Yuan Su

Raloxifene and estradiol benzoate both fully restore hippocampal choline acetyltransferase activity in ovariectomized rats.

Selective estrogen receptor modulators (SERMs) demonstrate tissue-specific estrogen receptor (ER) agonist or antagonist properties. Raloxifene, a prototypical SERM, has ER agonist properties in bone and on cholesterol metabolism but full antagonist properties in the uterus and breast. To characterize the ER agonist/antagonist profile of raloxifene in the brain, we have examined its effect on the activity of a known estrogen-responsive gene product, choline acetyltransferase (ChAT), in the hippocampus and other brain regions of 6-month-old ovariectomized (OVX) Sprague-Dawley rats. Three weeks post-ovariectomy, animals received estradiol benzoate (EB, 0.03 mg or 0.3 mg kg(-1) day(-1) for 3 or 10 days); raloxifene HCl (3.0 mg kg(-1) day(-1) for 3 or 10 days), tamoxifen (3.0 mg kg(-1) day(-1) for 10 days) or vehicle (20% CDX). As previously reported, ChAT activity decreased by approximately 20%-50% in the hippocampus of OVX compared with SHAM-operated control rats with no change in ChAT activity observed in the hypothalamus. Raloxifene or EB reversed the OVX-induced decrease in ChAT activity in the hippocampus but did not change ChAT activity in the hypothalamus. Animals that received combined EB (0.03 mg/kg) plus raloxifene (1 mg/kg) or tamoxifen alone (3.0 or 10 mg/kg) also showed increased hippocampal ChAT activity. Raloxifene failed to increase uterine weight and blocked the estrogen-induced increase in uterine weight, while another SERM, tamoxifen, increased uterine weight. These data demonstrate that raloxifene has estrogen-like properties on hippocampal ChAT activity in vivo, and suggest that benzothiophene SERMs may exert estrogen-like beneficial effects on cholinergic neurotransmission in brain without producing peripheral stimulation of breast or uterine tissue.

Geranylgeranyl pyrophosphate stimulates γ-secretase to increase the generation of Aβ and APP-CTFγ

Cleavage of the amyloid precursor protein (APP) by β‐ and γ‐secretases results in generation of the amyloid‐β protein (Aβ), which is characteristically deposited in the brain of Alzheimers disease patients. Inhibitors of 3‐hydroxy‐3‐methyl‐glutaryl (HMG)‐CoA reductase (the statins) reduce levels of cholesterol and isoprenoids such as geranylgeranyl pyrophosphate (GGPP). Previous studies have demonstrated that cholesterol increases and statins reduce Aβ levels mostly by regulating β‐secretase activity. In this study, we focused on the role of geranylgeranyl isopre‐noids GGPP and geranylgeraniol (GGOH) in regulating Aβ production. Our data show that the inhibition of GGPP synthesis by statins plays an important role in statin‐mediated reduction of Aβ secretion. Consistent with this finding, the geranylgeranyl isoprenoids preferentially increase the yield of Aβ of 42 residues (Aβ42) in a dose‐dependent manner. Our studies further demonstrated that geranylgeranyl iso‐prenoids increase the yield of APP‐CTFγ (a.k.a. AICD) as well as Aβ by stimulating γ‐secretase‐mediated cleavage of APP‐CTFα and APP‐CTFβ in vitm. Furthermore, GGOH increases the levels of the active γ‐secretase complex in the detergent‐insoluble membrane fraction along with its substrates, APP‐CTFα and APP‐CTFβ. Our results indicate that geranylgeranyl isoprenoids may be an important physiological facilitator of γ‐secretase activity that can foster production of the pathologically important Aβ42.— Zhou, Y., Suram, A., Venugopal, C., Prakasam, A., Lin, S., Su, Y., Li, B., Paul, S. M., Sambamurti, K. Geranylgeranyl pyrophos‐phate stimulates γ‐secretase to increase the generation of Aβ and APP‐CTFγ. FASEB J. 22, 47–54 (2008)

