Journal: Acta Neuropathologica Communications

Article Title: Blood-based CNS regionally and neuronally enriched extracellular vesicles carrying pTau217 for Alzheimer’s disease diagnosis and differential diagnosis

doi: 10.1186/s40478-024-01727-w

Figure Lengend Snippet: Development of novel, flow cytometry-based assays for CNS-specific and AD-associated markers on plasma EVs a example histograms showing populations of EVs which were positive for each marker after labeling with fluorophore-conjugated antibody; plasma EVs labeled using fluorophore-conjugated immunoglobulin G isotype control for the indicated marker target antibody; and plasma EVs incubated with dye (fluorophore only, no antibody) control experiment. b Histogram of remaining particles after depletion of EVs from plasma by ultracentrifugation. c Histogram of PBS incubated with fluorophore-conjugated antibody. d – f Summary data from experiments demonstrating specificity of EVs assays (n = 3) ( d ), linearity in different dilutions of EVs plasma samples (n = 3) ( e ), and stability of reference plasma (two replicates run each day on 5 separate days of the experiment) for GABRD, GPR162 and pTau217 ( f ). Positive particles were circled out using red boxes

Article Snippet: In short, anti-GABRD antibody (Proteintech, 15623-1-AP), or anti-GPR162 antibody (Proteintech, 15254-1-AP), or anti-NLGN3 antibody (Abcam, ab192880) was labeled with ZenonTM Alexa FluorTM 488 rabbit IgG Labeling Kit (Invitrogen, Z25302); anti-pTau217 antibody (Invitrogen, 44-744) was labeled with a ZenonTM Alexa FluorTM 647 rabbit IgG Labeling Kit (Z25308, Invitrogen).

Techniques: Flow Cytometry, Clinical Proteomics, Marker, Labeling, Control, Incubation

Journal: Acta Neuropathologica Communications

Article Title: Blood-based CNS regionally and neuronally enriched extracellular vesicles carrying pTau217 for Alzheimer’s disease diagnosis and differential diagnosis

doi: 10.1186/s40478-024-01727-w

Figure Lengend Snippet: GABRD and GPR162 expression in human brain a IHC analysis of the cortex, hippocampus, caudate and cerebellum. GABRD and GPR162 stained positively and strongly at a subset of neurons in the cortex, hippocampus, and less robustly in the caudate and cerebellum (n = 3). Scale bar = 100 μm. b Histone H3 was stained as internal control, and stained positively at nucleus, rather than other cellular components. Scale bar = 100 μm. c Histogram showed the expression abundance of markers in each brain region. * p < 0.05, ** p < 0.01, *** p < 0.001, one-tailed nonparametric ANOVA followed by Kruskal–Wallis test

Article Snippet: In short, anti-GABRD antibody (Proteintech, 15623-1-AP), or anti-GPR162 antibody (Proteintech, 15254-1-AP), or anti-NLGN3 antibody (Abcam, ab192880) was labeled with ZenonTM Alexa FluorTM 488 rabbit IgG Labeling Kit (Invitrogen, Z25302); anti-pTau217 antibody (Invitrogen, 44-744) was labeled with a ZenonTM Alexa FluorTM 647 rabbit IgG Labeling Kit (Z25308, Invitrogen).

Techniques: Expressing, Staining, Control, One-tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Blood-based CNS regionally and neuronally enriched extracellular vesicles carrying pTau217 for Alzheimer’s disease diagnosis and differential diagnosis

doi: 10.1186/s40478-024-01727-w

Figure Lengend Snippet: Characterization of EVs enriched by ultracentrifugation a EVs structure revealed by cryo-EM showed double layered membrane-bound vesicles with a diameter ≈100 nm. b NTA showed a population of EVs with a peak ≈100 nm (n = 3). Three lines with different colors represent three replicate experiments. c EVs and neuron marker proteins were present in the EVs fraction obtained by ultracentrifugation; meanwhile, the non-EVs proteins, albumin was absented in the EVs fraction. d Graph summarized the relative content of EVs/neuron marker proteins and non-EVs proteins of WB experiments (n = 3). e STORM imaging of neuron marker GABRD (green) with EVs marker CD9 (red) and pTau217 (violet) presence of EVs membranes together. Scale bar = 0.1 μm. f STORM imaging was performed to confirm GPR162 (green), CD9 (red) and pTau217 (violet) presence of EVs membranes together. Overlap of both markers with CD9 indicates their presence on EVs membranes. Scale bar = 0.1, μm. ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-tailed nonparametric ANOVA followed by Kruskal–Wallis test

