Journal: Journal of Biological Chemistry

Article Title: α-Synuclein Induces Alterations in Adult Neurogenesis in Parkinson Disease Models via p53-mediated Repression of Notch1

doi: 10.1074/jbc.m112.354522

Figure Lengend Snippet: FIGURE 2. Alterations in the expression of Notch1 signaling components in NPC-derived neural progeny expressing -syn. A, absolute quantification of Notch1 mRNA levels throughout neuronal differentiation, showing decay of signal as neuronal differentiation progress. Notch1 levels were significantly lower in -syn-overexpressing cells by day 4. B, quantitative real time PCR analysis of the mRNA levels of Notch1, Hes1, and Hes5 in NPC-derived neuronal progeny of cells infected with LV--syn in comparison with LV-control-infected cells at day 4. C, detection of -syn, Notch1, Hes1, and Hes5 levels by Western blotting at day 4 of neuronal differentiation. D, image analysis showing integrated pixel intensity of the immunoreactivity of Notch1 signaling components. ***, p 0.001 by paired Student’s t test. *, p 0.05 compared with LV-control-infected cells by one-way ANOVA with post hoc Tukey’s test.

Article Snippet: Double Immunocytochemical Analysis and Confocal Microscopy—Neuronal differentiated ARH-NPCs grown on coverslips were double labeled with polyclonal antibodies against -syn and Notch1 (1:1000) and detected with the Tyramide Signal AmplificationTM-Direct (Red) system (1:100; PerkinElmer Life Sciences), and the mouse monoclonal antibodies against tubulin III (Tuj1 clone; 1:100) or p53 (1:500), detected with FITC-conjugated secondary antibodies (1:75; Vector Laboratories, Burlingame, CA) (24).

Techniques: Expressing, Derivative Assay, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Infection, Comparison, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein Induces Alterations in Adult Neurogenesis in Parkinson Disease Models via p53-mediated Repression of Notch1

doi: 10.1074/jbc.m112.354522

Figure Lengend Snippet: FIGURE 3. Decreased endogenous -synuclein levels alter neuronal maturation in adult rat hippocampus neural progenitor cells. A, ARH-NPCs were infected with a lentiviral vector expressing a shRNA directed to the rat -syn (LV-shRNA--syn::GFP) or an shRNA specific for luciferase as control (LV-shRNA-Luc::GFP) at a MOI of 30. 48 h post-infection, when endogenous expression of -syn reached the lowest level, neuronal differentiation was started (day 0). Immunohistochemical analysis of the expression of tubulin III (as a marker of neuronal maturation), PCNA (as a marker of proliferation), active caspase 3 (apoptosis marker), and Notch1 was performed at days 0, 2, 4, and 8 of neuronal differentiation. Compared with LV-shRNA-Luc, the NPC-derived neural progeny from LV-shRNA--syn-infected cells showed an earlier appearance of more mature neuronal phenotypes as evidenced by longer -tubulin-immu- noreactive neurites, whereas no significant changes were detected in proliferation, apoptosis, or Notch1 expression. Scale bar, 10 m. B–E, quantitative image analysis showing levels of tubulin III, PCNA, active caspase 3, and Notch1. *, p 0.05 compared with LV-shRNA-Luc-infected cells by one-way ANOVA with post hoc Tukey’s test.

Article Snippet: Double Immunocytochemical Analysis and Confocal Microscopy—Neuronal differentiated ARH-NPCs grown on coverslips were double labeled with polyclonal antibodies against -syn and Notch1 (1:1000) and detected with the Tyramide Signal AmplificationTM-Direct (Red) system (1:100; PerkinElmer Life Sciences), and the mouse monoclonal antibodies against tubulin III (Tuj1 clone; 1:100) or p53 (1:500), detected with FITC-conjugated secondary antibodies (1:75; Vector Laboratories, Burlingame, CA) (24).

