Chien-liang Glenn Lin

Translational Control of Glial Glutamate Transporter EAAT2 Expression

Glutamate is the major excitatory neurotransmitter in the central nervous system. Its activity is carefully modulated in the synaptic cleft by glutamate transporters. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance. Reduced EAAT2 function could lead to accumulation of extracellular glutamate, resulting in a form of cell death known as excitotoxicity. In amyotrophic lateral sclerosis and Alzheimer disease, EAAT2 protein levels are significantly decreased in affected areas. EAAT2 mRNA levels, however, remain constant, indicating that alterations in EAAT2 expression are due to disturbances at the post-transcriptional level. In the present study, we found that some EAAT2 transcripts contained 5′-untranslated regions (5′-UTRs) greater than 300 nucleotides. The mRNAs that bear long 5′-UTRs are often regulated at the translational level. We tested this possibility initially in a primary astrocyte line that constantly expressed an EAAT2 transcript containing the 565-nt 5′-UTR and found that translation of this transcript was regulated by many extracellular factors, including corticosterone and retinol. Moreover, many disease-associated insults affected the efficiency of translation of this transcript. Importantly, this translational regulation of EAAT2 occurred in vivo (i.e. both in primary cortical neurons-astrocytes mixed cultures and in mice). These results indicate that expression of EAAT2 protein is highly regulated at the translational level and also suggest that translational regulation may play an important role in the differential EAAT2 protein expression under normal and disease conditions.

Molecular cloning, gene structure, expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3-18

Glutamate is an important amino acid implicated in energy metabolism, protein biosynthesis and neurotransmission. The Na(+)-dependent high-affinity excitatory amino acid transporter EAAT3 (EAAC1) facilitates glutamate uptake into most cells. Recently, a novel rat EAAT3-interacting protein called GTRAP3-18 has been identified by a yeast two-hybrid screening. GTRAP3-18 functions as a negative modulator of EAAT3-mediated glutamate transport. In order to further understand the function and regulation of GTRAP3-18, we cloned the mouse orthologue to GTRAP3-18 and determined its gene structure and its expression pattern. GTRAP3-18 encodes a 188-residue hydrophobic protein whose sequence is highly conserved amongst vertebrates. Mouse and human GTRAP3-18 genes contain three exons separated by two introns. The GTRAP3-18 gene is found on mouse chromosome 6D3 and on human chromosome 3p14, a susceptibility locus for cancer and epilepsy. GTRAP3-18 protein and RNA were found both in neuronal rich regions of the brain and in non-neuronal tissues such as the kidney, heart and skeletal muscle. Mouse GTRAP3-18 inhibited EAAT3-mediated glutamate transport in a dose-dependent manner. These studies show that GTRAP3-18 is a ubiquitously expressed protein that functions as a negative regulator of EAAT3 function.

Increased expression of cholesterol 24S‐hydroxylase results in disruption of glial glutamate transporter EAAT2 association with lipid rafts: a potential role in Alzheimer’s disease

J. Neurochem. (2010) 113, 978–989.

Human glioma cells and undifferentiated primary astrocytes that express aberrant EAAT2 mRNA inhibit normal EAAT2 protein expression and prevent cell death.

Abnormal splicing of astroglial glutamate transporter EAAT2 mRNA has been suggested to account for the loss of EAAT2 protein in amyotrophic lateral sclerosis (ALS) and Alzheimers disease (AD). We have identified several clones of human U251 glioma cells which express varying amounts of aberrantly spliced EAAT2 mRNA; these clones do not express any detectable EAAT2 protein. When the wild-type EAAT2 cDNA was expressed in each of these clones, we found that the amount of EAAT2 protein inversely correlated with the levels of endogenous aberrant EAAT2 mRNA. We also observed that ectopic expression of normal EAAT2 protein is toxic to U251 cells as well as to undifferentiated primary astrocytes. We conclude that expression of aberrant EAAT2 mRNA may be one possible mechanism to repress normal EAAT2 protein expression. The implication of this study for the mechanisms of EAAT2 protein loss in ALS and AD is discussed.

Increased glial glutamate transporter EAAT2 expression reduces visceral nociceptive response in mice

Visceral hypersensitivity is the leading complaint of functional bowel disorders. Central sensitization mediated by glutamate receptor activation is implicated in pathophysiology of visceral pain. The glial glutamate transporter EAAT2 is the principal mediator of glutamate clearance to terminate glutamate-mediated responses. Transgenic mice overexpressing human EAAT2 (EAAT2 mice), which exhibited a twofold enhanced glutamate uptake, showed 39% less writhing response to intraperitoneal acetic acid than nontransgenic littermates. Moreover, EAAT2 transgenic mice showed a 53-64% reduction in visceromotor response (VMR) to colorectal distension (CRD) in assessments of the response to graded increase in pressures. Corroborating the involvement of enhanced glutamate uptake, wild-type mice treated for 1 wk with ceftriaxone, an EAAT2 expression activator, showed a 49-70% reduction in VMR to CRD. Moreover, systemic pretreatment with the selective EAAT2 transporter blocker dihydrokainate reversed the ceftriaxone-blunted nociceptive response to CRD. However, the enhanced VMR to CRD produced by intracolonic ethanol was not significantly attenuated by 1-wk ceftriaxone pretreatment. The data suggest that enhanced glutamate uptake provides protective effects against colonic distension-induced nociception and represents an exciting new mechanistic approach leading to better therapeutic options to visceral pain disorders.

