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Signalling Adapter Expression Boosts Induced Neuron Functionality

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Review of “Signaling Adaptor Protein SH2B1 Enhances Neurite Outgrowth and Accelerates the Maturation of Human Induced Neurons” from Stem Cell Translational Medicine by Stuart P. Atkinson.

Direct reprogramming strategies aim to produce therapeutically relevant patient-specific cell types from somatic cells without first passing through the induced pluripotent stem cell (iPSC) stage. Previous studies have demonstrated the direct conversion of human cells to induced neurons (iNs) [1-5], although thus far each study has reported some level of functional deficit. To address these problems, a team lead by Ing-Ming Chiu from the National Health Research Institutes, Taiwan, have built on their work on the signaling adaptor protein SH2B1 which can enhance fibroblast growth factor 1-induced neurite outgrowth by modulation of the MEK-ERK1/2-STAT3-Egr1 pathway [6]. They now report on their study into the role for SH2B1, alongside miR124, BRN2, and MYT1L (S-IBM), in the enhancement of the conversion of human fibroblasts to functional neurons [7].

The group first reprogrammed Dox-inducible SH2B1-GFP human foetal fibroblasts (HFFs) towards iNs using IBM in the presence or absence of SH2B1 expression (S-IBM or IBM) (see Figure). 14 days after induction, S-IBM cells manifested typical neuronal morphology, with double the level of neurite outgrowth, and significantly more neuronal processes and branches compared to IBM-infected cells. This characteristic neural morphology was even more obvious at 28 days in the S-IBM cells, suggesting that SH2B1 may boost differentiation speed, or enhance maturation.

Further characterisation of S-IBM iNs found that 61% expressed the neurotransmitter GABA, 41% expressed Synapsin and they expressed significantly higher levels of neuronal markers (TuJ1, NeuN, and TH) than IBM iNs cells. The researchers noted spontaneous firing of action potentials in S-IBM iNs at day 14 with high peak amplitudes (+5268 mV) compared to IBM iNs, where only one of 4 cultures fired action potentials and at a lower amplitude (+10 mV). Analysis of stimulus-evoked action potentials found repetitive firings in the S-IBM iN populations at day 14, but only a single evoked spike in the IBM iNs, although the group did observed repetitive firings in both cultures by day 21, both spontaneously and stimulus-evoked. Resting membrane potential, measured by whole-cell current-clamp mode, was significantly different between the two groups, with a more negative resting membrane potential in S-IBM iNs (24569 mV) than in IBM iNs (22264 mV) at day 14, although by day 21 and 28 both iNs had membrane potentials close to that observed for regular neurons (~265 mV).

Overall, these authors have shown that expression of the signalling adapter protein SH2B1 enhances the generation of iNs by boosting reprogramming, enhancing neurite growth and shortening the neuronal maturation period. The authors posit that SH2B1 may promote FGF- [6] and GDNF-induced neurite outgrowth [8], factors which are both contained in the iN-defined culture media, although further work is required to fully delineate any such mechanism. This finding should prove useful to efforts to reprogram refractory somatic cell samples from patients with specific disease-related mutations, allowing them to be used for personalized regenerative medicine, drug screening or in vitro disease modeling.

References

  1. Ambasudhan R, Talantova M, Coleman R, et al. Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. Cell Stem Cell 2011;9:113-118.
  2. Caiazzo M, Dell'Anno MT, Dvoretskova E, et al. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 2011;476:224-227.
  3. Liu X, Li F, Stubblefield EA, et al. Direct reprogramming of human fibroblasts into dopaminergic neuron-like cells. Cell Res 2012;22:321-332.
  4. Marro S, Pang ZP, Yang N, et al. Direct lineage conversion of terminally differentiated hepatocytes to functional neurons. Cell Stem Cell 2011;9:374-382.
  5. Pang ZP, Yang N, Vierbuchen T, et al. Induction of human neuronal cells by defined transcription factors. Nature 2011;476:220-223.
  6. Lin WF, Chen CJ, Chang YJ, et al. SH2B1beta enhances fibroblast growth factor 1 (FGF1)-induced neurite outgrowth through MEK-ERK1/2-STAT3-Egr1 pathway. Cell Signal 2009;21:1060-1072.
  7. Hsu YC, Chen SL, Wang YJ, et al. Signaling Adaptor Protein SH2B1 Enhances Neurite Outgrowth and Accelerates the Maturation of Human Induced Neurons. Stem Cells Transl Med 2014;
  8. Zhang Y, Zhu W, Wang YG, et al. Interaction of SH2-Bbeta with RET is involved in signaling of GDNF-induced neurite outgrowth. J Cell Sci 2006;119:1666-1676.