Pretreatment of PC12 Cells with 17β-estradiol Prevents Aβ-Induced Down-Regulation of CREB Phosphorylation and Prolongs Inhibition of GSK-3β

It is believed that estrogen protects neurons against various toxicities like that from amyloid β (Aβ) in Alzheimer’s disease (AD). In the present study, we investigated the effects of Aβ_1–42 on the activities of cyclic-AMP response element-binding protein (CREB) and glycogen synthase kinase-3β (GSK-3β), two key proteins associated with learning and memory, and the effects of 17β-estradiol on Aβ_1–42-induced changes of CREB and GSK-3β in PC12 cells. We found that Aβ_1–42 induced a decrease in phosphorylation of CREB at Ser133 (CREB pS133) and caused a transient (30 min) up-regulation of the inhibitory GSK-3β phosphorylation at Ser9 (GSK-3β pS9), followed by down-regulation of GSK-3β pS9. Pretreatment of 17β-estradiol is needed for its protection against Aβ_1–42-induced changes of CREB. The protective role of 17β-estradiol against Aβ_1–42-induced down-regulation of CREB pS133 was abolished by the mitogen-activated protein kinase (MAPK) pathway inhibitor U0126. Furthermore, 17β-estradiol also prolonged the up-regulation of GSK-3β pS9 for at least 8 h. However, this action of 17β-estradiol was abrogated by PKA inhibitor H-89, AKT inhibitor LY294002, and MAPK inhibitor U0126. These results suggest that, while the protection of 17β-estradiol on CREB is MAPK dependent, its effect on GSK-3β integrates several pathways. These studies provide new insights into the role of estrogen in memory and AD.     

β-Lapachone ameliorization of experimental autoimmune encephalomyelitis

β-Lapachone is a naturally occurring quinine, originally isolated from the bark of the lapacho tree (Tabebuia avellanedae) which is currently being evaluated in clinical trials for the treatment of cancer. In addition, recent investigations suggest its potential application for treatment of inflammatory diseases. Multiple sclerosis (MS) is an autoimmune disorder characterized by CNS inflammation and demyelination. Reactive T cells including IL-17 and IFN-γ-secreting T cells are believed to initiate MS and the associated animal model system experimental autoimmune encephalomyelitis (EAE). IL-12 family cytokines secreted by peripheral dendritic cells (DCs) and CNS microglia are capable of modulating T-cell phenotypes. The present studies demonstrated that β-lapachone selectively inhibited the expression of IL-12 family cytokines including IL-12 and IL-23 by DCs and microglia, and reduced IL-17 production by CD4⁺ T-cells indirectly through suppressing IL-23 expression by microglia. Importantly, our studies also demonstrated that β-lapachone ameliorated the development on EAE. β-Lapachone suppression of EAE was associated with decreased expression of mRNAs encoding IL-12 family cytokines, IL-23R and IL-17RA, and molecules important in Toll-like receptor signaling. Collectively, these studies suggest mechanisms by which β-lapachone suppresses EAE and suggest that β-lapachone may be effective in the treatment of inflammatory diseases such as MS.     

Ratanasampil reduced levels of Aβ1-40 and Aβ1-42 and Aβ42/Aβ40 ratio in brains of Tg2576 mouse model of Alzheimer's disease

Objective To investigate the effect of ratanasampil (RNSP) which is traditional Chinese Tibet-Medicines on the β-amyloid peptide as a therapeutic target in Alzheimer's disease.Methods Thirteen female mice of 13-14 months old with Tg2576 transgene and ten BL6 × SJL mice as control were used in drug test All mice sacrificed after 8 weeks of RNSP treatment at 15-16 months of age.Open field activity and Y-maze tests were performed for animal behavioral change and memory ability.The β-amyloid peptides (Aβ1-40 and Aβ1-42) and β-amyloid precursor protein (APP) in Tg2576 mice brain were measured with western blot and enzyme linked immunosorbent assay (ELISA).Immunostaining with 1 E8 ( 1: 25 ) was carried out on brain sections of RNSP and vehicle-treated mice.Results The training times of Y-maze test decreased in RNSP-treated Tg2576 mice (P ＜0.01 ).After 2 months of RNSP treatment, a decrease in open field behavior was seen in Tg2576 mice ( P ＜ 0.05 ).The level of Aβ1-40 and Aβ1-42 in the brain was significantly decreased after RNSP treatment ( P ＜ 0.05 ).The Aβ42/Aβ40 ratio was significantly decreased after RNSP treatment as compared to vehicle-treated mice ( P ＜ 0.05 ) .But levels of APP in brain unchanged.Preliminary plaque counting of the sections showed reduced plaque numbers in the drug-treated mice in brain.Conclusions These results suggest that ratanasampil may reduce β-amyloid peptide production in brain to improve the learning and memory ability of Tg2576 mice of Alzheimer's disease.     

