Youming Lu

EPAC null mutation impairs learning and social interactions via aberrant regulation of miR-124 and Zif268 translation.

EPAC proteins are the guanine nucleotide exchange factors that act as the intracellular receptors for cyclic AMP. Two variants of EPAC genes including EPAC1 and EPAC2 are cloned and are widely expressed throughout the brain. But, their functions in the brain remain unknown. Here, we genetically delete EPAC1 (EPAC1(-/-)), EPAC2 (EPAC2(-/-)), or both EPAC1 and EPAC2 genes (EPAC(-/-)) in the forebrain of mice. We show that EPAC null mutation impairs long-term potentiation (LTP) and that this impairment is paralleled with the severe deficits in spatial learning and social interactions and is mediated in a direct manner by miR-124 transcription and Zif268 translation. Knockdown of miR-124 restores Zif268 and hence reverses all aspects of the EPAC(-/-) phenotypes, whereas expression of miR-124 or knockdown of Zif268 reproduces the effects of EPAC null mutation. Thus, EPAC proteins control miR-124 transcription in the brain for processing spatial learning and social interactions.

Synaptic Released Zinc Promotes Tau Hyperphosphorylation by Inhibition of Protein Phosphatase 2A (PP2A)

Background: Tangle distribution is largely overlapped with zinc-containing glutamatergic neurons. Synaptically released zinc may be involved in tau hyperphosphorylation. Results: Increased synaptic activity induces tau hyperphosphorylation by synaptic zinc through PP2A inhibition. Conclusion: Synaptic activity promotes tau hyperphosphorylation, and synaptically released zinc plays a central role. Significance: Therapies targeted to maintaining zinc homeostasis and moderating synaptic activity may benefit AD by reducing tauopathy. Hyperphosphorylated tau is the major component of neurofibrillary tangles in Alzheimer disease (AD), and the tangle distribution largely overlaps with zinc-containing glutamatergic neurons, suggesting that zinc released in synaptic terminals may play a role in tau phosphorylation. To explore this possibility, we treated cultured hippocampal slices or primary neurons with glutamate or Bic/4-AP to increase the synaptic activity with or without pretreatment of zinc chelators, and then detected the phosphorylation levels of tau. We found that glutamate or Bic/4-AP treatment caused tau hyperphosphorylation at multiple AD-related sites, including Ser-396, Ser-404, Thr-231, and Thr-205, while application of intracellular or extracellular zinc chelators, or blockade of zinc release by extracellular calcium omission almost abolished the synaptic activity-associated tau hyperphosphorylation. The zinc release and translocation of excitatory synapses in the hippocampus were detected, and zinc-induced tau hyperphosphorylation was also observed in cultured brain slices incubated with exogenously supplemented zinc. Tau hyperphosphorylation induced by synaptic activity was strongly associated with inactivation of protein phosphatase 2A (PP2A), and this inactivation can be reversed by pretreatment of zinc chelator. Together, these results suggest that synaptically released zinc promotes tau hyperphosphorylation through PP2A inhibition.

Phospholipase C-ε links Epac2 activation to the potentiation of glucose-stimulated insulin secretion from mouse islets of Langerhans

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is potentiated by cAMP-elevating agents, such as the incretin hormone glucagon-like peptide-1 (GLP-1), and cAMP exerts its insulin secretagogue action by activating both protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor designated as Epac2. Although prior studies of mouse islets demonstrated that Epac2 acts via Rap1 GTPase to potentiate GSIS, it is not understood which downstream targets of Rap1 promote the exocytosis of insulin. Here, we measured insulin secretion stimulated by a cAMP analog that is a selective activator of Epac proteins in order to demonstrate that a Rap1-regulated phospholipase C-epsilon (PLC-ε) links Epac2 activation to the potentiation of GSIS. Our analysis demonstrates that the Epac activator 8-pCPT-2’-O-Me-cAMP-AM potentiates GSIS from the islets of wild-type (WT) mice, whereas it has a greatly reduced insulin secretagogue action in the islets of Epac2 (-/-) and PLC-ε (-/-) knockout (KO) mice. Importantly, the insulin secretagogue action of 8-pCPT-2’-O-Me-cAMP-AM in WT mouse islets cannot be explained by an unexpected action of this cAMP analog to activate PKA, as verified through the use of a FRET-based A-kinase activity reporter (AKAR3) that reports PKA activation. Since the KO of PLC-ε disrupts the ability of 8-pCPT-2’-O-Me-cAMP-AM to potentiate GSIS, while also disrupting its ability to stimulate an increase of β-cell [Ca2+]i, the available evidence indicates that it is a Rap1-regulated PLC-ε that links Epac2 activation to Ca2+-dependent exocytosis of insulin.

