(C) PLOS One
This story was originally published by PLOS One and is unaltered.
. . . . . . . . . .



Synaptic components are required for glioblastoma progression in Drosophila [1]

['María Losada-Pérez', 'Instituto Cajal-Csic', 'Madrid', 'Mamen Hernández García-Moreno', 'Irene García-Ricote', 'Sergio Casas-Tintó', 'Iier-Instituto De Salud Carlosiii', 'Majadahonda']

Date: 2022-09

Glioblastoma (GB) is the most aggressive, lethal and frequent primary brain tumor. It originates from glial cells and is characterized by rapid expansion through infiltration. GB cells interact with the microenvironment and healthy surrounding tissues, mostly neurons and vessels. GB cells project tumor microtubes (TMs) contact with neurons, and exchange signaling molecules related to Wingless/WNT, JNK, Insulin or Neuroligin-3 pathways. This cell to cell communication promotes GB expansion and neurodegeneration. Moreover, healthy neurons form glutamatergic functional synapses with GB cells which facilitate GB expansion and premature death in mouse GB xerograph models. Targeting signaling and synaptic components of GB progression may become a suitable strategy against glioblastoma. In a Drosophila GB model, we have determined the post-synaptic nature of GB cells with respect to neurons, and the contribution of post-synaptic genes expressed in GB cells to tumor progression. In addition, we document the presence of intratumoral synapses between GB cells, and the functional contribution of pre-synaptic genes to GB calcium dependent activity and expansion. Finally, we explore the relevance of synaptic genes in GB cells to the lifespan reduction caused by GB advance. Our results indicate that both presynaptic and postsynaptic proteins play a role in GB progression and lethality.

Glioblastoma (GB) is the most frequent and aggressive type of brain tumor. It is originated from glial cells that expand and proliferate very fast in the brain. GB cells infiltrate and establish cell to cell communication with healthy neurons. Currently there is no effective treatment for GB and these tumors result incurable with an average survival of 16 months after diagnosis. Here we used a Drosophila melanogaster model to search for genetic suppressors of GB progression. The results show that genes involved in the formation of synapses are required for glial cell number increase, expansion of tumoral volume and premature death. Among these synaptic genes we found that post-synaptic genes that contribute to Neuron-GB interaction which validate previous findings in human GB. Moreover, we found electro dense structures between GB cells that are compatible with synapses and that expression of pre-synaptic genes, including brp, Lip-α and syt 1, is required for GB progression and aggressiveness. These results suggest a contribution of synapses between GB cells to disease progression, named as intratumoral synapses.

Funding: This research was supported by PID2019-110116GB-100 grant from the Spanish Ministerio de Ciencia e Innovación to S C-T and by a Postdoctoral Fellowship from the Comunidad de Madrid (2016-T2-BMD-1295) to M L-P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Losada-Pérez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Benefitting from the conserved nature of most synaptic components, we set out to dissect the pre- versus post-synaptic contributions to GB progression using a Drosophila model of the human disease, in which the pathological condition of each cell type can be genetically manipulated. Thus, in addition to demonstrating that neuron-glioblastoma synaptogenesis is a conserved mechanism in GB progression, we show that synapse-like structures are also formed intratumoral and identify several synaptic genes required for GB expansion and premature death.

Synapses elicit neurotransmission by mediating the clustering and fusion to the plasma membrane of neurotransmitters containing vesicles which release into the synaptic space [ 26 ]. The postsynaptic side is characterized by the accumulation of neurotransmitter receptors, including Glutamate receptors (GluR), the protein discs large (Dlg), orthologue of human PSD95 protein which mediates the clustering of postsynaptic molecules [ 27 ], and Synaptotagmin 4 (Syt 4), a vesicular calcium binding protein, directly implicated in retrograde signaling at synapses. Syt 4 is proposed to regulate calcium-dependent cargo trafficking within the postsynaptic compartment [ 28 ].

Brp is a well-studied component of the presynaptic component of the synapses in Drosophila that accumulates in mature active zones (AZ). Brp is the orthologue of human AZ protein ELKS/CAST/ERC, and it is required for synapse formation [ 21 ]. Lip-α is a presynaptic scaffolding protein, orthologue to several human genes including PPFIA1 (PTPRF interacting protein alpha 1) and PPFIA2 (PTPRF interacting protein alpha 2). Lip-α directly interacts with tyrosine phosphatase receptors and it is involved in synapse formation, anterograde synaptic vesicle transport, neuron development, synapse organization and axon guidance [ 22 – 24 ]. Finally, Syt 1 is a pre-synaptic vesicle calcium binding protein that functions as the fast calcium sensor for neurotransmitter release at synapses [ 25 ].