Techniques: Infection, Plasmid Preparation, Expressing, shRNA, Luciferase, Control, Immunohistochemical staining, Marker, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein Induces Alterations in Adult Neurogenesis in Parkinson Disease Models via p53-mediated Repression of Notch1

doi: 10.1074/jbc.m112.354522

Figure Lengend Snippet: FIGURE 4. p53 and -syn binding to Notch1 promoter. A, schematic representation of a proximal regulatory region in the rat Notch1 promoter depicting two potential p53 response elements (RE900 and RE500). Boxes show the sequence of the RE. The bold bigger font represents the core element, and bold smaller fonts show regulatory flanking sequences. Capital letters indicate conserved residues from the consensus site, and lowercase letters show deviation from consensus. Arrows indicate the approximate position of the primers used in PCR amplification. B, binding of endogenous p53 to the Notch1 promoter was assessed in vivo by ChIP assays in B103 cells infected with LV-control (LV-CT) or LV--syn. ChIP was performed using specific antibodies against p53 or control IgG. Input chromatin represents the portion of the enzymatic-sheared chromatin prior to immunoprecipitation. The immunoprecipitated chromatin was analyzed by PCR amplification with primers flanking each RE tested. C, quantification of the immunoprecipitated DNA was analyzed by quantitative real time PCR and is shown as a percentage of the total DNA input. D, EMSAs show shifts in mobility when increasing concentrations of human recombinant -syn (2 and 5 g) were incubated with both RE900 and RE500, suggesting direct binding to DNA. -Synuclein did not alter the mobility of a control double-stranded randomized oligonucleotide.

Article Snippet: Double Immunocytochemical Analysis and Confocal Microscopy—Neuronal differentiated ARH-NPCs grown on coverslips were double labeled with polyclonal antibodies against -syn and Notch1 (1:1000) and detected with the Tyramide Signal AmplificationTM-Direct (Red) system (1:100; PerkinElmer Life Sciences), and the mouse monoclonal antibodies against tubulin III (Tuj1 clone; 1:100) or p53 (1:500), detected with FITC-conjugated secondary antibodies (1:75; Vector Laboratories, Burlingame, CA) (24).

Techniques: Binding Assay, Sequencing, Amplification, In Vivo, Infection, Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, Recombinant, Incubation

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein Induces Alterations in Adult Neurogenesis in Parkinson Disease Models via p53-mediated Repression of Notch1

doi: 10.1074/jbc.m112.354522

Figure Lengend Snippet: FIGURE 5. p53 is a negative regulator of Notch1 promoter-driven transcription and -syn further represses its expression. A, schematic representation of Notch1 promoter fragments fused to luciferase-reporter vectors to test the effects of p53 on the transcription driven by Notch1 promoter. NP1002 expands from 1002 to 81 and contains both RE900 and RE500. NP580 contains the fragment 580 to 81 and only RE500. Promoter-less vector pGLuc Basic was used as control of basal luciferase expression. B, transfection of 293T cells with NP1002 or NP580 resulted in a more than 4-fold increase of luciferase expression in comparison with pGluc control plasmid. ***, p 0.001 by paired Student’s t test. C and D, p53 represses the transcription from NP1002 (C) and NP580 (D) as observed by a decrease in luciferase expression in cells transfected with 2 g of a plasmid expressing rat p53. Overexpression of -syn by infection of the cells with LV--syn aggravates the repression of transcription mediated by p53 only for NP1002. ***, p 0.001; **, p 0.01 compared with control cells by one-way ANOVA with post hoc Tukey’s test. E, transfection of 293T cells with increasing amounts of the p53-expression plasmid resulted in a 10% increase of cell death relative to mock-transfected cells.

Article Snippet: Double Immunocytochemical Analysis and Confocal Microscopy—Neuronal differentiated ARH-NPCs grown on coverslips were double labeled with polyclonal antibodies against -syn and Notch1 (1:1000) and detected with the Tyramide Signal AmplificationTM-Direct (Red) system (1:100; PerkinElmer Life Sciences), and the mouse monoclonal antibodies against tubulin III (Tuj1 clone; 1:100) or p53 (1:500), detected with FITC-conjugated secondary antibodies (1:75; Vector Laboratories, Burlingame, CA) (24).

