Natalia Filippova

Evidence for phosphorylation‐dependent internalization of recombinant human ρ1 GABAC receptors

1 Recombinant wild‐type or mutant human ρ1 GABA receptors were expressed in human embryonic kidney (HEK) 293 or monkey COS‐7 cells and studied using the patch clamp technique. 2 Standard whole‐cell recordings with 4 mM Mg‐ATP in the patch pipette induced a time‐dependent decrease in the GABA‐activated current (IGABA) amplitude that was not the result of a decrease in GABA sensitivity. In contrast, IGABA remained stable when recordings were obtained using the perforated patch configuration or with standard whole‐cell recording and no Mg‐ATP in the patch pipette. 3 The inhibitors of serine/threonine protein kinases KN‐62 (20 μM) or staurosporine (20 nM) prevented the time‐dependent decrease in the amplitude of IGABA seen in the presence of ATP. Alkaline phosphatase (220 U ml−1), when added to the patch pipette in the absence of ATP, induced a transient potentiation of IGABA. Although the protein kinase C (PKC) activator 4β‐phorbol 12‐myristate, 13‐acetate (PMA) did not reduce the amplitude of IGABA, inclusion of the catalytic domain of PKC in the recording pipette accelerated the time‐dependent decrease in current amplitude. These data suggest that phosphorylation is involved in the regulation of the amplitude of IGABA. 4 Mutation of the three PKC consensus sequences of the ρ1 receptor had no significant effect on the decline in IGABA, indicating that direct phosphorylation of these putative sites on the ρ1 receptor does not underlie the time‐dependent decrease in amplitude. 5 In COS‐7 cells transfected with wild‐type ρ1 receptors, the amplitude of IGABA had completely recovered to the original value when the same cells were repatched after 30‐40 min, indicating that the decline in IGABA was a reversible process. 6 The inhibitor of actin filament formation cytochalasin B, when added to the patch pipette in the absence of ATP, induced a time‐dependent inactivation suggesting that the actin cytoskeleton may play a role in the regulation of the amplitude. 7 Coincident with the decrease in the amplitude of IGABA, the cell capacitance significantly decreased in the presence of ATP in the patch pipette. This decrease in capacitance was not observed in the absence of Mg‐ATP. The decrease in the membrane surface area suggests that receptor internalization could be a potential mechanism for the observed inactivation. 8 At 32 °C, compared with 22 °C, the rate and magnitude of the decline was increased dramatically. In contrast, at 16 °C, no significant change in IGABA was observed over the 20 min recording time. This marked temperature sensitivity is consistent with receptor internalization as a mechanism for the time‐dependent decline in IGABA. 9 The specificity of the decrease in IGABA was assessed by coexpressing the voltage‐dependent potassium channel Kv1.4 along with the ρ1 receptor in HEK293 cells. The amplitude of the potassium current (IKv1.4) exhibited very little decrement in comparison to IGABA suggesting that the putative GABA receptor internalization was not the consequence of a non‐specific membrane retrieval.

Amyotrophic Lateral Sclerosis-linked Mutant SOD1 Sequesters Hu Antigen R (HuR) and TIA-1-related Protein (TIAR): IMPLICATIONS FOR IMPAIRED POST-TRANSCRIPTIONAL REGULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR*

Down-regulation of vascular endothelial growth factor (VEGF) in the mouse leads to progressive and selective degeneration of motor neurons similar to amyotrophic lateral sclerosis (ALS). In mice expressing ALS-associated mutant superoxide dismutase 1 (SOD1), VEGF mRNA expression in the spinal cord declines significantly prior to the onset of clinical manifestations. In vitro models suggest that dysregulation of VEGF mRNA stability contributes to that decline. Here, we show that the major RNA stabilizer, Hu Antigen R (HuR), and TIA-1-related protein (TIAR) colocalize with mutant SOD1 in mouse spinal cord extracts and cultured glioma cells. The colocalization was markedly reduced or abolished by RNase treatment. Immunoanalysis of transfected cells indicated that colocalization occurred in insoluble aggregates and inclusions. RNA immunoprecipitation showed a significant loss of VEGF mRNA binding to HuR and TIAR in mutant SOD1 cells, and there was marked depletion of HuR from polysomes. Ectopic expression of HuR in mutant SOD1 cells more than doubled the mRNA half-life of VEGF and significantly increased expression to that of wild-type SOD1 control. Cellular effects produced by mutant SOD1, including impaired mitochondrial function and oxidative stress-induced apoptosis, were reversed by HuR in a gene dose-dependent pattern. In summary, our findings indicate that mutant SOD1 impairs post-transcriptional regulation by sequestering key regulatory RNA-binding proteins. The rescue effect of HuR suggests that this impairment, whether related to VEGF or other potential mRNA targets, contributes to cytotoxicity in ALS.

