Hypertension impairs hippocampus-related adult neurogenesis, CA1 neuron dendritic arborization and long-term memory.

Hypertension is associated with neurodegenerative diseases and cognitive impairment. Several studies using spontaneous hypertensive rats to study the effect of hypertension on memory performance and adult hippocampal neurogenesis have reached inconsistent conclusions. The contradictory findings may be related to the genetic variability of spontaneous hypertensive rats due to the conventional breeding practices. The objective of this study is to examine the effect of hypertension on hippocampal structure and function in isogenic mice. Hypertension was induced by the '2 kidneys, 1 clip' method (2K1C) which constricted one of the two renal arteries. The blood pressures of 2K1C mice were higher than the sham group on post-operation day 7 and remained high up to day 28. Mice with 2K1C-induced hypertension had impaired long-term, but not short-term, memory. Dendritic complexity of CA1 neurons and hippocampal neurogenesis were reduced by 2K1C-induced hypertension on post-operation day 28. Furthermore, 2K1C decreased the levels of hippocampal brain-derived neurotrophic factor, while blood vessel density and activation status of astrocytes and microglia were not affected. In conclusion, hypertension impairs hippocampus-associated long-term memory, dendritic arborization and neurogenesis, which may be caused by down-regulation of brain-derived neurotrophic factor signaling pathways.

Transient ischemic attack induced by melted solid lipid microparticles protects rat brains from permanent focal ischemia.

This study aims to develop a transient ischemic attack (TIA) model in conscious animals and uses this model to investigate the effect of TIA on subsequent permanent ischemia. TIA was induced by injecting designed temperature-sensitive melted solid lipid microparticles with a melting point around body temperature into male Wistar rats via arterial cannulation. Neurologic deficit was monitored immediately after the injection without anesthesia. According to the clinical definition of TIA, rats were divided into neurologic symptom durations <24-h, 24-48-h and ≥48-h groups. The lipid microparticle-induced infarct volumes were small in the <24-h and 24-48-h groups, while the volumes were five times larger in the ≥48-h group. Permanent ischemic stroke was induced 3d after the induction of TIA by injecting a different kind of embolic particle manufactured by blending chitin and PLGA. The <24-h group had less severe neurologic deficits and smaller infarct volumes than that of 24-48-h and control (without prior lipid microparticle treatment) rats. Taken together, we successfully develop a TIA animal model which allows us to monitor the neurologic deficit in real-time. By adopting this model, we validate that TIA (<24h) preconditioning protects the brain from subsequent permanent ischemic stroke.

Chronic treadmill running protects hippocampal neurons from hypobaric hypoxia-induced apoptosis in rats.

This study was designed to examine the effects of chronic running exercise (Ex) on the hypobaric hypoxia-induced neuronal injury in the hippocampus. Male Wistar rats (9 weeks old) were caged in a hypoxic altitude chamber simulating the condition of 9,000 m high (0.303 atm) for 7h and the brains were examined at 0, 4, and 24h after treatment. Hypoxia challenge increased the levels of caspase 3 (mean ± SEM, % of baseline control, 121.9 ± 11.8, 152.3 ± 15.3, 141.6 ± 7.0 for 0, 4 and 24h, respectively, n=5) and induced apoptosis (cell number, 205.7 ± 8.8, 342.3 ± 33.4, 403.0 ± 12.2 for 0, 4 and 24h vs. 7.7 ± 1.4 baseline control, n=3) in the hippocampal CA1 pyramidal neurons. The expression levels (% of control for 0, 4 and 24h, respectively, n=5) of hypoxia inducible factor-1α (HIF-1α; 150.5 ± 8.1, 176.7 ± 11.1, 136.2 ± 13.3), neuronal nitric oxide synthase (nNOS; 163.4 ± 9.6, 194.5 ± 13.6, 163.7 ± 10.9) and inducible nitric oxide synthase (iNOS; 139.4 ± 9.5, 169.2 ± 13.3, 134.3 ± 13.0) and the degrees of microglia (cell number, 255.3 ± 48.2, 349.0 ± 57.3, 433.7 ± 42.4 vs. 57.7 ± 13.0 baseline control, n=3) and astrocyte (150.0 ± 9.7, 199.3 ± 10.8, 154.2 ± 4.7) activation were increased by the hypoxia treatment, indicating that the brain was under hypoxic, oxidative and inflammatory stresses. Furthermore, the protein levels of hippocampal brain-derived neurotrophic factor (BDNF; 76.0 ± 2.5, 76.1 ± 7.1, 69.3 ± 1.7 for 0, 4 and 24h, respectively, mean % of control ± SEM, n=5) were reduced by the hypoxia treatment. Four weeks of treadmill Ex before hypoxia treatment significantly reduced the hypoxia-induced apoptosis (p<0.001, n=3) in the hippocampal CA1 neurons. Ex decreased the hypoxia-induced elevations of HIF-1α (p<0.001, n=5), nNOS (p<0.001, n=5) and iNOS (p<0.001, n=5) levels and activation of microglia (p=0.005, n=3) and astrocyte (p<0.001, n=5) status; whereas the hypoxia-reduced BDNF protein levels (p=0.013, n=5) were restored. Taken together, our results show that chronic Ex protects hippocampal CA1 neurons against hypobaric hypoxia insult. Ex-enhanced bioenergetic adaptation and anti-oxidative capacity may prevent neurons from hypoxia-induced apoptosis. Furthermore, activation of the BDNF signaling pathway may be involved in the Ex-induced protection.

