Circadian Gene cry Controls Tumorigenesis through Modulation of Myc Accumulation in Glioblastoma Cells

  Citation: Jarabo, P.; de Pablo, C.; González-Blanco, A.; Casas-Tintó, S. Circadian Gene cry Controls Tumorigenesis through Modulation of Myc Accumulation in Glioblastoma Cells. Int. J. Mol. Sci. 2022,23, 2043. https://doi.org/ 10.3390/ijms23042043 Academic Editors: Nives Pe´cina-Šlaus and Ivana Jovˇcevska Received: 20 January 2022 Accepted: 8 February 2022 Published: 12 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Circadian Gene cry Controls Tumorigenesis through Modulation of Myc Accumulation in Glioblastoma Cells Patricia Jarabo *,† , Carmen de Pablo †, Amanda González-Blanco and Sergio Casas-Tintó* Instituto Cajal, CSIC, 28002 Madrid, Spain; [email protected] (C.d.P.); [email protected] (A.G.-B.) *Correspondence: [email protected] (P.J.); [email protected] (S.C.-T.) † These authors contributed equally to this work. Abstract: Glioblastoma (GB) is the most frequent malignant brain tumor among adults and currently there is no effective treatment. This aggressive tumor grows fast and spreads through the brain causing death in 15 months. GB cells display a high mutation rate and generate a heterogeneous population of tumoral cells that are genetically distinct. Thus, the contribution of genes and signaling pathways relevant for GB progression is of great relevance. We used a Drosophila model of GB that reproduces the features of human GB and describe the upregulation of the circadian gene cry in GB patients and in a Drosophila GB model. We studied the contribution of cry to the expansion of GB cells and the neurodegeneration and premature death caused by GB, and we determined that cry is required for GB progression. Moreover, we determined that the PI3K pathway regulates cry expression in GB cells, and in turn, cry is necessary and sufficient to promote Myc accumulation in GB. These results contribute to understanding the mechanisms underlying GB malignancy and lethality, and describe a novel role of Cry in GB cells. Keywords: cancer; neurodegeneration; glioma; Drosophila; disease model; PI3K; EGFR; genetics 1. Introduction Glioblastoma (GB) is the most common and aggressive type of glioma among all brain tumors, and it accounts for 57.3% of all gliomas [ 1 ]. It was classified in 2016 as a WHO grade IV diffuse oligodendroglial and astrocytic brain tumor, but the most recent classification (2021) includes these type of tumors in the “Gliomas, glioneuronal tumors, and neuronal tumors” group, termed as Glioblastoma, IDH-wildtype [ 2 ]. Despite current treatments, the median survival of GB patients is 15 months [ 3 ], and it is estimated that only 6.8% of patients survive five years after diagnosis [ 1 ]. To understand the genetic, molecular and cellular bases of gliomagenesis is fundamental for the development of effective therapies. In terms of histopathology and genetic expression, GB is a very heterogeneous type of tumor, even within the same patient [ 4 ]. However, there are common mutations in GB affecting different pathways that show mutual exclusion: the p53 pathway, the Rb pathway and components of the PI3K pathway [5]. Previous studies from our lab used a GB model in Drosophila, developed by Read and collaborators in 2009, that recapitulates key aspects of the disease both genetically and phenotypically [ 6 – 13 ]. This model is based on the expression of constitutively active forms of the epidermal growth factor receptor (EGFR λ ) and phosphatidyl inositol 3 kinase (PI3K) catalytic subunit (dp110 CAAX ) (orthologues of EGFR and PI3K catalytic subunit in Drosophila, respectively). We used the binary expression system Gal4/UAS [ 14 ] to express EGFR λ and PI3K dp110 CAAX specifically in glial cells under the control of repo-Gal4 driver [ 6 ]. The co-activation of EGFR and PI3K signaling pathways in Drosophila glial cells reproduces the cascade of signaling events that occurs in GB patients [ 6 ]. In consequence, GB cells upregulate myc expression, which is essential for tumoral transformation, and the glial tumor cell numbers increase along with the expansion of the glial membrane. As a result, Int. J. Mol. Sci. 2022,23, 