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 published breakthrough results in Neuron, detailing how NeuroD1, a protein involved in cell differentiation, could coax microglia into new neurons.

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

The team set 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,” the senior author 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,’” the author said. “Instead, our data strongly indicate that the previously observed conversion was actually due to the experimental artifacts from viral leakage.”

The assumed finding, the author 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,” the author 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, the author explained, appeared to be supported in the original paper, but when the 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 the author, 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,” the author 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 addressing concerns on NeuroD1-induced glia-to-neuron conversion.

https://www.cell.com/neuron/fulltext/S0896-6273(21)00944-2