Axonal BACE1 dynamics and targeting in hippocampal neurons: a role for Rab11 GTPase

BACKGROUND: BACE1 is one of the two enzymes that cleave amyloid precursor protein to generate Alzheimer's disease (AD) beta amyloid peptides. It is widely believed that BACE1 initiates APP processing in endosomes, and in the brain this cleavage is known to occur during axonal transport of APP. In addition, BACE1 accumulates in dystrophic neurites surrounding brain senile plaques in individuals with AD, suggesting that abnormal accumulation of BACE1 at presynaptic terminals contributes to pathogenesis in AD. However, only limited information is available on BACE1 axonal transport and targeting. RESULTS: By visualizing BACE1-YFP dynamics using live imaging, we demonstrate that BACE1 undergoes bi-directional transport in dynamic tubulo-vesicular carriers along axons in cultured hippocampal neurons and in acute hippocampal slices of transgenic mice. In addition, a subset of BACE1 is present in larger stationary structures, which are active presynaptic sites. In cultured neurons, BACE1-YFP is preferentially targeted to axons over time, consistent with predominant in vivo localization of BACE1 in presynaptic terminals. Confocal analysis and dual-color live imaging revealed a localization and dynamic transport of BACE1 along dendrites and axons in Rab11-positive recycling endosomes. Impairment of Rab11 function leads to a diminution of total and endocytosed BACE1 in axons, concomitant with an increase in the soma. Together, these results suggest that BACE1 is sorted to axons in endosomes in a Rab11-dependent manner. CONCLUSION: Our results reveal novel information on dynamic BACE1 transport in neurons, and demonstrate that Rab11-GTPase function is critical for axonal sorting of BACE1. Thus, we suggest that BACE1 transcytosis in endosomes contributes to presynaptic BACE1 localization

Pomegranate inhibits neuroinflammation and amyloidogenesis in IL-1β stimulated SK-N-SH cells

Purpose: Pomegranate fruit, Punica granatum L. (Punicaceae) and its constituents have been shown to inhibit inflammation. In this study we aimed to assess the effects of freeze-dried pomegranate (PWE) on PGE2 production in IL-1β stimulated SK-N-SH cells. Methods: An enzyme immuno assay (EIA) was used to measure prostaglandin E2 (PGE2) production from supernatants of IL-1β stimulated SK-N-SH cells. Expression of COX-2, phospho-IκB and phospho-IKK proteins were evaluated, while NF-κB reporter gene assay was carried out in TNFα-stimulated HEK293 cells to determine the effect of PWE on NF-κB transactivation. Levels of BACE-1 and Aβ in SK-N-SH cells stimulated with IL-1β were measured with an in cell ELISA. Results: PWE (25-200 µg/ml) dose dependently reduced COX-2 dependent PGE2 production in SK-N-SH cells stimulated with IL-1β. Phosphorylation of IκB and IKK were significantly (p<0.001) inhibited by PWE (50- 200 µg/ml). Our studies also show that PWE (50-200 µg/ml) significantly (p<0.01) inhibited NF-κB transactivation in TNFα-stimulated HEK293 cells. Furthermore PWE inhibited BACE-1 and Aβ expression in SK-N-SH cells treated with IL-1β. Conclusions: Taken together, our study demonstrates that pomegranate inhibits inflammation, as well as amyloidogenesis in IL-1β-stimulated SK-N-SH cells. We propose that pomegranate is a potential nutritional strategy in slowing the progression of neurodegenerative disorders like Alzheimer’s disease

Disposition of high dose busulfan in pediatric patients undergoing bone marrow transplantation

