Journal: bioRxiv

Article Title: Neuronal overexpression of mouse potassium channel subunit Kcnn1 in A53T α-synuclein mice more than doubles median survival time, associated with suppression of phospho-S129 α-synuclein formation

doi: 10.64898/2026.03.09.709927

Figure Lengend Snippet: Kaplan-Meier survival plots for A53T α-synuclein transgenic mice, A53T/Kcnn1-3copy mice, and A53T/Kcnn1-6copy mice. Median survival time was extended by 47% for the A53T/ Kcnn1-3copy mice and >100% for the A53T/Kcnn1-6copy mice. The two Kcnn1 transgenes are independent. See text for the different motor phenotypes of the A53T and A53T/Kcnn1-6copy mice.

Article Snippet: The mouse Kcnn1 cDNA sequence (NM_032397), was PCR-amplified from OriGene Technologies plasmid MR208601, omitting the 3’-terminal Myc-DDK tag sequence.

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Neuronal overexpression of mouse potassium channel subunit Kcnn1 in A53T α-synuclein mice more than doubles median survival time, associated with suppression of phospho-S129 α-synuclein formation

doi: 10.64898/2026.03.09.709927

Figure Lengend Snippet: Absence of serine129 phospho-α-synuclein (Psynuclein) immunostaining in 12 mo A53T/Kcnn1-6copy mouse as compared with A53T endstage (9 mo) mouse. A. Location of superior colliculus (purple). Sagittal and coronal cross-sections of brain are shown, to illustrate midbrain dorsal position in the sagittal section and paired left and right structures in the coronal views. B. Sagittal views of superior colliculus, from 20 µm sagittal sections ∼1.2 mm from midline, showing presence of Kcnn1 overexpression in A53T/Kcnn1-6copy 12 month old mouse is associated with absence of α-synuclein disease-associated Psynuclein. Upper panels, A53T endstage 9 month old mouse sagitally sectioned and immunostained to reveal copious Psynuclein (left) and endogenous levels of Kcnn1 (right). Lower panels, absence of Psynuclein associated with overexpression of Kcnn1. Red arrow likely denotes intermediate gray layer. Scale bar, 200 μm.

Article Snippet: The mouse Kcnn1 cDNA sequence (NM_032397), was PCR-amplified from OriGene Technologies plasmid MR208601, omitting the 3’-terminal Myc-DDK tag sequence.

Techniques: Immunostaining, Over Expression

Journal: bioRxiv

Article Title: Neuronal overexpression of mouse potassium channel subunit Kcnn1 in A53T α-synuclein mice more than doubles median survival time, associated with suppression of phospho-S129 α-synuclein formation

doi: 10.64898/2026.03.09.709927

Figure Lengend Snippet: Stereotactic injection of AAV9-CMV-Kcnn1 virus into the right superior colliculus of an asymptomatic 6 month old A53T mouse leads to overexpression of Kcnn1 in right superior colliculus (upper panel, outlined in light blue), associated with absence of Psynuclein (lower panel outlined in blue) observed after 54 days. Shown in upper and lower panels are separately immunostained neighboring 20 µm cryosections of flash-frozen brain. Upper panel, coronal section of brain showing right superior colliculus with clusters of fluorescent anti-Kcnn1-positive cells in what appear to be the bands of superficial, intermediate, and deep gray layers. Fluorescent cells are also observed beyond the boundaries of the superior colliculus, including crossing the midline and ventrally (see text). Lower panel, almost complete absence of Psynuclein staining in injected right superior colliculus region vs non-injected left (outlined and labeled in blue). (For detail on Psynuclein accumulation outside superior colliculus, see text). Scale bar 250 μm.

Article Snippet: The mouse Kcnn1 cDNA sequence (NM_032397), was PCR-amplified from OriGene Technologies plasmid MR208601, omitting the 3’-terminal Myc-DDK tag sequence.

