Clinical utility of neuronal cells directly converted from fibroblasts of patients for neuropsychiatric disorders: studies of lysosomal storage diseases and channelopathy

Abstract

Methodologies for generating functional neuronal cells directly from human fibroblasts [induced neuronal (iN) cells] have been recently developed, but the research so far has only focused on technical refinements or recapitulation of known pathological phenotypes. A critical question is whether this novel technology will contribute to elucidation of novel disease mechanisms or evaluation of therapeutic strategies. Here we have addressed this question by studying Tay-Sachs disease, a representative lysosomal storage disease, and Dravet syndrome, a form of severe myoclonic epilepsy in infancy, using human iN cells with feature of immature postmitotic glutamatergic neuronal cells. In Tay-Sachs disease, we have successfully characterized canonical neuronal pathology, massive accumulation of GM2 ganglioside, and demonstrated the suitability of this novel cell culture for future drug screening. In Dravet syndrome, we have identified a novel functional phenotype that was not suggested by studies of classical mouse models and human autopsied brains. Taken together, the present study demonstrates that human iN cells are useful for translational neuroscience research to explore novel disease mechanisms and evaluate therapeutic compounds. In the future, research using human iN cells with well-characterized genomic landscape can be integrated into multidisciplinary patient-oriented research on neuropsychiatric disorders to address novel disease mechanisms and evaluate therapeutic strategies.

Figure 1. GM2 ganglioside accumulation in iN cells from patients with Tay-Sachs disease

(A) Representative picture of GM2 ganglioside accumulation in iN cells derived from Tay-Sachs disease fibroblasts at day 20 after neuronal induction with lentivirus infection. Arrowhead indicates GM2 accumulation. (B) Quantification of MAP2-positive iN cells with GM2 accumulation per total MAP2-positive neuronal cells. Data are presented as mean ± S.E.M. of at least three independent experiments for each Tay-Sachs patient and control subject (14-23 neuronal cells per experiment). *p<0.05. (C) Representative pictures of GM2 ganglioside accumulation in MAP2-positive iN cells from a patient with Sandhoff disease. Arrowhead indicates GM2 accumulation. (D) Dose-dependent reduction of GM2 ganglioside accumulation in Tay-Sachs iN cells treated with N-Butyldeoxynojirimycin (NB-DNJ). Data are presented as mean ± S.E.M. of four independent experiments (16-24 neuronal cells per experiment). *p<0.05. Scale bars, 20 μm (A, C).

Figure 2. Altered patterns of action potential firings in iN cells from Dravet syndrome patients

(A) SCN1A staining in Dravet glutamatergic iN cells. (B) Representative traces of membrane potential changes induced by current injection in control and Dravet iN cells. Parameters for electrophysiological data analysis are shown to the right. Threshold was measured as the point of inflection leading to the rising phase of the action potential following the current stimulus. The difference in membrane potential between the threshold and the peak of the action potential was considered the amplitude. Duration was measured as the distance between the rising and the falling phases of the action potential at half the total amplitude. When present, afterhyperpolarization (AHP) amplitude was calculated as the difference between the threshold and the lowest point of the undershoot in the falling phase. Duration was measured as the width at half-amplitude. Rise time and decay time of action potentials were measured as the width from the threshold to half-amplitude and from the half-amplitude to the threshold, respectively. (C) No differences in action potential amplitude, threshold, afterhyperpolarization (AHP) amplitude, and decay time in Dravet iN cells compared to control iN cells. (D) Decrease in action potential durations and rise time in Dravet iN cells compared to control iN cells. **p<0.01, *p<0.05. (E) Effects of wild-type SCN1A overexpression on action potential rise time in Dravet iN cells. **p<0.01, *p<0.05. Scale bars, 20 μm (A). For C and D, data are presented as mean ± S.D. of at least four experiments per subject.