Ying Si

KSRP: A Checkpoint for Inflammatory Cytokine Production in Astrocytes

Chronic inflammation in the central nervous system (CNS) is a central feature of many neurodegenerative and autoimmune diseases. As an immunologically competent cell, the astrocyte plays an important role in CNS inflammation. It is capable of expressing a number of cytokines such as tumor necrosis factor alpha (TNF‐α) and interleukin‐1 beta (IL‐1β) that promote inflammation directly and through the recruitment of immune cells. Checkpoints are therefore in place to keep tight control over cytokine production. Adenylate/uridylate‐rich elements (ARE) in the 3′ untranslated region of cytokine mRNAs serve as a major checkpoint by regulating mRNA stability and translational efficiency. Here, we examined the impact of KH‐type splicing regulatory protein (KSRP), an RNA binding protein which destabilizes mRNAs via the ARE, on cytokine expression and paracrine phenotypes of primary astrocytes. We identified a network of inflammatory mediators, including TNF‐α and IL‐1β, whose expression increased 2 to 4‐fold at the RNA level in astrocytes isolated from KSRP−/− mice compared to littermate controls. Upon activation, KSRP−/− astrocytes produced TNF‐α and IL‐1β at levels that exceeded control cells by 15‐fold or more. Conditioned media from KSRP−/− astrocytes induced chemotaxis and neuronal cell death in vitro. Surprisingly, we observed a prolongation of half‐life in only a subset of mRNA targets and only after selective astrocyte activation. Luciferase reporter studies indicated that KSRP regulates cytokine gene expression at both transcriptional and post‐transcriptional levels. Our results outline a critical role for KSRP in regulating pro‐inflammatory mediators and have implications for a wide range of CNS inflammatory and autoimmune diseases.

Hu antigen R (HuR) is a positive regulator of the RNA-binding proteins TDP-43 and FUS/TLS: implications for amyotrophic lateral sclerosis.

Background: RNA processing abnormalities have been linked to amyotrophic lateral sclerosis (ALS). Results: HuR promotes the expression of the ALS-associated TDP-43 and FUS/TLS RNA-binding proteins. Conclusion: Through its regulation of TDP-43 and FUS/TLS, HuR potentially impacts a wide range of molecular and cellular phenotypes. Significance: A network of proteins exists to maintain RNA processing, and ALS-associated abnormalities may stem from disruption of this network. Posttranscriptional gene regulation is governed by a network of RNA-binding proteins (RBPs) that interact with regulatory elements in the mRNA to modulate multiple molecular processes, including splicing, RNA transport, RNA stability, and translation. Mounting evidence indicates that there is a hierarchy within this network whereby certain RBPs cross-regulate other RBPs to coordinate gene expression. HuR, an RNA-binding protein we linked previously to aberrant VEGF mRNA metabolism in models of SOD1-associated amyotrophic lateral sclerosis, has been identified as being high up in this hierarchy, serving as a regulator of RNA regulators. Here we investigated the role of HuR in regulating two RBPs, TDP-43 and FUS/TLS, that have been linked genetically to amyotrophic lateral sclerosis. We found that HuR promotes the expression of both RBPs in primary astrocytes and U251 cells under normal and stressed (hypoxic) conditions. For TDP-43, we found that HuR binds to the 3′ untranslated region (UTR) and regulates its expression through translational efficiency rather than RNA stability. With HuR knockdown, there was a shift of TDP-43 and FUS mRNAs away from polysomes, consistent with translational silencing. The TDP-43 splicing function was attenuated upon HuR knockdown and could be rescued by ectopic TDP-43 lacking the 3′ UTR regulatory elements. Finally, conditioned medium from astrocytes in which HuR or TDP-43 was knocked down produced significant motor neuron and cortical neuron toxicity in vitro. These findings indicate that HuR regulates TDP-43 and FUS/TLS expression and that loss of HuR-mediated RNA processing in astrocytes can alter the molecular and cellular landscape to produce a toxic phenotype.

