Rat Astrocytes Research Articles

Establishment and Preliminary Characterization of Three Astrocytic Cells Lines Obtained from Primary Rat Astrocytes by Sub-Cloning.

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenetic and epigenetic level. In parallel, we also studied the expression of the differentiation-related H1.0 linker histone variant to evaluate its possible modification in relation with transformation. We found that the most modified astrocytes (A-FC6) have epigenetic and chromosomal alterations typical of cancer, and that the other two clones (A-GS1 and A-VV5) have intermediate properties. Surprisingly, the differentiation-specific somatic histone H1.0 steadily increases from the normal astrocytes to the most transformed ones. As a whole, our results suggest that these three cell lines, together with the starting primary cells, constitute a potential model for studying glioma development.

Endothelin-1 decreases the expression of Ephrin-A and B subtypes in cultured rat astrocytes through ETB receptors

Ephrin family proteins are cell surface molecules that regulate several cellular functions through cell-cell interactions. During nervous tissue repair after injury, the expression of ephrin subtypes in astrocytes is altered, affecting the axonal elongation and migration of neuronal precursors. However, the mechanism regulating the expression of ephrin subtypes in astrocytes has not been investigated. Herein, we studied the effects of endothelin-1 (ET-1) on the expression of ephrin subtypes in cultured rat astrocytes. Our results showed that ET-1 (100 nM) treatment for 1−24 h reduced the expression of ephrin-A2, -A4, -B2, and -B3 mRNA and protein in astrocytes, whereas the expression of ephrin-A1, -A3, -A5, and -B1 mRNA were not affected. Sarafotoxin S6c, a selective ETB receptor agonist, decreased the expression of ephrin-A2, -A4, -B2, and -B3 in cultured astrocytes. The decrease in ephrin-A2, -A4, -B2, and -B3 expression by ET-1 treatment was reduced in the presence of BQ788, an ETB receptor antagonist, while FR139317, an ETA receptor antagonist, had no effects. These results suggest that ET-1 is a signaling molecule that downregulates ephrin-A2, -A4, -B2, and -B3 expression in astrocytes.

Lidocaine activates autophagy of astrocytes and ameliorates chronic constriction injury-induced neuropathic pain.

Lidocaine is a commonly used drug to alleviate neuropathic pain (NP). This work aims to investigate the mechanism of lidocaine in alleviating NP. Chronic constriction injury (CCI) rats were established by surgery to induce NP. We observed the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of rats. Immunofluorescence staining was performed to determine the LC3/glial fibrillary acidic protein (GFAP)-positive cells. Rat astrocytes were treated with lipopolysaccharide (LPS) to induce CCI, and then treated with lidocaine or 3-MA (autophagy inhibitor). CCK-8 was performed to detect cell proliferation. Western blot and enzyme-linked immunosorbent assay were performed to detect the level of protein and inflammatory factor. CCI rats exhibited a decrease of MWT and TWL, which was effectively abolished by lidocaine. Lidocaine enhanced the number of LC3/GFAP-positive cells in CCI rats. Moreover, lidocaine inhibited the expression of GFAP and p62, and enhanced LC3-II/LC3-I expression in the LPS-treated astrocytes. Lidocaine inhibited the level of TNF-α and IL-1β in the LPS-treated astrocytes. The influence conferred by lidocaine was effectively abolished by 3-MA. In conclusion, our work demonstrates that lidocaine activates autophagy of astrocytes and ameliorates CCI-induced NP. Thus, our study provides a further experimental basis for the mechanism of lidocaine to alleviate NP.

Targeting metabolic plasticity in glioma stem cells in vitro and in vivo through specific inhibition of c-Src by TAT-Cx43266-283.

