Journal: Cell reports

Article Title: Activity-dependent somatodendritic dopamine release in the substantia nigra autoinhibits the releasing neuron

doi: 10.1016/j.celrep.2021.108951

Figure Lengend Snippet: (A) Na v 1.1 Ab (secondary antibody only) and TH immunostaining in SNc after whole-cell recording; Na v 1.2 Ab immunostaining is illustrated in . Neurobiotin 488 was introduced with Na v 1.1 Ab via the recording pipette (n = 5 slices). Scale bar, 20 μm. (B) Time course of spontaneous SNc DA neuron firing frequency over 25 min with intracellular application of vehicle (n = 5), IgG (n = 5), or Na v 1.1 Ab + Na v 1.2 Ab (Na v Abs) (n = 7). (C) Spontaneous activity with vehicle, IgG, or Na v Abs in the pipette. (D) Summary of Na v Ab effects on spontaneous frequency (vehicle, 99% ± 23% of initial frequency, n = 5, p = 0.5; IgG, 79% ± 13%, n = 5, p = 0.15; Na v 1.1 Ab and Na v 1.2 Ab, 14% ± 5%, n = 7, **p < 0.01; paired t test). (E) Spontaneous activity with Na v Abs in the pipette; Δt is the time from resting membrane voltage (V m ) to AP threshold (AP threshold ). (F) Quantification of Δt when recorded with Na v Abs in the pipette (initial, 0.22 ± 0.07 s; final, 0.71 ± 0.04 s, ***p < 0.001). Bars are means ± SEM for n = 4; paired t test. (G) Response of SNc DA neurons recorded in current clamp during depolarizing current injection (+400 pA, 200 ms) after establishing whole-cell recording (initial) and the end of 25-min recording (final) with Na v Abs in the pipette. Intracellular application of Na v Abs decreased the number of APs evoked by current injection. (H) Time course of the effect of single-cell application of Na v Abs on AP number evoked during current injection (vehicle, n = 6; Na v Abs, n = 5). (I) AP number during current injection with vehicle or Na v Abs in the pipette (vehicle, 106% ± 9% of initial, n = 6, p = 0.4; Na v Abs, 64% ± 7%, n = 5, *p < 0.05; paired t test).

Article Snippet: The Na v 1.2 antibody (Alomone Labs, #ASC-002) was raised against the peptide (C)ASAESRDFSGAGGIGVFSE, corresponding to amino acid residues 467–485 of Na v 1.2 intracellular loop between domains I and II.

Techniques: Immunostaining, Transferring, Activity Assay, Injection

Journal: Cell reports

Article Title: Activity-dependent somatodendritic dopamine release in the substantia nigra autoinhibits the releasing neuron

doi: 10.1016/j.celrep.2021.108951

Figure Lengend Snippet: (A) Average time course of changes in D2IC amplitude with vehicle (n = 16), IgG (n = 12), or Na v 1.1 Ab + Na v 1.2 Ab (Na v Abs) (n = 12) in the recording pipette. (B) D2ICs after establishing whole-cell recording (initial) and at the end of the recording period (final). (C) Final D2IC amplitude recorded with vehicle, IgG, or Na v Abs (vehicle, 104% ± 7% of initial, n = 16, p = 0.6; IgG, 98% ± 11%, n = 12, p = 0.6; pooled Na v Abs, 51% ± 5%, n = 12, **p < 0.01; paired t test). (D) Examples of D2 currents induced by brief superfusion of quinpirole (250 nM, 15 s). Quinpirole was applied immediately after establishing whole-cell recording (initial) and at the end (final) with Na v Abs (Na v 1.1 + Na v 1.2 Abs or Na v 1.1 Ab) in the pipette. Gray bar indicates quinpirole duration. (E) Quantification of quinpirole-evoked D2 currents with vehicle, IgG, or Na v Abs (vehicle, 106% ± 16% of initial, n = 5, p = 0.6; IgG, 107% ± 11%, n = 5, p = 0.8; Na v 1.1 Ab and Na v 1.2 Ab [n = 4] or Na v 1.1 Ab [n = 2], 95% ± 8%, n = 6, p = 0.7; paired t test).

Article Snippet: The Na v 1.2 antibody (Alomone Labs, #ASC-002) was raised against the peptide (C)ASAESRDFSGAGGIGVFSE, corresponding to amino acid residues 467–485 of Na v 1.2 intracellular loop between domains I and II.

