New research shows that TSC2 mutations may disrupt brain development at very early stages by changing the genome’s output through altered DNA methylation and gene regulation in neural progenitor cells. Using patient-derived stem cells, researchers found that TSC2-mutant cells developed abnormally early, showing premature neuronal activity, altered cell fate decisions, and widespread changes in DNA methylation patterns. These findings suggest that the neurological features of tuberous sclerosis complex (TSC) may begin much earlier in development than previously recognized and could have important implications for future prenatal treatment strategies.
Early Neurodevelopmental Changes in TSC2-Mutant Cells
Tuberous sclerosis complex (TSC) is a genetic developmental disorder caused by loss-of-function mutations in the TSC1 or TSC2 genes, leading to overactivation of the mechanistic Target of Rapamycin (mTOR) signaling pathway. TSC is characterized by benign tumors in multiple organs and neurological symptoms including seizures, autism, and intellectual disabilities.
To better understand how TSC2 mutations affect early brain development, researchers used patient-derived induced pluripotent stem cells (iPSCs) engineered with TSC2 mutations. Both heterozygous (TSC2 +/LOF) and homozygous (TSC2 LOF/LOF) mutant cells showed significant developmental abnormalities in laboratory-grown brain organoids.
- Altered Cell Fate Decisions: At day 9 of differentiation, TSC2-mutant neural progenitor cells showed reduced expression of neural progenitor markers, suggesting disrupted developmental timing and altered lineage trajectories. The mutant organoids were also larger than wild-type controls.
- Premature Neuronal Activity: By day 10, TSC2-mutant cultures prematurely expressed neuronal markers and displayed early electrical activity, indicating that neural progenitor cells were adopting neuronal features sooner than expected.
DNA Methylation and Gene Regulation Changes
The study found that these developmental abnormalities were accompanied by significant changes in DNA methylation, an important mechanism that regulates gene activity during brain development.
- Differentially Methylated Regions (DMRs): Researchers identified hundreds of altered methylation sites in TSC2-mutant cells, including regions associated with neurodevelopmental genes. One example was the KCNC3 gene, involved in neurotransmitter regulation, which showed reduced methylation in TSC2 LOF/LOF cells.
- Changes in Gene Expression: RNA sequencing revealed that altered methylation patterns corresponded with changes in gene activity. KCNC3 expression, for example, was significantly increased in mutant cells, suggesting that methylation changes directly contributed to abnormal gene regulation.
- Reduced DNMT1 Protein Levels: TSC2 LOF/LOF organoids also showed decreased levels of DNMT1, an enzyme responsible for maintaining DNA methylation. This finding provides further evidence linking TSC2 mutations to disruptions in epigenetic regulation.
Implications for Future Treatments
The findings suggest that TSC2 mutations begin altering brain development much earlier than previously understood, potentially during the neural progenitor stage. By identifying widespread DNA methylation changes as a driver of abnormal neurodevelopment, the study provides new insight into the biological mechanisms underlying TSC-related neurological symptoms.
These discoveries may help guide future therapeutic strategies, including the possibility of earlier or even prenatal interventions aimed at preventing or reducing abnormal brain development in individuals with TSC.
Senior authors: Rebecca Ihrie, PhD, Professor of Pediatrics and Neurology, University of Colorado Anschutz, Aurora, Colorado and Kevin Ess, MD, PhD. Professor of Pediatrics and Neurology, University of Colorado Anschutz, Aurora, Colorado. TSC Clinic Director, Children’s Hospital Colorado.
Email addresses: rebecca.ihrie@cuanschutz.edu kevin.ess@childrenscolorado.org
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