Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination.

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is the most rapidly turned over mammalian enzyme. We have shown that its degradation is accelerated by ODC antizyme, an inhibitory protein induced by polyamines. This is a new type of enzyme regulation and may be a model for selective protein degradation. Here we report the identification of the protease responsible for ODC degradation. Using a cell-free degradation system, we demonstrate that immunodepletion of proteasomes from cell extracts causes almost complete loss of ATP- and antizyme-dependent degradation of ODC. In addition, purified 26S proteasome complex, but not the 20S proteasome, catalyses ODC degradation in the absence of ubiquitin. These results strongly suggest that the 26S proteasome, widely viewed as specific for ubiquitin-conjugated proteins, is the main enzyme responsible for ODC degradation. The 26S proteasome may therefore have a second role in ubiquitin-independent proteolysis.     

Functional mapping of single spines in cortical neurons in vivo

The individual functional properties and spatial arrangement of afferent synaptic inputs on dendrites have a critical role in the processing of information by neurons in the mammalian brain. Although recent work has identified visually-evoked local dendritic calcium signals in the rodent visual cortex, sensory-evoked signalling on the level of dendritic spines, corresponding to individual afferent excitatory synapses, remains unexplored. Here we used a new variant of high-resolution two-photon imaging to detect sensory-evoked calcium transients in single dendritic spines of mouse cortical neurons in vivo. Calcium signals evoked by sound stimulation required the activation of NMDA (N-methyl-D-aspartate) receptors. Active spines are widely distributed on basal and apical dendrites and pure-tone stimulation at different frequencies revealed both narrowly and widely tuned spines. Notably, spines tuned for different frequencies were highly interspersed on the same dendrites: even neighbouring spines were mostly tuned to different frequencies. Thus, our results demonstrate that NMDA-receptor-dependent single-spine synaptic inputs to the same dendrite are highly heterogeneous. Furthermore, our study opens the way for in vivo mapping of functionally defined afferent sensory inputs with single-synapse resolution.     

Independent regulation of calcium revealed by imaging dendritic spines

The dendritic spine is a basic structural unit of neuronal organization. It is assumed to be a primary locus of synaptic plasticity, and to undergo long-term morphological and functional changes, at least some of which are regulated by intracellular calcium concentrations. It is known that physiological stimuli can cause marked increases in intracellular calcium levels in hippocampal dendritic shafts, but it is completely unknown to what extent such changes in the dendrites would also be seen by calcium-sensing structures within spines. Will calcium levels in all spines change in parallel with the dendrite or will there be a heterogeneous response? This study, through direct visualization and measurement of intracellular calcium concentrations in individual living spines, demonstrates that experimentally evoked changes in calcium concentrations in the dendritic shaft ([Ca2+]d).     

Locally dynamic synaptic learning rules in pyramidal neuron dendrites

Long-term potentiation (LTP) of synaptic transmission underlies aspects of learning and memory. LTP is input-specific at the level of individual synapses, but neural network models predict interactions between plasticity at nearby synapses. Here we show in mouse hippocampal pyramidal cells that LTP at individual synapses reduces the threshold for potentiation at neighbouring synapses. After input-specific LTP induction by two-photon glutamate uncaging or by synaptic stimulation, subthreshold stimuli, which by themselves were too weak to trigger LTP, caused robust LTP and spine enlargement at neighbouring spines. Furthermore, LTP induction broadened the presynaptic-postsynaptic spike interval for spike-timing-dependent LTP within a dendritic neighbourhood. The reduction in the threshold for LTP induction lasted approximately 10 min and spread over approximately 10 microm of dendrite. These local interactions between neighbouring synapses support clustered plasticity models of memory storage and could allow for the binding of behaviourally linked information on the same dendritic branch.     

Transient aggregation of ubiquitinated proteins during dendritic cell maturation

Dendritic cells (DCs) are antigen-presenting cells with the unique capacity to initiate primary immune responses. Dendritic cells have a remarkable pattern of differentiation (maturation) that exhibits highly specific mechanisms to control antigen presentation restricted by major histocompatibility complex (MHC). MHC class I molecules present to CD8(+) cytotoxic T cells peptides that are derived mostly from cytosolic proteins, which are ubiquitinated and then degraded by the proteasome. Here we show that on inflammatory stimulation, DCs accumulate newly synthesized ubiquitinated proteins in large cytosolic structures. These structures are similar to, but distinct from, aggresomes and inclusion bodies observed in many amyloid diseases. Notably, these dendritic cell aggresome-like induced structures (DALIS) are transient, require continuous protein synthesis and do not affect the ubiquitin-proteasome pathway. Our observations suggest the existence of an organized prioritization of protein degradation in stimulated DCs, which is probably important for regulating MHC class I presentation during maturation.     

