Amyloid plaques composed of β-amyloid (Aβ) protein are a pathological hallmark of Alzheimer's disease. We here report the generation and characterization of a novel transgenic mouse model of Aβ ...toxicity. The rTg9191 mice harbor a transgene encoding the 695 amino-acid isoform of human amyloid precursor protein (APP) with the Swedish and London mutations (APPNLI) linked to familial Alzheimer's disease, under the control of a tetracycline-response element, as well as a transgene encoding the tetracycline transactivator, under the control of the promoter for calcium-calmodulin kinase IIα. In these mice, APPNLI is expressed at a level four-fold that of endogenous mouse APP and its expression is restricted to forebrain regions. Transgene expression was suppressed by 87% after two months of doxycycline administration. Histologically, we showed that (1) Aβ plaques emerged in cerebral cortex and hippocampus as early as 8 and 10.5-12.5 months of age, respectively; (2) plaque deposition progressed in an age-dependent manner, occupying up to 19% of cortex at ~25 months of age; and (3) neuropathology--such as abnormal neuronal architecture, tau hyperphosphorylation and misfolding, and neuroinflammation--was observed in the vicinity of neuritic plaques. Biochemically, we determined total Aβ production at varied ages of mice, and we showed that mice produced primarily fibrillar Aβ assemblies recognized by conformation-selective OC antibodies, but few non-fibrillar oligomers (e.g., Aβ*56) detectable by A11 antibodies. Finally, we showed that expression of the tetracycline transactivator resulted in reduced brain weight and smaller dentate-gyrus size. Collectively, these data indicate that rTg9191 mice may serve as a model for studying the neurological effects of the fibrillar Aβ assemblies in situ.
Here, we describe the generation of a novel transgenic mouse model of human tauopathy. The rTg(tau(P301L))4510 mouse expresses the P301L mutation in tau (4R0N) associated with frontotemporal dementia ...and parkinsonism linked to chromosome 17. Transgene expression was driven by a forebrain-specific Ca(2+) calmodulin kinase II promoter system resulting in high levels of expression in the hippocampus and neocortex. Importantly, transgene expression in this model is induced via the tetracycline-operon responsive element and is suppressed after treatment with doxycycline. Continued transgene expression in rTg(tau(P301L))4510 mice results in age-dependent development of many salient characteristics of hereditary human dementia. From an early age, immunohistochemical studies demonstrated abnormal biochemical processing of tau and the presence of pathological conformation- and phosphorylation-dependent epitopes. Neurofibrillary tangle (NFT) pathology was first observed in the neocortex and progressed into the hippocampus and limbic structures with increasing age. Consistent with the formation of NFTs, immunoblots indicated an age-dependent transition of accumulating tau species from Sarkosyl soluble 55 kDa to insoluble hyperphosphorylated 64 kDa. Ultrastructural analysis revealed the presence of straight tau filaments. Furthermore, the effects of tau(P301L) expression on spatial reference memory were longitudinally tested using the Morris water maze. Compared with nontransgenic age-matched control littermates, rTg(tau(P301L))4510 mice developed significant cognitive impairments from 4 months of age. Memory deficits were accompanied by gross forebrain atrophy and a prominent loss of neurons, most strikingly in hippocampal subdivision CA1. Collectively, these data describe a novel transgenic mouse that closely mimics human tauopathy and may represent an important model for the future study of tau-related neurodegenerative disease.
The ubiquitous nature of protein phosphorylation makes it challenging to map kinase-substrate relationships, which is a necessary step toward defining signaling network architecture. To trace the ...activity of individual kinases, we developed a semisynthetic reaction scheme, which results in the affinity tagging of substrates of the kinase in question. First, a kinase, engineered to use a bio-orthogonal ATPgammaS analog, catalyzes thiophosphorylation of its direct substrates. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues creates an epitope for thiophosphate ester-specific antibodies. We demonstrated the generality of semisynthetic epitope construction with 13 diverse kinases: JNK1, p38alpha MAPK, Erk1, Erk2, Akt1, PKCdelta, PKCepsilon, Cdk1/cyclinB, CK1, Cdc5, GSK3beta, Src and Abl. Application of this approach, in cells isolated from a mouse that expressed endogenous levels of an analog-specific (AS) kinase (Erk2), allowed purification of a direct Erk2 substrate.
