Abstract
GABAergic neuronal cell grafting has promise for treating a multitude of neurological disorders including epilepsy, age-related memory dysfunction, Alzheimer’s disease and schizophrenia. However, identification of an unlimited source of GABAergic cells is critical for advancing such therapies. Our previous study implied that reprogramming of bone marrow-derived mesenchymal stem cells (BMSCs) through overexpression of the Achaete-scute homolog 1 (Ascl1, also called Mash1) could generate GABAergic neuron-like cells. Here, we investigated mechanisms underlying the conversion of BMSCs into GABAergic cells. We inhibited γ-secretase (an enzyme that activates Notch signaling) with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) or manipulated the expression of Notch signaling components such as the recombination signal binding protein for immunoglobulin kappa J region (RBPJ), hairy and enhancer of split-1 (Hes1) or Mash1. We demonstrate that inhibition of γ-secretase through DAPT down-regulates RBPJ and Hes1, up-regulates Mash1 and results in an enhanced differentiation of BMSCs into GABAergic cells. On the other hand, RBPJ knockdown in BMSCs has no effect on Mash1 gene expression whereas Hes1 knockdown increases the expression of Mash1. Transduction of Mash1 in BMSCs also increases the expression of Hes1 but not RBPJ. Moreover, increased GABAergic differentiation in BMSCs occurs with concurrent Mash1 overexpression and Hes1-silencing. Thus, the Mash1-dependent Notch signaling pathway regulates GABAergic neuron-like differentiation of BMSCs. These results also suggest that genetic engineering of BMSCs is a useful avenue for obtaining GABAergic neuron-like donor cells for the treatment of neurological disorders.
Highlights
Gamma-amino butyric acid (GABA) is a chief inhibitory neurotransmitter that plays a principal role in reducing the neuronal excitability and synchronizing the firing of projection neuron ensembles in the mammalian central nervous system (CNS)
After bone marrow-derived mesenchymal stem cells (BMSCs) were treated with DAPT, Western blotting results (Fig. 2A) showed that the protein level of Notch signaling target gene – hairy and enhancer of split-1 (Hes1) and its effector – RBPJ were decreased (n=5/group, P < 0.01, vs. BMSCs), but the expression of Mash1 was increased in comparison to non-treated BMSCs (n=5/group, *P = 0.032, < 0.05; Fig. 2B). qRTPCR further confirmed that DAPT treatment inhibited the mRNA expression of Hes1 and RBPJ (n=4/group, P < 0.01, vs. BMSCs), in addition to inducing an upregulation of Mash1 mRNA expression in BMSCs (n=4/group, *P = 0.027, < 0.05; Fig. 3A)
To elucidate the regulatory role of Notch signaling in BMSCs, an RBPJ shRNA, Hes1 shRNA, or pGC-FU
Summary
Gamma-amino butyric acid (GABA) is a chief inhibitory neurotransmitter that plays a principal role in reducing the neuronal excitability and synchronizing the firing of projection neuron ensembles in the mammalian central nervous system (CNS). Grafting of GABAergic precursor cells into the hippocampus in a mouse model of temporal lobe epilepsy reduced the extent of spontaneous seizures and abnormal behavior [3]; transplantation of GABAergic cells into the spinal cord reversed mechanical hypersensitivity in a neuropathic pain model [4]; and introduction of new GABAergic cells into the limbic cortex in a hippocampal disinhibition model reversed psychosis-relevant features [6] Despite these exciting results, the clinical application of GABAergic precursor cell therapy is hampered by both difficulties in obtaining a sufficient amount of donor cells and ethical issues associated with the use of GABAergic precursor cells from fetal brains. In this study, we assessed which components of the Notch signaling pathway (RBPJ-Hes1Mash1) regulate the GABAergic neuron-like differentiation of BMSCs in vitro
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