Article Snippet: In short, anti-GABRD antibody (Proteintech, 15623-1-AP), or anti-GPR162 antibody (Proteintech, 15254-1-AP), or anti-NLGN3 antibody (Abcam, ab192880) was labeled with ZenonTM Alexa FluorTM 488 rabbit IgG Labeling Kit (Invitrogen, Z25302); anti-pTau217 antibody (Invitrogen, 44-744) was labeled with a ZenonTM Alexa FluorTM 647 rabbit IgG Labeling Kit (Z25308, Invitrogen).

Techniques: Cryo-EM Sample Prep, Membrane, Marker, Imaging, One-tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Blood-based CNS regionally and neuronally enriched extracellular vesicles carrying pTau217 for Alzheimer’s disease diagnosis and differential diagnosis

doi: 10.1186/s40478-024-01727-w

Figure Lengend Snippet: Performance of CNS-derived EVs markers in the discovery cohort a , e the ratio of GABRD + or GPR162 + EVs in each group. b , f The ratio of pTau217 + EVs in each group. c , g The ratio of GABRD + or GPR162 + - carrying pTau217 EVs in each group. d , h The corresponding size of distribution mode of GABRD + or GPR162 + -carrying pTau217 EVs in each group. i Integrative model combining age, the ratio of GABRD + or GPR162 + -carrying pTau217 EVs, the corresponding size of distribution mode of GABRD + or GPR162 + -carrying pTau217 EVs, distinguished AD from HC. j Integrative model distinguished AD from NAD. * p < 0.05, *** p < 0.001, **** p < 0.0001, one-tailed nonparametric ANOVA followed by Kruskal–Wallis test

Article Snippet: In short, anti-GABRD antibody (Proteintech, 15623-1-AP), or anti-GPR162 antibody (Proteintech, 15254-1-AP), or anti-NLGN3 antibody (Abcam, ab192880) was labeled with ZenonTM Alexa FluorTM 488 rabbit IgG Labeling Kit (Invitrogen, Z25302); anti-pTau217 antibody (Invitrogen, 44-744) was labeled with a ZenonTM Alexa FluorTM 647 rabbit IgG Labeling Kit (Z25308, Invitrogen).

Techniques: Derivative Assay, One-tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Blood-based CNS regionally and neuronally enriched extracellular vesicles carrying pTau217 for Alzheimer’s disease diagnosis and differential diagnosis

doi: 10.1186/s40478-024-01727-w

Figure Lengend Snippet: Performance of CNS-derived EVs markers in the validation cohort a , e the ratio of GABRD + or GPR162 + EVs in each group. b , f The ratio of pTau217 + EVs in each group. c , g The ratio of GABRD + or GPR162 + -carrying pTau217 EVs in each group. d , h The corresponding size of distribution mode of GABRD + or GPR162 + -carrying pTau217 EVs in each group. i Integrative model distinguished AD from HC. j Integrative model distinguished AD from NAD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-tailed nonparametric ANOVA followed by Kruskal–Wallis test

Article Snippet: In short, anti-GABRD antibody (Proteintech, 15623-1-AP), or anti-GPR162 antibody (Proteintech, 15254-1-AP), or anti-NLGN3 antibody (Abcam, ab192880) was labeled with ZenonTM Alexa FluorTM 488 rabbit IgG Labeling Kit (Invitrogen, Z25302); anti-pTau217 antibody (Invitrogen, 44-744) was labeled with a ZenonTM Alexa FluorTM 647 rabbit IgG Labeling Kit (Z25308, Invitrogen).

