Lesch-Nyhan disease (LND) is a rare monogenic disease caused by deficiency of the salvage pathway enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). LND is characterized by severe neuropsychiatric symptoms that currently cannot be treated. Predictive in vivo models are lacking for screening and evaluating candidate drugs because LND-associated neurological symptoms are not recapitulated in HGPRT-deficient animals. Here, we used human neural stem cells and neurons derived from induced pluripotent stem cells (iPSCs) of children affected with LND to identify neural phenotypes of interest associated with HGPRT deficiency to develop a target-agnostic–based drug screening system. We screened more than 3000 molecules and identified 6 pharmacological compounds, all possessing an adenosine moiety, that corrected HGPRT deficiency–associated neuronal phenotypes by promoting metabolism compensations in an HGPRT-independent manner. This included S -adenosylmethionine, a compound that had already been used as a compassionate approach to ease the neuropsychiatric symptoms in LND. Interestingly, these compounds compensate abnormal metabolism in a manner complementary to the gold standard allopurinol and can be provided to patients with LND via simple food supplementation. This experimental paradigm can be easily adapted to other metabolic disorders affecting normal brain development and functioning in the absence of a relevant animal model.

Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.

One of the major obstacles to the identification of therapeutic interventions for central nervous system disorders has been the difficulty in studying the step-by-step progression of diseases in neuronal networks that are amenable to drug screening. Recent advances in the field of human pluripotent stem cell (PSC) biology offers the capability to create patient-specific human neurons with defined clinical profiles using reprogramming technology, which provides unprecedented opportunities for both the investigation of pathogenic mechanisms of brain disorders and the discovery of novel therapeutic strategies via drug screening. Many examples not only of the creation of human pluripotent stem cells as models of monogenic neurological disorders, but also of more challenging cases of complex multifactorial disorders now exist. Here, we review the state-of-the art brain cell types obtainable from PSCs and amenable to compound-screening formats. We then provide examples illustrating how these models contribute to the definition of new molecular or functional targets for drug discovery and to the design of novel pharmacological approaches for rare genetic disorders, as well as frequent neurodegenerative diseases and psychiatric disorders.

Several studies have outlined a non-cell-autonomous contribution in Huntington Disease (HD). Mutant-HTT (mut-HTT) expressed solely in astrocytes is sufficient for recapitulating the neurological and peripheral symptoms of HD in mice. Our objective is to identify molecules that can rescue cellular dysfunctions mediated by mut-HTT in astrocytes derived from human pluripotent stem cells (hPSC). Herein, we describe a scalable and reproducible protocol to differentiate HD-hPSC into astrocytes. The cellular identity of these hPSC-derived glial cells was characterized by qPCR and immunocytochemistry (GFAP, S100B, Connexin-43, GLT-1). Astrocytes are the cell type primarily responsible for rapid removal of glutamate from the extracellular space. This process is required for the survival and normal function of neurons. We therefore measure the capacity of the hPSC-derived glial cells we generated to glutamate uptake in a sodium-dependent manner. Assessment of previously described dysfunctions in HD-hPSC-derived astrocytes is ongoing. Next, we addressed the issue of the quantification in HD-hPSC neural derivatives of the level of mut-HTT. We adapted a mut-HTT-targeting AlphaLISA assay for use in 384-well plate format for high throughput screening. This assay is CAG dependent and can measure mut-HTT in a broad variety of human samples. We demonstrated the specificity of this assay using allele-specific silencing of the mut-HTT (lentivirus and siRNA) and comparing WT and HD-hPSC-derived samples. This assay displays a good signal-to-background ratio and is sensitive enough to reliably detect mut in as few as 5000 HD cells. The feasibility of performing drug screening on neuronal or glial cells derived from HD-hPSC using this assay is currently tested.