Distinctive patterns of DNA methylation associated with Parkinson disease

Parkinson disease (PD) is a multifactorial neurodegenerative disorder with high incidence in the elderly, where environmental and genetic factors are involved in etiology. In addition, epigenetic mechanisms, including deregulation of DNA methylation have been recently associated to PD. As accurate diagnosis cannot be achieved pre-mortem, identification of early pathological changes is crucial to enable therapeutic interventions before major neuropathological damage occurs. Here we investigated genome-wide DNA methylation in brain and blood samples from PD patients and observed a distinctive pattern of methylation involving many genes previously associated to PD, therefore supporting the role of epigenetic alterations as a molecular mechanism in neurodegeneration. Importantly, we identified concordant methylation alterations in brain and blood, suggesting that blood might hold promise as a surrogate for brain tissue to detect DNA methylation in PD and as a source for biomarker discovery.     

Effects of the cholesterol-lowering compound methyl-beta-cyclodextrin in models of alpha-synucleinopathy

The aggregation of alpha-synuclein (alpha-syn) is believed to play a critical role in the pathogenesis of disorders such as dementia with Lewy bodies and Parkinson's disease. The function of alpha-syn remains unclear, although several lines of evidence suggest that alpha-syn is involved in synaptic vesicle trafficking, probably via lipid binding, and interactions with lipids have been shown to regulate alpha-syn aggregation. In this context, the main objective of this study was to determine whether methyl-beta-cyclodextrin (MbetaCD), a cholesterol-extracting agent, interfered with alpha-syn accumulation in models of synucleinopathy. For this purpose, we studied the effects of MbetaCD on the accumulation of alpha-syn in a transfected neuronal cell line and in transgenic mice. Immunoblot analysis showed that MbetaCD reduced the level of alpha-syn in the membrane fraction and detergent-insoluble fraction of transfected cells. In agreement with the in vitro studies, treatment of mice with MbetaCD resulted in decreased levels of alpha-syn in membrane fractions and reduced accumulation of alpha-syn in the neuronal cell body and synapses. Taken together, these results suggest that changes in cholesterol and lipid composition using cholesterol-lowering agents may be used as a tool for the treatment of synucleinopathies.     

Albendazole failure to control resistant nematodes in lambs: lack of effect of fasting-induced improvement on drug absorption

Enhanced plasma availability of albendazole sulphoxide (ABZSO), the active metabolite of albendazole (ABZ), has been described in feed-restricted sheep. The aim of the present work was to determine if the absorption-related pharmacokinetic changes derived from fasting animals prior to drug treatment would modify the clinical efficacy of ABZ against resistant gastrointestinal nematodes in lambs. Forty Corriedale lambs, naturally infected with resistant gastrointestinal nematodes, were divided into 4 groups. Controls were fed ad libitum and did not receive any drug treatment. Treated animals were fed ad libitum up to 30 min prior to treatment with ABZ (3.8 mg/kg) by the intraruminal route. The control (fasted) animals were not fed during the 24-hr period prior to the start of the experiment and did not receive any drug treatment. A second treated group of animals were fasted 24 hr prior to the treatment with ABZ, as previously described for the fed-treated group. Blood samples were collected over a period of 72 hr post-treatment from 6 animals in each treated group. Plasma samples were analyzed by high performance liquid chromatography. The pharmacokinetic parameters were statistically compared using parametric statistical tests. The estimation of the efficacy of the different treatments was performed by the fecal egg count reduction test (FECRT). Additionally, 4 animals randomly chosen from the control-fed and treated groups were killed 13 days post-treatment to evaluate the efficacy against different adult nematode parasites. The results were statistically compared by parametric and non-parametric tests. Significantly (P < 0.05) higher Cmax and AUC values were observed for both the ABZSO and ABZ-sulphone (ABZSO(2)) metabolites in the fasted compared to the fed animals. These kinetic results may be due to a fasting-induced delay in the GI transit time which increases ABZ dissolution and GI absorption. However, a poor ABZ efficacy (measured as FECRT), compatible with a high degree of nematode resistance, was obtained in both fed (48%) and fasted (49%) animals. Haemonchus contortus and Trichostrongylus colubriformis appeared as the more reluctant species with respect to ABZ treatment. The efficacy against H. contortus ranged between 37 (fed) and 54% (fasted) and against T. colubriformis between 0% (fed) and 16% (fasted). Under these experimental conditions, the fasting-induced improvement on ABZ systemic availability (>60%) did not improve its activity against nematodes with a high degree of resistance. However, the data described here for a highly resistant nematode population should not discourage the use of fasting as a practical and well-proven management tool for parasite control in ruminants.     

