Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm2. It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type and cholesterol 24S-hydroxylase knockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues

Visualizing Cholesterol in the Brain by On-Tissue Derivatization and Quantitative Mass Spectrometry Imaging.

Despite being a critical molecule in the brain, mass spectrometry imaging (MSI) of cholesterol has been under-reported compared to other lipids due to the difficulty in ionizing the sterol molecule. In the present work, we have employed an on-tissue enzyme-assisted derivatization strategy to improve detection of cholesterol in brain tissue sections. We report distribution and levels of cholesterol across specific structures of the mouse brain, in a model of Niemann-Pick type C1 disease, and during brain development. MSI revealed that in the adult mouse, cholesterol is the highest in the pons and medulla and how its distribution changes during development. Cholesterol was significantly reduced in the corpus callosum and other brain regions in the Npc1 null mouse, confirming hypomyelination at the molecular level. Our study demonstrates the potential of MSI to the study of sterols in neuroscience

New hyperekplexia mutations provide insight into glycine receptor assembly, trafficking, and activation mechanisms

Background: Hyperekplexia mutations have provided much information about glycine receptor structure and function. Results: Weidentified and characterized nine new mutations. Dominant mutations resulted in spontaneous activation, whereas recessive mutations precluded surface expression. Conclusion: These data provide insight into glycine receptor activation mechanisms and surface expression determinants. Significance: The results enhance our understanding of hyperekplexia pathology and glycine receptor structure-function. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A

Neuropeptide y modulates hippocampal neurogenesis

NPY and its predominant receptors Y1, Y2 and Y5 are abundantly expressed in the hippocampus and dentate and we hypothesised that some of NPY’s activity might be through its effects on neurogenesis, and sought to determine if NPY was neuroproliferative for cells of the hippocampus. We have demonstrated that NPY is proliferative for nestin-positive cells with stem/progenitor cell characteristics and for class III β-tubulin-positive neuroblasts via specific activation of Y1 receptors.  Using dentate and hippocampus-derived cultures (containing attached cells of the alveus and periventricular neurogenic margins) we have demonstrated that nestin-positive cells express Lewis-X, generate neurospheres and give rise to mature astrocytes and neurons on removal of the NPY growth stimulus.  NPY is only proliferative for neuroblasts within dentate-derived cultures.  Young adult Y1 receptor knockout mice and adult NPY knockout mice both display reduced subgranular zone cell proliferation in comparison to respective littermate controls, while the number of new neurons is also reduced in Y1-/- animals.  NPY knockout mice have a lower number of proliferating cells following chemoconvulsant seizures in comparison to wild type animals matched for both dose of chemoconvulsant and severity of seizures.  GABA and NPY are often co-expressed in interneurons, and GABAA receptor activation abolishes the proliferative effect of NPY, indicating that the balance in GABA/NPY tone in the hippocampus could regulate cell proliferation and differentiation. This work suggests that NPY modulates neurogenesis through development and into adulthood, and raises the intriguing possibility that much of NPY’s involvement in processes of learning or in depression is based upon its control of hippocampal neurogenesis.</p

NPY mediates basal and seizure-induced proliferation in the subcallosal zone

Stem cell niches exist around the lateral ventricle and in the subgranular layer of the dentate gyrus, supporting adult neurogenesis. Recently, a third germinal layer, the subcallosal zone has been identified supporting the generation of oligodendrocytes in the adult brain. We have previously described a proliferative role for neuropeptide Yon precursors in the dentate gyrus, caudal subventricular zone and subcallosal zone under basal conditions and in the dentate gyrus after seizures. Here we sought to determine a role for neuropeptide Y in seizure-induced proliferation in the subcallosal niche. Using the chemoconvulsant kainate and neuropeptide Y-/- mice with controls, we show an effect of neuropeptide Y on basal proliferation and demonstrate a significant reduction in seizure-induced proliferation in the subcallosal zone

Inhibition of 7α,26-dihydroxycholesterol biosynthesis selectively promotes midbrain dopaminergic neuron development.

