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    • While the results of the membrane based secretase assays are

    2018-10-24

    While the results of the membrane-based γ-secretase assays are not consistent with a direct effect on the core γ-secretase complex, there are indirect ways by which APP proteolysis may be altered that could go undetected in a cell-free assay. The specific APP processing paths leading to the generation of different Aβ species are determined by the dynamic shuttling between intracellular compartments of the membrane-bound substrates and the secretases, which must co-localize for proteolysis to occur (see review by Small and Gandy, 2006). These shuttling processes are amenable to pharmacological intervention, and indeed it was recently shown that small-molecule stabilization of the neuronal retromer complex, which traffics APP from endosomes to the Golgi, limits co-localization of APP and β-secretase in the endosomal membrane, thus causing a reduction in total Aβ peptide production (Mecozzi et al., 2014). More broadly, this study confirms the feasibility of unbiased phenotypic drug screening for modifiers of neurological disease in patient-derived neurons; an approach that has been a core aim of this technology since its inception (Khurana et al., 2015). In the small number of phenotypic drug screens in human stem cell models of neurodegeneration reported thus far, small molecules have been identified which rescued downregulated proteins in familial dysautonomia (Lee et al., 2012) and inhibited aberrant protein aggregation in amyotrophic lateral sclerosis (Burkhardt et al., 2013). Cortical neurons derived from patients carrying fAD mutations or more complex forms of AD, including TS21, faithfully reproduce pathological changes in disease-relevant proteins reported in vivo, without the need for artificial overexpression or exogenous toxic insult (Israel et al., 2012; Moore et al., 2015; Shi et al., 2012b; Yagi et al., 2011). Not only has this system previously provided mechanistic insight into AD Ozanimod and progression (Moore et al., 2015), but we have demonstrated here that the aberrant changes in Aβ production in AD human neurons provide a sensitized and relevant background for unbiased phenotypic screening, in a model system that is reproducible, scalable, and responsive to existing modulators of Aβ production. While cell-based phenotypic screens have traditionally utilized immortalized cell lines or overexpression systems, the ability to derive disease-relevant cell types from reprogrammed human cells is likely to be of additional benefit for phenotypic assays in neurodegenerative diseases, due to the high degree of cell-type- and cell-subtype-specific pathology (Mattson and Magnus, 2006). The two main avenues for the discovery and development of new medicines are target-based screening and cell-based phenotypic screening. In the first, a molecular target of interest in the disease is defined and validated prior to the screen, whereas in the second the phenotype of interest is screened for modifiers of which their underlying molecular mechanism of action is not yet known. While early drug discovery was almost entirely based on screening against known phenotypes, more recent advances in genomics and improved understanding of the molecular mechanisms underlying disease have shifted the focus toward more target-based approaches. However, the complex and multifactorial nature of human disorders, particularly those of the CNS, has hampered identification of relevant and specific singular drug targets, and perhaps explains why phenotypic screening still contributes to a greater proportion of first-in-class drugs (Swinney and Anthony, 2011). The lack of assumptions with a phenotypic approach also means there is the potential to uncover novel disease pathways. It has been argued that an initial approach using empirical phenotypic assays followed by hypothesis-driven target identification might provide an optimum combination of techniques for the identification and development of new treatments for complex human disorders (Swinney, 2013). Combined with the ability to derive disease-specific cell types from reprogrammed patient cells, phenotypic screening is once again in the spotlight as a powerful tool in the search for disease-modifying treatments.