Abstract
Despite their very close structural similarity, CxxC/S-type (class I) glutaredoxins (Grxs) act as oxidoreductases, while CGFS-type (class II) Grxs act as FeS cluster transferases. Here we ...show that the key determinant of Grx function is a distinct loop structure adjacent to the active site. Engineering of a CxxC/S-type Grx with a CGFS-type loop switched its function from oxidoreductase to FeS transferase. Engineering of a CGFS-type Grx with a CxxC/S-type loop abolished FeS transferase activity and activated the oxidative half reaction of the oxidoreductase. The reductive half-reaction, requiring the interaction with a second GSH molecule, was enabled by switching additional residues in the active site. We explain how subtle structural differences, mostly depending on the structure of one particular loop, act in concert to determine Grx function.
Ribbon synapses transmit information in sensory systems, but their development is not well understood. To test the hypothesis that ribbon assembly stabilizes nascent synapses, we performed ...simultaneous time-lapse imaging of fluorescently-tagged ribbons in retinal cone bipolar cells (BCs) and postsynaptic densities (PSD95-FP) of retinal ganglion cells (RGCs). Ribbons and PSD95-FP clusters were more stable when these components colocalized at synapses. However, synapse density on ON-alpha RGCs was unchanged in mice lacking ribbons (ribeye knockout). Wildtype BCs make both ribbon-containing and ribbon-free synapses with these GCs even at maturity. Ribbon assembly and cone BC-RGC synapse maintenance are thus regulated independently. Despite the absence of synaptic ribbons, RGCs continued to respond robustly to light stimuli, although quantitative examination of the responses revealed reduced frequency and contrast sensitivity.
The maintenance of synaptic transmission requires that vesicles be recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of ...central neurons, including a fast “kiss-and-run” mechanism that releases neurotransmitter through a fusion pore. Using an improved fluorescent reporter comprising pHluorin fused to synaptophysin, we find that only a slow mode of endocytosis (τ = 15 s) operates at hippocampal synapses when vesicle fusion is triggered by a single nerve impulse or short burst. This retrieval mechanism is blocked by overexpression of the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi, and it is associated with the movement of clathrin and vesicle proteins out of the synapse. These results indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of vesicle retrieval after physiological stimuli.
Sensory systems continually adjust the way stimuli are processed. What are the circuit mechanisms underlying this plasticity? We investigated how synapses in the retina of zebrafish adjust to changes ...in the temporal contrast of a visual stimulus by imaging activity in vivo. Following an increase in contrast, bipolar cell synapses with strong initial responses depressed, whereas synapses with weak initial responses facilitated. Depression and facilitation predominated in different strata of the inner retina, where bipolar cell output was anticorrelated with the activity of amacrine cell synapses providing inhibitory feedback. Pharmacological block of GABAergic feedback converted facilitating bipolar cell synapses into depressing ones. These results indicate that depression intrinsic to bipolar cell synapses causes adaptation of the ganglion cell response to contrast, whereas depression in amacrine cell synapses causes sensitization. Distinct microcircuits segregating to different layers of the retina can cause simultaneous increases or decreases in the gain of neural responses.
The visual system transmits information about fast and slow changes in light intensity through separate neural pathways. We used in vivo imaging to investigate how bipolar cells transmit these ...signals to the inner retina. We found that the volume of the synaptic terminal is an intrinsic property that contributes to different temporal filters. Individual cells transmit through multiple terminals varying in size, but smaller terminals generate faster and larger calcium transients to trigger vesicle release with higher initial gain, followed by more profound adaptation. Smaller terminals transmitted higher stimulus frequencies more effectively. Modeling global calcium dynamics triggering vesicle release indicated that variations in the volume of presynaptic compartments contribute directly to all these differences in response dynamics. These results indicate how one neuron can transmit different temporal components in the visual signal through synaptic terminals of varying geometries with different adaptational properties.
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). While most of the current treatment strategies focus on immune cell regulation, except for the ...drug siponimod, there is no therapeutic intervention that primarily aims at neuroprotection and remyelination. Recently, nimodipine showed a beneficial and remyelinating effect in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Nimodipine also positively affected astrocytes, neurons, and mature oligodendrocytes. Here we investigated the effects of nimodipine, an L-type voltage-gated calcium channel antagonist, on the expression profile of myelin genes and proteins in the oligodendrocyte precursor cell (OPC) line Oli-Neu and in primary OPCs. Our data indicate that nimodipine does not have any effect on myelin-related gene and protein expression. Furthermore, nimodipine treatment did not result in any morphological changes in these cells. However, RNA sequencing and bioinformatic analyses identified potential micro (mi)RNA that could support myelination after nimodipine treatment compared to a dimethyl sulfoxide (DMSO) control. Additionally, we treated zebrafish with nimodipine and observed a significant increase in the number of mature oligodendrocytes (*
≤ 0.05). Taken together, nimodipine seems to have different positive effects on OPCs and mature oligodendrocytes.
