Hypoxia promotes tau hyperphosphorylation with associated neuropathology in vascular dysfunction.

BACKGROUND: Hypertension-induced microvascular brain injury is a major vascular contributor to cognitive impairment and dementia. We hypothesized that chronic hypoxia promotes the hyperphosphorylation of tau and cell death in an accelerated spontaneously hypertensive stroke prone rat model of vascular cognitive impairment. METHODS: Hypertensive male rats (n = 13) were fed a high salt, low protein Japanese permissive diet and were compared to Wistar Kyoto control rats (n = 5). RESULTS: Using electron paramagnetic resonance oximetry to measure in vivo tissue oxygen levels and magnetic resonance imaging to assess structural brain damage, we found compromised gray (dorsolateral cortex: p = .018) and white matter (corpus callosum: p = .016; external capsule: p = .049) structural integrity, reduced cerebral blood flow (dorsolateral cortex: p = .005; hippocampus: p < .001; corpus callosum: p = .001; external capsule: p < .001) and a significant drop in cortical oxygen levels (p < .05). Consistently, we found reduced oxygen carrying neuronal neuroglobin (p = .008), suggestive of chronic cerebral hypoperfusion in high salt-fed rats. We also observed a corresponding increase in free radicals (NADPH oxidase: p = .013), p-Tau (pThr231) in dorsolateral cortex (p = .011) and hippocampus (p = .003), active interleukin-1ß (p < .001) and neurodegeneration (dorsolateral cortex: p = .043, hippocampus: p = .044). Human patients with subcortical ischemic vascular disease, a type of vascular dementia (n = 38; mean age = 68; male/female ratio = 23/15) showed reduced hippocampal volumes and cortical shrinking (p < .05) consistent with the neuronal cell death observed in our hypertensive rat model as compared to healthy controls (n = 47; mean age = 63; male/female ratio = 18/29). CONCLUSIONS: Our data support an association between hypertension-induced vascular dysfunction and the sporadic occurrence of phosphorylated tau and cell death in the rat model, correlating with patient brain atrophy, which is relevant to vascular disease.

MMP-9 inhibitors impair learning in spontaneously hypertensive rats.

Vascular cognitive impairment dementia (VCID) is a major cause of cognitive loss in the elderly. Matrix metalloproteinases (MMPs) are a family of proteases involved in remodeling the extracellular matrix in development, injury and repair. Blood-brain barrier (BBB) disruption due to inflammation mediated by MMPs is a mechanism of white matter injury. Currently there are no treatments besides the control of vascular risk factors. We tested two MMP-9 inhibitors that improved outcome in acute stroke: DP-460 and SB-3CT. We hypothesized that these inhibitors would have a beneficial effect in chronic stroke by reducing edema in white matter and improving behavioral outcomes. Spontaneously hypertensive stroke-prone rats (SHRSPs) with unilateral carotid artery occlusion (UCAO) fed a Japanese Permissive Diet (JPD) were used as a model of VCID. JPD was begun in the 12th week of life. Rats were treated with DP-460 (500 mg/kg) for 4 weeks, or SB-3CT (10 mg/kg) for 8 weeks, beginning at the UCAO/JPD onset. Rats treated with a dextrose or DMSO solution served as vehicle controls. Naïve SHRSPs on a standard diet served as sham control. Magnetic resonance imaging (MRI) analyses of the corpus callosum, external capsule, hippocampus and Morris water maze behavioral tests were conducted. We found an increase in body weight (p = 0.004) and blood pressure (p = 0.007) at 15 weeks with the DP-460 drug. SB-3CT increased body weight at 14 weeks (p = 0.015) and had significant but variable effects on blood pressure. Neither drug affected imaging parameters. Behavioral studies showed an impaired ability to learn with DP-460 (p<0.001) and no effect on learning with SB-3CT. Unchanged MMP-9 levels were detected in DP-460-treated rats via gel zymography. Our findings suggest that MMPs are not major factors in white matter damage in the SHRSP model of VCID and that drugs that are relatively selective for MMP-9 can interfere with learning.

