Valinomycin induced energy-dependent mitochondrial swelling, cytochrome c release, cytosolic NADH/cytochrome c oxidation and apoptosis

In valinomycin induced stimulation of mitochondrial energy dependent reversible swelling, supported by succinate oxidation, cytochrome c (cyto-c) and sulfite oxidase (Sox) [both present in the mitochondrial intermembrane space (MIS)] are released outside. This effect can be observed at a valinomycin concentration as low as 1 nM. The rate of cytosolic NADH/cyto-c electron transport pathway is also greatly stimulated. The test on the permeability of mitochondrial outer membrane to exogenous cyto-c rules out the possibility that the increased rate of exogenous NADH oxidation could be ascribed either to extensively damaged or broken mitochondria. Accumulation of potassium inside the mitochondria, mediated by the highly specific ionophore valinomycin, promotes an increase in the volume of matrix (evidenced by swelling) and the interaction points between the two mitochondrial membranes are expected to increase. The data reported and those previously published are consistent with the view that ‘‘respiratory contact sites’’ are involved in the transfer of reducing equivalents from cytosol to inside the mitochondria both in the absence and the presence of valinomycin. Magnesium ions prevent at least in part the valinomycin effects. Rather than to the dissipation of membrane potential, the pro-apoptotic property of valinomycin can be ascribed to both the release of cyto-c from mitochondria to cytosol and the increased rate of cytosolic NADH coupled with an increased availability of energy in the form of glycolytic ATP, useful for the correct execution of apoptotic program

EVIDENCE OF DIFFERENTIAL EXPRESSION RELATED TO THYROSINE HYDROXILASE GENES IN CHICK (GALLUS GALLUS) BRAIN STEM DURING EMBRYO DEVELOPMENT

By affecting efficiency of DOPA synthesis, tyrosine hydroxylase (TH) is the effective modulator of the limiting step of the biosynthetic pathways of catecholamines (dopamine, noradrenaline and adrenaline), highly conserved in vertebrate species. Catecholamines are involved in many physiological functions in the central and peripheral nervous systems as well as in the endocrine system, thus regulation of TH expression and activity are crucial for neuronal and hormonal functions that involve the entire dopaminergic, noradrenergic and adrenergic systems. THrelated expression is due to two non-allelic genes, called TH1 and TH2, reported in almost all vertebrates except placental mammalian, which have lost TH2 gene during evolution. THrelated genes have crucial ontogenetic roles, being linked to pathological onsets during embryo development. Here, we show the expression analysis of TH-related transcripts in brain stem of gallus gallus, as a key model of vertebrate central nervous system development. By real time RT-PCR assays, we assessed that TH1 and TH2 mRNAs show progressive increase during embryo development (from 8 to 21 days post fertilization) with differential trend. Moreover, a substantially different regulatory switch of expression was shown for the two genes when passing to the adult developmental phase. According to what stated in teleost fish, different expression patterns suggest different mechanism of transcriptional regulation related to potentially differing roles during development for TH1 and TH2 genes: based on our comparative results, TH1 mRNA expression in gallus gallus brain increases gradually during development reaching significantly high post-embryonic levels, whereas the TH2 mRNA seems to be more specifically linked to embryogenesis of vertebrate brain stem

Pro-inflammatory cytokines and their receptors expression in a mouse model of parkinson's like-disease

Upregulation of inflammatory response in the brain is associated with a number of neurodegenerative diseases, including Alzheimer‟s and Parkinson‟s disease (PD), amyotrophic lateral sclerosis, multiple sclerosis. In particular PD is a common neurodegenerative pathological state characterised by the degeneration of dopaminergic neurons in the substantia nigra (SN) pars compacta, determining reduced dopamine levels in the caudate-putamen (CP) which lead to movement malfunction. Despite intensive research, the cause of neuronal loss in PD is poorly understood. To study the specific cause of PD, researchers have used a variety of toxins as agents to bring about damage to the dopaminergic neurons, such as 6-hydroxydopamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), paraquat and rotenone. Among these, the MPTP poisoning leads to symptoms very closely matched to many features of Parkinsonian syndrome in both humans and animals. In this study we investigated, in an experimental MPTP mouse model of PD, the expression of pro-inflammatory cytokines interleukin (IL)-1 , tumor necrosis factor (TNF)- α and IL-6 and their receptors in the SN and CP, in order to evaluate their involvement in this neurodegenerative disease. In MPTP-treated animals we observed a significant increase in IL-1 , TNF- α and IL-6 mRNA expression levels both in the SN and CP in comparison with untreated mice. In addition, both mRNA and protein levels of IL-1RI, TNF- α RI and IL-6R were significantly enhanced in the SN of MPTP-treated mice in comparison to controls, whereas no significant differences were observed in the CP between treated and untreated mice. Overall, these results indicate a possible role of both pro-inflammatory cytokines and their receptors in the pathogenesis of PD

Tyrosine hydroxylase in chick embryo brain development

Tyrosine hydroxylase (TH) catalyses the first, rate limiting step in catecholamine biosynthesis. In vertebrates the catecholamines dopamine, noradrenaline and adrenaline play important roles in many physiological functions in the central and peripheral nervous systems as well as in the endocrine system. Thus the regulation of TH expression and activity is crucial for neuronal and hormonal functions that involve catecholamines. TH is regulated by many ways: among them, gene expression modulation, alternative RNA processing, allosteric modulation by polianions (mainly RNA), site-specific phosphorilation, feedback inhibition, ubiquitination. The importance of TH expression during embryonic development have been demonstrated by the targeted inactivation of the TH gene that determines, in mice, midgestional lethality. In most species is present a single TH mRNA, that following translation produces a single form of the protein. In some species, however, alternative splicing of the primary transcript can result in different forms of TH mRNA that encode for different protein isoforms with specific regulative properties. Considering the importance of the TH expression during embryonic development and the presence in many species of multiple TH protein/mRNA forms led us to study the enzyme expression during chick embryo brain development. By means of western blotting we found that in the chick embryo brain, together with the expected form of ~65 kDa, is present a TH protein showing a molecular weight of ~75 kDa from E (embryonic day) 8 to E14. The analysis of the cDNA sequence of TH obtained from chick embryo brains at E8-E14 revealed the possible presence of a mRNA splice variant that presents a nucleotide insertion which encodes for an additional aminoacidic sequence in the regulative N-terminal domain of the protein. We also found that TH immunoreactive bands showing an higher molecular weight were immunoreactive for ubiquitin, too. These results suggest that during chick embryo brain development is transiently present a TH protein of ~75 kDa; the possible mechanisms involved in this process are alternative splicing and/or ubiquitination