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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.

An improved understanding of the molecular mechanisms in synapse formation provides insight into both learning and memory and the etiology of neurodegenerative disorders. Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein methyltransferase that negatively regulates synaptic gene expression and inhibits neuronal differentiation. Despite its regulatory function in neurons, little is known about the CARM1 cellular location and its role in dendritic maturation and synapse formation. Here, we examined the effects of CARM1 inhibition on dendritic spine and synapse morphology in the rat hippocampus. CARM1 was localized in hippocampal post-synapses, with immunocytochemistry and electron microscopy revealing co-localization of CARM1 with post-synaptic density (PSD)-95 protein, a post-synaptic marker. Specific siRNA-mediated suppression of CARM1 expression resulted in precocious dendritic maturation, with increased spine width and density at sites along dendrites and induction of mushroom-type spines. These changes were accompanied by a striking increase in the cluster size and number of key synaptic proteins, including N-methyl-d-aspartate receptor subunit 2B (NR2B) and PSD-95. Similarly, pharmacological inhibition of CARM1 activity with the CARM1-specific inhibitor AMI-1 significantly increased spine width and mushroom-type spines and also increased the cluster size and number of NR2B and cluster size of PSD-95. These results suggest that CARM1 is a post-synaptic protein that plays roles in dendritic maturation and synaptic formation and that spatiotemporal regulation of CARM1 activity modulates neuronal connectivity and improves synaptic dysfunction.

Activity of neprilysin (NEP), the major protease which cleaves amyloid-β peptide (Aβ), is reportedly reduced in the brains of patients with Alzheimer’s disease (AD). Accumulation of Aβ generates reactive oxygen species (ROS) such as 4-hydroxynonenal (HNE), and then reduces activities of Aβ-degrading enzymes including NEP. Xanthorrhizol (Xan), a natural sesquiterpenoid, has been reported to possess antioxidant and anti-inflammatory properties. The present study examined the effects of Xan on HNE- or oligomeric Aβ42-induced oxidative modification of NEP protein. Xan was added to the HNE- or oligomeric Aβ42-treated SK-N-SH human neuroblastoma cells and then levels, oxidative modification and enzymatic activities of NEP protein were measured. Increased HNE levels on NEP proteins and reduced enzymatic activities of NEP were observed in the HNE- or oligomeric Aβ42-treated cells. Xan reduced HNE levels on NEP proteins and preserved enzymatic activities of NEP in HNE- or oligomeric Aβ42-treated cells. Xan reduced Aβ42 accumulation and protected neurones against oligomeric Aβ42-induced neurotoxicity through preservation of NEP activities. These findings indicate that Xan possesses therapeutic potential for the treatment of neurodegenerative diseases, including AD, and suggest a potential mechanism for the neuroprotective effects of antioxidants for the prevention of AD.

Fragile X syndrome (FXS) is caused by transcriptional silencing in neurons of the FMR1 gene product, fragile X mental retardation protein (FMRP), a repressor of dendritic mRNA translation. The lack of FMRP leads to dysregulation of synaptically driven protein synthesis and impairments of intellect, cognition, and behavior, a disorder that currently has no effective therapeutics. Fragile X mice were treated with chronic bryostatin-1, a relatively selective protein kinase ε activator with pharmacological profiles of rapid mGluR desensitization, synaptogenesis, and synaptic maturation/repairing. Differences in the major FXS phenotypes, synapses, and cognitive functions were evaluated and compared among the age-matched groups. Long-term treatment with bryostatin-1 rescues adult fragile X mice from the disorder phenotypes, including normalization of most FXS abnormalities in hippocampal brain-derived neurotrophic factor expression and secretion, postsynaptic density-95 levels, glycogen synthase kinase-3β phosphorylation, transformation of immature dendritic spines to mature synapses, densities of the presynaptic and postsynaptic membranes, and spatial learning and memory. Our results show that synaptic and cognitive function of adult FXS mice can be normalized through pharmacologic treatment and that bryostatin-1-like agents may represent a novel class of drugs to treat fragile X mental retardation even after postpartum brain development has largely completed.

