron, this likely translates into subsets of synapses differentially regulating protein turnover rates according to their activity state [5.Rangaraju V. et al.Local translation in neuronal compartments: how local is local?.EMBO Rep. 2017; 18: 693-711Google Scholar]. Alterations in protein turnover are accomplished by changes in the activity of protein degradation and protein synthesis machinery in the soma and, importantly, in neuronal processes and near synapses. The first data suggesting the capacity of neuronal processes to synthesize proteins came from early electron microscopy (EM) studies, showing polyribosomes in dendritic spines and in their near vicinity [6.Steward O. Levy W.B. Preferential localization of polyribosomes under the base of dendritic spines in granule cells of the dentate gyrus.J. Neurosci. 1982; 2: 284-291Google Scholar]. These experiments, however, lacked the ability to demonstrate the translational activity of these distally located ribosomes. Later, work on the CA1 hippocampal neuropil and retinal ganglion cell axons showed that the mRNA population in axons and dendrites is not limited to a small number of mRNAs but rather is a large and diverse group, containing up to ~5000 different mRNA species [7.Gumy L.F. et al.Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization.RNA. 2011; 17: 85-98Google Scholar, 8.Zivraj K.H. et al.Subcellular profiling reveals distinct and developmentally regulated repertoire of growth cone mRNAs.J. Neurosci. 2010; 30: 15464-15478Google Scholar, 9.Poon M.M. et al.Identification of process-localized mRNAs from cultured rodent hippocampal neurons.J. Neurosci. 2006; 26: 13390-13399Google Scholar, 10.Cajigas I.J. et al.The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging.Neuron. 2012; 74: 453-466Google Scholar, 11.Glock, C. et al. 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While these findings support the importance of local protein synthesis in shaping the local proteome with spatial and temporal precision, they also raise a number of questions related to the interplay between the complement of mRNAs in neuronal processes and the local translation machinery. So far, the axonal and dendritic transcriptomes have been investigated primarily by bulk-sequencing approaches on microdissected tissue, which fail to capture cell-to-cell and process-to-process heterogeneity. Recently, the combination of laser capture microdissection and droplet-based sequencing on dendrites and somata has revealed interesting differences and similarities between the dendritic and somatic transcriptomes and between excitatory and inhibitory neurons [14.Perez J.D. et al.Subcellular sequencing of single neurons reveals the dendritic transcriptome of gabaergic interneurons.Elife. 2021; 10: 1-26Google Scholar]. Nevertheless, laser capture microdissection is not precise enough to probe the spatial distribution of a large population of mRNAs relative to dendritic spines and synaptic boutons. In this regard, microscopy based approaches such as seqFISH+ [15.Eng C.H.L. et al.Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+.Nature. 2019; 568: 235-239Google Scholar] and ExSeq [16.Alon S. et al.Expansion sequencing: spatially precise in situ transcriptomics in intact biological systems.Science (80-. ). 2021; 371Google Scholar] hold promise for furthering our understanding of the subcellular distribution of mRNAs in neurons on a transcriptome-wide scale under basal conditions and during plasticity. A more complete understanding of how mRNAs map across neurons needs to be complemented with information on mRNA transport and release dynamics under baseline conditions and following changes in activity that promote plasticity. The 3′ and 5′untranslated regions (UTRs) contain elements important for the binding, transport, localization, and regulated translation of mRNAs [17.Fernandopulle M.S. et al.RNA transport and local translation in neurodevelopmental and neurodegenerative disease.Nat. Neurosci. 2021; : 1-11Google Scholar]. Pointing to the importance of translational regulation in neurons, the 3′ UTRs of neuronal mRNAs, particularly those localized to axons and dendrites, are significantly longer than those of other cell types [18.Tushev G. et al.Alternative 3′ UTRs modify the localization, regulatory potential, stability, and plasticity of mRNAs in neuronal compartments.Neuron. 2018; 98: 495-511.e6Google Scholar]. An important tier of regulation likely exists at the level of how mRNAs are packaged and combined together before being trafficked to distal locations [17.Fernandopulle M.S. et al.RNA transport and local translation in neurodevelopmental and neurodegenerative disease.Nat. Neurosci. 