Autism-Related Mutations in the CEP290 Gene Alter Cell Signaling at the Primary Cilium.

Friday, May 12, 2017: 5:00 PM-6:30 PM
Golden Gate Ballroom (Marriott Marquis Hotel)
M. Kilander and Y. C. Lin, Hussman Institute for Autism, Baltimore, MD
Background:  Whole exome sequencing has identified mutations in the centrosomal protein CEP290 in individuals with autism. This large protein is a crucial component in the formation and function of the primary cilium and thus is one of the main risk genes in ciliopathies, e.g. Joubert Syndrome, Meckel Syndrome and Bardet-Biedl Syndrome. Interestingly, defects in cerebellar development and autistic traits are occasionally observed in these ciliopathy patients, however, in combination with severe multi-organ dysfunctions phenotypes. The primary cilium is a microtubule rich cell protrusion important for cell proliferation, differentiation and migration. Moreover, the primary cilium serves as the confined compartment for selective cell signaling and for cell-environment communication. Sonic Hedgehog (Shh) signaling, a biological pathway necessary for proper tissue development and maintenance, is preferentially localized to the primary cilium and is essential for proliferation of granule cell progenitors (GCP) during cerebellar development. However, to date little is known about the role of the primary cilium in neurodevelopment and in the establishment of mature neural circuits. In addition, the role of CEP290 in regulating brain function is still unclear.

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 IncuCyteâ„¢ (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.