2043. https://doi.org/10.3390/ijms23042043 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022,23, 2043 2 of 16 GB progression causes a reduction in the number of synapses in neighboring neurons and premature death [ 6 , 9 , 15 ]. Furthermore, EGFR and PI3K pathway co-activation regulates processes such as progression and entry into the cell cycle and protein synthesis [6,7]. c-myc is one of the oncogenes most amplified in human cancer, including GB. About 60%–80% of human GB cases show elevated Myc levels [ 16 ]. Myc regulates cell proliferation, transcription, differentiation, apoptosis and cell migration. It is the point where EGFR and PI3K pathways converge; thus, Myc is considered essential for GB transformation [ 6 , 16 – 18 ]. Furthermore, in vitro and in vivo studies have shown that myc inhibition prevents glioma formation, inhibits cell proliferation and survival and even induces disease regression [ 16 , 19 ]. These features are conserved in Drosophila [6]. In the recent years, the study of alterations in circadian rhythm genes has emerged in different types of cancer, including GB [ 20 ]. Previous reports suggested that circadian rhythm genes play essential roles in different aspects of tumor progression. The central clock organizes the oscillations and rhythmicity of the physiological processes and modulates the expression of genes related to cell proliferation or differentiation, such as cell cycle components [21], proto-oncogenes and tumor suppressors [22]. In mammals, the structure responsible for coordinating circadian behavior throughout the body is the suprachiasmatic nucleus (SCN), located in the anterior region of the hypothalamus and made up of about 50,000 neurons in humans [ 23 ]. All the neurons that compose the central clock express the core circadian genes that control the oscillations that organize the cycles of the whole organism in absence of environmental cues. Furthermore, synchronization of the internal clock with light/dark cycles relies on cryptochrome protein (Cry), a blue light photopigment expressed in certain subsets of clock neurons. Cry is a receptor of near-UV/blue light and a regulator of gene expression that belongs to the group of DNA photolyases. It was suggested that the last universal common ancestor (LUCA) had one or several photolyases, supporting the evolutionary conservation of cryptochrome genes [ 24 ]. However, the mammalian gene that plays the role of Drosophila cry remains unknown. Interestingly, Drosophila Cry also acts as the mammalian Cry when expressed in peripheral clocks [ 25 ]. Besides, cry1 expression is androgen responsive, Cry1 regulates DNA repair and the G2/M transition and it is associated with poor outcome in prostate cancer and colorectal cancer. Regarding GB, studies in patients with primary gliomas show an association between a specific per1 variant with overall glioma risk. Several circadian genes, including cry1, exhibited differential expression in GB samples compared to control brains as described in the literature [ 26 , 27 ], and in human cancer gene expression databases (https://www.proteinatlas. org, accessed on 1 February 2022; https://cancer.sanger.ac.uk/, accessed on 10 January 2022). Besides, the expression of the circadian gene clk is significantly enhanced in high-grade gliomas and correlates with tumor progression [ 28 ]. Moreover, per1 and per2 expression increases the efficacy of radiotherapy also in GB cells [29]. Furthermore, high levels of cry1 inversely correlate with median survival in GB patients, acting as signal of poor prognosis (http://gepia.cancer-pku.cn/detail.php?gene= CRY1, accessed on 1 February 2022). Still, the functional mechanism of Cry in cancer susceptibility and carcinogenesis remains unsolved. Different studies show a relationship between Cry and Myc [ 30 ]; c-Myc levels decrease in cry1/cry2 null mutant mice [ 31 ]. Besides, cry1 expression is induced by Myc in GB cells in culture [32]. Taking into account the deregulation in the expression of circadian genes in tumor tissues and the pre-established relationship between Cry and myc, which is a key player in GB, here we show that cry is regulated by PI3K pathway, cry expression enhances Myc accumulation