Ying-Ju Sung

The fragile X mental retardation protein interacts with U-rich RNAs in a yeast three-hybrid system ☆

We recently identified several ESTs that bind to the fragile X mental retardation protein (FMRP) in vitro. To determine whether they interacted in vivo we performed three-hybrid screens in a Saccharomyces cerevisiae histidine auxotroph. We demonstrate that two of the ESTs support growth on histidine and transduce beta-galactosidase activity when co-expressed with FMRP under selective growth conditions. In contrast, the iron response element (IRE) RNA does not. Likewise, the ESTs do not support growth or transduce beta-galactosidase activity when co-expressed with the iron response element binding protein (IRP). Each EST is relatively small and has 40% identity with a sequence in FMR1 mRNA harboring FMRP binding determinants. Interestingly, while neither the ESTs contain a G-quartet structural motif they do contain U-rich sequences that are found in mRNA with demonstrated in vitro binding and in vivo association with FMRP. This indicates that U-rich elements comprise another motif recognized by FMRP.

Activation and retrograde transport of protein kinase G in rat nociceptive neurons after nerve injury and inflammation

Nerve injury elicits both universal and limited responses. Among the former is regenerative growth, which occurs in most peripheral neurons, and among the latter is the long-term hyperexcitability that appears selectively in nociceptive sensory neurons. Since positive injury signals communicate information from the site of an injury to the cell body, we hypothesize that a nerve injury activates both universal and limited positive injury signals. Studies in Aplysia indicate that protein kinase G is a limited signal that is responsible for the induction of long-term hyperexcitability. Given that long-term hyperexcitability contributes to chronic pain after axotomy in rodent neuropathic pain models, we investigated its underlying basis in the rat peripheral nervous system. Using biochemical assays, Western blots, and immunocytochemistry we found that the Type 1alpha protein kinase G is the predominant isoform in the rat periphery. It is present primarily in axons and cell bodies of nociceptive neurons, including populations that are isolectin B4-positive, isolectin B4-negative, and those that express transient receptor potential vanilloid receptor-1. Surprisingly, protein kinase G is not present in the facial nerve, which overwhelmingly contains axons of motor neurons. Crushing the sciatic nerve or a cutaneous sensory nerve activates protein kinase G in axons and results in its retrograde transport to the neuronal somata in the DRG. Preventing the activation of protein kinase G by injecting Rp-8-pCPT-cGMPS into the crush site abolished the transport. The protein kinase A inhibitor Rp-8-pCPT-cAMPS had no effect. Extracellular signal-related kinases 42/44 are also activated and transported after nerve crush, but in both motor and sensory axons. Chronic pain has been linked to long-term hyperexcitability following a nerve inflammation in several rodent models. We therefore injected complete Freunds adjuvant into the hindpaw to induce an inflammation and found that protein kinase G was activated in the sural nerve and transported to the DRG. In contrast, the extracellular signal-related kinases in the sensory axons were not activated by the complete Freunds adjuvant. These studies support the idea that the extracellular signal-related kinases are universal positive axonal signals and that protein kinase G is a limited positive axonal signal. They also establish the association between protein kinase G, the induction of long-term hyperexcitability, and chronic pain in rodents.

Pathways that elicit long-term changes in gene expression in nociceptive neurons following nerve injury: contributions to neuropathic pain

Abstract Chronic neuropathic pain following nerve injury or inflammation is mediated by transcription-dependent changes in neurons that comprise the nociceptive pathway. Among these changes is often a long-term hyperexcitiability (LTH) in primary nociceptors that persists long after the lesion has healed. LTH is manifest by a reduction in threshold and an increased tendency to fire action potentials. This increased excitability activates higher order neurons in the pathway, leading to the perception of pain. Efforts to ameliorate chronic pain would therefore benefit if we understood how LTH is induced, but studies toward this goal are impeded by the complexity and heterogeneity of vertebrate nervous systems. Fortunately, LTH is an evolutionarily conserved mechanism that underlies defensive behaviors across phyla, including invertebrates. Thus, the same electrophysiological changes that underlie LTH in vertebrate nociceptive neurons are seen in their counterparts in the experimentally favorable mollusk Aplysia californica. Nociceptive neurons of Aplysia are readily accessible and large enough to approach using a variety of cell and molecular approaches not possible in higher organisms. Studies of the molecular cascades activated by injury to Aplysia peripheral nerves has focused on a group of positive injury signals that are retrogradely transported from the injury site in the axon to the cell nucleus where they regulate gene transcription. One of these, protein kinase G, is activated by nitric oxide synthetase and its activation in axons is required for the induction of LTH after injury. This pathway, and the transcriptional events that it activates, are targets for therapeutic intervention for chronic pain.

