Bryostatin 1, a potent activator of protein kinase C epsilon (PKCɛ), has been shown to reverse synaptic loss and facilitate synaptic maturation in animal models of Alzheimer’s disease (AD), Fragile X, stroke, and other neurological disorders. In a single-dose (25 μg/m2) randomized double-blind Phase IIa clinical trial, bryostatin levels reached a maximum at 1-2 h after the start of infusion. In close parallel with peak blood levels of bryostatin, an increase of PBMC PKCɛ was measured (p = 0.0185) within 1 h from the onset of infusion. Of 9 patients with a clinical diagnosis of AD, of which 6 received drug and 3 received vehicle within a double-blind protocol, bryostatin increased the Mini-Mental State Examination (MMSE) score by +1.83±0.70 unit at 3 h versus –1.00±1.53 unit for placebo. Bryostatin was well tolerated in these AD patients and no drug-related adverse events were reported. The 25 μg/m2 administered dose was based on prior clinical experience with three Expanded Access advanced AD patients treated with bryostatin, in which return of major functions such as swallowing, vocalization, and word recognition were noted. In one Expanded Access patient trial, elevated PKCɛ levels closely tracked cognitive benefits in the first 24 weeks as measured by MMSE and ADCS-ADL psychometrics. Pre-clinical mouse studies showed effective activation of PKCɛ and increased levels of BDNF and PSD-95. Together, these Phase IIa, Expanded Access, and pre-clinical results provide initial encouragement for bryostatin 1 as a potential treatment for AD.

Xanthorrhizol, a natural sesquiterpenoid isolated from the rhizome of Curcuma xanthorrhiza Roxb (Zingiberaceae), has antibacterial activities and protective effects against cisplatin-induced hepatotoxicity. In this study, we investigated the activities of xanthorrhizol as an antioxidant or antiinflammatory agent using neuronal and microglial cells. Xanthorrhizol had potent neuroprotective effects on glutamate-induced neurotoxicity and reactive oxygen species (ROS) generation in the murine hippocampal HT22 cell line. Also, xanthorrhizol inhibited H(2)O(2)-induced lipid peroxidation in rat brain homogenates. The properties of xanthorrhizol as an antiinflammatory agent were investigated in microglial activation by lipopolysaccharide. It reduced the expression of cyclooxygenase-2 and the inducible nitric oxide synthase, which consequently resulted in the reduction of nitric oxide. The production of proinflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha in activated microglial cells, was reduced by xanthorrhizol. These results suggest that xanthorrhizol could be an effective candidate for the treatment of Alzheimer's disease- and other neurological disease-related ROS and inflammation.

During central nervous system development, growth factors and their associated receptor protein tyrosine kinases regulate many neuronal functions such as neurite extension and dendrite maturation. Hepatocyte growth factor (HGF) and its receptor, c-Met, can promote formation of neurites and enhance elaboration of dendrites in mature neurons, but their effects on the early stages of dendrite maturation in hippocampal neurons and the signaling pathways by which they promote dendrite formation have not been studied. Exogenous HGF treatment effectively enhanced the phosphorylation and activation of c-Met in cultured hippocampal neurons at 4 days in vitro. HGF treatment increased the number of dendrites and promoted dendrite elongation in these neurons. Consistent with these results, HGF activated Akt, which phosphorylates glycogen synthase kinase-3β (GSK-3β) to inactivate it, and reduced phosphorylation of microtubule-associated protein 2 (MAP2), which can promote microtubule polymerization and dendrite elongation when dephosphorylated. Conversely, pharmacological inhibition of c-Met with its specific inhibitor, PHA-665752, or genetic knock-down of c-Met with short hairpin RNAs (shRNAs) suppressed HGFinduced phosphorylation of Akt and GSK-3β, increased phosphorylation of MAP2, and reduced dendrite number and length in cultured hippocampal neurons. Moreover, suppressing c-Met with PHA-665752 or by shRNA decreased MAP2 expression. Inhibiting Akt activity with the phosphoinositide-3-kinase inhibitor LY294002 or Akt inhibitor X suppressed HGF-induced phosphorylation of GSK-3β, increased MAP2 phosphorylation, and blocked the ability of HGF to enhance dendritic length. These observations indicate that HGF and c-Met can regulate the early stages of dendrite maturation via activation of the Akt/GSK-3β pathway.