Mutant tristetraprolin: a potent inhibitor of malignant glioma cell growth

Malignant gliomas rely on the production of certain critical growth factors including VEGF, interleukin (IL)-6 and IL-8, to fuel rapid tumor growth, angiogenesis, and treatment resistance. Post-transcriptional regulation through adenine and uridine-rich elements of the 3′ untranslated region is one mechanism for upregulating these and other growth factors. In glioma cells, we have shown that the post-transcriptional machinery is optimized for growth factor upregulation secondary to overexpression of the mRNA stabilizer, HuR. The negative regulator, tristetraprolin (TTP), on the other hand, may be suppressed because of extensive phosphorylation. Here we test that possibility by analyzing the phenotypic effects of a mutated form of TTP (mt-TTP) in which 8 phosphoserine residues were converted to alanines. We observed a significantly enhanced negative effect on growth factor expression in glioma cells at the post-transcriptional and transcriptional levels. The protein became stabilized and displayed significantly increased antiproliferative effects compared to wild-type TTP. Macroautophagy was induced with both forms of TTP, but inhibition of autophagy did not affect cell viability. We conclude that glioma cells suppress TTP function through phosphorylation of critical serine residues which in turn contributes to growth factor upregulation and tumor progression.

Transforming Growth Factor Beta (TGF-β) Is a Muscle Biomarker of Disease Progression in ALS and Correlates with Smad Expression.

We recently identified Smads1, 5 and 8 as muscle biomarkers in human ALS. In the ALS mouse, these markers are elevated and track disease progression. Smads are signal transducers and become activated upon receptor engagement of ligands from the TGF-β superfamily. Here, we sought to characterize ligands linked to activation of Smads in ALS muscle and their role as biomarkers of disease progression. RNA sequencing data of ALS muscle samples were mined for TGF-β superfamily ligands. Candidate targets were validated by qRT-PCR in a large cohort of human ALS muscle biopsy samples and in the G93A SOD1 mouse. Protein expression was evaluated by Western blot, ELISA and immunohistochemistry. C2C12 muscle cells were used to assess Smad activation and induction. TGF-β1, 2 and 3 mRNAs were increased in ALS muscle samples compared to controls and correlated with muscle strength and Smads1, 2, 5 and 8. In the G93A SOD1 mouse, the temporal pattern of TGF-β expression paralleled the Smads and increased with disease progression. TGF-β1 immunoreactivity was detected in mononuclear cells surrounding muscle fibers in ALS samples. In muscle cells, TGF-β ligands were capable of activating Smads. In conclusion, TGF-β1, 2 and 3 are novel biomarkers of ALS in skeletal muscle. Their correlation with weakness in human ALS and their progressive increase with advancing disease in the ALS mouse suggest that they, as with the Smads, can track disease progression. These ligands are capable of upregulating and activating Smads and thus may contribute to the Smad signaling pathway in ALS muscle.

HuR promotes the molecular signature and phenotype of activated microglia: Implications for amyotrophic lateral sclerosis and other neurodegenerative diseases

In neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), chronic activation of microglia contributes to disease progression. Activated microglia produce cytokines, chemokines, and other factors that normally serve to clear infection or damaged tissue either directly or through the recruitment of other immune cells. The molecular program driving this phenotype is classically linked to the transcription factor NF‐κB and characterized by the upregulation of proinflammatory factors such as IL‐1β, TNF‐α, and IL‐6. Here, we investigated the role of HuR, an RNA‐binding protein that regulates gene expression through posttranscriptional pathways, on the molecular and cellular phenotypes of activated microglia. We performed RNA sequencing of HuR‐silenced microglia and found significant attenuation of lipopolysaccharide‐induced IL‐1β and TNF‐α inflammatory pathways and other factors that promote microglial migration and invasion. RNA kinetics and luciferase reporter studies suggested that the attenuation was related to altered promoter activity rather than a change in RNA stability. HuR‐silenced microglia showed reduced migration, invasion, and chemotactic properties but maintained viability. MMP‐12, a target exquisitely sensitive to HuR knockdown, participates in the migration/invasion phenotype. HuR is abundantly detected in the cytoplasmic compartment of activated microglia from ALS spinal cords consistent with its increased activity. Microglia from ALS‐associated mutant SOD1 mice demonstrated higher migration/invasion properties which can be blocked with HuR inhibition. These findings underscore an important role for HuR in sculpting the molecular signature and phenotype of activated microglia, and as a possible therapeutic target in ALS and other neurodegenerative diseases.