TGF-ß induces TIAF1 self-aggregation via type II receptor-independent signaling that leads to generation of amyloid ß plaques in Alzheimer's disease.

The role of a small transforming growth factor beta (TGF-ß)-induced TIAF1 (TGF-ß1-induced antiapoptotic factor) in the pathogenesis of Alzheimer's disease (AD) was investigated. TIAF1 physically interacts with mothers against DPP homolog 4 (Smad4), and blocks SMAD-dependent promoter activation when overexpressed. Accordingly, knockdown of TIAF1 by small interfering RNA resulted in spontaneous accumulation of Smad proteins in the nucleus and activation of the promoter governed by the SMAD complex. TGF-ß1 and environmental stress (e.g., alterations in pericellular environment) may induce TIAF1 self-aggregation in a type II TGF-ß receptor-independent manner in cells, and Smad4 interrupts the aggregation. Aggregated TIAF1 induces apoptosis in a caspase-dependent manner. By filter retardation assay, TIAF1 aggregates were found in the hippocampi of nondemented humans and AD patients. Total TIAF1-positive samples containing amyloid ß (Aß) aggregates are 17 and 48%, respectively, in the nondemented and AD groups, suggesting that TIAF1 aggregation occurs preceding formation of Aß. To test this hypothesis, in vitro analysis showed that TGF-ß-regulated TIAF1 aggregation leads to dephosphorylation of amyloid precursor protein (APP) at Thr668, followed by degradation and generation of APP intracellular domain (AICD), Aß and amyloid fibrils. Polymerized TIAF1 physically interacts with amyloid fibrils, which would favorably support plaque formation in vivo.

Associations of polybrominated diphenyl ethers (PBDEs) in breast milk and dietary habits and demographic factors in Taiwan.

The aim of this study was to examine levels of PBDEs in breast milk associated with seafood consumptions of Taiwanese mothers. Our participants were selected from healthy women recruited between December 2000 and November 2001 from a medical center in central Taiwan. The congeners of PBDEs in 20 milk samples were analyzed by a gas chromatograph with a high resolution mass detector. The mean level of BDE47 in breast milk from mothers with pre-pregnant BMI <22.0kg/m2 had a significantly higher magnitude compared to those with pre-pregnant BMI > or = 22.0kg/m2 (1.59 vs. 0.995ng/g lipid, p=0.041). We did not find significant correlations between PBDEs exposure levels and women's age, parity, blood pressure, annual household income, and education level. Women who ate more fish and meat did not show significantly higher PBDE levels than those who ate less, but a significant difference in PBDE levels was demonstrated between the higher (2.15ng/g lipid) and lower (3.98ng/g lipid) shellfish consuming subjects (p=0.002) after an adjustment for the confounders. The ratios of PCB153/BDE47, PCB153/BDE153, and PCB153/PBDEs were significantly correlated with frequent consumption of fish and shellfish. The PCB153/BDE153 ratio was not associated with the other dietary habits (i.e. meat). The ratios of PCB153/PBDEs may therefore be a new indicator for exposure as a result of seafood consumption.