Techniques: Transferring

Journal: Pharmacological research

Article Title: CircRNA CDR1as affects functional repair after spinal cord injury and regulates fibrosis through the SMAD pathway.

doi: 10.1016/j.phrs.2024.107189

Figure Lengend Snippet: Fig. 4. CDR1as regulates NSCs differentiation and microglia polarization. NSCs differentiation was identified by immunofluorescence. GFAP staining was used to detect astrocytes and MAP2 was used to detect nerve cells (A-B). The differentiation was further identified by PCR. The characteristics of nerve cells included MAP2, Nestin and TUJ1, and the characteristics of astrocytes were GFAP (C). iNOS (D) and Arg-1 (E) were detected by immunofluorescence after the BV2 model was established in vitro. Quantitative analysis of immunofluorescence staining (F). Expression of proinflammatory cytokines iNOS, IL-6, TNF-α (G) and anti-inflammatory cytokines Arg-1, CD206 and IL-4 (H) by PCR. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The immunofluorescence steps of NSCs and BV2 differentiation are consistent with the identification of fibroblast, and primary antibodies were: inducible nitric oxide synthase (iNOS,6022, Proteintech, Rosemont, USA), Arginase-1 (Arg-1, 16001–1-AP, Proteintech), GFAP (3670, CST), and microtubule-associated protein 2 (MAP2, 17490–1-AP, Proteintech).

Techniques: Immunofluorescence, Staining, In Vitro, Expressing

Journal: Nature Communications

Article Title: CYFIP1 governs the development of cortical axons by modulating calcium availability

doi: 10.1038/s41467-025-65801-0

Figure Lengend Snippet: a CYFIP1 RNA immunoprecipitation (RNA-IP) from DIV 3 WT cortical neurons. Histogram showing relative enrichment of the mRNAs over the non-specific IgG, measured by RT-qPCR of the eluate. The values were normalized for the input and mHprt1 mRNA and expressed as fold change over the non-specific IgG of each mRNA ( n = 4 embryos; mean ± SEM; One-Way ANOVA p < 0.0001; mMap1b mRNA p = 0.0390, mCacna1c mRNA p = 0.0054, mCacna1e mRNA p = 0.0078, mCacna1i mRNA p = 0.0009, mCacng2 mRNA p = 0.9997, mCacnb3 mRNA p = 0.9983). b Total mRNA levels of the Ca 2+ channels in DIV 3 WT and Cyfip1 +/- cortical neurons. Histograms represent mCacna1c , mCacna1e , mCacna1i , mCacng2, mCacnb3 and mCyfip1 mRNA levels, normalized to mH3f3 levels and expressed as a fold change over WT (WT n = 6/7 embryos, Cyfip1 +/- n = 7 embryos; mean ± SEM; Two-tailed Multiple Mann-Whitney test, mCacna1c mRNA p = 0.0766, mCacna1e mRNA p = 0.0435, mCacna1i mRNA p = 0.0202, mCacng2 mRNA p = 0.6282, mCacnb3 mRNA p = 0.5343, mCyfip1 mRNA p = 0.0034). c Left, representative Western Blot showing CYFIP1, Ca V 1.2 (CACNA1C), Ca V 2.3 (CACNA1E), Ca V 3.3 (CACNA1I), Ca V γ2 (CACNG2/Stargazin) and Ca V β3 (CACNB3) in membrane-enriched fractions from WT and Cyfip1 +/- DIV 3 cortical neurons. The molecular weight of each protein is indicated in kDa. Right, histogram representing Ca V 1.2, Ca V 2.3, Ca V 3.3, Ca V γ2, Ca V β3 and CYFIP1 protein expression levels in membrane-enriched fractions from WT and Cyfip1 +/- DIV 3 cortical neurons. Protein levels were normalized to Coomassie staining (WT n = 4 embryos, Cyfip1 +/- n = 7/8 embryos; mean ± SEM; Two-tailed Multiple unpaired t -test, Ca V 1.2 p = 0.0338, Ca V 2.3 p = 0.0281, Ca V 3.3 p = 0.0129, Ca V γ2 p = 0.2574, Ca V β3 p = 0.6259, CYFIP1 p = 0.0137). d–f Representative images from WT and Cyfip1 +/- DIV 3 cortical neurons stained for Ca V 1.2, Ca V 2.3, Ca V 3.3 (magenta) and βIII-Tubulin (green) (scale bar 20 μm). Histograms show the fluorescence intensity of each calcium channel normalized to βIII-Tubulin in the total neuron (left) and in the axon (right), expressed as a percentage over WT (Ca V 1.2: WT n = 4 embryos, Cyfip1 +/- n = 5 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.1111, axon p = 0.4127; Ca V 2.3: WT n = 5 embryos, Cyfip1 +/- n = 4 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.0635, axon p = 0.0159; Ca V 3.3: WT n = 4 embryos, Cyfip1 +/- n = 4 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.0286, axon p = 0.0286). Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used: mouse anti-βIII Tubulin (1:200, BioLegend, #801201), rabbit anti-Ca V 1.2 (CACNA1C) (1:100, Alomone Labs, #ACC003), rabbit anti-Ca V 2.3 (CACNA1E) (1:100, Alomone Labs, #ACC006), and rabbit anti-Ca V 3.3 (CACNA1I) (1:100, Alomone Labs, #ACC009).

