05 Jul 2019

Astrocytes are devoted nurturers of neurons—facilitating synaptic transmission, maintaining the blood-brain barrier, and repairing injuries are but a few of their ministrations. As if that weren’t enough, scientists described an instance where astrocytes made the ultimate sacrifice: by leaving their identity behind and becoming something else entirely. At the joint symposia Neurodegenerative Diseases: New Insights and Therapeutic Opportunities, and Neural Environment in Disease: Glial Responses and Neuroinflammation, held June 16–21 in Keystone, Colorado, Don Cleveland of the University of California, San Diego, reported on an experimental protocol whereby dialing down expression of a single gene transformed astrocytes into dopaminergic neurons within the substantia nigra of the mouse brain. No infusion of cells needed.

The in situ neuronal converts pumped out dopamine, and rescued motor deficits in a model of Parkinson’s disease, Cleveland reported. Researchers at the conference peppered Cleveland with questions and potential pitfalls of this approach. Perhaps only by converting to neurons could astrocytes—forever the wallflowers of brain research—spark this much excitement at a neurodegeneration meeting.

In fact, Cleveland kicked off his Keystone talk by extolling the virtues of antisense oligonucleotide therapy, which has resulted in an approved drug for spinal muscular atrophy, and is in various stages of clinical development to take down a cadre of disease-related proteins including huntingtin, tau, and more. But Cleveland suggested ASOs could be put to use to accomplish a different feat. Rather than knock down this or that disease protein, ASOs could give rise to brand-new neurons in the diseased, aging brain.

Researchers led by Fred Gage of the Salk Institute in La Jolla, California, previously developed protocols that could convert fibroblasts directly into neurons in a dish by adding in the right mix of transcription factors (Jun 2011 news). Across the street at UCSD, Cleveland’s colleague Xiang-Dong Fu subsequently simplified the conversion process. He found that turning down expression of a single protein—pyrimidine-tract-binding (PTB)—triggered fibroblasts to transform into bona fide neurons that sprouted axons and fired action potentials (Jan 2013 news on Xue et al., 2013; for review, see Hu et al., 2018). PTB is an RNA-binding protein. At Keystone, Cleveland presented the results of his collaboration with Fu, which took the logical, if ambitious, next step of sparking the conversion of astrocytes into neurons within the brain.

To pull off this form of “identity theft,” as Cleveland called it, the researchers targeted PTB with an ASO. In culture, this oligonucleotide induced the differentiation of mouse or human astrocytes into cells that expressed typical neuronal markers, and that differentiated into different kinds of neurons. A small proportion of the converted neurons expressed tyrosine hydroxylase (Th), a marker of dopaminergic neurons.

Could they orchestrate this conversion within the brain? To find out, the researchers injected wild-type mice with an adeno-associated virus (AAV) expressing a small-hairpin RNA (shRNA) to inhibit PTB expression, and a red fluorescent protein to mark infected cells. Fu lab postdoc Hao Qian and colleagues’ expression system glowed red in infected astrocytes, and remained red even if the cells converted into neurons. Cleveland reported at Keystone that one month after injection, 80 percent of red cells expressed neuronal, not astrocyte, markers. A quarter of the converted cells also expressed Th, suggesting they could make dopamine. In animals injected with a control virus expressing a nonspecific shRNA along with the red fluorescent protein, the marked cells remained astrocytes.

Next, the scientists tested the strategy in a mouse model of PD, in which they obliterated dopaminergic neurons on one side of the substantia nigra by injecting the neurotoxin 6-OHDA. Following ablation, the researchers injected the PTB-lowering or control virus directly into the substantia nigra. A month later, they found that while a neuronal desert surrounded the toxic lesion in mice receiving the control, converted neurons abounded in those with the PTB-lowering virus. The treatment raised neuronal numbers up to a third of their original levels. The 6-OHDA lesion docked dopamine levels in the nearby striatum, which receives dopamine from nigral neurons, by 80 percent. Treatment with the PTB-lowering virus restored dopamine levels back up to 66 percent of normal.