Dysregulated cholesterol metabolism has been linked to neurodegeneration. We previously found that free, non-esterified, 7α,(25R)26-dihydroxycholesterol (7α,26-diHC), also known as 7α,27-dihydroxycholesterol, was significantly elevated in the cerebrospinal fluid (CSF) of Parkinson's disease patients. In this study we investigated the role of 7α,26-diHC in mouse and human midbrain dopamine (mDA) neuron development and survival. We report that 7α,26-diHC induces apoptosis in mouse midbrain progenitor cultures and reduces the number of mDA neurons in hESC-derived cultures and in mouse progenitor cultures. Voriconazole, an oxysterol 7α-hydroxylase (CYP7B1) inhibitor, but not the non-azole inhibitor metyrapone, increases the number of mDA neurons. Moreover, voriconazole prevents the loss of mDA neurons induced by 7α,26-diHC in hESC-derived cultures and in mouse progenitor cultures. These effects on mDA neurons are specific since neither 7α,26-diHC nor voriconazole alter the number of Islet1+ oculomotor neurons in human and mouse midbrain progenitor cultures. Furthermore, our results suggest that elevated 24(S),25-epoxycholesterol (24,25-EC), which has been shown to promote mDA neurogenesis, may be partially responsible for the effect of voriconazole on mDA neurons. The findings presented in this study suggest that voriconazole, and/or other azole CYP7B1 inhibitors, could be utilised as protective agents for mDA neurons and may have implications for Parkinson's disease therapy development

Dozrávání rajčat z pozdních sběrů

In this diploma thesis "Ripening tomatoes from the late harvest" were investigated supporting options of the post harvest of the completely green fruit, which remained unharvested in the later stages of harvest. Fruits were transferred from the field conditions to the temperature of 20 °C and afterwards were successively treated with ethylene as the supportive maturing factor, which helped them to get to the suitable conditions for subsequent maturation. Two varieties of tomatoes were used for the speed of ripening. At the end of the cultivation period, both varieties of tomatoes 'Expres' and 'Premio' were treated by the three concentrations of exogenous ethylene, 2 100 ppm, 7 360 ppm and 2 200 ppm. Technologically important parameters such as the size and weight of fruit, fruit color, fruit firmness, soluble solids and titration acid were evaluated during the post-harvest storage (21 days). Influence of ethylene in concentrations up to 7 360 ppm has proven effective post-harvest treatment that supports ripening of originally unripe fruit at the end of the growing season. Also differences in the investigated varieties were proved, the 'Premio' variety was after the ethylene treatment sensitive in parameters, which were monitored

Neuropeptide Y is important for basal and seizure induced precursor cell proliferation in the hippocampus

We have shown that neuropeptide Y (NPY) regulates neurogenesis in the normal dentate gyrus (DG) via Y(1) receptors (Howell, O.W., Scharfman, H.E., Herzog, H., Sundstrom, L.E., Beck-Sickinger, A. and Gray, W.P. (2003) Neuropeptide Y is neuroproliferative for post-natal hippocampal precursor cells. J Neurochem, 86, 646-659; Howell, O.W., Doyle, K., Goodman, J.H., Scharfman, H.E., Herzog, H., Pringle, A., Beck-Sickinger, A.G. and Gray, W.P. (2005) Neuropeptide Y stimulates neuronal precursor proliferation in the post-natal and adult dentate gyrus. J Neurochem, 93, 560-570). This regulation may be relevant to epilepsy, because seizures increase both NPY expression and precursor cell proliferation in the DG. Therefore, the effects of NPY on DG precursors were evaluated in normal conditions and after status epilepticus. In addition, potentially distinct NPY-responsive precursors were identified, and an analysis performed not only of the DG, but also the caudal subventricular zone (cSVZ) and subcallosal zone (SCZ) where seizures modulate glial precursors. We show a proliferative effect of NPY on multipotent nestin cells expressing the stem cell marker Lewis-X from both the DG and the cSVZ/SCZ in vitro. We confirm an effect on proliferation in the cSVZ/SCZ of Y(1) receptor(-/-) mice and demonstrate a significant reduction in basal and seizure-induced proliferation in the DG of NPY(-/-) mice