Fanny Chun Fun Ip

Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction

Here we describe an important involvement of Cdk5/p35 in regulating the gene expression of acetylcholine receptor (AChR) at the neuromuscular synapse. Cdk5 and p35 were prominently expressed in embryonic muscle, and concentrated at the neuromuscular junction in adulthood. Neuregulin increased the p35-associated Cdk5 kinase activity in the membrane fraction of cultured C2C12 myotubes. Co-immunoprecipitation studies revealed the association between Cdk5, p35 and ErbB receptors in muscle and cultured myotubes. Inhibition of Cdk5 activity not only blocked the NRG-induced AChR transcription, but also attenuated ErbB activation in cultured myotubes. In light of our finding that overexpression of p35 alone led to an increase in AChR promoter activity in muscle, Cdk5 activation is sufficient to mediate the up-regulation of AChR gene expression. Taken together, these results reveal the unexpected involvement of Cdk5/p35 in neuregulin signaling at the neuromuscular synapse.

Cloning and expression of a novel neurotrophin, NT-7, from carp.

Neurotrophins have been demonstrated to play important roles in the development and functioning of the nervous system. This family of proteins consists of four homologous members in mammals: NGF, BDNF, NT-3, and NT-4/5. A new member, called NT-6, was recently cloned from the platyfish Xiphophorus maculatus. This protein shares closer structural relationship to NGF than the other neurotrophins, but contains a characteristic insertion of 22 amino acids that constituted the heparin-binding domain. Here we report the cloning of a novel neurotrophin from the fish Cyprinus carpio (carp), which shared about 66% amino acid identity to Xiphophorus NGF and NT-6. The neurotrophin, designated NT-7, possesses structural characteristics common to all known neurotrophins, such as the presence of six conserved cysteine residues and the flanking conserved sequences. In addition, there is an insertion of 15 amino acids at the position corresponding to that observed for NT-6. The neurotrophic activity of NT-7 was demonstrated by its ability to promote neurite outgrowth and neuronal survival of chick dorsal root ganglia. Phosphorylation assay of various Trk receptors overexpressed in fibroblasts suggested that NT-7 could activate TrkA but not TrkB or TrkC. Northern blot analysis revealed that NT-7 was predominantly expressed in peripheral tissues, though weak expression was also detected in the brain. Like NT-6, this novel neurotrophin might represent yet another NGF-like neurotrophin in lower vertebrates.

Tyk2/STAT3 Signaling Mediates β-Amyloid-Induced Neuronal Cell Death: Implications in Alzheimer's Disease

One of the pathological hallmarks of Alzheimers disease (AD) is deposition of extracellular amyloid-β (Aβ) peptide, which is generated from the cleavage of amyloid precursor protein (APP). Accumulation of Aβ is thought to associate with the progressive neuronal death observed in AD. However, the precise signaling mechanisms underlying the action of Aβ in AD pathophysiology are not completely understood. Here, we report the involvement of the transcription factor signal transducer and activator of transcription 3 (STAT3) in mediating Aβ-induced neuronal death. We find that tyrosine phosphorylation of STAT3 is elevated in the cortex and hippocampus of APP/PS1 transgenic mice. Treatment of cultured rat neurons with Aβ or intrahippocampal injection of mice with Aβ both induces tyrosine phosphorylation of STAT3 in neurons. Importantly, reduction of either the expression or activation of STAT3 markedly attenuates Aβ-induced neuronal apoptosis, suggesting that STAT3 activation contributes to neuronal death after Aβ exposure. We further identify Tyk2 as the tyrosine kinase that acts upstream of STAT3, as Aβ-induced activation of STAT3 and caspase-3-dependent neuronal death can be inhibited in tyk2−/− neurons. Finally, increased tyrosine phosphorylation of STAT3 is also observed in postmortem brains of AD patients. Our observations collectively reveal a novel role of STAT3 in Aβ-induced neuronal death and suggest the potential involvement of Tyk2/STAT3 signaling in AD pathophysiology.

Expression of Eph receptors in skeletal muscle and their localization at the neuromuscular junction

The participation of ephrins and Eph receptors in guiding motor axons during muscle innervation has been well documented, but little is known about their expression and functional significance in muscle at later developmental stages. Our present study investigates the expression and localization of Eph receptors and ephrins in skeletal muscle. Prominent expression of EphA4, EphA7, and ephrin-A ligands was detected in muscle during embryonic development. More importantly, both EphA4 and EphA7, as well as ephrin-A2, were localized at the neuromuscular junction (NMJ) of adult muscle. Despite their relative abundance, they were not localized at the synapses during embryonic stages. The concentration of EphA4, EphA7, and ephrin-A2 at the NMJ was observed at postnatal stages and the synaptic localization became prominent at later developmental stages. In addition, expression of Eph receptors was increased by neuregulin and after nerve injury. Furthermore, we demonstrated that overexpression of EphA4 led to tyrosine phosphorylation of the actin-binding protein cortactin and that EphA4 was coimmunoprecipitated with cortactin in muscle. Taken together, our findings indicate that EphA4 is associated with the actin cytoskeleton. Since actin cytoskeleton is critical to the formation and stability of NMJ, the present findings raise the intriguing possibility that Eph receptors may have a novel role in NMJ formation and/or maintenance.

