Fig.1. Discovery and characterization of GALC folding-assistant and psychosine-reducing small molecule agents for globoid-cell leukodystrophy (GLD). (a) From newborn, brain cortices, brain-derived cell line (145M-Twi) from GLD mouse model (Twitcher) were established and shown to accumulate high-levels of psychosine (Ribbens et al 2014). (b) Using LC-MS/MS throughput screening, psychosine-reducing small molecules were identified. (c) In another high-throughput screening (HTS), using the 145M-Twi cell line expressing the human GALC, we identified several GALC-folding assisting molecules. Both HTS were quantitative and performed in multiple concentrations of the screened libraries (e.g. seven concentration or the psychosine-reducing library).
Funding: NIH/NINDS R61NS118407-02
2. Investigation of Pathogenic Cascades in LSDs as potential therapeutic targets
From a clinical perspective, patients suffering from LSDs present involvement of multiple organs and systems, predominantly affecting the central nervous system. From a cellular and molecular standpoint, the general lysosomal dysfunction may disturb several molecular pathways ultimately resulting in cell death. In LSDs, specific molecular mechanisms resulting in apoptosis have not been understood.
Fig.2. Pathogenic cascades were observed in cultured patients’ cells with neurological lysosomal diseases. (a) Sketch depicts the increased lysosomal membrane permeability (LMP) in lysosomal storage diseases (LSDs) and subsequent pathogenic cascades to activating the caspase-dependent apoptotic signaling pathway. (b) Lysosomal membrane stability assay. Using real-time confocal immunofluorescence, live skin fibroblasts from three patients with MPS II, IVA, and VI and control were treated with low concentrations of acridine orange, AO (0.5 ug/mL for 15 min). AO localizes in lysosomes at low concentrations, emitting red fluorescence at acidic pH. Under blue light (488 nm), AO is photo-oxidized, and loss of lysosomal integrity is visualized by the loss of red signal (lysosomes) and increase in the green signal (cytoplasm and nuclei). MPS II, IVA, and VI fibroblasts showed decreased lysosomal membrane stability (demonstrated by the rapid loss of red signal loss) when compared to the control cell line (upper panel) over 80 seconds of blue-light exposure time. An assay based on lysosomal membrane stability assay described in Brunk UT et al. 1997. (b) Using specific antibodies for LC3-II (red), a key protein in autophagy (text), a control (A, B), and an MPS-II patient cell line (C, D) are shown. A specific antibody against LAMP-1 is shown as a lysosomal marker (green). In comparison to control (A, B), the MPS-II fibroblasts showed increased expression of LC3-II, which is adjacent to the LAMP1 location (C). LC3-II location is more appreciable at higher magnification panels (control B; MPS-II D).
The current advances in LSD treatment are exemplified by enzyme replacement therapy (ERT), which consists of weekly or biweekly intravenous administrations of the recombinant enzymes deficient in the patients. Although generally well-tolerated, the intrathecally delivery of ERT showed limited CNS biodistribution in neuronopathic forms of mucopolysaccharidosis (MPS-III). Therefore, ERT agents are large molecules and are incapable of crossing the blood-brain barrier (BBB). The lack of reliable and efficacious delivery across the BBB severely limits the ERT and other therapies to treat neurological symptoms. Hence, the development of delivery strategies for therapeutic agents for LSDs is an unmet and urgent need and will have a substantial impact on affected patients with these and other neurodegenerative disorders.