in GB cells and it is necessary for GB progression. Int. J. Mol. Sci. 2022,23, 2043 3 of 16 2. Results 2.1. Cry Expression in Glioblastoma To determine if cry expression was affected in glioma samples, we extracted RNA from the heads of 7-day-old adult control and glioma flies. Quantitative RT-PCR results (see Table 1in Materials and Methods) indicate that cry mRNA levels are 50 times higher in glioma samples as compared to controls (Figure 1A). This result goes in line with the data retrieved from TCGA-GBM dataset (at http://gliovis.bioinfo.cnio.es/, accessed on 1 February 2022) that indicate a significant increase of cry1 mRNA levels (RNA-seq) in GB samples, as compared to non-tumor tissue. Next, to determine if cry upregulation occurs in GB cells, we used a specific reporter line that generates a green fluorescent protein (GFP) tagged form of Cry (GFP-Cry) and visualized adult brains in confocal microscopy. The images show the GFP signal (Cry) and glial membrane marked in red with myristoylated red fluorescent protein (mRFP) (Figure 1B–E,B’–E’). The quantification of GFP-Cry and mRFP co-localization is higher in glioma samples than in controls (Figure 1B,C,F) suggesting an accumulation of Cry in glioma cells. This signal is restored to control levels upon cry knockdown by means of RNAi expression in glial or glioma cells (Figure 1D–F). Next, we analyzed human mRNA expression databases for Glioblastoma multiforme (http://gliovis.bioinfo.cnio.es/, accessed on 10 January 2022). The results indicate that cry in GB patients is transcriptionally upregulated in primary tumors (Figure 1G) and cry1 upregulation correlates with worse prognosis (Figure 1H). Moreover, cry1 is also upregulated in secondary GB (Figure 1I) and correlates with poor prognosis in secondary GB patients (Figure 1J). All together, these results indicate that cry is transcriptionally upregulated in GB cells in Drosophila and patients and suggest a role in GB malignancy and aggressiveness. 2.2. Cry Mediates GB Progression and Neurodegeneration To determine the contribution of cry to GB progression, we used a previously validated protocol to quantify tumor progression and the associated neurodegeneration in Drosophila [ 7 , 9 , 11 ]. We stained adult control brains and compared them with GB, GB + cryRNAi and wt brains expressing cryRNAi in glial cells. We used a specific antibody against repo to visualize the nuclei of all glial cells and quantified the number of glial cells in the confocal images (Figure 2A–E). The results indicate that GB samples have a significant increase in the number of glial cells compared to control samples, but this increase depends on cry expression (Figure 2A–C,E). Besides, knockdown of cry in normal glia does not alter the number of glial cells (Figure 2D,E). In addition, we quantified the volume of glial membrane. We used Imaris software to measure the volume of the red signal that corresponds to a myristoilated form of RFP (mRFP) expressed in glial cells under the control of repo-Gal4. The quantification of the volume show a significant expansion of glial membrane in GB compared to control samples, but this increase depends on cry expression (Figure 2A’–C’,F). Again, knockdown of cry in normal glia does not alter the volume of glial membrane (Figure 2D’,F). These results suggest that cry expression is required for GB progression, but not for normal glia development. Int. J. Mol. Sci. 2022,23, 2043 4 of 16 Int. J. Mol. Sci. 2022, 23, 2043 3 of 17 2. Results 2.1. cry Expression in Glioblastoma To determine if cry expression was affected in glioma samples, we extracted RNA from the heads of 7-day-old adult control and glioma flies. Quantitative RT-PCR results (see Table 1 in Materials and Methods) indicate that cry mRNA levels are 50 times higher in glioma samples as compared to controls (Figure 1A). This result goes in line with the data retrieved from TCGA-GBM dataset (at http://gliovis.bioinfo.cnio.es/, accessed on February 1st 2022) that indicate a significant increase of cry1 mRNA levels (RNA-seq) in GB samples, as compared to non-tumor tissue. Figure 1. Circadian gene cry is upregulated in both human GB samples and GB Drosophila model. ( A ) RT-qPCR analysis