Ongoing Projects

Based on a previous cell-based high-throughput screening (HTS) assay developed by our lab (Geng et al. 2011), a quantitative cell-based HTS assay for globoid-cell leukodystrophy (GLD), known mostly as Krabbe disease, we developed two different quantitative cell-based HTS assay have been developed b-galactocerebrosidase (GALC). GLD is rapidly progressive, invariably fatal LSDs manifested mostly in infancy (Wenger D et al. 2001). GLD is a classical neurological lysosomal storage disease (LSD) caused by the deficiency of galactocerebrosidase (GALC), a lysosomal hydrolase catalyzing the removal of galactose from two sphingolipids galactosylceramide and galactosylsphingosine, mostly known as psychosine. In affected patients with GLD, psychosine is found at high levels in both the central (CNS) and peripheral nervous system (PNS). Psychosine is present at low levels in myelin-forming cells in a physiological state. However, in GLD, psychosine becomes extremely cytotoxic at high concentrations, resulting in the loss of oligodendrocytes (CNS), Schwann cells (PNS), and subsequent diffuse demyelination and leukodystrophy.

Using the brain-derived cell line established in our lab from the cortices of the GLD murine model, the Twitcher (145M-Twi), we developed and implemented two throughput screening assays and identified two classes of potential therapeutic molecules for GLD (Fig.1a). First, we developed and implemented a live cell-based throughput LC-MS/MS assay for psychosine (Fig.1b). Through this high-throughput screening (HTS) assay, we identified molecules that reduce their levels to a nearly physiological range. Second, expressing a common human GALC misfolding mutation in the 145M-Twi cell line, we developed and implemented a cell-based HTS assay and identified molecules that assist in folding and prevent mutant GALC degradation, enhancing its residual activity (Fig.1c). Through validation assays, the small molecule ‘hits’ showed in vitro and in vivo effects on psychosine and GALC residual activity.

Here, we are examining whether psychosine-reducing and GALC-folding assistant molecules are therapeutic agents able to reduce psychosine levels resulting in the arrest and even prevention of the rapid demyelination in GLD. We developed two cell-based assays for characterizing compounds using differentiated neural cells from induced-neural stem cells (iNSC) established from four early-onset and four late-onset GLD patients' fibroblasts. The iNSC cells will be differentiated into oligodendrocytes to further study several synthesized analogs from two classes of small molecules. In collaboration with NIH/NCATS. We are currently performing SAR activities and analog synthesis, followed by in vitro screening of analogs in cell-based assays to test their efficacy in reducing cytotoxic GALC-substrates, such as psychosine (Fig.1d). In vitro ADME and in vivo PK/PD will also be assessed for top small molecule ‘hits.’ Subsequently, we will evaluate and prioritize top analogs based on neurobehavioral outcomes, morphometric myelin assessments, and BBB penetration using established GLD mouse models (Fig.1e).

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.

The accumulation of glycosphingolipids and glycosaminoglycans in LSDs can potentially induce LMP with the subsequent release of lysosomal proteolytic enzymes into the cytosol, demonstrated to activate several signaling pathways leading to apoptosis. Additionally, substantial evidence supports LMP as the primary event rather than occurring at later stages of apoptosis (Boya P et al. 2008). LMP involves the selective release of lysosomal hydrolases and proteases, particularly cathepsins, that translocate from the lysosomal lumen into the cytosol (Li W et al. 2004); (Fig.2a). These cathepsins remain partially active at neutral pH and function as apoptotic trigger elements by directly activating caspases and calpains. Cathepsins B (CB) and (CD) were shown to cleave BH-3-only proteins (Bid), which are B-cell lymphoma-2 (Bcl-2)-family proteins that are activated by proteolytic cleavage (Cirman T et al. 2004; fig.2a). The activated “truncated” Bid (_tBid) promotes the assembly of Bak (Bcl-2 antagonist/killer-1) and Bax (Bcl-2 –associated X-protein) oligomers in the mitochondrial outer membrane, initiating its permeabilization. The mitochondrial outer membrane permeabilization (MOMP) results in cytochrome c release into the cytosol, which then activates caspases-3 and -7, effector caspases that orchestrate the dismantling of diverse cell structures (Boya et al. 2008). Although not currently recognized as a pathogenic cascade in LSD, LMP will be examined separately. It may potentially consist of a common cascade resulting in the activation of apoptotic events in LSDs. The examination of skin fibroblast lines from patients with various LSDs may offer a unique opportunity to evaluate any variation of LMP among these diseases. At low concentrations (0.5-1.5 mg/mL), AO, a lysosomotropic metachromatic fluorochrome, accumulated in acidic organelles, including lysosomes emitting red signal visualized by live-cell confocal fluorescence microscopy or measured by flow cytometry. AO was also shown to emit green fluorescence at low concentrations in the cytosol and nucleus. Thus, AO-treated cells manifest decreased red fluorescence and increased green fluorescence after LMP. I will also evaluate LMP using specific antibodies against CB and CD to demonstrate the relocalization of these cathepsins from lysosomes to the cytosol as described in preliminary results (Fig.2b).

Lysosomes are the endpoint for the autophagy pathway. Macroautophagy is a catabolic pathway responsible for the turnover of long-lived cytosolic proteins and organelles. Basically, autophagic cargo is sequestered by double-membrane vesicles (autophagosomes) and is ultimately degraded after fusion with lysosomes (Settembre C et al. 2008). In addition to its crucial role during cellular stress, autophagy may also function as a pathway that results in apoptosis caused by the impairment of autophagosome-lysosome fusion or by exacerbating an uncontrolled autophagy response. To evaluate autophagy, we measure the expression levels of two autophagic vesicular markers: LC3, both in its soluble (LC3-I) and phosphatidylethanolamine (PE)-conjugated form (LC3-II), and Beclin-1 (34). Confocal immunofluorescence assays with specific antibodies to LC3-II and Beclin, using the lysosomal-associate-membrane (LAMP1 and 2), as lysosomal labeling antibodies, are tools to assess the level of autophagy flux (Fig.2c). As mentioned above, we also perform immunoblots of lysosomal fractions from cultured skin fibroblast from the LSDs and controls. The LC3-II/LC3-I and Beclin are considered reliable makers to evaluate autophagy.

3. GALC-containing EVs as a tool to deliver ERT agents and Treat Neuronopathic Processes in Krabbe disease