Vascular tight junction disruption and angiogenesis in spontaneously hypertensive rat with neuroinflammatory white matter injury.

Vascular cognitive impairment is a major cause of dementia caused by chronic hypoxia, producing progressive damage to white matter (WM) secondary to blood-brain barrier (BBB) opening and vascular dysfunction. Tight junction proteins (TJPs), which maintain BBB integrity, are lost in acute ischemia. Although angiogenesis is critical for neurovascular remodeling, less is known about its role in chronic hypoxia. To study the impact of TJP degradation and angiogenesis during pathological progression of WM damage, we used the spontaneously hypertensive/stroke prone rats with unilateral carotid artery occlusion and Japanese permissive diet to model WM damage. MRI and IgG immunostaining showed regions with BBB damage, which corresponded with decreased endothelial TJPs, claudin-5, occludin, and ZO-1. Affected WM had increased expression of angiogenic factors, Ki67, NG2, VEGF-A, and MMP-3 in vascular endothelial cells and pericytes. To facilitate the study of angiogenesis, we treated rats with minocycline to block BBB disruption, reduce WM lesion size, and extend survival. Minocycline-treated rats showed increased VEGF-A protein, TJP formation, and oligodendrocyte proliferation. We propose that chronic hypoxia disrupts TJPs, increasing vascular permeability, and initiating angiogenesis in WM. Minocycline facilitated WM repair by reducing BBB damage and enhancing expression of TJPs and angiogenesis, ultimately preserving oligodendrocytes.

The neuropathology and cerebrovascular mechanisms of dementia.

The prevalence of dementia is increasing in our aging population at an alarming rate. Because of the heterogeneity of clinical presentation and complexity of disease neuropathology, dementia classifications remain controversial. Recently, the National Plan to address Alzheimer's Disease prioritized Alzheimer's disease-related dementias to include: Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia, vascular dementia, and mixed dementias. While each of these dementing conditions has their unique pathologic signature, one common etiology shared among all these conditions is cerebrovascular dysfunction at some point during the disease process. The goal of this comprehensive review is to summarize the current findings in the field and address the important contributions of cerebrovascular, physiologic, and cellular alterations to cognitive impairment in these human dementias. Specifically, evidence will be presented in support of small-vessel disease as an underlying neuropathologic hallmark of various dementias, while controversial findings will also be highlighted. Finally, the molecular mechanisms shared among all dementia types including hypoxia, oxidative stress, mitochondrial bioenergetics, neuroinflammation, neurodegeneration, and blood­brain barrier permeability responsible for disease etiology and progression will be discussed.

Attenuation of mitochondrial and nuclear p38α signaling: a novel mechanism of estrogen neuroprotection in cerebral ischemia.

P38 mitogen-activated protein kinase (MAPK) is a pro-apoptotic and pro-inflammatory protein that is activated in response to cellular stress. While p38 is known to be activated in response to cerebral ischemia, the precise role of p38 and its isoforms in ischemia-induced neuronal apoptosis remains unclear. In the current study, we examined the differential activation and functional roles of p38α and p38ß MAPK isoforms in short-term ovariectomized female rats treated with either the neuroprotective ovarian hormone 17beta-estradiol (E2) or placebo in a model of global cerebral ischemia (GCI). GCI induced biphasic activation of total p38 in the hippocampal CA1, with peaks at 30 min and 1 day after 10-min ischemia-reperfusion. Further study demonstrated that activated p38α, but not p38ß, translocated to the nucleus 30 min and 3 h post reperfusion, and that this event coincided with increased phosphorylation of activating transcription factor 2 (ATF2), a p38 target protein. Intriguingly, activated p38α was also enhanced in mitochondrial fractions of CA1 neurons 1 day after GCI, and there was loss of mitochondrial membrane potential, as well as enhanced cytochrome c release and caspase-3 cleavage at 2 days post GCI. Importantly, E2 prevented the biphasic activation of p38, as well as both nuclear and mitochondrial translocation of p38α after GCI, and these findings correlated with attenuation of mitochondrial dysfunction and delayed neuronal cell death in the hippocampal CA1. Furthermore, administration of a p38 inhibitor was able to mimic the neuroprotective effects of E2 in the hippocampal CA1 region by preventing nuclear and mitochondrial translocation of p38α, loss of mitochondrial membrane potential, and neuronal apoptosis. As a whole, this study suggests that changes in subcellular localization of the activated p38α isoform are required for neuronal apoptosis following GCI, and that E2 exerts robust neuroprotection, in part, through dual inhibition of activation and subcellular trafficking of p38α.