2021; : 1-11Google Scholar]. Work in yeast has shown that mRNA transport granules can comprise multiple functionally related RNAs in, what effectively represent, instruction packages for coordinated metabolic responses [19.Lange S. et al.Simultaneous transport of different localized mRNA species revealed by live-cell imaging.Traffic. 2008; 9: 1256-1267Google Scholar]. Biochemical analysis of neuronal RNA granules has led to the identification of hundreds of granule-associated transcripts [20.De Graeve F. Bessé F. Neuronal RNP granules: from physiological to pathological assemblies.Biol. Chem. 2018; 399: 623-635Google Scholar], and both multiplexed [21.Gao Y. et al.Multiplexed dendritic targeting of α calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein RNAs by the A2 pathway.Mol. Biol. Cell. 2008; 19: 2311-2327Google Scholar] and multicopy [22.Donlin-Asp P.G. et al.Differential regulation of local mRNA dynamics and translation following long-term potentiation and depression.Proc. Natl. Acad. Sci. 2021; 118e2017578118Google Scholar] targeting of different mRNAs in neuronal dendrites have been reported. Other reports have suggested that RNA transport granules in neurons contain single mRNAs [23.Batish M. et al.Neuronal mRNAs travel singly into dendrites.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 4645-4650Google Scholar]. A potential explanation for this discordance may be that these studies were performed on a small cohort of mRNAs. The degree of single versus multiplex mRNA packaging within single transport granules remains to be investigated on an transcriptome-wide scale. In addition to mRNAs, RNA granules contain ribosomes along with translational repressors, and their integrity can be modulated in response to neuronal depolarization [24.Krichevsky A.M. Kosik K.S. Neuronal RNA granules: A link between RNA localization and stimulation-dependent translation.Neuron. 2001; 32: 683-696Google Scholar]. Studies of long-term potentiation (LTP) and long-term depression have shown that, following plasticity, RNAs are rapidly released from RNA granules, accumulate in the vicinity of dendritic spines, and undergo 3′ UTR cleavage, polyadenylation and on-site translation [22.Donlin-Asp P.G. et al.Differential regulation of local mRNA dynamics and translation following long-term potentiation and depression.Proc. Natl. Acad. Sci. 2021; 118e2017578118Google Scholar,25.Udagawa T. et al.Bidirectional control of mRNA translation and synaptic plasticity by the cytoplasmic polyadenylation complex.Mol. Cell. 2012; 47: 253-266Google Scholar,26.Fontes M.M. et al.Activity-dependent regulation of alternative Cleavage and Polyadenylation during Hippocampal Long-Term Potentiation.Sci. Rep. 2017; 7: 1-13Google Scholar] (Figure 1). Conversely, local translational repression can be achieved through the release and maturation of pre-miRNAs following N-methyl-D-aspartate (NMDA) receptor stimulation [27.Sambandan S. et al.Activity-dependent spatially localized miRNA maturation in neuronal dendrites.Science (80-. ). 2017; 355: 634-637Google Scholar]. The combination of these mechanisms allows for the decoding of spatially complex plasticity patterns into specific translational profiles that precisely remodel the synaptic proteome. At first glance, the abundance of RNA transcripts in neuronal processes might seem to contrast with the low abundance of polyribosomes in axons and dendrites [28.Ostroff L.E. et al.Shifting patterns of polyribosome accumulation at synapses over the course of hippocampal long-term potentiation.Hippocampus. 2018; 28: 416-430Google Scholar]. The notion that neuronal processes, and in particular axons, are depleted of ribosomes historically has come from histological analyses by Nissl staining and EM [29.Peters A. et al.The Fine Structure of the Nervous System: Neurons and Their Supporting Cells. W. B. Saunders, 1976Google Scholar]. However, recent work has reaffirmed what was inferred from the robust translational capacity and shown that ribosomes are found ubiquitously in vertebrate pre- and post-synaptic termini [12.Hafner A.S. et al.Local protein synthesis is a ubiquitous feature of neuronal pre- and postsynaptic compartments.Science (80-. ). 2019; 364eaau3644Google Scholar,13.Shigeoka T. et al.Dynamic axonal translation in developing and mature visual circuits.Cell. 2016; 166: 181-192Google Scholar]. A potential reason for this apparent discrepancy is that, while polyribosomes (recognized as 3+ ribosomes) can be detected with confidence by EM, monosomes cannot be unambiguously distinguished from other electron-dense cytoplasmic particles. Resolving these apparently contrasting observations, it was recently shown that monosomes are predominant in neuronal processes and are actively translating [30.Biever A. et al.Monosomes actively translate synaptic mRNAs in neuronal processes.Science (80-. ). 