Techniques: Derivative Assay, Biomarker Discovery, One-tailed Test

Journal: bioRxiv

Article Title: BDNF controls cognitive processes related to neuropsychiatric manifestations via autophagic regulation of p62 and GABA A receptor trafficking

doi: 10.1101/334466

Figure Lengend Snippet: (A) Surface biotinylation of primary cortical neurons prepared from WT, Bdnf +/- , or Bdnf +/- ; p62 +/- mice, analyzed by Western blot using the indicated antibodies. Surface levels of expression were normalized by the total levels of expression for each genotype. Surface levels of α5-GABA A R were significantly reduced in Bdnf +/- neurons compared to the other genotypes, whereas those of NR1 subunit of glutamate receptors were equivalent across the three genotypes. The assays were performed in triplicate. *p<0.05 (Kruskal-Wallis test). (B) BS3 cross-linking assays using the PFC extracts from WT, Bdnf +/- , or Bdnf +/- ; p62 +/- mice. Levels of non-crosslinked α5-GABA A R (~50 kDa) were normalized to levels of α-Tubulin at each time point. Differences in the levels of non-crosslinked α5-GABA A R at a given time point versus those of the control sample (No Xlink: no cross-linker added) represent the amount of surface α5-GABA A R that underwent mobility shift toward a higher molecular weight range due to covalent cross-linking with anonymous cell surface proteins. Cross-linking reaction reached plateau in 2–3 h in our assay conditions. Calculated surface α5-GABA A R levels (= levels at No Xlink – levels at 3 h Xlink) in Bdnf +/- mice were significantly reduced compared to the other genotypes. *p<0.05 (Kruskal-Wallis test). See also for cross-linking assays with α1- and α2-GABA A Rs. (C) The amplitude of startle response and the percentage of PPI were evaluated for WT ( n = 10), Bdnf +/- ( n = 10), and Bdnf +/- ; p62 +/- mice ( n = 10). No significant difference in startle response (left panel; F 2,27 = 0.378, P = 0.9243, one-way ANOVA). PPI was reduced in Bdnf +/- mice compared to WT mice, and rescued to control levels in Bdnf +/- ; p62 +/- mice for the prepulse–pulse pair of trials (p82–120 and p86–120 dB) (right panel; F 2,27 = 9.775, P < 0.001, two-way ANOVA with repeated measures; *p<0.05, Bonferroni post-hoc test). (D) Schematic diagram of the rule shift assay: Mice were habituated to food, feeding apparatus, different odor cues (O1, O2, etc.; e.g ., coriander versus garlic powder) and texture cues (TA, TB, etc.; e.g ., fine versus coarse digging media) prior to testing, and then food-deprived a day before the assays. Mice were initially trained in a sequence of trials to associate a food reward with a specific stimulus ( i.e ., either an odor or a digging medium; a stimulus associated with food reward is shown in red). A varying combination of stimulus and food reward was presented to mice per trial. Eight consecutive correct responses to the food reward were considered reaching criterion ( i.e ., successful establishment of association between the stimulus and the food reward), and the number of trials to reach criterion were scored for each mouse tested, before and after rule shifting ( e.g ., from an odor cue to a different texture cue to predict reward). (E) Numbers of trials to criterion were scored for WT ( n = 6), Bdnf +/- ( n = 6), and Bdnf +/- ; p62 +/- mice ( n = 6) during the initial association phase, as well as the rule shift phase of the assays. No significant difference during the initial association phase ( F 2,15 = 1.25, P = 0.934). Statistical significance during the rule shift phase: F 2,15 = 9.93, P < 0.001 (one-way ANOVA); ** p <0.01 (Bonferroni post-hoc test). During the rule shift phase, Bdnf +/- mice made a greater number of perseverative errors than the other genotypes. *p<0.05, **p<0.01 (Kruskal-Wallis test).

Article Snippet: The primary antibodies used were anti-α1-GABA A receptor (rabbit, 1:5,000, abcam), anti-α2-GABA A receptor (rabbit, 1:400, Alomone Labs), anti-α5-GABA A receptor (rabbit, 1:1,000, R&D Systems), anti-NR1 (rabbit monoclonal [1.17.2.6], 1:1,000, Millipore), anti-p62 (guinea pig, 1:1,000, MBL), anti-LC3B (rabbit, 1:1,000, Novus), anti-GAPDH (mouse, 1:1,000, abcam), and anti-α-Tubulin (mouse monoclonal [B-5-1-2], 1:8,000, Sigma).