Tumor microenvironment and the communication between tumoral cells and neurons are crucial for GB progression and patient survival [ 3 – 5 , 14 – 17 ]. In addition, neuronal activity can also stimulate GB growth. Activity-dependent release of neuroliglin-3 (NLGN3) is required for GB progression in xenografts models, and NLGN3 induces the expression of synaptic proteins in glioma cells [ 18 ]. Moreover, GB samples show synaptic gene enrichment [ 19 ] and glioma cells form functional glutamate synapses with neighboring neurons, where GB cells are post-synaptic [ 18 – 20 ]. These studies also demonstrated that pharmacological or genetic inhibition of these electrical signals reduces growth and invasion of the tumor [ 19 , 20 ].

In the last decade, Drosophila melanogaster has emerged as a reliable in vivo GB model that reproduces the features of human GB [ 3 – 9 ]. The GB condition is experimentally elicited by the expression of constitutively active forms of EGFR (Epidermal Growth Factor Receptor) and PI3K (Phosphoinositide 3-kinase) in glial cells, which are the two most common mutations in patients [ 9 ]. This experimental model has been previously used to study the contribution of RIO kinases [ 8 ], vesicle transport [ 6 ], the human kinase STK17A orthologue (Drak) [ 10 , 11 ], circadian rhythms [ 12 ] and several metabolic pathways in GB [ 13 ]. Consequently, the Drosophila model of GB is well characterized and suitable to study cellular properties of GB in vivo.

Glioblastoma (GB) is the most lethal and aggressive tumor of the Central Nervous System. GB has an incidence of 3/100,000 adults per year [ 1 ], and accounts for 52% of all primary brain tumors. GB originates from glial cells or glial progenitors and causes death within 16 months after diagnosis [ 2 ] due to the low efficacy of standard treatments such as chemotherapy, radiotherapy or surgical resection.

All experiments including different genotypes were done in parallel under the same experimental conditions, with the exception of viability analysis where each genotype was normalized with their parallel control. Data were analysed and plotted using GraphPad Prism v7.0.0 and Excel (viability assays). A D’Agostino & Pearson normality tests were performed and data with normal distributions were analysed using a two-tailed T-test with Welch-correction. If data had multiple comparisons, a One-way ANOVA with Bonferroni posthoc-test was used. Data that did not pass normality testing were submitted to a two-tailed Mann-Whitney U-test or where the data had multiple comparisons a Kruskal-Wallis test and Dunnett’s post hoc-test. Error bars represent Standard Error of the Mean, significance values are: ***p≤0.0001, ** p≤0.001, *p≤0.005, ns = non-significant.

For survival analyses of adult flies, males and females were analyzed separately. 0–5 day old adult flies raised at restricted temperature were put at 29°C in groups of 10 animals per vial and were monitored blinded every 2–3 days; each experiment was done at least three times.

Flies were crossed at restricted temperature (17°C, to inactivate the UAS/Gal4 system with tub-Gal80ts) for 4 days then progeny was transfer at 29°C (when the UAS/Gal4 system is active and the glioblastoma develops). The number of adult flies emerged from the pupae were counted for each genotype. The number of control flies was considered 100% viability and all genotypes are represented relative to controls. Experiments were performed in triplicates.

IMARIS quantification (Imaris 6.3.1 software): The number of glial cells (Repo+) and the number of synaptic active sites was quantified by using the spots tool. The tumor volume was quantified using the surface tool. We selected a minimum size and threshold for the puncta or surface in the control samples of each experiment to establish the conditions. Then we applied the same conditions to the analysis of each corresponding experimental sample.

Fluorescent images were acquired by confocal microscopy (LEICA TCS SP5) and were processed using Fiji (Image J 1.50e). These images were quantified with Fiji (Image J 1.50e) or Imaris 6.3.1 (Bitplane) software. The images of the ultra-fine slices were taken with a Transmission electron microscopy JEM1010 (Jeol) with a CMOS TemCam F416 (TVIPS) camera and processed with Adobe Photoshop CS4. Figures were assembled using Adobe Photoshop CS4 and Adobe Illustrator CS4.