2020; 367Google Scholar] (Figure 1). Historically, monosome translation has been considered negligible in mammalian cells (see [31.Heyer E.E. Moore M.J. Redefining the translational status of 80S monosomes.Cell. 2016; 164: 757-769Google Scholar] for a study in yeast) but in neurons it appears to be a key process. The advantage of monosome over polysome translation in neurons remains unclear, it could represent an adaptation to the small compartment size or a means to diversify the protein products with a limited ribosome population; alternatively, it could be adapted to translate specific mRNAs present in dendrites. Another important aspect of mRNA translation concerns the ribosomes themselves – the protein and RNA composition of the ribosome complex and their PTMs. It is still not fully understood how heterogeneous individual ribosomes in neuronal processes are in terms of their composition. It is well established that the 40S and 60S ribosomal subunits form and mature in the nucleolus and nucleoplasm prior to nuclear export and cytoplasmic assembly into the 80S ribosome. However, several sequencing studies have detected a large number of mRNAs coding for ribosomal proteins (RPs) in neuronal processes [10.Cajigas I.J. et al.The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging.Neuron. 2012; 74: 453-466Google Scholar,13.Shigeoka T. et al.Dynamic axonal translation in developing and mature visual circuits.Cell. 2016; 166: 181-192Google Scholar,18.Tushev G. et al.Alternative 3′ UTRs modify the localization, regulatory potential, stability, and plasticity of mRNAs in neuronal compartments.Neuron. 2018; 98: 495-511.e6Google Scholar], suggesting local translation. Furthermore, in cultured neurons the RPs half-lives are variable, raging from 3 to 9 days [4.Dörrbaum A.R. et al.Local and global influences on protein turnover in neurons and glia.Elife. 2018; 7e34202Google Scholar], thus suggesting that their degree of incorporation into fully assembled ribosomes may vary. This is consistent with the extra ribosomal function of certain RPs [32.Warner J.R. McIntosh K.B. How common are extraribosomal functions of ribosomal proteins?.Mol. Cell. 2009; 34: 3-11Google Scholar], but it also raises the possibility that ribosome composition in neuronal compartments may be more heterogeneous than previously thought. Related to this, recent work suggests that RP mRNAs are translated in dendrites and axons and has identified a population of nascent ribosomal proteins that dynamically exchange with mature, preassembled ribosomes [33.Fusco, C.M. et al. Neuronal ribosomes dynamically exchange ribosomal proteins in a context-dependent manner. bioRxiv Published online March 25, 2021. https://doi.org/10.1101/2021.03.25.437026.Google Scholar]. Another study has showed that axonally translated ribosomal subunits are incorporated into axonal ribosomes in a nucleolus-independent fashion and that interfering with this process reduces branching of Xenopus retinal ganglion cell axons [34.Shigeoka T. et al.On-site ribosome remodeling by locally synthesized ribosomal proteins in axons.Cell Rep. 2019; 29: 3605-3619.e10Google Scholar]. Arguments against the view of a ribosome with invariant composition are also based on the observation that ribosomopathies caused by mutations in different RPs lead to specific phenotypes across different tissues [35.Guo H. Specialized ribosomes and the control of translation.Biochem. Soc. Trans. 2018; 46: 855-869Google Scholar]. These observations have led to the idea of specialized ribosomes that, by incorporating different RPs, may display distinct properties such as transcript preference and translation rate [36.Xue S. Barna M. Specialized ribosomes: a new frontier in gene regulation and organismal biology.Nat. Rev. Mol. Cell Biol. 2012; 13: 355-369Google Scholar]. However, direct evidence in support of the specialized ribosome hypothesis, in particular due to heterogeneity in RP composition, remains limited [35.Guo H. Specialized ribosomes and the control of translation.Biochem. Soc. Trans. 