Deprivation of pantothenic acid elicits a movement disorder and azoospermia in a mouse model of pantothenate kinase-associated neurodegeneration.

We asked whether a movement disorder could be elicited by deprivation of pantothenic acid (PA; vitamin B5), the substrate for the enzyme pantothenate kinase 2 (PANK2), which is deficient in the inherited neurological disorder PKAN (pantothenate kinase-associated neurodegeneration formerly called Hallervorden-Spatz syndrome). This study was undertaken because mice made null for Pank2 failed to show the neurological manifestations of the human disease. Wild-type and Pank2 mutant mice were fed pantothenic acid-deficient diets and were monitored for general health, fertility and movement compared with animals on control diets over time. Mice of both genotypes on PA-deficient diets exhibited poor grooming, greying of fur and decreased body weight. With PA deprivation, wild-type mice manifested azoospermia (a phenotype also seen in Pank2 mice) as well as a movement disorder with a low-lying pelvis and slow steps. Rear limbs appeared to drag and occasionally extended into unnatural postures for 16-17 s duration, possibly indicative of dystonia. Movement disruption probably also occurs in PA-deprived Pank2 mutant mice, but they died precipitously before undergoing detailed analysis. Remarkably, restoration of dietary PA led to recovery of general health and grooming, weight gain, reversal of the movement disorder, and reappearance of mature sperm within 4 weeks. This study confirms the primacy of PA metabolism in the mechanism of disease in PKAN. PA deprivation provides a useful phenocopy for PKAN and allows us to test pharmacological and other interventional strategies in the treatment of this devastating disease.

Expression, localisation and hormone regulation of the human copper transporter hCTR1 in placenta and choriocarcinoma Jeg-3 cells.

Copper is an essential trace element necessary for normal growth and development. During pregnancy, copper is transported from the maternal circulation to the fetus by mechanisms which have not been clearly elucidated. The copper uptake protein, hCTR1 is predicted to play a role in copper transport in human placental cells. This study has examined the expression and localisation of hCTR1 in human placental tissue and Jeg-3 cells. In term placental tissue the hCTR1 protein was detected as a 105 kDa protein, consistent with the size of a trimer which may represent the functional protein. A 95 kDa band, possibly representing the glycosylated protein, was also detected. hCTR1 was localised within the syncytiotrophoblast layer and the fetal vascular endothelial cells in the placental villi and interestingly was found to be localised toward the basal plasma membrane. It did not co-localise with either the Menkes or the Wilson copper transporting ATPases. Using the placental cell line Jeg-3, it was shown that the 35 kDa monomer was absent in the extracts of cells exposed to insulin, estrogen or progesterone and in cells treated with estrogen an additional 65 kDa band was detected which may correspond to a dimeric form of the protein. The 95 kDa band was not detected in the cultured cells. These results provide novel insights indicating that hormones have a role in the formation of the active hCTR1 protein. Furthermore, insulin altered the intracellular localisation of hCTR1, suggesting a previously undescribed role of this hormone in regulating copper uptake through the endocytic pathway.

Compulsive exercise acutely upregulates rat hippocampal brain-derived neurotrophic factor.

This study was to examine the effects of treadmill exercise on the expression of brain-derived neurotrophic factor (BDNF) in rat hippocampus. After 1-wk treadmill familiarization, animals in exercise groups received a 4-wk exercise training or an acute exercise. They were sacrificed 2 h or 2 d after exercise and their hippocampal BDNF mRNA and protein levels were determined. We demonstrated that 1) hippocampal BDNF mRNA and protein levels were both elevated in response to exercise training at 2 h after the last run but not after 2 d; 2) an acute moderate exercise (1 or 3 d) increased BDNF protein levels; 3) acute severe exercise increased BDNF protein and mRNA levels in animals under a familiarization regimen, while suppressed the BDNF mRNA level in rats without treadmill familiarization, paralleling the stress effect of immobilization/water exposure. We conclude that compulsive treadmill exercise with pre-familiarization acutely upregulates rat hippocampal BDNF gene expression.

Antibody to severe acute respiratory syndrome (SARS)-associated coronavirus spike protein domain 2 cross-reacts with lung epithelial cells and causes cytotoxicity.