Ratanasampil reduced levels of Aβ1-40 and Aβ1-42 and Aβ42/Aβ40 ratio in brains of Tg2576 mouse model of Alzheimer's disease

Objective To investigate the effect of ratanasampil (RNSP) which is traditional Chinese Tibet-Medicines on the β-amyloid peptide as a therapeutic target in Alzheimer's disease.Methods Thirteen female mice of 13-14 months old with Tg2576 transgene and ten BL6 × SJL mice as control were used in drug test All mice sacrificed after 8 weeks of RNSP treatment at 15-16 months of age.Open field activity and Y-maze tests were performed for animal behavioral change and memory ability.The β-amyloid peptides (Aβ1-40 and Aβ1-42) and β-amyloid precursor protein (APP) in Tg2576 mice brain were measured with western blot and enzyme linked immunosorbent assay (ELISA).Immunostaining with 1 E8 ( 1: 25 ) was carried out on brain sections of RNSP and vehicle-treated mice.Results The training times of Y-maze test decreased in RNSP-treated Tg2576 mice (P ＜0.01 ).After 2 months of RNSP treatment, a decrease in open field behavior was seen in Tg2576 mice ( P ＜ 0.05 ).The level of Aβ1-40 and Aβ1-42 in the brain was significantly decreased after RNSP treatment ( P ＜ 0.05 ).The Aβ42/Aβ40 ratio was significantly decreased after RNSP treatment as compared to vehicle-treated mice ( P ＜ 0.05 ) .But levels of APP in brain unchanged.Preliminary plaque counting of the sections showed reduced plaque numbers in the drug-treated mice in brain.Conclusions These results suggest that ratanasampil may reduce β-amyloid peptide production in brain to improve the learning and memory ability of Tg2576 mice of Alzheimer's disease.     

β-Amyloid formation by myocytes of leptomeningeal vessels

Ultrastructural study of the leptomeningeal vessels of three subject with Alzheimer's disease (AD) shows that -amyloid deposits in the media of arteries and arterioles are produced by smooth muscle cells. It appears that the soluble -protein secreted by sarcolemmal vesicles of the muscle cell polymerizes into amyloid fibrils in basal lamina. Myocytes trapped in amyloid deposits degenerate and die. The most common and severe degeneration of smooth muscle cells in seen in the external and medial zone of the vascular media. In more advanced stages of amyloidotic changes, the internal zone of media is also involved. The media of vessels with severe changes consists of amyloid deposits and cell debris. Amyloid fibrils around the dead myocytes also undergo degradation. They lose their fibrillar appearance and become floccular, granular, amorphous proteinous material; however, this material is continually positive in immunostaining for -amyloid. This study suggests that amyloid formation by smooth muscle cells involves a secretory path. Our data indicate that the smooth muscle cell secretes nonfibrillar -protein or -protein containing peptides and that conversion of nonfibrillar into fibrillar -amyloid takes place in the environment of the basement membrane.Supported in part by funds from the New York State Office of Mental Retardation and Developmental Disabilities and a grant from the National Institutes of Health, National Institute of Aging No. PO1-AGO-4220     

β-Band correlates of the fMRI BOLD response

Oscillatory activity in the β-band (15-30 Hz) has been studied in detail in the sensorimotor cortex. It has been postulated that β-activity acts as a localized gating of cortical activity. Here, the induced oscillatory response in the β-band is measured by magnetoencephalography, and the hemodynamic response is measured by fMRI. We assess the linearity of the responses to stimuli of varying duration in the primary motor cortex and to a sinusoidal drifting grating of varying contrast amplitude and drift frequency in the visual cortex. In this way, we explore the nature of β-oscillations and their relationship with hemodynamic effects. Excellent spatial colocalization of BOLD and β-activity in both central and lateral (MT) visual areas and sensorimotor areas suggests that the two are intimately related. In contrast to the BOLD response, the level of β-desynchronization is not modulated by stimulus contrast or by stimulus duration, consistent with a gating role. The amplitude of β-desynchronization in the central visual area is however modulated by drift frequency, and this seems to parallel the modulation in BOLD amplitude at the same location.     