BackgroundGlioblastoma is the most aggressive primary brain tumour and has a very poor prognosis. Inhibition of c-Src activity in glioblastoma stem cells (GSCs, responsible for glioblastoma lethality) and primary glioblastoma cells by the peptide TAT-Cx43266–283 reduces tumorigenicity, and boosts survival in preclinical models. Because c-Src can modulate cell metabolism and several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells, here we explored the inhibition of advantageous GSC metabolic plasticity by the c-Src inhibitor TAT-Cx43266-283.MethodsMetabolic impairment induced by the c-Src inhibitor TAT-Cx43266-283 in vitro was assessed by fluorometry, western blotting, immunofluorescence, qPCR, enzyme activity assays, electron microscopy, Seahorse analysis, time-lapse imaging, siRNA, and MTT assays. Protein expression in tumours from a xenograft orthotopic glioblastoma mouse model was evaluated by immunofluorescence.FindingsTAT-Cx43266–283 decreased glucose uptake in human GSCs and reduced oxidative phosphorylation without a compensatory increase in glycolysis, with no effect on brain cell metabolism, including rat neurons, human and rat astrocytes, and human neural stem cells. TAT-Cx43266-283 impaired metabolic plasticity, reducing GSC growth and survival under different nutrient environments. Finally, GSCs intracranially implanted with TAT-Cx43266–283 showed decreased levels of important metabolic targets for cancer therapy, such as hexokinase-2 and GLUT-3.InterpretationThe reduced ability of TAT-Cx43266-283–treated GSCs to survive in metabolically challenging settings, such as those with restricted nutrient availability or the ever-changing in vivo environment, allows us to conclude that the advantageous metabolic plasticity of GSCs can be therapeutically exploited through the specific and cell-selective inhibition of c-Src by TAT-Cx43266-283.FundingSpanish Ministerio de Economía y Competitividad (FEDER BFU2015-70040-R and FEDER RTI2018-099873-B-I00), Fundación Ramón Areces. Fellowships from the Junta de Castilla y León, European Social Fund, Ministerio de Ciencia and Asociación Española Contra el Cáncer (AECC).

Regulation and role of Acyl-CoA synthetase 4 in glial cells

Acyl-CoA synthetase 4 (Acsl4), an enzyme involved in arachidonic acid (AA) metabolism, participates in physiological and pathological processes such as steroidogenesis and cancer. The role of Acsl4 in neurons and in nervous system development has also been documented but little is known regarding its functionality in glial cells. In turn, several processes in glial cells, including neurosteroidogenesis, stellation and AA uptake, are regulated by cyclic adenosine monophosphate (cAMP) signal. In this context, the aim of this work was to analyze the expression and functional role of Acsl4 in primary rat astrocyte cultures and in the C6 glioma cell line by chemical inhibition and stable silencing, respectively. Results show that Acsl4 expression was regulated by cAMP in both models and that cAMP stimulation of steroidogenic acute regulatory protein mRNA levels was reduced by Acsl4 inhibition or silencing. Also, Acsl4 inhibition reduced progesterone synthesis stimulated by cAMP and also affected cAMP-induced astrocyte stellation, decreasing process elongation and increasing branching complexity. Similar effects were observed for Acsl4 silencing on cAMP-induced C6 cell morphological shift. Moreover, Acsl4 inhibition and silencing reduced proliferation and migration of both cell types. Acsl4 silencing in C6 cells reduced the capacity for colony proliferation and neurosphere formation, the latter ability also being abolished by Acsl4 inhibition. In sum, this work presents novel evidence of Acsl4 involvement in neurosteroidogenesis and the morphological changes of glial cells promoted by cAMP. Furthermore, Acsl4 participates in migration and proliferation, also affecting cell self-renewal. Altogether, these findings provide insights into Acsl4 functions in glial cells.

Regulation of the KATP-JNK gap junction signaling pathway by immunomodulator astragaloside IV attenuates neuropathic pain

Background and objectivesGap junctions play a pivotal role in contributing to the formation of astroglial networks and in chronic pain. However, the mechanisms underlying the dysfunction of astroglial gap junctions...