Phosphoregulation of the RNA-binding Protein Hu Antigen R (HuR) by Cdk5 Affects Centrosome Function

Background: Regulation of protein function by phosphorylation is an important mechanism to control many cellular processes. Results: We found that the mRNA-binding protein HuR is phosphorylated by Cdk5 at the serine 202 residue. Conclusion: The aberrant phosphorylation of HuR at Ser-202 affects centrosome function and induces arrest of cell cycle progression. Significance: This work emphasizes HuR phosphorylation as a novel molecular target in cancer. Hu antigen R (HuR) is an mRNA-binding protein belonging to the ELAV family. It is highly expressed in cancer and involved in cell survival and proliferation. The impact of post-translational regulation of HuR and resulting cellular effects are poorly understood. In the current report, we describe a direct interaction between HuR and Cdk5 in glioma. We determined that Cdk5 specifically phosphorylates HuR at the serine 202 residue in the unique hinge region. The molecular consequences of this interaction are an altered HuR ability to bind, stabilize, and promote translation of mRNAs. At the cellular level, the anomalous HuR phosphorylation at this site evokes robust defects in centrosome duplication and cohesion as well as arrest of cell cycle progression. Subcellular fractionation and immunofluorescence technique confirm a direct integration of HuR and Cdk5 with centrosomes. We propose that HuR stores mRNA in the centrosome and that HuR phosphorylation by Cdk5 controls de novo protein synthesis in near proximity to centrosomes and, thus, impacts centrosome function.

Recombinant GABAC receptors expressed in rat hippocampal neurons after infection with an adenovirus containing the human ρ1 subunit

1 A recombinant adenovirus was generated with the human ρ1 GABAC receptor subunit (adeno‐ρ). Patch‐clamp and antibody staining were employed to confirm functional expression of recombinant ρ1 receptors after infection of human embryonic kidney cells (HEK293 cell line), human embryonic retinal cells (911 cell line), dissociated rat hippocampal neurons and cultured rat hippocampal slices. 2 Standard whole‐cell recording and Western blot analysis using ρ1 GABAC receptor antibodies revealed that recombinant ρ1 receptors were expressed in HEK293 and 911 cells after adeno‐ρ infection and exhibited properties similar to those of ρ1 receptors after standard transfection. 3 Cultured rat hippocampal neurons (postnatal day (P)3‐P5) did not show a native GABAC‐like current. After adeno‐ρ infection, however, a GABAC‐like current appeared in 70‐90 % of the neurons. 4 Five days after infection, expression of GABAC receptors in hippocampal neurons significantly decreased native GABAA receptor currents from 1200 ± 300 to 150 ± 70 pA (n= 10). The native glutamate‐activated current was unchanged. 5 Hippocampal slices (P8) did not show a native GABAC‐like current, although recombinant ρ1 receptors could be expressed in cultured hippocampal slices after adeno‐ρ infection. 6 These data indicate that an adenovirus can be used to express recombinant GABAC receptors in hippocampal neurons. This finding could represent an important step towards the gene therapy of CNS receptor‐related diseases.

The role of Src family kinases in growth and migration of glioma stem cells

Src family kinases (SFKs) are highly expressed and active in clinical glioblastoma multiforme (GBM) specimens. SFKs inhibitors have been demonstrated to inhibit proliferation and migration of glioma cells. However, the role of SFKs in glioma stem cells (GSCs), which are important for treatment resistance and recurrence, has not been reported. Here, we examined the expression pattern of individual members of SFKs and their functional role in CD133+ GSCs in comparison to primary glioma cells. We found that Fyn, c-Src and Yes were robustly expressed in GSCs while Lck was absent. Knockdown of c-Src, Yes or treatment with the SFK inhibitor dasatinib inhibited the migration of GSCs, but had no impact on their growth or self-renewal. These results suggest that SFKs represent an effective target for GSC migration but not for their growth.

Growth factor dependent regulation of centrosome function and genomic instability by HuR.

