PROJECT SUMMARY
Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by an epigenetic silencing of the X-
linked FMR1 gene, which leads to loss of its protein product fragile X messenger ribonucleoprotein (FMRP).
Currently, FXS is the largest single-gene contributor to autism affecting about 1 in 4,000 males and 1 in 7,000
females in the U.S. Despite FXS being a monogenic disorder, FMRP controls many aspects of
neurodevelopment leading FXS patients to present a wide range of symptoms that include intellectual disability,
language impairment, anxiety, hyperactivity, and aggression. Many studies investigating the molecular
mechanisms affected in FXS have been performed in Fmr1 knockout (KO) rodent models and drosophila.
However, many of the treatments that proved to be successful in animal models failed in human clinical trials,
leaving a large unmet need for treatment targeting FXS. Human induced pluripotent stem cells (iPSCs) have
been a useful model in the study of neurodevelopmental disorders and studies have shown that FXS iPSC
derived neurons have defects in neuronal maturation and differentiation. Recently, 3-dimensional cortical
organoids (3DOs) derived from iPSCs have become an increasingly promising tool for the study of human
neurodevelopment because they resemble human brain formation and can be cultured for more than one year.
However, a study examining the effect of FMRP deficiency throughout development has not been done.
Therefore, I propose to study cortical development, electrical activity, and genetic signatures using 3DOs derived
from FMR1 deficient iPSC lines at different developmental stages. My preliminary data already shows increased
cell proliferation in FXS patient-derived 3DOs, which corresponds to previous findings in FXS neural progenitor
cells (NPCs), and hyperexcitability, which is consistent with our previous studies in FXS 2D neurons. I have also
found upregulation of a putative FMRP target SPTBN1 (Spectrin Beta, Non-Erythrocytic 1) across three
developmental stages. I hypothesize that FMRP deficiency leads to dysregulation of SPTBN1, a gene important
for neuronal maturation, which results in disrupted development of FXS cortical organoids. To test this
hypothesis, in the first aim I propose to identify developmental deficits in FXS 3DOs by performing
immunohistochemistry of developmental markers in early, middle, and late developmental time-points along with
the analysis of electrical activity using calcium imaging. In the second aim, I propose to determine whether and
how FMRP regulates SPTBN1 and investigate how dysregulation of SPTBN1 may contribute to developmental
deficits that we have found in FXS 3DOs. I also plan to confirm these previous results and identify novel
differentially expressed genes through single nucelus RNA sequencing (snRNAseq) at different developmental
time points. Overall, this project will further the understanding of the developmental and molecular mechanisms
disrupted in FXS patients throughout development and lead to better-targeted treatments.