Hippocampal astrocytes in juvenile rats with persistent epilepsy: the role of cannabinoid receptor type 2 in regulating MAPK pathway

Hippocampal astrocytes in juvenile rats with persistent epilepsy: the role of cannabinoid receptor type 2 in regulating MAPK pathway

Neuroprotective potential of isothiocyanates in an in vitro model of neuroinflammation

Isothiocyanates (ITCs), present as glucosinolate precursors in cruciferous vegetables, have shown anti-inflammatory, antioxidant and anticarcinogenic activities. Here, we compared the effects of three different ITCs on ROS production and on the expression of matrix metalloproteinase (MMP)-2 and -9, which represent important pathogenetic factors of various neurological diseases. Primary cultures of rat astrocytes were activated by LPS and simultaneously treated with different doses of Allyl isothiocyanate (AITC), 2-Phenethyl isothiocyanate (PEITC) and 2-Sulforaphane (SFN). Results showed that SFN and PEITC were able to counteract ROS production induced by H2O2. The zymographic analysis of cell culture supernatants evidenced that PEITC and SFN were the most effective inhibitors of MMP-9, whereas, only SFN significantly inhibited MMP-2 activity. PCR analysis showed that all the ITCs used significantly inhibited both MMP-2 and MMP-9 expression. The investigation on the mitogen-activated protein kinase (MAPK) signaling pathway demonstrated that ITCs modulate MMP transcription by inhibition of extracellular-regulated protein kinase (ERK) activity. Results of this study suggest that ITCs could be promising nutraceutical agents for the prevention and complementary treatment of neurological diseases associated with MMP involvement.

Inflammatory factors and amyloid β-induced microglial polarization promote inflammatory crosstalk with astrocytes.

The immunological responses are a key pathological factor in Alzheimer’s disease (AD). We hypothesized that microglial polarization alters microglia-astrocyte immune interactions in AD. M1 and M2 microglia were isolated from primary rat microglia and were confirmed to secrete pro-inflammatory and anti-inflammatory factors, respectively. Primary rat astrocytes were co-cultured with M1 or M2 microglial medium. M1 microglial medium increased astrocyte production of pro-inflammatory factors (interleukin [IL]-1β, tumor necrosis factor α and IL-6), while M2 microglial medium enhanced astrocyte production of anti-inflammatory factors (IL-4 and IL-10). To analyze the crosstalk between microglia and astrocytes after microglial polarization specifically in AD, we co-cultured astrocytes with medium from microglia treated with amyloid-β (Aβ) alone or in combination with other inflammatory substances. Aβ alone and Aβ combined with lipopolysaccharide/interferon-γ induced pro-inflammatory activity in M1 microglia and astrocytes, whereas IL-4/IL-13 inhibited Aβ-induced pro-inflammatory activity. Nuclear factor κB p65 was upregulated in M1 microglia and pro-inflammatory astrocytes, while Stat6 was upregulated in M2 microglia and anti-inflammatory astrocytes. These results provide direct evidence that microglial polarization governs communication between microglia and astrocytes, and that AD debris alters this crosstalk.

Metformin alleviates high glucose-induced ER stress and inflammation by inhibiting the interaction between caveolin1 and AMPKα in rat astrocytes

Hyperglycemia-induced endoplasmic reticulum (ER) stress and inflammatory response afflict neuropathological diseases (such as epilepsy and Alzheimer’s disease). Astrocytes are the critical cells that mediate brain inflammation in this process. Metformin is a kind of hypoglycemic drugs widely used in clinical practice, which has anti-inflammatory and antioxidant effects. However, the biological mechanism of metformin in regulating inflammation and ER stress induced by hyperglycemia remains unclear. Therefore, in this study, rat primary astrocytes were preincubated with metformin and AMPK agonist AICAR for 1 h prior to administration of high glucose (33 mM glucose). Our findings indicated that metformin treatment inhibited the elevated ER stress and inflammation in high glucose-treated astrocytes. Moreover, metformin inhibited the formation of caveolin1/AMPKα complex. Additionally, the effects of AICAR on astrocytes were similar to metformin. In conclusion, metformin reduced high glucose-induced ER stress and inflammation by inhibiting the interaction between caveolin1 and AMPKα, suggesting that the caveolin1/AMPKα complex may be a potential therapeutic target for metformin.