Techniques: Western Blot, Expressing, Mobility Shift, Molecular Weight, Shift Assay, Sequencing

Journal: bioRxiv

Article Title: BDNF controls cognitive processes related to neuropsychiatric manifestations via autophagic regulation of p62 and GABA A receptor trafficking

doi: 10.1101/334466

Figure Lengend Snippet: BS3 cross-linking assays using the PFC extracts from WT ( n = 3) and Bdnf +/- mice ( n = 3). Levels of non-crosslinked α1- and α2-GABA A R (~50 kDa) were normalized to levels of α-Tubulin at each time point. Differences in the levels of non-crosslinked α1- or α2-GABA A R at a given time point versus those of the control sample (No Xlink: no cross-linker added) represent the amounts of surface GABA A R that underwent mobility shift toward a higher molecular weight range due to covalent crosslinking with anonymous cell surface proteins. No significant differences in the surface levels of α1- and α2-GABA A R were observed in the PFC of WT versus Bdnf +/- mice.

Article Snippet: The primary antibodies used were anti-α1-GABA A receptor (rabbit, 1:5,000, abcam), anti-α2-GABA A receptor (rabbit, 1:400, Alomone Labs), anti-α5-GABA A receptor (rabbit, 1:1,000, R&D Systems), anti-NR1 (rabbit monoclonal [1.17.2.6], 1:1,000, Millipore), anti-p62 (guinea pig, 1:1,000, MBL), anti-LC3B (rabbit, 1:1,000, Novus), anti-GAPDH (mouse, 1:1,000, abcam), and anti-α-Tubulin (mouse monoclonal [B-5-1-2], 1:8,000, Sigma).

Techniques: Mobility Shift, Molecular Weight

Journal: Scientific Reports

Article Title: Reduced GABAergic transmission in the ventrobasal thalamus contributes to thermal hyperalgesia in chronic inflammatory pain

doi: 10.1038/srep41439

Figure Lengend Snippet: We did no detect significant changes in protein expression of the GABA A R gamma2 subunit (( A ) ~45 KD) or delta subunit (( B ) ~55 KD) in CFA-induced inflammatory pain rats. Na + , K + -ATPase was used as the reference in the Western blotting. Control: normal saline injection into hindpaws of mice. CFA-1D or CFA-7D: day 1 or day 7 after CFA injection to hindpaws of mice. Error bars indicated SEMs, unpaired t test.

Article Snippet: Membranes were blocked with 5% milk for 30 min at the room temperature and incubated at 4 °C overnight with rabbit anti-GABA A R primary antibodies (for the gamma2 subunit, 1:1000, Novus; for the delta subunit, 1:1000, Merck Millipore) and anti-GAD65 primary antibodies (1:5000, Abcam).

Techniques: Expressing, Western Blot, Injection

Journal: Scientific Reports

Article Title: Reduced GABAergic transmission in the ventrobasal thalamus contributes to thermal hyperalgesia in chronic inflammatory pain

doi: 10.1038/srep41439

Figure Lengend Snippet: ( A – C ) GABA A R gamma2 subunit knockout using the Cre-LoxP system. Immunofluorescent double staining of GABA A R gamma2 subunits (red) with AAV-Cre vector (green) into the VB of Gabrg2 tm2Lusc /J mice. Gamma2 subunits were knocked-out in AAV-Cre-transfected thalamic relay cells. ( D ) and ( E ) Localization and expression of AAV-Cre/AAV-eGFP in the right VB at Bregma −1.7 mm. ( F ) GABA A R gamma2 subunit expression in AAV-Cre/AAV-eGFP-injected Gabrg2 tm2Lusc /J mice with Western blot. The protein level of gamma2 subunits significantly decreased in AAV-Cre-injected, but not AAV-eGFP-injected mice. ( G ) Thalamic GABA A R gamma2 subunit knockout delayed the recovery from CFA-induced inflammatory pain (Left paw, CFA-14D and CFA-21D) without affecting contralateral PWLs ( H ) or acute inflammatory pain (CFA-1D). Error bars indicated SEMs, * P < 0.05, ** P < 0.01, two-way ANOVA with Bonferroni post-test. PWL: paw withdrawal latency.

Article Snippet: Membranes were blocked with 5% milk for 30 min at the room temperature and incubated at 4 °C overnight with rabbit anti-GABA A R primary antibodies (for the gamma2 subunit, 1:1000, Novus; for the delta subunit, 1:1000, Merck Millipore) and anti-GAD65 primary antibodies (1:5000, Abcam).

Techniques: Knock-Out, Double Staining, Plasmid Preparation, Transfection, Expressing, Injection, Western Blot