Yun Fei Lu

CaM kinase Iα–induced phosphorylation of Drp1 regulates mitochondrial morphology

Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Iα (CaMKIα). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKIα is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

Oxytocin improves long-lasting spatial memory during motherhood through MAP kinase cascade

Oxytocin is an essential hormone for mammalian labor and lactation. Here, we show a new function of oxytocin in causing plastic changes in hippocampal synapses during motherhood. In oxytocin-perfused hippocampal slices, one-train tetanus stimulation induced long-lasting, long-term potentiation (L-LTP) and phosphorylation of cyclic AMP–responsive element binding protein (CREB), and MAP kinase inhibitors blocked these inductions. An increase in CREB phosphorylation and L-LTP induced by one-train tetanus were observed in the multiparous mouse hippocampus without oxytocin application. Furthermore, intracerebroventricular injection of oxytocin in virgin mice improved long-term spatial learning in vivo, whereas an injection of oxytocin antagonist in multiparous mice significantly inhibited the improved spatial memory, L-LTP and CREB phosphorylation. These findings indicate that oxytocin is critically involved in improving hippocampus-dependent learning and memory during motherhood in mice.

Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

Cdk5-dependent regulation of glucose-stimulated insulin secretion

Tight glycemic control in individuals with diabetes mellitus is essential to prevent or delay its complications. Present treatments to reduce hyperglycemia mainly target the ATP-sensitive K+ (KATP) channel of pancreatic beta cells to increase insulin secretion. These current approaches are often associated with the side effect of hypoglycemia. Here we show that inhibition of the activity of cyclin-dependent kinase 5 (Cdk5) enhanced insulin secretion under conditions of stimulation by high glucose but not low glucose in MIN6 cells and pancreatic islets. The role of Cdk5 in regulation of insulin secretion was confirmed in pancreatic beta cells deficient in p35, an activator of Cdk5. p35-knockout mice also showed enhanced insulin secretion in response to a glucose challenge. Cdk5 kinase inhibition enhanced the inward whole-cell Ca2+ channel current and increased Ca2+ influx across the L-type voltage-dependent Ca2+ channel (L-VDCC) upon stimulation with high glucose in beta cells, but had no effect on Ca2+ influx without glucose stimulation. The inhibitory regulation by Cdk5 on the L-VDCC was attributed to the phosphorylation of loop II-III of the α1C subunit of L-VDCC at Ser783, which prevented the binding to SNARE proteins and subsequently resulted in a decrease of the activity of L-VDCC. These results suggest that Cdk5/p35 may be a drug target for the regulation of glucose-stimulated insulin secretion.

Protein Therapy: In Vivo Protein Transduction by Polyarginine (11R) PTD and Subcellular Targeting Delivery

Protein Therapy is a newly developed method, which allows proteins, peptides and biologically active compounds to penetrate across the plasma membrane of eukaryotic cells by a polyarginine (most efficiently by 11-arginine, 11R) protein transduction domain. This method enables us to control the localization of targeted substances in subcellular compartments, such as the nuclei, mitochondria and post-synaptic density. The method is very efficient and applicable not only to cultured cells but also to tissue slices and the whole animal. Brain, heart, skeletal muscle, liver, pancreas and lymphocytes are efficient target organs and tissues for Protein Therapy. The method is therefore a very useful strategy in the post-genomic era. In this mini-review, the development of Protein Therapy and its application for cancer cells and neuroscience study will be shown.

Enhanced synaptic transmission and reduced threshold for LTP induction in fyn‐transgenic mice

To elucidate the physiological role of Fyn, we analysed the properties of synaptic transmission and synaptic plasticity in hippocampal slices of mice overexpressing either wild‐type Fyn (w‐Fyn) or its constitutively active mutant (m‐Fyn). These fyn‐transgenes were driven by the calcium/calmodulin‐dependent protein kinase IIα promoter which turned on in the forebrain neurons including hippocampal pyramidal cells and in late neural development. In the hippocampal slices expressing m‐Fyn the paired‐pulse facilitation was reduced and the basal synaptic transmission was enhanced. A weak theta‐burst stimulation, which was subthreshold for the induction of long‐term potentiation (LTP) in control slices, elicited LTP in CA1 region of the slices expressing m‐Fyn. When a relatively strong stimulation was applied, the magnitude of LTP in m‐Fyn slices was similar to that in control slices. By contrast, the basal synaptic transmission and the threshold for the induction of LTP were not altered in the slices overexpressing wild‐type Fyn. To examine the effect of expression of m‐Fyn on GABAergic inhibitory system, we applied bicuculline, a GABAA receptor blocker, to the hippocampal slices. The ability of bicuculline to enhance excitatory postsynaptic potentials was attenuated in slices expressing m‐Fyn, suggesting that the overexpression of m‐Fyn reduced the GABAergic inhibition. The enhancement of synaptic transmission and the reduction of GABAergic inhibition may contribute to the enhanced seizure susceptibility in the mice expressing m‐Fyn. Thus, these results suggest that regulation of Fyn tyrosine kinase activity is important for both synaptic transmission and plasticity.