Differential effects of oral and transdermal menopausal hormone therapy on prostacyclin and thromboxane in platelets.

Abstract Menopausal hormone therapies (MHT) may increase thrombotic risk but modulate endothelial function and reduce development of vascular lesions. This study compared effects of MHT on prostanoid-modulated adenosine triphosphate (ATP) secretion from platelets in relationship with endothelial reactive hyperemia (RH) index and carotid intima medial thickness (CIMT). Participants were healthy, recently menopausal women of the Kronos Early Estrogen Prevention Study (KEEPS) randomized to one of three treatments: oral conjugated equine estrogen (oCEE, 0.45 mg/day), transdermal 17ß-estradiol (tE2, 50 µg/day) each with intermittent oral progesterone or placebo pills and patch (PL). Prostacyclin and thromboxane A2 were assessed by quantification of their stable metabolites (6-keto-prostaglandin F1α, 6-k-PGF1α; thromboxane B2, TXB2), using ELISA. Dense granule ATP secretion from activated platelets was determined by bioluminescence; RH and CIMT were determined by fingertip tonometry and ultrasound, respectively. After 48 months of treatment, platelet content of 6-k-PGF1α and TXB2 was significantly lower in oCEE compared to the PL. Inhibition of ATP secretion by exogenous activation of cAMP associated with platelet 6-k-PGF1α (r = -0.41, P = 0.04) and TXB2 (r = 0.71, P = 0.0005) only in the oCEE group. Serum and platelet content of 6-k-PGF1α and TXB2 associated positively in the PL and tE2 groups. Serum 6-k-PGF1α positively associated with RH in the oCEE group (r = 0.73, P = 0.02), while serum TXB2 positively associated with CIMT in the tE2 group (r = 0.64, P = 0.01). Thus, oCEE and tE2 differentially affect prostanoid-mediated platelet secretory pathways but alone would not account for an increased thrombotic risk for oral MHT. Furthermore, platelet-derived prostanoids may contribute to RH and vascular remodeling in healthy menopausal women.

Estrogen and cerebrovascular regulation in menopause.

Estrogen (E2), classically viewed as a reproductive steroid hormone, has non-reproductive functions throughout the body including in the brain and vasculature. Studies report diminished neuroprotection with declining E2 levels, corresponding with higher incidence of cerebrovascular and neurological disease. However, the effects of menopausal hormone therapy (MHT) on the cerebral vasculature and brain function remain controversial. This review will focus on evidence of 17ß-estradiol actions in the cerebral vasculature, with a particular emphasis on the vasoactive, anti-inflammatory, anti-oxidant, metabolic and molecular properties. Controversies surrounding MHT in relation to cerebrovascular disease and stroke risk will be discussed, particularly the emerging evidence from clinical trials supporting the critical period hypothesis of estrogen protection.

Thrombogenic microvesicles and white matter hyperintensities in postmenopausal women.