Analysis of calcium channels in single spines using optical fluctuation analysis

Most synapses form on small, specialized postsynaptic structures known as dendritic spines. The influx of Ca2+ ions into such spines--through synaptic receptors and voltage-sensitive Ca2+ channels (VSCCs)--triggers diverse processes that underlie synaptic plasticity. Using two-photon laser scanning microscopy, we imaged action-potential-induced transient changes in Ca2+ concentration in spines and dendrites of CA1 pyramidal neurons in rat hippocampal slices. Through analysis of the large trial-to-trial fluctuations in these transients, we have determined the number and properties of VSCCs in single spines. Here we report that each spine contains 1-20 VSCCs, and that this number increases with spine volume. We are able to detect the opening of a single VSCC on a spine. In spines located on the proximal dendritic tree, VSCCs normally open with high probability (approximately 0.5) following dendritic action potentials. Activation of GABA(B) receptors reduced this probability in apical spines to approximately 0.3 but had no effect on VSCCs in dendrites or basal spines. Our studies show that the spatial distribution of VSCC subtypes and their modulatory potential is regulated with submicrometre precision.     

Transmitter-evoked local calcium release stabilizes developing dendrites

In the central nervous system, dendritic arborizations of neurons undergo dynamic structural remodelling during development. Processes are elaborated, maintained or eliminated to attain the adult pattern of synaptic connections. Although neuronal activity influences this remodelling, it is not known how activity exerts its effects. Here we show that neurotransmission-evoked calcium (Ca(2+)) release from intracellular stores stabilizes dendrites during the period of synapse formation. Using a ballistic labelling method to load cells with Ca(2+) indicator dyes, we simultaneously monitored dendritic activity and structure in the intact retina. Two distinct patterns of spontaneous Ca(2+) increases occurred in developing retinal ganglion cells--global increases throughout the arborization, and local 'flashes' of activity restricted to small dendritic segments. Blockade of local, but not global, activity caused rapid retraction of dendrites. This retraction was prevented locally by focal uncaging of caged Ca(2+) that triggered Ca(2+) release from internal stores. Thus, local Ca(2+) release is a mechanism by which afferent activity can selectively and differentially regulate dendritic structure across the developing arborization.     

LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite

Structural remodelling of synapses and formation of new synaptic contacts has been postulated as a possible mechanism underlying the late phase of long-term potentiation (LTP), a form of plasticity which is involved in learning and memory. Here we use electron microscopy to analyse the morphology of synapses activated by high-frequency stimulation and identified by accumulated calcium in dendritic spines. LTP induction resulted in a sequence of morphological changes consisting of a transient remodelling of the postsynaptic membrane followed by a marked increase in the proportion of axon terminals contacting two or more dendritic spines. Three-dimensional reconstruction revealed that these spines arose from the same dendrite. As pharmacological blockade of LTP prevented these morphological changes, we conclude that LTP is associated with the formation of new, mature and probably functional synapses contacting the same presynaptic terminal and thereby duplicating activated synapses.     

Induction of dendritic spines by an extracellular domain of AMPA receptor subunit GluR2

Synaptic transmission from excitatory nerve cells in the mammalian brain is largely mediated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors located at the surface of dendritic spines. The abundance of postsynaptic AMPA receptors correlates with the size of the synapse and the dimensions of the dendritic spine head. Moreover, long-term potentiation is associated with the formation of dendritic spines as well as synaptic delivery of AMPA receptors. The molecular mechanisms that coordinate AMPA receptor delivery and spine morphogenesis are unknown. Here we show that overexpression of the glutamate receptor 2 (GluR2) subunit of AMPA receptors increases spine size and density in hippocampal neurons, and more remarkably, induces spine formation in GABA-releasing interneurons that normally lack spines. The extracellular N-terminal domain (NTD) of GluR2 is responsible for this effect, and heterologous fusion proteins of the NTD of GluR2 inhibit spine morphogenesis. We propose that the NTD of GluR2 functions at the cell surface as part of a receptor-ligand interaction that is important for spine growth and/or stability.     

Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites

For nerve cells to develop their highly polarized form, appropriate structural molecules must be targeted to either axons or dendrites. This could be achieved by the synthesis of structural proteins in the cell body and their sorting to either axons or dendrites by specific transport mechanisms. For dendrites, an alternative possibility is that proteins could be synthesized locally in the dendritic cytoplasm. This is an attractive idea because it would allow regulation of the production of structural molecules in response to local demand during dendritic development. The feasibility of dendritic protein synthesis is suggested both by the existence of dendritic polyribosomes and by the recent demonstration that newly synthesized RNA is transported into the dendrites of neurons differentiating in culture. However, to date there has been no demonstration of the selective synthesis of an identified dendrite-specific protein in the dendritic cytoplasm. Here, we use in situ hybridization with specific complementary DNA probes to show that messenger RNA for the dendrite-specific microtubule-associated protein MAP2 (refs 3-5) is present in dendrites in the developing brain. By contrast the mRNA for tubulin, a protein present in both axons and dendrites is located exclusively in neuronal cell bodies.     

Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2

Autism spectrum disorders comprise a range of neurodevelopmental disorders characterized by deficits in social interaction and communication, and by repetitive behaviour. Mutations in synaptic proteins such as neuroligins, neurexins, GKAPs/SAPAPs and ProSAPs/Shanks were identified in patients with autism spectrum disorder, but the causative mechanisms remain largely unknown. ProSAPs/Shanks build large homo- and heteromeric protein complexes at excitatory synapses and organize the complex protein machinery of the postsynaptic density in a laminar fashion. Here we demonstrate that genetic deletion of ProSAP1/Shank2 results in an early, brain-region-specific upregulation of ionotropic glutamate receptors at the synapse and increased levels of ProSAP2/Shank3. Moreover, ProSAP1/Shank2(-/-) mutants exhibit fewer dendritic spines and show reduced basal synaptic transmission, a reduced frequency of miniature excitatory postsynaptic currents and enhanced N-methyl-d-aspartate receptor-mediated excitatory currents at the physiological level. Mutants are extremely hyperactive and display profound autistic-like behavioural alterations including repetitive grooming as well as abnormalities in vocal and social behaviours. By comparing the data on ProSAP1/Shank2(-/-) mutants with ProSAP2/Shank3αβ(-/-) mice, we show that different abnormalities in synaptic glutamate receptor expression can cause alterations in social interactions and communication. Accordingly, we propose that appropriate therapies for autism spectrum disorders are to be carefully matched to the underlying synaptopathic phenotype.     

Dendritic spikes as a mechanism for cooperative long-term potentiation

Strengthening of synaptic connections following coincident pre- and postsynaptic activity was proposed by Hebb as a cellular mechanism for learning. Contemporary models assume that multiple synapses must act cooperatively to induce the postsynaptic activity required for hebbian synaptic plasticity. One mechanism for the implementation of this cooperation is action potential firing, which begins in the axon, but which can influence synaptic potentiation following active backpropagation into dendrites. Backpropagation is limited, however, and action potentials often fail to invade the most distal dendrites. Here we show that long-term potentiation of synapses on the distal dendrites of hippocampal CA1 pyramidal neurons does require cooperative synaptic inputs, but does not require axonal action potential firing and backpropagation. Rather, locally generated and spatially restricted regenerative potentials (dendritic spikes) contribute to the postsynaptic depolarization and calcium entry necessary to trigger potentiation of distal synapses. We find that this mechanism can also function at proximal synapses, suggesting that dendritic spikes participate generally in a form of synaptic potentiation that does not require postsynaptic action potential firing in the axon.     