Calcineurin inhibitors, FK506 and cyclosporin A, suppress the NMDA receptor-mediated potentials and LTP, but not depotentiation in the rat hippocampus

The effects of FK506, a Ca2+/calmodulin-dependent phosphatase 2B (calcineurin) inhibitor, on the NMDA receptor-mediated potentials and synaptic plasticity were investigated in the CA1 region of the rat hippocampus. Bath application of FK506 (50 microM) produced a 45% inhibition on the NMDA receptor-mediated potentials. FK506 also inhibited the induction of long-term potentiation (LTP), but had no effect on the depotentiation in the CA1 hippocampus. Cyclosporin A (100 microM), another calcineurin inhibitor, mimicked the effects of FK506 on the NMDA responses and synaptic plasticity. These results suggest that FK506 inhibits the activity of NMDA receptors via the involvement of calcineurin. The differential effects of FK506 on LTP and depotentiation may attribute to the partial inhibition on the activity of NMDA receptors and the subsequent attenuation of intracellular Ca2+ increase.

Changes in the expression of novel Cdk5 activator messenger RNA (p39nck5ai mRNA) during rat brain development.

We previously reported that a neuron-specific Cdk5 activator, p35nck5ai, was most prominent in the newborn rat brain. In the adult brain, the expression decreased in most regions except hippocampus and primary olfactory cortex. A novel neuron-specific Cdk5 activator, p39nck5ai, has been recently cloned. To clarify whether two activators were differentially distributed throughout brain development, in this study, we examined the spatial and temporal expression of p39nck5ai in the development rat brain. Northern blot analysis showed that p39nck5ai expression was low in 15-day old fetuses and newborn, and was most prominent in the 1-3 week-old rat brains. In the adult rat brain, expression declined to the same level as in newborn rat brain. In situ hybridization showed that p39nck5ai mRNA was weakly expressed in all neurons of all regions in the newborn rat brain and the transcriptional level was highest in all regions in the 3 week-old rat brain. In the adult, expression was decreased in most neurons except Purkinje and granule cells in the cerebellum which retained high levels. These results suggest that p35nck5a and p39nck5ai may have different functional roles in distinct brain regions during different states of the rat brain development.

HIV-1 Inhibits Long-Term Potentiation and Attenuates Spatial Learning

Although memory deficits have been clearly documented in patients with human immunodeficiency virus type‐1 (HIV‐1) infection, the physiological basis of this dysfunction is poorly understood. We focused on Tat, a viral protein released from HIV‐1–infected cells and investigated its effect on spatial learning in adult mice. An intracerebroventricular injection of Tat leads to attenuation of spatial learning accompanied by suppression of long‐term potentiation (LTP), the cellular basis of spatial learning, in hippocampal cornu ammonis 1 pyramidal neurons. Tat facilitates extrasynaptic but not synaptic N‐methyl‐D‐aspartate (NMDA) receptor activity. Taken together, these data provide strong evidence that the Tat pathway underlies the development of memory dysfunction in patients with HIV‐1 infection and suggest a causal relationship between Tat, the facilitation of extrasynaptic NMDA receptor activity, inhibition of LTP, and attenuation of spatial learning.

Inhibition of excitatory neuronal cell death by cell-permeable calcineurin autoinhibitory peptide

In glutamate‐mediated excitatory neuronal cell death, immunosuppressants (FK506, Cys‐A) are powerful agents that protect neurons from apoptosis. Immunosuppressants inhibit two types of enzyme, calcium/calmodulin‐dependent protein phosphatase (calcineurin: CaN), and peptidyl‐prolyl cis‐trans‐isomerase (PPIase) activity such as the FKBP family. In this study, we used a protein transduction approach to determine the functional role of CaN and to produce a potential therapeutic agent for glutamate‐mediated neuronal cell death. We created a novel cell‐permeable CaN autoinhibitory peptide using the 11 arginine protein transduction domain. This peptide was highly efficient at transducing into primary culture neurons, potently inhibited CaN phosphatase activities, and inhibited glutamate‐mediated neuronal cell death. These results showed that CaN plays an important role in excitatory neuronal cell death and cell‐permeable CaN autoinhibitory peptide could be a new drug to protect neurons from excitatory neuronal death.