OBJECTIVE: To determine the association of conventional cardiovascular risk factors, markers of platelet activation, and thrombogenic blood-borne microvesicles with white matter hyperintensity (WMH) load and progression in recently menopausal women. METHODS: Women (n = 95) enrolled in the Mayo Clinic Kronos Early Estrogen Prevention Study underwent MRI at baseline and at 18, 36, and 48 months after randomization to hormone treatments. Conventional cardiovascular risk factors, carotid intima-medial thickness, coronary arterial calcification, plasma lipids, markers of platelet activation, and thrombogenic microvesicles were measured at baseline. WMH volumes were calculated using a semiautomated segmentation algorithm based on fluid-attenuated inversion recovery MRI. Correlations of those parameters with baseline WMH and longitudinal change in WMH were adjusted for age, months past menopause, and APOE ε4 status in linear regression analysis. RESULTS: At baseline, WMH were present in all women. The WMH to white matter volume fraction at baseline was 0.88% (0.69%, 1.16%). WMH volume increased by 122.1 mm(3) (95% confidence interval: -164.3, 539.5) at 36 months (p = 0.003) and 155.4 mm(3) (95% confidence interval: -92.13, 599.4) at 48 months (p < 0.001). These increases correlated with numbers of platelet-derived and total thrombogenic microvesicles at baseline (p = 0.03). CONCLUSION: Associations of platelet-derived, thrombogenic microvesicles at baseline and increases in WMH suggest that in vivo platelet activation may contribute to a cascade of events leading to development of WMH in recently menopausal women.

Considerations of sex and gender differences in preclinical and clinical trials.

Women continue to be underrepresented in clinical trials, particularly in Phases I and II of experimental drug studies in spite of legislative guidelines in the USA, Canada, the European Union, Australia, and Japan requiring the inclusion of women in clinical trials. As such, women remain a vulnerable population subject to the adverse effects of pharmacological therapies. Thus, women experience higher rates of adverse drug reactions than do men and for women of reproductive age or who may be pregnant, therapeutic options may be limited. This chapter provides a brief history of inclusion of sex and gender as variables in clinical trials, summarizes governmental legislation for consideration of sex and gender in clinical trials and provides specific examples of drugs which have been withdrawn from the market because of side effects in women. Additional information related to sex and gender in preclinical testing, trial design, challenges to recruitment of women for clinical trials and statistical methods for analysis of data also is considered.

Acetylation of the pro-apoptotic factor, p53 in the hippocampus following cerebral ischemia and modulation by estrogen.

BACKGROUND: Recent studies demonstrate that acetylation of the transcription factor, p53 on lysine(373) leads to its enhanced stabilization/activity and increased susceptibility of cells to stress. However, it is not known whether acetylation of p53 is altered in the hippocampus following global cerebral ischemia (GCI) or is regulated by the hormone, 17ß-estradiol (17ß-E(2)), and thus, this study examined these issues. METHODOLOGY/PRINCIPAL FINDINGS: The study revealed that Acetyl p53-Lysine(373) levels were markedly increased in the hippocampal CA1 region after GCI at 3 h, 6 h and 24 h after reperfusion, an effect strongly attenuated by 17ß-E(2). 17ß-E(2) also enhanced interaction of p53 with the ubiquitin ligase, Mdm2, increased ubiquitination of p53, and induced its down-regulation, as well as attenuated elevation of the p53 transcriptional target, Puma. We also observed enhanced acetylation of p53 at a different lysine (Lys(382)) at 3 h after reperfusion, and 17ß-E(2) also markedly attenuated this effect. Furthermore, administration of an inhibitor of CBP/p300 acetyltransferase, which acetylates p53, was strongly neuroprotective of the CA1 region following GCI. In long-term estrogen deprived (LTED) animals, the ability of 17ß-E(2) to attenuate p53 acetylation was lost, and intriguingly, Acetyl p53-Lysine(373) levels were markedly elevated in sham (non-ischemic) LTED animals. Finally, intracerebroventricular injections of Gp91ds-Tat, a specific NADPH oxidase (NOX2) inhibitor, but not the scrambled tat peptide control (Sc-Tat), attenuated acetylation of p53 and reduced levels of Puma following GCI. CONCLUSIONS/SIGNIFICANCE: The studies demonstrate that p53 undergoes enhanced acetylation in the hippocampal CA1 region following global cerebral ischemia, and that the neuroprotective agent, 17ß-E(2), markedly attenuates the ischemia-induced p53 acetylation. Furthermore, following LTED, the suppressive effect of 17ß-E(2) on p53 acetylation is lost, and p53 acetylation increases in the hippocampus, which may explain previous reports of increased sensitivity of the hippocampus to ischemic stress following LTED.