2018; 46: 855-869Google Scholar]. Its conclusive demonstration will likely require purification of ribosomes with different compositions followed by assays showing different properties or different populations of mRNAs translated. Future efforts in this context should focus on addressing three main questions. First, mechanistically, how are these ribosomes remodeled outside the nucleus? For example, are established ribosome biogenesis factors locally recruited to remodel and regulate ribosomes? Second, how prevalent and diverse are locally remodeled ribosomes? Is their assembly elicited in response to changes in neuronal activity? Third, is there a specific translational pattern associated with different ribosome types? There is a well-established and increasingly growing literature pointing to local protein synthesis as a main contributor to the synaptic proteome, along with protein transport from somata. By contrast, what happens to proteins after they have been synthesized or localized to their ultimate subcellular compartment remains poorly understood. Are these proteins transported back to the soma to be degraded? Are they locally degraded? Or, are they secreted? The general consensus is that most cytosolic and nuclear proteins, short-lived and misfolded proteins are degraded by the proteasome, while long-lived and membrane proteins are commonly degraded by the lysosomal pathway [37.Zhao J. et al.FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells.Cell Metab. 2007; 6: 472-483Google Scholar]. The proteasome, a multiprotein macromolecular machine, degrades substrates that are usually tagged by ubiquitin chains; collectively this system is referred to as the ubiquitin–proteasome system (UPS). For an in depth review on proteasome biology and its function in neurons, see [38.Türker F. et al.The proteasome and its role in the nervous system.Cell Chem. Biol. 2021; Google Scholar]. The proteasome plays a key role in the maintenance of neuronal function [39.Mabb A.M. Ehlers M.D. Ubiquitination in postsynaptic function and plasticity.Annu. Rev. Cell Dev. 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Furthermore, CaMKIIα redistribution into post-synaptic termini was shown to be required for activity-dependent degradation of ubiquitylated proteins at post-synaptic sites. Underscoring the importance of the spatial dimension for synaptic protein turnover, one possible explanation for this surprising translocation mechanism might be that it keeps proteasomes away from synaptic substrates under basal conditions and then rapidly recruits them following changes induced by neuronal activity. Because ubiquitination is necessary for substrate degradation by the 26S proteasome, its translocation into spines might represent an additional layer of regulation specific to this cell type. The findings discussed above indicate a role for local UPS activity in synapse biology and physiology. Nevertheless, to date we are missing a full understanding of how ubiquitin activating and conjugating enzymes, ligases, deubiquitinating enzymes (DUBs), and the proteasome coordinate to regulate synaptic proteins under basal conditions and upon changes elicited by activity. Recent evidence indicates that proteasome abundance and assembly can be adapted to meet cellular challenges [50.Rousseau A. Bertolotti A. An evolutionarily conserved pathway controls proteasome homeostasis.Nature. 2016; 536: 184-189Google Scholar]. This mechanism and its high degree of evolutionary conservation point to the fact that the proteasome itself is an important regulatory hub of the UPS. The factors involved in proteasome biogenesis are well characterized from yeast to mammals [51.Rousseau A. Bertolotti A. Regulation of proteasome assembly and activity in health and disease.Nat. Rev. Mol. Cell Biol. 2018; 19: 697-712Google Scholar,52.Wendler P. Enenkel C. Nuclear transport of yeast proteasomes.Front. Mol. 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Recently, it was shown that in neurons up to 40% of the cellular 20S proteasomes exist in a membrane-associated form, breaking down intracellular proteins into peptides that are released directly into the extracellular space and may regulate neuronal activity (Figure 1) [58.Ramachandran K.V. Margolis S.S. A mammalian nervous-system-specific plasma membrane proteasome complex that modulates neuronal function.Nat. Struct. Mol. Biol. 2017; 24: 419-430Google Scholar,59.Ramachandran K.V. et al.Activity-dependent degradation of the nascentome by the neuronal membrane proteasome.Mol. Cell. 2018; 71: 169-177.e6Google Scholar]. At present it remains unclear how the neuronal membrane proteasome is able to interact with membranes, whether it is present as a peripheral membrane protein closely interacting with a channel able to release peptides into the extracellular space or whether it is inserted into the