Methods and compositions for treatment of lipid storage disorders

[0060] Materials and methods

[0061] Dulbecco's Modified Eagle medium (DMEM), trypsin, L-glutamine, gentamicin, and NuPage gels and buffers were from Invitrogen (Carlsbad, CA), while FBS was from Hyclone, ThermoScientific (Rockford, IL). Monoclonal anti-vimentin (V9), conjugated anti-mouse-IgG and anti-rabbit-IgG antibodies were from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). Anti-GAPDH antibody was from Millipore (Billerica, MA), and anti-Rab9 polyclonal antibody has been described elsewhere [27]. Filipin was from Polysciences, Inc. (Warrington, PA). Both Lumilight Plus substrate and FuGENET 6 transfection reagent were from Roche Diagnostics (Indianapolis, IN). [9,10-3H(N)]oleic acid (15Ci / mmol) was obtained from NEN Life Science Products (Boston, MA) and LDL was from EMD Biosciences Inc. (La Jolla, CA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

[0062] Cell Culture and Transfection

[0063] Human wild-type fibroblast (GM05387), NPCl...

[0096] We also studied the PKC activator bryostatin 1 using probes for free cholesterol (Philippines), glycosphingolipid levels (VTB), and ganglioside motility (CTB) in human NPC1 cells treated 48-72 hours later. and DCPLA on the Niemann-Pick C disease phenotype.

[0097] Materials and methods

[0098] Table 1: Human NPC1 cell lines

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[0101] Table 2: Reagents

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[0103] general method

[0104] Cells were seeded in 6-well plates and treated with the appropriate compound (dissolved in DMSO) at the indicated doses for 48 hours daily. Control cells received DMSO. After 48 hr, cells were transferred to coverslips with fresh compound and grown for an additional 24 hr. Coverslips were collected and processed for microscopy.

[0105] Assay protocol

[0106] Filipinos: To detect unesterified cholesterol in lysosomes, fix cells in formalin at 4 °C for 30 min, wash 2X in 0.9% NaCl for 5 min, incubate with 0.01% Filipinos in PBS at room temperature...

[0117] Niemann-Pick C disease is a severe inherited lipid deposition that leads to neurodegeneration and early childhood death. The biochemical and cellular events that lead to neurodegeneration are currently poorly understood. However, PKC activation has been shown to restore blocked lipid transport pathways and lead to a reduction of stored lipid material in NPC endosomes / lysosomes. Thus, treatment of NPC1 mice with bryostatin 1, a natural product activator of PKC, should lead to an amelioration of disease progression in this animal model.

[0118] A total of 30 C57B16NPC1 mice, mixed sexes, were used. These mice were divided into 5 groups, 5 mice in each group, 1-5 groups.

[0119] drug research

[0120] One vial of 2 mg bryostatin 1 (>95% purity) from Aphios (Woburn, MA) was dissolved in 5% DMSO, 20% Solutol, and 75% saline solution and used as the study drug. Negative (vehicle) controls were 5% DMSO, 20% Solutol and 75% saline solution. DCP-LA from Sigma-Aldrich: 5 m...