Techniques: Injection, Virus, Over Expression, Staining, Labeling

Journal: bioRxiv

Article Title: Neuronal overexpression of mouse potassium channel subunit Kcnn1 in A53T α-synuclein mice more than doubles median survival time, associated with suppression of phospho-S129 α-synuclein formation

doi: 10.64898/2026.03.09.709927

Figure Lengend Snippet: Absence of Psynuclein from right superior colliculus along ∼1 mm of rostro-caudal length of the AAV9-CMV-Kcnn1-injected mouse shown in (Mouse 1). The right and left superior colliculus are outlined in blue in all sections. Note that the shape of the outline is slightly altered from frame to frame to account for its position relative to surrounding landmarks (not shown) and compared with The Mouse Brain in Stereotaxic Coordinates, Franklin and Paxinos, 2007 . Coronal sections shown are spaced every 200 µm rostrocaudally. Consecutive 20 µm coronal sections of brain were taken, beginning at an arbitrary point several 100 µm rostral to superior colliculus and proceeding into the superior colliculus. Every 10 th section (sxn) was stained for Psynuclein. There is absence of Psynuclein from the injected right superior colliculus along the entire cut region. Section #89 appears to contain an artefact in its left aspect. Scale bar 250 μm. Figure 4B. A53T mouse injected with injection solution lacking virus (Control). Sections were collected and immunostained as in A. Superior colliculi circled in blue. There is symmetric presence of Psynuclein in the superior colliculi and outside of it. Scale bar 250 μm.

Article Snippet: The mouse Kcnn1 cDNA sequence (NM_032397), was PCR-amplified from OriGene Technologies plasmid MR208601, omitting the 3’-terminal Myc-DDK tag sequence.

Techniques: Injection, Staining, Virus, Control

Journal: bioRxiv

Article Title: Neuronal overexpression of mouse potassium channel subunit Kcnn1 in A53T α-synuclein mice more than doubles median survival time, associated with suppression of phospho-S129 α-synuclein formation

doi: 10.64898/2026.03.09.709927

Figure Lengend Snippet: Magnified views of corresponding regions of left and right superior colliculus from an AAV9 CMV-Kcnn1 virus-injected mouse at level of intermediate gray layers, immunostained with anti-Kcnn1 and anti-P-synuclein, showing near complete absence of Psynuclein in the presence of overexpressed Kcnn1. Displayed are 400 µm X 400 µm squares, taken from coronal section #60 (Kcnn1) and section #59 (Psynuclein) of Mouse 1, with their centers ∼1100 microns from either side of the midline. Anti-Kcnn1 (upper panels) shows brighter Kcnn1-immunoreactive cells on the virus-injected right side vs much less bright cells (endogenously expressing Kcnn1) on the uninjected left side. The number of Kcnn1-immunostained cells was roughly equal on the two sides (data not shown). Anti-Psynuclein (lower panels) shows copious intracellular Psynuclein on the left, in the setting of endogenous Kcnn1, and also that Psynuclein extends into processes and that a portion may be in glia or neuropil. On the right, in the setting of overexpressed Kcnn1, only four or five flecks of Psynuclein are visible, the rightmost of which appears to be intracellular. Scale bar 20 µm.

Article Snippet: The mouse Kcnn1 cDNA sequence (NM_032397), was PCR-amplified from OriGene Technologies plasmid MR208601, omitting the 3’-terminal Myc-DDK tag sequence.