Oxidative Stress Modulates Apoptotic Substrate Phagocytosis by Primary Rat Astrocytes

Brain astrocytes are commonly believed to be relatively tolerant to oxidative stress. In this work, we studied the effect of oxidative stress on the ability of rat brain astrocytes in primary culture to clean apoptotic substrates. Using fluorescent microscopy, we demonstrated that the ingestion of rat retinal rod outer segments (ROS), used here as a phagocytic substrate, dramatically decreased in astrocytes preincubated with various hydroperoxides. The ability of primary astrocytes to ingest ROS also depended on the substrate oxidative status. The ingestion of “oxidized” ROS preparations preincubated with hydrogen peroxide was sharply reduced compared to “non-oxidized” ROS. We also showed that natural and synthetic antioxidants (α-tocopherol, Trolox) efficiently protected the phagocytic function of astrocytes from oxidative damage. These data suggest that the phagocytic function of rat brain primary astrocytes is extremely vulnerable to oxidative stress, while oxidative stress tolerance characterizes not all functions of these glial cells.

Hydrogen Sulfide Protects Against Ammonia-Induced Neurotoxicity Through Activation of Nrf2/ARE Signaling in Astrocytic Model of Hepatic Encephalopathy.

Objective: Hepatic encephalopathy (HE) characterized by neuropsychiatric abnormalities is a major complication of cirrhosis with high mortality. However, the pathogenesis of HE has not been fully elucidated. This study aimed to determine endogenous hydrogen sulfide (H2S) in the blood of HE patients and investigate the role of H2S in an astrocytic model of HE.Methods: Patients with and without HE were recruited to determine plasma H2S levels and blood microbial 16S rRNA gene. Rat astrocytes were employed as a model of HE by treatment of NH4Cl. Exogenous H2S was preadded. Cell viability was measured by Cell Counting Kit-8 (CCK-8) assay, and cell death was evaluated by lactate dehydrogenase (LDH) release. Apoptosis was determined by Hoechst 33342/Propidium Iodide (PI) Double Staining and Western blot analysis of apoptosis-related protein expression. Intracellular reactive oxygen species (ROS) levels were assessed by flow cytometer. Expressions of Nrf2 and its downstream regulated genes were examined by immunofluorescence staining and Western blot, respectively. Nrf2 gene knockdown was performed by antisense shRNA of Nrf2 gene.Results: There was a significant decrease in H2S levels in cirrhotic patients with HE compared with without HE. Blood microbiota analyses revealed that certain strains associated with H2S production were negatively correlated with HE. In vitro, H2S markedly attenuated NH4Cl-induced cytotoxicity, oxidative stress, and apoptosis. This effect was mediated by Nrf2/ARE signaling, and knockdown of Nrf2 expression abolished the antagonistic effect of H2S on NH4Cl-induced neurotoxicity in astrocytes.Conclusion: Levels of H2S and bacteria associated with H2S production are decreased in HE, and H2S functions as the neuroprotector against NH4Cl-induced HE by activating Nrf2/ARE signaling of astrocytes.

