Living on the edge: The functional organization of metabotropic glutamate receptors at excitatory synapses

The brain is a tremendously complex organ that consists of an intricate network of billions of nerve cells, also called neurons. Neurons are the main communicators of the brain and by sending signals from one neuron to the other they can trigger actions, ultimately underlying processes such as learning and memory. Neurons form synapses, which are special contact sites between two neurons where the communication takes place. Like in a handshake, but instead of greeting each other one neuron transfers a signal which is received by receptors on the other neuron. The signal activates the receptors which initiates a signaling cascade in the receiving neuron. The focus of this thesis is on metabotropic glutamate receptors (mGluRs), which are critical modulators of synaptic signaling on longer time scales. Synaptic signaling is tightly regulated and two aspects are essential for proper and efficient signal transmission: (1) the receiving receptors need to be positioned at the correct location, in close proximity to the released signal, to receive and respond, and (2) the activated receptors need to be rapidly deactivated in order to prevent overstimulation in the receiving neurons. This thesis aims at understanding how mGluR organization is regulated and how this tunes synaptic signaling. We used a combination of novel molecular tools, advanced imaging techniques and functional read-outs to directly examine this. We discovered that mGluRs are organized in dynamic clusters surrounding the area where signals are released, which is regulated by a specific part of the receptor. Furthermore, we show that Shank proteins regulate the uptake and recycling of mGluRs to prevent overstimulation after activation. Together, this thesis presents exciting novel insights in the functional organization of mGluRs at synapses, crucial for a better understanding of the brain in both health an disease

SCN1A-deficient excitatory neuronal networks display mutation-specific phenotypes

Dravet syndrome is a severe epileptic encephalopathy, characterized by (febrile) seizures, behavioural problems and developmental delay. Eighty per cent of patients with Dravet syndrome have a mutation in SCN1A, encoding Nav1.1. Milder clinical phenotypes, such as GEFS+ (generalized epilepsy with febrile seizures plus), can also arise from SCN1A mutations. Predicting the clinical phenotypic outcome based on the type of mutation remains challenging, even when the same mutation is inherited within one family. This clinical and genetic heterogeneity adds to the difficulties of predicting disease progression and tailoring the prescription of anti-seizure medication. Understanding the neuropathology of different SCN1A mutations may help to predict the expected clinical phenotypes and inform the selection of best-fit treatments. Initially, the loss of Na+-current in inhibitory neurons was recognized specifically to result in disinhibition and consequently seizure generation. However, the extent to which excitatory neurons contribute to the pathophysiology is currently debated and might depend on the patient clinical phenotype or the specific SCN1A mutation. To examine the genotype-phenotype correlations of SCN1A mutations in relation to excitatory neurons, we investigated a panel of patient-derived excitatory neuronal networks differentiated on multi-electrode arrays. We included patients with different clinical phenotypes, harbouring various SCN1A mutations, along with a family in which the same mutation led to febrile seizures, GEFS+ or Dravet syndrome. We hitherto describe a previously unidentified functional excitatory neuronal network phenotype in the context of epilepsy, which corresponds to seizurogenic network prediction patterns elicited by proconvulsive compounds. We found that excitatory neuronal networks were affected differently, depending on the type of SCN1A mutation, but did not segregate according to clinical severity. Specifically, loss-of-function mutations could be distinguished from missense mutations, and mutations in the pore domain could be distinguished from mutations in the voltage sensing domain. Furthermore, all patients showed aggravated neuronal network responses at febrile temperatures compared with controls. Finally, retrospective drug screening revealed that anti-seizure medication affected GEFS+ patient- but not Dravet patient-derived neuronal networks in a patient-specific and clinically relevant manner. In conclusion, our results indicate a mutation-specific excitatory neuronal network phenotype, which recapitulates the foremost clinically relevant features, providing future opportunities for precision therapies.</p