Techniques: Expressing, Luciferase, Plasmid Preparation, Control, Transfection, Comparison, Over Expression, Infection

Journal: Journal of Biological Chemistry

Article Title: α-Synuclein Induces Alterations in Adult Neurogenesis in Parkinson Disease Models via p53-mediated Repression of Notch1

doi: 10.1074/jbc.m112.354522

Figure Lengend Snippet: FIGURE 7. Knockdown of p53 expression partially rescues the repression of Notch1 transcription induced by -syn. A, delivery of specific siRNA directed toward rat p53 by lentiviral vectors (LV-si p53) to ARH-NPCs resulted in 65% reduction of p53 protein levels 48 h post-infection. B, quantitative real time PCR analysis of the mRNA levels of Notch1 in NPC-derived neuronal progeny of cells infected with LV--syn in comparison with LV-control-infected cells and co-infected with LV-si p53 to knock down p53 expression, or LV-si Luc as control for double viral infection. **, p 0.01 by paired Student’s t test. C, immuno- histochemical analysis of p53 and Notch1 expression in the NPC-derived neuronal cells infected with LV--syn or control and with or without knocking down of p53 expression (si p53 or si Luc). Compared with LV--syn/si Luc cells, the cells that received si p53 show higher expression of Notch1, suggesting release of transcriptional repression. Scale bar, 20 m. D and E, quantitative image analysis showing levels of p53 and Notch1. F, expression of human -syn in cells infected with the different lentiviral constructs assayed, showing robust expression also in cells co-infected with si p53 and si Luc. * and #, p 0.05 compared with control cells by one-way ANOVA with post hoc Tukey’s test.

Article Snippet: Double Immunocytochemical Analysis and Confocal Microscopy—Neuronal differentiated ARH-NPCs grown on coverslips were double labeled with polyclonal antibodies against -syn and Notch1 (1:1000) and detected with the Tyramide Signal AmplificationTM-Direct (Red) system (1:100; PerkinElmer Life Sciences), and the mouse monoclonal antibodies against tubulin III (Tuj1 clone; 1:100) or p53 (1:500), detected with FITC-conjugated secondary antibodies (1:75; Vector Laboratories, Burlingame, CA) (24).

Techniques: Knockdown, Expressing, Infection, Real-time Polymerase Chain Reaction, Derivative Assay, Comparison, Control, Construct

Journal: Frontiers in Molecular Neuroscience

Article Title: Age-Related Transcriptional Deregulation of Genes Coding Synaptic Proteins in Alzheimer's Disease Murine Model: Potential Neuroprotective Effect of Fingolimod

doi: 10.3389/fnmol.2021.660104

Figure Lengend Snippet: Levels of mRNAs coding for synaptic proteins in 12-month-old cortex: effect of AβPP and FTY720. Levels of mRNAs measured with real-time PCR in the cerebral cortex of 12-month-old (old adult) mice as described in Materials and methods. mRNA levels in AβPP-expressing mice were compared with control animals that did not inherit the transgene. Effect of FTY720 administration was assessed against vehicle-treated animals in each group. Snap25 , synaptosomal-associated protein, 25kDa; Stx1a , syntaxin 1A; Vamp1 , vesicle-associated membrane protein 1; Nrxn1 , neurexin 1; Cplx1 , complexin 1; Syt1 , synaptotagmin 1; Syp , synaptophysin; Syn1 , synapsin 1; Nefl , neurofilament light; Nefm , neurofilament medium; APP − , animals without V717I AβPP transgene; APP + , mice expressing V717I AβPP transgene. * p < 0.05 vs. corresponding controls, two-way ANOVA followed by Tukey's post-hoc test (horizontal bars: effect of AβPP; significances marked over FTY720 values: effect of FTY720 treatment within each animal group). n = 3–6, measured in triplicate ( n = 6–9 for Cplx1, Snap25, Syn1 , and Nefl ) ± SEM.

