Journal: Frontiers in Neuroscience

Article Title: Silencing of Glucocerebrosidase Gene in Drosophila Enhances the Aggregation of Parkinson's Disease Associated α-Synuclein Mutant A53T and Affects Locomotor Activity

doi: 10.3389/fnins.2018.00081

Figure Lengend Snippet: Absence of transcriptional changes in α-syn expression in dG2/dG2 RNAi flies. (A) RT-PCR confirms knockdown of GBA1 mRNA in flies with dG2/dG2 (in presence/absence of α-syn-WT, A30P, A53T) with single copy of GMR-Gal4 driver compared to respective control flies. This confirms significant transcriptional downregulation of GBA even in the absence of eye phenotype (in flies with single copy of GMR). Corresponding densitometric analysis of RT-PCR was done by normalizing controls to 100% and experimental flies (dG2/dG2 with/without α-syn) are compared against their respective controls. (B) The effect of GBA silencing on α-syn protein expression, immunoblotting was done by whole tissue lysis extraction method. Further densitometric analysis revealed no change in α-syn protein level in flies with dG2/dG2 expressing α-syn (WT, A30P, and A53T) under GMR-Gal4 drive compared to control flies expressing α-syn (WT, A30P, and A53T) alone. Corresponding densitometric graph where controls (GMR with/without α-syn) normalized to 100% is shown. (C) RT-PCR results show no changes in the transcriptional level of α-syn during GBA silencing. In densitometric analysis of α-syn RT-PCR, controls (GMR with/without α-syn) are normalized to 100% to compare experimental flies (dG2/dG2 with/without α-syn) against their respective controls. Graph represents mean ± S.E.M. for 3 independent experiments. Differences in means were compared by one-way ANOVA followed by the Newman-Keuls Multiple Comparison post hoc test. *** represents p < 0.0001.

Article Snippet: Fixed brains were stained with mouse anti-Tyrosine Hydroxylase (TH) antibody (Mouse monoclonal, Immunostar)/ anti-α-syn antibody (Syn1- Rabbit polyclonal, BD biosciences)/anti- α-syn filament antibody (Rabbit polyclonal, Abcam) at 1:200 dilution for 48 hr.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Lysis

Journal: Frontiers in Neuroscience

Article Title: Silencing of Glucocerebrosidase Gene in Drosophila Enhances the Aggregation of Parkinson's Disease Associated α-Synuclein Mutant A53T and Affects Locomotor Activity

doi: 10.3389/fnins.2018.00081

Figure Lengend Snippet: Triton-soluble and triton-insoluble α-syn level in dG2/dG2 RNAi flies expressing WT, A30P, and A53T synuclein. Western blot of triton-soluble and insoluble fraction of 0 day (A) and 30 day (B) old fly head lysates probed for α-syn showed increase in triton-insoluble α-syn levels in dG2/dG2 A53Tmutant flies compared to corresponding A53T control flies at 30days. In the corresponding densitometric analysis, controls (GMR with/without α-syn) are normalized to 100% and experimental flies (dG2/dG2 with/without α-syn) are compared against their respective controls. Triton-Insoluble α-syn was also normalized to corresponding triton-soluble α-syn. Graph represents mean ± S.E.M. for 3 independent experiments. Differences in means were compared by one-way ANOVA followed by the Newman-Keuls Multiple Comparison post hoc test. * represents p < 0.05.

Article Snippet: Fixed brains were stained with mouse anti-Tyrosine Hydroxylase (TH) antibody (Mouse monoclonal, Immunostar)/ anti-α-syn antibody (Syn1- Rabbit polyclonal, BD biosciences)/anti- α-syn filament antibody (Rabbit polyclonal, Abcam) at 1:200 dilution for 48 hr.