Techniques: Virus, Injection, Expressing

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: Chemical structure of ( a ) ORG25543 and ( b ) RPIGLYT2-82. c Glycine dose-responses in the presence of different concentrations of RPI-GLYT2-82 showing non-competitive inhibition. A Brown-Forsythe ANOVA test with Dunnett’s T3 multiple comparisons determined there is no statistically significant difference in the glycine EC 50 across all four conditions ( p = 0.49, n = 5 per condition). There is a significant reduction in I/I 300 between 0 nM RPI-GLYT2-82 and 600 nM ( p < 0.0001, n = 5) and 1.8 µM RPI-GLYT2−82 ( p < 0.0001, n = 5), determined by an ordinary one-way ANOVA with Dunnett’s multiple comparisons. There is no significant reduction in I/I 300 between 0 nM and 200 nM RPI-GLYT2-82 ( p = 0.07, n = 5). d RPI-GLYT2-82 concentration-dependent inhibition of glycine transport. The difference between hGlyT2 WT ( n = 6) and hGlyT2 Δ185 ( n = 5) is not significant ( p = 0.45), determined by a two-tailed unpaired T-test. e ORG25543 concentration-dependent inhibition of glycine transport. The difference between hGlyT2 WT ( n = 6) and hGlyT2 Δ185 ( n = 5) is not statistically significantly ( p = 0.60), determined by a two-tailed unpaired T-test. f RPI-GLYT2-82 washes out within 5-min (WT not fitted due to full reversal before the first time point) ( n = 5 for both conditions). g ORG25543 has prolon g ed inhibitory effects with minimal restoration of glycine-induced currents ( n = 5 for both conditions). h RPI-GLYT2-82 inhibits GlyT2 ( n = 5) with minimal effect on GlyT1 ( n = 5) ( p < 0.0001) determined by a two-tailed unpaired T-test. i The RPI-GLYT2-82 IC 50 at 20 mM Na + (IC 50 = 159 nM (95% CI: 81 to 238 nM), n = 5) is significantly lower than 100 mM Na + (IC 50 = 279 nM (95% CI: 182 to 376 nM), n = 5) ( p = 0.04). j The ORG25543 IC 50 at 20 mM Na + (IC 50 = 18 nM (95% CI: 6.8 to 29 nM), n = 5) is not significantly different to 100 mM Na + (IC 50 = 17.6 nM (95% CI: 5.5 to 30 nM), n = 5) ( p > 0.9999). Data in i , j analysed by a two-way ANOVA with Tukey’s multiple comparisons. k Normalised glycine-dependent transport currents showing comparable transport by hGlyT2 WT ( n = 5) and hGlyT2 Δ185 ( n = 5). ( p = 0.78), determined by a two-tailed unpaired T-test. n indicates biological replicates. Error bars represent ± SD from the mean. Source data are provided as a Source Data file.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Inhibition, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: Cryo-EM density maps of hGlyT2 Δ185 in ( a ) substrate-free state (purple map contour level = 0.04 in ChimeraX), bound to b substrate glycine (pink map contour level = 0.046), c ORG25543 (green map contour level = 0.055 in ChimeraX), and d RPI-GLYT2-82 (blue map contour level = 0.038 in ChimeraX). Lipid densities shown in light yellow, densities corresponding to sterols built into the models shown in dark yellow. e-h Surface representation of substrate-free, substrate-bound, and inhibitor-bound structures of hGlyT2 Δ185 viewed parallel to the membrane. e Slice view of hGlyT2 Δ185 in substrate-free form. Closed extracellular vestibule around W215 (blue) and open intracellular pathway are displayed. f Slice view of hGlyT2 Δ185 showing glycine binding pocket (cyan, compound density shown at map contour level = 0.046 in ChimeraX) g Slice view of hGlyT2 Δ185 showing ORG25543 binding pocket (orange, compound density shown at map contour level = 0.055 in ChimeraX). h Slice view of hGlyT2 Δ185 showing RPI-GLYT2-82 binding pocket (dark purple, compound density shown at map contour level = 0.038 in ChimeraX). Residues W215 (TM1, blue), R216 (TM1, blue), P543 (EL4, yellow), and D633 (TM10, orange) are shown as sticks in e-h ; TM6 shown in green in f .

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Cryo-EM Sample Prep, Membrane, Binding Assay