Article Snippet: Mm01198853_m1 ( Cplx1 ), Mm01315666_m1 ( Nefl ), Mm00456201_m1 ( Nefm ), Mm00660298_m1 ( Nrxn1 ), Mm01276449_m1 ( Snap25 ), Mm00444008_m1 ( Stx1a ), Mm00449772_m1 ( Syn1 ), Mm00436850_m1 ( Syp ), Mm00436858_m1 ( Syt1 ), Mm01185107_g1 ( Vamp1 ).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Membrane

Journal: Frontiers in Molecular Neuroscience

Article Title: Age-Related Transcriptional Deregulation of Genes Coding Synaptic Proteins in Alzheimer's Disease Murine Model: Potential Neuroprotective Effect of Fingolimod

doi: 10.3389/fnmol.2021.660104

Figure Lengend Snippet: Levels of mRNAs coding for synaptic proteins in 12-month-old hippocampus: effect of AβPP and FTY720. Levels of mRNAs measured with real-time PCR in the hippocampus of 12-month-old (old adult) mice as described in Materials and methods. mRNA levels in AβPP-expressing mice were compared with control animals that did not inherit the transgene. Effect of FTY720 administration was assessed against vehicle-treated animals in each group. Snap25 , synaptosomal-associated protein, 25kDa; Stx1a , syntaxin 1A; Vamp1 , vesicle-associated membrane protein 1; Nrxn1 , neurexin 1; Cplx1 , complexin 1; Syt1 , synaptotagmin 1; Syp , synaptophysin; Syn1 , synapsin 1; Nefl , neurofilament light; Nefm , neurofilament medium; APP − , animals without V717I AβPP transgene; APP + , mice expressing V717I AβPP transgene. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. corresponding controls, two-way ANOVA followed by Tukey's post-hoc test (horizontal bars: effect of AβPP; significances marked over FTY720 values: effect of FTY720 treatment within each animal group). n = 3–6, measured in triplicate ( n = 7–14 for Cplx1, Syp, Syn1, Vamp1 , and Nefl ) ± SEM.

Article Snippet: Mm01198853_m1 ( Cplx1 ), Mm01315666_m1 ( Nefl ), Mm00456201_m1 ( Nefm ), Mm00660298_m1 ( Nrxn1 ), Mm01276449_m1 ( Snap25 ), Mm00444008_m1 ( Stx1a ), Mm00449772_m1 ( Syn1 ), Mm00436850_m1 ( Syp ), Mm00436858_m1 ( Syt1 ), Mm01185107_g1 ( Vamp1 ).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Membrane

Journal: Journal of neuroinflammation

Article Title: Electroacupuncture remediates glial dysfunction and ameliorates neurodegeneration in the astrocytic α-synuclein mutant mouse model.

doi: 10.1186/s12974-015-0302-z

Figure Lengend Snippet: Fig. 3 Expression of α-syn in astrocytes in the SN and striatum of A53T mice. a Representative immunofluorescent photomicrographs showing α-syn (red) and GFAP (green) costaining in the SN of A53T mice. The α-syn immunoreactivity was restricted to GFAP-expressing astrocytes. b and c Immunoblot analysis of expression of α-syn proteins in the midbrain and striatum of A53T mice. Representative immunoblots are shown above the quantified data. A53T mice were sacrificed for subsequent immunoblot analysis at 1 or 2 months after the birth or at the paralysis development in the symptomatic A53T mice. Data are expressed as means ± SEM (n = 4 per group). ***P < 0.001 vs. 1 or 2 months

Article Snippet: Primary antibodies against GFAP (1:1000, Sigma-Aldrich USA, St. Louis, MO), ionized calcium-binding adaptor molecule-1 (Iba1) (1:1000, Wako Chemicals USA, Richmond, VA), tyrosine hydroxylase (TH) (1:1000, Sigma-Aldrich USA, St. Louis, MO), α-syn (C20 & 211, 1:1000, Santa Cruz Biotech, Santa Cruz, CA), and secondary Alexa 488- or Alex 568- conjugated antibodies (1:1000, Invitrogen) were used to visualize the staining.

Techniques: Expressing, Western Blot

Journal: Journal of neuroinflammation

Article Title: Electroacupuncture remediates glial dysfunction and ameliorates neurodegeneration in the astrocytic α-synuclein mutant mouse model.

doi: 10.1186/s12974-015-0302-z

Figure Lengend Snippet: Fig. 4 EA decreased exogenous α-syn expression in the midbrain and striatum of A53T mice. a and b Effects of EA on α-syn protein expression in the midbrain (a) and striatum (b) of A53T and nTg control mice. Representative immunoblots are shown above the quantified data. Note that expression level of α-syn proteins in both the midbrain and striatum was markedly reduced by EA. c and d Effects of EA on α-syn mRNA expression in the midbrain (c) and striatum (d) of A53T and nTg control mice. The α-syn mRNA level in midbrain and striatal tissue was detected by real-time PCR. Data are expressed as means ± SEM (n = 4 per group). #P < 0.05, ##P < 0.01 vs. A53T group