of complete brains of 7-day-old adult flies from repo-Gal4 >UAS-LacZ (Control) and repo-Gal4 >UAS-dEGFR λ , UAS-dp110 CAAX (Glioma) genotypes in LD conditions at ZT6 for the circadian gene cry (t-test) in n= 90. ( B – E ) Confocal microscopy images of brains of 7-day-old adult flies from ( B )repo-Gal4 > UAS-LacZ (Control), ( C )repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX (Glioma), ( D )repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX , UAS-cryRNAi (Glioma CryRNAi) and ( E )repo-Gal4 > UAS-cryRNAi (Glia CryRNAi) after using ( B’ – E” ) magnifications of the brain lobe central region, the reporter GFP-Cry in green and the glial membrane are marked in red. ( F ) Colocalization between GFP-Cry signal and the glial membrane (mRFP). Statistical analysis in at least n= 16 (ANOVA, post-hoc Bonferroni). ( G ) Data on overexpression of cry1 in human primary gliomas and GB against normal tissue. ( H ) Graph showing a lower life expectancy in those patients with primary GB and cry1 overexpressed compared to patients with primary GB with low expression of cry1. ( I ) Data on cry1 expression in human secondary GB compared to normal tissue. ( J ) Graph showing a lower life expectancy in those patients with secondary GB and cry1 overexpressed compared to patients with secondary GB with low expression of cry1. Images obtained from gliovis.bioinfo.cnio.es based on the 2016 classification of brain tumors (scale bar, 100 µ m in ( B – E ) and 20 µ m in ( B’ – E’ ) (n.s. not significant, ** p-value < 0.01, *** p-value < 0.001). Int. J. Mol. Sci. 2022,23, 2043 5 of 16 Int. J. Mol. Sci. 2022, 23, 2043 5 of 17 Figure 2. Ectopic downregulation of cry prevents GB tumorigenesis and effects. (A–D) Confocal microscopy images of brains from 7-day-old adult flies with the following genotypes: (A) repo-Gal4 > UAS-LacZ (Control), (B) repo-Gal4 > UAS-dEGFRλ, UAS-dp110 CAAX (Glioma), (C) repo-Gal4 > UASdEGFRλ, UAS-dp110 CAAX, UAS-cryRNAi (Glioma CryRNAi) and (D) repo-Gal4 > UAS-cryRNAi (Glia CryRNAi) with glial nuclei marked in green with anti-repo (scale bar, 100 µm). (A’–D’) Glial membrane is shown in red by the expression of mRFP. (E) Quantification of glial cells number and (F) quantification of glial membrane volume. Statistical analysis for at least n = 11 per genotype (ANOVA, post-hoc Bonferroni). (G–J) Confocal images of adult NMJ of 7-day-old flies from (G) repo-Gal4 > UAS-LacZ (Control) (H) repo-Gal4 > UAS-dEGFRλ, UAS-dp110CAAX (Glioma), (I) repo-Gal4 > UAS-dEGFRλ, UAS-dp110 CAAX, UAS-cryRNAi (Glioma CryRNAi) and (J) repo-Gal4 > UAS-cryRNAi (Glia CryRNAi) genotypes. Active zones are visualized by nc82 (anti-Brp) antibody and marked in Figure 2. Ectopic downregulation of cry prevents GB tumorigenesis and effects. ( A – D ) Confocal microscopy images of brains from 7-day-old adult flies with the following genotypes: ( A )repo-Gal4 > UAS-LacZ (Control), ( B )repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX (Glioma), ( C )repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX , UAS-cryRNAi (Glioma CryRNAi) and ( D )repo-Gal4 > UAS-cryRNAi (Glia CryRNAi) with glial nuclei marked in green with anti-repo (scale bar, 100 µ m). ( A’ – D’ ) Glial membrane is shown in red by the expression of mRFP. ( E ) Quantification of glial cells number and ( F ) quantification of glial membrane volume. Statistical analysis for at least n= 11 per genotype (ANOVA, post-hoc Bonferroni). ( G – J ) Confocal images of adult NMJ of 7-day-old flies from ( G ) repo-Gal4 > UAS-LacZ (Control) ( H )repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX (Glioma),( I )repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX , UAS-cryRNAi (Glioma CryRNAi) and ( J )repo-Gal4 >UAS-cryRNAi (Glia CryRNAi) genotypes. Active zones are visualized by nc82 (anti-Brp) antibody and marked in green (scale bar, 25 µ m). ( K ) Quantification and statistical analysis of active zones in at least n= 17 per genotype (ANOVA, post-hoc Bonferroni). ( L ) Graph shows a survival assay of repo-Gal4 > UAS-LacZ (Control, grey), repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX (Glioma, black), repo-Gal4 > UAS-dEGFR λ , UAS-cryRNAi, (Glioma CryRNAi, dark green) and repo-Gal4 > UAS-cryRNAi, (Glia