The present work identifies AEG-1 as a novel modulator of astrocyte injury responses by regulating astrocyte migration and proliferation. Our in vivo brain injury mouse model and in vitro cell migration assays provides evidence for the induction of AEG-1 expression during gliosis in injured astrocytes and a subsequent alteration in subcellular distribution consequent to injury. We also report the nucleolar localization of AEG-1 during astrocyte wound healing, which provides direction for future mechanistic studies.
Reactive astrogliosis, a prerequisite for CNS wound healing, orchestrates events that lead to glial scar formation, which is critical for maintaining CNS homeostasis
. While AEG-1 was first described as an HIV-1-inducible transcript in astrocytes
, this is the first report that describes a critical role of AEG-1 in reactive astrogliosis, the first line of defense for any CNS injury
. The in vivo brain injury mouse model provides evidence for the induction of AEG-1 expression during gliosis, however, AEG-1 levels in injured astrocytes remained unchanged in the in vitro wound-healing model. This distinction may be due to the difference in the complexities of the two models. In the intact CNS brain injury model there are elevated levels of AEG-1-inducing agents, such as TNFα and interleukin 1β (IL-1β), secreted by other non-neural cells such as activated microglia
, in contrast to the confluent layer of pure astrocyte cultures of the in vitro wound-healing model. AEG-1 induction at the wound site in the in vivo brain injury mouse model colocalized with reactive astrocytes, indicating a possible involvement of AEG-1 in the injury responses of astrocytes.
During gliosis, astrocytes undergo multitude of changes in their gene expression patterns, secretion profiles and morphological traits that lead to a reactive phenotype capable of mediating wound healing
[17, 38, 39]. AEG-1 induction following injury can alter many intracellular signaling pathways, such as nuclear factor (NF)κB
[8, 10], Wnt
, cyclic adenosine monophosphate
[5, 41], mitogen activated protein kinases (MAPK)
 and phosphatidylinositol 3-kinases-AKT
. It is interesting to note that AEG-1 expression in astrogliomas has been shown to induce CXCL8 expression
 and matrix metalloproteinase-9 production
, both of which show a dramatic increase during gliosis
[43, 44]. Reactive astrogliosis is often associated with deregulated glutamate clearance, which is responsible for toxicity to neurons
[45, 46]. Interestingly, AEG-1 has been shown to suppress the expression of excitatory amino acid transporter-2 in astrogliomas
[47–49]. Besides the altered secretory profiles of reactive astrocytes, structural changes such as elevated expression of GFAP, vimentin and nestin are observed during reactive astrogliosis
. These can also be modulated by AEG-1, by functioning as a coactivator for signaling molecules, such as NFκB and MAPK. AEG-1 has been reported to physically interact with NFκB p65 subunit and activate NFκB responsive genes, most of which are overexpressed during gliosis
Recruitment of astrocytes to the site of injury is an important primary step for initiation of reactive astrogliosis
. Here, we report for the first time AEG-1-mediated regulation of human astrocyte migration. As a metastasis adhesion protein, increased cytoplasmic AEG-1 expression has been shown to promote augmented migration, invasion and metastasis of cancer cells such as neuroblastoma and malignant gliomas, facilitating anchorage-independent growth and survival in the secondary site
[1, 52]. The in vitro wound-healing assay utilized in this study allows characterization of the innate injury-induced subcellular changes in AEG-1 localization excluding the confounding factors present in an intact CNS. Here, we report higher cytoplasmic AEG-1 levels and significantly reduced nuclear levels following injury, which is similar to AEG-1 localization in highly invasive cancer cells
. While AEG-1 has been shown to mediate metastasis as an adhesion protein, the interacting counterpart on other cells has yet to be identified and further mechanistic studies are lacking
. However, AEG-1 was previously reported to induce matrix metalloproteinase production in glioma cells, thereby facilitating glioma invasion
. A study in breast cancer revealed that AEG-1 promotes epithelial-mesenchymal transition and enhanced migration by upregulating mesenchymal markers, downregulating epithelial markers, and inducing nuclear accumulation of NFκB
[5, 54]. It has been reported that ectopic expression of AEG-1 could augment anchorage-independent growth of non-tumorigenic melanocytes and immortalized astrocytes
, whereas AEG-1 knockdown reduced cell viability and promoted apoptosis in prostate cancer cells
Our study demonstrated reduced astrocyte migration following AEG-1 knockdown. The decrease in migratory capacities was sustained over 5 days indicating that AEG-1 may also have influenced astrocyte proliferation. Astrocytic hyperproliferation is yet another hallmark of reactive astrogliosis
. AEG-1 knockdown also reduced astrocyte proliferation as assayed by change in Ki67 and PCNA expression. Nuclear protein Ki67 is expressed in all cell cycle phases except G0, whereas nuclear protein PCNA levels are highest during the G1/S phase of the cell cycle
. Therefore, the decrease in both Ki67 and PCNA positive astrocytes following AEG-1 knockdown reported here implies G1/S phase cell cycle arrest following AEG-1 knockdown, indicating a plausible role of AEG-1 during these cell cycle phases, and further studies are warranted. It remains to be seen whether overexpression of AEG-1 will enhance the ability of astrocytes to participate in gliosis.
