Journal: The Journal of Neuroscience

Article Title: Role of the glycine receptor ß subunit in synaptic localization and pathogenicity in severe startle disease

doi: 10.1523/jneurosci.0837-23.2023

Figure Lengend Snippet: Figure 1. GlyRβ is highly expressed in the spinal cord and brainstem. Immunohistochemical stainings of Glrb+/+ (A) and Glrbeos/eos (B) spinal cord sections for mEos4b-GlyRβ (green, anti-mEos-488 antibody) and GlyRα1 (magenta, mAb2b). C, mEos4b-GlyRβ (converted mEos4b fluorescence, shown in green) signal in spinal cord tissue of Glrbeos/eos after clearing, imaging, and 3D reconstruction (xyz represent the axis in 3 dimensions), d, dorsal; v, ventral spinal cord. The gray transparent shell marks the region of interest. Yellow dotted rectangle marks slice of spinal cord shown in C1. Scale bar refers to 100 µm. (C1) Spinal cord image (front view, for position see yellow dotted rectangle in (C) with the photoconverted mEos4b signal (green). Scale bar 100 µm. (C2) Detailed mEos4b-GlyRβ (green) signal with top view (from dorsal), front view (dorsal to ventral axis is marked by a white arrow), and side view (dorsal to ventral axis is marked by a white arrow). Scale bar 200 µm. D, Immunohistochemical stainings of Glrbeos/eos brainstem section with higher magnifications (10× and 40×) of the hypoglossal nucleus XII (D1). Stained are mEos4b-GlyRβ (green, anti-mEos-488) and GlyRα1 (magenta, mAb2b). Scale bars refer to 500 µm in the overview images and 10× magnification and 50 µm in 40× magnification.

Article Snippet: The primary antibodies against different glycine receptor subunits were GlyRα1 (AB5052, RRID:AB_91659, 1:1,000, Merck), GlyRα2 (NBP3-03685, 1:1,000, Novus Biologicals), and pan-α-GlyR (146008, 1:1,000, Synaptic Systems).

Techniques: Immunohistochemical staining, Imaging, Staining

Journal: The Journal of Neuroscience

Article Title: Role of the glycine receptor ß subunit in synaptic localization and pathogenicity in severe startle disease

doi: 10.1523/jneurosci.0837-23.2023

Figure Lengend Snippet: Figure 2. High GlyRβ expression in areas of the periaqueductal gray (PAG) and the thalamic and hypothalamic nuclei. A,B, Immunohistochemical stainings of Glrbeos/eos midbrain and forebrain sections with higher magnifications (10× and 40×) of the periaqueductal gray (PAG) and the hippocampus (HC), thalamus (TH), and hypothalamus (HY). Stained are mEos4b-GlyRβ (green, anti-mEos-488) and GlyRα1 (magenta, mAb2b). Scale bars refer to 500 µm in overview and 10× magnification and 50 µm in 40× magnification (A1 PAG; B1 thalamus; B2 hypothalamus; B3 hippocampus).

Article Snippet: The primary antibodies against different glycine receptor subunits were GlyRα1 (AB5052, RRID:AB_91659, 1:1,000, Merck), GlyRα2 (NBP3-03685, 1:1,000, Novus Biologicals), and pan-α-GlyR (146008, 1:1,000, Synaptic Systems).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: The Journal of Neuroscience