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Western Blot, Membrane, Molecular Weight, Expressing, Staining, Fluorescence

Journal: Nature Communications

Article Title: CYFIP1 governs the development of cortical axons by modulating calcium availability

doi: 10.1038/s41467-025-65801-0

Figure Lengend Snippet: CYFIP1, potentially interacting with the RNA binding proteins HuD and HuR (previously identified as CYFIP1 interactors), is implicated in regulating the mRNA stability of calcium channel subunits Cacna1c , Cacna1e and Cacna1i . In Cyfip1 +/- neurons, reduced CYFIP1 levels result in a decrease protein abundance of the regulated calcium channel subunits, consequently leading to a decrease in intracellular and mitochondrial calcium concentration. Low levels of calcium ions may affect mitochondria polarity and motility, both of which we found impaired in Cyfip1 +/- axons. The decreased calcium concentration and the mitochondrial defects concur in reducing axonal growth observed in Cyfip1 +/- neurons. By restoring the intracellular calcium homeostasis, both the axonal growth and mitochondrial defects are rescued. Created in BioRender. https://BioRender.com/xquv8cy .

Article Snippet: The following primary antibodies were used: mouse anti-βIII Tubulin (1:200, BioLegend, #801201), rabbit anti-Ca V 1.2 (CACNA1C) (1:100, Alomone Labs, #ACC003), rabbit anti-Ca V 2.3 (CACNA1E) (1:100, Alomone Labs, #ACC006), and rabbit anti-Ca V 3.3 (CACNA1I) (1:100, Alomone Labs, #ACC009).

Techniques: RNA Binding Assay, Quantitative Proteomics, Concentration Assay

Journal: Neuroscience letters

Article Title: Mangiferin alleviates 6-OHDA-induced Parkinson's disease by inhibiting AKR1C3 to activate Wnt signaling pathway.

doi: 10.1016/j.neulet.2023.137608

Figure Lengend Snippet: Fig. 2. The inhibition of AKR1C3 activation of the Wnt signaling pathway in PC12 Cells by mangiferin. (A) Assessment of AKR1C3 expression levels using RT-qPCR. (B) Molecular docking simulations to investigate the binding affinity and interaction sites between mangiferin and AKR1C3 protein. (C) Measurement of gene expression levels of GSK-3β, Wnt1, and β-catenin using RT-qPCR. (D) Western blot analysis to examine the protein expression levels of p-GSK-3β, GSK-3β, Wnt1, β-catenin, and AKR1C3. *P<0.05, **P<0.01, ***P<0.001 vs the control group, #P<0.05, ##P<0.01, ###P<0.001, 6-OHDA+MG (50 μM) group vs 6-OHDA group, n=3. 6-OHDA: 6- hydroxydopamine, 100 μmol/L; MG: mangiferin, 50 μmol/L, one-way ANOVA.

Article Snippet: Primary antibodies are listed as follow: TH (1:5000, 25859-1-AP, proteintech), α-synuclein (1:1000, ab212184, Abcam), phosphate- GSK-3β (1:1000, AF2016, Affinity), GSK-3β (1:2000, ab93926, Abcam), AKR1C3 (1:2000, 11194-1-AP, proteintech), wnt1 (1:2000, 27935-1-AP, proteintech), β-catenin (1:10000, 51067-2-AP, proteintech), caspase-3 (1:2000, ab184787, Abcam), GAPDH (1:10000, ab8245, Abcam).

