The invasion of tumor cells into brain tissue is a pathologic hallmark of malignant gliomas and contributes to treatment failures. Diffuse glioblastomas contain numerous microglial cells, which enhance the progression of gliomas; however, factors responsible for invasionpromoting role of microglia are unknown. Transforming growth factor-b (TGF-b) can enhance tumor growth, invasion, angiogenesis and immunosuppression. Antagonizing TGF-b activity has been shown to inhibit tumor invasion in vitro and tumorigenicity, but a systemic inhibition or lack of TGF-b signaling results in acute inflammation and disruption of immune system homeostasis. We developed plasmid-transcribed small hairpin RNAs (shRNAs) to downregulate the TGF-b type II receptor (TbIIR) expression, which effectively inhibited cytokine-induced signaling pathways and transcriptional responses in transiently transfected human glioblastoma cells. Silencing of TbIIR abolished TGF-b-induced glioblastoma invasiveness and migratory responses in vitro. Moreover, tumorigenicity of glioblastoma cells stably expressing TbIIR shRNAs in nude mice was reduced by 50%. Microglia strongly enhanced glioma invasiveness in the co-culture system, but this invasion-promoting activity was lost in glioma cells stably expressing shTbRII, indicating a crucial role of microglia-derived TGF-b in tumor-host interactions. Our results demonstrate a successful targeting of TGF-b-dependent invasiveness and tumorigenicity of glioblastoma cells by RNAi-mediated gene silencing.

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Development of the nervous system is carried out by complex gene expression programs that are regulated at both transcriptional and translational level. In addition, quality control mechanisms such as the TP53-mediated apoptosis or neuronal activity-stimulated survival ensure successful neurogenesis and formation of functional circuitries. In the nucleolus, production of ribosomes is essential for protein synthesis. In addition, it participates in chromatin organization and regulates the TP53 pathway via the ribosomal stress response. Its tight regulation is required for maintenance of genomic integrity. Mutations in several ribosomal components and trans-acting ribosomal biogenesis factors result in neurodevelopmental syndromes that present with microcephaly, autism, intellectual deficits and/or progressive neurodegeneration. Furthermore, ribosomal biogenesis is perturbed by exogenous factors that disrupt neurodevelopment including alcohol or Zika virus. In this review, we present recent literature that argues for a role of dysregulated ribosomal biogenesis in pathogenesis of various neurodevelopmental syndromes. We also discuss potential mechanisms through which such dysregulation may lead to cellular pathologies of the developing nervous system including insufficient proliferation and/or loss of neuroprogenitors cells, apoptosis of immature neurons, altered neuronal morphogenesis, and neurodegeneration.

The nucleolus serves as a principal site of ribosome biogenesis but is also implicated in various non-ribosomal functions, including negative regulation of the pro-apoptotic transcription factor p53. Although disruption of the nucleolus may trigger the p53-dependent neuronal death, neurotoxic consequences of a selective impairment of ribosome production are unclear. Here, we report that in rat forebrain neuronal maturation is associated with a remarkable expansion of ribosomes despite postnatal down-regulation of ribosomal biogenesis. In cultured rat hippocampal neurons, inhibition of the latter process by knockdowns of ribosomal proteins S6, S14, or L4 reduced ribosome content without disrupting nucleolar integrity, cell survival, and signaling responses to the neurotrophin brain-derived neurotrophic factor. Moreover, reduced general protein synthesis and/or formation of RNA stress granules suggested diminished ribosome recruitment to at least some mRNAs. Such a translational insufficiency was accompanied by impairment of brain-derived neurotrophic factor-mediated dendritic growth. Finally, RNA stress granules and smaller dendritic trees were also observed when ribosomal proteins were depleted from neurons with established dendrites. Thus, a robust ribosomal apparatus is required to carry out protein synthesis that supports dendritic growth and maintenance. Consequently, deficits of ribosomal biogenesis may disturb neurodevelopment by reducing neuronal connectivity. Finally, as stress granule formation and dendritic loss occur early in neurodegenerative diseases, disrupted homeostasis of ribosomes may initiate and/or amplify neurodegeneration-associated disconnection of neuronal circuitries.The ribosome is the principal component of the protein synthesis machinery. Most steps of ribosomal biogenesis occur in the nucleolus starting with the RNA-polymerase-1 (pol 1) 2 -mediated transcription of rRNA genes (1). In addition, pol 1 activity is required for the maintenance of the nucleolus. Therefore, inhibition of pol 1 not only blocks ribosomal biogenesis but also perturbs non-ribosomal functions of the nucleolus, including negative regulation of the pro-apoptotic transcription factor p53 (2). Although pol 1 initiates ribosome production, many additional steps are required for that process, including rRNA processing and binding to ribosomal proteins (RPs) (3). Consequently, depletion of various RPs, including S6 or S14, disrupts ribosomal biogenesis without interfering with nucleolar integrity (4). Impaired ribosome production and/or ribosomal deficits are documented in various neurodegenerative diseases, including Alzheimer, Huntington, Parkinson, amyotrophic lateral sclerosis (ALS), and fronto-temporal lobe dementia (FTLD) (5-13). Reduced ribosomal biogenesis may also contribute to neurodevelopmental disorders. Thus, microcephaly and neurodevelopmental delays are present in Bowen-Conradi syndrome that is caused by deficiency of the ribosomal biogenesis factor EMG1 or in patients with a newly described RPL10 ribos...