We have previously shown that nicotine, the addictive component of tobacco products, alters the blood-brain barrier (BBB) Na(+),K(+),2Cl(-) cotransporter (NKCC) during in vitro hypoxia-aglycemia ...exposure. Attenuation of abluminal NKCC suggests that accumulation of ions in the brain extracellular fluid would result in an increase of fluid or cytotoxic edema in the brain during hypoxia-aglycemia or stroke conditions. To further investigate whether nicotine products have the potential to worsen stroke outcome by increasing edema formation, two separate models to mimic stroke conditions were utilized to decipher the effects of short-term and long-term administrations of nicotine products on brain edema following stroke. Oxygen glucose deprivation (OGD) was studied in rat hippocampal slices with short-term or long-term exposure to nicotine and cigarette smoke constituents. During short-term exposure, the presence of nicotine at a concentration mimicking heavy smokers increased water content of hippocampal slices during OGD. Furthermore, long-term 1-week administration of nicotine increased water content in hippocampal slices that could be attenuated with nicotine acetylcholine receptor (nAChR) antagonists, suggesting nicotine increase edema during OGD via nAChRs. A second model of focal ischemia, middle cerebral artery occlusion, showed an increase of infarct size during short-term exposure to nicotine and an increase of edema during both short-term and long-term administration of nicotine, compared with saline controls. These findings support the paradigm that nicotine products not only increase the incidence of stroke but also have the potential to worsen stroke outcome by increased edema formation.
Transgenic mouse models that independently express mutations in amyloid precursor protein (APP) and tau have proven useful for the study of the neurological consequences of amyloid-β (Aβ) plaque and ...neurofibrillary tangle pathologies. Studies using these mice have yielded essential discoveries with regard to specific aspects of neuronal dysfunction and degeneration that characterize the brain during Alzheimer's disease (AD) and other age-dependent tauopathies. Most recent transgenic studies have focused on the creation of regulatable models that allow the temporal control of transgene expression. To study a more complete model of AD pathology, we designed a new regulatable transgenic mouse that harbors both APP and tau transgenes. Here, we present a novel transgenic mouse model, rTg3696AB, which expresses human APPNLI and tauP301L driven by the CaMKII promoter system. Subsequent generation of Aβ and 4R0N tau in the brain resulted in the development of three neuropathological features of AD: Aβ plaques, neurofibrillary tangles, and neurodegeneration. Importantly, transgene expression in these mice is regulatable, permitting temporal control of gene expression and the investigation of transgene suppression.
Accurate nuclear position is essential for each daughter cell to receive one DNA complement. In budding yeast, a surveillance mechanism known as the spindle position checkpoint ensures that exit from ...mitosis only occurs when the anaphase nucleus is positioned along the mother-bud axis. We identified the protein kinase Kin4 as a component of the spindle position checkpoint.
KIN4 prevents exit from mitosis in cells with mispositioned nuclei by inhibiting the mitotic exit network (MEN), a GTPase signaling cascade that promotes exit from mitosis. Kin4 is active in cells with mispositioned nuclei and predominantly localizes to mother cells, where it is ideally situated to inhibit MEN signaling at spindle pole bodies (SPBs) when anaphase spindle elongation occurs within the mother cell.
Nicotine, a major constituent of tobacco smoke, has important effects on brain recovery after focal ischemia (Wang et al., 1997). The purpose of this work is to systematically test the effects of ...nicotine during stroke conditions on blood-brain barrier (BBB) potassium transport, protein expression of the Na,K,2Cl-cotransporter (NKCC), and cell signaling pathways that control NKCC activity at the BBB. Confluent bovine brain microvessel endothelial cells (BBMECs) were exposed to both a hypoxic/aglycemic (H/A) environment to model BBB function during stroke conditions and nicotine and cotinine (N/C) to model plasma levels seen in smokers. BBMECs exhibit both Na,K-ATPase and NKCC activity (60 and 34 nmol/min/g, respectively) that contribute to 98% of the K(+) uptake in cultured endothelial cells. An adaptive up-regulation of NKCC activity was identified to occur on the basolateral surface of the BBB after in vitro stroke conditions. Twenty-four hours of N/C exposure, at doses equivalent to plasma levels of smokers, combined with 6 h of H/A, reduced NKCC protein expression and total NKCC activity (shown by bumetanide-sensitive (86)RB uptake) compared with 6 h of H/A alone (P < 0.01). Basolateral K(+) transport was found to be modulated by nicotinic acetylcholine receptors expressed at the BBB. NKCC activity on the basolateral surface of the BBB is controlled by an ongoing phosphorylation/dephosphorylation processes. We have identified a potential mechanism in altered BBB response to stroke conditions with prior N/C exposure directly implicating damage to brain-to-blood K(+) transport mediated at the BBB and perhaps neuronal recovery after stroke.