Transmission electron microscopy (TEM) was performed in CNS of 3rd instar larvae with horseradish peroxidase (HRP) genetically driven to glial cells (repo-Gal4>UAS-HRP CD2). Brains were fixed in 4% formaldehyde in PBS for 30 min at room temperature, and washed in PBS, followed by an amplification of HRP signal using the ABC kit (Vector Laboratories) at room temperature. After developing with DAB, brains were washed with PBS and fixed with 2% glutaraldehyde, 4% formaldehyde in PBS for 1h at room temperature. After washing in a phosphate buffer the samples were postfixed with OsO4 1% in 0.1 M 7phosphate buffer, 1% K3[Fe(CN)6] 1h at 4°C. After washing in dH2O, Brains were incubated with tannic acid in PBS for 1 min at room temperature then washed in PBS for 5min and dH2O 2x5min. Then the samples were stained with 2% uranyl acetate in H2O for 1h at room temperature in darkness followed by 3 washes in H2O2d. Brains dehydrated in ethanol series (30%, 50%, 70%, 95%, 3x100% 10 min each at 4°C). Infiltration: samples were incubated in EtOH:propylene’s OXID (1:1;V.V) for 5 min, propylene’s OXID 2x10min, propylene’s OXID:Epon (1:1) for 45 min, Epon 100% in agitation for 1 h and Epon 100% in agitation overnight. Then change to Epon 100% for 2–3 h. After, the samples were encapsulated in BEEM and incubated 48h at 60°C for polymerization. Finally, the samples were cut in ultra-fine slices for TEM imaging [ 33 ].

Third-instar larval brains, were dissected in phosphate-buffered saline (PBS), fixed in 4% formaldehyde for 30 min, washed in PBS + 0.1 or 0.3% Triton X-100 (PBT), and blocked in PBT + 5% BSA for 1 hour. Samples were incubated overnight with primary antibodies diluted in block solution, washed in, incubated with secondary antibodies diluted in block solution for 2 hours and washed in PBT. Fluorescent labeled samples were mounted in Vectashield mounting medium with DAPI (Vector Laboratories).

Fly stocks used were UAS-lacZ (BL8529), UAS-myr-RFP (BL7119), UAS-CD8GFP (BL 5137), repo-Gal4 (BL7415), tub-gal80ts (BL7019), elav-Gal4 (BL8760), elav-lexA (BL52676), UAS-CD2:HRP (BL8763), UAS-Syt1-GFP (BL6926), lexAop-nSyb-spGFP1-10UAS-CD4-spGFP11 (BL64315), UAS-nSyb-spGFP1-10lexAop-CD4-spGFP11 (BL64314), UAS-mLexA-VP16-NFAT lexAop-rCD2-GFP (CaLexA, BL66542), UAS-Cameleon2.1 (BL 6901) UAS-Syb RNAi (BL38234), UAS-Liprin-alpha RNAi (BL53868), UAS-Syt1 RNAi (BL31289), UAS-Syt4 RNAi (BL39016), UAS-Brp RNAi (BL25891), UAS-Brp RNAi80449 (BL80449, only for survival tests), UAS-ShiTS (BL 44222), Df(2)cl-h4 (BL6304), GluRIIA-GFP (BL23757), TRE-RFP (BL-59011), UAS-Kir2.1 (BL 6595 and 6596), UAS-nAChRα1 RNAi (BL 28688), UAS-nAChRα4 RNAi (BL 31985), UAS-KCNQ RNAi (BL 27252) and UAS-Caβ1 RNAi (BL 29575) from the Bloomington Stock Center ( https://bdsc.indiana.edu/index.html ); UAS-yellowRNAi (KK106068), UAS-GluRIIA-RNAi (KK101686), UAS-Dlg-RNAi (KK109274) and UAS-Bruchpilot-RNAi (KK104630), UAS-Shaker RNAi (KK104474) and UAS-ShakingB RNAi (GD24578) from the Vienna Drosophila Resource Centre ( https://stockcenter.vdrc.at/control/main ); UAS-dEGFRλ and UAS-dp110CAAX gifted by R. Read; UAS-Liprinα-GFP [ 29 ], GluRIIA-RFP (Genomic fragment containing the GluRIIA gene including 1.2kb sequence upstream of the start codon and the sequence for red fluorescent protein (RFP) inserted in the C terminus after Ser893) [ 30 ] and Df Δ22 [ 31 ] gifted by S.J. Sigrist;UAS-ihog-RFP [ 32 ] gifted by I. Guerrero, UAS-grnd Minos gifted by P. Leopold, C57-Gal4 gifted by Lori L. Wallrath, UAS-TNT gifted by Carolina Gómez Diaz.