Smads as muscle biomarkers in amyotrophic lateral sclerosis.

To identify molecular signatures in muscle from patients with amyotrophic lateral sclerosis (ALS) that could provide insight into the disease process and serve as biomarkers.

Muscle microRNA signatures as biomarkers of disease progression in amyotrophic lateral sclerosis

ALS is a fatal neurodegenerative disorder of motor neurons leading to progressive atrophy and weakness of muscles. Some of the earliest pathophysiological changes occur at the level of skeletal muscle and the neuromuscular junction. We previously identified distinct mRNA patterns, including members of the Smad and TGF-β family, that emerge in muscle tissue at the earliest (pre-clinical) stages. These patterns track disease progression in the mutant SOD1 mouse and are present in human ALS muscle. Because miRNAs play a direct regulatory role in mRNA expression, we hypothesized in this study that there would be distinct miRNA patterns in ALS muscle appearing in early stages that could track disease progression. We performed next-generation miRNA sequencing on muscle samples from G93A SOD1 mice at early (pre-clinical) and late (symptomatic) stages, and identified distinct miRNA patterns at both stages with some overlap. An Ingenuity Pathway Analysis predicted effects on a number of pathways relevant to ALS including TGF-β signaling, axon guidance signaling, and mitochondrial function. A subset of miRNAs was validated in the G93A SOD1 mouse at four stages of disease, and several appeared to track disease progression, including miR-206. We assessed these miRNAs in a large cohort of human ALS and disease control samples and found that some had similar changes but were not specific for ALS. Surprisingly, miR-206 levels did not change overall compared to normal controls, but did correlate with changes in strength of the muscle biopsied. In summary, we identified distinct miRNA patterns in ALS muscle that reflected disease stage which could potentially be used as biomarkers of disease activity.

Mast cells and neutrophils mediate peripheral motor pathway degeneration in ALS

Neuroinflammation is a recognized pathogenic mechanism underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS), but the inflammatory mechanisms influencing peripheral motor axon degeneration remain largely unknown. A recent report showed a pathogenic role for c-Kit-expressing mast cells mediating inflammation and neuromuscular junction denervation in muscles from SOD1G93A rats. Here, we have explored whether mast cells infiltrate skeletal muscles in autopsied muscles from ALS patients. We report that degranulating mast cells were abundant in the quadriceps muscles from ALS subjects but not in controls. Mast cells were associated with myofibers and motor endplates and, remarkably, interacted with neutrophils forming large extracellular traps. Mast cells and neutrophils were also abundant around motor axons in the extensor digitorum longus muscle, sciatic nerve, and ventral roots of symptomatic SOD1G93A rats, indicating that immune cell infiltration extends along the entire peripheral motor pathway. Postparalysis treatment of SOD1G93A rats with the tyrosine kinase inhibitor drug masitinib prevented mast cell and neutrophil infiltration, axonal pathology, secondary demyelination, and the loss of type 2B myofibers, compared with vehicle-treated rats. These findings provide further evidence for a yet unrecognized contribution of immune cells in peripheral motor pathway degeneration that can be therapeutically targeted by tyrosine kinase inhibitors.

Abberrant Posttranscriptional Regulation and Protein Degradtion of TDP-43 and FUS to Stress Respone in ALS (IN9-1.008)

