Airway Inflammation and Lung Function in Sickle Cell Disease.

Rationale: Asthma is a common comorbid condition in sickle cell disease (SCD). However, obstructive lung disease is prevalent in SCD, independent of a diagnosis of asthma. It is speculated that the heightened state of inflammation in SCD, involving pathways distinct from allergic asthma, may underlie the SCD-specific obstructive disease. Objective: The objective of the study was to compare airway and systemic inflammatory markers between SCD patients with pulmonary manifestations and patients with allergic asthma, and correlate the discriminating inflammatory markers with clinical measures of pulmonary disease. Materials and Methods: In a pilot translational study conducted at the Children's Hospital at Montefiore, 15 patients with SCD, and history of asthma, airway obstruction, or airway hyper-reactivity, and 15 control patients with allergic asthma 6-21 years of age were recruited. Inflammatory markers, including peripheral blood T helper cell subsets, serum and exhaled breath condensate (EBC) cytokines and chemokines of the Th-1/Th-17, Th-2, and monocytic pathways, and serum cysteinyl leukotrienes B4 (LTB4), were quantified, compared between the study groups, and correlated with atopic sensitization, pulmonary function tests, and markers of hemolysis. Results: White blood cells (P < 0.05) and monocytes (P < 0.001) were elevated in the SCD group, while atopic characteristics were higher in the control asthma group. Tumor necrosis factor-alpha (P < 0.01), interferon gamma inducible protein (IP)-10 (P < 0.05), and interleukin-4 (P < 0.01) in serum and monocyte chemotactic protein (MCP)-1 in EBC were higher in the SCD group (P ≤ 0.05). Forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) in patients with SCD inversely correlated with serum IP-10 and LTB4 levels. Conclusions: Compared with atopic asthmatic patients, inflammatory markers involving Th-1, Th-2, and monocytic pathways were higher in the SCD group, among which Th-1 measures correlated with pulmonary function deficits.

Potential role of α-synuclein in neurodegeneration: studies in a rat animal model.

Neuronal protein α-synuclein (α-syn) is an essential player in the development of neurodegenerative diseases called synucleinopathies. A spontaneous autosomal recessive rat model for neurodegeneration was developed in our laboratory. These rats demonstrate progressive increases in α-syn in the brain mesencephalon followed by loss of dopaminergic terminals in the basal ganglia (BG) and motor impairments. The severity of pathology is directly related to the overexpression of α-syn and parallel decrease in dopamine (DA) level in the striatum (ST) of affected rats. The neurodegeneration in this model is characterized by the presence of perikarya and neurites Lewis bodies (LB) and diffuse marked accumulation of perikaryal α-syn in the substantia nigra (SN), brain stem (BS), and striatum (ST) along with neuronal loss. Light and ultrastructural analyses revealed that the process of neuronal degeneration is a 'dying back' type. The disease process is accompanied by gliosis and release of inflammatory cytokines. This neurodegeneration is a multisystemic disease and implicate α-syn as a major factor in the pathogenesis of this inherited autosomal recessive animal model. Decrease dopamine (DA) and overexpression of α-syn in the brain mesencephalon may provide a naturally occurring animal model for Parkinson's disease (PD) and other synucleinopathies that reproduces significant pathological, neurochemical, and behavioral features of the human disease.

Absence of amyloid ß oligomers at the postsynapse and regulated synaptic Zn2+ in cognitively intact aged individuals with Alzheimer's disease neuropathology.

BACKGROUND: Early cognitive impairment in Alzheimer Disease (AD) is thought to result from the dysfunctional effect of amyloid beta (Aß) oligomers targeting the synapses. Some individuals, however, escape cognitive decline despite the presence of the neuropathologic features of AD (Aß plaques and neurofibrillary tangles). We term this group Non-Demented with AD Neuropathology or NDAN. The present study illustrates one putative resistance mechanism involved in NDAN cases which may suggest targets for the effective treatment of AD. RESULTS: Here we describe the localization of Aß oligomers at the postsynapse in hippocampi from AD cases. Notably, however, we also found that while present in soluble fractions, Aß oligomers are absent from hippocampal postsynapses in NDAN cases. In addition, levels of phosphorylated (active) CREB, a transcription factor important for synaptic plasticity, are normal in NDAN individuals, suggesting that their synapses are functionally intact. Analysis of Zn2+ showed that levels were increased in both soluble fractions and synaptic vesicles in AD hippocampi, paralleled by a decrease of expression of the synaptic vesicle Zn2+ transporter, ZnT3. Conversely, in NDAN individuals, levels of Zn2+ in soluble fractions were significantly lower than in AD, whereas in synaptic vesicles the levels of Zn2+ were similar to AD, but accompanied by preserved expression of the ZnT3. CONCLUSIONS: Taken together, these data illustrate that despite substantial AD neuropathology, Aß oligomers, and increased synaptic vesicle Zn2+, susceptible brain tissue in these aged NDAN individuals features, as compared to symptomatic AD subjects, significantly lower total Zn2+ levels and no association of Aß oligomers with the postsynapse, which collectively may promote the maintenance of intact cognitive function.

Brain serum amyloid P levels are reduced in individuals that lack dementia while having Alzheimer's disease neuropathology.

The neuropathological signs of Alzheimer's disease (AD) include beta amyloid plaques and neurofibrillary tangles. There is a significant population of individuals that have these key hallmarks but show no signs of cognitive impairment, termed non-demented with AD neuropathology (NDAN). The protective mechanism allowing these individuals to escape dementia is unknown. Serum amyloid P (SAP) is a serum protein associated with wound repair that is elevated in the brains of Alzheimer's patients and binds to amyloid plaques. Using immunoblotting and immunohistochemistry, we evaluated SAP levels in postmortem samples of hippocampus and frontal cortex in age-matched controls, AD, and NDAN individuals. AD individuals had significantly increased SAP levels compared to normal controls, while NDAN samples had no significant difference in SAP levels compared to normal controls. Our results suggest that low levels of SAP in plaques marks the brains of individuals that escape dementia despite the presence of beta amyloid plaques and tangles.

Selective, quantitative measurement of releasable synaptic zinc in human autopsy hippocampal brain tissue from Alzheimer's disease patients.

Aberrant central nervous system zinc homeostasis has been reported in Alzheimer's disease (AD). However, there are conflicting reports describing zinc concentration either increased or decreased in the brain of AD patients. Such discrepancies may be due to differences in the brain area examined, zinc detection method, and/or tissue composition. Furthermore, detection and measurement of the releasable zinc pool in autopsy tissue is difficult and usually unreliable. Obtaining an adequate assessment of this releasable zinc pool is of particular significance in AD research in that zinc can coordinate with and stabilize toxic amyloid beta oligomers, which are believed to play a key role in AD neuropathology. In addition, zinc released into the synaptic cleft can interact with the postsynaptic neurons causing altered signaling and synaptic dysfunction, which is a well established event in AD. The method presented here combines two approaches, biochemical fractionation and atomic absorption spectrophotometry, to allow, in addition to extracellular zinc concentration, the reliable and quantitative measurement of zinc specifically localized in synaptic vesicles, which contain the majority of the neuronal releasable zinc. Using this methodology, we found that synaptic vesicle zinc concentrations were increased in AD hippocampi compared to age-matched controls and that this increase in releasable zinc matched increased concentration of zinc in the extracellular space.

Increased insertion of glutamate receptor 2-lacking alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors at hippocampal synapses upon repeated morphine administration.

Evidence suggests that the long-term adaptations in the hippocampus after repeated drug treatment may parallel its role during memory formation. The neuroplasticity that subserves learning and memory is also believed to underlie addictive processes. We have reported previously that repeated morphine administration alters local distribution of endocytic proteins at hippocampal synapses, which could in turn affect expression of glutamate receptors. Glutamatergic systems, including alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), are believed to be involved in opiate-induced neuronal and behavioral plasticity, although the mechanisms underlying these effects are only beginning to be understood. The present study further examines the effects of repeated morphine administration on the expression and composition of AMPARs and the functional ramifications. Twelve hours after the last morphine injection, we observed an increased expression of AMPARs lacking glutamate receptor (GluR) 2 in hippocampal synaptic fractions. Immunoblotting studies show that 12 h after morphine treatment, GluR1 subunits are increased at the postsynaptic density (PSD) and at extrasynaptic sites, whereas GluR3 subunits are only increased at the PSD, and they show how this alters receptor subunit composition. In addition, we provide electrophysiological evidence that AMPARs are switched to Ca(2+)-permeable (GluR2-lacking) at the synapse 12 h after repeated morphine treatment, affecting the magnitude of long-term depression at hippocampal neurons. We propose that morphine-induced changes in glutamatergic synaptic transmission in the hippocampus may play an important role in the neuroadaptations induced by repeated morphine administration.