Article Title: Role of the glycine receptor ß subunit in synaptic localization and pathogenicity in severe startle disease

doi: 10.1523/jneurosci.0837-23.2023

Figure Lengend Snippet: Figure 3. Oscillator mice with severe startle disease show unaltered synaptic localization of GlyRβ. A, Breeding scheme of Glra1+/spdot/Glrbeos/eos mice. Glrbeos/eos mice have a knock-in of an mEos4b tag in exon 2 of Glrb (Maynard et al., 2021). Homozygous Glra1spdot/spdot mice harbor a microdeletion in exon 8 of Glra1 leading to an early STOP codon and thus resulting in a lack of GlyRα1 expression. B, Weight diagram of Glra1+/+/Glrbeos/eos (n = 20), Glra1+/spdot/Glrbeos/eos (n = 50), and Glra1spdot/spdot/Glrbeos/eos (n = 20) mice from day 10 to 21. Marked is the symptom onset of homozygous oscillator mice. Note, the number of Glra1spdot/spdot/Glrbeos/eos animals decreased from 20 to 7 after day 14 (onset of symptoms) until day 19 due to premature death. C, Immunocytochemical stainings of mixed primary spinal cord neuronal cultures of Glra1+/+/Glrbeos/eos and Glra1spdot/spdot/Glrbeos/eos mice. Neurons were stained with antibodies against mEos4b-GlyRβ (green), GlyR pan-α (magenta), and gephyrin (yellow). Arrows point to colocalization of stained proteins. Scale bars refer to 20 µm and 2 µm in the magnifications. Quantification of puncta/100 microns dendrite of GlyRβ (D), GlyRα (E), gephyrin (F), GlyRβ–GlyRα (G), GlyRβ–gephyrin (H), GlyRα–gephyrin (I) as well as extrasynaptic GlyRβ (J) and GlyRα (K) in Glra1+/+/Glrbeos/eos compared to Glra1spdot/spdot/Glrbeos/eos neurons (N = 3 independent experiments). Data are shown as mean ± SEM and individual data points. Glra1+/

Article Snippet: The primary antibodies against different glycine receptor subunits were GlyRα1 (AB5052, RRID:AB_91659, 1:1,000, Merck), GlyRα2 (NBP3-03685, 1:1,000, Novus Biologicals), and pan-α-GlyR (146008, 1:1,000, Synaptic Systems).

Techniques: Knock-In, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: Role of the glycine receptor ß subunit in synaptic localization and pathogenicity in severe startle disease

doi: 10.1523/jneurosci.0837-23.2023

Figure Lengend Snippet: Figure 4. Partial compensation of GlyRα expression by GlyRα2 in oscillator mice. A–C, Representative Western blots of spinal cord (SC), brain stem (BS), and cortex (CX) tissue homogenates from Glra1+/+/Glrbeos/eos and Glra1spdot/spdot/Glrbeos/eos mice. Expression of GlyRα1, all GlyRα subunits (pan-α), and GlyRα2 (48 kDa) are shown. GAPDH (30 kDa) served as housekeeping protein. Quantification of normalized expression levels of GlyRα1 (A), all GlyRα subunits (pan-α) (B) and GlyRα2 (C) to GAPDH level in tissue samples of Glra1+/+/Glrbeos/eos and Glra1spdot/spdot/Glrbeos/eos

Article Snippet: The primary antibodies against different glycine receptor subunits were GlyRα1 (AB5052, RRID:AB_91659, 1:1,000, Merck), GlyRα2 (NBP3-03685, 1:1,000, Novus Biologicals), and pan-α-GlyR (146008, 1:1,000, Synaptic Systems).

Techniques: Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: Role of the glycine receptor ß subunit in synaptic localization and pathogenicity in severe startle disease

doi: 10.1523/jneurosci.0837-23.2023

Figure Lengend Snippet: Figure 5. GlyRβ expression is driven by GlyRα2 in oscillator mice with severe startle dis- ease. Immunohistochemical staining of slices from Glra1spdot/spdot/Glrbeos/eos mice. Spinal cord (A,B) sections stained for mEos4b-GlyRβ (green) and GlyRα1 or GlyRα2 (magenta). Scale bars refer to 500 µm. (C) Whole-cell recordings of glycinergic currents from wild-type and oscillator spinal cord dissociated neurons at 300 µM glycine. (D) Dose–response curves for glycine (3, 10, 30, 60, 100, 300, 600 µM glycine), N = 3 independent biological replicates with n = 11–13 cells recorded. Level of significance ****p < 0.0001.