Chitosan-coated rosmarinic acid nanoemulsion nasal administration protects against LPS-induced memory deficit, neuroinflammation, and oxidative stress in Wistar rats

Rosmarinic acid (RA) lipid-nanotechnology-based delivery systems associate with mucoadhesive biopolymers for nasal administration has arisen as a new promising neuroprotective therapy for neurodegenerative disorders (ND). We have previously demonstrated the glioprotective effect of chitosan-coated RA nanoemulsions (RA CNE) against lipopolysaccharide (LPS)-induced damage in rat astrocyte primary culture. Here, we further investigate the protective effect of RA CNE nasal administration on LPS-induced memory deficit, neuroinflammation, and oxidative stress in Wistar rats, since these in vivo studies were crucial to understand the impact of developed delivery systems in the RA neuroprotective effects. The animals were treated through nasal route with RA CNE (2 mg·mL−1), free RA (2 mg·mL−1), blank CNE, and saline (control and LPS groups) administrations (n.a., 100 μL per nostril) twice a day (7 a.m./7 p.m.) for six days. On the sixth day, the animals received the last treatments and LPS was intraperitoneally (i.p.) administrated (250 μg·kg−1). Overall results, proved for the first time that the RA CNE nasal administration elicits a neuroprotective effect against LPS-induced damage, which was associated with increased 1.6 times recognition index, decreased 5.0 and 1.9 times in GFAP+ cell count and CD11b expression, respectively, as well as increased 1.7 times SH in cerebellum and decreased 3.9 times TBARS levels in cerebral cortex in comparison with LPS group. RA CNE treatment also facilitates RA bioavailability in the brain, confirmed by RA quantification. Free RA also demonstrates a protective effect in some studied parameters, although no RA was quantified in the brain.

Astrocytes from cortex and striatum show differential responses to mitochondrial toxin and BDNF: implications for protection of striatal neurons expressing mutant huntingtin

BackgroundEvidence shows significant heterogeneity in astrocyte gene expression and function. We previously demonstrated that brain-derived neurotrophic factor (BDNF) exerts protective effects on whole brain primary cultured rat astrocytes treated with 3-nitropropionic acid (3NP), a mitochondrial toxin widely used as an in vitro model of Huntington’s disease (HD). Therefore, we now investigated 3NP and BDNF effects on astrocytes from two areas involved in HD: the striatum and the entire cortex, and their involvement in neuron survival.MethodsWe prepared primary cultured rat cortical or striatal astrocytes and treated them with BDNF and/or 3NP for 24 h. In these cells, we assessed expression of astrocyte markers, BDNF receptor, and glutamate transporters, and cytokine release. We prepared astrocyte-conditioned medium (ACM) from cortical and striatal astrocytes and tested its effect on a cellular model of HD.ResultsBDNF protected astrocytes from 3NP-induced death, increased expression of its own receptor, and activation of ERK in both cortical and striatal astrocytes. However, BDNF modulated glutamate transporter expression differently by increasing GLT1 and GLAST expression in cortical astrocytes but only GLT1 expression in striatal astrocytes. Striatal astrocytes released higher amounts of tumor necrosis factor-α than cortical astrocytes in response to 3NP but BDNF decreased this effect in both populations. 3NP decreased transforming growth factor-β release only in cortical astrocytes, whereas BDNF treatment increased its release only in striatal astrocytes. Finally, we evaluated ACM effect on a cellular model of HD: the rat striatal neuron cell line ST14A expressing mutant human huntingtin (Q120) or in ST14A cells expressing normal human huntingtin (Q15). Neither striatal nor cortical ACM modified the viability of Q15 cells. Only ACM from striatal astrocytes treated with BDNF and ACM from 3NP + BDNF-treated striatal astrocytes protected Q120 cells, whereas ACM from cortical astrocytes did not.ConclusionsData suggest that cortical and striatal astrocytes respond differently to mitochondrial toxin 3NP and BDNF. Moreover, striatal astrocytes secrete soluble neuroprotective factors in response to BDNF that selectively protect neurons expressing mutant huntingtin implicating that BDNF modulation of striatal astrocyte function has therapeutic potential against neurodegeneration.Graphical abstract

Expression and Distribution Pattern of Pnn in Ischemic Cerebral Cortex and Cultured Neural Cells Exposed to Oxygen-Glucose Deprivation.

