Two separate groups have recently shown that in mouse models, inactivation of a single gene was enough to directly convert other cell types in the brain into neurons.

When the gene, which codes for the RNA binding protein PTB, was suppressed in the brain, it led to the transformation of astrocytes into neurons that were able to transmit light-induced neuronal signals from the eye to the cerebral cortex, and alleviate symptoms of Parkinson’s disease, respectively.

A team from the Chinese Academy of Sciences’ Institute of Neuroscience reported the direct reprogramming of support cells into visual system and midbrain dopaminergic neurons, respectively, in Cell in April 2020.

And investigators at the University of California at San Diego (UCSD) presented their Parkinson’s disease data in the June 25, 2020, issue of Nature and at the annual meeting of the International Society of Stem Cell Research (ISSCR) last week.

Don Cleveland, chair of the department of cellular and molecular medicine at UCSD and a co-author of the Nature paper, gave an overview of the discovery and its history in a Friday ISSCR session on neural cell identity.

For all the brain’s enormous complexity, the basics of neuronal fate determination are surprisingly simple. In 2010, researchers first demonstrated that it was possible to produce functional neurons by suppressing the production of two RNA-binding proteins.

Digging into the mechanism, scientists realized that “there appear to be two three-gene circuits that tell you you’re a neuron,” Cleveland said.

Furthermore, “if you look at the astrocyte, it’s already set in the second position,” with one of those circuits already active.

That led to the hypothesis that, perhaps, it would be possible to generate neurons from astrocytes, without a more stem cell-like intermediate, by manipulating a single gene – PTB.

In the studies now published in Nature, Cleveland and his colleagues first demonstrated that in cell culture, PTB down-regulation led to the stable formation of neurons. The team then decided to test the approach in an animal model of chemically induced PD.

The team showed that when they reduced dopaminergic neurons in the substantia nigra, the region most affected by PD, by roughly 90%, and then silenced PTB activity with a short hairpin RNA, a month after the treatment neuronal levels had bounced back up to about a third of normal levels.

That increase was enough to restore motor function – “all of those mice, over a five-month period, have a disease reversal,” Cleveland said.

Further experiments showed that about a quarter of the neurons generated after PTB silencing “have at least aspects of a dopamine neuron,” he said.

The team then used antisense oligonucleotides (ASOs) to silence PTB, to test whether direct reprogramming could be induced in a therapeutically viable method.

Using a PTB-ASO, they saw partial disease reversal.

One mouse of seven “didn’t get better,” Cleveland elaborated, “and we probably failed to inject the ASO” in a way that allowed it to reach its target.

At his ISSCR presentation, Cleveland speculated that “maybe this is the tip of an iceberg where we can generate this more broadly in neurodegenerative disease.”

That possibility is also suggested by the work published in April in Cell, where the investigators used PTB silencing via CRISPR/Cas9 to generate distinct neuronal cell types from two different support cells – dopaminergic neurons from astrocytes in the midbrain, and retinal ganglion cells from Muller glia in the eye.

Clinical potential

For clinical purposes, much about PTB’s potential remains TBD.

When asked in the Q&A session following his talk whether in PD patients, diseased astrocytes would not simply generate diseased neurons, Cleveland acknowledged that “absolutely, that’s a key question.”

Even if such neurons were initially healthy, another key question is whether α-synuclein, which spreads via retrograde transport during PD, would spread to newly generated neurons and ultimately damage them

Tilo Kunath, group leader in the Centre for Regenerative Medicine, University of Edinburgh, nevertheless called the results reported by Cleveland and his colleagues “remarkable,” and pointed out several factors that increase their chances of ultimately leading to advances in Parkinson’s disease treatment.

The data appear to be robust, Kunath said – it “fits and agrees well with several papers on PTB biology and the gene networks that regulate formation of neurons.”

The fact that the April Cell paper reported “very similar biology using a different technique” also suggests that the findings are reproducible, he added.

The ASOs used by Cleveland and his colleagues to suppress PTB are a clinically proven strategy. The first antisense drug, Vitravene (fomivirsen), was approved more than two decades ago, in 1998.

But the technology’s full potential was recognized by the broader biomedical research community with the 2016 approval of Spinraza (nusinersen, Ionis Pharmaceuticals Inc./Biogen Inc.).

Ionis and Biogen are developing ASOs for multiple other indications, including PD itself, which is being targeted via an LLRK2-targeting ASO. Of their experimental therapies, furthest on path to approval, in phase III, is tominersen (IONIS-Httrx/RG-6042) – a drug candidate that got its start in a collaboration between Cleveland’s lab and Ionis scientists.

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