DOI: 10.1002/alz.076331 ISSN: 1552-5260

A risk variant in TTC3 modifies the actin cytoskeleton organization and PI3K‐Akt signaling in iPSC‐derived forebrain neurons

Holly N. Cukier, Carolina L. Duarte, Juliana Laverde‐Paz, Shaina A. Simon, Derek Van Booven, Amanda T. Miyares, Jeffery M. Vance, Margaret A. Pericak‐Vance, Anthony J. Griswold, Derek M. Dykxhoorn
  • Psychiatry and Mental health
  • Cellular and Molecular Neuroscience
  • Geriatrics and Gerontology
  • Neurology (clinical)
  • Developmental Neuroscience
  • Health Policy
  • Epidemiology



We identified a rare, nonsynonymous variant in the tetratricopeptide repeat domain 3 (TTC3) gene that segregated in a non‐Hispanic white late onset Alzheimer disease (LOAD) family (Kohli, et al, 2016). This missense alteration, rs377155188 (p.S1038C), is predicted to be deleterious and is extremely rare. Studies have reported that cortical TTC3 expression is reduced in LOAD patients and negatively correlated with AD neuropathology.


To understand the mechanism by which the TTC3 p.S1038C may contribute to LOAD risk, CRISPR/Cas9 genome edited induced pluripotent stem cells (iPSCs) were developed that were homozygous for the variant to examine cellular and transcriptional consequences in iPSC‐derived neuronal cells (Laverde‐Paz, et al, 2021).


Quantitative PCR and western blot analysis demonstrated that TTC3 levels were decreased in edited compared to unedited iPSCs, as well as differentiated neurons. In growing neuronal precursor cells (NPCs), cells with the TTC3 variant recovered more quickly from a scratch wound. Since there is evidence that modulation of TTC3 affects neurite growth, morphological measures of axon formation were assessed using the Incucyte Zoom. Studies demonstrate an increase in neurite outgrowth, which phenotypically corresponds with previous studies of a decrease in TTC3 function. This phenotype was tempered by treatment with Cytochalasin D, an inhibitor of actin polymerization. Additionally, TTC3 ubiquitinates phosphorylated AKT and regulates AKT signaling. The edited cells were found to have an increase in phosphorylated AKT (pAKT) relative to total AKT. RNA‐seq of day 70 neurons identified 979 genes that were differentially expressed (FDR<0.05). This included known AD genes (BACE1) and genes in AD GWAS loci (ADAMTS1, MAF, NCK2). KEGG pathway analysis identified differential expression in PI3K‐Akt signaling pathway components, in which TTC3 has been previously implicated, as well as the axon guidance pathway, the GABAergic synapse pathway, and the Wnt signaling pathway.


Combined, these results suggest that the TTC3 p.S1038C variant causes a loss of function. Utilizing a CRISPR genome edited iPSC carrying a homozygous alteration in TTC3, we were able to identify potential mechanisms by which TTC3 may contribute to LOAD risk.

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