Both viral effect and immune-mediated mechanism are involved in the pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. In this study, we showed that in SARS patient sera there were autoantibodies (autoAbs) that reacted with A549 cells, the type-2 pneumocytes, and that these autoAbs were mainly IgG. The autoAbs were detectable 20 days after fever onset. Tests of non-SARS-pneumonia patients did not show the same autoAb production as in SARS patients. After sera IgG bound to A549 cells, cytotoxicity was induced. Cell cytotoxicity and the anti-epithelial cell IgG level were positively correlated. Preabsorption and binding assays indicated the existence of cross-reactive epitopes on SARS-CoV spike protein domain 2 (S2). Furthermore, treatment of A549 cells with anti-S2 Abs and IFN-gamma resulted in an increase in the adherence of human peripheral blood mononuclear cells to these epithelial cells. Taken together, we have demonstrated that the anti-S2 Abs in SARS patient sera cause cytotoxic injury as well as enhance immune cell adhesion to epithelial cells. The onset of autoimmune responses in SARS-CoV infection may be implicated in SARS pathogenesis.

Effects of Tremella mesenterica on steroidogenesis in MA-10 mouse Leydig tumor cells.

Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as food and crude medicine to improve several kinds of symptoms in Chinese society for a long time. Recent studies have illustrated that the fractions of fruiting bodies of TM exhibit a significant hypoglycemic activity in diabetic mouse models, which usually suffer from sexual dysfunction. In a previous study, we showed that TM reduced plasma testosterone production in normal rats without any positive effect in diabetic rats. It evolved a question of TM directly regulating Leydig cell steroidogenesis. In this study, MA-10 mouse Leydig tumor cells were treated with vehicle, different dosages of TM with or without human chorionic gonadotropin (hCG 50 ng/ml) to clarify the effects. Results showed that TM at different dosages (0.01-10 mg/ml) did not have any effect on MA-10 cell steroidogenesis (p > 0.05). In the presence of hCG, there was an inhibitory trend that TA suppressed MA-10 cell progesterone production at 3 hr treatment with a statistically significant difference by the 10 mg/ml TM (p < 0.05). In time course effect, TM alone did not have any effect on MA-10 cell steroidogenesis from at 1, 2, 3, 6 and 12 hr (p > 0.05). However, TM did reduce hCG-treated MA-10 cell progesterone production at 1, 2 and 3 hr (p < 0.05), respectively. To determine whether TM would have adverse effects on MA-10 cell steroidogenesis in the presence of hCG, MTT assay and recovery studies were conducted. MTT assay indicated that TM had no effect on surviving cells. In addition, with the removal of TM, and then the addition of hCG (2 and 4 hr), progesterone levels were restored within 4 hr. Taken together, present studies suggested that TM suppressed hCG-treated steroidogenesis in MA-10 cells without any toxicity effect.

Dietary rescue of fumble--a Drosophila model for pantothenate-kinase-associated neurodegeneration.

Hallervorden-Spatz syndrome (HSS) is a devastating neurological disease, characterized by iron accumulation in the globus pallidus in the basal ganglia. Most HSS cases are caused by mutations in one of the four human pantothenate kinases (PANK2). This PANK2-caused subgroup of HSS is sometimes referred as PKAN (pantothenate-kinase-associated neurodegeneration). No effective treatment for PKAN or HSS is currently available. fumble, a Drosophila mutant that carries a mutation in Drosophila Pank, has many features similar to those of PKAN patients. In this study, we used fumble as a model to evaluate various compounds or nutritional products for their possible therapeutic efficacy. While no product was found to dramatically improve the symptoms, GKE (containing Ginkgo biloba extract and flavone) and vitamin E showed statistically significant beneficial effects. Our studies indicate that pantothenate is of limited value in alleviating fumble phenotypes and also suggest that some compounds might have deleterious effects.

Mislocalisation of hephaestin, a multicopper ferroxidase involved in basolateral intestinal iron transport, in the sex linked anaemia mouse.

