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.

mGluR5 is transiently confined in perisynaptic nanodomains to shape synaptic function.

The unique perisynaptic distribution of postsynaptic metabotropic glutamate receptors (mGluRs) at excitatory synapses is predicted to directly shape synaptic function, but mechanistic insight into how this distribution is regulated and impacts synaptic signaling is lacking. We used live-cell and super-resolution imaging approaches, and developed molecular tools to resolve and acutely manipulate the dynamic nanoscale distribution of mGluR5. Here we show that mGluR5 is dynamically organized in perisynaptic nanodomains that localize close to, but not in the synapse. The C-terminal domain of mGluR5 critically controlled perisynaptic confinement and prevented synaptic entry. We developed an inducible interaction system to overcome synaptic exclusion of mGluR5 and investigate the impact on synaptic function. We found that mGluR5 recruitment to the synapse acutely increased synaptic calcium responses. Altogether, we propose that transient confinement of mGluR5 in perisynaptic nanodomains allows flexible modulation of synaptic function.

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.

Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons.

Axon formation critically relies on local microtubule remodeling and marks the first step in establishing neuronal polarity. However, the function of the microtubule-organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human-induced pluripotent stem cell (iPSC)-derived neurons. Depleting centrioles, the core components of centrosomes, in unpolarized human neuronal stem cells results in various axon developmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule-associated Trim46 proteins, suppressed expression of growth cone proteins, and affected growth cone morphologies. Live-cell imaging of microtubules reveals that centriole loss impairs axonal microtubule reorganization toward the unique parallel plus-end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC-derived neurons, thereby laying the foundation for further axon development and function.

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons.

The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.

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.

Microglia innately develop within cerebral organoids.

Cerebral organoids are 3D stem cell-derived models that can be utilized to study the human brain. The current consensus is that cerebral organoids consist of cells derived from the neuroectodermal lineage. This limits their value and applicability, as mesodermal-derived microglia are important players in neural development and disease. Remarkably, here we show that microglia can innately develop within a cerebral organoid model and display their characteristic ramified morphology. The transcriptome and response to inflammatory stimulation of these organoid-grown microglia closely mimic the transcriptome and response of adult microglia acutely isolated from post mortem human brain tissue. In addition, organoid-grown microglia mediate phagocytosis and synaptic material is detected inside them. In all, our study characterizes a microglia-containing organoid model that represents a valuable tool for studying the interplay between microglia, macroglia, and neurons in human brain development and disease.

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.