Pinin (Pnn), a multifunctional protein, participates in embryonic development as well as in cellular apoptosis, proliferation, and migration through regulating mRNA alternative splicing and gene transcription. Previous studies have shown that Pnn plays important roles in neural system development and the expression level of Pnn in astrocytes is altered by ischemic stress and associated with cellular apoptosis. In the present study, we further utilized primary cultured rat neurons and astrocytes with oxygen-glucose deprivation (OGD) and a mouse model with middle cerebral artery occlusion (MCAO)-induced ischemic stroke to examine the effect of ischemic stress on Pnn expression and distribution in different types of neural cells. Under normoxia, Pnn is mainly localized in the nuclear speckle of primary cultured neurons. The expression level of Pnn was increased after the OGD treatment and then decreased in the reoxygenation period. Moreover, the cytoplasmic expression of Pnn was observed in neurons with OGD and reoxygenation (OGD/R). Unlike that in neurons, the Pnn expression in astrocytes was decreased after OGD treatment and then gradually increased during the reoxygenation period. Of interest, the nuclear–cytoplasmic translocation of Pnn was not observed in astrocytes with OGD/R. In the MCAO mouse model, the neuronal expression of Pnn in the peri-ischemic region was reduced by three days post induction of ischemic stroke. However, the Pnn expression in astrocytes was not altered. Moreover, the nuclear speckle distribution of Pnn in neurons was also diminished following ischemic stroke. In conclusion, the Pnn expression and distribution after OGD and during reoxygenation showed distinct manners in neurons and astrocytes, implying that Pnn may play different roles in different types of neural cells in the stress response to ischemic injury.

Cognitive Training as a Potential Activator of Hippocampal Neurogenesis in the Rat Model of Sporadic Alzheimer's Disease.

There is a growing body of evidence that interventions like cognitive training or exercises prior to the manifestation of Alzheimer’s disease (AD) symptoms may decelerate cognitive decline. Nonetheless, evidence of prevention or a delay of dementia is still insufficient. Using OXYS rats as a suitable model of sporadic AD and Wistar rats as a control, we examined effects of cognitive training in the Morris water maze on neurogenesis in the dentate gyrus in presymptomatic (young rats) and symptomatic (adult rats) periods of development of AD signs. Four weeks after the cognitive training, we immunohistochemically estimated densities of quiescent and amplifying neuronal progenitors, neuronal-lineage cells (neuroblasts and immature and mature neurons), and astrocytes in young and adult rats, and the amyloid precursor protein and amyloid-β in adult rats. Reference memory was defective in OXYS rats. The cognitive training did not affect neuronal-lineage cells’ density in either rat strain either at the young or adult age, but activated neuronal progenitors in young rats and increased astrocyte density and downregulated amyloid-β in adult OXYS rats. Thus, to activate adult neurogenesis, cognitive training should be started before first neurodegenerative changes, whereas cognitive training accompanying amyloid-β accumulation affects only astrocytic support.

Activated microglia-derived macrophage-like cells exacerbate brain edema after ischemic stroke correlate with astrocytic expression of aquaporin-4 and interleukin-1 alpha release