CryRNAi, light green) flies and statistical analysis in n= 90 (Mantel-Cox test) (n.s. not significant, *** p-value < 0.001, **** p-value < 0.0001). Int. J. Mol. Sci. 2022,23, 2043 6 of 16 Next, we studied the impact of GB progression and cry expression in neighboring neurons. We counted the number of synapses in motor neurons of adult neuromuscular junction (NMJ), a standardized tissue to study neurodegeneration [ 9 , 11 , 33 ]. To visualize synapses, we used an anti-Brp antibody (nc82) to detect active zones in the neurons, and counted the number of synapses in control samples, GB, GB + cryRNAi and normal glia +cryRNAi (Figure 2G–J). The quantification of synapse number (Figure 2K) shows that GB induction provokes a significant reduction in the number of synapses as compared to control samples, compatible with a neurodegenerative process. This effect was previously described [ 7 , 9 , 11 ] as a consequence of GB progression. Moreover, cry knockdown in GB prevents the reduction in the number of synapses, and cryRNAi expression in normal glial cells does not cause any detectable change in the number of synapses. Finally, we aimed to determine the systemic effect of cry. We expressed cryRNAi in glia or GB cells, and we analyzed the life span of adult flies. The results show that GB causes a significant reduction of life span and a premature death, which is prevented by cryRNAi expression in GB cells. Moreover, cryRNAi expression in normal glial cells does not reduce lifespan but causes a significant increase in the average lifespan (Figure 2L). 2.3. Signaling Pathway to Control Cry Upregulation To decipher the specific signaling pathway responsible for cry transcriptional activation in GB cells, we analyzed the contribution of the two main pathways activated in this model of GB, EGFR and PI3K. Both pathways converge in the expression of the gene myc (see Figure 3A for detailed genetic epistasis in GB). Thus, we analyzed the contribution of PI3K, EGFR and myc to cry upregulation. We measured the fluorescent signal of GFP-cry reporter in control adult brains (Figure 3B–B”) and compared it with adult brains upon expression of the constitutively active forms of PI3K (Figure 3C–C”) or EGFR (Figure 3D–D”) in glial cells (under the control of repo-Gal4). In addition, we analyzed the GFP-cry signal in glial cells upon myc upregulation (Figure 3E–E”). We quantified in the confocal images the signal of GFP that co-localizes with glial membranes (mRFP) (Figure 3F). The results indicate that PI3K expression is sufficient to increase GFP-cry signal but not EGFR or myc overexpression. These results suggest that PI3K upregulation induces cry transcription, and EGFR or myc expression do not induce cry expression in glial cells. 2.4. Cry Regulates Myc Expression in Glial Cells Next, to determine the epistatic relation between cry and myc, we analyzed Myc protein accumulation in glial cells upon cry expression. First, to analyze if Cry is sufficient to cause an increase in Myc protein levels, we used a specific antibody against Myc and analyzed Myc signal levels upon cry overexpression, myc overexpression or cry + myc overexpression in glial cells (Figure 4A–D’). The quantification of Myc surface signal that coincides with glial cells (anti-repo) showed that cry expression in glia is sufficient to increase Myc protein signal in glial cells, comparable to myc upregulation. In addition, cry + myc upregulation show a summation effect on the increase of Myc protein levels (Figure 4E). To conclude if cry is required for myc expression in GB, we quantified glial Myc signal in the control, cry RNAi, GB, GB + cryRNAi and cry upregulation (Figure 4F–J’). The quantifications indicate that cryRNAi in glial cells does not reduce the amount of Myc in glial cells (Figure 4K). In addition, GB condition triggers the number of Myc positive glial cells, as well as cry upregulation in glial cells (Figure 4K). Finally, cryRNAi expression in GB cells prevents the accumulation of Myc in GB cells. Taking all these results together, we conclude that cry is sufficient to trigger Myc accumulation in glial cells, and cry expression is necessary for Myc accumulation in GB condition. Int. J. Mol. Sci. 