Autophagy is a cellular degradation process for cellular aggregates and unneeded cellular compartments including damaged mitochondria, ER, and peroxisomes. Melanosome is cellular organelle that is the cellular site of generation, storage and transports of melanin in melanocytes. Despite potential importance of autophagy, the role of autophagy in melanogenesis and melanosome autophagy are largely unknown. In here, we identified 3′-hydroxydaidzein (3′-ODI) as an autophagy inducer from a phytochemical library screening. Treatment with 3′-ODI significantly reduced α-MSH-mediated melanogenesis but efficiently increased autophagy both in melanoma cells and melanocytes. Furthermore, inhibition of autophagy significantly reduced the anti-melanogenic effects of 3′-ODI in α-MSH-stimulated melanoma cells. Taken together, these results suggest that autophagy mediates anti-melanogenic activity of 3′-ODI.

Neuropsychiatric disorders such as depression are frequently associated with Alzheimer's disease (AD) and the degeneration of cholinergic basal forebrain neurons and reductions in acetylcholine that occur in AD have been identified as potential mediators of these secondary neuropsychiatric symptomologies. Indeed, removal of cholinergic innervation to the hippocampus via selective immunolesions of septohippocampal cholinergic neurons induces dysfunction of the hypothalamic-pituitary-adrenocortical (HPA) axis and decreases glucocorticoid receptor expression (GR). A subsequent study showed that loss of cholinergic input decreases the activity of the catalytic subunit of protein kinase A (PKAc) and lessens the interaction of protein kinase A (PKA) with GR. Because cross-coupling between nuclear factor-κB (NF-κB) p65 and GR depends on PKA signaling, the present study was conducted to evaluate the status of NF-κB as well as interactions of PKA with NF-κB in the hippocampus following cholinergic denervation. Expression of cytosolic NF-κB p65 was diminished and IκB was degraded in the hippocampus of cholinergic immunolesioned rats compared to the controls. Immunolesions also increased NF-κB p65 Ser276 phosphorylation, as well as interactions between PKAc and NF-κB p65. These results indicate that loss of cholinergic input to the hippocampus results in decreased PKA activity and increased NF-κB activity. Such altered signaling may contribute to psychiatric symptoms, including depression, in patients with AD.

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-3beta (GSK-3beta) 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 HGF-induced phosphorylation of Akt and GSK-3beta, 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-3beta, 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-3beta 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.

SPIN90 is a widely expressed Nck-binding protein that contains one Src homology 3 (SH3) domain, three Pro-rich motifs, and a serine/threonine-rich region, and is known to participate in sarcomere assembly during cardiac myocyte differentiation. We used in vitro binding assays and yeast two-hybrid screening analysis to identify Nck, betaPIX, Wiscott-Aldrich syndrome protein (WASP), and ERK1 as SPIN90-binding proteins. It appears that betaPIX, WASP, and SPIN90 form a complex that interacts with Nck in a manner dependent upon cell adhesion to extracellular matrix. The betaPIX.WASP.SPIN90.Nck interaction was abolished in suspended and cytochalasin D-treated cells, but was recovered when cells were replated on fibronectin-coated dishes. The SPIN90.betaPIX.WASP complex was stable, even in suspended cells, suggesting SPIN90 serves as an adaptor molecule to recruit other proteins to Nck at focal adhesions. In addition, we found that overexpression of the SPIN90 SH3 domain or Pro-rich region, respectively, abolished SPIN90.Nck and SPIN90.betaPIX interactions, resulting in detachment of cells from extracellular matrix. SPIN90 was phosphorylated by ERK1, which was, itself, activated by cell adhesion and platelet-derived growth factor. Such phosphorylation of SPIN90 likely promotes the interaction of the SPIN90.betaPIX.WASP complex and Nck. It thus appears that the interaction of the betaPIX.WASP.SPIN90 complex with Nck is crucial for stable cell adhesion and can be dynamically modulated by SPIN90 phosphorylation that is dependent on cell adhesion and ERK activation.

