Actin cytoskeleton dynamics in stem cells from autistic individuals

Abstract

Several lines of indirect evidence, such as mutations or dysregulated expression of genes related to cytoskeleton, have suggested that cytoskeletal dynamics, a process essential for axons and dendrites development, is compromised in autism spectrum disorders (ASD). However, no study has yet examined whether cytoskeleton dynamics is functionally altered in cells from ASD patients. Here we investigated the regulation of actin cytoskeleton dynamics in stem cells from human exfoliated deciduous teeth (SHEDs) of 13 ASD patients and 8 control individuals by inducing actin filament depolymerization and then measuing their reconstruction upon activation of the RhoGTPases Rac, Cdc42 or RhoA. We observed that stem cells from seven ASD individuals (53%) presented altered dymanics of filament reconstruction, including a patient recently studied by our group whose iPSC-derived neuronal cells show shorten and less arborized neurites. We also report potentially pathogenic genetic variants that might be related to the alterations in actin repolymerization dynamics observed in some patient-derived cells. Our results suggest that, at least for a subgroup of ASD patients, the dynamics of actin polymerization is impaired, which might be ultimately leading to neuronal abnormalities.

Figure 1

Dynamics of actin re-polymerization on SHEDs. At left, representative images of cells with depolymerized actin filaments right after incubation with ROCKi (time 0 h) at 20x magnification (A), for the control sample F5541 (B) and for the ASD sample F7511 (C). At right, representative images of cells with re-polymerized filaments after 60 minutes of Cdc42 activation treatment at 20x magnification (D), for the control sample F5541 (E), and for the ASD sample F7511 (F). It is possible to notice much more cells with a large cell body due to stress fibers formation in the control sample (E), whereas in patient, there are much more cells with a thin cell body with no stress fiber formation (F). Scale bar: A and D: 50um; B,C, E and F: 200um.

Figure 2

Adjusted models for actin re-polymerization dynamics. Graphs show estimated average of the percentage of cells with reconstituted actin filaments along the time and confidence interval for control (C) samples (blue), all ASD samples (red), ASD samples allocated in group 1 by clustering analysis (purple) or in group 2 (orange) for each treatment. In upper left, it is indicated the p-values obtained by Generalized Additive Model for Location Scale and Shape analysis for each comparison.

Figure 3

Predicted molecular alterations in actin polymerization regulatory pathway. (A–C) Schematic representation of possible responses upon treatment with the different activators, depending on the location of a molecular alteration. Red crosses symbolize alteration in any molecule represented inside the balloon. Red arrows indicate abnormal regulation from that point of the pathway to the next, while green arrows, represent normal regulation. (D) Summary of the alterations found for each of the ASD individuals that presented abnormal regulation of actin reconstitution in any of the treatments and the predicted alteration in the regulatory pathway accordingly to the model described above.

Figure 4

RhoGTPases protein expression. (A) Western-blots showing protein expression of RhoA, Rac1/2/3, Cdc42 and b-actin in SHEDs from ASD individuals whose functional assay results indicated an alteration in RhoGTPases or downstream of them (F2688-1 and F2749-1), plus 3 control samples and 3 other ASD samples. (B) Average of the relative expression of the RhoGTPases in controls, ASD samples and F2749-1 (RhoGTPases protein levels were normalized by b-actin levels and then normalized to the control sample F7647-1).