2022,23, 2043 7 of 16 Int. J. Mol. Sci. 2022, 23, 2043 7 of 17 Figure 3. PI3K upregulates the levels of cry. (A) Scheme of EGFR (blue) and PI3K (yellow) signaling pathways involved in GB tumoral transformation with Myc as convergence point (green) (modified from [6]). (B–E) Confocal microscopy images of brains from 7-day-old adult flies with the following genotypes: (B) repo-Gal4 > UAS-LacZ (Control), (C) repo-Gal4 > UAS-dp110CAAX (PI3K), (C) repo-Gal4 > UAS-dEGFRλ (EGFR) and (D) repo-Gal4 > UAS-dmyc (Myc) using the reporter GFP-cry visualized in green (scale bar, 100 µm) and B’–E’) the glial membrane is marked in red by the expression of mRFP. Green and red signal merge produces the yellow signal. (F) Co-localization between GFP-cry and the glial membrane (mRFP) and statistical analysis in at least n = 16 (ANOVA, post-hoc Bonferroni) ( n.s. not significant, * p-value < 0.05, *** p-value < 0.001). 2.4. cry Regulates myc Expression in Glial Cells Next, to determine the epistatic relation between cry and myc, we analyzed Myc protein accumulation in glial cells upon cry expression. First, to analyze if Cry is sufficient to cause an increase in Myc protein levels, we used a specific antibody against Myc and analyzed Myc signal levels upon cry overexpression, myc overexpression or cry + myc overexpression in glial cells (Figure 4A–D’). The quantification of Myc surface signal that coincides with glial cells (anti-repo) showed that cry expression in glia is sufficient to increase Myc protein signal in glial cells, comparable to myc upregulation. In addition, cry + myc upregulation show a summation effect on the increase of Myc protein levels (Figure 4E). To conclude if cry is required for myc expression in GB, we quantified glial Myc signal in the control, cry RNAi, GB, GB + cryRNAi and cry upregulation (Figure 4F–J’). The quantifications indicate that cryRNAi in glial cells does not reduce the amount of Myc in glial cells (Figure 4K). In addition, GB condition triggers the number of Myc positive glial cells, Figure 3. PI3K upregulates the levels of cry. ( A ) Scheme of EGFR (blue) and PI3K (yellow) signaling pathways involved in GB tumoral transformation with Myc as convergence point (green) (modified from [ 6 ]). ( B – E ) Confocal microscopy images of brains from 7-day-old adult flies with the following genotypes: ( B )repo-Gal4 > UAS-LacZ (Control), ( C )repo-Gal4 > UAS-dp110 CAAX (PI3K), ( C )repo-Gal4 > UAS-dEGFR λ (EGFR) and ( D )repo-Gal4 > UAS-dmyc (Myc) using the reporter GFP-cry visualized in green (scale bar, 100 µ m) and B’ – E’ ) the glial membrane is marked in red by the expression of mRFP. Green and red signal merge produces the yellow signal. ( F ) Co-localization between GFP-cry and the glial membrane (mRFP) and statistical analysis in at least n= 16 (ANOVA, post-hoc Bonferroni) ( n.s. not significant, * p-value < 0.05, *** p-value < 0.001). 2.5. Cry Contribution to GB Progression To investigate the contribution of Cry to glioma progression, we determined the number of glial cells and volume of glial membrane network in control adult brain, GB (PI3K + EGFR), PI3K + cry,EGFR + cry or myc + cry expressed in glial cells (Figure 5A–E’). The quantification showed that all these genetic combinations cause an increase in the number of glial cells as compared to control brains (Figure 5F). However, only the GB condition provoked an expansion of the glial membrane volume, and the combination of PI3K + cry,EGFR + cry or myc + cry showed a volume of glial membrane comparable to control brains (Figure 5G). To further determine the contribution of cry to GB expansion, we analyzed the contribution of single gene upregulation in glial cells for cry or myc, and the combination of cry + myc expression (Figure 5H–K’). The quantification of glial cell number showed that cry or myc expression alone, or in combination, is sufficient to increase the number of glial cells with respect to control samples (Figure 5L). Nevertheless, none of these genetic modifications is sufficient to expand glial membrane volume (Figure 5M). Int. J. Mol. Sci. 2022,23, 2043 8 of 16 These results suggest that cry or myc are sufficient to trigger glial cell number increase in adult