Epac2‐dependent mobilization of intracellular Ca2+ by glucagon‐like peptide‐1 receptor agonist exendin‐4 is disrupted in β‐cells of phospholipase C‐ɛ knockout mice

Calcium can be mobilized in pancreatic β‐cells via a mechanism of Ca2+‐induced Ca2+ release (CICR), and cAMP‐elevating agents such as exendin‐4 facilitate CICR in β‐cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide‐specific phospholipase C‐ɛ (PLC‐ɛ) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC‐ɛ gene expression in mice disrupts the action of exendin‐4 to facilitate CICR in the β‐cells of these mice. Thus, in the present study, in which wild‐type (WT) C57BL/6 mouse β‐cells were loaded with the photolabile Ca2+ chelator NP‐EGTA, the UV flash photolysis‐catalysed uncaging of Ca2+ generated CICR in only 9% of the β‐cells tested, whereas CICR was generated in 82% of the β‐cells pretreated with exendin‐4. This action of exendin‐4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A (6‐Bnz‐cAMP‐AM) or Epac2 (8‐pCPT‐2′‐O‐Me‐cAMP‐AM) selectively. However, in β‐cells of PLC‐ɛ KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin‐4 was partly effective, 6‐Bnz‐cAMP‐AM was fully effective, and 8‐pCPT‐2′‐O‐Me‐cAMP‐AM was without significant effect. Importantly, transduction of PLC‐ɛ KO β‐cells with recombinant PLC‐ɛ rescued the action of 8‐pCPT‐2′‐O‐Me‐cAMP‐AM to facilitate CICR, whereas a K2150E PLC‐ɛ with a mutated Ras association (RA) domain, or a H1640L PLC‐ɛ that is catalytically dead, were both ineffective. Since 8‐pCPT‐2′‐O‐Me‐cAMP‐AM failed to facilitate CICR in WT β‐cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction ‘module’ comprised of Epac2, Rap and PLC‐ɛ exists in β‐cells, and that the activities of Epac2 and PLC‐ɛ are key determinants of CICR in this cell type.

DAPK1–p53 Interaction Converges Necrotic and Apoptotic Pathways of Ischemic Neuronal Death

Necrosis and apoptosis are two distinct types of mechanisms that mediate ischemic injury. But a signaling point of convergence between them has yet to be identified. Here, we show that activated death-associated protein kinase 1 (DAPK1), phosphorylates p53 at serine-23 (pS23) via a direct binding of DAPK1 death domain (DAPK1DD) to the DNA binding motif of p53 (p53DM). We uncover that the pS23 acts as a functional version of p53 and mediates necrotic and apoptotic neuronal death; in the nucleus, pS23 induces the expression of proapoptotic genes, such as Bax, whereas in the mitochondrial matrix, pS23 triggers necrosis via interaction with cyclophilin D (CypD) in cultured cortical neurons from mice. Deletion of DAPK1DD (DAPK1DDΔ) or application of Tat-p53DM that interrupts DAPK1–p53 interaction blocks these dual pathways of pS23 actions in mouse cortical neurons. Thus, the DAPK1–p53 interaction is a signaling point of convergence of necrotic and apoptotic pathways and is a desirable target for the treatment of ischemic insults.

The MT2 receptor stimulates axonogenesis and enhances synaptic transmission by activating Akt signaling

The MT2 receptor is a principal type of G protein-coupled receptor that mainly mediates the effects of melatonin. Deficits of melatonin/MT2 signaling have been found in many neurological disorders, including Alzheimer’s disease, the most common cause of dementia in the elderly, suggesting that preservation of the MT2 receptor may be beneficial to these neurological disorders. However, direct evidence linking the MT2 receptor to cognition-related synaptic plasticity remains to be established. Here, we report that the MT2 receptor, but not the MT1 receptor, is essential for axonogenesis both in vitro and in vivo. We find that axon formation is retarded in MT2 receptor knockout mice, MT2-shRNA electroporated brain slices or primary neurons treated with an MT2 receptor selective antagonist. Activation of the MT2 receptor promotes axonogenesis that is associated with an enhancement in excitatory synaptic transmission in central neurons. The signaling components downstream of the MT2 receptor consist of the Akt/GSK-3β/CRMP-2 cascade. The MT2 receptor C-terminal motif binds to Akt directly. Either inhibition of the MT2 receptor or disruption of MT2 receptor-Akt binding reduces axonogenesis and synaptic transmission. Our data suggest that the MT2 receptor activates Akt/GSK-3β/CRMP-2 signaling and is necessary and sufficient to mediate functional axonogenesis and synaptic formation in central neurons.