We studied the pharmacokinetics of busulfan (16 mg/kg) in 16 pediatric patients affected by malignant and nonmalignant disorders between 6 months and 19 years of age (mean ± SD, 5.7 ± 6.5 years) who were undergoing allogeneic (15 patients) and autologous (one patient) bone marrow transplantation. In all children, the conditioning regimen consisted of busulfan given orally at a dose of 1 mg/kg every 6 hours for 16 doses (total dose, 16 mg/kg), associated with other drugs. The pharmacokinetics of busulfan was studied during the 6‐hour dosing interval on the third day of therapy by use of a high‐performance liquid chromatographie assay. The value for the time to reach maximum concentration, expressed as mean ± SD, was 1.1 ± 0.5 hour; maximum concentration was 609.6 ± 225.3 ng/ml; steady‐state concentration was 358.9 ± 135.5 ng/ml; area under the plasma concentration–time curve was 2153.6 ± 813.1 ng · hr/ml; oral clearance was 0.535 ± 0.226 L/hr/kg; and half‐life was 2.4 ± 0.8 hours. Age‐related differences in busulfan disposition were observed. The mean busulfan oral clearance in a group of 10 patients with an age range from 6 months to 3 years was 0.619 L/hr/kg, whereas six patients whose ages ranged from 7 to 19 years had a oral clearance of 0.396 L/hr/kg. The half‐lives for busulfan during infancy decrease continuously until early childhood but were prolonged in older children. No significant relationship between systemic exposure to busulfan and drug effect was observed. Clinical Pharmacology and Therapeutics (1993) 54, 45–52; doi: © 1993 American Society for Clinical Pharmacology and Therapeutic

Disposition of high‐dose busulfan in pediatric patients undergoing bone marrow transplantation

We studied the pharmacokinetics of busulfan (16 mg/kg) in 16 pediatric patients affected by malignant and nonmalignant disorders between 6 months and 19 years of age (mean ± SD, 5.7 ± 6.5 years) who were undergoing allogeneic (15 patients) and autologous (one patient) bone marrow transplantation. In all children, the conditioning regimen consisted of busulfan given orally at a dose of 1 mg/kg every 6 hours for 16 doses (total dose, 16 mg/kg), associated with other drugs. The pharmacokinetics of busulfan was studied during the 6‐hour dosing interval on the third day of therapy by use of a high‐performance liquid chromatographie assay. The value for the time to reach maximum concentration, expressed as mean ± SD, was 1.1 ± 0.5 hour; maximum concentration was 609.6 ± 225.3 ng/ml; steady‐state concentration was 358.9 ± 135.5 ng/ml; area under the plasma concentration–time curve was 2153.6 ± 813.1 ng · hr/ml; oral clearance was 0.535 ± 0.226 L/hr/kg; and half‐life was 2.4 ± 0.8 hours. Age‐related differences in busulfan disposition were observed. The mean busulfan oral clearance in a group of 10 patients with an age range from 6 months to 3 years was 0.619 L/hr/kg, whereas six patients whose ages ranged from 7 to 19 years had a oral clearance of 0.396 L/hr/kg. The half‐lives for busulfan during infancy decrease continuously until early childhood but were prolonged in older children. No significant relationship between systemic exposure to busulfan and drug effect was observed. Clinical Pharmacology and Therapeutics (1993) 54, 45–52; doi: © 1993 American Society for Clinical Pharmacology and Therapeutic

Nitrite and nitrate plasma levels, as markers for nitric oxide synthesis, in thrombotic thrombocytopenic purpura (TTP). A case-control study

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Evaluation of two buflomedil tablet formulations in patients with atherosclerotic disease.

