Galactolipid deficiency in the early pathogenesis of neuronal ceroid lipofuscinosis model Cln8mnd : implications to delayed myelination and oligodendrocyte maturation.

AIMS: CLN8 deficiency underlies one of a group of devastating childhood neurodegenerative disorders, the neuronal ceroid lipofuscinoses. The function of the CLN8 protein is currently unknown, but a role in lipid metabolism has been proposed. In human CLN8 diseased brains, alterations in lipid composition have been detected. To further investigate the connection of CLN8 to lipid metabolism, we characterized the lipid composition of early symptomatic Cln8-deficient mouse (Cln8(mnd)) brains. METHODS: For lipid profiling, Cln8(mnd) cerebral cortical tissue was analysed by liquid chromatography/mass spectrometry. Galactolipid synthesis was measured through enzyme activity and real-time mRNA expression analyses. Based on the findings, myelination and white matter integrity were studied by immunohistochemistry, stereological methods, electron microscopy and magnetic resonance imaging. The development of myelin-forming oligodendrocytes was also studied in vitro. RESULTS: Sphingolipid profiling showed a selective reduction in myelin-enriched galactolipids. The mRNA expression and activity of UDP-galactose:ceramide galactosyltransferase (CGT), the key enzyme in the galactolipid synthesis, was reduced in the Cln8(mnd) brain. Expression of oligodendrocyte markers suggests a maturation defect. The amount of myelin was reduced in 1-month-old Cln8(mnd) mice, but reached normal levels by 5 months of age. The level of Cln8 gene expression followed the developmental pattern of myelin formation and was high in primary oligodendrocytes. CONCLUSIONS: Taken together, these observations suggest that galactolipid deficiency and delayed myelin maturation characterize the early CLN8 disease pathogenesis through a maturation defect of oligodendrocytes.

Detection of residual aneuploid leukemic cells by "continuous gating".

BACKGROUND: A new method for the detection of residual aneuploid leukemic cells in bone marrow by flow cytometry is described. This method is based on the analysis of FCM derived list-mode-datasets with a new software called "Continuous Gating". The program is able to decrease the detection level of aneuploid tumor cells by analyzing groups of cells with comparable antigen density and scatter properties. METHODS: Aneuploid acute lymphocytic leukemia cells with a known CD34 expression were diluted with diploid bone marrow cells to a concentration of 10, 1, 0.1, 0.05, and 0.01%. Each sample was measured in a FACScan flow cytometer, after staining with CD34 Moab and propidium iodide. Listmode-data were analyzed with the new "Windows"-based "Continuous Gating" software. A gate was set in the DNA parameter, defining the channels in which the aneuploid G0/G1-peak of possible residual tumor-cells should be found. Ten thousand overlapping gates of size 200 x 200 channels (out of 1,023 x 1,023 channels) were set automatically by the program into the side-scatter (SSC)/CD34 dot-plot, calculating the percentage of aneuploid G0/G1-phase cells for every specific gate. RESULTS: The results are plotted in a contour-plot. In dot-plot gates with less than 20 cells, the calculation of the percentage of aneuploid cells was declared invalid and the area in the contour-plot was marked. Detection of residual aneuploid cells, based on a defined expression of CD34 and granularity (SSC), was possible down to a contamination of 0.1%. CONCLUSIONS: The new "Continuous Gating" software can be used for the automated detection of aneuploid leukemic cells, if the density of a certain surface-marker is slightly different from normal cells.