Up-regulation of apoptosis and regeneration genes in the dorsal root ganglia during cisplatin treatment

Cisplatin is an effective anti-neoplastic drug, but its use is dose-limited due to its association with severe peripheral neurotoxicity. The neurotoxic effect of cisplatin is believed to result from its accumulation in the dorsal root ganglia (DRG), although the mechanism is not completely understood. We used a rat model of cisplatin neurotoxicity to examine changes in gene expression in the DRG. The results indicate that cisplatin affects the expression of several genes associated with apoptosis (Cdkn1a, Ckap2, Bid3, S100a8, S100a9), inflammation (S100a8, S100a9, Cd163, Mmp9), and nerve growth and regeneration (Mmp9, Gfap, Fabp7). The differential regulation of some of these genes may directly contribute to the neurotoxic effect of cisplatin, while others are likely to be representative of the subsequent cellular response to contain damage and initiate recovery. As such, the identified genes may represent candidate processes and pathways that should be considered as targets for therapeutic intervention in cisplatin-induced neuropathy.

Regulating a translational regulator: mechanisms cells use to control the activity of the fragile X mental retardation protein

Fragile X syndrome results from the loss of a normal cellular protein, FMRP. FMRP is an RNA binding protein, and it is likely that altering the way FMRP’s messenger RNA (mRNA) targets are processed results in the clinical features associated with the disease. Using complementary DNA microarray screening, a number of brain-derived mRNAs that interact directly with FMRP in vitro and associate with FMRP-containing mRNPs in vivo have been identified. These target messages encode RNA-binding proteins, transcription factors, neuronal receptors, cytoskeletal proteins, a few enzymes as well as several unknown proteins. For a subset of these mRNAs it has been shown that modulating FMRP levels in cultured cells correspondingly affects their expression. In addition, several modes by which cells modulate FMRP activity have been described; these include posttranscriptional processing and posttranslational modification. Here, the most recent results concerning the biochemical activities of FMRP and how they are affected by various modifications are reviewed. The data lead to a model signaling mechanism by which FMRP normally regulates the expression of its target mRNAs.

cJun and CREB2 in the Postsynaptic Neuron Contribute to Persistent Long-Term Facilitation at a Behaviorally Relevant Synapse

Basic region leucine zipper (bZIP) transcription factors regulate gene expression critical for long-term synaptic plasticity or neuronal excitability contributing to learning and memory. At sensorimotor synapses of Aplysia, changes in activation or expression of CREB1 and CREB2 in sensory neurons are required for long-term synaptic plasticity. However, it is unknown whether concomitant stimulus-induced changes in expression and activation of bZIP transcription factors in the postsynaptic motor neuron also contribute to persistent long-term facilitation (P-LTF). We overexpressed various forms of CREB1, CREB2, or cJun in the postsynaptic motor neuron L7 in cell culture to examine whether these factors contribute to P-LTF. P-LTF is evoked by 2 consecutive days of 5-HT applications (2 5-HT), while a transient form of LTF is produced by 1 day of 5-HT applications (1 5-HT). Significant increases in the expression of both cJun and CREB2 mRNA in L7 accompany P-LTF. Overexpressing each bZIP factor in L7 did not alter basal synapse strength, while coexpressing cJun and CREB2 in L7 evoked persistent increases in basal synapse strength. In contrast, overexpressing cJun and CREB2 in sensory neurons evoked persistent decreases in basal synapse strength. Overexpressing wild-type cJun or CREB2, but not CREB1, in L7 can replace the second day of 5-HT applications in producing P-LTF. Reducing cJun activity in L7 blocked P-LTF evoked by 2 5-HT. These results suggest that expression and activation of different bZIP factors in both presynaptic and postsynaptic neurons contribute to persistent change in synapse strength including stimulus-dependent long-term synaptic plasticity.

mRNAs encoding the Aplysia homologues of fasciclin‐I and β‐thymosin are expressed only in the second phase of nerve injury and are differentially segregated in axons regenerating in vitro and in vivo

Studies using Aplysia californica have demonstrated that transcription after nerve injury occurs during a rapid, transient first phase and a delayed, prolonged second phase. Although the second phase is especially important for regeneration, the mRNAs produced during this phase have not been identified. We characterized two such mRNAs following axotomy. One encodes a novel fasciclin‐I homologue, Aplysia fasciclin‐like protein (apFasP), and the other encodes Aplysia β‐thymosin (apβT). In addition to mRNA synthesis, proteins required for regeneration must be available at the site of growth, and the transport and local translation of certain extrasomatic mRNAs aids in this process. We found apβT and apFasP proteins and mRNA at growth cones in vitro. However, only the mRNA for apβT was present in regenerating axons in vivo. This implies that the membrane protein apFasP is supplied by rapid transport from the soma, whereas the soluble apβT is synthesized locally.

