We have identified a stromal cell-derived inducing activity (SDIA) that promotes neural differentiation of mouse ES cells. SDIA accumulates on the surface of PA6 stromal cells and induces efficient neuronal differentiation of cocultured ES cells in serum-free conditions without use of either retinoic acid or embryoid bodies. BMP4, which acts as an antineuralizing morphogen in Xenopus, suppresses SDIA-induced neuralization and promotes epidermal differentiation. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained from SDIA-treated ES cells. When transplanted, SDIA-induced dopaminergic neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase expression. Neural induction by SDIA provides a new powerful tool for both basic neuroscience research and therapeutic applications.
The role of inhibitory Golgi cells in cerebellar function was investigated by selectively ablating Golgi cells expressing human interleukin-2 receptor alpha subunit in transgenic mice, using the immunotoxin-mediated cell targeting technique. Golgi cell disruption caused severe acute motor disorders. These mice showed gradual recovery but retained a continuing inability to perform compound movements. Optical and electrical recordings combined with immunocytological analysis indicated that elimination of Golgi cells not only reduces GABA-mediated inhibition but also attenuates functional NMDA receptors in granule cells. These results demonstrate that synaptic integration involving both GABA inhibition and NMDA receptor activation is essential for compound motor coordination. Furthermore, this integration can adapt after Golgi cell elimination so as not to evoke overexcitation by the reduction of NMDA receptors.
Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein () or repeat expansions in Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS.
The physiological role of striatal cholinergic interneurons was investigated with immunotoxin-mediated cell targeting (IMCT). Unilateral cholinergic cell ablation caused an acute abnormal turning behavior. These mice showed gradual recovery but displayed abnormal turning by both excess stimulation and inhibition of dopamine actions. In the acute phase, basal ganglia function was shifted to a hyperactive state by stimulation and suppression of striatonigral and striatopallidal neurons, respectively. D1 and D2 dopamine receptors were then down-regulated, relieving dopamine-predominant synaptic perturbation but leaving a defect in controlling dopamine responses. The acetylcholine-dopamine interaction is concertedly and adaptively regulated for basal ganglia synaptic integration.
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