Although, a number of AEG-1-interacting proteins have been identified
[10, 11, 13], a complete understanding of the biological functions and biochemical characteristics, particularly the molecular triggers for differential sub-cellular localization and its subsequent effects on cellular mechanisms remains elusive to date. AEG-1 localization to the nucleolus has been reported in a few isolated cancer types, but is most frequently detected either in the nucleoplasm or cytoplasm of many metastatic tumors
[9, 57]. Here, we report injury-induced colocalization of AEG-1 with nucleolar protein fibrillarin, a component of the small nuclear ribonucleoprotein complex that is required for processing of the pre-rRNA molecules
[58, 59]. This suggests a novel role of AEG-1 in pre-rRNA processing and assembly. Aberrant ribosome biogenesis leads to p53-dependent G1 arrest, and therefore is crucial for cell survival and proliferation
[60–62]. Recently, cytoplasmic AEG-1 has been identified as a RNA-binding protein, which provides survival advantage to cancer cells under conditions of stress by blocking Rad51 nuclear accumulation
[12, 63]. Furthermore, AEG-1 has been shown to physically interact with Staphylococcal nuclease domain-containing protein 1, a component of the RNA-induced silencing complex assembly, and to regulate microRNA processing and function in hepatocellular carcinoma
. However, proteomic studies failed to identify a DNA-binding domain on the AEG-1 protein. Together these findings imply a potential role as a scaffolding protein of the small nuclear ribonucleoprotein complex mediating either transcription and/or processing of the pre-rRNA molecules. The role of AEG-1 in the nucleolus has not been investigated to date in either cancer or non-cancerous cells. Therefore, the current finding of AEG-1 colocalization with fibrillarin provides a physiological basis for future studies. Additional studies into the physical interaction between AEG-1 and nucleolar-processing complex components and mechanism of participation in pre-rRNA processing are necessary to determine the role of AEG-1 in astrocytic wound-healing responses.
Restricted HIV-1 infection of astrocytes contributes to HIV-1-associated neuropathies via multiple mechanisms, including reactive astrogliosis and glutamate excitotoxicity
. In conjunction to the previous findings on AEG-1, as a mediator of glutamate excitotoxicity, this study identifies yet another mechanism of AEG-1-mediated regulation of HIV-1-associated neuropathies, via regulation of reactive astrogliosis. In addition, we have previously reported that disruption of astroglial-neuronal interactions and secretion of neurotoxic cytokines and chemokines during astrogliosis can also contribute towards neuronal cell atrophy
. Yet another important molecular change during reactive astrogliosis is the increase production of reactive oxygen species such as nitric oxide, nitric oxide synthase, super oxide dismutase and glotathione
[61–63]. Interestingly, previous studies on AEG-1 in glioma have revealed AEG-1-mediated protection against glocuse deprivation-induced cytotoxicity
[49, 64]. Also, AEG-1 increased the chemotherapeutic resistance of breast cancer cells by increasing the expression ot multidrug resistance gene 1, thereby increasing the efflux of toxic waste from the cells
[53, 65, 66]. Hence, further studies on AEG-1-induced alterations in the astrocyte secretary profiles are currently ongoing.
The oncogenic Ha-Ras pathway has been implicated to induce AEG-1 expression in cancers
; however, the pathway involved in the induction of AEG-1 in normal non-cancerous cells such as astrocytes has yet to be investigated. We are currently investigating many of the molecular triggers and modulators of reactive astrogliosis as plausible inducers of AEG-1 in human astrocytes, including cytokines and growth factors, ischemia-associated mediators like hypoxia
, glucose-privation, and mediators of innate immunity such as LPS
 and other toll-like receptor agonists. Thus, additional works to elucidate the molecular pathways involved in AEG-1 induction in human astrocytes during reactive astrogliosis are warranted.