Imaging of optical reporters of neural activity across large populations of neurones is a widely used approach for investigating the function of neural circuits in slices and in vivo. Major ...challenges in analysing such experiments include the automatic identification of neurones and synapses, extraction of dynamic signals, and assessing the temporal and spatial relationships between active units in relation to the gross structure of the circuit. We have developed an integrated set of software tools, named SARFIA, by which these aspects of dynamic imaging experiments can be analysed semi-automatically. Key features are image-based detection of structures of interest using the Laplace operator, determining the positions of units in a layered network, clustering algorithms to classify units with similar functional responses, and a database to store, exchange and analyse results across experiments. We demonstrate the use of these tools to analyse synaptic activity in the retina of live zebrafish by multi-photon imaging of SyGCaMP2, a genetically encoded synaptically localised calcium reporter. By simultaneously recording activity across tens of bipolar cell terminals distributed throughout the IPL we made a functional map of the ON and OFF signalling channels and found that these were only partially separated. The automated detection of signals across many neurones in the retina allowed the reliable detection of small populations of neurones generating “ectopic” signals in the “ON” and “OFF” sublaminae. This software should be generally applicable for the analysis of dynamic imaging experiments across hundreds of responding units.
Sensory processing can be tuned by a neuron’s integration area, the types of inputs, and the proportion and number of connections with those inputs. Integration areas often vary topographically to ...sample space differentially across regions. Here, we highlight two visual circuits in which topographic changes in the postsynaptic retinal ganglion cell (RGC) dendritic territories and their presynaptic bipolar cell (BC) axonal territories are either matched or unmatched. Despite this difference, in both circuits, the proportion of inputs from each BC type, i.e., synaptic convergence between specific BCs and RGCs, remained constant across varying dendritic territory sizes. Furthermore, synapse density between BCs and RGCs was invariant across topography. Our results demonstrate a wiring design, likely engaging homotypic axonal tiling of BCs, that ensures consistency in synaptic convergence between specific BC types onto their target RGCs while enabling independent regulation of pre- and postsynaptic territory sizes and synapse number between cell pairs.
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•Bipolar cells (BCs) and ganglion cells (GCs) can vary topographically in size•Changes in BC size do not always scale with that of their postsynaptic GCs•Proportion of synapses from each BC type with partner GC is invariant of location•BC axonal tiling promotes maintenance of stereotypic BC-GC connectivity patterns
Yu et al. show that the density of excitatory synapses on retinal output neurons is invariant of retinal location, even though connectivity between pre- and postsynaptic cell pairs can vary. These results have implications for understanding topographic variations in processing of visual stimuli and for understanding consistency of CNS wiring diagrams.
Redox regulation of specific cysteines via oxidoreductases of the thioredoxin family is increasingly being recognized as an important signaling pathway. Here, we demonstrate that the cytosolic ...isoform of the vertebrate-specific oxidoreductase Glutaredoxin 2 (Grx2c) regulates the redox state of the transcription factor SP-1 and thereby its binding affinity to both the promoter and an enhancer region of the CSPG4 gene encoding chondroitin sulfate proteoglycan nerve/glial antigen 2 (NG2). This leads to an increased number of NG2 glia during in vitro oligodendroglial differentiation and promotes migration of these wound healing cells. On the other hand, we found that the same mechanism also leads to increased invasion of glioma tumor cells. Using in vitro (human cell lines), ex vivo (mouse primary cells), and in vivo models (zebrafish), as well as glioblastoma patient tissue samples we provide experimental data highlighting the Yin and Yang of redox signaling in the central nervous system and the enzymatic Taoism of Grx2c.
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•CSPG4 promoter binding of the transcription factor SP-1 depends on glutaredoxin 2•Cytosolic glutaredoxin 2 inhibits NG2 glia differentiation into oligodendrocytes•Migration and wound healing capacity of NG2 glia is increased by glutaredoxin 2•Glutaredoxin 2 increases invasion of human glioblastoma cells in vitro and in vivo
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