Emerging Roles of Primary Cilia in Glioma

Primary cilia are microtubule-based organelles that are typically present on cells during the G0 or G1-S/G2 phases of the cell cycle. Recent studies of glioblastoma (GBM) biopsies, a brain tumor that is notorious for its aggressive growth and resistance to treatment, show that many cells in the tumor lack cilia. At this point, it remains unclear whether primary cilia promote or suppress glioma tumorigenesis. In this review, we will discuss the different roles that have been proposed for primary cilia in glioma and how cilia may contribute to the resistance of these tumors to current therapies

Infiltrative and drug-resistant slow-cycling cells support metabolic heterogeneity in glioblastoma

Metabolic reprogramming has been described in rapidly growing tumors, which are thought to mostly contain fast‐cycling cells (FCCs) that have impaired mitochondrial function and rely on aerobic glycolysis. Here, we characterize the metabolic landscape of glioblastoma (GBM) and explore metabolic specificities as targetable vulnerabilities. Our studies highlight the metabolic heterogeneity in GBM, in which FCCs harness aerobic glycolysis, and slow‐cycling cells (SCCs) preferentially utilize mitochondrial oxidative phosphorylation for their functions. SCCs display enhanced invasion and chemoresistance, suggesting their important role in tumor recurrence. SCCs also demonstrate increased lipid contents that are specifically metabolized under glucose‐deprived conditions. Fatty acid transport in SCCs is targetable by pharmacological inhibition or genomic deletion of FABP7, both of which sensitize SCCs to metabolic stress. Furthermore, FABP7 inhibition, whether alone or in combination with glycolysis inhibition, leads to overall increased survival. Our studies reveal the existence of GBM cell subpopulations with distinct metabolic requirements and suggest that FABP7 is central to lipid metabolism in SCCs and that targeting FABP7‐related metabolic pathways is a viable therapeutic strategy

Neuronal sensitivity to TDP-43 overexpression is dependent on timing of induction

Ubiquitin-immunoreactive neuronal inclusions composed of TAR DNA binding protein of 43 kDa (TDP-43) are a major pathological feature of frontotemporal lobar degeneration (FTLD-TDP). In vivo studies with TDP-43 knockout mice have suggested that TDP-43 plays a critical, although undefined role in development. In the current report, we generated transgenic mice that conditionally express wild-type human TDP-43 (hTDP-43) in the forebrain and established a paradigm to examine the sensitivity of neurons to TDP-43 overexpression at different developmental stages. Continuous TDP-43 expression during early neuronal development produced a complex phenotype, including aggregation of phospho-TDP-43, increased ubiquitin immunoreactivity, mitochondrial abnormalities, neurodegeneration and early lethality. In contrast, later induction of hTDP-43 in the forebrain of weaned mice prevented early death and mitochondrial abnormalities while yielding salient features of FTLD-TDP, including progressive neurodegeneration and ubiquitinated, phospho-TDP-43 neuronal cytoplasmic inclusions. These results suggest that neurons in the developing forebrain are extremely sensitive to TDP-43 overexpression and that timing of TDP-43 overexpression in transgenic mice must be considered when distinguishing normal roles of TDP-43, particularly as they relate to development, from its pathogenic role in FTLD-TDP and other TDP-43 proteinopathies. Finally, our adult induction of hTDP-43 strategy provides a mouse model that develops critical pathological features that are directly relevant for human TDP-43 proteinopathies

Abnormal levels of Gadd45alpha in developing neocortex impair neurite outgrowth.

To better understand the short and long-term effects of stress on the developing cerebral cortex, it is necessary to understand how early stress response genes protect or permanently alter cells. One family of highly conserved, stress response genes is the growth arrest and DNA damage-45 (Gadd45) genes. The expression of these genes is induced by a host of genotoxic, drug, and environmental stressors. Here we examined the impact of altering the expression of Gadd45alpha (Gadd45a), a member of the Gadd45 protein family that is expressed throughout the developing cortices of mice and humans. To manipulate levels of Gadd45a protein in developing mouse cortex, we electroporated cDNA plasmids encoding either Gadd45a or Gadd45a shRNA to either overexpress or knockdown Gadd45a levels in the developing cortices of mice, respectively. The effects of these manipulations were assessed by examining the fates and morphologies of the labeled neurons. Gadd45a overexpression both in vitro and in vivo significantly impaired the morphology of neurons, decreasing neurite complexity, inducing soma hypertrophy and increasing cell death. Knockdown of Gadd45a partially inhibited neuronal migration and reduced neurite complexity, an effect that was reversed in the presence of an shRNA-resistant Gadd45a. Finally, we found that shRNA against MEKK4, a direct target of Gadd45a, also stunted neurite outgrowth. Our findings suggest that the expression of Gadd45a in normal, developing brain is tightly regulated and that treatments or environmental stimuli that alter its expression could produce significant changes in neuronal circuitry development

Gadd45a expression is critical for development of normal neuron morphology in neocortex.