Techniques: Inhibition, Activation Assay, Expressing, Quantitative RT-PCR, Binding Assay, Gene Expression, Western Blot, Control

Journal: Neuroscience letters

Article Title: Mangiferin alleviates 6-OHDA-induced Parkinson's disease by inhibiting AKR1C3 to activate Wnt signaling pathway.

doi: 10.1016/j.neulet.2023.137608

Figure Lengend Snippet: Fig. 3. Weakening of the Protective Effect of mangiferin on PD by AKR1C3 Overexpression in PC12 Cells. (A) The influence of AKR1C3 overexpression on mitochondrial membrane potential using JC-1 staining. (B) The impact of AKR1C3 overexpression on intracellular reactive oxygen species (ROS) levels. (C) The effects of AKR1C3 overexpression on TH protein and α-synuclein protein. *P<0.05, **P<0.01, ***P<0.001 vs the control group, #P<0.05, ##P<0.01, ###P<0.001, 6- OHDA+MG (50 μM) group vs 6-OHDA group, n=3. PD: Parkinson’s disease; ROS, reactive oxygen species; TH: tyrosine hydroxylase; 6-OHDA: 6-hydroxydopamine, 100μmol/L; oeNC: over-expression negtive control; MG: mangiferin, 50μmol/L, one- way ANOVA.

Article Snippet: Primary antibodies are listed as follow: TH (1:5000, 25859-1-AP, proteintech), α-synuclein (1:1000, ab212184, Abcam), phosphate- GSK-3β (1:1000, AF2016, Affinity), GSK-3β (1:2000, ab93926, Abcam), AKR1C3 (1:2000, 11194-1-AP, proteintech), wnt1 (1:2000, 27935-1-AP, proteintech), β-catenin (1:10000, 51067-2-AP, proteintech), caspase-3 (1:2000, ab184787, Abcam), GAPDH (1:10000, ab8245, Abcam).

Techniques: Over Expression, Membrane, Staining, Control

Journal: Neuroscience letters

Article Title: Mangiferin alleviates 6-OHDA-induced Parkinson's disease by inhibiting AKR1C3 to activate Wnt signaling pathway.

doi: 10.1016/j.neulet.2023.137608

Figure Lengend Snippet: Fig. 4. Suppression of AKR1C3-mediated activation of the Wnt signaling by mangiferin in PC12 Cells. (A) Measurement of gene expression levels of GSK-3β, Wnt1, and mangiferin-catenin in AKR1C3-overexpressing cells using RT-qPCR. (B) Analysis of the protein expression levels of p-GSK-3mangiferin ,GSK-3β,Wnt1, and β- catenin through Western blotting in AKR1C3-overexpressing cells. *P<0.05, **P<0.01, ***P<0.001 vs the control group, #P<0.05, ##P<0.01, ###P<0.001, 6-OHDA+MG (50 μM) group vs 6-OHDA group, n=3. PD: Parkinson’s disease; TH: tyrosine hydroxylase; oeNC: over-expression negtive control; 6-OHDA: 6-hydroxydopamine, 100 μmol/L; MG: mangiferin, 50 μmol/L, one-way ANOVA.

Article Snippet: Primary antibodies are listed as follow: TH (1:5000, 25859-1-AP, proteintech), α-synuclein (1:1000, ab212184, Abcam), phosphate- GSK-3β (1:1000, AF2016, Affinity), GSK-3β (1:2000, ab93926, Abcam), AKR1C3 (1:2000, 11194-1-AP, proteintech), wnt1 (1:2000, 27935-1-AP, proteintech), β-catenin (1:10000, 51067-2-AP, proteintech), caspase-3 (1:2000, ab184787, Abcam), GAPDH (1:10000, ab8245, Abcam).

Techniques: Activation Assay, Gene Expression, Quantitative RT-PCR, Expressing, Western Blot, Control, Over Expression

Journal: Neuroscience letters

Article Title: Mangiferin alleviates 6-OHDA-induced Parkinson's disease by inhibiting AKR1C3 to activate Wnt signaling pathway.

doi: 10.1016/j.neulet.2023.137608

Figure Lengend Snippet: Fig. 7. Attenuation of mangiferin's improvement on motor behavior in PD mice with AKR1C3 activation in vivo. (A) Flowchart depicting the establishment of the PD mouse model, drug treatment, behavioral testing, and biological evaluation. day 0 was considered the day when stereotaxic injections of lentivirus or lentiviral empty vector were administered to the lesion side of the striatum, and mice were raised for three weeks. From days 14 to 28, Mangiferin was administered i.p. at a dose of 30mg/kg/day for 14 days, and behavioral training and experiments were conducted from days 21 to 28. At the end of the experiment, animals were euthanized with excess isoflurane anesthesia. The substantia nigra was extracted for the measurement of MDA and SOD levels via ELISA, and WB and RT-qPCR was applied. (B) Impact of mangiferin on fatigue stick-turning time in PD mouse model. (C) Effects of mangiferin on pole-