Results

We performed a Drosophila biased genetic screening to search for relevant genes related to GB progression. We selected 2000 genes involved in cell to cell communication, and we used VDRC UAS-RNAi lines to knockdown the expression of such genes encoding transmembrane, secreted and cell to cell communication proteins. In addition, we used the previously validated EGFR/PI3K model [5,6,9,16]. GB induction in larvae causes premature death and animals do not reach adulthood. We took advantage of this unequivocal phenotype as a read-out, quantifying the number of adult flies that emerged from each experiment. We obtained 25 RNAi lines that rescued the lethality caused by the GB. Among the suppressors, we found well known mediators of GB progression such as Frizzled1 (Fz1) or Gryzun (Gry) and PI3K signaling pathway members [5,6,9]. These genes validate the experiment as positive controls. Most RNAi lines, as well as negative controls UAS-yellow RNAi or UAS-beta-galactosidase (lacZ), did not rescue GB-induced pupal lethality however, we found RNAi lines against synaptic genes, such as Liprin-α (Lip-α) and synaptotagmin1 (syt 1) that rescue GB-induced lethality (Fig 1A). These results motivated this study to determine the contribution of synaptic components to GB progression.

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. GB cells form synapses with neurons. A) Histogram showing the percentage of viability of flies when glioblastoma (GB) is induced alone (GB+lacZ or GB+yellowRNAi) or combined with PI3K, gry, fz, lip α or syt 1 knockdown in GB cells by RNAi. Percentage corresponds to the number of adult flies that emerged from the pupae for each genotype, relative to the controls (siblings without repoGal4, considered 100% of viability). Absolute numbers and percentages are in S1 Table, B top left) confocal image of GRASP+ signal in larval brain when presynaptic Syb-GFP 1-10 fragment is expressed in neurons and CD4-GFP 11 fragment is expressed in GB cells; B bottom left) magnification of above; B top right) Diagram of GRASP technique; B bottom right) confocal image of GRASP–signal when presynaptic Syb-GFP 1-10 fragment is expressed in glioma cells and CD4-GFP 11 fragment is expressed in neurons; Synaptic contracts are shown in green (GFP), glial membrane are in red (repo>myrRFP) and all nuclei (DAPI) are in blue. C) Confocal image of larval GB brain, carrying the protein-trap GluRIIA-RFP (green), showing glial membrane (repo>GFP, red) surrounding healthy tissue (neurons, not red). Dotted lines mark the limit between neurons and glioma cells. GluRIIA postsynaptic protein is detected in both GB and healthy tissue (C and C”). DAPI staining nuclei is in blue. D) GluRIID staining (green) in control and GB larval brains, presented with glial membrane or glial membrane and DAPI in the bottom images. D’) Number of GluRIID-positive dots overlapping with glial membranes in control and GB (No. = 6 brain lobes) samples. Statistic: Unpaired T-Test (* p<0.05). E) High magnification confocal image showing GB membrane (red) and Neuron-Glia GRASP signal (green) in the proximity of Brp signal (blue). F) High magnification confocal image showing GB membrane (red) and Neuron-Glia GRASP signal (green) in the proximity of GluRIID signal (blue). Scale bars: 100μm (B up), 10μm (B down), 20μm (C), 5μm (D and E) and 1μm (F). Raw numbers and complete genotypes are in S1 Table. https://doi.org/10.1371/journal.pgen.1010329.g001

