UC San Diego

Rett syndrome (RTT) is a X-linked neurodevelopmental disorder caused by mutations in the gene encoding the transcriptional regulator methyl CpG binding protein 2 (MeCP2). It is not fully understood whether the genes regulated by MeCP2 contribute to the neuronal RTT phenotype. Recently, we identified Neurotrypsin as a gene repressed by MeCP2. Neurotrypsin is a serine protease, which may play a synaptic role by processing the synaptic regulator agrin. Altered levels of neurotrypsin's proteolytic activity lead to major synaptic and cognitive defects. Given neurotrypsin's synaptic role, MeCP2's regulatory function on Neurotrypsin expression, and MeCP2 dysfunction's involvement in causing RTT, we wondered whether Neurotrypsin expression is altered in RTT. To start addressing this question, we used two different clones of human induced pluripotent stem cell (hiPSC)- derived neural stem cells (NSCs) from a male RTT patient carrying a nonsense MECP2 mutation (RTT-Q83X). RTT-Q83X NSCs showed no detectable MeCP2. Basal levels of Neurotrypsin expression were variable between the RTT-Q83X NSC clones; however, both clones exhibited increased Neurotrypsin expression, relative to control cells, during differentiation. Additionally, fluorescence-activated cell sorting (FACS)-purified RTT-Q83X neurons also exhibited Neurotrypsin upregulation. To mimic the RTT-Q83X neurons, we knocked-down MeCP2 in FACS-purified control neurons. Surprisingly, Neurotrypsin was downregulated, suggesting a more complex regulatory mechanism for Neurotrypsin expression than anticipated. In conclusion, we found Neurotrypsin upregulation in neurons derived from a RTT patient. This could potentially lead to increased levels of neurotrypsin and neurotrypsin-dependent cleavage of agrin, which might contribute to the synaptic defects observed in RTT-derived neurons and ultimately the disease