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: a Cartoon representation of substrate-free hGlyT2 Δ185 , glycine-bound hGlyT2 Δ185 , ORG25543 -bound hGlyT2 Δ185 , and RPI-GLYT2-82-bound hGlyT2 Δ185 . b Upper panel is a magnified view of collapsed Na1 site, usual Na1 site-forming residues shown with corresponding densities (contour level = 0.030 in ChimeraX). Lower panel is of Cl – (green) modelled in substrate-free hGlyT2 Δ185 with coordinating residues (mean coordination distance of 3.1 Å ± 0.4) and densities shown (contour level = 0.030 in ChimeraX). c Upper panel is a magnified view of Na + (purple) modelled into Na2 with coordinating residues (mean coordination distance of 2.9 Å ± 0.4) with densities (contour level = 0.046 in ChimeraX). Lower panel is a magnified view of Cl – (green) modelled in glycine-bound hGlyT2 Δ185 with coordinating residues (mean coordination distance of 3.2 Å ± 0.5) and densities (contour level = 0.046 in ChimeraX). d Upper panel is a magnified view of Na + (purple) modelled into the density at Na1 with coordination residues (contour level = 0.050 in ChimeraX). ORG25543 shown in orange and water molecules shown in red (with corresponding densities). Lower panel is a magnified view of Cl – (green) modelled in ORG25543 -bound hGlyT2 Δ185 with coordinating residues (mean coordination distance of 2.8 Å ± 0.3) and densities shown (contour level = 0.050 in ChimeraX). e Cartoon representation of RPI-GLYT2-82-bound hGlyT2 Δ185 . Upper panel is a magnified view of Na + (purple) modelled into the density at Na1 with coordination residues (contour level = 0.045 in ChimeraX). RPI-GLYT2-82 is in magenta and water molecules in red (with corresponding densities). Lower panel shows Cl – (green) modelled in RPI-GLYT2-82-bound hGlyT2 Δ185 with coordinating residues (mean coordination distance of 2.9 Å ± 0.5) and densities (contour level = 0.045 in ChimeraX).

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques:

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: a Cryo-EM density of glycine (contour level = 0.042 in ChimeraX). b Glycine (cyan) bound to the central site of hGlyT2. Interacting residues shown in sticks, H-bonds and ionic interactions shown as dashed grey lines. Na + modelled into Na2 shown as a green circle, modelled Cl – shown as a purple circle, and modelled water molecule shown as a red circle. c Glycine binds in distinct poses in GlyT2 and GlyT1. Glycine-bound hGlyT2 (pink) overlayed with glycine-bound GlyT1 (PDB ID 8WFI, grey). Interacting residues shown as sticks (hGlyT2 = pink, hGlyT1 = black). d Cryo-EM density of ORG25543 (contour level = 0.055 in ChimeraX). e Cryo-EM density of RPI-GLYT2-82 (contour level = 0.04 in ChimeraX). f ORG25543 (orange) bound to hGlyT2 Δ185 (green). Interacting residues shown as sticks and H-bonds and ionic interactions shown as grey dashed lines. N213 is likely involved in a weak hydrogen bond interaction with the polarised C–H of the methyl group attached to positively charged ammonium nitrogen of R4 substituent. g RPI-GLYT2-82 (purple) bound to hGlyT2 Δ185 (blue). Interacting residues shown as sticks, and H-bonds and ionic interactions shown as grey dashed lines. h , i Chemical structure of h ORG25543 and i RPI-GLYT2-82 with key chemical substituents R1 (blue), R2 (orange), R3 (green), and R4 (yellow) outlined. j ORG25543 concentration-dependant inhibition of glycine transport by hGlyT2 WT (black) ( n = 6), hGlyT2 W215F (red) ( n = 5) and hGlyT2 D633E (blue) ( n = 5). k Reversibility of ORG25543 inhibition of glycine transport currents mediated by hGlyT2 WT ( n = 5), hGlyT2 W215F ( n = 5) and hGlyT2 D633E ( n = 5). l RPI-GLYT2-82 dose-response curves of hGlyT2 WT ( n = 5), hGlyT2 W215F ( n = 6) and hGlyT2 D633E ( n = 5). The IC 50 values for hGlyT2 W215F and hGlyT2 D633E are greater than the maximal tested dose ( > 10 µM). m RPI-GLYT2-82 is a reversible inhibitor of glycine transport by hGlyT2 WT ( n = 5), hGlyT2 W215F ( n = 5), and hGlyT2 D633E ( n = 5). n refers to biological replicates with error bars representing ± SD from the mean. Source data are provided as a Source Data file.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Cryo-EM Sample Prep, Concentration Assay, Inhibition