Methyl-β-cyclodextrin but not retinoic acid reduces EAAT3-mediated glutamate uptake and increases GTRAP3-18 expression

The Na+‐dependent glutamate transporter EAAT3 facilitates glutamate uptake into neurons as well as many other cell types. GTRAP3‐18 (JWA, Arl6ip5) is a novel protein that interacts with EAAT3 and negatively modulates EAAT3‐mediated glutamate uptake. Previous studies suggest that retinoic acid (RA) decreases Na+‐dependent glutamate uptake and increases GTRAP3‐18 protein expression. However, the RA used in those studies was complexed with methyl‐β‐cyclodextrin (MeβCD). In the present study we found that MeβCD, but not RA, significantly reduced Na+‐dependent EAAT3‐mediated [3H]glutamate uptake in human embryonic kidney 293 (HEK293) cells. MeβCD also significantly increased GTRAP3‐18 protein expression in HEK293 cells as well as in rat hypothalamic neuron cultures. Intracerebroventricular administration of MeβCD to the mouse brain resulted in a significant increase in GTRAP3‐18 immunoreactivity in the hippocampus and cerebral cortex. In conclusion, we have shown that MeβCD reduces EAAT3‐mediated glutamate uptake and induces the expression of GTRAP3‐18 protein.

RNA oxidation: A contributing factor or an epiphenomenon in the process of neurodegeneration

In the past decade, RNA oxidation has caught the attention of many researchers, working to uncover its role in the pathogenesis of neurodegenerative diseases. It has been well documented that RNA oxidation is involved in a wide variety of neurological diseases and is an early event in the process of neurodegeneration. The analysis of oxidized RNA species revealed that at least messenger RNA (mRNA) and ribosomal RNA (rRNA) are damaged in several neurodegenerative diseases, including Alzheimers disease and amyotrophic lateral sclerosis (ALS). The magnitude of the RNA oxidation, at least in mRNA, is significantly high at the early stage of the disease. Oxidative damage to mRNA is not random but selective and many oxidized mRNAs are related to the pathogenesis of the disease. Several studies have suggested that oxidative modification of RNA affects the translational process and consequently produces less protein and/or defective protein. Furthermore, several proteins have been identified to be involved in handling of damaged RNA. Although a growing body of studies suggests that oxidative damage to RNA may be associated with neuron deterioration, further investigation and solid evidence are needed. In addition, further uncovering of the consequences and cellular handling of the oxidatively damaged RNA should be important focuses in this area and may provide significant insights into the pathogenesis of neurodegenerative diseases.