A Novel Mechanism of Spine Damages in Stroke via DAPK1 and Tau

Synaptic spine loss is one of the major preceding consequences of stroke damages, but its underlying molecular mechanisms remain unknown. Here, we report that a direct interaction of DAPK1 with Tau causes spine loss and subsequently neuronal death in a mouse model with stroke. We found that DAPK1 phosphorylates Tau protein at Ser262 (pS262) in cortical neurons of stroke mice. Either genetic deletion of DAPK1 kinase domain (KD) in mice (DAPK1-KD−/−) or blocking DAPK1-Tau interaction by systematic application of a membrane permeable peptide protects spine damages and improves neurological functions against stroke insults. Thus, disruption of DAPK1-Tau interaction is a promising strategy in clinical management of stroke.

Opposite effects of two estrogen receptors on tau phosphorylation through disparate effects on the miR-218/PTPA pathway

The two estrogen receptors (ERs), ERα and ERβ, mediate the diverse biological functions of estradiol. Opposite effects of ERα and ERβ have been found in estrogen‐induced cancer cell proliferation and differentiation as well as in memory‐related tasks. However, whether these opposite effects are implicated in the pathogenesis of Alzheimers disease (AD) remains unclear. Here, we find that ERα and ERβ play contrasting roles in regulating tau phosphorylation, which is a pathological hallmark of AD. ERα increases the expression of miR‐218 to suppress the protein levels of its specific target, protein tyrosine phosphatase α (PTPα). The downregulation of PTPα results in the abnormal tyrosine hyperphosphorylation of glycogen synthase kinase‐3β (resulting in activation) and protein phosphatase 2A (resulting in inactivation), the major tau kinase and phosphatase. Suppressing the increased expression of miR‐218 inhibits the ERα‐induced tau hyperphosphorylation as well as the PTPα decline. In contrast, ERβ inhibits tau phosphorylation by limiting miR‐218 levels and restoring the miR‐218 levels antagonized the attenuation of tau phosphorylation by ERβ. These data reveal for the first time opposing roles for ERα and ERβ in AD pathogenesis and suggest potential therapeutic targets for AD.

A novel mechanism of memory loss in Alzheimer's disease mice via the degeneration of entorhinal-CA1 synapses.

The entorhinal cortex (EC) is one of the most vulnerable brain regions that is attacked during the early stage of Alzheimer’s disease (AD). Here, we report that the synaptic terminals of pyramidal neurons in the EC layer II (ECIIPN) directly innervate CA1 parvalbumin (PV) neurons (CA1PV) and are selectively degenerated in AD mice, which exhibit amyloid-β plaques similar to those observed in AD patients. A loss of ECIIPN–CA1PV synapses disables the excitatory and inhibitory balance in the CA1 circuit and impairs spatial learning and memory. Optogenetic activation of ECIIPN using a theta burst paradigm rescues ECIIPN–CA1PV synaptic defects and intercepts the decline in spatial learning and memory. These data reveal a novel mechanism of memory loss in AD mice via the selective degeneration of the ECIIPN–CA1PV pathway.

Therapeutic Intervention of Learning and Memory Decays by Salidroside Stimulation of Neurogenesis in Aging

Cognition in all mammals including human beings declines during aging. The cellular events responsible for this decay involve a reduction of neurogenesis in the dentate gyrus. Here, we show that treatment with a nature product from a traditional Chinese medicine, namely salidroside restores the capacity of the dentate gyrus to generate new neurons and intercepts learning and memory decays in mice during aging. We uncover that new neurons in aging mice have functional features of an adult granule neuron by forming excitatory synapses with their putative targeting neurons. Genetic inhibition of synaptic transmission from new neurons abolishes the therapeutic effects of salidroside in behavioral tests. We also identify that salidroside targets CREB transcription for the survival of new neurons in the dentate gyrus of old mice. Thus, salidroside is therapeutically effective against learning and memory decays via stimulation of CREB-dependent functional neurogenesis in aging.