brains, but not to expand the volume of glial membrane network. Int. J. Mol. Sci. 2022, 23, 2043 8 of 17 as well as cry upregulation in glial cells (Figure 4K). Finally, cryRNAi expression in GB cells prevents the accumulation of Myc in GB cells. Taking all these results together, we conclude that cry is sufficient to trigger Myc accumulation in glial cells, and cry expression is necessary for Myc accumulation in GB condition. Figure 4. Cry increases glial Myc protein levels in physiological and GB conditions. (A–D) Confocal microscopy images of brains from 7-day-old adult flies with the following genotypes: (A) repo-Gal4 > UAS-LacZ (Control), (B) repo-Gal4 > UAS-cry (Cry), (C) repo-Gal4 > UAS-dmyc (Myc) and (D) repoGal4 > UAS-dmyc, UAS-cry (MycCry) with Myc marked in magenta (anti-Myc). (A’–D’) Glial nuclei marked in green (anti-Repo) (scale bar, 25 µm). (E) Glial Myc quantification and statistical analysis for at least n = 9 per genotype (ANOVA, post-hoc Bonferroni). (F–J) Confocal microscopy images of brains 7-day-old adult flies from (F) repo-Gal4 > UAS-LacZ (Control), (G) repo-Gal4 > UAS-cryRNAi (CryRNAi), (H) repo-Gal4 > UAS-dEGFRλ, UAS-dp110 CAAX, UAS-cryRNAi (Glioma CryRNAi), (I) repo-Gal4 > UAS-dEGFRλ, UAS-dp110 CAAX (Glioma) and (J) repo-Gal4 > UAS-cry (Cry); Myc is marked in magenta (anti-Myc) (F’–J’) and glial nuclei are marked in green (anti-Repo) (scale bar, 25 µm). (K) Glial Myc quantification and statistical analysis for at least n = 12 per genotype (ANOVA, post-hoc Bonferroni) ( n.s. not significant, ** p-value < 0.01, *** p-value <0.001). 2.5. cry Contribution to GB Progression To investigate the contribution of Cry to glioma progression, we determined the number of glial cells and volume of glial membrane network in control adult brain, GB (PI3K + EGFR), PI3K + cry, EGFR + cry or myc + cry expressed in glial cells (Figure 5A-E’). The quantification showed that all these genetic combinations cause an increase in the number of glial cells as compared to control brains (Figure 5F). However, only the GB condition provoked an expansion of the glial membrane volume, and the combination of PI3K + cry, EGFR + cry or myc + cry showed a volume of glial membrane comparable to control brains (Figure 5G). To further determine the contribution of cry to GB expansion, we analyzed the contribution of single gene upregulation in glial cells for cry or myc, and the combination of cry + myc expression (Figure 5H–K’). The quantification of glial cell number showed that cry or myc expression alone, or in combination, is sufficient to increase the number of glial cells with respect to control samples (Figure 5L). Nevertheless, none of these genetic modifications is sufficient to expand glial membrane volume (Figure 5M). These results suggest that cry or myc are sufficient to trigger glial cell number increase in adult brains, but not to expand the volume of glial membrane network. Figure 4. Cry increases glial Myc protein levels in physiological and GB conditions. ( A – D ) Confocal microscopy images of brains from 7-day-old adult flies with the following genotypes: ( A )repo-Gal4 > UAS-LacZ (Control), ( B )repo-Gal4 > UAS-cry (Cry), ( C )repo-Gal4 > UAS-dmyc (Myc) and ( D )repoGal4 > UAS-dmyc, UAS-cry (MycCry) with Myc marked in magenta (anti-Myc). ( A’ – D’ ) Glial nuclei marked in green (anti-Repo) (scale bar, 25 µ m). ( E ) Glial Myc quantification and statistical analysis for at least n= 9 per genotype (ANOVA, post-hoc Bonferroni). ( F – J ) Confocal microscopy images of brains 7-day-old adult flies from ( F )repo-Gal4 > UAS-LacZ (Control), ( G )repo-Gal4 > UAS-cryRNAi (CryRNAi), ( H )repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX , UAS-cryRNAi (Glioma CryRNAi), ( I ) repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX (Glioma) and ( J )repo-Gal4 > UAS-cry (Cry); Myc is marked in magenta (anti-Myc) ( F’ – J’ ) and glial nuclei are marked in green (anti-Repo) (scale bar, 25 µ m). ( K ) Glial Myc quantification and statistical analysis for at least n= 12 per genotype (ANOVA, post-hoc Bonferroni) ( n.s. not significant, ** p-value < 0.01, *** p-value <0.001). 