The bioequivalence of a 600-mg methocel tablet containing buflomedil hydrochloride in sustained-release form was determined relative to a 300-mg CAP/carbovax-coated tablet of buflomedil hydrochloride in immediate-release form. The tablets were given to 20 patients in a double-blind placebo- controlled clinical study with cross-over between the administration plans. The 300-mg tablets were given b.i.d., at 8 a.m. and 8 p.m. while the 600-mg tablets were taken once a day at 8 a.m. (+placebo at 8 p.m.). Plasma samples were collected at appropriate times up to 24 h after administration and were analysed for buflomedil with a validated high-performance liquid chromatographic procedure. Results showed an overall significant mean difference in absorption rate between the two formulations. The mean t(max) (5.5 \ub1 3.5 h) for the 600-mg tablet was longer (P<0.001) than the t(max) value (1.8 \ub1 0.8 h) seen after administration of the first 300-mg tablet. Analysis of AUC((O- 1e)) values indicated that the sustained-release preparation (32.1 \ub1 20.7 \u3bcg/ml h) was not significantly different from the 300-mg tablet b.i.d. (28.7 \ub1 16.0 \u3bcg/ml h). Furthermore, it was seen that single administration of a 600-mg sustained-release tablet of buflomedil hydrochloride delivered the same amount of total drug as a 300-mg tablet given twice a day

Ca2+ influx through store-operated Ca2+ channels reduces Alzheimer disease β-amyloid peptide secretion.

Alzheimer disease (AD), the leading cause of dementia, is characterized by the accumulation of β-amyloid peptides (Aβ) in senile plaques in the brains of affected patients. Many cellular mechanisms are thought to play important roles in the development and progression of AD. Several lines of evidence point to the dysregulation of Ca(2+) homeostasis as underlying aspects of AD pathogenesis. Moreover, direct roles in the regulation of Ca(2+) homeostasis have been demonstrated for proteins encoded by familial AD-linked genes such as PSEN1, PSEN2, and APP, as well as Aβ peptides. Whereas these studies support the hypothesis that disruption of Ca(2+) homeostasis contributes to AD, it is difficult to disentangle the effects of familial AD-linked genes on Aβ production from their effects on Ca(2+) homeostasis. Here, we developed a system in which cellular Ca(2+) homeostasis could be directly manipulated to study the effects on amyloid precursor protein metabolism and Aβ production. We overexpressed stromal interaction molecule 1 (STIM1) and Orai1, the components of the store-operated Ca(2+) entry pathway, to generate cells with constitutive and store depletion-induced Ca(2+) entry. We found striking effects of Ca(2+) entry induced by overexpression of the constitutively active STIM1(D76A) mutant on amyloid precursor protein metabolism. Specifically, constitutive activation of Ca(2+) entry by expression of STIM1(D76A) significantly reduced Aβ secretion. Our results suggest that disruptions in Ca(2+) homeostasis may influence AD pathogenesis directly through the modulation of Aβ production

Ca2+ influx through store-operated Ca2+ channels reduces Alzheimer disease β-amyloid peptide secretion.

Alzheimer disease (AD), the leading cause of dementia, is characterized by the accumulation of β-amyloid peptides (Aβ) in senile plaques in the brains of affected patients. Many cellular mechanisms are thought to play important roles in the development and progression of AD. Several lines of evidence point to the dysregulation of Ca(2+) homeostasis as underlying aspects of AD pathogenesis. Moreover, direct roles in the regulation of Ca(2+) homeostasis have been demonstrated for proteins encoded by familial AD-linked genes such as PSEN1, PSEN2, and APP, as well as Aβ peptides. Whereas these studies support the hypothesis that disruption of Ca(2+) homeostasis contributes to AD, it is difficult to disentangle the effects of familial AD-linked genes on Aβ production from their effects on Ca(2+) homeostasis. Here, we developed a system in which cellular Ca(2+) homeostasis could be directly manipulated to study the effects on amyloid precursor protein metabolism and Aβ production. We overexpressed stromal interaction molecule 1 (STIM1) and Orai1, the components of the store-operated Ca(2+) entry pathway, to generate cells with constitutive and store depletion-induced Ca(2+) entry. We found striking effects of Ca(2+) entry induced by overexpression of the constitutively active STIM1(D76A) mutant on amyloid precursor protein metabolism. Specifically, constitutive activation of Ca(2+) entry by expression of STIM1(D76A) significantly reduced Aβ secretion. Our results suggest that disruptions in Ca(2+) homeostasis may influence AD pathogenesis directly through the modulation of Aβ production