Piccolino is required for ribbon architecture at cochlear inner hair cell synapses and for hearing.

Cochlear inner hair cells (IHCs) form specialized ribbon synapses with spiral ganglion neurons that tirelessly transmit sound information at high rates over long time periods with extreme temporal precision. This functional specialization is essential for sound encoding and is attributed to a distinct molecular machinery with unique players or splice variants compared to conventional neuronal synapses. Among these is the active zone (AZ) scaffold protein piccolo/aczonin, which is represented by its short splice variant piccolino at cochlear and retinal ribbon synapses.

Microglia actively remove NR1 autoantibody-bound NMDA receptors and associated post-synaptic proteins in neuron microglia co-cultures.

Autoantibodies against the NR1 subunit of NMDA receptors (NMDARs) have been shown to promote crosslinking and internalization of bound receptors in NMDAR encephalitis (NMDARE). This internalization-mediated loss of NMDARs is thought to be the major mechanism leading to pathogenic outcomes in patients. However, the role of bound autoantibody in engaging the resident immune cells, microglia, remains poorly understood.

Differential Modes of Action of α1- and α1γ2-Autoantibodies Derived from Patients with GABAR Encephalitis.

Autoantibodies against central nervous system proteins are increasingly being recognized in association with neurologic disorders. Although a growing number of neural autoantibodies have been identified, a causal link between specific autoantibodies and disease symptoms remains unclear, as most studies use patient-derived CSF-containing mixtures of autoantibodies. This raises questions concerning mechanism of action and which autoantibodies truly contribute to disease progression.

Synaptic logistics: The presynaptic scaffold protein Piccolo a nodal point tuning synaptic vesicle recycling, maintenance and integrity.

Properly working synapses are one important guarantor for a functional and healthy brain. They are small, densely packed structures, where information is transmitted through the release of neurotransmitters from synaptic vesicles (SVs). The latter cycle within the presynaptic terminal as they first fuse with the plasma membrane to deliver their neurotransmitter, and afterwards become recycled and prepared for a new release event.

Organization of Presynaptic Autophagy-Related Processes.

Brain synapses pose special challenges on the quality control of their protein machineries as they are far away from the neuronal soma, display a high potential for plastic adaptation and have a high energy demand to fulfill their physiological tasks. This applies in particular to the presynaptic part where neurotransmitter is released from synaptic vesicles, which in turn have to be recycled and refilled in a complex membrane trafficking cycle. Pathways to remove outdated and damaged proteins include the ubiquitin-proteasome system acting in the cytoplasm as well as membrane-associated endolysosomal and the autophagy systems.

Patient-Derived Anti-NMDAR Antibody Disinhibits Cortical Neuronal Networks through Dysfunction of Inhibitory Neuron Output.

Anti-NMDA receptor (NMDAR) encephalitis is a severe neuropsychiatric disorder associated with autoantibodies against NMDARs, which cause a variety of symptoms from prominent psychiatric and cognitive manifestations to seizures and autonomic instability. Previous studies mainly focused on hippocampal effects of these autoantibodies, helping to explain mechanistic causes for cognitive impairment. However, antibodies' effects on higher cortical network function, where they could contribute to psychosis and/or seizures, have not been explored in detail until now.

Encephalitis patient-derived monoclonal GABAA receptor antibodies cause epileptic seizures.

Autoantibodies targeting the GABAA receptor (GABAAR) hallmark an autoimmune encephalitis presenting with frequent seizures and psychomotor abnormalities. Their pathogenic role is still not well-defined, given the common overlap with further autoantibodies and the lack of patient-derived mAbs. Five GABAAR mAbs from cerebrospinal fluid cells bound to various epitopes involving the α1 and γ2 receptor subunits, with variable binding strength and partial competition.

In vitro zinc supplementation alters synaptic deficits caused by autism spectrum disorder-associated Shank2 point mutations in hippocampal neurons.

Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders characterised by deficits in social interactions and repetitive behaviours. ASDs have a strong genetic basis with mutations involved in the development and function of neural circuitry. Shank proteins act as master regulators of excitatory glutamatergic synapses, and Shank mutations have been identified in people with ASD.

BSN (bassoon) and PRKN/parkin in concert control presynaptic vesicle autophagy.

Maintaining the integrity and function of the presynaptic neurotransmitter release apparatus is a demanding process for a post-mitotic neuron; the mechanisms behind it are still unclear. BSN (bassoon), an active zone scaffolding protein, has been implicated in the control of presynaptic macroautophagy/autophagy, a process we recently showed depends on poly-ubiquitination of synaptic proteins. Moreover, loss of BSN was found to lead to smaller synaptic vesicle (SV) pools and younger pools of the SV protein SV2.

Parkin contributes to synaptic vesicle autophagy in Bassoon-deficient mice.

Mechanisms regulating the turnover of synaptic vesicle (SV) proteins are not well understood. They are thought to require poly-ubiquitination and degradation through proteasome, endo-lysosomal or autophagy-related pathways. Bassoon was shown to negatively regulate presynaptic autophagy in part by scaffolding Atg5.

Loss of Piccolo Function in Rats Induces Cerebellar Network Dysfunction and Pontocerebellar Hypoplasia Type 3-like Phenotypes.

Piccolo, a presynaptic active zone protein, is best known for its role in the regulated assembly and function of vertebrate synapses. Genetic studies suggest a further link to several psychiatric disorders as well as Pontocerebellar Hypoplasia type 3 (PCH3). We have characterized recently generated Piccolo KO ( ) rats.

