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. Lysosomal membrane permeabilization (LMP) has been shown to be a primary event, preceding other hallmarks of apoptosis including mitochondrial membrane permeabilization and caspase activation. Given the majority of LSDs are neurological disorders, the use of disease-relevant cells will be crucial, especially as sphingolipids are mostly accumulated in neuronal cells. Therefore, in collaboration with Akira Sawa MD, PhD from Psychiatry and Neuroscience, we are currently establishing fibroblast-derived induced-neuronal (iN) cells from patients with LSDs. The iN cells from LSD will be crucial to investigate pathogenic cascades of neurological relevance. The aim is to investigate to confirm LMP in different LSDs and investigate the molecular mechanisms causing LMP and its downstream events in iN cells from LSD patients. Tackling common intervention points shared by several LSDs is highly desirable, as these diseases are individually rare, but collectively common inborn organelle disorders. Therefore, LMP may also play an important role in the pathogenesis of LSDs and other neurodegenerative diseases, and consequently a potential therapeutic target for these conditions.

Fig.5. Increased autophagy marked in cultured fibroblasts from neuronopathic MPS-II

Fig.5. Increased autophagy marked in cultured fibroblasts from neuronopathic MPS-II. Using specific antibody for LC3-II (red), a key protein in autophagy (text), a control (A, B) and a MPS-II patient cell line (C,D) are shown. Specific antibody for 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 LAMP1 location (C). LC3-II location is more appreciable at higher magnification panels (control B; MPS-II D).

Fig.6. Increased lysosomal membrane permeabilization (LMP) in cultured fibroblasts from LSDs

Fig.6. Increased lysosomal membrane permeabilization (LMP) in cultured fibroblasts from LSDs. Using real-time confocal immunofluorescence, live skin fibroblasts from three patients with MPS II, IVA and VI and a control were treated with low concentrations of acridine orange, AO (0.5 mg/mL for 15 min). At low concentrations, AO localizes in lysosomes, 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 (shown by the rapid loss of red signal loss) when compared to control cell line (upper panel) over 80 seconds blue-light exposure time. Assay based on lysosomal membrane stability assay described in Brunk UT et al. [6].

Fig.7. Disease-cell models for neurological LSDs

Fig.7. Disease-cell models for neurological LSDs. In our laboratory, we are neurologically-relevant disease cell models to study neuropathogenesis of lysosomal storage diseases and also identify potential targets. Briefly, primary fibroblasts obtained in clinic from patients affected with different LSDs are established. Later, these cells are transfected with the defined transcription factors (red box) which allow us to directly generate induced-neural stem cells (iNSCs). The co-transfection with the EGFP markers allowed us to select specific clones and further track the differentiated neuroglial cells. These cells can be further differentiated into mature neurons, oligodendrocytes and other glial cells under conditioned media with specific growth factors. These cells have showed increased accumulated sphingolipids in specific LSDs as here depicted in the histograms from iNSCs and differentiated neurons from GLD patients. In addition, here we depict iNSCs-derived neural cells cultured in a microfluidic chamber to separate in the matured neurons and their axonal process from mature oligodendrocytes. In this setting, we can study both the myelination process along with the intra-axonal trafficking.