Neuronal dopamine transporter activity, density and methamphetamine inhibition are differentially altered in the nucleus accumbens and striatum with no changes in glycosylation in rats behaviorally sensitized to methamphetamine.

Animals sensitized to methamphetamine (METH) have altered dopaminergic systems, including dopamine transporter (DAT) activity. We investigated the effects induced by a sensitizing dose (5 mg/kg, i.p. per day for 5 days) of METH on rat behavior, DA transport by the DAT, DAT density, and inhibition of DA transport by METH in both the nucleus accumbens and striatum. We further investigated possible changes to glycosylation of the DAT after METH sensitization. The dosing paradigm caused an increased stereotyped response in rats treated with METH compared with saline controls. In animals treated with METH, DA transport velocities were increased by 6.4% in the nucleus accumbens and decreased by 21% in the striatum. Western blots demonstrated that DAT density was unchanged in the nucleus accumbens of METH-treated animals, but striatal DAT density was decreased by 20%. Further studies investigating METH inhibition of DA transport found that in the nucleus accumbens of METH-treated animals, the IC(50) was shifted to a larger value (from 0.81 to 1.45 microM). In the striatum, the IC(50) was decreased by 19% (from 1.00 to 0.81 microM) in METH-treated animals. Studies using glycosidase treatments and Western blots revealed that glycosylation was effectively removed by N-glycanase and neuraminidase, but not O-glycosidase or alpha-mannosidase. These studies also suggest that glycosylation was not altered in METH-treated animals. This study demonstrates that in animals sensitized to METH, the DAT is differentially regulated in different areas of the brain important for drug abuse, and that DA transport changes induced by METH are not due to DAT density, but to changes in the kinetics of the DAT. Additionally, this study suggests that glycosylation may not play a role in DAT activity changes after METH exposure.

Differential effects of Zn2+ on the kinetics and cocaine inhibition of dopamine transport by the human and rat dopamine transporters.

Zn2+ may play a major role in the modulation of neurotransmission because it modulates membrane receptors and channels. Recent literature has shown Zn2+ inhibits dopamine transport by the dopamine transporter (DAT), the main target of cocaine and some other drugs of abuse. Cocaine inhibits DAT and modulation of the DAT by Zn2+ may alter effects of cocaine on dopamine neurotransmission. This study investigates how Zn2+ changes DAT kinetics and its inhibition by cocaine. Steady-state and pre-steady-state kinetics of DAT activity were investigated using rotating disk electrode voltammetry. Values of KM and Vmax in hDAT and effects of cocaine match those in the literature. Zn2+ allosterically inhibited transport in the human DAT (hDAT) with a KI=7.9+/-0.42 microM. Removal of endogenous Zn2+ with penicillamine in hDAT increased transport values. In contrast, Zn2+ did not alter transport by rat DAT (rDAT), with KM and Vmax values of 1.2+/-0.49 microM and 15.7+/-2.57 pmol/(sx10(6) cells), respectively, and removal of Zn2+ did not increase dopamine transport values. Zn2+ allosterically reduced the inhibition by cocaine in hDAT. Results of pre-steady-state studies demonstrated that Zn2+ increases the second order binding rate constant for dopamine to hDAT (3.5 fold to 19.2x10(6) M-1 s-1 for hDAT). In rat striatal homogenates Zn2+ increased initial dopamine transport velocity and decreased cocaine inhibition providing evidence for differences in sensitivity to Zn2+ between the three different preparations. Modulation of the DAT by Zn2+ needs to be assessed further in development of cocaine antagonists.

Methylphenidate analogs with behavioral differences interact differently with arginine residues on the dopamine transporter in rat striatum.

The methylphenidate analogs N-methyl-4-methyl-methylphenidate and N-benzylmethylphenidate are believed to interact differently with the dopamine transporter (DAT) in vitro and in vivo. Herein, we report that methylphenidate and N-methyl-4-methyl-methylphenidate, but not N-benzylmethylphenidate, protect the rat striatal DAT from the arginine-selective chemical modifying agent, phenylglyoxal. This suggests that methylphenidate and N-methyl-4-methyl-methylphenidate, but not N-benzylmethylphenidate, interact with the guanidine groups of arginine residues in the DAT of rat striatum. This differential interaction may, at least in part, explain the in vitro and in vivo differences between N-methyl-4-methyl-methylphenidate and N-benzylmethylphenidate.