Fanny C.F. Ip

Blockade of EphA4 signaling ameliorates hippocampal synaptic dysfunctions in mouse models of Alzheimer's disease

Significance Synaptic loss and dysfunction is associated with cognitive impairment in Alzheimer’s disease (AD). However, the pathophysiological mechanisms underlying synaptic impairment are largely unknown. Here, we reveal a previously unidentified signaling pathway whereby activation of a receptor tyrosine kinase EphA4 is critical for synaptic dysfunctions in AD. Proof-of-concept studies undertaken in both in vitro and in vivo systems demonstrate its importance in mediating the deficit of synaptic transmission and hippocampal long-term potentiation in AD models. Specifically, blocking the EphA4-dependent pathway through knockdown studies or the use of small-molecule inhibitors effectively rescues the impaired synaptic transmission induced by Aβ and reverses impaired synaptic plasticity in AD mouse models. Thus, this study reveals a new disease-modifying therapeutic intervention for AD. Alzheimer’s disease (AD), characterized by cognitive decline, has emerged as a disease of synaptic failure. The present study reveals an unanticipated role of erythropoietin-producing hepatocellular A4 (EphA4) in mediating hippocampal synaptic dysfunctions in AD and demonstrates that blockade of the ligand-binding domain of EphA4 reverses synaptic impairment in AD mouse models. Enhanced EphA4 signaling was observed in the hippocampus of amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mouse model of AD, whereas soluble amyloid-β oligomers (Aβ), which contribute to synaptic loss in AD, induced EphA4 activation in rat hippocampal slices. EphA4 depletion in the CA1 region or interference with EphA4 function reversed the suppression of hippocampal long-term potentiation in APP/PS1 transgenic mice, suggesting that the postsynaptic EphA4 is responsible for mediating synaptic plasticity impairment in AD. Importantly, we identified a small-molecule rhynchophylline as a novel EphA4 inhibitor based on molecular docking studies. Rhynchophylline effectively blocked the EphA4-dependent signaling in hippocampal neurons, and oral administration of rhynchophylline reduced the EphA4 activity effectively in the hippocampus of APP/PS1 transgenic mice. More importantly, rhynchophylline administration restored the impaired long-term potentiation in transgenic mouse models of AD. These findings reveal a previously unidentified role of EphA4 in mediating AD-associated synaptic dysfunctions, suggesting that it is a new therapeutic target for this disease.

Astragaloside IV and Cycloastragenol Stimulate the Phosphorylation of Extracellular Signal-Regulated Protein Kinase in Multiple Cell Types

Two Chinese herb-derived small molecule telomerase activators, astragaloside IV (AG-IV) and cycloastragenol (CAG), have recently been shown to improve the proliferative response of CD8+ T lymphocytes from HIV-infected patients by upregulating telomerase activity. Here, we examined the signaling mechanism of AG-IV and CAG. Telomerase activity in human embryonic kidney HEK293 fibroblasts was increased upon treatment with increasing concentrations of AG-IV or CAG. Both compounds induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) in a time- and dose-dependent manner in HEK293 cells and HEK-neo keratinocytes. AG-IV and CAG also stimulated ERK phosphorylation in other cell lines of lung, brain, mammary, endothelial, and hematopoietic origins. Use of selective inhibitors and dominant negative mutants revealed the involvement of c-Src, MEK (ERK kinase), and epidermal growth factor receptor in CAG-induced ERK phosphorylation. Our data indicate that AG-IV and CAG may exert their cellular effects through the activation of the Src/MEK/ERK pathway.