Would this be sufficient to stop the motor problems caused by the neurotoxic lesion? Cleveland reported that the ablation caused mice to walk in circles, reflecting loss of motor control on one side. However, animals treated with the PTB-lowering virus stopped circling two to three months later, while those receiving a control virus didn’t. Notably, the benefits of the PTB-lowering virus lasted up to one and a half years, while animals treated with the control virus still displayed the circling behavior.

Trying to link this improvement to induced neurons, the researchers next added another gene to their PTB-lowering virus. They loaded on a mutagenized version of a muscarinic acetylcholine receptor that deactivates neurons when it encounters the synthetic peptide CNO. Essentially, this addition allowed the researchers to deactivate converted neurons at will. Cleveland reported that while the PTB-lowering/mACh virus rescued the circling phenotype in lesioned mice, they relapsed back into circling when the researchers deactivated the converted neurons with CNO. The mice recovered mobility a second time after the injected CNO wore off. In all, the findings suggested that the conversion of astrocytes into neurons not only restored dopaminergic function in the 6-OHDA-poisoned mice, but also provided a durable rescue of motor deficits caused by the ablation.

Excitement was palpable in the room as researchers barraged Cleveland with questions. Charles Meshul of Oregon Health Sciences University in Portland asked whether the converted neurons formed functional circuits with the striatum. Cleveland said that while patch-clamp experiments suggest the neurons are functional and dopamine levels suggest they pump out the neurotransmitter, it is unclear whether they form connections akin to those of native nigral neurons. Fu later told Alzforum that they are addressing this question by measuring newly formed synapses between the converted neurons and existing circuits in the brain. Martin Kampmann of the University of California, San Francisco, wondered what triggered the astrocytes to become dopaminergic neurons, instead of some other type of neuron. Cleveland speculated that different astrocytic subtypes might be inclined to transform into distinct neuronal subsets upon PTB inhibition, but local cues within the nigra, where the virus was injected, likely also steered the converts into the dopaminergic type.

Others wondered about potential downsides of trading astrocytes for neurons. Cleveland acknowledged that he had yet to investigate consequences of lowering the astrocyte pool, and noted that any company developing ASOs against PTB should run dosing studies to evaluate side effects.

Others questioned how the strategy would work in the context of true PD, as opposed to an acute neurotoxic lesion. Cleveland said that remains to be seen. More work needs to be done in genetic models of the disease, he said. Later, he told Alzforum that in PD patients, the neuronal converts would have to withstand an ongoing neurodegenerative environment. Therefore, he sees astrocyte conversion working best in combination with other treatments that target the processes driving neurodegeneration.

Could this strategy assuage neuronal loss in other diseases? Cleveland said possibly, yes, although a targeted approach of replacing lost dopaminergic neurons in the substantia nigra has the highest chance of success. Aaron Gitler of Stanford University was impressed by Fu and Cleveland’s findings. He agreed that neuronal replacement could theoretically work for PD, but would be hard-pressed to bring back functional motor neurons lost in ALS. Motor neurons must sprout axons that form connections over vast distances, and Cleveland agreed that might be asking too much of a newly minted neuron.—Jessica Shugart

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References

News Citations

1. Turning Human Fibroblasts Into Neurons; Making Safer Stem Cells 5 Jun 2011
2. Stem Cells: Simpler to Make, Easier on the Immune System 17 Jan 2013

Paper Citations

1. Xue Y, Ouyang K, Huang J, Zhou Y, Ouyang H, Li H, Wang G, Wu Q, Wei C, Bi Y, Jiang L, Cai Z, Sun H, Zhang K, Zhang Y, Chen J, Fu XD. Direct Conversion of Fibroblasts to Neurons by Reprogramming PTB-Regulated MicroRNA Circuits. Cell. 2013 Jan 9; PubMed.
2. Hu J, Qian H, Xue Y, Fu XD. PTB/nPTB: master regulators of neuronal fate in mammals. Biophys Rep. 2018;4(4):204-214. Epub 2018 Aug 28 PubMed.

Further Reading

No Available Further Reading