1H, 13C, and 15N backbone resonance assignments of the L124D mutant of StAR-related lipid transfer domain protein 4 (StARD4)

Protein-mediated cholesterol trafficking is central to maintaining cholesterol homeostasis in cells. START (Steroidogenic acute regulatory protein-related lipid transfer) domains constitute a sterol and lipid binding motif and the START domain protein StARD4 typifies a small family of mammalian sterol transport proteins. StARD4 consists of a single START domain and has been reported to act as a general cholesterol transporter in cells. However, the structural basis of cholesterol uptake and transport is not well understood and no cholesterol-bound START domain structures have been reported. We have undertaken the study of cholesterol binding and transport by StARD4 using solution state NMR spectroscopy. To this end, we report nearly complete ¹H, ¹⁵N, and ¹³C backbone resonance assignments of an inactive but well behaved mutant (L124D) of StARD4.     

Coordinate regulation of RARgamma2, TBP, and TAFII135 by targeted proteolysis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells

Background  

Treatment of mouse F9 embryonal carcinoma cells with all-trans retinoic acid (T-RA) induces differentiation into primitive endodermal type cells. Differentiation requires the action of the receptors for all trans, and 9cis-retinoic acid (RAR and RXR, respectively) and is accompanied by growth inhibition, changes in cell morphology, increased apoptosis, proteolytic degradation of the RARγ2 receptor, and induction of target genes.     

New class of potent antinociceptive and antiplatelet 10H-phenothiazine-1-acylhydrazone derivatives

In this work, we reported the synthesis and evaluation of the analgesic, antiinflammatory, and antiplatelet properties of new phenothiazine-attached acylhydrazone derivatives (6), designed exploring the molecular hybridization approach between antipsychotic chlorpromazine (4) and other heterocyclic derivatives (3) and (5) developed at LASSBio. Target compounds were synthesized in very good yields exploiting diphenylamine (7) as starting material, through regioselective functionalization of the C-1 position of 10H-phenothiazine ring. The evaluation of platelet antiaggregating profile lead us to identify a new potent prototype of antiplatelet derivative, that is (6a) (IC(50)=2.3 microM), which acts in arachidonic acid pathway probably by inhibition of platelet COX-1 enzyme. Additionally, the change of para-substituent group of acylhydrazone framework permitted us to identify hydrophilic carboxylate derivative (6g) and hydrophobic bromo derivative (6b) as two new leads of analgesics more active than dipyrone used as standard and with selective peripheral or central mechanism of action.     

Effects of alpha-synuclein immunization in a mouse model of Parkinson's disease

Abnormal folding of alpha-synuclein (alpha-syn) is thought to lead to neurodegeneration and the characteristic symptoms of Lewy body disease (LBD). Since previous studies suggest that immunization might be a potential therapy for Alzheimer's disease, we hypothesized that immunization with human (h)alpha-syn might have therapeutic effects in LBD. For this purpose, halpha-syn transgenic (tg) mice were vaccinated with halpha-syn. In mice that produced high relative affinity antibodies, there was decreased accumulation of aggregated halpha-syn in neuronal cell bodies and synapses that was associated with reduced neurodegeneration. Furthermore, antibodies produced by immunized mice recognized abnormal halpha-syn associated with the neuronal membrane and promoted the degradation of halpha-syn aggregates, probably via lysosomal pathways. Similar effects were observed with an exogenously applied FITC-tagged halpha-syn antibody. These results suggest that vaccination is effective in reducing neuronal accumulation of halpha-syn aggregates and that further development of this approach might have a potential role in the treatment of LBD.     

Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells

Many proteins require the binding of trace metals such as Ca, Fe, Cu, or Zn, which may modulate their structure, function, or activity. To determine if there were any overall changes in metalloprotein distribution or metal concentration during the process of macrophage differentiation we induced human myeloid HL-60 leukemia cells with phorbol 12-myristate 13-acetate (PMA) and quantitatively mapped their metal content using hard X-ray fluorescence micro-analysis. We found a transient increase in the zinc content of HL-60 cell nuclei during the early stages of differentiation induction. This finding was confirmed by spectrofluorometry in HL-60 cells and extended to U-937 leukemia cells. A role for protein kinase C-beta (PKC-beta) in this process was established by examining zinc content in an HL-60 variant, HL-525, which is PKC-beta deficient, and in HL-525 cells in which PKC-beta was restored by stable overexpression. Chemical chelation of both Cu and Zn served to inhibit macrophage differentiation in HL-60 cells, indicating a requirement for these metals during this process. Finally, we demonstrate that growth of HL-60 cells in a low-zinc environment removes their susceptibility to PMA-induced differentiation, and that this capacity can be partially restored by the addition of exogenous zinc.