Pre- and postsynaptic nanostructures increase in size and complexity after induction of long-term potentiation.

Synapses, specialized contact sites between neurons, are the fundamental elements of neuronal information transfer. Synaptic plasticity involves changes in synaptic morphology and the number of neurotransmitter receptors, and is thought to underlie learning and memory. However, it is not clear how these structural and functional changes are connected. We utilized time-lapse super-resolution STED microscopy of organotypic hippocampal brain slices and cultured neurons to visualize structural changes of the synaptic nano-organization of the postsynaptic scaffolding protein PSD95, the presynaptic scaffolding protein Bassoon, and the GluA2 subunit of AMPA receptors by chemically induced long-term potentiation (cLTP) at the level of single synapses. We found that the nano-organization of all three proteins increased in complexity and size after cLTP induction. The increase was largely synchronous, peaking at ∼60 min after stimulation. Therefore, both the size and complexity of individual pre- and post-synaptic nanostructures serve as substrates for tuning and determining synaptic strength.

Nedd4-2-dependent regulation of astrocytic Kir4.1 and Connexin43 controls neuronal network activity.

Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.

S-SCAM is essential for synapse formation.

Synapse formation is critical for the wiring of neural circuits in the developing brain. The synaptic scaffolding protein S-SCAM/MAGI-2 has important roles in the assembly of signaling complexes at post-synaptic densities. However, the role of S-SCAM in establishing the entire synapse is not known. Here, we report significant effects of RNAi-induced S-SCAM knockdown on the number of synapses in early stages of network development in vitro. In vivo knockdown during the first three postnatal weeks reduced the number of dendritic spines in the rat brain neocortex. Knockdown of S-SCAM in cultured hippocampal neurons severely reduced the clustering of both pre- and post-synaptic components. This included synaptic vesicle proteins, pre- and post-synaptic scaffolding proteins, and cell adhesion molecules, suggesting that entire synapses fail to form. Correspondingly, functional and morphological characteristics of developing neurons were affected by reducing S-SCAM protein levels; neurons displayed severely impaired synaptic transmission and reduced dendritic arborization. A next-generation sequencing approach showed normal expression of housekeeping genes but changes in expression levels in 39 synaptic signaling molecules in cultured neurons. These results indicate that S-SCAM mediates the recruitment of all key classes of synaptic molecules during synapse assembly and is critical for the development of neural circuits in the developing brain.

Molecular Dissection of Neurobeachin Function at Excitatory Synapses.

Spines are small protrusions from dendrites where most excitatory synapses reside. Changes in number, shape, and size of dendritic spines often reflect changes of neural activity in entire circuits or at individual synapses, making spines key structures of synaptic plasticity. Neurobeachin is a multidomain protein with roles in spine formation, postsynaptic neurotransmitter receptor targeting and actin distribution. However, the contributions of individual domains of Neurobeachin to these functions is poorly understood. Here, we used mostly live cell imaging and patch-clamp electrophysiology to monitor morphology and function of spinous synapses in primary hippocampal neurons. We demonstrate that a recombinant full-length Neurobeachin from humans can restore mushroom spine density and excitatory postsynaptic currents in neurons of Neurobeachin-deficient mice. We then probed the role of individual domains of Neurobeachin by comparing them to the full-length molecule in rescue experiments of knockout neurons. We show that the combined PH-BEACH domain complex is highly localized in spine heads, and that it is sufficient to restore normal spine density and surface targeting of postsynaptic AMPA receptors. In addition, we report that the Armadillo domain facilitates the formation of filopodia, long dendritic protrusions which often precede the development of mature spines, whereas the PKA-binding site appears as a negative regulator of filopodial extension. Thus, our results indicate that individual domains of Neurobeachin sustain important and specific roles in the regulation of spinous synapses. Since heterozygous mutations in Neurobeachin occur in autistic patients, the results will also improve our understanding of pathomechanism in neuropsychiatric disorders associated with impairments of spine function.