<p>E15.5 mouse cerebral cortex was electroporated with constructs designed to reduce or increase Gadd45a levels in electroporated cells. Each construct was co-electroporated with vectors encoding red (RFP) or green (GFP) fluorescent reporter protein. RFP or GFP-positive neurons were analyzed at P14 or P10 respectively (n = 4–6 brains/group from two separate litters). (<b>A and B</b>) Although most Gadd45a shRNA-treated neurons (<b>B</b>) reached the upper layers of neocortex, there were significantly more cells distributed beneath upper layers compared to RFP alone (<b>A</b>) (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044207#pone.0044207.s008" target="_blank">Fig. S8</a>). Higher magnification of control neurons (<b>A’ and A”</b>) and Gadd45a shRNA-treated (<b>B’ and B”</b>) reveals that the dendritic processes of neurons expressing Gadd45a shRNA are less arborized than those of neurons expressing RFP alone. Arrows indicate somas of RFP positive neurons. (<b>C</b>) Migration of neurons to the upper layers (2/3) of neocortex is unaffected by overexpression of Gadd45a. Left panels show neurons in control brains. Right panels show neurons overexpressing Gadd45a. (<b>C’</b>) Higher magnification images show examples of Gadd45a-AU1 neurons (right panels) with hypertrophied, multipolar shaped somas compared to control neurons (left panels). Scale bars = 50 µm (<b>A, A”</b>).</p

Knockdown of MEKK4 reduces neurite complexity.

<p>E15.5 mice were electroporated with cDNA encoding GFP (control) or GFP plus MEKK4 shRNA (MEKK4 knockdown). At E16.5, GFP-positive regions of cortex were isolated, cultured 6DIV, fixed, imaged and analyzed. (<b>A</b>) Example images of neurons (control and MEKK4 knockdown) converted for Sholl analyses. (<b>B</b>) Sholl analyses of the cells in (A) reveals that neurites produced by MEKK4 shRNA-transfected cells are less complex than control neurons. n = number of cells analyzed.</p

Gadd45a regulates neurite outgrowth from cortical neurons in vitro.

<p>(<b>A</b>) Method used to transfect, culture and analyze transfected cortical neurons. E15.5 mice were electroporated with cDNA encoding GFP and different Gadd45a constructs. At E16.5, GFP-positive regions of cortex were isolated, cultured 6DIV, fixed, imaged and analyzed. Sholl analyses were performed by centering concentric rings with increasing radii of 10 um over the soma center and counting the number of times the dendritic processes intersected the rings. (<b>B-D</b>) Images of GFP-positive cells converted for Sholl analyses. Examples of control (pLKO +GFP) (<b>B</b>), Gadd45a knockdown (Gadd45a shRNA + GFP) (<b>C</b>), and Gadd45a overexpressing (Gadd45a -AU1+GFP) (<b>D</b>) neurons. Scale bar = 20 µm. (<b>C and E</b>) Gadd45a knockdown results in significantly fewer neurites. (<b>D and F</b>) Gadd45a overexpression results in hypertrophied somas, and significantly fewer distal processes (∼70 µm –200 um from the soma). The significant difference within the first 10 µm is likely due to the increase in soma size (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044207#pone.0044207.s006" target="_blank">Fig. S6</a>). Note: Gadd45a overexpression (blue) was compared to two different controls, GFP (green) and pLKO (black, same data presented in (E)). (<b>E</b>) Electroporation of a Gadd45a-shRNA resistant construct (R- Gadd45a (blue line)) is able to significantly restore neuronal morphology compared to Gadd45a shRNA treated neurons. (<b>E</b> and <b>F</b>) Lines with asterisks indicate ranges where data was averaged for posthoc analysis (bar graphs). Sholl data was analyzed using two-way ANOVA with repeated measures while the averaged data was analyzed by Fisher’s PLSD post-hoc test. ns = not significant.</p

PCM1 Depletion Inhibits Glioblastoma Cell Ciliogenesis and Increases Cell Death and Sensitivity to Temozolomide

A better understanding of the molecules implicated in the growth and survival of glioblastoma (GBM) cells and their response to temozolomide (TMZ), the standard-of-care chemotherapeutic agent, is necessary for the development of new therapies that would improve the outcome of current GBM treatments. In this study, we characterize the role of pericentriolar material 1 (PCM1), a component of centriolar satellites surrounding centrosomes, in GBM cell proliferation and sensitivity to genotoxic agents such as TMZ. We show that PCM1 is expressed around centrioles and ciliary basal bodies in patient GBM biopsies and derived cell lines and that its localization is dynamic throughout the cell cycle. To test whether PCM1 mediates GBM cell proliferation and/or response to TMZ, we used CRISPR/Cas9 genome editing to generate primary GBM cell lines depleted of PCM1. These PCM1-depleted cells displayed reduced AZI1 satellite protein localization and significantly decreased proliferation, which was attributable to increased apoptotic cell death. Furthermore, PCM1-depleted lines were more sensitive to TMZ toxicity than control lines. The increase in TMZ sensitivity may be partly due to the reduced ability of PCM1-depleted cells to form primary cilia, as depletion of KIF3A also ablated GBM cells' ciliogenesis and increased their sensitivity to TMZ while preserving PCM1 localization. In addition, the co-depletion of KIF3A and PCM1 did not have any additive effect on TMZ sensitivity. Together, our data suggest that PCM1 plays multiple roles in GBM pathogenesis and that associated pathways could be targeted to augment current or future anti-GBM therapies