Fluorescent sensors are an essential part of the experimental toolbox of the life sciences, where they are used ubiquitously to visualize intra- and extracellular signaling. In the brain, optical ...neurotransmitter sensors can shed light on temporal and spatial aspects of signal transmission by directly observing, for instance, neurotransmitter release and spread. Here we report the development and application of the first optical sensor for the amino acid glycine, which is both an inhibitory neurotransmitter and a co-agonist of the N-methyl-D-aspartate receptors (NMDARs) involved in synaptic plasticity. Computational design of a glycine-specific binding protein allowed us to produce the optical glycine FRET sensor (GlyFS), which can be used with single and two-photon excitation fluorescence microscopy. We took advantage of this newly developed sensor to test predictions about the uneven spatial distribution of glycine in extracellular space and to demonstrate that extracellular glycine levels are controlled by plasticity-inducing stimuli.
Objective
Assess occurrence of the dendritic spine scaffolding protein Drebrin as a pathophysiologically relevant autoantibody target in patients with recurrent seizures and suspected encephalitis as ...leading symptoms.
Methods
Sera of 4 patients with adult onset epilepsy and suspected encephalitis of unresolved etiology and equivalent results in autoantibody screening were subjected to epitope identification. We combined a wide array of approaches, ranging from immunoblotting, immunoprecipitation, mass spectrometry, subcellular binding pattern analyses in primary neuronal cultures, and immunohistochemistry in brains of wild‐type and Drebrin knockout mice to in vitro analyses of impaired synapse formation, morphology, and aberrant neuronal excitability by antibody exposure.
Results
In the serum of a patient with adult onset epilepsy and suspected encephalitis, a strong signal at ∼70kDa was detected by immunoblotting, for which mass spectrometry revealed Drebrin as the putative antigen. Three other patients whose sera also showed strong immunoreactivity around 70kDa on Western blotting were also anti‐Drebrin‐positive. Seizures, memory impairment, and increased protein content in cerebrospinal fluid occurred in anti‐Drebrin‐seropositive patients. Alterations in cerebral magnetic resonance imaging comprised amygdalohippocampal T2‐signal increase and hippocampal sclerosis. Diagnostic biopsy revealed T‐lymphocytic encephalitis in an anti‐Drebrin‐seropositive patient. Exposure of primary hippocampal neurons to anti‐Drebrin autoantibodies resulted in aberrant synapse composition and Drebrin distribution as well as increased spike rates and the emergence of burst discharges reflecting network hyperexcitability.
Interpretation
Anti‐Drebrin autoantibodies define a chronic syndrome of recurrent seizures and neuropsychiatric impairment as well as inflammation of limbic and occasionally cortical structures. Immunosuppressant therapies should be considered in this disorder. ANN NEUROL 2020;87:869–884
Stable function of networks requires that synapses adapt their strength to levels of neuronal activity, and failure to do so results in cognitive disorders. How such homeostatic regulation may be ...implemented in mammalian synapses remains poorly understood. Here we show that the phosphorylation status of several positions of the active-zone (AZ) protein RIM1 are relevant for synaptic glutamate release. Position RIMS1045 is necessary and sufficient for expression of silencing-induced homeostatic plasticity and is kept phosphorylated by serine arginine protein kinase 2 (SRPK2). SRPK2-induced upscaling of synaptic release leads to additional RIM1 nanoclusters and docked vesicles at the AZ and is not observed in the absence of RIM1 and occluded by RIMS1045E. Our data suggest that SRPK2 and RIM1 represent a presynaptic phosphosignaling hub that is involved in the homeostatic balance of synaptic coupling of neuronal networks.
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•RIM is required for induction of presynaptic homeostatic plasticity (PHP)•The kinase SRPK2 regulates basal synaptic vesicle release and PHP•SRPK2 controls the number of RIM nanoclusters and of docked synaptic vesicles•Phosphorylation of RIM1 at serine 1045 increases release and occludes PHP
The amount of neurotransmitters released in response to an action potential can be tuned by altering the number of release sites. Müller et al. show that the kinase SRPK2 is involved in regulating this process via the presynaptic active-zone protein RIM. Phosphorylation status changes of RIM dynamically modulate synaptic strength.
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