Journal: NPJ Parkinson's Disease

Article Title: Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity

doi: 10.1038/s41531-023-00444-w

Figure Lengend Snippet: a Spontaneous excitatory (sEPSC) and inhibitory postsynaptic currents (sIPSC) measured in DIV14-18 hippocampal neurons from SNCA −/− rats transduced with αS WT or αS S129A (schematics created with BioRender.com). b Representative sEPSC traces. c Representative sIPSC traces. Scale bars, X -axis = 1 s and Y -axis = 100 pA. d sEPSC frequency in SNCA −/− neurons expressing WT αS or αS S129A. e Cumulative frequency distribution of data shown in d , expressed as percentage. f sEPSC amplitude in SNCA −/− neurons expressing αS WT or αS S129A. g Cumulative frequency distribution plot of data shown in f , expressed as percentage. Total number of individual events analyzed in panels e and g : αS WT = 1228; αS S129A = 1701. Total number of individual cells across two independent neuronal cultures ( N = 2 ) recorded in sEPSC experiments: WT αS = 21 and αS S129A n = 12. h , i sIPSC frequency between conditions as bar charts and cumulative distribution, respectively. j sIPSC amplitude in SNCA −/− neurons expressing WT or S129A αS. k Cumulative frequency distribution of data shown in j . Total number of individual events analyzed in panels i and k : αS WT = 302; αS S129A = 585. Total number of individual cells across two independent neuronal cultures recorded in sIPSC experiments: αS WT = 19 and αS S129A = 15. l , m The excitatory/inhibitory ratio of the amplitude as a bar chart and cumulative frequency distribution, respectively. E/I amplitude ratios were derived from f and j . Respective averages in f (αS WT or αS S129A) were divided by respective individual values (αS WT or αS S129A) in j to obtain E/I amplitude ratios. Each circle represents an individual cell. Recordings performed on at least three different days. Unpaired t -tests with Welch’s correction; two-tailed; mean ± SD; ns not significant; *** p < 0.001; **** p < 0.0001.

Article Snippet: Briefly, 293-T cells were transfected with αS WT or S129A plasmids along with pMD2.G and psPAX2 (packaging plasmids: Addgene #12259 and #12260, respectively).

Techniques: Transduction, Expressing, Derivative Assay, Two Tailed Test

Journal: NPJ Parkinson's Disease

Article Title: Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity

doi: 10.1038/s41531-023-00444-w

Figure Lengend Snippet: a – f Generation of the S129A knock-in mutation in mice. a Genomic structure of the mouse SNCA gene. Exons are depicted after transcript variant SNCA-201 (ENSMUST00000114268.5), with coding and non-coding regions shown as filled or open boxes, respectively. Exon 5 is boxed with red dashed line. b A knock-in strategy using CRISPR-Cas9 and ssODN was employed to generate the S129A mutation in Exon 5 of the endogenous SNCA gene. Relative positions of sgRNA (orange horizontal line), ssODN (blue horizontal line), and the S129A mutation (red vertical line) are indicated. c – f ES cell clone screening and mouse genotyping. A 3-primer PCR strategy (primers indicated with black and red arrows) was designed to distinguish WT and mutant alleles ( c , d ). For genotyping, a common pair of primers (indicated with green arrows) was used for PCR followed by sequencing to distinguish different genotypes ( c , e , f ). g Total mouse brain homogenates from indicated genotypes. WB for total αS, pS129 (D1R1R) and GAPDH. h Input and out current curve from hippocampal slices (CA1 region) of indicated mouse genotypes. N = 3 animals each, n = 16 (WT) or 17 (S129A KI ) individual slices. i Paired-pulse facilitation of WT and S129A KI hippocampal slices. Inter-stimulation intervals as indicated. N = 3 animals each, n = 16 (WT) or 17 (S129A KI ) individual slices. Unpaired t -tests for 20, 40, 60, 100, 200, and 500 ms with Welch’s correction; two-tailed; mean ± SD; ns not significant; * p < 0.05; ** p < 0.01. j Short-term plasticity assessed by multi-pulse events. Values normalized to first excitatory post synaptic current (EPSP). N = 3 animals each, n = 16 (WT) or 17 (S129A KI ) individual slices. Unpaired t -tests for pulses 2, 3, 4, 5, 6, 7, and 8 with Welch’s correction; two-tailed; mean ± SD; ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. k , l Long Term Potentiation (LTP) of WT and S129A KI . LTP induced by standard 100 Hz stimulation. N = 3 animals each, n = 8 (WT) or 10 (S129A KI ) individual slices. Unpaired t -tests for panel l (values at 60 min from K) with Welch’s correction; two-tailed; mean ± SD; ** p < 0.01.

Article Snippet: Briefly, 293-T cells were transfected with αS WT or S129A plasmids along with pMD2.G and psPAX2 (packaging plasmids: Addgene #12259 and #12260, respectively).

Techniques: Knock-In, Mutagenesis, Variant Assay, CRISPR, Sequencing, Two Tailed Test