A high-throughput screening platform to facilitate treatment development in Rett syndrome

Rett syndrome (RTT) is a rare X-linked progressive neurodevelopmental disorder affecting predominantly females with no cure and a prevalence of ~1 in 10,000 female birth worldwide. Before mutations in the methyl-CpG binding protein 2 (MECP2) gene were identified to cause classic RTT, there were suggestions that RTT is a mitochondrial disease. Being an essential organelle for all eukaryotic cells, the mitochondria produce energy, buffer calcium, and regulate the generation of reactive oxygen species. Indeed, accumulated reports documented mitochondrial abnormalities in RTT patient biopsies, and animal models and human stem cell models of RTT, including reduced ATP production, altered mitochondrial structure, increased systemic oxidative stress, abnormal calcium activity, mtDNA copy number, and deficiencies in mitochondrial enzyme activity. While it remains unclear how loss of MECP2 function leads to wide-ranging mitochondrial deficits, improving mitochondrial function could still bring benefits to RTT patients. After defining the mitochondrial membrane potential deficit in astrocytes differentiated from RTT patient-specific induced pluripotent stem cells (iPSC), we established a novel high-throughput screening (HTS) platform based on the JC-10 mitochondrial membrane potential (MMP) assay, which served as a rapid primary readout. All primary hits were subsequently validated by independent functional assays to confirm their effects on mitochondrial health. Using this system, we performed a small-molecule screening of 1,134 selected US Food and Drug Administration (FDA)-approved drugs and a small interfering RNA (siRNA) screening of 336 genes upregulated in RTT astrocytes and identified candidate drugs and candidate genes that reversed the MMP deficits in RTT astrocytes. Among the candidate drug hits, isradipine, a dihydropyridine calcium-channel blocker, provided preliminary evidence of neuroprotective effects both in vitro and in vivo. Among the candidate gene hits, LRRC17, a gene encoding a secreted protein, emerged as a strong candidate mediator whose elevated levels are strongly associated with and likely contribute to various observed cellular deficits. siRNA knockdown of LRRC17 not only rescued mitochondrial dysfunction in RTT astrocytes but also reversed deficits in neurons cultured in astrocyte-conditioned media. Our study provides new insights into mitochondrial dysfunction in RTT and establishes an HTS platform for the initial identification of novel therapeutic targets for follow-up studies.