Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71

Alzheimer's disease (AD) is a neurodegenerative disorder in which oxidative stress has been implicated as an important event in the progression of the pathology. In particular, it has been shown that protein modification by reactive oxygen species (ROS) occurs to a greater extent in AD than in control brain, suggesting a possible role for oxidation-related decrease in protein function in the process of neurodegeneration. Oxidative damage to proteins, assessed by measuring the protein carbonyl content, is involved in several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, neuronal death. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. Previously, we used our proteomics approach, which successfully substitutes for labor-intensive immunochemical analysis, to detect proteins and identified creatine kinase, glutamine synthase and ubiquitin carboxy-terminal hydrolase L-1 as specifically oxidized proteins in AD brain. In this report we again applied our proteomics approach to identify new targets of protein oxidation in AD inferior parietal lobe (IPL). The dihydropyrimidinase related protein 2 (DRP-2), which is involved in the axonal growth and guidance, showed significantly increased level in protein carbonyls in AD brain, suggesting a role for impaired mechanism of neural network formation in AD. Additionally, the cytosolic enzyme alpha-enolase was identified as a target of protein oxidation and is involved the glycolytic pathway in the pathological events of AD. Finally, the heat shock cognate 71 (HSC-71) revealed increased, but not significant, oxidation in AD brain. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain.     

Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB,glutamine synthase,and ubiquitin carboxy-terminal hydrolase L-1

Oxidative alterations of proteins by reactive oxygen species (ROS) have been implicated in the progression of aging and age-related neurodegenerative disorders such as Alzheimer's disease (AD). Protein carbonyls, a marker of protein oxidation, are increased in AD brain, indicating that oxidative modification of proteins is relevant in AD. Oxidative damage can lead to several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, to neuronal death. Identification of specific targets of protein oxidation represents a crucial step in establishing a relationship between oxidative modification and neuronal death in AD, and was partially achieved previously in our laboratory through immunochemical detection of creatine kinase BB and beta-actin as specifically oxidized proteins in AD brain versus control brain. However, this process is laborious, requires the availability of specific antibodies, and, most importantly, requires a reasonable guess as to the identity of the protein in the first place. In this study, we present the first proteomics approach to identify specifically oxidized proteins in AD, by coupling 2D fingerprinting with immunological detection of carbonyls and identification of proteins by mass spectrometry. The powerful techniques, emerging from application of proteomics to neurodegenerative disease, reveal the presence of specific targets of protein oxidation in Alzheimer's disease (AD) brain: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain. Proteomics offers a rapid means of identifying oxidatively modified proteins in aging and age-related neurodegenerative disorders without the limitations of the immunochemical detection method.     

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.     

Experimental design considerations: a determinant of acute neonatal toxicity

Few studies have investigated the potential developmental differences resulting from treating neonatal rat pups in either split-litter or whole-litter (nested) experimental designs. We directly compared rat pups dosed with triethyl lead (TEL) via both split-litter (representing all dosage groups within a single litter) and nested (all pups randomly assigned to a single litter receive the same dose) designs. The nested design was chosen to produce a uniform behavioral pattern across pups within each litter, whereas the split-litter design was chosen to promote pup competition and differential maternal care. On postpartum day 5, pups were administered either 12, 13, 14, or 15 mg/kg TEL, with each design represented by 12 litters. Although the LD50 values for the two designs were not significantly different, there were significantly more deaths in the 12 mg/kg dosage group within the split-litter design than in the nested design group. Preweaning survival times for split-litter dosed animals were also decreased. In addition, significant growth reduction (7-16%) was observed in the split-litter group, relative to the nested design animals during the preweaning period. These results suggest that neonatal toxicity is not independent from experimental design considerations, and that the factors of littermate competition and/or pup-induced maternal care deserve further study.     