Neuroprotective effect of hydroxypropyl-β-cyclodextrin in hypoxia-ischemia

Neonatal cerebral ischemic injury is a common and debilitating pathology for which there is currently no known purely pharmacological treatments that are effective when delivered immediately after injury. Cyclodextrins are cyclic oligosaccharides that can remove cholesterol from cell membranes and thereby affect receptor function. Cyclodextrins have previously been shown to be neuroprotective in vitro. We showed that hydroxypropyl-β-cyclodextrin is neuroprotective in rats in vivo when delivered by intraperitoneal injection 30 min following hypoxia-ischemia, when assessed 15 days after surgery. A single dose of 1 g/kg hydroxypropyl-β-cyclodextrin reduced brain infarction size by 28.57% compared with control (P<0.001). We also report that the same compound reduces neuronal excitability in hippocampal slices and propose that hydroxypropyl-β-cyclodextrin is neuroprotective by reducing excitotoxicity in the delayed phase of brain damage.     

Induction of the C-terminal proteolytic cleavage of AβPP by statins

Statins are drugs commonly used to inhibit cholesterol synthesis, with the goal of reducing vascular diseases such as myocardial infarction and stroke. Statins have also been suggested as a therapeutic option for Alzheimer's disease (AD), although their benefit in AD remains controversial. We have previously shown that the intracellular C-terminal cleavage of the amyloid-β protein precursor (AβPP) is a major contributor to the neuronal toxicity seen in AD, and that this cleavage can be induced by amyloid-β. We now report that certain brain permeable statins are also able to induce the C-terminal cleavage of AβPP and associated cell death, whereas other statins do not. This statin effect on AβPP exceeded the effects of all other FDA-approved drugs in a library composed of these compounds, suggesting that this effect on AβPP cleavage is unique to a subset of the statins. Furthermore, the greatest effect occurred with cerivastatin, which has previously been shown to be the statin associated with the greatest risk of rhabdomyolysis. These results may have implications for the choice of which statins to evaluate in AD therapeutic trials; furthermore, the results may inform statin choice in individuals who are at high risk for the development of AD, such as those with an apolipoprotein E ε4 allele.     

Study of memapsin 2 (β-secretase) and strategy of inhibitor design

The discovery that β-secretase is a membrane-anchored aspartic protease memapsin 2 has stimulated much interest in the design and testing of its inhibitors for the treatment of Alzheimer’s disease. This article discusses the strategy for the development of such inhibitor drugs. Enzymology and structural determination tools have permitted the design of memapsin 2 inhibitors with high potency and in a size range possible for penetration of the blood-brain barrier. Transgenic Alzheimer’s mice have been used to show that when memapsin 2 inhibitors are transported to the brain, they effectively reduce the production of amyloid β. Although development of a clinical candidate of memapsin 2 inhibitor drug remains a very challenging undertaking, the progress so far lends some optimism for future prospects.     