Inhibition of Aβ production and APP maturation by a specific PKA inhibitor

Alzheimers disease is characterized pathologically by extracellular amyloid β protein (Aβ) deposition in the brain. The Aβ peptide, a 39–42 amino acid fragment, is derived from defined proteolysis of the amyloid precursor protein (APP) [Glenner et al., Appl. Pathol. 2 (1984) 357–369; Selkoe, Neuron 6 (1991) 487–498] and is the primary component of senile plaques. Although it is known that intracellular APP is subjected to posttranslational modification, the molecular mechanism that regulates the APP processing is not completely clear. In the present study, we demonstrates that H89, a specific inhibitor for cAMP dependent protein kinase A (PKA), inhibits Aβ production and APP secretion in a dose dependent manner in cells stably transfected with human APP bearing a ‘Swedish mutation’. Concurrent with the effect, H89 inhibits C‐terminal fragment of the APP. We also found that the PKA inhibitor abolishes the mature form of intracellular APP and accumulates the immature form. Finally, direct administration of H89 into brains of transgenic mice overexpressing human APP shows that the compound inhibits Aβ production in the hippocampal region. Our data suggests that PKA plays an important role in the maturation of APP associated with APP processing.

Overexpression of GSK3βS9A Resulted in Tau Hyperphosphorylation and Morphology Reminiscent of Pretangle-Like Neurons in the Brain of PDGSK3β Transgenic Mice

It has been demonstrated that GSK3β is involved in Alzheimer Disease (AD) pathogenesis. In order to understand the underlying mechanism, we have generated and characterized transgenic mice in which the constitutively active human GSK3β(with S9A mutation) was overexpressed in the brain under the control of the platelet-derived growth factor (PDGF) B-chain promoter. Varying levels of human GSK3βS9A transgene protein expression was observed in six of the seven founders generated. Line 3083, 3107, 3112 and 3125 displayed higher GSK3βS9A protein expression levels. Immunostaining analysis demonstrated that transgene expression was observed mainly in cortex and hippocampus of transgenic brain. Expression of human GSK3β transgene did not significantly change the brain total GSK3β protein levels in any of the generated mouse lines, as comparing to age matched wild type mice. Although significant kinase activity was detected in human GSK3βS9A transgene protein extracted from brains of all six expressing lines, significant increase in total GSK3βS9A kinase activity was observed only in the offspring of line 3083 and 3107. By analyzing the offspring from several transgenic mouse lines, including lines other than 3083 and 3107, it was found that overexpressed constitutively active human GSK3βS9A resulted in hyperphosphorylation of tau and morphology reminiscent of pretangle-like neurons in cortex and hippocampus.

Regulation of amyloid precursor protein expression and secretion via activation of ERK1/2 by hepatocyte growth factor in HEK293 cells transfected with APP751

The increased intracellular levels and aberrant processing of the amyloid precursor protein (APP) are associated with beta-amyloid peptide (A beta) production, cerebrovascular amyloid deposition, and amyloid plaque formation. Here we report that APP level, soluble APP (sAPP) secretion, and A beta production in HEK293 cells transfected with either wild-type APP(751) or APP(751) carrying the Swedish mutation are all elevated by hepatocyte growth factor (HGF). We investigated the potential molecular mechanisms underlying the HGF effect. Our data show that HGF stimulated extended activation of extracellular signal-regulated protein kinases (ERK1/2). Pretreatment of cells with inhibitors (UO126 or PD98059) for MEK, the upstream kinase of ERK1/2, abolished ERK1/2 activation evoked by HGF, and abrogated HGF-induced increases in APP levels and sAPP secretion. In addition, transient expression of active MEK1 activated ERK1/2 and increased intracellular APP levels and sAPP secretion. Inhibition of ERK1/2 activity, however, failed to block HGF-stimulated A beta production. Consistently, transient expression of active MEK1 did not increase A beta accumulation. Taken together, these results suggest that: (1) HGF regulates the intracellular levels of APP and the secretion of sAPP and A beta; (2) the modulation of APP levels and sAPP secretion induced by HGF is mediated via the MEK1/ERK1/2 signaling pathway; (3) HGF-stimulated A beta production is independent of ERK activity and, therefore, independent of HGF-evoked elevation of intracellular APP levels.

RIFLE: A novel ring zinc finger‐leucine‐rich repeat containing protein, regulates select cell adhesion molecules in PC12 cells

Cell adhesion molecules play a critical role in cell contacts, whether cell–cell or cell–matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger‐leucine‐rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase‐3α/β (GSK‐3α/β) resulting in the inhibition of GSK‐3 kinase activity and increase of β‐catenin levels. RIFLE expression also is associated with elevated E‐cadherin protein levels but not N‐cadherin. The regulation of these cell adhesion‐associated molecules by RIFLE is accompanied by a significant increase in cell–cell and cell–matrix adhesion. Moreover, increase in cell–cell adhesion but not cell–matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK‐3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells.