Autism spectrum disorder (ASD) is increasingly linked with dysfunction of the cerebellum, but the details, to date, have been murky. Now, a rare genetic syndrome known as tuberous sclerosis complex (TSC) is providing a glimpse.
TSC includes features of ASD in about half of all cases. Previous brain autopsies have shown that patients with TSC, as well as patients with ASD in general, have reduced numbers of Purkinje cells, the main type of neuron that communicates out of the cerebellum.
In a 2012 mouse study, team led by Mustafa Sahin, MD, at Boston Children’s Hospital, knocked out a TSC gene (Tsc1) in Purkinje cells. They found social deficits and repetitive behaviors in the mice, together with abnormalities in the cells.
The new study, published last week in Molecular Psychiatry, takes the research into human cells, for the first time creating cerebellar cells known as Purkinje cells from patients with TSC.
Led by first author Maria Sundberg, PhD, in Sahin’s lab, the team first created induced pluripotent stem cells from blood cells or skin cells taken from three patients. They then differentiated these into neural progenitor cells and finally Purkinje cells, analogous to what happens during early brain formation.
“Developmentally, stem-cell derived neurons are close to a fetal state, recapitulating early differentiation of cells,” Sundberg explains.
Purkinje cell abnormalities
The cells showed several differences when compared with Purkinje cells derived from unaffected people and with cells whose TSC mutation was corrected using CRISPR-Cas9 gene editing. These differences may help explain how ASD develops at the molecular level.
Right off the bat, cells with the TSC gene defect were harder to differentiate from neural progenitor cells, suggesting that TSC may impair the early development of cerebellar tissue.
The diseased cells also had structural abnormalities in dendrites (the projections neurons use to take in signals) and signs of impaired development of synapses (junctions with other neurons).
“The cells are bigger and fire less than control cells — exactly what we see in the mouse model,” adds Sahin, who directs the Translational Neuroscience Center at Boston Children’s.
At the molecular level, the cells showed over-activation of a cell growth pathway known as mTOR. Accordingly, the team treated the cells with rapamycin, an mTOR inhibitor that is already used clinically to shrink TSC-related tumors and prevent TSC-related seizures. The added rapamycin enabled more Purkinje precursor cells to develop, improved the functioning of their synapses and increased their tendency to fire.
Commonalities with Fragile X syndrome?
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. FMRP is known to help regulate synapse function, so it may contribute to the abnormalities in Purkinje cell functioning in TSC.
“These conditions may have a common downstream pathway,” says Sahin.
The analysis also showed reduced production of two proteins important for neuron-to-neuron communication at synapses: synaptophysin and a glutamate receptor protein.
A platform for studying autism
The study was the first to create human Purkinje cells using TSC patients’ own cells. In future studies, Sahin and colleagues hope to generate these cells in larger numbers to compare those derived from patients with TSC alone with those who also have ASD. They also hope to use the Purkinje cell platform to study other genetic disorders with ASD features, including Fragile X and SHANK3 mutation, and to test potential drugs.
Sundberg also plans to create other types of neurons for modeling ASD. “Looking at cell-type-specific changes is very important,” she says. “In TSC, we know that in different cell types, the mutation causes different effects.”
The fine print
The study was supported by U.S. Army Medical Research Tuberous Sclerosis Complex Research Program (W81XWH-15-1-0189), Nancy Lurie Marks Family Foundation, the Harvard Stem Cell Institute, and the Children’s Hospital Boston Translational Research Program, the National Institutes of Health (R25 NS07068207S1, UL1 TR000043, R21 NS093540-01), the Iris and Jumming Le Foundation and the Rockefeller University Center for Clinical and Translational Science.
Ivan Tochitsky (Boston Children’s) and David Buchholz (The Rockefeller University, New York) were co-second authors on the paper. Other authors were Ville Kujala of the Harvard John A. Paulson School of Engineering and Applied Sciences; Kellen Winden, Kush Kapur, Deniz Cataltepe, Daria Turner, Min-Joon Han and Clifford J. Woolf of Boston Children’s; and Mary E. Hatten (Rockefeller University). Sahin and Sundberg are also affiliated with the Harvard Stem Cell Institute.