Increasing evidence has linked autism spectrum disorder (ASD) with dysfunction of brain cerebellum, but the details have been unclear. In a new study published in Molecular Psychiatry , at Boston Children's Hospital used stem cell technology to create cerebellum cells known as Purkinje cells from patients with tuberous sclerosis complex (TSC). The lab-grown cells showed several characteristics that may help explain how ASD develops at the molecular level.
TSC is associated with ASD in about half of all cases. Previous brain autopsies have shown that patients with TSC, as well as patients with ASD, have reduced numbers of Purkinje cells , the main type of neuron that communicates out of the cerebellum. In a 2012 mouse study, Sahin and colleagues knocked out TSC gene (Tsc1) in Purkinje cells and found social deficits and repetitive behaviors in the mice, together with abnormalities in the cells.
In the new study, Mustafa Sahin, director of the Translational Research Center at Boston Children's and colleagues, studied their Purkinje cells derived from three patients with TSC (two had ASD symptoms, and all three had epilepsy). "Developmentally, stem-cell derived neurons are close to a fetal state, recapitulating early differentiation of cells," says Maria Sundberg, first author of the study.
To make the cells, Sundberg first created induced pluripotent stem cells from patients' blood cells or skin cells, then differentiated these into neural progenitor cells and finally Purkinje cells . The team then compared them with Purkinje cells derived from unaffected people (parents or gender-matched controls) and with cells whose TSC mutation was corrected using CRISPR-Cas9 gene editing. "We saw changes," says Sahin. "The cells are bigger and fire less than control cells."
Purkinje cells with the TSC genetic defect were harder to differentiate from neural progenitor cells, suggesting that TSC may impair the early development of cerebellar tissue . On examination, the patient-derived Purkinje cells showed structural abnormalities in dendrites (the projections neurons used to take in signals) and signs of impaired development of synapses (junctions with other neurons).
The TSC Purkinje cells also showed over-activation of a cell growth pathway called mTOR. Accordingly, the team treated the cells with rapamycin , an inhibitory factor that is used clinically in TSC to reduce the size of TSC-related tumors and prevent TSC-related seizures. Added to patient-derived cells in culture, rapamycin enabled the development of more Purkinje precursor cells , improved the functioning of their synapses and increased their tendency to fire.
Finally, the researchers compared what genes were being "turned on" in Purkinje cells from TSC patients versus controls. Unexpectedly, the patient-derived cells showed reduced production of FMRP , a protein that is associated with Fragile X syndrome, a common genetic cause of ASD and intellectual disability, and also known to help regulate synapse function. The analysis also showed reduced production of two proteins important for neuron-to-neuron communication at synapses: synaptophysin and a glutamate receptor protein.
"Looking at cell-type-specific changes is very important," Sundberg says. "In TSC, we know that in different cell types, the mutation causes different effects."