Article Snippet: Primary antibodies against GFAP (1:1000, Sigma-Aldrich USA, St. Louis, MO), ionized calcium-binding adaptor molecule-1 (Iba1) (1:1000, Wako Chemicals USA, Richmond, VA), tyrosine hydroxylase (TH) (1:1000, Sigma-Aldrich USA, St. Louis, MO), α-syn (C20 & 211, 1:1000, Santa Cruz Biotech, Santa Cruz, CA), and secondary Alexa 488- or Alex 568- conjugated antibodies (1:1000, Invitrogen) were used to visualize the staining.

Techniques: Expressing, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal of neuroinflammation

Article Title: Electroacupuncture remediates glial dysfunction and ameliorates neurodegeneration in the astrocytic α-synuclein mutant mouse model.

doi: 10.1186/s12974-015-0302-z

Figure Lengend Snippet: Fig. 5 EA reduced the astrogliosis in the midbrain of A53T mice. a Immunofluorescent images illustrating effects of EA on GFAP immunostaining in the SN of A53T and control nTg mice. GFAP-containing astrocytes were visualized by GFAP immunostaining. b Immunoblots illustrating the effects of EA on GFAP protein expression in the midbrain. Representative immunoblots are shown above the quantified data. c and d Effects of EA on GFAP mRNA expression in the midbrain (c) and striatum (d) of A53T and control nTg mice. α-syn mRNAs were detected determined by quantitative real-time PCR. Data are expressed by means ± SEM (n = 4 per group). **P < 0.01, ***P < 0.001 vs. nTg group. #P < 0.05, ###P < 0.001 vs. A53T group

Article Snippet: Primary antibodies against GFAP (1:1000, Sigma-Aldrich USA, St. Louis, MO), ionized calcium-binding adaptor molecule-1 (Iba1) (1:1000, Wako Chemicals USA, Richmond, VA), tyrosine hydroxylase (TH) (1:1000, Sigma-Aldrich USA, St. Louis, MO), α-syn (C20 & 211, 1:1000, Santa Cruz Biotech, Santa Cruz, CA), and secondary Alexa 488- or Alex 568- conjugated antibodies (1:1000, Invitrogen) were used to visualize the staining.

Techniques: Immunostaining, Control, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Decreased LIN28B in preeclampsia impairs human trophoblast differentiation and migration

doi: 10.1096/fj.201801163R

Figure Lengend Snippet: LIN28B is predominant LIN28 paralog expressed in human placenta. A, B) qPCR analysis of LIN28A and LIN28B mRNA expression in term human placenta (A) and isolated primary DCs, CTs, and STs (B) normalized to GAPDH. C–E) Representative LIN28B immunohistochemistry images in term human placenta of invasive trophoblasts (C), villous trophoblasts (D), and nonimmune rabbit IgG–only control (E). F) In situ protein LIN28B expression in DCs, CTs, and STs of placentas from normotensive pregnancies. Scale bars, 35 μm; n = 10 placental sections (F). Results represent means ± sem. *P < 0.05 vs. LIN28A (A, B) vs. DC (F).

Article Snippet: cDNA was synthesized using M-MuLV reverse transcriptase according to the manufacturer’s instructions (M0253L; New England Biolabs, Ipswich, MA, USA) and relative mRNA expression levels were determined using TaqMan Fast Advanced Mastermix (4366072; Thermo Fisher Scientific) and TaqMan real-time quantitative PCR (qPCR) primers/probes [(LIN28A: HS_00702808_s1, LIN28B: HS_01013729_m1, TNF-α: HS_01113624_g1, SYN-1: HS_01926764_u1, ITGβ4: HS_00236216_m1, apelin receptor early endogenous ligand (ELABELA): HS_04274421_m1; Thermo Fisher Scientific)] with the Quantstudio 3 qPCR machine (Thermo Fisher Scientific).

Techniques: Expressing, Isolation, Immunohistochemistry, Control, In Situ