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: bioRxiv

Article Title: Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

doi: 10.1101/2021.08.23.457423

Figure Lengend Snippet: a , Enhanced neuronal activity of glutamatergic cells upon co-culture with hPSC-astrocytes. Multi-electrode array (MEA) analysis was performed at day 8 of co-culture show. b-d . Quantification of spikes (b), active electrodes (c) and reduced coefficient of variation between recorded wells (d) in co-culture as compared to glutamatergic neurons only (n=6 wells per group). e , Immunoreactivity for synaptic marker synapsin-1 (SYN1) expressed by motor neurons co-cultured with astrocytes for 21 days versus neuron-only cultures. f , MEA recording (1 out of 48 electrodes represented) to visualize sporadic spikes in-between bursts in neuronal mono-culture versus higher synchrony in co-cultures. g , MEA analysis shows reduced inter burst interval when motor neurons are co-cultured with hPSC-astrocytes (unpaired t-test, p=0.18, nMN = 11; nMN + Astro = 6 wells). h , Video-microscopy and quantification of RFP + neurite length over the course of 12 days in neuron-only versus neuron-astrocyte co-cultures. i3-neurons were co-cultured with astrocytes in a ratio of 1 astrocyte per 3 neurons (1:3). Images obtained with IncuCyte S3 live imaging system (each dot represents a well of a 24-well plate; and for each dot 4 images were taken and analyzed). Neurite length is normalized to cell body clusters. Note the clear separation of co-cultures from day 2 onward. i , Representative images of RFP + i3-neurons and image mask of neurites and cell bodies (NeuroTrack software). j . Uptake of glutamate by iPSC-astrocytes is comparable to controls (primary mouse astrocytes and human iCell astrocytes (FUJIFILM CDI). k , Secretion of human complement C3 by hPSC-astrocytes into the culture medium after stimulation with inflammatory cytokines (Interleukin-1b, TNF-alpha and C1q) for 24 h. l , Calcium transients in iPSC-astrocyte cultures after stimulation with KCL, ATP and L-glutamate. m . Periodic acid-Schiff stain of intracellular glycogen in hPSC-astrocytes and controls (iCell Astro from FUJIFILM CDI). Scale bar, 100 µm ( e, h, m ).

Article Snippet: Primary antibodies used for immunocytochemistry are as follows: mouse anti-PAX6 1:200 (561462, BD Biosciences), rabbit anti-FABP7 (BLBP) 1:200 (ABN14, EMD Millipore), rabbit anti-CD44 1:400 (ab157107, Abcam), rat anti-CD44 1:100 (A25528), mouse anti-VIMENTIN 1:500 (M0725, DAKO), rabbit anti-NFIA 1:250 (NBP-1-81406, Novus), rabbit anti-S100-beta 1:100 (ab52642, Abcam), mouse anti-TUJ1 1:1000 (801201, BioLegend), rabbit anti-GFAP 1:1000 (Z0334, Dako), rabbit anti-synapsin-1 1:500 (106-011, Synaptic Systems), rabbit anti-ASPM 1:100 (NB-100-227, Novus), rabbit anti-FAT1 1:100 (HPA023882 Millipore Sigma) and mouse anti-Pan-Neuronal Marker (PNM) 1:200 (MAB2300, EMD Millipore).

Techniques: Activity Assay, Co-Culture Assay, Marker, Cell Culture, Microscopy, Imaging, Software, Staining