Phosphorylation and function of α4β2 receptor

The neuronal nicotinic acetylcholine receptor (nAChR) alpha4 and beta2 subunits expressed in heterologous expression systems assemble into high- and low-affinity receptors (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003), which reflects the assembly of two distinct subunit stoichiometries of alpha4beta2 receptor (Nelson et al., 2003). The high-affinity receptor ([alpha4]2[beta2]3) is about 100-fold more sensitive to ACh than the low-affinity receptor ([alpha4]3[beta2]2) (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003). Recent evidence implicated 14-3-3 proteins as modulators of the relative abundance of nAChR subunits in the endoplasmic reticulum (ER), where ligand-gated ion channels assemble. The 14-3-3 proteins influence ER-to-plasma membrane trafficking of multimeric cell-surface proteins (O'Kelly et al., 2002). 14-3-3 proteins bind components of these multimeric proteins, and this interaction overrides dibasic COP1 retention signal to permit forward transport of the protein (O'Kelly et al., 2002). In the case of alpha4beta2 nAChRs, 14-3-3 binds the alpha4 subunit, and this association is dependent on phosphorylation of a serine residue within a protein kinase A(PKA) consensus sequence in the large cytoplasmic domain of the alpha4 subunit, which is also a binding motif recognized by 14-3-3 (Jeancloss et al., 2001; O'Kelly et al., 2002). The interplay among PKA, alpha4 subunits, and 14-3-3 proteins increases cell-surface expression of alpha4beta2 nAChRs by increasing steady-state levels of the alpha4 subunit available for assembly with beta2 subunits (Jeancloss et al., 2001). Because it is not known how 14-3-3-dependent changes in the steady-state levels of the alpha4 subunit might affect the functional type of alpha4beta2 receptors, we have investigated the effects of mutations of the 14-3-3 binding motif in the alpha4 subunit on alpha4beta2 nAChR function.     

Selective loss of Purkinje cells from the rat cerebellum caused by acrylamide and the responses ofβ-glucuronidase andβ-galactosidase

Summary Acrylamide (30mg/kg) given daily to rats five times each week for 3 weeks leads to progressive loss of Purkinje cells. The necrotic cells begin to be visible from the third day and their numbers reach a peak at the time when the dosing ceases at 18 days. They are less frequent thereafter, but are still visible almost 3 weeks later in small numbers. The density of Purkinje cells per millimeter falls to about 70% of normal at the 7th day, and a similar degree of reduction of the neuronal marker enzyme, -galactosidase, is found over the same time scale. By contrast, while there is a brisk macrophage/microglial response in the molecular layer to the loss of the Purkinje cell dendrites, the increase in -glucuronidase activity is relatively minor and is not significantly different from normal until after the 21st day. These responses are discussed in the context of the use of lysosomal enzyme activities in the assay of certain neurotoxic lesions.     

Upregulation of AKT attenuates amyloid-β-induced cell apoptosis

Overproduction and accumulation of amyloid-β (Aβ) have been proposed to be an initiating factor of neuron loss in Alzheimer's disease (AD). AKT is a pivotal molecule in regulating neuronal survival, however, it is still not known whether upregulation of AKT can protect the cells from the Aβ-induced apoptosis. By using cell viability assay and flow cytometry, we demonstrated in the present study that overexpression of AKT could significantly attenuate the cell apoptosis induced by Aβ1-42, whereas simultaneous inhibition of PI3 K, the immediate upstream stimulator of AKT, abolished the protective effect of AKT in HEK293 cells. Upregulation of AKT restored the Aβ-induced alterations of the mitochondria-related Bcl-2 family members (including Bcl-xL, Bcl-w, Bad, and Bax) and suppressed the activation of caspase-3 and JNK. Our data suggest that upregulation of AKT could be a promising therapeutic strategy for arresting Aβ toxicity in AD patients.     

17β-oestradiol regulation of gonadotrophin-releasing hormone neuronal excitability

17β-Oestradiol (E(2)) is essential for cyclical gonadotrophin-releasing hormone (GnRH) neuronal activity and secretion. In particular, E(2) increases the excitability of GnRH neurones during the afternoon of pro-oestrus in the rodent, which is associated with increased synthesis and secretion of GnRH. It is well established that E(2) regulates the activity of GnRH neurones through both presynaptic and postsynaptic mechanisms. E(2) significantly modulates the mRNA expression of numerous ion channels in GnRH neurones and alters the associated endogenous conductances, including potassium (K(ATP) , A-type) currents and low-voltage T-type and high-voltage L-type calcium currents. Notably, K(ATP) channels are critical for maintaining GnRH neurones in a hyperpolarised state for recruiting the T-type calcium channels, which are important for burst firing in GnRH neurones. In addition, there are other critical channels contributing to burst firing pattern, including the small conductance Ca(2+) -activated K(+) channels that may be modulated by E(2) . Despite these advances, the cellular mechanisms underlying the cyclical GnRH neuronal activity and GnRH release are largely unknown. Ultimately, the ensemble of both pre- and postsynaptic targets of the actions of E(2) will dictate the excitability and activity pattern of GnRH neurones.     