2.6. Cry Upregulation in Glial Cells Causes Synapse Loss and Premature Death It was previously described that GB progression induces synapse loss, an early symptom of neurodegeneration. To determine the contribution of cry to synapse loss, we counted the number of active zones in motor neurons of adult neuromuscular junction in the control, GB (PI3K + EGFR), PI3K + cry,EGFR + cry or myc + cry samples (Figure 5N–R). The quantification of the number of active zones showed that the expression in glial cells of GB (PI3K + EGFR), PI3K + cry,EGFR + cry or myc + cry is sufficient to reduce the number of synapses in NMJ neurons (Figure 5S). Finally, to evaluate the systemic effect of GB and glial expression of PI3K + cry,EGFR + cry or my c + cry, we analyzed the lifespan of adult individuals. The results show that GB causes a premature death, as previously described in Drosophila and mice Xenografts [ 8 , 9 , 11 ], glial upregulation of EGFR + cry or myc + cry causes a significant reduction of lifespan but less aggressive than GB, and PI3K + cry upregulation in glial cells does not reduce lifespan (Figure 5T). Int. J. Mol. Sci. 2022,23, 2043 9 of 16 Int. J. Mol. Sci. 2022, 23, 2043 9 of 17 Figure 5. EGFR-cry co-expression induces glial cells number increase, synapse number and survival reduction. (A–E) Confocal microscopy images of brains from 7-day-old adult flies with the following genotypes: (A) repo-Gal4 > UAS-LacZ (Control), (B) repo-Gal4 > UAS-dEGFRλ, UAS-dp110 CAAX (Glioma), (C) repo-Gal4 > UAS-dp110 CAAX, UAS-cry (PI3KCry), (D) repo-Gal4 > UAS-dEGFRλ, UAS-cry (EGFRCry) and (E) repo-Gal4 > UAS-dmyc, UAS-cry (MycCry) with glial nuclei marked in green (antiRepo) (scale bar, 100 µm). (A’–E’) Glial membrane is visualized in red by the expression. (F) Glial cells number and (G) glial membrane volume quantification and statistical analysis for at least n = 12 per genotype (ANOVA, post-hoc Bonferroni). (H–K) Confocal images of adult brains of 7-dayold flies from (H) repo-Gal4 > UAS-LacZ (Control) (I) repo-Gal4 > UAS-cry (Cry), (J) repo-Gal4 > UASdmyc (Myc) and (K) repo-Gal4 > UAS-cry, UAS-dmyc (MycCry) genotypes with glial nuclei marked in green (anti-Repo) (scale bar, 100 µm) (H’–K’) and glial membrane shown in red (mRFP). (L) Glial cells number and (M) glial membrane volume quantification and statistical analysis for at least n = 9 per genotype (ANOVA, post-hoc Bonferroni). (N–R) Confocal microscopy images of NMJ of 7Figure 5. EGFR-cry co-expression induces glial cells number increase, synapse number and survival reduction. ( A – E ) Confocal microscopy images of brains from 7-day-old adult flies with the following genotypes: ( A )repo-Gal4 > UAS-LacZ (Control), ( B )repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX (Glioma), ( C )repo-Gal4 >UAS-dp110 CAAX , UAS-cry (PI3KCry), ( D )repo-Gal4 > UAS-dEGFR λ , UAS-cry (EGFRCry) and ( E )repo-Gal4 > UAS-dmyc, UAS-cry (MycCry)with glial nucleimarked in green (anti-Repo) (scale bar, 100 µ m). ( A’ – E’ ) Glial membrane is visualized in red by the expression. ( F ) Glial cells number and ( G ) glial membrane volume quantification and statistical analysis for at least n= 12 per genotype (ANOVA, post-hoc Bonferroni). ( H – K )Confocal imagesof adultbrainsof 7-day-old flies from ( H )repo-Gal4 > UAS-LacZ (Control) ( I )repo-Gal4 > UAS-cry (Cry),( J )repo-Gal4 > UAS-dmyc (Myc) and ( K )repo-Gal4 >UAS-cry, UAS-dmyc (MycCry) genotypes with glial nuclei marked in green (anti-Repo) (scale bar, 100 µ m) ( H’ – K’ ) and glial membrane shown in red (mRFP). ( L ) Glial cells number and ( M ) glial membrane volume quantification and statistical analysis for at least n= 9 per genotype (ANOVA, post-hoc Bonferroni). ( N – R ) Confocal microscopy images of NMJ of 7-day-old adult flies from ( N )repo-Gal4 > UAS-LacZ (Control), ( O )repo-Gal4 > UAS-dEGFR λ , UAS-dp110 CAAX (Glioma), ( P )repo-Gal4 > UAS-dp110 CAAX , UAS-cry (PI3KCry), ( Q )repo-Gal4 > UAS-dEGFR λ , UAS-cry (EGFRCry) and ( R )repo-Gal4 > UAS-dmyc, UAS-cry (MycCry). Active zones are marked with anti-Brp (nc-82) visualized in green (nc82, anti-Brp) (scale bar, 25 µ m). ( S ) Quantification and statistical analysis of active zones in at least n= 13 per genotype (ANOVA, post-hoc Bonferroni). 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