Experience-dependent and cell-type-specific spine growth in the neocortex

Functional circuits in the adult neocortex adjust to novel sensory experience, but the underlying synaptic mechanisms remain unknown. Growth and retraction of dendritic spines with synapse formation and elimination could change brain circuits. In the apical tufts of layer 5B (L5B) pyramidal neurons in the mouse barrel cortex, a subset of dendritic spines appear and disappear over days, whereas most spines are persistent for months. Under baseline conditions, new spines are mostly transient and rarely survive for more than a week. Transient spines tend to be small, whereas persistent spines are usually large. Because most excitatory synapses in the cortex occur on spines, and because synapse size and the number of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are proportional to spine volume, the excitation of pyramidal neurons is probably driven through synapses on persistent spines. Here we test whether the generation and loss of persistent spines are enhanced by novel sensory experience. We repeatedly imaged dendritic spines for one month after trimming alternate whiskers, a paradigm that induces adaptive functional changes in neocortical circuits. Whisker trimming stabilized new spines and destabilized previously persistent spines. New-persistent spines always formed synapses. They were preferentially added on L5B neurons with complex apical tufts rather than simple tufts. Our data indicate that novel sensory experience drives the stabilization of new spines on subclasses of cortical neurons. These synaptic changes probably underlie experience-dependent remodelling of specific neocortical circuits.     

Postsynaptic translation affects the efficacy and morphology of neuromuscular junctions

Long-term synaptic plasticity may be associated with structural rearrangements within the neuronal circuitry. Although the molecular mechanisms governing such activity-controlled morphological alterations are mostly elusive, polysomal accumulations at the base of developing dendritic spines and the activity-induced synthesis of synaptic components suggest that localized translation is involved during synaptic plasticity. Here we show that large aggregates of translational components as well as messenger RNA of the postsynaptic glutamate receptor subunit DGluR-IIA are localized within subsynaptic compartments of larval neuromuscular junctions of Drosophila melanogaster. Genetic models of junctional plasticity and genetic manipulations using the translation initiation factors eIF4E and poly(A)-binding protein showed an increased occurrence of subsynaptic translation aggregates. This was associated with a significant increase in the postsynaptic DGluR-IIA protein levels and a reduction in the junctional expression of the cell-adhesion molecule Fasciclin II. In addition, the efficacy of junctional neurotransmission and the size of larval neuromuscular junctions were significantly increased. Our results therefore provide evidence for a postsynaptic translational control of long-term junctional plasticity.     

Dendritic spines as individual neuronal compartments for synaptic Ca2+ responses.

The possibility that postsynaptic spines on neuronal dendrites are discrete biochemical compartments for Ca(2+)-activated processes involved in synaptic plasticity is a widely proposed concept that has eluded experimental demonstration. Using microfluorometry on CA3 neurons in hippocampal slices, we show here that with weak presynaptic stimulation of associative/commissural fibres, Ca2+ accumulates in single postsynaptic spines but not in the parent dendrite. Stronger stimulation also promotes changes in dendrites. The NMDA-receptor antagonist AP-5 blocks changes in Ca2+ in spines. Sustained steep Ca2+ gradients between single spines and the parent dendrite, often lasting several minutes, develop with repeated stimulation. The observed compartmentalization allows for the specificity, cooperativity and associativity displayed by memory models such as long-term potentiation.     

Repetitive motor learning induces coordinated formation of clustered dendritic spines in vivo

Many lines of evidence suggest that memory in the mammalian brain is stored with distinct spatiotemporal patterns. Despite recent progresses in identifying neuronal populations involved in memory coding, the synapse-level mechanism is still poorly understood. Computational models and electrophysiological data have shown that functional clustering of synapses along dendritic branches leads to nonlinear summation of synaptic inputs and greatly expands the computing power of a neural network. However, whether neighbouring synapses are involved in encoding similar memory and how task-specific cortical networks develop during learning remain elusive. Using transcranial two-photon microscopy, we followed apical dendrites of layer 5 pyramidal neurons in the motor cortex while mice practised novel forelimb skills. Here we show that a third of new dendritic spines (postsynaptic structures of most excitatory synapses) formed during the acquisition phase of learning emerge in clusters, and that most such clusters are neighbouring spine pairs. These clustered new spines are more likely to persist throughout prolonged learning sessions, and even long after training stops, than non-clustered counterparts. Moreover, formation of new spine clusters requires repetition of the same motor task, and the emergence of succedent new spine(s) accompanies the strengthening of the first new spine in the cluster. We also show that under control conditions new spines appear to avoid existing stable spines, rather than being uniformly added along dendrites. However, succedent new spines in clusters overcome such a spatial constraint and form in close vicinity to neighbouring stable spines. Our findings suggest that clustering of new synapses along dendrites is induced by repetitive activation of the cortical circuitry during learning, providing a structural basis for spatial coding of motor memory in the mammalian brain.     