Functional organization of postsynaptic glutamate receptors

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in the brain, responsible for mediating the vast majority of excitatory transmission in neuronal networks. The AMPA- and NMDA-type ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the fast synaptic responses, while metabotropic glutamate receptors (mGluRs) are coupled to downstream signaling cascades that act on much slower timescales. These functionally distinct receptor sub-types are co-expressed at individual synapses, allowing for the precise temporal modulation of postsynaptic excitability and plasticity. Intriguingly, these receptors are differentially distributed with respect to the presynaptic release site. While iGluRs are enriched in the core of the synapse directly opposing the release site, mGluRs reside preferentially at the border of the synapse. As such, to understand the differential contribution of these receptors to synaptic transmission, it is important to not only consider their signaling properties, but also the mechanisms that control the spatial segregation of these receptor types within synapses. In this review, we will focus on the mechanisms that control the organization of glutamate receptors at the postsynaptic membrane with respect to the release site, and discuss how this organization could regulate synapse physiology

Functional organization of postsynaptic glutamate receptors

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in the brain, responsible for mediating the vast majority of excitatory transmission in neuronal networks. The AMPA- and NMDA-type ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the fast synaptic responses, while metabotropic glutamate receptors (mGluRs) are coupled to downstream signaling cascades that act on much slower timescales. These functionally distinct receptor sub-types are co-expressed at individual synapses, allowing for the precise temporal modulation of postsynaptic excitability and plasticity. Intriguingly, these receptors are differentially distributed with respect to the presynaptic release site. While iGluRs are enriched in the core of the synapse directly opposing the release site, mGluRs reside preferentially at the border of the synapse. As such, to understand the differential contribution of these receptors to synaptic transmission, it is important to not only consider their signaling properties, but also the mechanisms that control the spatial segregation of these receptor types within synapses. In this review, we will focus on the mechanisms that control the organization of glutamate receptors at the postsynaptic membrane with respect to the release site, and discuss how this organization could regulate synapse physiology

Living on the edge: The functional organization of metabotropic glutamate receptors at excitatory synapses

The brain is a tremendously complex organ that consists of an intricate network of billions of nerve cells, also called neurons. Neurons are the main communicators of the brain and by sending signals from one neuron to the other they can trigger actions, ultimately underlying processes such as learning and memory. Neurons form synapses, which are special contact sites between two neurons where the communication takes place. Like in a handshake, but instead of greeting each other one neuron transfers a signal which is received by receptors on the other neuron. The signal activates the receptors which initiates a signaling cascade in the receiving neuron. The focus of this thesis is on metabotropic glutamate receptors (mGluRs), which are critical modulators of synaptic signaling on longer time scales. Synaptic signaling is tightly regulated and two aspects are essential for proper and efficient signal transmission: (1) the receiving receptors need to be positioned at the correct location, in close proximity to the released signal, to receive and respond, and (2) the activated receptors need to be rapidly deactivated in order to prevent overstimulation in the receiving neurons. This thesis aims at understanding how mGluR organization is regulated and how this tunes synaptic signaling. We used a combination of novel molecular tools, advanced imaging techniques and functional read-outs to directly examine this. We discovered that mGluRs are organized in dynamic clusters surrounding the area where signals are released, which is regulated by a specific part of the receptor. Furthermore, we show that Shank proteins regulate the uptake and recycling of mGluRs to prevent overstimulation after activation. Together, this thesis presents exciting novel insights in the functional organization of mGluRs at synapses, crucial for a better understanding of the brain in both health an disease

Membrane trafficking and positioning of mGluRs at presynaptic and postsynaptic sites of excitatory synapses

The plethora of functions of glutamate in the brain are mediated by the complementary actions of ionotropic and metabotropic glutamate receptors (mGluRs). The ionotropic glutamate receptors carry most of the fast excitatory transmission, while mGluRs modulate transmission on longer timescales by triggering multiple intracellular signaling pathways. As such, mGluRs mediate critical aspects of synaptic transmission and plasticity. Interestingly, at synapses, mGluRs operate at both sides of the cleft, and thus bidirectionally exert the effects of glutamate. At postsynaptic sites, group I mGluRs act to modulate excitability and plasticity. At presynaptic sites, group II and III mGluRs act as auto-receptors, modulating release properties in an activity-dependent manner. Thus, synaptic mGluRs are essential signal integrators that functionally couple presynaptic and postsynaptic mechanisms of transmission and plasticity. Understanding how these receptors reach the membrane and are positioned relative to the presynaptic glutamate release site are therefore important aspects of synapse biology. In this review, we will discuss the currently known mechanisms underlying the trafficking and positioning of mGluRs at and around synapses, and how these mechanisms contribute to synaptic functioning. We will highlight outstanding questions and present an outlook on how recent technological developments will move this exciting research field forward

mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function

The subsynaptic organization of group I mGluRs modulates their activation and downstream signaling, essential for synaptic transmission and plasticity. Here, the authors describe how the C-terminal domain of mGluR5 controls its dynamic organization in perisynaptic nanodomains, and prevents mGluR5 form entering the synapse, allowing mGluR5 to finely tune synaptic signalling