Article Snippet: The primary antibodies against different glycine receptor subunits were GlyRα1 (AB5052, RRID:AB_91659, 1:1,000, Merck), GlyRα2 (NBP3-03685, 1:1,000, Novus Biologicals), and pan-α-GlyR (146008, 1:1,000, Synaptic Systems).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: The Journal of Neuroscience

Article Title: Role of the glycine receptor ß subunit in synaptic localization and pathogenicity in severe startle disease

doi: 10.1523/jneurosci.0837-23.2023

Figure Lengend Snippet: Figure 6. Synaptic localization of GlyRβ in shaky mice. A, Breeding scheme of Glra1+/shy/Glrbeos/eos mice. Intercrossings result in animals homozygous for the shaky allele harboring a missense mutation Q177K in exon 6 of the Glra1 gene. B, Weight diagram of Glra1+/+/Glrbeos/eos (n = 19), Glra1+/shy/Glrbeos/eos (n = 23), and Glra1shy/shy/Glrbeos/eos (n = 16) mice from day 10 to 21. Marked is the symptom onset of homozygous shaky mice. C, Immunocytochemical stainings of mixed primary spinal cord neuronal cultures of Glra1+/+/Glrbeos/eos and Glra1shy/shy/Glrbeos/eos

Article Snippet: The primary antibodies against different glycine receptor subunits were GlyRα1 (AB5052, RRID:AB_91659, 1:1,000, Merck), GlyRα2 (NBP3-03685, 1:1,000, Novus Biologicals), and pan-α-GlyR (146008, 1:1,000, Synaptic Systems).

Techniques: Mutagenesis

Journal: The Journal of Neuroscience

Article Title: Role of the glycine receptor ß subunit in synaptic localization and pathogenicity in severe startle disease

doi: 10.1523/jneurosci.0837-23.2023

Figure Lengend Snippet: Figure 7. Presynaptic GlyRα in VGAT-positive clusters is only slightly affected in oscillator and shaky mutant mice. A,B, Immunocytochemical staining of mixed primary spinal cord neuronal cultures of Glra1+/+/Glrbeos/eos, Glra1spdot/spdot/Glrbeos/eos, and Glra1shy/shy/Glrbeos/eos mice. Neurons were stained with antibodies against GlyRα (magenta) and VGAT (yellow). Colocalization of stained proteins is marked by arrowheads. Scale bars refer to 20 µm and 2 µm in the zoomed images. Quantification of the puncta/100 µm dendrite of VGAT (C,D), GlyRα-VGAT (E,F) (N = 3 independent experiments). Data are shown as mean ± SEM and individual data points (n = 113–119 for Glra1+/+/Glrbeos/eos, n = 101–108 for Glra1spdot/spdot/Glrbeos/eos, n = number of den- drites). Level of significance: *p < 0.05.

Article Snippet: The primary antibodies against different glycine receptor subunits were GlyRα1 (AB5052, RRID:AB_91659, 1:1,000, Merck), GlyRα2 (NBP3-03685, 1:1,000, Novus Biologicals), and pan-α-GlyR (146008, 1:1,000, Synaptic Systems).

Techniques: Mutagenesis, Staining

Journal: The Journal of Neuroscience

Article Title: Role of the glycine receptor ß subunit in synaptic localization and pathogenicity in severe startle disease