Serine-Arginine Protein Kinase SRPK2 Modulates the Assembly of the Active Zone Scaffolding Protein CAST1/ERC2.

Neurons release neurotransmitters at a specialized region of the presynaptic membrane, the active zone (AZ), where a complex meshwork of proteins organizes the release apparatus. The formation of this proteinaceous cytomatrix at the AZ (CAZ) depends on precise homo- and hetero-oligomerizations of distinct CAZ proteins. The CAZ protein CAST1/ERC2 contains four coiled-coil (CC) domains that interact with other CAZ proteins, but also promote self-assembly, which is an essential step for its integration during AZ formation.

Critical role for Piccolo in synaptic vesicle retrieval.

Loss of function of the active zone protein Piccolo has recently been linked to a disease, Pontocerebellar Hypoplasia type 3, which causes brain atrophy. Here, we address how Piccolo inactivation in rat neurons adversely affects synaptic function and thus may contribute to neuronal loss. Our analysis shows that Piccolo is critical for the recycling and maintenance of synaptic vesicles.

Autism-associated Shank3 mutations alter mGluR expression and mGluR-dependent but not NMDA receptor-dependent long-term depression.

SHANK3 is a postsynaptic structural protein localized at excitatory glutamatergic synapses in which deletions and mutations have been implicated in patients with autism spectrum disorders (ASD). The expression of Shank3 ASD mutations causes impairments in ionotropic glutamate receptor-mediated synaptic responses in neurons, which is thought to underlie ASD-related behaviors, thereby indicating glutamatergic synaptopathy as one of the major pathogenic mechanisms. However, little is known about the functional consequences of ASD-associated mutations in Shank3 on another important set of glutamate receptors, group I metabotropic glutamate receptors (mGluRs).

N-methyl-D-aspartate receptor dysfunction by unmutated human antibodies against the NR1 subunit.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is the most common autoimmune encephalitis related to autoantibody-mediated synaptic dysfunction. Cerebrospinal fluid-derived human monoclonal NR1 autoantibodies showed low numbers of somatic hypermutations or were unmutated. These unexpected germline-configured antibodies showed weaker binding to the NMDAR than matured antibodies from the same patient.

A Multiple Piccolino-RIBEYE Interaction Supports Plate-Shaped Synaptic Ribbons in Retinal Neurons.

Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones.

Cognitive impairment and autistic-like behaviour in SAPAP4-deficient mice.

In humans, genetic variants of DLGAP1-4 have been linked with neuropsychiatric conditions, including autism spectrum disorder (ASD). While these findings implicate the encoded postsynaptic proteins, SAPAP1-4, in the etiology of neuropsychiatric conditions, underlying neurobiological mechanisms are unknown. To assess the contribution of SAPAP4 to these disorders, we characterized SAPAP4-deficient mice.

Light-Activated ROS Production Induces Synaptic Autophagy.

The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin-dependent tagging and degradation through endo-lysosomal and autophagy pathways. Yet, it remains unclear which of these pathways are used, when they become activated, and whether SVs are cleared en masse together with SV proteins or whether both are degraded selectively. Equally puzzling is how quickly these systems can be activated and whether they function in real-time to support synaptic health.

Shank and Zinc Mediate an AMPA Receptor Subunit Switch in Developing Neurons.

During development, pyramidal neurons undergo dynamic regulation of AMPA receptor (AMPAR) subunit composition and density to help drive synaptic plasticity and maturation. These normal developmental changes in AMPARs are particularly vulnerable to risk factors for Autism Spectrum Disorders (ASDs), which include loss or mutations of synaptic proteins and environmental insults, such as dietary zinc deficiency. Here, we show how Shank2 and Shank3 mediate a zinc-dependent regulation of AMPAR function and subunit switch from GluA2-lacking to GluA2-containing AMPARs.

Dietary Zinc Supplementation Prevents Autism Related Behaviors and Striatal Synaptic Dysfunction in Exon 13-16 Mutant Mice.

The SHANK family of synaptic proteins (SHANK1-3) are master regulators of the organizational structure of excitatory synapses in the brain. Mutations in are prevalent in patients with autism spectrum disorders (ASD), and loss of one copy of causes Phelan-McDermid Syndrome, a syndrome in which Autism occurs in >80% of cases. The synaptic stability of SHANK3 is highly regulated by zinc, driving the formation of postsynaptic protein complexes and increases in excitatory synaptic strength.

The Exocyst Component Exo70 Modulates Dendrite Arbor Formation, Synapse Density, and Spine Maturation in Primary Hippocampal Neurons.

Neurons are highly polarized cells displaying an elaborate architectural morphology. The design of their dendritic arborization and the distribution of their synapses contribute importantly to information processing in the brain. The growth and complexity of dendritic arbors are driven by the formation of synapses along their lengths.

LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells.

Information on protein-protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double-readout bioluminescence-based two-hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence-based co-precipitation (LuC).

N-terminal SAP97 isoforms differentially regulate synaptic structure and postsynaptic surface pools of AMPA receptors.

The location and density of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is controlled by scaffolding proteins within the postsynaptic density (PSD). SAP97 is a PSD protein with two N-terminal isoforms, α and β, that have opposing effects on synaptic strength thought to result from differential targeting of AMPA receptors into distinct synaptic versus extrasynaptic locations, respectively. In this study, we have applied dSTORM super resolution imaging in order to localize the synaptic and extrasynaptic pools of AMPA receptors in neurons expressing α or βSAP97.