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: a RPI-GLYT2-82 reduced response rate to von Frey stimulus in chronic constriction injury (CCI) mice. Two-way ANOVA with Dunnett’s multiple comparison post-hoc test comparing vehicle and every other group showed significant effects of time ( p < 0.001; F (5, 149) = 27.3) and treatment ( p < 0.001; F (3, 28) = 48.3), and an interaction effect ( p < 0.001; F (21, 196) = 6.30). A 50 mg/kg (i.p.) dose of RPI-GLYT2-82 demonstrated efficacy against mechanical allodynia, significant at 2 and 3 hours post-injection compared to vehicle ( p = 0.003, p < 0.001, respectively) A 100 mg/kg dose of RPI-GLYT2-82 showed a similar trend at 2 and 3 hours ( p = 0.001, p < 0.001, respectively). b RPI-GLYT2-82 reduced response rate to acetone stimulus in CCI mice. Two-way ANOVA with Dunnett’s multiple comparison post-hoc test was conducted between CCI model mice administered vehicle and every other group showing significant main effects of time ( p < 0.0001; F (4, 93) = 25.8) and treatment ( p < 0.0001; F (3, 26) = 21.8, and an interaction effect ( p < 0.0001; F (11, 93) = 4.4). Both 50 and 100 mg/kg (i.p.) significantly reduced cold allodynia between 0.5 and 6-hours postinjection compared to vehicle ( p < 0.05-0.001). Gabapentin was also significant at 1-6 hours ( p < 0.05-0.001). c In PSNL mice, RPI-GLYT2-82 reduced von Frey response rates. Two-way ANOVA showed significant main effects of time ( p < 0.001; F (4, 46) = 12.7) and treatment ( p < 0.001; F (2, 13) = 20.8), and an interaction effect ( p < 0.001; F (14, 91) = 5.86). A 50 mg/kg (i.p.) dose reduced mechanical allodynia, significant at 90 minutes and 2-hours post-injection compared to vehicle ( p = 0.02, p = 0.03, respectively). d – f No deficits were observed at 50 mg/kg RPI-GLYT2-82. At 250 mg/kg, RPI-GLYT2-82 caused transient reductions in d rotarod performance, e grip strength (at 30 minutes but fully recovered by 60 minutes), and f sedation. g – i RPI-GLYT2−82 (50 mg/kg and 250 mg/kg) examined against positive morphine control (10 mg/kg) and vehicle (saline). Activity of compounds using whole-body plethysmography, measuring ( g ) respiratory frequency, h min volume, and i tidal volume. Two-way ANOVA with Dunnett’s multiple comparison post-hoc test conducted between mice administered saline and every other group. j RPI-GLYT2-82 did not increase preference in a conditioned place preference paradigm. One-way ANOVA with Dunnett’s multiple comparison post-hoc test conducted between mice administered vehicle and every other group (F (3, 25) = 3.03; p = 0.0482). RPI-GLYT2-82 50 mg/kg ( n = 8, p = 0.78) and 150 mg/kg ( n = 8, p = 0.15) compared to positive 10 mg/kg morphine control ( n = 6, p = 0.0287) and vehicle (5% solutol, 95% PBS) ( n = 7). a – i Data shown as mean ± SEM. J Individual replicates shown with error bars representing ± SEM from the mean. Significance is denoted as: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. n = biological replicates. Source data are provided as a Source Data file.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Comparison, Injection, Control, Saline, Activity Assay, Conditioned Place Preference

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: Schematic showing conformational differences between inhibitor-bound outward-open, glycine-bound inward-occluded and substrate-free inward-open states of GlyT2. ORG25543 (orange) and RPI-GLYT2-82 (magenta) lock GlyT2 in an outward-open conformation. Key binding site residues stabilizing the compounds, L211, W215, R216, P543, and D633, shown as sticks. The slow dissociation rate of ORG25543 prohibits the transporter cycling to an inward-open state. RPI-GLYT2-82 binding to GlyT2 is reversible allowing the transporter to sample other conformational states after release. Glycine is bound to GlyT2 in the inward-occluded state, S479 forming a key interaction to stabilize glycine in the central pocket. In the substrate-free inward-open state, the extracellular gate formed by R216 and D633 closes access to the extracellular side and W215 occupies the allosteric site. Cl – (green circle) is bound in all states, Na + (purple circle) in Na1 is bound only in the inhibitor-bound states, and Na + in Na2 is observed only in the substrate-bound state. Transmembrane helices TM1, TM6, and TM10 shown in blue, green, and orange, respectively, and EL4 is shown in yellow.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Binding Assay