Brain edema following brain infarction affects mobility and mortality. The mechanisms underlying this process remain to be elucidated. Animal studies have shown that aquaporin-4 (AQP4) expression in astrocytes increases after stroke, and its deletion significantly reduces brain swelling. Recently, two kinds of cells, resident microglia-derived macrophage-like cells (MG-MΦ) and bone marrow-derived macrophages (BM-MΦ), have been reported to accumulate in the ischemic core and stimulate adjacent astrocytes. Therefore, we hypothesized that these cells play crucial roles in the expression of AQP4 and ultimately lead to exacerbated brain edema. To verify this hypothesis, we investigated the role of MG- or BM-MΦ in brain edema using a rat model of transient middle cerebral artery occlusion and rat astrocyte primary cultures. AQP4 expression significantly increased in the peri-infarct tissue at 3–7 days post-reperfusion (dpr) and in the core tissue at 5 and 7 dpr, which synchronized with the expression of Iba1, Il1a, Tnf, and C1qa mRNA. Interleukin (IL)-1α treatment or coculture with MG- and BM-MΦ increased AQP4 expression in astrocytes, while an IL-1 receptor type I antagonist reduced these effects. Furthermore, aggravated animals exhibited high expression of Aqp4 and Il1a mRNA in the ischemic core at 7 dpr, which led to the exacerbation of brain edema. MG-MΦ signature genes were highly expressed in the ischemic core in aggravated rats, while BM-MΦ signature genes were weakly expressed. These findings suggest that IL-1α produced by MG-MΦ induces astrocytic AQP4 expression in the peri-infarct and ischemic core tissues, thereby exacerbating brain edema. Therefore, the regulation of MG-MΦ may prevent the exacerbation of brain edema.

Interleukin-1β Drives Cellular Senescence of Rat Astrocytes Induced by Oligomerized Amyloid β Peptide and Oxidative Stress

Background: Alzheimer's disease (AD) is the leading cause of dementia. With no reliable treatment that delays or reverses the progress of AD, effective medical drugs, and interventions for AD treatment are in urgent need. Clinical success for patients thus relies on gaining a clearer understanding of AD pathogenesis to feed the development of novel and potent therapy strategies. It is well-established that inflammatory processes are involved in the pathology of AD, and recent studies implicated senescence of glial cells as an important player in the progression of AD.Methods: We did a preliminary screen in rat astrocytes for the five most abundant inflammatory factors in neuroinflammation, namely IL-1β, IL-6, IL-8, TGF-β1, and TNF-α, and found that IL-1β could efficiently induce cellular senescence. After that, SA-β-gal staining, immunofluorescence, ELISA, qRT-PCR, and immunoblotting were used to explore the underlying mechanism through which IL-1β mediates cellular senescence of rat astrocytes.Results: IL-1β-induced cellular senescence of rat astrocytes was accompanied by increased total and phosphorylated tau. Further experiments showed that both oligomerized amyloid β (Aβ) and H2O2 treatment can induce cellular senescence in rat astrocytes and increase the production and secretion of IL-1β from these cells. Subsequent mechanistic study revealed that activation of NLRP3 mediates Aβ and H2O2-induced maturation and secretion of IL-1β.Conclusion: Our results suggest that IL-1β mediates senescence in rat astrocytes induced by several common adverse stimuli in AD, implicating IL-1β and NLRP3 as valuable diagnostic biomarkers and therapeutic targets for AD.

The Laminin-Induced Phosphorylation of PKCδ Regulates AQP4 Distribution and Water Permeability in Rat Astrocytes.

In astrocytes, the water-permeable channel aquaporin-4 (AQP4) is concentrated at the endfeet that abut the blood vessels of the brain. The asymmetric distribution of this channel is dependent on the function of dystroglycan (DG), a co-expressed laminin receptor, and its associated protein complex. We have demonstrated that the addition of laminin to astrocytes in culture causes the clustering of AQP4, DG, and lipid rafts. The last, in particular, have been associated with the initiation of cell signaling. As laminin binding to DG in muscle cells can induce the tyrosine phosphorylation of syntrophin and laminin requires tyrosine kinases for acetylcholine receptor clustering in myotubes, we asked if signal transduction might also be involved in AQP4 clustering in astrocytes. We analyzed the timecourse of AQP4, DG, and monosialotetrahexosylganglioside (GM1) clustering in primary cultures of rat astrocytes following the addition of laminin, and determined that the clustering of DG precedes that of AQP4 and GM1. We also showed that laminin induces the formation of phosphotyrosine-rich clusters and that the tyrosine kinase inhibitor, genistein, disrupts the laminin-induced clustering of both β-DG and AQP4. Using the Kinexus antibody microarray chip, we then identified protein-serine kinase C delta (PKCδ) as one of the main proteins exhibiting high levels of tyrosine phosphorylation upon laminin treatment. Selective inhibitors of PKC and siRNA against PKCδ disrupted β-DG and AQP4 clustering, and also caused water transport to increase in astrocytes treated with laminin. Our results demonstrate that the effects of laminin on AQP4 localization and function are relayed, at least in part, through PKC signaling.