BACKGROUND: Hephaestin is a multicopper ferroxidase required for basolateral transport of iron from enterocytes. Sex linked anaemia (sla) mice have a defect in the release of iron from intestinal enterocytes into the circulation due to an interstitial deletion in the hephaestin gene (heph). RESULTS: We have demonstrated that hephaestin is primarily localised to a supranuclear compartment in both intestinal enterocytes and in cultured cells. In normal intestinal enterocytes, hephaestin was also present on the basolateral surface. In sla mice, hephaestin was present only in the supranuclear compartment. In contrast, the iron permease Ireg1 localised to the basolateral membrane in both control and sla mice. CONCLUSION: We suggest that mislocalisation of hephaestin likely contributes to the functional defect in sla intestinal epithelium.

The association of a cystatin C gene polymorphism with late-onset Alzheimer's disease and vascular dementia.

A polymorphism in the cystatin C (CST3) gene was suggested to associate with Alzheimer's disease (AD). In the present study we attempted to determine the association between CST3 polymorphism and AD or vascular dementia (VD), and whether such effects are dependent of the APOE4 allele. The polymorphisms of CST3 genotype were determined using polymerase chain reactions (PCR) followed by gel electrophoresis in 124 AD, 70 VD, and 115 control individuals. No statistical difference in CST3B allele frequencies was observed among all three groups. Associations between CST3B/B genotype and AD patients older than 75-year-old, or VD patients younger than 75-year-old were evident. The APOE4 allele alone significantly increased the odds for the developing AD, but not VD. A logistic regression analysis revealed that either CST3 or its interaction with APOE4 were not significant predictors of AD. However, a synergistic association of CST3 and APOE4 alleles was observed in predicting VD patients. These results suggest that CST3 might interact with APOE4 on conferring vascular pathologies.

Brain region-dependent increases in beta-amyloid and apolipoprotein E levels in hypercholesterolemic rabbits.

Recent studies indicate a possible link between serum cholesterol level, beta-amyloid (Abeta) peptide concentrations, and the incidence of Alzheimer's disease (AD). In the present report, the effects of dietary cholesterol on Abeta and apolipoprotein E (APOE) levels in several brain regions were examined using diet-induced hypercholesterolemic rabbits as the animal model. Increased dietary cholesterol levels increased Abeta concentrations in temporal cortex (p = 0.02). A similar trend was observed in the frontal cortex (p = 0.06), yet not in the cerebellum. Interestingly, the regional levels of Abeta in the hypercholesterolemic rabbit paralleled the amyloid pathology observed in AD brain. Elevated APOE levels were also noticed in temporal (p < 0.01) and frontal (p < 0.01) cortices, but not in cerebellum, in the rabbit fed with cholesterol-abundant diet. These results suggest that high serum cholesterol levels could induce the elevation of brain APOE, which may play a role in aggravating the Abeta accumulation.

Striatal formation of 6-hydroxydopamine in mice treated with pargyline, pyrogallol and methamphetamine.

Formation of 6-hydroxydopamine (6-OHDA) has been posited in the striatum following methamphetamine treatment and plays a critical role in methamphetamine-induced nigrostriatal dopaminergic toxicity. We used high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to determine the formation of 6-OHDA by the treatments of methamphetamine combined with pargyline, a monoamine oxidase inhibitor, and pyrogallol, a catechol-O-methyl-transferase inhibitor, in female C57BL/6J mouse striatum. A substantial amount of 6-OHDA (9.9 +/- 0.7 pg/mg wet tissue) was detected in mice treated with pargyline (100 mg/kg) and pyrogallol (25 mg/kg) in combination. Greater striatal 6-OHDA levels were observed in mice treated with combined pargyline, pyrogallol and methamphetamine (50 mg/kg) as compared to mice treated with combined pargyline and pyrogallol. However, mice treated with pargyline and pyragollol in combination followed by one and two doses of methamphetamine exhibited comparable striatal 6-OHDA levels (23.2 +/- 4.3, 27.3 +/- 1.3 pg/mg wet tissue) in our protocol. We conclude that blockade of the primary metabolic pathways of dopamine by inhibiting both monoamine oxidase and catechol-O-methyl-transferase activities is sufficient to induce 6-OHDA formation in the striatum. Acute 6-OHDA accumulation in the striatum can be potentiated by methamphetamine, a potent dopamine releaser, administration following such metabolic inhibitions.