doi: 10.1523/jneurosci.0837-23.2023

Figure Lengend Snippet: Figure 8. Super-resolution imaging reveals smaller synapse sizes in oscillator compared to wild-type and shaky mutant mice. A, Spinal cord slice of a wild-type Glra1+/+/Glrbeos/eos mouse (4 µm nominal thickness) expressing fluorescent mEos4b-GlyRβ (green), and immunolabeled for gephyrin (red). Scale bar 5 µm. B, Super-resolution imaging of glycinergic spinal cord synapses in wild-type, oscillator, and shaky mutants. Representative PALM reconstructions of mEos4b-GlyRβ clusters in spinal cord tissue slices from Glra1+/+/Glrbeos/eos, Glra1spdot/spdot/Glrbeos/eos and Glra1shy/shy/Glrbeos/eos mice. Scale bar 1 µm. (B1) Enlarged images of dotted squares in B. (C) Quantification of mEos4b-GlyRβ cluster sizes in rendered super-resolution images from Glra1+/+/Glrbeos/eos (n = 14 fields of view), Glra1spdot/spdot/Glrbeos/eos (n = 16), and Glra1shy/shy/Glrbeos/eos mice (n = 14) . Significance level ****p < 0.0001; n.s. = not significant.

Article Snippet: The primary antibodies against different glycine receptor subunits were GlyRα1 (AB5052, RRID:AB_91659, 1:1,000, Merck), GlyRα2 (NBP3-03685, 1:1,000, Novus Biologicals), and pan-α-GlyR (146008, 1:1,000, Synaptic Systems).

Techniques: Imaging, Mutagenesis, Expressing, Immunolabeling

Journal: Frontiers in Molecular Neuroscience

Article Title: Polyclonal glycine receptor aAbs: a challenge for personalized epitope characterization

doi: 10.3389/fnmol.2026.1747209

Figure Lengend Snippet: Overview of GlyR α 1/α3 constructs. (A) Overall structure of the heteromeric GlyR pentamer (side and top views), with α-subunits in blue and the β subunit in dark gray (PDB: 7MLY; ). (B) The subunit interface between two GlyR α-subunits (indicated by different blue colors, light blue and blue) is depicted. Within the darker blue α-subunit (right subunit), the epitopes of commercial antibodies are labeled in red, and residues different between GlyRα1 and GlyRα3 are marked in yellow (as in C ) and labeled by residue numbers in the enlarged extracellular domain (ECD) of a single subunit. (C) Sequence alignment of ECD of GlyRα1 (UniProt P23415-1) and GlyRα3L (UniProt O75311-1), including the N-terminal domain (NTD), transmembrane 2–3 loop (TM2-3 loop), and the C-terminal domain (CTD). Residues differing between GlyRα1 and GlyRα3 are displayed in bold letters, and missing residues in the alignment are marked by a dot. Residues or small domains exchanged between GlyRα1 and α3 are labeled with a yellow background. The commonly proposed aAb-binding epitope is marked by a gray box. The blue box indicates the location of the neutralizing peptides used. The epitopes of the commercial antibodies, mAb2b (GlyRα1-specific) and mAb4a (pan-α), are depicted in red. Amino acid residue numbers refer to mature proteins. Secondary elements are labeled above the sequence, including two α-helices and 10 β-sheets. (D) Sequences of the neutralizing peptides: a mAb4a-like epitope identified in a patient with aAbs against GlyRβ; the mAb4a epitope N-terminally elongated; and the mAb4a N-terminally shortened combined with C-terminal elongation. (E) Scheme of GlyRα1/α3L Constructs. Exchanges of amino acids between GlyRα1 (blue) and GlyRα3 (orange) are marked, as well as existing epitopes for commercial antibodies mAb2b and mAb4a (red dotted box).

Article Snippet: For live-cell staining, transfected cells were incubated with patient sera diluted 1:50 or a commercial monoclonal antibody against GlyR α 1 (mAb2b, mouse: 146111 or rabbit: 146118, Synaptic Systems, Göttingen, Germany) diluted 1:500 in cell medium for 2 h. After fixation for 10 min using 4% paraformaldehyde (PFA) with 4% sucrose in phosphate-buffered saline (PBS; pH 7.4), the cells were blocked with 5% goat serum in PBS for 30 min.

Techniques: Construct, Labeling, Residue, Sequencing, Binding Assay