Article Snippet: Primary antibodies are listed as follow: TH (1:5000, 25859-1-AP, proteintech), α-synuclein (1:1000, ab212184, Abcam), phosphate- GSK-3β (1:1000, AF2016, Affinity), GSK-3β (1:2000, ab93926, Abcam), AKR1C3 (1:2000, 11194-1-AP, proteintech), wnt1 (1:2000, 27935-1-AP, proteintech), β-catenin (1:10000, 51067-2-AP, proteintech), caspase-3 (1:2000, ab184787, Abcam), GAPDH (1:10000, ab8245, Abcam).

Techniques: Activation Assay, In Vivo, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Neuroscience letters

Article Title: Mangiferin alleviates 6-OHDA-induced Parkinson's disease by inhibiting AKR1C3 to activate Wnt signaling pathway.

doi: 10.1016/j.neulet.2023.137608

Figure Lengend Snippet: Fig. 8. Attenuation of mangiferin's protective effect in PD with AKR1C3 activation in vivo. (A) The impact of AKR1C3 activation on MDA and SOD levels in vivo. (B) The effects of AKR1C3 activation on TH protein and α-synuclein protein in vivo. *P<0.05, **P<0.01, ***P<0.001 vs the control group, #P<0.05, ##P<0.01, 6- OHDA+MG (50 μM) group vs 6-OHDA group, n=6. PD: Parkinson’s disease; oeNC: over-expression negtive control; TH: tyrosine hydroxylase; 6-OHDA: 6- hydroxydopamine, 100μmol/L; MG: mangiferin, 50μmol/L, one-way ANOVA.

Article Snippet: Primary antibodies are listed as follow: TH (1:5000, 25859-1-AP, proteintech), α-synuclein (1:1000, ab212184, Abcam), phosphate- GSK-3β (1:1000, AF2016, Affinity), GSK-3β (1:2000, ab93926, Abcam), AKR1C3 (1:2000, 11194-1-AP, proteintech), wnt1 (1:2000, 27935-1-AP, proteintech), β-catenin (1:10000, 51067-2-AP, proteintech), caspase-3 (1:2000, ab184787, Abcam), GAPDH (1:10000, ab8245, Abcam).

Techniques: Activation Assay, In Vivo, Control, Over Expression

Journal: Neuroscience letters

Article Title: Mangiferin alleviates 6-OHDA-induced Parkinson's disease by inhibiting AKR1C3 to activate Wnt signaling pathway.

doi: 10.1016/j.neulet.2023.137608

Figure Lengend Snippet: Fig. 9. Suppression of Wnt signaling activation by AKR1C3 overexpression in vivo effects of mangiferin. (A) Measurement of gene expression levels of GSK-3β, Wnt1, and β-catenin in AKR1C3 overexpressing cells using RT-qPCR. (B) Analysis of protein expression levels of p-GSK-3β, GSK-3β, Wnt1, and β-catenin through Western blotting in AKR1C3 overexpressing cells. *P<0.05, **P<0.01, ***P<0.001 vs the control group, #P<0.05, ##P<0.01, ###P<0.001, 6-OHDA+MG(50μM) group vs 6-OHDA group, n=6. oeNC: over-expression negtive control; 6-OHDA: 6-hydroxydopamine, 100 μmol/L; MG: mangiferin, 50 μmol/L, one-way ANOVA.

Article Snippet: Primary antibodies are listed as follow: TH (1:5000, 25859-1-AP, proteintech), α-synuclein (1:1000, ab212184, Abcam), phosphate- GSK-3β (1:1000, AF2016, Affinity), GSK-3β (1:2000, ab93926, Abcam), AKR1C3 (1:2000, 11194-1-AP, proteintech), wnt1 (1:2000, 27935-1-AP, proteintech), β-catenin (1:10000, 51067-2-AP, proteintech), caspase-3 (1:2000, ab184787, Abcam), GAPDH (1:10000, ab8245, Abcam).

Techniques: Activation Assay, Over Expression, In Vivo, Gene Expression, Quantitative RT-PCR, Expressing, Western Blot, Control