Auger-electron cascades,charge potential and microdosimetry of iodine-125

Summary This paper is a contribution to the microdosimetry of I-125. It shows microdosimetric spectra of individual and average disintegrations of I-125 for various target sizes and gives evidence for the relative contributions of energy-deposition events of low and high LET. It further presents information on the relative efficiencies of Auger-electrons and multiple charges in terms of local energy deposition, e.g. to model targets of DNA, and discusses their radiobiological implications, e.g. the microdosimetric understanding of the different efficiencies of specific and random incorporations of I-125.When I-125 is specifically incorporated into DNA, most of the energy deposition events are very large, e.g. above 40 keV/µm for a simulated target volume of 20 nm diameter, regardless of the number and energy of Auger electrons emitted. Therefore it is not necessary, for the discussion of the radiobiological implications, to distinguish between different classes of disintegrations.For unspecific, homogeneous incorporation of I-125 somewhere into tissue, about 20% of the dose to critical targets of 25 nm diameter is made up by disintegrations that happen to occur within these targets. When assuming that other critical targets and target structures can be neglected, this part of the dose will be equally effective as in the case of specific incorporation of I-125 into such target models. In addition, there are the normal, low-LET radiation effects from the other, 80% large fraction of the dose. With this information, for the biological systems and end points for which a short section of the elemental chromatine fiber can be taken as the relevant critical target, it is shown that the expected D₃₇ value for homogeneous unspecific incorporation of I-125 can be estimated when the D₃₇ for specific incorporation in DNA is known.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthdayOn leave of absence from Institute of Nuclear Physics, PL-31342 Cracow, PolandStaff member Commission of the European Communities     

Synaptodendritic recovery following HIV Tat exposure: Neurorestoration by phytoestrogens

HIV‐1 infects the brain and, despite antiretroviral therapy, many infected individuals suffer from HIV‐1‐associated neurocognitive disorders (HAND). HAND is associated with dendritic simplification and synaptic loss. Prevention of synaptodendritic damage may ameliorate or forestall neurocognitive decline in latent HIV‐1 infections. The HIV‐1 transactivating protein (Tat) is produced during viral latency in the brain and may cause synaptodendritic damage. This study examined the integrity of the dendritic network after exposure to HIV‐1 Tat by labeling filamentous actin (F‐actin)‐rich structures (puncta) in primary neuronal cultures. After 24 h of treatment, HIV‐1 Tat was associated with the dendritic arbor and produced a significant reduction of F‐actin‐labeled dendritic puncta as well as loss of dendrites. Pre‐treatment with either of two plant‐derived phytoestrogen compounds (daidzein and liquiritigenin), significantly reduced synaptodendritic damage following HIV‐1 Tat treatment. In addition, 6 days after HIV‐1 Tat treatment, treatment with either daidzein, or liquiritigenin enhanced recovery, via the estrogen receptor, from HIV‐1 Tat‐induced synaptodendritic damage. These results suggest that either liquiritigenin or daidzein may not only attenuate acute synaptodendritic injury in HIV‐1 but may also promote recovery from synaptodendritic damage.

     

Identification of D3 and σ Receptors in the Rat Striatum and Nucleus Accumbens Using (±)-7-Hydroxy-N,N-Di-n-[3H]Propyl-2-Aminotetralin and Carbetapentane

Abstract: Cross-reactions between dopamine D₃ and σ receptor ligands were investigated using (±)-7-hydroxy-N,N-di-n-[³H]propyl-2-aminotetralin [(±)-7-OH-[³H]DPAT], a putative D₃-selective radioligand, in conjunction with the unlabeled σ ligands 1,3-di(2-tolyl)guanidine (DTG), carbetapentane, and R(?)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane [R(?)-PPAP]. In transfected CCL1.3 mouse fibroblasts expressing the human D₃ receptor, neither DTG nor carbetapentane (0.1 µM) displaced (±)-7-OH-[³H]DPAT binding. R(?)-PPAP (0.1 µM) displaced 39.6 ± 1.0% of total (±)-7-OH-[³H]DPAT binding. In striatal and nucleus accumbens homogenates, (±)-7-OH-[³H]DPAT labeled a single site (15–20 fmol/mg of protein) with high (1 nM) affinity. Competition analysis with carbetapentane defined both high- and low-affinity sites in striatal (35 and 65%, respectively) and nucleus accumbens (59 and 41%, respectively) tissue, yet R(?)-PPAP identified two sites in equal proportion. Carbetapentane and R(?)-PPAP (0.1 µM) displaced ～20–50% of total (±)-7-OH-[³H]DPAT binding in striatum, nucleus accumbens, and olfactory tubercle in autoradiographic studies, with the nucleus accumbens shell subregion exhibiting the greatest displacement. To determine directly (+)-7-OH-[³H]DPAT binding to σ receptors, saturation analysis was performed in the cerebellum while masking D₃ receptors with 1 µM dopamine. Under these conditions (+)-7-OH-[³H]DPAT labeled σ receptors with an affinity of 24 nM. These results suggest that (a) (±)-7-OH-[³H]DPAT binds D₃ receptors with high affinity in rat brain and (b) a significant proportion of (±)-7-OH-[³H]DPAT binding consists of σ₁ sites and the percentages of these sites differ among the subregions of the striatum and nucleus accumbens.     

