Fluorescence visualization of newly synthesized proteins in mammalian cells.
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Animals↗
Biomedical subjects
Erin M Schuman
Publications and source records attributed to Erin M Schuman.
Dendritic protein synthesis, synaptic plasticity, and memory.
Considerable evidence suggests that the formation of long-term memories requires a critical period of new protein synthesis. Recently, the notion that some of these newly synthesized proteins originate through local translation in neuronal dendrites has gained some traction. Here, we review the experimental support for this idea and highlight some of the key questions outstanding in this area.
Animals↗
Synaptic regulation of translation of dendritic mRNAs.
The selective localization of protein synthetic machinery at postsynaptic sites makes it possible for the synthesis of particular proteins to be regulated by synaptic signals. Here we consider how the structure of the machinery constrains synthetic capacity and the evidence that mRNA translation is locally controlled by synaptic signals. Since the discovery of protein synthetic machinery at synaptic sites on dendrites (Steward and Levy, 1982), substantial progress has been made in identifying dendritic mRNAs and in showing that dendritic protein synthesis is critical for persistent synaptic modifications like long-term potentiation (LTP) and long-term depression (LTD). Although many pieces of the puzzle have been identified, major questions remain. Here we focus on one of the unknowns: how translational activity at synapses is regulated and whether regulation involves upregulation or downregulation of overall translation or differential regulation of the translation of particular transcripts. It is useful to begin by considering constraints imposed by the nature of the protein synthetic machinery at synapses.
Animals↗
Activity-dependent dynamics and sequestration of proteasomes in dendritic spines.
The regulated degradation of proteins by the ubiquitin proteasome pathway is emerging as an important modulator of synaptic function and plasticity. The proteasome is a large, multi-subunit cellular machine that recognizes, unfolds and degrades target polyubiquitinated proteins. Here we report NMDA (N-methyl-D-aspartate) receptor-dependent redistribution of proteasomes from dendritic shafts to synaptic spines upon synaptic stimulation, providing a mechanism for local protein degradation. Using a proteasome-activity reporter and local perfusion, we show that synaptic stimulation regulates proteasome activity locally in the dendrites. We used restricted photobleaching of individual spines and dendritic shafts to reveal the dynamics that underlie proteasome sequestration, and show that activity modestly enhances the entry rate of proteasomes into spines while dramatically reducing their exit rate. Proteasome sequestration is persistent, reflecting an association with the actin-based cytoskeleton. Together, our data indicate that synaptic activity can promote the recruitment and sequestration of proteasomes to locally remodel the protein composition of synapses.
Actins↗
Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).
In both normal and pathological states, cells respond rapidly to environmental cues by synthesizing new proteins. The selective identification of a newly synthesized proteome has been hindered by the basic fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable. We describe here a technology, based on the cotranslational introduction of azide groups into proteins and the chemoselective tagging of azide-labeled proteins with an alkyne affinity tag, to separate and identify, specifically, the newly synthesized proteins in mammalian cells. Incorporation of the azide-bearing amino acid azidohomoalanine is unbiased, not toxic, and does not increase protein degradation. As a first demonstration of the method, we report the selective purification and identification of 195 metabolically labeled proteins with multidimensional liquid chromatography in-line with tandem MS. Furthermore, in combination with leucine-based mass tagging, candidates were immediately validated as newly synthesized proteins. The identified proteins, synthesized in a 2-h window, possess a broad range of biochemical properties and span most functional gene ontology categories. This technology makes it possible to address the temporal and spatial characteristics of newly synthesized proteomes in any cell type.
Amino Acids↗
Miniature neurotransmission stabilizes synaptic function via tonic suppression of local dendritic protein synthesis.
Activity deprivation in neurons induces a slow compensatory scaling up of synaptic strength, reflecting a homeostatic mechanism for stabilizing neuronal activity. Prior studies have focused on the loss of action potential (AP) driven neurotransmission in synaptic homeostasis. Here, we show that the miniature synaptic transmission that persists during AP blockade profoundly shapes the time course and mechanism of homeostatic scaling. A brief blockade of NMDA receptor (NMDAR) mediated miniature synaptic events ("minis") rapidly scales up synaptic strength, over an order of magnitude faster than with AP blockade alone. The rapid scaling induced by NMDAR mini blockade is mediated by increased synaptic expression of surface GluR1 and the transient incorporation of Ca2+-permeable AMPA receptors at synapses; both of these changes are implemented locally within dendrites and require dendritic protein synthesis. These results indicate that NMDAR signaling during miniature synaptic transmission serves to stabilize synaptic function through active suppression of dendritic protein synthesis.
Action Potentials↗
Single-trial learning of novel stimuli by individual neurons of the human hippocampus-amygdala complex.