Techniques: Expressing, Western Blot

Journal: Frontiers in Neuroscience

Article Title: Silencing of Glucocerebrosidase Gene in Drosophila Enhances the Aggregation of Parkinson's Disease Associated α-Synuclein Mutant A53T and Affects Locomotor Activity

doi: 10.3389/fnins.2018.00081

Figure Lengend Snippet: Increased neurodegeneration in dG2/dG2 flies expressing WT, A30P, and A53T synuclein. (A) Representative image of a projected Z-series of 30 day-old control fly brain with TH-Gal4 driver stained with anti-Tyrosine Hydroxylase (TH) to identify dopaminergic (DA) neurons. DA neurons within each cluster are indicated by labels. (B) Relative number of DA neurons within the PPL1 cluster of 30 day-old dG2/dG2 RNAi flies under control of TH-Gal4 driver expressing WT, A30P, A53T synuclein ( n = 16), compared to age-matched corresponding control flies without dG2/dG2 RNAi ( n = 16). There was also significant decrease in number of DA neurons in flies expressing α-syn (WT/A30P/A53T) compared to control TH/+ flies. Differences in means were compared by one-way ANOVA followed by the Newman-Keuls Multiple Comparison post hoc test. ** represents p < 0.001, * p < 0.05. (C) Representative image of projected Z-series of PPL1 cluster in 30 day-old fly brain with under control of TH-Gal4 driver co-stained with anti-TH (green) and anti-syn antibody (red) confirms expression of α-syn in DA neurons in different genotypes. (D) Representative immunofluorescent staining of 30 day-old fly brain expressing WT, A30P, and A53T synuclein (TH-Gal4 UAS transgenes) with/without GBA silencing stained with conformational antibody (anti-α-synuclein filament antibody) for α-syn aggregates.

Article Snippet: Fixed brains were stained with mouse anti-Tyrosine Hydroxylase (TH) antibody (Mouse monoclonal, Immunostar)/ anti-α-syn antibody (Syn1- Rabbit polyclonal, BD biosciences)/anti- α-syn filament antibody (Rabbit polyclonal, Abcam) at 1:200 dilution for 48 hr.

Techniques: Expressing, Staining

Journal: Frontiers in Neuroscience

Article Title: Silencing of Glucocerebrosidase Gene in Drosophila Enhances the Aggregation of Parkinson's Disease Associated α-Synuclein Mutant A53T and Affects Locomotor Activity

doi: 10.3389/fnins.2018.00081

Figure Lengend Snippet: Sleep Behavior in dG2/dG2 GBA RNAi flies expressing wildtype and mutant α-synuclein. Sleep behavior in young flies (0–3days) with and without dG2/dG2 silencing, expressing wildtype (WT), A30P and A53T mutant synuclein using TH-Gal4 is represented as total length of sleep, fragmentation in sleep indicated by sleep bout number, and the average length of sleep in 12 h light (Day) and 12 h dark (Night) cycles (A,B) . Activity index represents fly activity level during the wake periods in day, night and in total (C) . GBA silenced A53T old flies showed pathogenic symptoms such as increased daytime sleep, with an increased number of bouts (D) and a decreased night sleep with decreased night bout length (E) . These flies also show marked reduction in wake activity during day, night and in total (F) . Bars represent mean values of at least three independent experiments ± the standard error of the mean from 16 flies that were individually recorded in each experiment conducted using drosophila activity monitor. Differences in means were compared by one-way ANOVA followed by the Newman-Keuls Multiple Comparison post hoc test (between TH/+ vs. dG2/dG2;TH/+, dG2/dG2 vs. dG2/dG2;WT/TH, dG2/dG2; A30P/TH and dG2/dG2;A53T and WT/TH vs. dG2/dG2;WT/TH and A30P vs. dG2/dG2;A30P/TH and A53T vs. dG2/dG2;A53T/TH). *** represents p < 0.0001, ** p < 0.001, and * p < 0.05.

Article Snippet: Fixed brains were stained with mouse anti-Tyrosine Hydroxylase (TH) antibody (Mouse monoclonal, Immunostar)/ anti-α-syn antibody (Syn1- Rabbit polyclonal, BD biosciences)/anti- α-syn filament antibody (Rabbit polyclonal, Abcam) at 1:200 dilution for 48 hr.