Abstract 2635: Role of LRRC15, a candidate immunotherapy target, in cell adhesion, migration and spheroid formation in osteosarcoma cells

Abstract Background: Leucine-rich repeat containing 15 (LRRC15), a transmembrane protein, has been found to be highly expressed in stromal cells in multiple solid tumors especially tumors of mesenchymal origin (e.g. sarcoma, glioma, melanoma) [1]. LRRC15 has been nominated as a candidate target for immunotherapy with antibody drug conjugates in cancers, notably osteosarcomas that strongly express this surface marker. In addition, LRRC15 has been found to be significantly upregulated during osteogenic induction [2] and TGFβ induction in mesenchymal stem cells [3]. The present study explores the functional role of LRRC15 in osteosarcoma (OS) cells. Methods: To delineate the role of LRRC15 in OS cells, we generated inducible shRNA knockdown derivatives of high LRRC15 expressing OS cells (SAOS2/HuO9) and induced LRRC15 in low expressing OS cells (HOS & U2OS). We explored various cell-cell and cell-ECM interactions using invasion, migration, adhesion, 3D colony formation (Matrigel), spheroid formation and spheroid migration assays. Results: LRRC15 expression and knockdown in different OS cells were confirmed by transcriptional, flow cytometric and western blot analysis We found that expression of LRRC15 varies significantly among osteosarcoma (OS) cell-lines. Here we report that inducible knockdown of LRRC15 in high expressing OS cells significantly reduces collagen type 1 based adhesion (using extracellular matrix screening array), migration, invasion and 3D colony formation using Matrigel. Enlarged colonies were observed in TGFβ treated HOS cells in Matrigel. Spheroids were formed using ultra low attachment plates with SaOS2/HuO9/HOS & U2OS cells. Knockdown of LRRC15 significantly affects the spheroid compaction in high LRRC15 expressing cells (SaOS2 & HuO9). A spheroid-based migration assay on collagen type 1 coated plates revealed significantly reduced migratory phenotype in LRRC15-knockdown SAOS2 cells. Conclusion: Taken together, these results suggest that LRRC15 plays a key role in cell-cell and cell-ECM interactions of OS cells providing insights relevant to biological function of LRRC15 in osteosarcoma cells. 1. Satoh, K., M. Hata, and H. Yokota, A novel member of the leucine-rich repeat superfamily induced in rat astrocytes by beta-amyloid. Biochem Biophys Res Commun, 2002. 290(2): p. 756-62. 2. Wang, Y., et al., LRRC15 promotes osteogenic differentiation of mesenchymal stem cells by modulating p65 cytoplasmic/nuclear translocation. Stem Cell Res Ther, 2018. 9(1): p. 65. 3. Purcell, J.W., et al., LRRC15 Is a Novel Mesenchymal Protein and Stromal Target for Antibody-Drug Conjugates. Cancer Res, 2018. 78(14): p. 4059-4072. Citation Format: Sanjit Mukherjee, Konrad Huppi, Robert L. Walker, Jack Zhu, Marbin Pineda, James W. Purcell, Lee J. Helman, Paul S. Meltzer. Role of LRRC15, a candidate immunotherapy target, in cell adhesion, migration and spheroid formation in osteosarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2635.