Autism-Related Mutations in the CEP290 Gene Alter Cell Signaling at the Primary Cilium.
Objectives: Here, we test the hypothesis that autism-associated mutations in CEP290 alter the function and molecular structure of primary cilium on cerebellar GCPs and ultimately affect their proliferation or differentiation during critical stages of brain development.
Methods: We employ live cell imaging, immunocytochemistry and molecular techniques to assess the changes in morphological, proliferative and cell signaling mechanisms caused by mutations in CEP290. Using the IncuCyte™ (Essen Bioscience) automated cell monitoring system we are able to perform detailed analysis on changes in proliferation and migration, as well as neurite formation and establishment in cell cultures expressing CEP290 mutant proteins.
Results: Overexpressed CEP290 wildtype as well as mutant proteins localize to the base of the primary cilium in NIH/3T3 cells and do not perturb ciliary formation. However, we find that cell proliferation rates are affected. When using a Shh response reporter, Glix8-EGFP, we observe that cells expressing CEP290 mutants show defects in response to Shh stimulation. Moreover, fluorescence recovery after photobleaching (FRAP) analysis of the mobility of proteins present in the primary cilium indicates that mutant CEP290 might play a role in disruption of the dynamics of the ciliary molecular signaling platform needed for proper Shh pathway activation.
Conclusions: Our present research suggest a link between autism-associated mutations in the CEP290 gene and abnormal ciliary protein dynamics and Shh signaling. Thus, our ongoing investigation will aim to provide novel insight on the function of the primary cilium in neurological conditions and in regulating brain development.