Lipid and protein composition of Niemann-Pick type A brain rafts

Lipid rafts, or DRM (Detergent Resistant Membrane) are supermolecular organizations created by lateral spontaneous segregation of specific lipids (particularly sphingolipids and cholesterol) and proteins in restricted areas of the plasma membrane. They represent structural and functional units involved in different pathways of signalling and in cellular adhesion [2]. In the nervous system, they are involved in normal cell functions and in the pathogenesis of neurodegenerative diseases. In fact, alterations of molecular composition and organization within DRM have been related to these pathological events. This hypothesis is based on two important observations: the first one is that certain proteins, whose transformation in aberrant isoforms causes the onset of neuronal pathologies, are enriched in these domains (for example, the amyloid precursor protein and the prion protein); the second one regards the fact that numerous neurodegenerative diseases show altered sphingolipid metabolism. The relatively large number of papers dedicated to the preparation of a lipid domain fraction from brain or brain substructures obviously reflects the interest for the supposed role of lipid domains in neuronal development and in the pathogenesis of neurodegenerative diseases [3]. However, studies on DRM have been carried out mainly using cell cultures and the suitability of methods for the preparation of DRM fractions from brain tissues has never been systematically defined. The cellular approach is important and useful when we want to study the structural and biochemical properties of DRM isolated from specific cellular types, reducing as much as possible the contamination by other cells, but this model is very simplified and reductive to study the effects of neurodegeneration on DRM in the brain. In fact, it is known that neurodegeneration involves more than one cellular type and can cause the loss of neurons, demyelination events or changes in the structure of the whole cerebral tissue. We set up an experimental condition for the preparation of a sphingolipid- and cholesterol-enriched membrane fraction from mouse brain. To reach this result we performed several DRM preparations starting from a brain lysate prepared varying only the ratio between the protein and the detergent content and maintaining unaltered all the other parameters. For all the experimental conditions assayed we worked at 4°C in presence of Triton X-100, as detergent, at a final concentration of 1%. Starting from different conditions of brain lysis the effects on the DRM preparation have been carefully checked by a detailed characterization of lipid and protein content of the low density fractions obtained and we could define a restricted range of values for which it has been possible to effectively isolate a low density fraction characterized by a protein and lipid content and by relative enrichments typical of the so called DRM. Once this procedure has been set up, we tried to apply it on an available model of neurodegenerative disease. This model consists in an acid sphingomyelinase knockout mouse (ASMKO), which develops a neurodegenerative pathology resembling the human Niemann-Pick type A disease. These mice accumulate sphingomyelin in the reticuloendothelial system of liver, spleen, bone marrow and lung and in the brain. First of all, we did a comparative study of the lipid and protein composition of wild type (WT) and ASMKO brain at different time of development. Since sphingomyelin is one of the main components of lipid rafts, we analysed how the composition of these Niemann-Pick A brain rafts changed, as a consequence of the highly increased amount of sphingomyelin.