Techniques: Expressing, Mutagenesis, Activity Assay

Journal: STAR Protocols

Article Title: Protocol for generating human cerebral organoids from two-dimensional cultures of pluripotent stem cells bypassing embryoid body aggregation

doi: 10.1016/j.xpro.2025.103678

Figure Lengend Snippet: Characterization of the main cell types in the hCOs at maturation (A) hCOs immunostained for cell cycle marker Ki67 (green) and neural precursor marker Sox2 (green), different neuronal markers (DCX, βIIITubulin and MAP2) (red) and synaptic marker (SYN1) (green). Cell nuclei were identified by Höechst 33258 staining (blue). VZs are marked with white dashed lines. Scale bar = 100 μm. (B) Whole-mount immunohistochemistry of hCOs stained for Sox2 (red) and MAP2 (green). Cell nuclei were identified by Höechst 33258 staining (blue). VZs are marked with white dashed lines. Scale bar = 100 μm. hCOs, human cerebral organoids.

Article Snippet: Rabbit polyclonal anti-Synapsin 1 (Syn1) (1:200) , Merck (Millipore) , Cat#AB1543; RRID: AB_2200400.

Techniques: Marker, Staining, Immunohistochemistry

Journal: STAR Protocols

Article Title: Protocol for generating human cerebral organoids from two-dimensional cultures of pluripotent stem cells bypassing embryoid body aggregation

doi: 10.1016/j.xpro.2025.103678

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Synapsin 1 (Syn1) (1:200) , Merck (Millipore) , Cat#AB1543; RRID: AB_2200400.

Techniques: Recombinant, Knock-Out, Software

Journal: International Journal of Molecular Sciences

Article Title: High-Salt Diet Accelerates Neuron Loss and Anxiety in APP/PS1 Mice Through Serpina3n

doi: 10.3390/ijms252111731

Figure Lengend Snippet: HS diet induces neuron reduction in AD mice. ( A ) Thioflavin T staining was used to detect S-plaque in mice brains. No S-plaque was observed in the HS/ND-treated wt mice; n = 5 in each group. Scale bar = 50 μm. ( B , C ) Nissl staining was performed to detect the neuron number of HS/ND-treated wt mice. Neuron numbers were counted in the CA1 region of the hippocampus in each group. Scale bar = 50 μm. ( D , E ) Thioflavin T staining was used to detect S-plaque in the brains of HS/ND-treated AD mice. The number of S-plaques in parietal cortex, hippocampus, and prefrontal cortex was counted; n = 5 in each group. Scale bar = 50 μm. ( F ) Representative Nissl staining of hippocampus in each group. CA1 regions are magnified on the right. The immunohistochemistry for NeuN in the hippocampus of each group is also shown. Between the two green dotted lines is the neurons in the CA1 region. Scale bar = 50 μm. ( G ) The Nissl body-positive neuron numbers were counted from the CA1 region of brain slides from five mice in each group; n = 5 in each group. The scatter bar chart shows the integrated density of NeuN-positive particles in each brain slide from five mice, which was measured using Image J software; n = 5 in each group. ( H ) Immunohistochemistry for syn1 staining in the hippocampus of each group. Between the two green dotted lines is the neurons in the Or, Rad, and Mol regions; scale bar = 20 μm. ( I ) The scatter bar chart shows the integrated density of Syn1-positive particles from Or, Rad, and Mol, respectively. Each brain slide from five mice was measured using Image J software; n = 5 in each group. ( J , K ) Western blot analysis for caspase 3, Bax, Syn1, and β-tubulin 3; β-actin was used as an internal control. The data are the mean ± SE; n = 4 biologically independent animals. Blue represent the ND treated mice, red represent the HS treated mice.

Article Snippet: The sections were incubated with rabbit anti-NeuN (1:500, 14H6L24, Invitrogen, CA, USA) or mouse anti-syn1 (1:200, 7H10G6, Novus, CA, USA) overnight at 4 °C.

Techniques: Staining, Immunohistochemistry, Software, Western Blot, Control