Neurons produce synaptic contacts with glioma cells It was recently described that neurons establish functional synapses with glioblastoma cells in mouse xenografts [19,20]. In these studies, GB cells are postsynaptic with respect to neurons, however our results from the screening indicate that presynaptic genes are also involved in GB-induced lethality (Fig 1A). Our previous results suggested that neurons and GB cells establish an intimate contact with neurons, compatible with synaptic distance [5]. Therefore, we wondered if GB cells were pre- or postsynaptic in the Drosophila GB model. We used a modified version of the GFP reconstitution across synaptic partners (GRASP) technique [35] to determine synaptic contacts between GB cells and neurons. This technique allows the identification of pre- and postsynaptic cells (see Materials and Methods). The confocal images of larvae brains show that GFP signal is reconstituted (GRASP+) if presynaptic Syb-GFP 1-10 fragment is expressed in neurons, under the control of the specific neuronal enhancer elav-lexA, and CD4-GFP 11 fragment is expressed in GB cells under the control of the specific glial enhancer repo-Gal4 [36] (Fig 1B). In addition, we co-expressed a myristoylated form of Red Fluorescent Protein (myrRFP) in glial cells under the control of the UAS/Gal4 system to visualize GB cells membranes. In contrast, GFP does not reconstitute when GB cells express the presynaptic component of GRASP, and neurons express the post-synaptic component (Fig 1B). These results indicate that neurons (pre-synaptic) establish synapses with GB cells (post-synaptic) in Drosophila. These contacts occur in a unidirectional manner, therefore validating previous results in other GB model systems. Next, to further explore the postsynaptic role of GB cells, we studied the expression of the post-synaptic Glutamate receptor II (GluRII) gene in GB cells. We used the GluRIIA-RFP protein trap transgenic line to monitor the expression and localization of GluRIIA protein [29] (S1A–S1A” Fig). Confocal images (Fig 1C–1C”) show GB tissue (red) and not-tumoral healthy tissue (not-red), and we observed the presence of GluRIIA-RFP signal in GB tissue (Fig 1C”). Moreover, to confirm the presence of GluRII protein in the membranes of GB cells, we used a validated antibody against the GluRIID subunit. Confocal images of control larval brain samples showed GluRIID dotted signals through the brain revealing glutamatergic synapses (Fig 1D). We used confocal images from control larvae brains and GB brains, and quantified with IMARIS the total number of GluRIID dots, and the number of GluRIID positive dots that overlap with glial membranes (mRFP). We aimed to compare the green signal of GluRIID antibody that overlaps with the red signal from the glial membrane. In the glioma samples, the images and quantifications show that green signal overlapping with red signal increases. In the image corresponding to glioma, the green pattern reproduces the pattern of glial membrane, and we did not observe an increase of GluRIID signal out of glial membrane. We have considered the total GluRIID signal to make the ratio shown in Fig 1D´. The results show that less than 10% of the total GluRIID signal corresponds to glial membranes in control samples, whereas the number of GluRIID positive signals in the GB membrane reaches 40% of total synapses (Fig 1D and 1D’). In summary, our data suggest that GluRIID protein accumulates in GB cells. To further determine the nature of these synaptic structures, we co-stained GB larval samples with a specific monoclonal antibody that recognizes the pre-synaptic protein Bruchpilot (Brp), or an antibody against GluRIID and analyzed the relative position with GRASP signal. High magnification confocal images show GB membrane (red) and Neuron-Glia GRASP signal (green) in the proximity of Brp signal (blue in Fig 1E) or GluRIID signal (blue in Fig 1F). In both cases, proteins appear at less than 1 micrometer distance (Fig 1E and 1F) compatible with the formation of synapses. These results suggest that pre- and post-synaptic proteins accumulate in the GB-neuron contact region.