Membrane trafficking and positioning of mGluRs at presynaptic and postsynaptic sites of excitatory synapses

The plethora of functions of glutamate in the brain are mediated by the complementary actions of ionotropic and metabotropic glutamate receptors (mGluRs). The ionotropic glutamate receptors carry most of the fast excitatory transmission, while mGluRs modulate transmission on longer timescales by triggering multiple intracellular signaling pathways. As such, mGluRs mediate critical aspects of synaptic transmission and plasticity. Interestingly, at synapses, mGluRs operate at both sides of the cleft, and thus bidirectionally exert the effects of glutamate. At postsynaptic sites, group I mGluRs act to modulate excitability and plasticity. At presynaptic sites, group II and III mGluRs act as auto-receptors, modulating release properties in an activity-dependent manner. Thus, synaptic mGluRs are essential signal integrators that functionally couple presynaptic and postsynaptic mechanisms of transmission and plasticity. Understanding how these receptors reach the membrane and are positioned relative to the presynaptic glutamate release site are therefore important aspects of synapse biology. In this review, we will discuss the currently known mechanisms underlying the trafficking and positioning of mGluRs at and around synapses, and how these mechanisms contribute to synaptic functioning. We will highlight outstanding questions and present an outlook on how recent technological developments will move this exciting research field forward

Shank Proteins Couple the Endocytic Zone to the Postsynaptic Density to Control Trafficking and Signaling of Metabotropic Glutamate Receptor 5

Activation of postsynaptic metabotropic glutamate receptors (mGluRs) modulates neuronal excitability and synaptic plasticity, while deregulation of mGluR signaling has been implicated in neurodevelopmental disorders. Overstimulation of mGluRs is restricted by the rapid endocytosis of receptors after activation. However, how membrane trafficking of mGluRs at synapses is controlled remains poorly defined. We find that in hippocampal neurons, the agonist-induced receptor internalization of synaptic mGluR5 is significantly reduced in Shank knockdown neurons. This is rescued by the re-expression of wild-type Shanks, but not by mutants unable to bind Homer1b/c, Dynamin2, or Cortactin. These effects are paralleled by a reduction in synapses associated with an endocytic zone. Moreover, a mutation in SHANK2 found in autism spectrum disorders (ASDs) similarly disrupts these processes. On the basis of these findings, we propose that synaptic Shank scaffolds anchor the endocytic machinery to govern the efficient trafficking of mGluR5 and to balance the surface expression of mGluRs to efficiently modulate neuronal functioning

Shank Proteins Couple the Endocytic Zone to the Postsynaptic Density to Control Trafficking and Signaling of Metabotropic Glutamate Receptor 5

Activation of postsynaptic metabotropic glutamate receptors (mGluRs) modulates neuronal excitability and synaptic plasticity, while deregulation of mGluR signaling has been implicated in neurodevelopmental disorders. Overstimulation of mGluRs is restricted by the rapid endocytosis of receptors after activation. However, how membrane trafficking of mGluRs at synapses is controlled remains poorly defined. We find that in hippocampal neurons, the agonist-induced receptor internalization of synaptic mGluR5 is significantly reduced in Shank knockdown neurons. This is rescued by the re-expression of wild-type Shanks, but not by mutants unable to bind Homer1b/c, Dynamin2, or Cortactin. These effects are paralleled by a reduction in synapses associated with an endocytic zone. Moreover, a mutation in SHANK2 found in autism spectrum disorders (ASDs) similarly disrupts these processes. On the basis of these findings, we propose that synaptic Shank scaffolds anchor the endocytic machinery to govern the efficient trafficking of mGluR5 and to balance the surface expression of mGluRs to efficiently modulate neuronal functioning