Environmental Enrichment Alters Nicotine-Mediated Locomotor Sensitization and Phosphorylation of DARPP-32 and CREB in Rat Prefrontal Cortex

Exposure within an environmental enrichment paradigm results in neurobiological adaptations and decreases the baseline of locomotor activity. The current study determined activation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32) and CREB (cAMP response element binding protein), and locomotor activity in rats raised in enriched (EC), impoverished (IC), and standard (SC) conditions following repeated administration of nicotine or saline. In the saline-control group, the basal phosphorylation state of DARPP-32 at Threonine-34 site (pDARPP-32 Thr34) in the prefrontal cortex (PFC) was lower in EC compared to IC and SC rats, which was positively correlated with their respective baseline activities. While nicotine (0.35 mg/kg, freebase) produced locomotor sensitization across all housing conditions when the nicotine-mediated locomotor activity was expressed as a percent change from their respective saline control, EC rats displayed greater sensitization to nicotine than IC and SC rats. Consistent with the behavioral findings, repeated nicotine injection increased pDARPP-32 Thr34 in PFC of EC and IC rats and in nucleus accumbens of EC rats; however, the magnitude of change from saline control in nicotine-induced enhancement of pDARPP-32 Thr34 in PFC was strikingly increased in EC rats relative to IC rats. Moreover, EC rats had lower basal phosphorylation levels of CREB at serine 133 in PFC and nucleus accumbens compared to IC and SC rats, whereas the nicotine-induced increase in phosphorylated CREB-Ser133 was more pronounced in PFC of EC rats relative to IC and SC rats. Collectively, these findings suggest innovative insights into advancing our understanding of the molecular mechanisms of enrichment-induced changes in the motivational effects of nicotine, and aiding in the identification of new therapeutic strategies for tobacco smokers.     

Upregulation of (+)-7-Hydroxy-N,N-di-n-[3H]Propyl-2-Aminotetralin Binding Following Intracerebroventricular Administration of a Nitric Oxide Generator

Nitric oxide modulation of dopamine D₂ and D₃ receptor binding was examined using [¹²⁵I]epidepride (D₂) and (+)7-hydroxy-N,N-di-n-[³H]propyl-2-aminotetralin([³H](+)-7-OH-DPAT, D₃). Nitric oxide, generated by i.c.v. injection of S-nitroso-N-acetyl-penicillamine (SNAP; 5 g or 10 g), significantly increased the density of [³H](+)-7-OH-DPAT binding sites (39% and 134%, respectively) in the striatum 24 hours post-injection in the absence of Gpp(NH)p, representing an upregulation of either D₃, receptors or high affinity D₂ receptors. In the presence of 10 M Gpp(NH)p, D₃ receptor upregulation was maintained in both the 5 g (increased 35%) and 10 g SNAP (increased 44%) groups. [³H](+)-7-OH-DPAT binding was reduced in both striatum and nucleus accumbens in the presence of 10 M Gpp(NH)p compared to binding in the absence of Gpp(NH)p, suggesting an upregulation of D₃ receptors. Administration of SNAP did not alter total specific [¹²⁵I]epidepride binding in either brain region. These data suggest that; 1) D₃ receptor density is modified following nitric oxide generation, and 2) the density of high affinity D₂ receptors identified by [³H](+)-7-OH-DPAT increases in the striatum, but decreases in the nucleus accumbens.