Researchers Convert Astrocytes to Neurons In Vivo to Treat… : Neurology Today – LWW Journals

Posted: May 8, 2020 at 4:51 am

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A mouse study shows that select transcription factors to the striatum can effectively and safely convert astrocytes to neurons to treat Huntington's disease.

Delivering two transcription factors to the striatum in a mouse model of Huntington's disease can safely convert astrocytes into neurons with high efficiency, according to a new study in the February 27 issue of Nature Communications.

The neurons grow to and wire up with their targets in the globus pallidus and substantia nigra, and remaining astrocytes proliferate to replace those that have been converted. The treatment extends the lifespan and improves the motor behavior of the mice.

What is exciting about this study is that the authors have clearly made cells that do what they are supposed to do, namely replace dying neurons in existing circuits, said Roger Barker, PhD, professor of clinical neuroscience and honorary consultant in neurology at the University of Cambridge and at Addenbrooke's Hospital, who was not involved in the work. I think the challenge of scaling up this strategy to the human Huntington's disease brain is pretty substantial, but nonetheless, this is an important discovery.

The new study, led by Gong Chen, PhD, builds on discoveries beginning in the mid-2000s showing that a small number of exogenously applied transcription factors could transform skin fibroblasts into stem cells, which could then be further converted to become virtually any cell type. That discovery was quickly followed by advances in direct reprogramming, in which one cell type is directly converted into another, skipping the stem cell intermediate.

Most of that work has taken place in vitro, and most attempts to use the strategy therapeutically have depended on transplantation of stem cells or newly converted cells.

We tried stem cell transplants to the mouse brain 10 years ago, but we couldn't find a lot of functional neurons, said Dr. Chen, professor at Guangdong-Hong Kong-Macau Institute of CNS Regeneration of Jinan University in Guangzhou, China.

It was also clear that anything you do in vitro, you eventually have to transplant, and that didn't seem to be a very promising technology, so I said, Let's try this in vivo, and put transcriptions factors directly into the mouse brain.

Dr. Chen initially tried introducing the transcription factor neurogenin 2, but the efficiency of conversion of astrocytes to neurons was very low, so he turned to the transcription factor NeuroD1, which Dr. Chen's group had previously shown could convert astrocytes into excitatory glutamatergic neurons.

In the current study, in order to generate GABAergic neurons, the team combined NeuroD1 with another transcription factor, D1x2, based on previous work showing its importance for generating GABAergic neurons.

The team packed the genes for the transcription factors into a recombinant adeno-associated virus vector (rAAV 2/5) and used an astrocyte-specific promoter to drive the transgene expression so that it preferentially expresses in astrocytes. They first injected the vector into the normal mouse striatum.

Surprisingly, this strategy worked very well at high efficiency, Dr. Chen said. After seven days, all transfected cells expressed astrocyte markers, indicating a high level of specificity in the vector. Of those cells, 81 percent co-expressed the two transcription factors. By 30 days, 73 percent of the cells expressing the transcription factors now expressed neuronal, rather than astrocytic markers, and were primarily GABAergic in character.

Next, Dr. Chen asked whether the remaining astrocytes could repopulate to replace those lost to conversion. Using immunostaining for astrocytes and neurons, as well as other techniques, the team found that the neuron/astrocyte ratio was unchanged, and that some remaining astrocytes could be found at different stages of cell division, suggesting the process facilitated astrocyte proliferation.

Dr. Chen then turned to the R6/2 mouse, the most common mouse model of Huntington's disease. He treated mice at 2 months of age, just as they began to show motor symptoms

As in the wild-type mice, astrocytes were converted to GABAergic neurons at high efficiency without altering the neuron/astrocyte ratio. The researchers observed similar results in a less-severe HD mouse model as well. Treated mice had only about half the degree of striatal atrophy as untreated mice. The converted neurons still contained aggregated huntingtin protein, but less than in native neurons, and similar to the reduced amount found in astrocytes in the mouse brain.

The real test of any cell therapy in neurodegenerative disease is whether the new cells can link into the existing circuits and provide functional benefit, feats that have been hard to achieve with transplanted fetal cells or stem cells.

Examining striatal slices from the treated mice, Dr. Chen found that the converted neurons displayed electrical properties largely identical to those of normal neurons, including resting potential, action potential threshold, firing amplitude, and firing frequency. They integrated into local circuits and behaved similarly to the native neurons around them. By tracking a marker contained in the AAV gene construct, they showed that converted neurons projected axons to the two basal ganglia targets of medium spiny neurons in the striatum, the globus pallidus and the substantia nigra.

Finally, Dr. Chen found that stride length and travel distance were both significantly improved in treated mice, though still falling below those of wild-type mice, and lifespan was significantly extended.

There were no hints of tumors in the mice, Dr. Chen noted. He suggested that in situ conversion is likely intrinsically safer in this regard than using stem cell-derived neurons, since a proliferative astrocyte is being converted into a non-proliferative neuron, with no residual pool of unconverted and potentially tumorigenic stem cells. We are actually reducing the tumor risk, he said.

Why the converted neurons developed appropriate neuronal connections is an important unanswered question, Dr. Chen said. He suggested there were two important factorsfirst, the astrocytes from which they arose are likely developmentally related to neighboring neurons, and thus may express similar position markers that help guide them to the right targets, just like the native neurons. Second, those remaining neurons may also provide guide tracks for the newly growing axons.

This conversion technique is not limited to Huntington's disease, he stressed, noting that his team last year published a paper showing promise in ischemic stroke, and work is underway to test its potential in Alzheimer's disease, Parkinson's disease, spinal cord injury, and ALS. He is also moving on to testing in non-human primates, setting the stage for eventual human trials.

I think eventually we will want to correct the Huntington's mutation as well, Dr. Chen said, for instance by using CRISPR, but he pointed out that while that strategy can repair diseased neurons, it cannot make new ones, like astrocyte-to-neuron conversion can.

This study is really elegantly done, commented Veronica Garcia, PhD, who has studied astrocytes derived from induced pluripotent stem cells from Huntington's disease patients as a postdoctoral scientist working with Clive Svendsen, PhD, in the Regenerative Medicine Institute at Cedars-Sinai Medical Center in Los Angeles.

The conversion efficiency is similar between wild-type and disease models, suggesting that the disease process is not interfering with the conversion, she said.

Astrocyte depletion does not seem to be a problem, at least in the short term, but Dr. Garcia noted there is a limit on the number of divisions astrocytes appear able to undergo, after which they lose the ability to proliferate. That may be a problem for chronic treatment, she suggested. Nonetheless, these results really look promising for therapeutic development.

The concept of trying to reprogram cells in situ to take on the phenotype of the cells that are lost is not new, commented Dr. Barker, but being able to do it with any degree of efficiency, to make enough cells to make a significant difference, has been problematic. For that reason, and because the cells grow to their target sites and make connections, these results are surprising.

A major hurdle for clinical trials, he noted, will be scaling up to the human striatum, which has approximately 100 times the volume of that in the mouse. Delivering the vector to such a large volume will be a significant challenge, he said, along with determining whether this approach will really work in a disease that affects many different brain structures such as in HD.

Dr. Chen is co-founder of NeuExcell Therapeutics Inc, which will develop clinical trials in the future. Drs. Barker and Garcia disclosed no conflicts.

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Researchers Convert Astrocytes to Neurons In Vivo to Treat... : Neurology Today - LWW Journals

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