The ability to distinguish novel from familiar stimuli allows nervous systems to rapidly encode significant events following even a single exposure to a stimulus. This detection of novelty is necessary for many types of learning. Neurons in the medial temporal lobe (MTL) are critically involved in the acquisition of long-term declarative memories. During a learning task, we recorded from individual MTL neurons in vivo using microwire electrodes implanted in human epilepsy surgery patients. We report here the discovery of two classes of neurons in the hippocampus and amygdala that exhibit single-trial learning: novelty and familiarity detectors, which show a selective increase in firing for new and old stimuli, respectively. The neurons retain memory for the stimulus for 24 hr. Thus, neurons in the MTL contain information sufficient for reliable novelty-familiarity discrimination and also show rapid plasticity as a result of single-trial learning.
Action Potentials↗
MicroRNA: microRNAs reach out into dendrites.
A recent study has shown that miR-134, a brain-specific microRNA, is present in dendrites where it represses the local synthesis of the protein kinase LimK1; this is a novel form of translational regulation in dendrites and may have important physiological implications.
Dendrites↗
Online detection and sorting of extracellularly recorded action potentials in human medial temporal lobe recordings, in vivo.
Understanding the function of complex cortical circuits requires the simultaneous recording of action potentials from many neurons in awake and behaving animals. Practically, this can be achieved by extracellularly recording from multiple brain sites using single wire electrodes. However, in densely packed neural structures such as the human hippocampus, a single electrode can record the activity of multiple neurons. Thus, analytic techniques that differentiate action potentials of different neurons are required. Offline spike sorting approaches are currently used to detect and sort action potentials after finishing the experiment. Because the opportunities to record from the human brain are relatively rare, it is desirable to analyze large numbers of simultaneous recordings quickly using online sorting and detection algorithms. In this way, the experiment can be optimized for the particular response properties of the recorded neurons. Here we present and evaluate a method that is capable of detecting and sorting extracellular single-wire recordings in realtime. We demonstrate the utility of the method by applying it to an extensive data set we acquired from chronically implanted depth electrodes in the hippocampus of human epilepsy patients. This dataset is particularly challenging because it was recorded in a noisy clinical environment. This method will allow the development of "closed-loop" experiments, which immediately adapt the experimental stimuli and/or tasks to the neural response observed.
Action Potentials↗
Dopaminergic stimulation of local protein synthesis enhances surface expression of GluR1 and synaptic transmission in hippocampal neurons.
The use-dependent modification of synapses is strongly influenced by dopamine, a transmitter that participates in both the physiology and pathophysiology of animal behavior. In the hippocampus, dopaminergic signaling is thought to play a key role in protein synthesis-dependent forms of synaptic plasticity. The molecular mechanisms by which dopamine influences synaptic function, however, are not well understood. Using a GFP-based reporter, as well as a small-molecule reporter of endogenous protein synthesis, we show that dopamine D1/D5 receptor activation stimulates local protein synthesis in the dendrites of hippocampal neurons. We also identify the GluR1 subunit of AMPA receptors as one protein upregulated by dopamine receptor activation, with increased incorporation of surface GluR1 at synaptic sites. The insertion of new GluRs is accompanied by an increase in the frequency of miniature synaptic events. Together, these data suggest a local protein synthesis-dependent activation of previously silent synapses as a result of dopamine receptor stimulation.
Animals↗
Local translational control in dendrites and its role in long-term synaptic plasticity.
Local protein synthesis in dendrites has emerged as a key mechanism contributing to enduring forms of synaptic plasticity. Although the translational capability of dendrites has been appreciated for over 20 years, it is only recently that significant progress has been made in elucidating mechanisms that contribute to its regulation. It is clear from work over the last few years that the control of translation in dendrites is complex, involving a host of unique (and often surprising) mechanisms that can operate together or in parallel to tightly control gene expression in time and space. Here, we discuss the strategies used by neurons to regulate translation in dendrites and how these are implemented in the service of long-term information storage.
Animals↗
Light-mediated inhibition of protein synthesis.
The regulation of protein synthesis is vital for a host of cell biological processes, but investigating roles for protein synthesis have been hindered by the inability to selectively interfere with it. To inhibit protein synthesis with spatial and temporal control, we have developed a photo-releasable anisomycin compound, N-([6-bromo-7-hydroxycoumarin-4-yl]methyloxycarbonyl)anisomycin (Bhc-Aniso), that can be removed through exposure to UV light. The area of protein synthesis inhibition can be restricted to a small light-exposed region or, potentially, the volume of two-photon excitation if a pulsed IR laser is the light source. We have tested the compound's effectiveness with an in vitro protein-translation system, CHO cells, HEK293 cells, and neurons. The photo-released anisomycin can inhibit protein synthesis in a spatially restricted manner, which will enable the specific inhibition of protein synthesis in subsets of cells with temporal and spatial precision.
Animals↗
Synaptic protein degradation by the ubiquitin proteasome system.
Synaptic plasticity -- the modulation of synaptic strength between a presynaptic terminal and a postsynaptic dendrite -- is thought to be a mechanism that underlies learning and memory. It has become increasingly clear that regulated protein synthesis is an important mechanism used to regulate the protein content of synapses that results in changes in synaptic strength. Recent experiments have highlighted a role for the opposing process, that is, regulated protein degradation via the ubiquitin-proteasome system, in synaptic plasticity. These recent findings raise exciting questions as to how proteasomal activity can regulate synapses over different temporal and spatial scales.