Role of Rac1 GTPase in NADPH oxidase activation and cognitive impairment following cerebral ischemia in the rat.

BACKGROUND: Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat. METHODOLOGY/PRINCIPAL FINDINGS: Our studies revealed that NADPH oxidase activity and superoxide (O(2)(-)) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O(2)(-) production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated "in situ" O(2)(-) production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7-9 d post reperfusion as compared to vehicle-treated animals. CONCLUSIONS/SIGNIFICANCE: The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.

Estrogen attenuates ischemic oxidative damage via an estrogen receptor alpha-mediated inhibition of NADPH oxidase activation.

The goal of this study was to elucidate the mechanisms of 17beta-estradiol (E(2)) antioxidant and neuroprotective actions in stroke. The results reveal a novel extranuclear receptor-mediated antioxidant mechanism for E(2) during stroke, as well as a hypersensitivity of the CA3/CA4 region to ischemic injury after prolonged hypoestrogenicity. E(2) neuroprotection was shown to involve a profound attenuation of NADPH oxidase activation and superoxide production in hippocampal CA1 pyramidal neurons after stroke, an effect mediated by extranuclear estrogen receptor alpha (ERalpha)-mediated nongenomic signaling, involving Akt activation and subsequent phosphorylation/inactivation of Rac1, a factor critical for activation of NOX2 NADPH oxidase. Intriguingly, E(2) nongenomic signaling, antioxidant action, and neuroprotection in the CA1 region were lost after long-term E(2) deprivation, and this loss was tissue specific because the uterus remained responsive to E(2). Correspondingly, a remarkable loss of ERalpha, but not ERbeta, was observed in the CA1 after long-term E(2) deprivation, with no change observed in the uterus. As a whole, the study reveals a novel, membrane-mediated antioxidant mechanism in neurons by E(2) provides support and mechanistic insights for a "critical period" of E(2) replacement in the hippocampus and demonstrates a heretofore unknown hypersensitivity of the CA3/CA4 to ischemic injury after prolonged hypoestrogenicity.

Rapid estrogen signaling in the brain.

Estrogen has multiple actions in the brain to modulate homeostasis, synaptic plasticity/cognition and neuroprotection. While many of these actions undoubtedly involve mediation via the classical genomic mechanism of regulation of transcription of genes via estrogen nuclear receptors, there has been growing interest in the rapid nongenomic effects of estrogen and the role they may play in the neural actions of estrogen. In this review, we will focus on these rapid nongenomic actions of estrogen in the brain and discuss the potential physiological significance of these actions. The evidence for rapid estrogen regulation of cell signaling pathways, including calcium, ion channel and kinase signaling pathways in the brain will be reviewed, as will evidence derived from plasma-membrane impermeable estrogen-peptide conjugates in the regulation of these cell signaling pathways. Evidence supporting classical and nonclassical estrogen receptor localization to the plasma membrane of neurons will also be reviewed, including the putative new membrane estrogen G-protein-coupled receptor, GPR30. Precisely how membrane estrogen receptors couple to kinase signaling pathways is unclear, but we will discuss the latest findings on estrogen receptor-interacting scaffold proteins, such as MNAR/PELP1, striatin and p130Cas, which are capable of linking estrogen receptors and kinases such as Src and PI3K, to potentially mediate estrogen-induced kinase signaling. Finally, we will review the growing evidence that rapid membrane-mediated effects of estrogen play an important physiological role in the neural actions of estrogen in the brain, including estrogen feedback control and modulation of homeostasis, regulation of synaptic plasticity/cognition, and estrogen-mediated neuroprotection.