Apolipoprotein E polymorphism in various dementias in Taiwan Chinese population.

To verify the association between APOE4 frequency and various dementias in Taiwan Chinese individuals, APOE genotypes were determined in patients with dementia of Alzheimer's type (AD), vascular dementia (VD), dementias due to other general medical conditions (OD), and dementia of Alzheimer's type with cerebral vascular disease (mixed type dementia; MD). Only AD patients exhibited higher APOE4 frequency (OR = 2.95, p = 0.001) than controls after Bonferroni correction to control the overall type I error rate for the multiple testing. No such difference was observed among VD, OD, MD and control groups. The lack of association between VD and APOE4 allele frequency suggests that APOE4 allele does not associate with cerebrovascular pathology related dementia in Taiwan Chinese.

hGFRalpha-4: a new member of the GDNF receptor family and a candidate for NBIA.

Hallervorden-Spatz syndrome (neurodegeneration with brain iron accumulation type 1; OMIM entry 234200) is a rare inherited neurodegenerative disease. In this article, evidence for a newly identified gene as a candidate for Hallervorden-Spatz syndrome is given. Previously Hallervorden-Spatz syndrome was mapped to a 4-cm region in 20p12.3-13. During positional cloning efforts a new member of the glial-derived neurotrophic factor receptor family was discovered in this region. Like other members of this receptor family, this new gene is predicted to be secreted and glycosyl-phosphatidylinositol linked, and it maintains conserved cysteine residues. However, cDNA and genomic studies in both humans and mice indicate that this gene lacks the sequence corresponding to exons 2 and 3 in other family members. In situ hybridization reveals that it is expressed primarily in the brain and bladder in the embryonic mouse. Mutation analysis of patients with Hallervorden-Spatz syndrome revealed two potentially significant amino acid changes in two patients but failed to identify mutations in the remaining 10 subjects. The implication of these findings for the relationship between this gene and Hallervorden-Spatz syndrome is discussed.

Reduction of cortical amyloid beta levels in guinea pig brain after systemic administration of physostigmine.

Overproduction of the peptide amyloid beta (Abeta) is thought to be a critical pathogenetic event in Alzheimer's disease (AD). Decreasing A production may therefore slow or halt the progression of AD. In vitro work has indicated that cholinergic muscarinic receptor agonists may reduce cellular production of Abeta. Here we show that systemic administration of physostigmine, an acetylcholinesterase inhibitor, lowers Abeta levels in vivo. Guinea pigs treated for 10 days with s.c. physostigmine had levels of cortical AbetaN-40 and N-42 which were 57% and 72%, respectively, of those in control animals. Levels of cortical beta-amyloid precursor protein were not significantly affected by drug treatment. These results suggest that cholinergic therapy may affect the course of AD by limiting Abeta accumulation.

The copper transporter CTR1 provides an essential function in mammalian embryonic development.

Copper serves as an essential cofactor for a variety of proteins in all living organisms. Previously, we described a human gene (CTR1;SLC31A1) that encodes a high-affinity copper-uptake protein and hypothesized that this protein is required for copper delivery to mammalian cells. Here, we test this hypothesis by inactivating the Ctr1 gene in mice by targeted mutagenesis. We observe early embryonic lethality in homozygous mutant embryos and a deficiency in copper uptake in the brains of heterozygous animals. Ctr1(-/-) embryos can be recovered at E8.5 but are severely developmentally retarded and morphologically abnormal. Histological analysis reveals discontinuities and variable thickness in the basement membrane of the embryonic region and an imperfect Reichert's membrane, features that are likely due to lack of activity in the collagen cross-linking cupro-enzyme lysyl oxidase. A collapsed embryonic cavity, the absence of an allantois, retarded mesodermal migration, and increased cell death are also apparent. In the brains of heterozygous adult mice, which at 16 months are phenotypically normal, copper is reduced to approximately half compared with control littermates, implicating CTR1 as the required port for copper entry into at least this organ. A study of the spatial and temporal expression pattern of Ctr1 during mouse development and adulthood further shows that CTR1 is ubiquitously transcribed with highest expression observed in the specialized epithelia of the choroid plexus and renal tubules and in connective tissues of the eye, ovary, and testes. We conclude that CTR1 is the primary avenue for copper uptake in mammalian cells.