The appropriate regulation of the actin cytoskeleton is essential for cell movement, changes in cell shape, and formation of membrane protrusions like lamellipodia and filopodia. Moreover, several regulatory proteins affecting actin dynamics have been identified in the motile regions of cells. Here, we provide evidence for the involvement of SPIN90 in the regulation of actin cytoskeleton and actin comet tail formation. SPIN90 was distributed throughout the cytoplasm in COS-7 cells, but exposing the cells to platelet-derived growth factor (PDGF) caused a redistribution of SPIN90 to the cell cortex and the formation of lamellipodia (or membrane ruffles), both of which were dramatically inhibited in SPIN90-knockdown cells. In addition, the binding of the C terminus of SPIN90 with both the Arp2/3 complex (actin-related proteins Arp 2 and Arp 3) and G-actin activates the former, leading to actin polymerization in vitro. And when coexpressed with phosphatidylinositol 4-phosphate 5 kinase, SPIN90 was observed within actin comet tails. Taken these findings suggest that SPIN90 participates in reorganization of the actin cytoskeleton and in actin-based cell motility.The actin cytoskeleton plays key roles in cell motility and morphology, intracellular organization, membrane trafficking, and the intracellular movement of a variety of pathogens (1, 2). Many actin-based structures, especially those involved in membrane protrusion, are assembled through the coordinated polymerization and cross-linking of actin monomers into actin filaments that in turn form orthogonal or parallel filament networks (3). In that regard, the dynamics of actin stress fibers, filopodia, and lamellipodia (or membrane ruffles) are tightly regulated by various nucleation-promoting factors (WASP 4 family proteins) and actin nucleation proteins (e.g. the Arp2/3 complex) (4, 5). Among these components, the Arp2/3 complex localizes at the leading edge of cells, where the actin cytoskeleton is nucleated and reorganized (6, 7). The major activators of the complex include the WASP family proteins, which contain a conserved VCA domain composed of one or two VPH domains, which bind actin monomers, a central region, and an A domain, which binds to the Arp2/3 complex (8). In addition, cortactin, a filamentous actin-associated protein, binds to the Arp2/3 complex via an A domain at its N terminus and stimulates nucleation of actin filaments, ultimately promoting formation and stabilization of actin filament networks (9). In similar fashion, Abp1, an F-actin binding protein, also associates with the Arp2/3 complex and stimulates actin nucleation (10).The Arp2/3 complex also plays a role in the intracellular motility of pathogens and vesicles. For instance, the pathogenic bacterium Listeria monocytogenes utilizes actin polymerization mediated by the Arp2/3 complex to move within the cytoplasm of infected host cells. Likewise, movement of intracellular vesicles (i.e. endosomes) is dependent upon actin polymerization mediated by the Arp2/3 complex, which localizes al...

HuD protein is an RNA-binding protein involved in post-transcriptional regulation of gene expression for synaptogenesis, neuronal differentiation, and learning and memory, and is up-regulated and redistributed by a protein kinase C (PKC)-dependent pathway in neurons. Here, we show a PKC-regulated mechanism on HuD-mediated mRNA stability and expression of several neurotrophic factors (NTFs) in cultured hippocampal neurons. HuD pull-down assays showed that HuD is associated with brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin (NT)-3 mRNAs. Reduction of HuD expression with short hairpin RNAs decreased BDNF, NGF, and NT-3 mRNAs and NTFs expression. Bryostatin, a PKC activator, treatment enhanced their association with HuD and increased these transcripts' stability. Bryostatin induced HuD phosphorylation, which was inhibited by Ro 32-0432, a specific PKC inhibitor. Activated PKC specifically phosphorylated coactivator-associated arginine methyltransferase 1 (CARM1), which methylates HuD and negatively modulates HuD-mRNA interactions during neuronal differentiation, and inhibited its methyltransferase activity, resulting in decrease in CARM1-mediated HuD methylation. Furthermore cotreatment of bryostatin and AMI-1, a specific CARM1 inhibitor, potentiated PKC-dependent HuD-mRNA interactions and enhanced dendritic arborization. These results demonstrate that PKC may play an important role in neuronal differentiation and synaptogenesis via stimulating HuD-mediated mRNA stability and inhibiting CARM1 in hippocampal neurons.