The mRNA binding protein HuR is over expressed in cancer cells and contributes to disease progression through post-transcriptional regulation of mRNA. The regulation of HuR and how this relates to glioma is the focus of this report. SRC and c-Abl kinases regulate HuR sub-cellular trafficking and influence accumulation in the pericentriolar matrix (PCM) via a growth factor dependent signaling mechanism. Growth factor stimulation of glioma cell lines results in the associate of HuR with the PCM and amplification of centrosome number. This process is regulated by tyrosine phosphorylation of HuR and is abolished by mutating tyrosine residues. HuR is overexpressed in tumor samples from patients with glioblastoma and associated with a reduced survival. These findings suggest HuR plays a significant role in centrosome amplification and genomic instability, which contributes to a worse disease outcome.

Hu Antigen R (HuR) multimerization contributes to glioma disease progression

Among primary brain cancers, gliomas are the most deadly and most refractory to current treatment modalities. Previous reports overwhelmingly support the role of the RNA-binding protein Hu antigen R (HuR) as a positive regulator of glioma disease progression. HuR expression is consistently elevated in tumor tissues, and a cytoplasmic localization appears essential for HuR-dependent oncogenic transformation. Here, we report HuR aggregation (multimerization) in glioma and the analysis of this tumor-specific HuR protein multimerization in clinical brain tumor samples. Using a split luciferase assay, a bioluminescence resonance energy transfer technique, and site-directed mutagenesis, we examined the domains involved in HuR multimerization. Results obtained with the combination of the split HuR luciferase assay with the bioluminescence resonance energy transfer technique suggested that multiple (at least three) HuR molecules come together during HuR multimerization in glioma cells. Using these data, we developed a model of HuR multimerization in glioma cells. We also demonstrate that exposing glioma cells to the HuR inhibitor tanshinone group compound 15,16-dihydrotanshinone-I or to the newly identified compound 5 disrupts HuR multimerization modules and reduces tumor cell survival and proliferation. In summary, our findings provide new insights into HuR multimerization in glioma and highlight possible pharmacological approaches for targeting HuR domains involved in cancer cell-specific multimerization.

Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment

Objective: MLN4924, a pharmacological inhibitor of cullin neddylation, resulted in glioma cell apoptosis, deregulation of the S-phase of DNA synthesis and thus, offers great potential for the treatment of brain tumours. However, targeting the neddylation pathway with an MLN4924 treatment stabilized the hypoxia-inducible factor 1A (HIF1A), which is one of the main transcriptional enhancers of the immune checkpoint molecule PDL1 (programmid death ligand-1) in cancer cells. The influence of immune checkpoint molecules on glioma progression has recently been discovered; PDL1 overexpression in gliomas corresponds to a significant shortening of patient survival and a decrease of the anti-tumour immune response. We hypothesize that i) PDL1 is up-regulated in gliomas after treatment with MLN4924 and induces T-cell energy; ii) co-utilization of the PD1/PDL1 blockage with MLN4924 therapy may reduce T-cell energy and may engage MLN4924-induced tumour disruption with the immune response. Methods: PDL1 expression and its immunosuppressive role in gliomas, glioma microenvironments, and after treatments with MLN4924 were assessed by utilizing methods of immunohistochemistry, molecular biology, and biochemistry. Results: We confirmed PDL1 overexpression in clinical brain tumour samples, PDGx and established glioma cell lines, extracellular media from glioma cells, and CSF (cerebrospinal fluid) samples from tumour-bearing mice. Our primary T-cell based assays verified that the up-regulation of PDL1 in tumour cells protects gliomas from T-cell treatment and reduces T-cell activation. We found that a pharmacological inhibitor of cullin neddylation, MLN4924, exhibited strong cytotoxicity towards PDGx and established glioma cell lines, in vitro, with an IC50’s range from 0.2 to 3 uM. However, we observed a significant increase of HIF1A and PDL1 in mRNA and protein levels in all glioma cell lines after treatment with MLN4924. The MLN4924-dependent induction of PDL1 in gliomas resulted in T-cell energy, which was blocked by a blockage of the PD1/PDL1 interaction. Conclusion: We conclude that i) PDL1 up-regulation in gliomas and the glioma microenvironment is an important chemotherapeutic target; ii) MLN4924 therapy, combined with a blockage of the PD1/PDL1 pathway, should be considered as a potential strategy for glioma treatment.