New Secoiridoid Glucosides from Ligustrum lucidum Induce ERK and CREB Phosphorylation in Cultured Cortical Neurons

Two new secoiridoid glucosides, namely iso-oleonuezhenide (1) and methyloleoside 7-ethyl ester (2), along with five known ones, oleonuezhenide (3), nuezhenide (4), oleuropein (5), G13 (6), and jaspolyside methyl ester (7), were isolated from the fruits of Ligustrum lucidum. Their structures were assigned based on 1H-NMR, 13C-NMR, and 2D-NMR analyses, in combination with HR-MS experiments and the comparison with literature data of related compounds, as well as on chemical experiments. We have examined the ability of these compounds to activate ERK and CREB in cultured cortical neurons. Our studies demonstrate that compound 1 induces ERK and CREB phosphorylation in primary cortical neurons in a dose- and temporal-dependent manner, suggesting its bioactivity on neurons.

Coronin 6 Regulates Acetylcholine Receptor Clustering through Modulating Receptor Anchorage to Actin Cytoskeleton

The maintenance of a high density of neurotransmitter receptors at the postsynaptic apparatus is critical for efficient neurotransmission. Acetylcholine receptors (AChRs) are neurotransmitter receptors densely packed on the postsynaptic muscle membrane at the neuromuscular junction (NMJ) via anchoring onto the actin cytoskeletal network. However, how the receptor-associated actin is coordinately regulated is not fully understood. We report here that Coronin 6, a newly identified member of the coronin family, is highly enriched at adult NMJs and regulates AChR clustering through modulating the interaction between receptors and the actin cytoskeletal network. Experiments with cultured myotubes reveal that Coronin 6 is important for both agrin- and laminin-induced AChR clustering. Furthermore, Coronin 6 forms a complex with AChRs and actin in a manner dependent on its C-terminal region and a conserved Arg29 residue at the N terminus, both of which are critical for the cytoskeletal anchorage of AChRs. Importantly, in vivo knockdown of Coronin 6 in mouse skeletal muscle fibers leads to destabilization of AChR clusters. Together, these findings demonstrate that Coronin 6 is a critical regulator of AChR clustering at the postsynaptic region of the NMJs through modulating the receptor-anchored actin cytoskeleton.

Cycloastragenol Is a Potent Telomerase Activator in Neuronal Cells: Implications for Depression Management

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression-like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression.

Agrin regulates growth cone turning of Xenopus spinal motoneurons.

The pivotal role of agrin in inducing postsynaptic specializations at neuromuscular junctions has been well characterized. Increasing evidence suggests that agrin is also involved in neuronal development. In this study, we found that agrin inhibited neurite extension and, more importantly, a gradient of agrin induced repulsive growth-cone turning in cultured Xenopus spinal neurons. Incubation with a neutralizing antibody to agrin or expression of the extracellular domain of muscle-specific kinase, a component of the agrin receptor complex, abolished these effects of agrin. Agrin-induced repulsive growth-cone turning requires the activity of PI3-kinase and Ca2+ signaling. In addition, the expression of dominant-negative Rac1 inhibited neurite extension and blocked agrin-mediated growth-cone turning. Taken together, our findings suggest that agrin regulates neurite extension and provide evidence for an unanticipated role of agrin in growth-cone steering in developing neurons.

Anemoside A3 ameliorates experimental autoimmune encephalomyelitis by modulating T helper 17 cell response

Anemoside A3 (AA3) is a natural triterpenoid glycoside isolated from the root of Pulsatilla chinensis (Bunge) Regel. We previously showed that AA3 exhibits cognitive-enhancing and neuroprotective properties. In the present study, we demonstrated that AA3 modulates inflammatory responses by regulating prostaglandin E receptor 4 signaling. Because prostaglandin E receptor 4 is involved in the pathophysiology of experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), we assessed the beneficial effect of AA3 in EAE mice. AA3 treatment significantly reduced clinical severity and inflammatory infiltrates in the spinal cord of EAE mice. In vitro studies revealed that AA3 inhibited the T cell response toward the encephalitogenic epitope of myelin oligodendrocyte glycoprotein (MOG). AA3 significantly downregulated the expressions of certain Th1 and Th17 cytokines in activated T cells re-stimulated by MOG. Moreover, AA3 inhibited the activation of STAT4 and STAT3, which are the transcription factors pivotal for Th1 and Th17 lineage differentiation, respectively, in activated T cells. Pharmacological analysis further suggested that AA3 reduced Th17 cell differentiation and expansion. In conclusion, AA3 exerts an immunomodulatory effect in EAE, demonstrating its potential as a therapeutic agent for MS in humans.