Synaptogenesis Regulates Axotomy-Induced Activation of c-Jun–Activator Protein-1 Transcription

The activator protein-1 (AP1) transcription complex remains active for long periods after axotomy, but its activity diminishes during target contact. This raises the possibility that the function of this complex is regulated by the synaptic connections. Using Aplysia californica, we found that crushing peripheral nerves in vivo enhanced AP1 binding in the sensory neurons that lasted for weeks and then declined as regeneration was completed. The AP1 complex in Aplysia is a c-Jun homodimer. Its activation, after axotomy, is mediated by Aplysia c-Jun–N-terminal kinase (apJNK), which enters the nucleus of sensory neurons and phosphorylates c-Jun at Ser-73 (p73-c-Jun). Active AP1 in the sensory neurons did not mediate apoptosis and was not involved in the appearance of the long-term hyperexcitability that develops in these cells after axotomy, and blocking the activation of apJNK in vitro did not influence neurite outgrowth. In contrast, the levels of activated apJNK and p73-c-Jun declined markedly when sensory neurons formed synapses with motor neuron L7 in vitro. Furthermore, inhibiting the pathway accelerated synaptogenesis between sensory neurons and L7. These data suggest that positive and negative modulation of the JNK–c-Jun–AP1 pathway functions in alerting the nucleus to the loss and gain of synapses, respectively.

A novel inhibitor of active protein kinase G attenuates chronic inflammatory and osteoarthritic pain

Abstract Activating PKG-1&agr; induces a long-term hyperexcitability (LTH) in nociceptive neurons. Since the LTH correlates directly with chronic pain in many animal models, we tested the hypothesis that inhibiting PKG-1&agr; would attenuate LTH-mediated pain. We first synthesized and characterized compound N46 (N-((3R,4R)-4-(4-(2-fluoro-3-methoxy-6-propoxybenzoyl)benzamido)pyrrolidin-3-yl)-1H-indazole-5-carboxamide). N46 inhibits PKG-1&agr; with an IC50 of 7.5 nmol, was highly selective when tested against a panel of 274 kinases, and tissue distribution studies indicate that it does not enter the CNS. To evaluate its antinociceptive potential, we used 2 animal models in which the pain involves both activated PKG-1&agr; and LTH. Injecting complete Freunds adjuvant (CFA) into the rat hind paw causes a thermal hyperalgesia that was significantly attenuated 24 hours after a single intravenous injection of N46. Next, we used a rat model of osteoarthritic knee joint pain and found that a single intra-articular injection of N46 alleviated the pain 14 days after the pain was established and the relief lasted for 7 days. Thermal hyperalgesia and osteoarthritic pain are also associated with the activation of the capsaicin-activated transient receptor protein vanilloid-1 (TRPV1) channel. We show that capsaicin activates PKG-1&agr; in nerves and that a subcutaneous delivery of N46 attenuated the mechanical and thermal hypersensitivity elicited by exposure to capsaicin. Thus, PKG-1&agr; appears to be downstream of the transient receptor protein vanilloid-1. Our studies provide proof of concept in animal models that a PKG-1&agr; antagonist has a powerful antinociceptive effect on persistent, already existing inflammatory pain. They further suggest that N46 is a valid chemotype for the further development of such antagonists.

GABAAergic stimulation modulates intracellular protein arginine methylation.

Changes in cytoplasmic pH are known to regulate diverse cellular processes and influence neuronal activities. In neurons, the intracellular alkalization is shown to occur after stimulating several channels and receptors. For example, it has previously demonstrated in P19 neurons that a sustained intracellular alkalinization can be mediated by the Na(+)/H(+) antiporter. In addition, the benzodiazepine binding subtypes of the γ-amino butyric acid type A (GABAA) receptor mediate a transient intracellular alkalinization when they are stimulated. Because the activities of many enzymes are sensitive to pH shift, here we investigate the effects of intracellular pH modulation resulted from stimulating GABAA receptor on the protein arginine methyltransferases (PRMT) activities. We show that the major benzodiazepine subtype (2α1, 2β2, 1γ2) is constitutively expressed in both undifferentiated P19 cells and retinoic acid (RA) differentiated P19 neurons. Furthermore stimulation with diazepam and, diazepam plus muscimol produce an intracellular alkalinization that can be detected ex vivo with the fluorescence dye. The alkalinization results in significant perturbation in protein arginine methylation activity as measured in methylation assays with specific protein substrates. Altered protein arginine methylation is also observed when cells are treated with the GABAA agonist muscimol but not an antagonist, bicuculline. These data suggest that pH-dependent and pH-independent methylation pathways can be activated by GABAAergic stimulation, which we verified using hippocampal slice preparations from a mouse model of fragile X syndrome.