In Alzheimer's disease (AD) transgenic mice, activation of synaptogenic protein kinase C e (PKCe) was found to prevent synaptotoxic amyloid-β (Aβ)-oligomer elevation, PKCe deficits, early synaptic loss, cognitive deficits, and amyloid plaque formation. In humans, to study the role of PKCe in the pathophysiology of AD and to evaluate its possible use as an early AD-biomarker, we examined PKCe and Aβ in the brains of autopsy-confirmed AD patients (n = 20) and age-matched controls (AC, n = 19), and in skin fibroblast samples from AD (n = 14), non-AD dementia patients (n = 14), and AC (n = 22). Intraneuronal Aβ levels were measured immunohistochemically (using an Aβ-specific antibody) in hippocampal pyramidal cells of human autopsy brains. PKCe was significantly lower in the hippocampus and temporal pole areas of AD brains, whereas Aβ levels were significantly higher. The ratio of PKCe to Aβ in individual CA1 pyramidal cells was markedly lower in the autopsy AD brains versus controls. PKCe was inversely correlated with Aβ levels in controls, whereas in AD patients, PKCe showed no significant correlation with Aβ. In autopsy brains, PKCe decreased as the Braak score increased. Skin fibroblast samples from AD patients also demonstrated a deficit in PKCe compared to controls and an AD-specific change in the Aβ-oligomer effects on PKCe. Together, these data demonstrate that the relationship between Aβ levels and PKCe is markedly altered in AD patients' brains and skin fibroblasts, reflecting a loss of protective effect of PKCe against toxic Aβ accumulation. These changes of PKCe levels in human skin fibroblasts may provide an accurate, non-invasive peripheral AD biomarker.

In the yeast two-hybrid screening, we have isolated a cDNA clone from a human heart library using Nck Src homology 3 (SH3) domains as bait. The full-length cDNA, which encoded 722 amino acids, was identified as a VIP54-related gene containing an SH3 domain, proline-rich motifs, a serine/threonine-rich region, and a long C-terminal hydrophobic region. We refer to this protein as SPIN90 (SH3 ProteinInteracting with Nck, 90 kDa). The amino acid sequence of the SH3 domain has the highest homology with those of Fyn, Yes, and c-Src. SPIN90 was broadly expressed in human tissues; in particular, it was highly expressed in heart, brain, and skeletal muscle, and its expression was developmentally regulated during cardiac myocyte differentiation. SPIN90 is able to bind to the first and third SH3 domains of Nck, in vitro, and is colocalized with Nck at sarcomere Z-discs within cardiac myocytes. Moreover, treatment with antisera raised against SPIN90 disrupted sarcomere structure, suggesting that this protein may play an important role in the maintenance of sarcomere structure and/or in the assembly of myofibrils into sarcomeres.

The cellular localization of alpha3beta1 integrin isoforms was examined in cultured neonatal myocytes at selected times during development using double immunofluorescence assays. The distribution of alpha3A subunits began as diffuse and patternless, but as the cells matured, the distribution assumed a sarcomeric banding pattern, and alpha3A appeared to be localized in costameres - sarcolemmal regions adjacent to the Z-disks. Alpha-actinin, a component of the Z-disk, was localized in the same intracellular regions. Temporal analysis of the incorporation of the alpha3A subunit and other myofibrillar proteins into sarcomeres revealed that alpha3A was integrated into sarcomeres following incorporation of alpha-actinin and myosin heavy chain (MHC) but prior to that of desmin. This suggests that alpha3A integrins are incorporated into a pre-existing myofibrillar structure, and it is unlikely that alpha3A integrins participate in the initial assembly of myofibrillar proteins. The alpha3B, beta1A and beta1D subunits were also localized in costameres, where they formed alpha3Abeta1A, alpha3Abeta1D and alpha3Bbeta1A heterodimers. The alpha3Bbeta1D heterodimer, however, was not found in cardiac myocytes. The antisera raised against the cytoplasmic domains of alpha3A, alpha3B, beta1A and beta1D caused disruption of sarcomere structure. Thus, the myofibril-extracellular matrix linkages mediated by isoforms of alpha3beta1 integrin may play a crucial role in the stabilization of myofibril assembly and in the maintenance of sarcomere structure. Co-immunoprecipitation experiments revealed that beta1A, but not beta1D, interacts with the Nck signaling protein, suggesting that Nck participates in downstream signaling triggered by beta1A and that the beta1A-mediated signaling pathway is distinct from that of beta1D.

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.

Removal of thecholinergic innervation to the hippocampus via selective immunolesions of septohippocampal cholinergic neurons induces dysfunction of the hypothalamic–pituitary–adrenocortical (HPA) axis and decreases glucocorticoid receptor (GR) mRNA. This study examined whether removal of the cholinergic innervation decreased GR protein levels and induced changes in the interaction between GR and the cytoplasmic catalytic subunit of protein kinase A (PKAc) in the hippocampus. In lesioned animals, GR protein levels were markedly decreased in the nucleus, but not in the cytosol of hippocampal neurons, whereas mineralocorticoid receptor (MR) levels remained unchanged in both the nucleus and cytosol. PKAc levels did not differ between lesioned and control groups, but PKAc activity was reduced in lesion tissue compared with the controls. The interaction between GR and PKAc was also decreased in the hippocampus without cholinergic input. These results indicate that degeneration of septohippocampal cholinergic neurons leads to reduced PKAc activity in the hippocampus which, in turn, alters GR signaling. The altered GR signaling induced by the degeneration of basal forebrain cholinergic neurons may contribute to dysfunction of the HPA axis in aged animals and patients with Alzheimer's disease (AD) and lead to neuropsychiatric symptoms that occur throughout the course of AD. © 2011 Wiley Periodicals, Inc.