We report here that in rat and human neuroprogenitor cells as well as rat embryonic cortical neurons Zika virus (ZIKV) infection leads to ribosomal stress that is characterized by structural disruption of the nucleolus. The anti-nucleolar effects were most pronounced in postmitotic neurons. Moreover, in the latter system, nucleolar presence of ZIKV capsid protein (ZIKV-C) was associated with ribosomal stress and apoptosis. Deletion of 22 C-terminal residues of ZIKV-C prevented nucleolar localization, ribosomal stress and apoptosis. Consistent with a casual relationship between ZIKV-C-induced ribosomal stress and apoptosis, ZIKV-C-overexpressing neurons were protected by loss-of-function manipulations targeting the ribosomal stress effector Tp53 or knockdown of the ribosomal stress mediator RPL11. Finally, capsid protein of Dengue virus, but not West Nile virus, induced ribosomal stress and apoptosis. Thus, anti-nucleolar and pro-apoptotic effects of protein C are flavivirus-species specific. In the case of ZIKV, capsid protein-mediated ribosomal stress may contribute to neuronal death, neurodevelopmental disruption and microcephaly.

The transcription factor BMAL1/ARNTL is a non-redundant component of the clock pathway that regulates circadian oscillations of gene expression. Loss of BMAL1 perturbs organismal homeostasis and usually exacerbates pathological responses to many types of insults by enhancing oxidative stress and inflammation. Surprisingly, we observed improved locomotor recovery and spinal cord white matter sparing in Bmal1 −/− mice after T9 contusive spinal cord injury (SCI). While acute loss of neurons and oligodendrocytes was unaffected, Bmal1 deficiency reduced the chronic loss of oligodendrocytes at the injury epicenter 6 weeks post SCI. At 3 days post-injury (dpi), decreased expression of genes associated with cell proliferation, neuroinflammation and disruption of the blood spinal cord barrier (BSCB) was also observed. Moreover, intraspinal extravasation of fibrinogen and immunoglobulins was decreased acutely at dpi 1 and subacutely at dpi 7. Subacute decrease of hemoglobin deposition was also observed. Finally, subacutely reduced levels of the leukocyte marker CD45 and even greater reduction of the pro-inflammatory macrophage receptor CD36 suggest not only lower numbers of those cells but also their reduced inflammatory potential. These data indicate that Bmal1 deficiency improves SCI outcome, in part by reducing BSCB disruption and hemorrhage decreasing cytotoxic neuroinflammation and attenuating the chronic loss of oligodendrocytes. Contusive spinal cord injury (SCI), which represents most clinical SCI cases, involves the primary injury and a secondary injury cascade that progresses hours to months post-SCI 1. Secondary injury is mediated by multiple pathophysiological mechanisms, exacerbating the injury and leading to greater functional loss 1. In thoracic contusive SCI, functional deficits are driven by white matter (WM) damage 2. Acute loss of axons and oligodendrocytes (OLs), neuroinflammation, protracted loss of OLs and insufficient myelin repair are major, functionally meaningful components of the SCI-associated WM damage 3. Oxidative stress is a major contributor to WM injury by driving neurotoxic neuroinflammation and directly causing OL death 4,5. SCI-activated inflammatory cells including microglia/macrophages are a major source of reactive oxygen species (ROS) and pro-oxidant cytokines that kill OLs 3,4,6. At least in rodents, lost OLs are robustly replaced by OL precursor cell

S100A6 (calcyclin) is a calcium binding protein with two EF-hand structures expressed mostly in fibroblasts and epithelial cells. We have established a NIH 3T3 fibroblast cell line stably transfected with siRNA against S100A6 to examine the effect of S100A6 deficiency on non-transformed cell physiology. We found that NIH 3T3 fibroblasts with decreased level of S100A6 manifested altered cell morphology and proliferated at a much slower pace than the control cells. Cell cycle analysis showed that a large population of these cells lost the ability to respond to serum and persisted in the G0/G1 phase. Furthermore, fibroblasts with diminished S100A6 level exhibited morphological changes and biochemical features of cellular senescence as revealed by beta-galactosidase and gelatinase assays. Also, S100A6 deficiency induced changes in the actin cytoskeleton and had a profound impact on cell adhesion and migration. Thus, we have shown that the S100A6 protein is involved in multiple aspects of fibroblast physiology and that its presence ensures normal fibroblast proliferation and function.