The Importance of Preclinical Trial Timing – a Potential Reason for the Disconnect between mouse Studies and Human Clinical Trials in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive degeneration of motor neurons in the spinal cord, motor cortex, and brainstem. There is currently no effective treatment for ALS. Transgenic mice expressing familiar ALS-linked mutant superoxide dismutase (SOD1) have been commonly used for preclinical trials; however, successful trails in mice mostly fail in subsequent human trials. Most preclinical trials start treatment before the onset of symptoms, while ALS patients in clinical trials have surpassed the onset of disease. In addition, ALS is not only a multi-factorial disease but also a multi-systemic disease that affects several cell types [1]. Therefore, therapies should be aimed at the interception of multiple mechanisms. In the present study, we investigated (1) whether the therapeutic agents, which were previously tested starting before disease onset, still showed protective effects when the treatments were initiated after disease onset and (2) whether combination therapies offered better effects after disease onset. Minocycline, which inhibits microglia activation and apoptotic cascade, was previously found to be effective at ameliorating motor impairment and increasing the lifespan of SOD1G93A mice when administered at 5 weeks of age [2]. However, minocycline fails in human trials [3]. We assessed whether similar beneficial effects could be seen when treatment began at 90 days of age. This time point was chosen because SOD1G93A mice [4] exhibited 10~20% decline in motor function at this age, which may be equivalent to the stage that ALS patients first visit a doctor. The results showed no delay in impaired motor function (Fig. 1A&B, Mino) and no extension in survival (Fig. 1C&D, Mino). This result is consistent with a recent report [5]. Ceftriaxone, which reduces excitotoxicity via activation of glial glutamate transporter EAAT2, was found to slow disease progression in SOD1G93A mice when administered at 12 weeks of age [6]. Ceftriaxone is currently in clinical trials for ALS. We found that when ceftriaxone was administered starting from 90 days of age, treatment no longer slowed disease progression (Fig. 1A&B, Cfx) and survival (Fig. 1C&D, Cfx). We then assessed whether the combined treatments of ceftriaxone and minocycline, starting at 90 days of age, would have protective effects. The results showed no delay in impaired motor function (Fig. 1A&B, Double) but a significant increase in survival by ~12 days (Fig. 1C&D, Double). Figure 1 Effects of single and combination treatments on disease onset and survival of SOD1G93A mice. SOD1G93A mice (B6SJL-Tg(SOD1-G93A)1Gur/J with high copy number of the mutant human SOD1 gene, Jackson Laboratory) were used in this study (the cited previous ... Vitamin E, which reduces oxidative damage, can delay disease onset when administered at 30 days of age, but has no effect in extending the lifespan of SOD1G93A mice [7], only slightly slowing disease progression in human ALS trials [8,9]. We found that when mice were treated with vitamin E starting at 30 days of age and then with ceftriaxone and minocycline starting at 90 days of age, a significant delay in motor dysfunction by ~18 days (Fig. 1A&B, Triple D30) and an extension in survival by ~16 days (Fig. 1C&D, Triple D30) were observed. Furthermore, we assessed the combined three treatments starting at 90 days of age. This triple treatment resulted in greater improvement of motor performance than that seen in the double treatment (ceftriaxone and minocycline) (Fig. 1A&B, Triple D90). The prolonged survival for the triple treatment was similar to that seen in the double treatment (Fig. 1C&D, Triple D90). To confirm that the protective effects of combined treatments were due to the inhibition of targeted mechanisms, we performed immunofluorescent staining on lumbar spinal cord sections prepared from treated SOD1G93A mice and wild-type littermates at 115 days of age. As shown in Fig. 2A, EAAT2 protein levels were restored in treated mice (in the double and triple groups). Oxidative damage as measured RNA oxidation (15A3 staining) was also reduced [7]. Astrogliosis (GFAP staining) and microglial activation (CD11b staining) in SOD1G93A mice were increased compared to that seen in wild-type littermates. There was even greater enhanced glial activation by the cocktail treatments - the double and triple groups, which is consistent with a recent report [5]. Furthermore, to confirm that the delayed disease onset and prolonged survival were associated with reduced neuronal loss, we performed cresyl violet staining on lumbar spinal cord adjacent sections. A significant reduction of motor neuron loss was observed in the triple groups (Fig. 2B). Figure 2 Effects of combination treatments on oxidative damage, EAAT2 expression, glial activation, and motor neuron loss in SOD1G93A mice. Immunofluorescent (A) and Cresyl violet (B) stainings were performed on lumbar spinal cord sections prepared from treated ... Transition from preclinical mouse studies to human clinical trials is difficult for most diseases. As the first step, proper design of preclinical trials is very important. The results of this study suggest that inappropriate pre-symptomatic treatment in mice may partially provide explanations for the failure of some clinical trials in ALS patients. In addition, combination treatments may serve as a therapeutic strategy for human trials. The agents that have been unsuccessful in human trials may prove to be more beneficial when used in combination with one another.

A novel noncoding RNA rescues mutant SOD1-mediated cell death

Transgenic mice expressing mutant Cu2+/ Zn2+ superoxide dismutase SOD1(G93A) develop similar clinical and pathological phenotypes to amyotro‐phic lateral sclerosis (ALS) patients. Here, we utilize representational difference analysis to identify the transcripts that are up‐regulated in the presymptomatic stage of SOD1(G93A) mice. Unexpectedly, three predominant clones were 18S or 28S ribosomal RNA (rRNA) segments. One of these clones corresponded to a capped and polyadenylated transcript containing a large portion of 18S rRNA, named MSUR1 (mutant SOD1‐up‐regulated RNA 1). In vitro expression experiments show that MSUR1 is able to rescue SOD1(G93A)‐mediated cell death. Expression of MSUR1 significantly reduces SOD1(G93A)‐induced free radical levels and oxidative damage. Further, MSUR1 can reduce hydrogen peroxide‐mediated cytotoxicity. MSUR1 does not encode a protein, suggesting its role as a functional noncoding RNA. It is widely expressed in various tissues. Searching the database of GenBank revealed that a large number of expressed sequence tag (EST) clones contain large portions of rRNAsequence, potentially indicating a heretofore overlooked class of mRNAs with functional significance.—Chang Y., Stockinger, M. P., Tashiro, H., Glenn Lin C. A novel noncoding RNA rescues mutant SOD1‐mediated cell death. FASEB J. 22, 691–702 (2008)