Autoinhibition of X11/Mint scaffold proteins revealed by the closed conformation of the PDZ tandem

Members of the X11/Mint family of multidomain adaptor proteins are composed of a divergent N terminus, a conserved PTB domain and a pair of C-terminal PDZ domains. Many proteins can interact with the PDZ tandem of X11 proteins, although the mechanism of such interactions is unclear. Here we show that the highly conserved C-terminal tail of X11α folds back and inserts into the target-binding groove of the first PDZ domain. The binding of this tail occludes the binding of other target peptides. This autoinhibited conformation of X11 requires that the two PDZ domains and the entire C-terminal tail be covalently connected to form an integral structural unit. The autoinhibited conformation of the X11 PDZ tandem provides a mechanistic explanation for the unique target-binding properties of the protein and hints at potential regulatory mechanisms for the X11–target interactions.

Identification and Characterization of Differentially Expressed Genes in Denervated Muscle

Denervation results in a series of changes in skeletal muscle. To elucidate the molecular basis underlying these changes, it is important to identify the profile of altered gene expression in skeletal muscle following nerve injury. In the present study, we have examined the differentially expressed genes in denervated gastrocnemius muscle using RNA fingerprinting by arbitrarily primed PCR. Eight differentially expressed mRNA transcripts have been identified. A bilateral regulatory profile can be observed for the up-regulated genes in both denervated and contralateral control muscle following unilateral sciatic nerve injury. The temporal expression profiles of the denervation-regulated genes in muscle during development, together with their dependency on nerve activity, suggest potential functional roles following nerve injury in vivo. In particular, the identification of two apoptosis-related genes in denervated muscle provides molecular evidence that the apoptotic process is likely to be involved in the intricate changes that lead to muscle atrophy. Our findings not only allow the identification of novel genes, but also suggest possible functions for some known genes in muscle following nerve injury. Taken together, these findings provide important insights into our understanding of the molecular events in denervated muscle and suggest that the differentially expressed genes may play potential roles during muscle denervation and regeneration.

The expression profiles of neurotrophins and their receptors in rat and chicken tissues during development

Neurotrophic factors are target-derived proteins that promote the survival and differentiation of the innervating neurons. Increasing evidence indicate the involvement of these factors and receptors during the formation and maturation of the neuromuscular junction. To gain further insight on the expression pattern of these factors and receptors in developing spinal cord and skeletal muscle during the critical stages of synapse formation, a systematic study was performed with chicken and rat tissues using Northern blot analysis. The expression of all the neurotrophins was detected in skeletal muscle early in development, coincidental with the appearance of their corresponding receptors in the spinal cord. Taken together, the similar regulatory patterns observed in both rat and chicken tissues suggest that the potential roles of neurotrophins at the neuromuscular synapse are conserved throughout evolution.

Ephexin1 Is Required for Structural Maturation and Neurotransmission at the Neuromuscular Junction

The maturation of neuromuscular junctions (NMJs) requires the topological transformation of postsynaptic acetylcholine receptor (AChR)-containing structures from a simple plaque to an elaborate structure composed of pretzel-like branches. This maturation process results in the precise apposition of the presynaptic and postsynaptic specializations. However, little is known about the molecular mechanisms underlying the plaque-to-pretzel transition of AChR clusters. In this study, we identify an essential role for the RhoGEF ephexin1 in the maturation of AChR clusters. Adult ephexin1(-/-) mice exhibit severe muscle weakness and impaired synaptic transmission at the NMJ. Intriguingly, when ephexin1 expression is deficient in vivo, the NMJ fails to mature into the pretzel-like shape, and such abnormalities can be rescued by re-expression of ephexin1. We further demonstrate that ephexin1 regulates the stability of AChR clusters in a RhoA-dependent manner. Taken together, our findings reveal an indispensible role for ephexin1 in regulating the structural maturation and neurotransmission of NMJs.

Induction of Cdk5 activity in rat skeletal muscle after nerve injury.

Cyclin-dependent kinase 5 (Cdk5) was originally identified as a serine/threonine kinase and subsequently demonstrated to play a critical role in the development of CNS. We recently reported the novel function of Cdk5 in the neuregulin signaling pathway during the development of neuromuscular junction (NMJ). Here, we report the regulation of Cdk5 and p35 in rat skeletal muscle after nerve injury. Northern blot analysis revealed that Cdk5 and p35 transcripts were up-regulated in muscle after nerve denervation. The temporal profiles for the regulation of Cdk5 and p35 transcripts were different, suggesting that these changes in gene transcription might be regulated by different mechanism. Our finding on the ability of tetrodotoxin to induce p35 transcript in muscle suggested that electrical activity could regulate p35 expression. In addition to the induction of mRNA expression, the total Cdk5 and p35-associated kinase activity in muscle increased prominently after nerve denervation. Taken together, our findings suggest that Cdk5 and p35 may play important physiological roles in muscle regeneration following nerve injury.

Identification of Genes Induced by Neuregulin in Cultured Myotubes

The formation of the neuromuscular junction (NMJ) involves a series of inductive interactions between motor neurons and muscle fibers. The neural signals proposed to induce the mRNA expression of acetylcholine receptors in muscle include neuregulin (NRG). In the present study, we have employed RNA fingerprinting by arbitrarily primed PCR analysis to identify the differentially expressed transcripts following NRG treatment in cultured myotubes. Nine partial cDNA fragments were isolated; the mRNA expression of eight of these genes was found to be up-regulated by NRG. The spatial and temporal expression profiles of these NRG-regulated genes in rat tissues during development suggest potential functional roles during the formation of NMJ in vivo. Our findings not only allowed the identification of novel genes, but also suggested possible functions for some known genes that are consistent with their potential roles at the NMJ. Furthermore, the identification of G-protein beta1 subunit and G-protein-coupled receptor as NRG-regulated genes has provided the first demonstration that activation of the NRG signaling pathway can induce the expression of components in the G-protein signaling cascade.