A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes

The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. It comprises one catalytic 20S proteasome and two axially positioned 19S regulatory complexes. The 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7 (refs 4, 5), but the mechanism responsible for the assembly of such a complex structure remains elusive. Here we report two chaperones, designated proteasome assembling chaperone-1 (PAC1) and PAC2, that are involved in the maturation of mammalian 20S proteasomes. PAC1 and PAC2 associate as heterodimers with proteasome precursors and are degraded after formation of the 20S proteasome is completed. Overexpression of PAC1 or PAC2 accelerates the formation of precursor proteasomes, whereas knockdown by short interfering RNA impairs it, resulting in poor maturation of 20S proteasomes. Furthermore, the PAC complex provides a scaffold for alpha-ring formation and keeps the alpha-rings competent for the subsequent formation of half-proteasomes. Thus, our results identify a mechanism for the correct assembly of 20S proteasomes.     

A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal

The 26S proteasome is the chief site of regulatory protein turnover in eukaryotic cells. It comprises one 20S catalytic complex (composed of four stacked rings of seven members) and two axially positioned 19S regulatory complexes (each containing about 18 subunits) that control substrate access to the catalytic chamber. In most cases, targeting to the 26S proteasome depends on tagging of the substrate with a specific type of polyubiquitin chain. Recognition of this signal is followed by substrate unfolding and translocation, which are presumably catalysed by one or more of six distinct AAA ATPases located in the base-a ring-like 19S subdomain that abuts the axial pore of the 20S complex and exhibits chaperone activity in vitro. Despite the importance of polyubiquitin chain recognition in proteasome function, the site of this signal's interaction with the 19S complex has not been identified previously. Here we use crosslinking to a reactive polyubiquitin chain to show that a specific ATPase subunit, S6' (also known as Rpt5), contacts the bound chain. The interaction of this signal with 26S proteasomes is modulated by ATP hydrolysis. Our results suggest that productive recognition of the proteolytic signal, as well as proteasome assembly and substrate unfolding, are ATP-dependent events.     

Compartmentalized dendritic plasticity and input feature storage in neurons

Although information storage in the central nervous system is thought to be primarily mediated by various forms of synaptic plasticity, other mechanisms, such as modifications in membrane excitability, are available. Local dendritic spikes are nonlinear voltage events that are initiated within dendritic branches by spatially clustered and temporally synchronous synaptic input. That local spikes selectively respond only to appropriately correlated input allows them to function as input feature detectors and potentially as powerful information storage mechanisms. However, it is currently unknown whether any effective form of local dendritic spike plasticity exists. Here we show that the coupling between local dendritic spikes and the soma of rat hippocampal CA1 pyramidal neurons can be modified in a branch-specific manner through an N-methyl-d-aspartate receptor (NMDAR)-dependent regulation of dendritic Kv4.2 potassium channels. These data suggest that compartmentalized changes in branch excitability could store multiple complex features of synaptic input, such as their spatio-temporal correlation. We propose that this 'branch strength potentiation' represents a previously unknown form of information storage that is distinct from that produced by changes in synaptic efficacy both at the mechanistic level and in the type of information stored.     

Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated.

At excitatory synapses in the central nervous system, the number of glutamate molecules released from a vesicle is much larger than the number of postsynaptic receptors. But does release of a single vesicle normally saturate these receptors? Answering this question is critical to understanding how the amplitude and variability of synaptic transmission are set and regulated. Here we describe the use of two-photon microscopy to image transient increases in Ca2+ concentration mediated by NMDA (N-methyl-D-aspartate) receptors in single dendritic spines of CA1 pyramidal neurons in hippocampal slices. To test for NMDA-receptor saturation, we compared responses to stimulation with single and double pulses. We find that a single release event does not saturate spine NMDA receptors; a second release occurring 10 ms later produces approximately 80% more NMDA-receptor activation. The amplitude of spine NMDA-receptor-mediated [Ca2+] transients (and the synaptic plasticity which depends on this) may thus be sensitive to the number of quanta released by a burst of action potentials and to changes in the concentration profile of glutamate in the synaptic cleft.