The antioxidant and anti-inflammatory properties of the marine red alga Neorhodomela aculeate (N. aculeata) Masuda were investigated with neuronal and microglial cells. Extracts of N. aculeata had potent neuroprotective effects on glutamate-induced neurotoxicity and inhibited reactive oxygen species (ROS) generation in the murine hippocampal HT22 cell line. Also, extracts of N. aculeata inhibited H2O2-induced lipid peroxidation in rat brain homogenates. The properties of the extract as an anti-inflammatory agent were investigated in microglial activation by interferon-gamma (IFN-gamma): it reduced the inducible nitric oxide synthase that consequently resulted in the reduction of nitric oxide. These results suggest that the marine red alga N. aculeata could be considered as a potential source for reducing reactive oxygen species and inflammation related to neurological diseases.

It has been reported that inflammatory processes are associated with the pathophysiology of Alzheimer's disease (AD), and the treatment of AD using anti-inflammatory agents slows the progress of AD. Marine algae have been utilized in food products as well as in medicine products for a variety of purposes. In this study, we investigated the neuroprotective effects of methanol extracts of Ulva conglobata (U. conglobata), a marine algae, on glutamate-induced neurotoxicity in the murine hippocampal HT22 cell line and the anti-inflammatory effects on interferon gamma (IFN-gamma)-induced microglial activation in BV2 cells. U. conglobata methanol extracts significantly attenuated the neurotoxicity induced by glutamate in HT22 cells and inhibited nitric oxide production induced by IFN-gamma in BV2 cells. U. conglobata methanol extract treatments were also examined and it was found that they almost completely suppressed the expression of the proinflammatory enzyme cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). These results suggest that U. conglobata possesses therapeutic potential for combating neurodegenerative diseases associated with neuroinflammation.

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.

Epidemiological studies suggest that the treatments of anti-inflammatory agents and anti-oxidants slow the progress of neurological diseases. Lignans are anti-oxidants and phytoestrogens found in a variety of plants. In this study, we investigated the neuroprotective effect of macelignan on glutamate-induced neurotoxicity and reactive oxygen species (ROS) in murine hippocampal HT22 cell line. Macelignan significantly attenuated the ROS production and neurotoxicity induced by glutamate in HT22 cell. Also, the properties of macelignan as an anti-inflammatory agent were investigated in microglials activation by lipopolysaccharide (LPS). It potently suppressed the expression of cyclooxygenase-2 and inducible nitric oxide synthase, that consequently resulted in the reduction of nitric oxide in LPS-treated microglial cells. It also significantly suppressed the production of pro-inflammatory cytokine tumor necrosis factor-alpha and interleukin-6. These results suggest that macelignan possesses therapeutic potentials against neurodegenerative diseases with oxidative stress and neuroinflammation.

Cardiac hypertrophy is characterized by remodeling of the extracellular matrix (ECM). Integrins are cell-surface molecules that link the ECM to the cellular cytoskeleton where they play roles as signaling molecules and transducers of mechanical force. To clarify the possible roles of integrins in cardiac myocyte hypertrophy, we investigated the cellular localization and expression of ECM proteins and integrins in both normal cardiac myocytes and phenylephrine-induced hypertrophic myocytes. Addition of phenylephrine (PE) to cultured neonatal cardiac myocytes induced sarcomeric organization, increase in cell size, and synthesis of the hypertrophic marker, atrial natriuretic factor (ANF). In particular, fibronectin and collagen underwent dramatic localization changes during PE-induced cardiac hypertrophy. Significant changes were noted in the cellular localization of the respective collagen and fibronectin receptors, integrin alpha1 and alpha5, from diffuse to a sarcomeric banding pattern. Expression levels of integrins were also increased during hypertrophy. Treatment with okadaic acid (OA), an inhibitor of protein phosphatase 2A (PP2A), resulted in inhibition of hypertrophic response. These results suggest that dephosphorylation of integrin beta1 may be important in the induction of cardiac hypertrophy.