Objective: To further analyze molecular interactions and posttranscriptional regulation of TDP-43, FUS with HuR in the presence of mutant SOD1 and its impact on protein degradation. Background The genetic link of TDP-43 and FUS with familial and sporadic ALS underscores the importance of RNA binding proteins (RBP) and post-transcriptional regulation in the pathogenesis of ALS. HuR, another RBP, shares structural and functional similarities to TDP-43 and FUS. These RBPs provide an important contribution to stress response. We have observed aberrant cross-regulation and protein degradation of a network of RBPs in associated with mutant SOD1. Recent identification of UBQLN 2 mutation accentuated abnormal protein degradation in ALS pathogenesis. Design/Methods: HuR was upregulated ectopically or knocked down using siRNA in primary astrocytes or U251 cells expressing wild-type or mutant SOD1. Cells were subjected to oxidative, hypoxic and hormonal stress. TDP-43 39UTR regulation was measured by a luciferase reporter. Protein stability was analyzed by Western blot after treating cells with cycloheximide for different time intervals. Ubiquitination was quantified with Western blot and an ELISA kit. Results: Following oxidative stress, we observed cytoplasmic association and stress granules of TDP43 and FUS with HuR in G93A astrocytes. In both U251 and astrocytes, TDP-43 and FUS are downregulated by HuR silencing. In the presence of mutant SOD1, their protein half life significantly increased compared to wt cells. With stress induction, we observed accumulation of these RBPs and significantly increased ubiliquitination. Knockdown of TDP-43 and FUS had no effect on HuR protein and RNA level. There was increasing neuronal toxicity when co-cultured with those conditional medium. Conclusions: 1. The posttranscriptional regulation and protein stability of a network of RBPs may play a role in the pathogenesis of ALS. 2. ALS-associated mutant SOD1 and HuR dysfunction may block the ubiquitin proteasome pathway of these RBPs that leads to their aberrant degradation. Supported by: NIH/NINDS Grant NS057664, NS064133 and a merit review from the Department of Veterans Affairs. Disclosure: Dr. Lu has nothing to disclose. Dr. Zheng has nothing to disclose. Dr. Si has nothing to disclose. Dr. Chen has nothing to disclose. Dr. Luo has received personal compensation for activities with XenoPort as an employee. Dr. Oh has nothing to disclose. Dr. King has nothing to disclose.

Abstract 4070: Mutant tristetraprolin enhances growth factor downregulation and inhibition of cell growth in malignant glioma

Background: Tristetraprolin (TTP) negatively regulates short-lived mRNAs by binding to AU-rich elements in the 3′ untranslated region (3′UTR). A number of mRNA targets, including vascular endothelial growth factor (VEGF), and interleukin (IL)-8, play an important role in tumor progression. We have shown previously that TTP downregulates IL-8 and VEGF in malignant glioma (MG) cells via RNA destabilization. In primary glioblastoma tumors, TTP is heavily phosphorylated which may render the protein less active. Hypothesis: We postulate that the destabilizing effect of TTP on target growth factor mRNAs is held in check by hyperphosphorylation which leads to altered subcellular localization and protein stability. Methods: Wild-type TTP and two mutant forms in which select serines were exchanged with alanines were cloned with a Flag epitope into pTRE2 and pEGFP vectors. U251 Tet On MG cells were transfected with pTRE2–TTP plasmids to produce stable clones. TTP expression was induced using doxycycline. Cellular location was assessed by fluorescence microscopy and Western blotting. Protein stability was measured by assessment of TTP levels following cycloheximide treatment. VEGF and IL-8 mRNA and protein levels were measured by qRT-PCR and ELISA. For assessment of RNA half-lives, cells were treated with actinomycin D. Cell growth, proliferation and cell viability were assessed using biochemical assays, soft agar, and Trypan Blue method. Results: Both IL-8 and VEGF mRNA levels were significantly lowered in the mutant TTP clones compared to wild-type. Half-lives of these mRNAs were also shortened, and ELISA analysis showed a correspondingly greater decrease in protein expression. There was a marked impact on cellular phenotype with greater inhibition of proliferation and loss of cell viability. A Soft agar colony formation was decreased by five-fold in mutant clones versus wild-type and a greater than ten-fold decrease versus the parent cell line. With TNF-α, there was a significant shift of mutant TTP to the nuclear compartment compared to WT TTP. Treatment with cycloheximide revealed that the WT TTP was less stable compared to dephosphorylated mutant. Conclusion: Removal of key serine residues from TTP enhances its function as an RNA destabilizer of VEGF and IL-8 mRNs and as a negative regulator of tumor cell growth in MG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4070.