Animals↗
Role for a cortical input to hippocampal area CA1 in the consolidation of a long-term memory.
A dialogue between the hippocampus and the neocortex is thought to underlie the formation, consolidation and retrieval of episodic memories, although the nature of this cortico-hippocampal communication is poorly understood. Using selective electrolytic lesions in rats, here we examined the role of the direct entorhinal projection (temporoammonic, TA) to the hippocampal area CA1 in short-term (24 hours) and long-term (four weeks) spatial memory in the Morris water maze. When short-term memory was examined, both sham- and TA-lesioned animals showed a significant preference for the target quadrant. When re-tested four weeks later, sham-lesioned animals exhibited long-term memory; in contrast, the TA-lesioned animals no longer showed target quadrant preference. Many long-lasting memories require a process called consolidation, which involves the exchange of information between the cortex and hippocampus. The disruption of long-term memory by the TA lesion could reflect a requirement for TA input during either the acquisition or consolidation of long-term memory. To distinguish between these possibilities, we trained animals, verified their spatial memory 24 hours later, and then subjected trained animals to TA lesions. TA-lesioned animals still exhibited a deficit in long-term memory, indicating a disruption of consolidation. Animals in which the TA lesion was delayed by three weeks, however, showed a significant preference for the target quadrant, indicating that the memory had already been adequately consolidated at the time of the delayed lesion. These results indicate that, after learning, ongoing cortical input conveyed by the TA path is required to consolidate long-term spatial memory.
Animals↗
Regulation of dendritic protein synthesis by miniature synaptic events.
We examined dendritic protein synthesis after a prolonged blockade of action potentials alone and after a blockade of both action potentials and miniature excitatory synaptic events (minis). Relative to controls, dendrites exposed to a prolonged blockade of action potentials showed diminished protein synthesis. Dendrites in which both action potentials and minis were blocked showed enhanced protein synthesis, suggesting that minis inhibit dendritic translation. When minis were acutely blocked or stimulated, an immediate increase or decrease, respectively, in dendritic translation was observed. Taken together, these results reveal a role for miniature synaptic events in the acute regulation of dendritic protein synthesis in neurons.
Action Potentials↗
A proteasome-sensitive connection between PSD-95 and GluR1 endocytosis.
Synaptic transmission at excitatory synapses can be regulated by changing the number of synaptic glutamate receptors (GluRs) through endocytosis and exocytosis. The endocytosis of GluRs has recently been shown to require the activity of the ubiquitin-proteasome system (UPS): proteasome inhibitors or dominant negative forms of ubiquitin block the ligand-stimulated internalization of GluRs. We have examined whether PSD-95 is a potential target of the UPS. Following neurotransmitter stimulation, PSD-95 levels are negatively correlated with the magnitude of internalized GluR1 in individual neurons. Neurotransmitter stimulation also results in a proteasome-dependent decrease in dendritic PSD-95. Consistent with the idea that PSD-95 degradation is important for GluR internalization, overexpression of PSD-95 can inhibit neurotransmitter-stimulated GluR1 endocytosis. If PSD-95 is a direct target for proteasomal degradation, then the polyubiquitination of PSD-95 is expected. Using experimental conditions that favor the detection of polyubiquitination, however, no ubiquitination of PSD-95 was detected. It is possible that the polyubiquitination of PSD-95 is short-lived and thus difficult to detect. Alternatively, the regulation of PSD-95 levels by the proteasome important for ligand-stimulated GluR endocytosis may be accomplished via an intermediate protein.
Animals↗
Ubiquitin-mediated proteasome activity is required for agonist-induced endocytosis of GluRs.
Recent studies documenting a role for local protein synthesis in synaptic plasticity have lead to interest in the opposing process, protein degradation, as a potential regulator of synaptic function. The ubiquitin-conjugation system identifies, modifies, and delivers proteins to the proteasome for degradation. We found that both the proteasome and ubiquitin are present in the soma and dendrites of hippocampal neurons. As the trafficking of glutamate receptors (GluRs) is thought to underlie some forms of synaptic plasticity, we examined whether blocking proteasome activity affects the agonist-induced internalization of GluRs in cultured hippocampal neurons. Treatment with the glutamate agonist AMPA induced a robust internalization of GluRs. In contrast, brief pretreatment with proteasome inhibitors completely prevented the internalization of GluRs. To distinguish between a role for the proteasome and a possible diminution of the free ubiquitin pool, we expressed a chain elongation defective ubiquitin mutant (UbK48R), which causes premature termination of polyubiquitin chains but, importantly, can serve as a substrate for mono-ubiquitin-dependent processes. Expression of K48R in neurons severely diminished AMPA-induced internalization establishing a role for the proteasome. These data demonstrate the acute (e.g., minutes) regulation of synaptic function by the ubiquitin-proteasome pathway in mammalian neurons.
Animals↗