High- and low-sensitivity subforms of α4β2 and α3β2 nAChRs

Previous studies in other laboratories have shown that alpha4beta2 nicotinic acetylcholine receptor (nAChR) exhibits a biphasic concentration-response relationship for ACh with low and high EC50 components, and that the low EC50 component can be augmented by decreasing the alpha4:beta2 message ratio or incubating overnight in nicotine or at low temperature (Zwart and Vijverberg, 1998; Covernton and Connolly, 2000; Buisson and Bertrand, 2001; Nelson et al., 2003; Zhou et al., 2003). In the process of cloning ferret nAChR subunits, we found alpha4 and beta2 messages with long untranslated regions (UTRs), as well as those with no UTRs. Combinations of these messages revealed that the presence of UTRs influenced the ability to exclusively express high-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChRs. Injection of oocytes with alpha4 and beta2 RNAs lacking UTRs (1:1 ratio) led to expression of a biphasic concentration-response relationship for ACh with EC50 values of 0.5 (high sensitivity) and 114 microM(low sensitivity). Decreasing the alpha4:beta2 message ratio to as much as 1:120 increased the high-sensitivity component slightly, but the ACh concentration response remained biphasic. In contrast, injection of messages with UTRs (1:1 ratio) led to expression of a monophasic concentration response to ACh and a high-sensitivity EC50 value of 2.3 microM, as shown in Fig. 1.     

Intraneuronal accumulation of Aβ42 induces age-dependent slowing of neuronal transmission in Drosophila

Beta amyloid (Aβ₄₂)-induced dysfunction and loss of synapses are believed to be major underlying mechanisms for the progressive loss of learning and memory abilities in Alzheimer’s disease (AD). The vast majority of investigations on AD-related synaptic impairment focus on synaptic plasticity, especially the decline of long-term potentiation of synaptic transmission caused by extracellular Aβ₄₂. Changes in other aspects of synaptic and neuronal functions are less studied or undiscovered. Here, we report that intraneuronal accumulation of Aβ₄₂ induced an age-dependent slowing of neuronal transmission along pathways involving multiple synapses.     

Lack of dystrophin functionally affects α3β2/β4-nicotinic acethylcholine receptors in sympathetic neurons of dystrophic mdx mice

In the sympathetic superior cervical ganglion (SCG), nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission. We previously demonstrated that in SCG neurons of mdx mice, an animal model for Duchenne muscular dystrophy, lack of dystrophin causes a decrease, compared to the wild-type, in post-synaptic nAChRs containing the α3 subunit associated with β2 and/or β4 (α3β2/β4-nAChRs), but not in those containing the α7 subunit. Here we show, by whole cell patch-clamp recordings from cultured SCG neurons, that both nicotine and acetylcholine-evoked currents through α3β2/β4-nAChRs are significantly reduced in mdx mice compared to the wild-type, while those through α7-nAChR are unaffected. This reduction associates with that of protein levels of α3, β2 and β4 subunits. Therefore, we suggest that, in mdx mouse SCG neurons, lack of dystrophin, by specifically affecting membrane stabilization of α3β2/β4-nAChRs, could determine an increase in receptor internalization and degradation, with consequent reduction in the fast intraganglionic cholinergic transmission.     

Analysis of TGF-β2 and TGF-β3 expression in the hydrocephalic H-Tx rat brain

Introduction Transforming growth factor- (TGF-) is an important cytokine with modulatory actions in the nervous system. The development of hydrocephalus in mouse models resulting from the overexpression of TGF-1 has previously been described, but the mechanism by which this occurs remains obscure.Methods In order to evaluate the role of TGF- in hydrocephalus, we used SYBR Green I-based real-time quantitative RT-PCR method and Western blot analysis to analyze the TGF-2 and TGF-3 mRNA and protein expressions in the cerebral cortex of the H-Tx rat, a model of congenital hydrocephalus.Results The hydrocephalic H-Tx rat expressed significantly higher TGF-3 levels than their normal siblings (p<0.01) at 7 and 14 days of age. This difference became insignificant when analyzed at 21 days of age. On the other hand, such a difference has not been observed in the TGF-2 levels in the hydrocephalic H-Tx rat.Conclusions These results suggest that TGF-2 and TGF-3 expression may be modulated differently in the hydrocephalus, and TGF-3 may contribute to the development of hydrocephalus in this rat model.