Impaired formation of high-order gephyrin oligomers underlies gephyrin dysfunction-associated pathologies

Kim, Seungjoon; Kang, Mi Sun; Park, Dongseok; Lee, Ae-Ree; Betz, Heinrich; Ko, Jaewon; Chang, Iksoo; Um, Ji Won

doi:10.1016/j.isci.2021.102037

Cited by 8 publications

(9 citation statements)

References 42 publications

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“…eGFP, mCherry, mEos2 and other fluorescent proteins can be fused with gephyrin and expressed in cells via transfection or infection [6, 13, 14] . Fluorescent gephyrin chimeras have been successfully used for synapse studies both in fixed and live cell cultures, [6, 13, 14] as well as in conditional expression in transgenic animals, [15] but an inherent drawback of induced secondary gephyrin expression are morphological and/or functional effects in cells [15–17] . To circumvent these drawbacks, two alternative methods of genetic tagging were applied.…”

Section: Introductionmentioning

confidence: 99%

“…[6,13,14] Fluorescent gephyrin chimeras have been successfully used for synapse studies both in fixed and live cell cultures, [6,13,14] as well as in conditional expression in transgenic animals, [15] but an inherent drawback of induced secondary gephyrin expression are morpho-logical and/or functional effects in cells. [15][16][17] To circumvent these drawbacks, two alternative methods of genetic tagging were applied. The first method involves knock-in mice expressing mRFP-gephyrin where expression levels, subcellular distribution of the endogenous protein and synaptic function are largely preserved.…”

Section: Introductionmentioning

confidence: 99%

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A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**

et al. 2022

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Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding "Sylite"-a probe with outstanding signal-tobackground ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In superresolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**

et al. 2022

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“…Gephyrin has functional links with several synaptic proteins, mutations of which have been reported in various neurodevelopmental disorders ( Choii and Ko, 2015 ; Kim et al, 2021 ). Half dosage of GLRA3 may contribute to impair gephyrin-mediated aggregation and post-synaptic clustering.…”

Section: Resultsmentioning

confidence: 99%

Neurodevelopmental Disorders in Patients With Complex Phenotypes and Potential Complex Genetic Basis Involving Non-Coding Genes, and Double CNVs

et al. 2021

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Neurodevelopmental disorders (NDDs) are a heterogeneous class of brain diseases, with a complex genetic basis estimated to account for up to 50% of cases. Nevertheless, genetic diagnostic yield is about 20%. Array-comparative genomic hybridization (array-CGH) is an established first-level diagnostic test able to detect pathogenic copy number variants (CNVs), however, most identified variants remain of uncertain significance (VUS). Failure of interpretation of VUSs may depend on various factors, including complexity of clinical phenotypes and inconsistency of genotype-phenotype correlations. Indeed, although most NDD-associated CNVs are de novo, transmission from unaffected parents to affected children of CNVs with high risk for NDDs has been observed. Moreover, variability of genetic components overlapped by CNVs, such as long non-coding genes, genomic regions with long-range effects, and additive effects of multiple CNVs can make CNV interpretation challenging. We report on 12 patients with complex phenotypes possibly explained by complex genetic mechanisms, including involvement of antisense genes and boundaries of topologically associating domains. Eight among the 12 patients carried two CNVs, either de novo or inherited, respectively, by each of their healthy parents, that could additively contribute to the patients’ phenotype. CNVs overlapped either known NDD-associated or novel candidate genes (PTPRD, BUD13, GLRA3, MIR4465, ABHD4, and WSCD2). Bioinformatic enrichment analyses showed that genes overlapped by the co-occurring CNVs have synergistic roles in biological processes fundamental in neurodevelopment. Double CNVs could concur in producing deleterious effects, according to a two-hit model, thus explaining the patients’ phenotypes and the incomplete penetrance, and variable expressivity, associated with the single variants. Overall, our findings could contribute to the knowledge on clinical and genetic diagnosis of complex forms of NDD.

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“…The Gephyrin.FingR intrabodies used here were designed to recognize trimeric gephyrin [ 33 ]. It may well be that synapses designated as gephyrin-negative in fact have low levels of gephyrin, or gephyrin in its monomeric form, although gephyrin trimerization is thought to be necessary for its synaptic localization [ 102 , 103 ]. Importantly, the antibodies used for immuno-EM analysis in the current study could detect monomeric gephyrin and still revealed a subpopulation of somatic GABAergic inputs that lacked immunolabeling.…”

Section: Discussionmentioning

confidence: 99%

Gephyrin-Lacking PV Synapses on Neocortical Pyramidal Neurons

Kuljis

Ray

Wegner

et al. 2021

IJMS

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Gephyrin has long been thought of as a master regulator for inhibitory synapses, acting as a scaffold to organize γ-aminobutyric acid type A receptors (GABAARs) at the post-synaptic density. Accordingly, gephyrin immunostaining has been used as an indicator of inhibitory synapses; despite this, the pan-synaptic localization of gephyrin to specific classes of inhibitory synapses has not been demonstrated. Genetically encoded fibronectin intrabodies generated with mRNA display (FingRs) against gephyrin (Gephyrin.FingR) reliably label endogenous gephyrin, and can be tagged with fluorophores for comprehensive synaptic quantitation and monitoring. Here we investigated input- and target-specific localization of gephyrin at a defined class of inhibitory synapse, using Gephyrin.FingR proteins tagged with EGFP in brain tissue from transgenic mice. Parvalbumin-expressing (PV) neuron presynaptic boutons labeled using Cre- dependent synaptophysin-tdTomato were aligned with postsynaptic Gephyrin.FingR puncta. We discovered that more than one-third of PV boutons adjacent to neocortical pyramidal (Pyr) cell somas lack postsynaptic gephyrin labeling. This finding was confirmed using correlative fluorescence and electron microscopy. Our findings suggest some inhibitory synapses may lack gephyrin. Gephyrin-lacking synapses may play an important role in dynamically regulating cell activity under different physiological conditions.

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Impaired formation of high-order gephyrin oligomers underlies gephyrin dysfunction-associated pathologies

Cited by 8 publications

References 42 publications

A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**

A Versatile Synthetic Affinity Probe Reveals Inhibitory Synapse Ultrastructure and Brain Connectivity**

Neurodevelopmental Disorders in Patients With Complex Phenotypes and Potential Complex Genetic Basis Involving Non-Coding Genes, and Double CNVs

Gephyrin-Lacking PV Synapses on Neocortical Pyramidal Neurons

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