Filipin complex-reactive brain lesions: a cautionary tale.

OBJECTIVE: Filipin complex is an autooxidation-prone fluorescent histochemical stain used in the diagnosis of Niemann-Pick Disease Type C (NP-C), a neurodegenerative lysosomal storage disorder. It is also widely used by researchers examining the distribution and accumulation of unesterified cholesterol in cell and animal models of neurodegenerative diseases including NP-C and Sanfilippo syndrome (mucopolysaccharidosis IIIA; MPS IIIA). Recently, it has been suggested to be useful in studying Alzheimer's and Huntington's disease. Given filipin's susceptibility to photobleaching, we sought to establish a quantitative biochemical method for free cholesterol measurement. METHODS: Brain tissue from mice with MPS IIIA was stained with filipin. Total and free cholesterol in brain homogenates was measured using a commercially available kit and a quantitative LC-MS/MS assay was developed. Gangliosides GM1, GM2 and GM3 were also quantified using LC-MS/MS. RESULTS: As anticipated, the MPS IIIA mouse brain displayed large numbers of filipin-positive intra-cytoplasmic inclusions, presumptively endo-lysosomes. Challenging the prevailing dogma, however, we found no difference in the amount of free cholesterol in MPS IIIA mouse brain homogenates cf. control tissue, using either the fluorometric kit or LC-MS/MS assay. Filipin has previously been reported to bind to GM1 ganglioside, however, this lipid does not accumulate in MPS IIIA cells/tissues. Using a fluorometric assay, we demonstrate for the first time that filipin cross-reacts with both GM2 and GM3 gangliosides, explaining the filipin-reactive inclusions observed in MPS IIIA brain cells. CONCLUSION: Filipin is not specific for free cholesterol, and positive staining in any setting should be interpreted with caution.

Synthesis and characterization of a PAMAM dendrimer nanocarrier functionalized by SRL peptide for targeted gene delivery to the brain.

Blood-brain barrier inhibits most of drugs and genetic materials from reaching the brain. So, developing high efficiency carriers for gene and drug delivery to the brain, is the challenging area in pharmaceutical sciences. This investigation aimed to target DNA to brain using Serine-Arginine-Leucine (SRL) functionalized PAMAM dendrimers as a novel gene delivery system. The SRL peptide was linked on G4 PAMAM dendrimers using bifunctional PEG. DNA was then loaded in these functionalized nanoparticles and their physicochemical properties and cellular uptake/distribution evaluated by AFM, NMR, FTIR and fluorescence and confocal microscopy. Also, biodistribution and brain localization of nanoparticles were studied after IV injection of nanoparticles into rat tail. Unmodified nanoparticles were used as control in all evaluations. In vitro studies showed that SRL-modified nanoparticles have good transfection efficacy and low toxicity. Results also showed that SRL is a LRP ligand and SRL-modified nanoparticles internalized by clathrin/caveolin energy-dependent endocytosis to brain capillary endothelial cells. After intravenous administration, the SRL-modified nanoparticles were able to cross the blood-brain barrier and enter the brain parenchyma. Our result showed that, SRL-modified nanoparticles provide a safe and effective nanocarrier for brain gene delivery.