“Disappointing” results reveal potential neural repair approach ineffective

Microglia are the central nervous system’s main line of defense: immune cells that tend to and dispose of harmful waste, damaged neurons, the invasive pathogens that manage to cross the blood-brain barrier and more. While neurons have limited regenerative capacity, microglia can repopulate to their original numbers even after significant injury — making them a potential ideal source candidate for experimentally inducing neuronal growth. In 2019, researchers in Japan published breakthrough results in Neuron, detailing how NeuroD1, a protein involved in cell differentiation, could coax microglia into new neurons.

Now, researchers in China have found that not only does NeuroD1 not induce microglia-to-neuron conversion, but also that the protein induces microglia death. They published their results on Dec 6^th in Neuron.

The team, led by Bo Peng, professor in the Institute for Translational Brain Research at Fudan University, sent out to investigate the molecular mechanisms underpinning the original finding, since microglia and neurons descend from different cellular lineages. 

“This finding was unexpected and hotly debated, since the extent to which NeroD1 can exert cross-lineage reprograming of microglia, from a myeloid lineage, to neurons, a neuroectodermal lineage, was unclear,” Peng said. “In this study, we unexpectedly found that NeuroD1 cannot convert microglia to neurons. Instead, NeuroD1 induces microglial cell death.”

The researchers applied a rigid lineage tracing protocol to follow the cellular differentiation progression in mice, as well as to monitor the effect of lentiviral vectors — an inert virus package used to carry NeuroD1 to the central nervous system — on the process. They validated their observations through live cell imaging and pharmacological approaches.

“Disappointingly, our results do not support the ‘microglia-to-neuron conversion,’” Peng said. “Instead, our data strongly indicate that the previously observed conversion was actually due to the experimental artifacts from viral leakage.”

The assumed finding, Peng said, was likely due to NeuroD1’s actual role: triggering microglial cell death. Neurons are unaffected by NeuroD1 so their numbers will stay the same, while microglia cell numbers decrease. However, due to the low purity of the microglia and the viral leakage, it could appear that while microglia cells were decreasing, non-microglia cells were increasing, leading to the conclusion in vitro that microglia were converting to neurons.

“All great and astonishing findings must be demonstrated by utmost stringent evidence,” Peng said. “The ‘microglia-to-neuron’ conversion should be verified following three principles: 1) unambiguous microglial-based lineage tracing and lack of lentiviral leakage, along with well-designed controls; 2) unambiguous live cell imaging to show how an individual microglial cell converts to a neuron; and 3) upon microglial depletion, there should be no or few microglia-converted neurons.”

The last point, Peng explained, appeared to be supported in the original paper, but when his team replicated the experiment, they found that even when 98.9% of microglia cells were killed, numerous “microglia-converted neurons” were still observed. Such a finding suggests that the converted neurons were mislabeled cells rather than the desired neurons.   

While NeuroD1 may not facilitate microglia-to-neuron conversion, according to Peng, it may be a helpful tool in microglia transplantation. In some illnesses and injuries, microglia cells may become less efficient or die off, but they can be replaced with donor microglia cells. However, it can become a problem if the transplanted microglia overpopulate or if the body reacts poorly to them.

“We propose that NeuroD1, which induced microglial death, can serve as a ‘switch-off’ control if transplanted microglia control is lost or if they become pathogenic,” Peng said.

The researchers plan to continue investigating NeuroD1 uses, as well as potential cell conversions.

Note: Previous studies showed NeuroD1-induced astrocyte-to-neuron conversion. However, recent study led by Chun-Li Zhang at UTSW demonstrated that the NeuroD1-induced astrocyte-to-neuron conversion was also experimental artifacts by viral leakage (Wang et al., 2021, Cell). Both of these two papers were accepted in September, addressing concerns on NeuroD1-induced glia-to-neuron conversion.

Contributors include Yanxia Rao, Siling Du, Baozhi Yang, Yuqing Wang, Ruofan Li and Ti-Fei Yuan, Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine; Yuxin Li, Xiangjuan Du, Yang He, Yafei Wang, Xin Zhou and Ying Mao, Department of Neurosurgery, Huashan Hospital, Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Fudan University; and Tian Zhou, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences. Du, who is currently affiliated with the Neuroscience Graduate Program in Washington University’s School of Medicine, Yang and Wang are also affiliated with Fudan University; Yang and Wang are also affiliated with the School of Basic Medical Sciences, Jinzhou Medical University.

The National Key R&D Program of China (2017YFC0111202), the National Natural Science Foundation of China (31922027, 32000678, 32170958 and 32000727), the Program of Shanghai Academic/Technology Research Leader (21XD1420400), the Shanghai Pilot Program for Basic Research (21TQ014), The Innovative Research Team of High-Level Local University in Shanghai, and Shenzhen Science and Technology Research Program (JCYJ20180507182033219) funded this research.

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Related papers:

(1) Rao Y., Du S., Yang B., Wang Y., Li Y., Li R., Zhou T., Du X., He Y., Wang Y., Zhou X., Yuan T.-F., Mao Y. and Peng B. (2021) NeuroD1 induces microglial apoptosis and cannot induce microglia-to-neuron cross-lineage reprogramming, Neuron, 109. 

(2) Matsuda, T., Irie, T., Katsurabayashi, S., Hayashi, Y., Nagai, T., Hamazaki, N., Adefuin, A.M.D., Miura, F., Ito, T., Kimura, H., et al. (2019). Pioneer Factor NeuroD1 Rearranges Transcriptional and Epigenetic Profiles to Execute Microglia-Neuron Conversion. Neuron 101, 472-485 e477. 

Recent publications from Bo Peng Lab:

(1) Rao Y.*, Du S., Yang B., Wang Y., Li Y., Li R., Zhou T., Du X., He Y., Wang Y., Zhou X., Yuan T.-F.*, Mao Y.* and Peng B.* (2021) NeuroD1 induces microglial apoptosis and cannot induce microglia-to-neuron cross-lineage reprogramming, Neuron, 109.

(2) Huang Y.^#, Xu, Z.^#, Xiong S., Sun F., Qin G., Hu G., Wang J., Zhao L., Liang Y.-X., Wu T., Lu Z., Humayun M.S., So K.-F., Pan Y., Li N., Yuan T.-F.*, Rao Y.* and Peng B.* (2018). Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion. Nature Neuroscience 21, 530-540.

(3) Xu Z.^#, Rao Y.^#, Huang Y., Zhou T., Feng R., Xiong S., Yuan T.F., Qin S., Lu Y., Zhou X., Li X., Qin B., Mao Y., and Peng B.* (2020). Efficient strategies for microglia replacement in the central nervous system. Cell Reports 32, 108041.

(4) Huang Y.^#, Xu Z.^#, Xiong S., Qin G., Sun F., Yang J., Yuan T.F., Zhao L., Wang K., Liang Y.X., Fu L., Wu T., Lu Z., So K.F., Rao Y.* and Peng B.* (2018) Dual origins of retinal microglia in the model of microglia repopulation. Cell Discovery 4, 9.

(5) Xu, Z., Zhou, X., Peng, B.*, and Rao, Y.* (2021). Microglia replacement by bone marrow transplantation (Mr BMT) in the central nervous system of adult mice. STAR Protocols 2, 100666.

(6) Xu, Z., Rao, Y.*, and Peng, B.* (2021). Protocol for microglia replacement by peripheral blood (Mr PB). STAR Protocols 2, 100613.

(7) Xu, Z., Peng, B.*, and Rao, Y.* (2021). Microglia replacement by microglia transplantation (Mr MT) in the adult mouse brain. STAR Protocols 2, 100665.