Presynaptic proteins are required for the enhanced GB calcium activity To further explore the presynaptic condition of GB cells, we measured the accumulation of intracellular calcium by knocking down the presynaptic genes brp, syt 1 or lip-α in GB cells. The confocal images as well as the quantification of CaLexA intensity signal showed that the knockdown of these presynaptic genes prevented the increase of CaLexA signal in GB cells (Fig 4A and 4B). This suggests that presynaptic genes are required for the induction of calcium accumulation and calcium-dependent activity in GB cells. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Presynaptic proteins are required for Calcium influx in GB. A) Quantifications of CaLexA signal in GB brains, and GB brains upon presynaptic genes brp, syt 1 or lip-ɑ knockdown by RNAi. No.≥ 8 brain lobes. Statistics: Bonferroni’s Multiple Comparison Test (***p<0.0001, ns = non-significant). B) Representative confocal images of CaLex signal in GB brains, and GB brains upon presynaptic genes brp, syt 1 or lip-ɑ knockdown by RNAi. C) Histogram showing the viability of control flies (100%) compared with flies where brp (134.26% and 108.5%), lip (121.26%) α or syt 1 (103.19%) are downregulated in normal glia. No.≥ 106 animals. D) Quantification of the number of glial cells, glial membrane volume and DAPI volume, per larval brain lobe in controls, and animals where brp, lip α or Syt 1 are downregulated in glia. No.≥ 8 brain lobes. Statistics: Dunnett’s Multiple Comparison Test (***p<0.0001, *p<0.005). Scale bars: 50 μm. Raw numbers and complete genotypes are in S1 Table. https://doi.org/10.1371/journal.pgen.1010329.g004 To ensure that presynaptic proteins have a specific role in GB progression and are not required for normal glia development we analyzed the viability of animals where lip-α, brp or syt 1 are knocked down in glial cells (through all developmental stages under the control of repo-Gal4). The quantification shows that, in all cases, the percentage of animals that reach adulthood compared with controls (the siblings) is similar or even higher (Fig 4C), and therefore, we conclude that downregulation of lip-α, brp or syt 1 in glial cells does not affect viability and therefore, are not required for vital functions during development. Additionally, we dissected brains of third instar larvae and quantified the number of glial cells, the volume of glial membrane and brain size (Fig 4D). The results show that the number of glial cells was normal in all cases with the exception of the expression of Brp RNAi BL. In addition, we measured the total volume of glial cells membrane marked with myrRFP. The quantification of RFP volume indicates that the expression of Brp RNAi VDRC and syt 1 RNAi did not cause any effect during development; however, Brp RNAi BL and Lip-α RNAi expression in glial cells caused a reduction of the total volume of glial cells membranes (Fig 4D). Finally, we measured the total brain volume marked with DAPI, brain volume was reduced upon Brp BL RNAi, Lip-α RNAi and syt 1 RNAi expression, but not upon Brp RNAi VDRC expression (Fig 4D). These data unveil a role of these synaptic proteins in the biology of normal glial cells that was unknown hereto.

JNK pathway activation depends on presynaptic gene expression We have previously described that JNK activation is required for GB progression [5] so we wondered if this pathway was also related to the synaptic components. To answer this question, we overexpressed the dominant negative form of the JNK receptor Grindewall (Grnd Minos) [61] in GB samples. Upon downregulation of the JNK pathway, Lip-α-GFP accumulation and tumor volume were reduced compared with GB samples (Figs 5A, 5A’, 6A and 6B). Moreover, we quantified the total of lip-GFP spots as well as the ratio of Lip-GFP spots related to glial membrane and found that downregulation of JNK pathway reduces Lip-α accumulation in GB cells (Fig 6C and 6D). Finally we analyzed JNK pathway activation with the TRE-RFP reporter [62] in control brains, GB and GB with presynaptic genes (brp, syt 1 or lip-α) downregulated (Fig 6E). The results indicate that the JNK pathway is upregulated in GB in line with our previous results [5,16]. In addition, downregulation of brp, syt 1 or lip-α on GB rescue JNK activation to control levels. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. Presynaptic proteins are necessary for JNK upregulation in GB. A) Representative confocal images of Lip-α-GFP accumulation in the membranes of GB cells that express a dominant negative form of Grnd, grnd Minos. Lip-GFP is shown in green and GB membrane is shown in magenta. B) Quantification of glial membrane marked by mRFP in control brains, GB, and GB with grnd minos expressed in glia. No.≥ 5 animals. C) Quantification of Lip-α-GFP spots in control brains, GB, and GB with grnd minos expressed in glia. No.≥ 5 animals. D) Quantification of Lip-α-GFP signal normalized with glial membrane in control brains, GB, and GB with grnd minos expressed in glia. No.≥ 5 animals. Statistics in B-D: Bonferroni’s Multiple Comparison Test (**p<0.001; ***p<0.0001) E) Quantification of TRE-FRP signal intensity per cell in control, GB, GB that expresses Brp lip-α or syt 1 RNAi. Statistics: Dunnett’s Multiple Comparison Test (***p<0.0001; ns = not significant). No.≥ 5 images from 5 brain lobes. Scale bar: 15 μm. Raw numbers and complete genotypes are in S1 Table. https://doi.org/10.1371/journal.pgen.1010329.g006

[END]
---
[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010329

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.