Smoking tobacco, including cigarettes, has been associated with an increased incidence and relative risk for cerebral infarction in both men and women. Recently, we have shown that nicotine and ...cotinine attenuate abluminal (brain facing) K(+) uptake mediated by the Na,K,2Cl-cotransporter (NKCC) in bovine brain microvessel endothelial cells (BBMECs) after hypoxic/aglycemic exposure (stroke conditions). The purpose of the current study was to explore the effects of nicotine and tobacco smoke chemicals on K(+) movement through the blood-brain barrier during both hypoxia/aglycemia and reoxygenation. BBMECs were exposed to nicotine/cotinine, nicotine-containing cigarette smoke extract (N-CSE), or nicotine-free cigarette smoke extract (NF-CSE) in quantities designed to mimic plasma concentrations of smokers. Stroke conditions were mimicked in vitro in BBMECs through 6 h of hypoxia/aglycemia with or without 12 h of reoxygenation, after which NKCC-mediated K(+) uptake and paracellular integrity were measured with (86)Rb and (14)Csucrose, respectively. In addition, K(+) concentrations in brain extracellular fluid were estimated in (86)Rb-injected rats that were administered nicotine, N-CSE, or NF-CSE and on whom global ischemia/reperfusion by in vivo four-vessel occlusion was performed. Both in vitro and in vivo paradigms showed nicotine, the major alkaloid present in tobacco smoke, to be the determining factor of an inhibited response of abluminal NKCC in BBMECs during and after stroke conditions. This was measured as a decrease in abluminal brain endothelial cell NKCC activity and as an increase in brain extracellular K(+) concentration measured as the brain extracellular fluid (86)Rb/plasma ratio after in vivo four-vessel occlusion with reperfusion.
Based on the premise that similar surface properties between the adhesive and the substrate would yield a strong adhesive bond, copolymers of acrylic acid (AA) and 2-ethylhexyl acrylate (EHA), ...P(AA-co-EHA), were designed and synthesized for buccal mucoadhesion. A series of linear copolymers with varying feed ratios of the two monomers (AA and EHA) were synthesized through free radical copolymerization at 69±0.5°C using azobis(isobutyronitrile) (AIBN) as initiator. The reactions were carried out in THF under nitrogen for 24 h. The glass transition temperatures,
T
g, of the copolymers were determined using DSC. The adhesion studies were conducted to determine the effects of copolymer composition, contact time between the substrate and the adhesive, and crosshead speed on mucoadhesive performance of the copolymer films using a computer interfaced Instron material testing system. The glass transition temperature of the copolymers decreased with increasing EHA content. Wet glass surface as substrate was shown not to be a good substrate model for adhesion determination studies. The copolymer composed of 46:54 mol.% AA:EHA (an almost 1:1 ratio in the repeat units) yielded the highest mucoadhesive force in contact with porcine buccal mucosa which was significantly greater (
P<0.05) than that of poly(acrylic acid) (PAA) (used as positive control). The mucoadhesive force for all copolymers studied was significantly (
P<0.05) greater than that of the negative control (backing material without copolymer film) except for the EHA homopolymer. Crosshead speed increased mucoadhesive force linearly and had a more pronounced effect on the mucoadhesive performance than time of contact between the adhesive and the substrate.
Highlights • Our sample includes veterans with and without posttraumatic stress disorder (PTSD). • We examine if inflammatory markers are associated with hippocampal volume. • Higher sTNF-RII, but ...not IL-6, was associated with reduced hippocampal volume. • Neither current nor past PTSD diagnoses were associated with sTNF-RII or IL-6. • More severe PTSD symptoms were associated with elevated sTNF-RII and lower IL-6.
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