Identification of new EphA4 inhibitors by virtual screening of FDA-approved drugs

The receptor tyrosine kinase, erythropoietin-producing hepatocellular A4 (EphA4), was recently identified as a molecular target for Alzheimer’s disease (AD). We found that blockade of the interaction of the receptor and its ligands, ephrins, alleviates the disease phenotype in an AD transgenic mouse model, suggesting that targeting EphA4 is a potential approach for developing AD interventions. In this study, we identified five FDA-approved drugs—ergoloid, cyproheptadine, nilotinib, abiraterone, and retapamulin—as potential inhibitors of EphA4 by using an integrated approach combining virtual screening with biochemical and cellular assays. We initially screened a database of FDA-approved drugs using molecular docking against the ligand-binding domain of EphA4. Then, we selected 22 candidate drugs and examined their inhibitory activity towards EphA4. Among them, five drugs inhibited EphA4 clustering induced by ephrin-A in cultured primary neurons. Specifically, nilotinib, a kinase inhibitor, inhibited the binding of EphA4 and ephrin-A at micromolar scale in a dosage-dependent manner. Furthermore, nilotinib inhibited the activation of EphA4 and EphA4-dependent growth cone collapse in cultured hippocampal neurons, demonstrating that the drug exhibits EphA4 inhibitory activity in cellular context. As demonstrated in our combined computational and experimental approaches, repurposing of FDA-approved drugs to inhibit EphA4 may provide an alternative fast-track approach for identifying and developing new treatments for AD.

Diarylheptanoids from Rhizomes of Alpinia officinarum Inhibit Aggregation of α-Synuclein

Two new diarylheptanoids, alpinin A (1) and alpinin B (2), together with 18 known diarylheptanoids (3-20), were isolated from the rhizomes of Alpinia officinarum. Their structures were elucidated by comprehensive spectroscopic analysis, including high-resolution mass spectrometry, infrared spectroscopy, and one- and two-dimensional nuclear magnetic resonance spectroscopy. Structurally, alpinin A is a new member of the small family of oxa-bridged diarylheptanoids and contains the characteristic 2,6-cis-configured tetrahydropyran motif (C1-C5 oxa bridge). The absolute configuration of alpinin A was confirmed by asymmetric total synthesis of the enantiomer (ent-1), corroborating the assignment of the molecular structure. The absolute configuration of alpinin B was determined on the basis of the analysis of the circular dichroism exciton chirality spectrum. We evaluated the inhibitory activity of all isolated diarylheptanoids against α-synuclein aggregation at 10 μM. Alpinins A and B significantly inhibited α-synuclein aggregation by 66 and 67%, respectively.

NRG induces membrane targeting of G??z in muscle: implication in myogenesis

The neuregulins (NRGs) constitute a family of trophic factors that are known to play critical roles during neural development. We recently reported that G&bgr; subunit regulates NRG-mediated signaling and gene transcription in cultured C2C12 myotubes. In this study, we demonstrated that NRG treatment of C2C12 myotubes stimulates a rapid translocation of G&agr;z protein to the plasma membranes. In addition, G&agr;z protein is localized to the postsynaptic regions at adult neuromuscular junctions and is prominently expressed in rat skeletal muscle during early postnatal stages. Interestingly, we found that expression of the constitutively activated G&agr;z in C2C12 myoblasts attenuates myogenic differentiation. Taken together, our observations reveal an unanticipated role of G&agr;z in mediating the actions of NRG during neural development.