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. 2023 Nov:131:182-195.
doi: 10.1016/j.neurobiolaging.2023.07.007. Epub 2023 Jul 17.

An Alzheimer's disease risk variant in TTC3 modifies the actin cytoskeleton organization and the PI3K-Akt signaling pathway in iPSC-derived forebrain neurons

Holly N Cukier  1 , Carolina L Duarte  2 , Mayra J Laverde-Paz  2 , Shaina A Simon  2 , Derek J Van Booven  2 , Amanda T Miyares  3 , Patrice L Whitehead  2 , Kara L Hamilton-Nelson  2 , Larry D Adams  2 , Regina M Carney  4 , Michael L Cuccaro  5 , Jeffery M Vance  1 , Margaret A Pericak-Vance  1 , Anthony J Griswold  5 , Derek M Dykxhoorn  6
Affiliations

An Alzheimer's disease risk variant in TTC3 modifies the actin cytoskeleton organization and the PI3K-Akt signaling pathway in iPSC-derived forebrain neurons

Holly N Cukier et al. Neurobiol Aging. 2023 Nov.

Abstract

A missense variant in the tetratricopeptide repeat domain 3 (TTC3) gene (rs377155188, p.S1038C, NM_003316.4:c 0.3113C>G) was found to segregate with disease in a multigenerational family with late-onset Alzheimer's disease. This variant was introduced into induced pluripotent stem cells (iPSCs) derived from a cognitively intact individual using CRISPR genome editing, and the resulting isogenic pair of iPSC lines was differentiated into cortical neurons. Transcriptome analysis showed an enrichment for genes involved in axon guidance, regulation of actin cytoskeleton, and GABAergic synapse. Functional analysis showed that the TTC3 p.S1038C iPSC-derived neuronal progenitor cells had altered 3-dimensional morphology and increased migration, while the corresponding neurons had longer neurites, increased branch points, and altered expression levels of synaptic proteins. Pharmacological treatment with small molecules that target the actin cytoskeleton could revert many of these cellular phenotypes, suggesting a central role for actin in mediating the cellular phenotypes associated with the TTC3 p.S1038C variant.

Keywords: Alzheimer’s disease (AD); Induced pluripotent stem cells (iPSCs); Tetratricopeptide repeat domain 3 (TTC3) gene.

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Conflict of interest statement

Disclosure statement The authors report no conflict of interest.

Figures

Figure 1.
Figure 1.. Transcriptional analysis of day 70 neurons.
A. Volcano plot of the 381 upregulated and 597 down regulated genes that were significant between the neurons with and without the TTC3 alteration. Genes marked in red have a FDR < 0.05 and FC +/−1.25. B. Heat map of the 978 significantly differentially expressed genes with a false discovery rate (FDR) < 0.05 and fold change (FC) +/−1.25 C. KEGG pathway analysis showing cellular functions overrepresented in the DEGs from the parental compared to TTC3 p.S1038C derived neurons.
Figure 2.
Figure 2.. Neuronal Precursor Cell Migration and Cytoskeleton Assessment
A-C. Scratch wound assay on D30 neuron precursor cells recording cell migration for three days. Migration rate was measured as the wound area relative to the spatial cell density outside of the wound (Wound Density) and the confluence of cells within the wound region (Wound Confluence). Scale Bars: 300 μm. D-E. Endpoint tracking of migrating cells using the μ-Slide Chemotaxis assay. The start point for each tracked cell is located in the center of the diagram and the vectorial displacement is delineated in the direction of movement. The Y-axis represents movement along the chemoattractant gradient, which is higher towards the top of the diagram, and the X-axis represents movement perpendicular to the chemoattractant gradient. F. Chemotaxis analysis. G. Representative images of D30 neurons. Blue: Nuclear counterstain DAPI, Green: Actin filament stain Phalloidin, Scale Bars: 125 μm. H. Immunofluorescent assessment of actin filament organization and cytoskeleton pattern analysis of D30 neurons. Ch1: Phalloidin. *: p-value<0.05, **: p-value<0.01, ***: p-value<0.001, ****: p-value<0.0001
Figure 3.
Figure 3.. iPSC-Derived Neuron Cytoskeleton Assessment and Neurite Formation
A. Representative images of D70 neurons. Blue: Nuclear counterstain DAPI, Green: Actin filament stain Phalloidin, Red: β-tubulin 3 (TUBB3). Scale Bars: 125 μm C-F. Immunofluorescent assessment of actin filament organization and cytoskeleton pattern analysis of D70 neurons Ch1: Phalloidin, Ch3: TUBB3. C-E. Representative images and quantification of neurite formation recorded from D30 to D35 and presented as neurite length per surface area. The intersection of two masked neurites in an image is presented as branch points/cell body cluster count. Scale Bars: 150 μm. *: p-value<0.05, **: p-value<0.01, ***: p-value<0.001, ****: p-value<0.0001
Figure 4.
Figure 4.. Markers of Neuronal Differentiation and Synaptic Vesicle Formation
A-C. Immunofluorescent detection of markers of neuronal differentiation on D70 neurons. Scale Bars A & C: 10 μm. Scale Bars B: 20 μm. D-G. Western blot analysis and quantification of the expression of β-tubulin 3 and synaptic vesicle markers Synapsin 1 and Synaptophysin. β-tubulin 3 (TUBB3), Microtubule Associated Protein 2 (MAP2), Synapsin 1 (SYN1) and Synaptophysin (SYP). *: p-value<0.05, **: p-value<0.01, ***: p-value<0.001, ****: p-value<0.0001
Figure 5.
Figure 5.. Cytoskeleton Rearrangement Assay of Neuronal Precursor Cells and iPSC-Derived Neurons
A. Representative images of D30 neuron progenitor cells. Blue: Nuclear counterstain DAPI, Green: Actin filament stain Phalloidin. Scale Bars: 125 μm B. Representative images of D70 neurons. Blue: Nuclear counterstain DAPI, Green: Actin filament stain Phalloidin, Red: β-tubulin 3. Scale Bars: 125 μm C-D. Comparison of cytoskeleton characteristics that were significantly different between D30 (C-D) and D70 (E-F) TTC3 pS1038C cells and unedited cells after treatment with Cytochalasin D (CytoD), Jasplakinolide (Jaspla) and Y27632. Ch1: Phalloidin, Ch3: β-tubulin 3. *: p-value<0.05, ***: p-value<0.001.
Figure 6.
Figure 6.. Migration and Neurite Formation of TTC3 pS1038C Bearing Neuronal Precursor Cells after Treatment with Actin Disrupting Agents
A-E. Scratch wound assay on D30 neuron precursor cells recording cell migration 24 hours before and 24 hours after treatment with Cytochalasin D (CytoD), Jasplakinolide (Jaspla) and Y27632. Migration rate was measured as the wound area relative to the spatial cell density outside of the wound (Wound Density) throughout the duration of the experiment (C-D) and at endpoint (E). Scale Bars: 300 μm F-H. Representative images and quantification of neurite formation after treatment with Cytochalasin D (CytoD), Jasplakinolide (Jaspla) and Y27632, recorded from D30 to D35 and presented as neurite length per surface area. The intersection of two masked neurites in an image presented as branch points/cell body cluster count. Scale Bars: 150 μm. *: p-value<0.05, **: p-value<0.01, ****: p-value<0.0001.
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