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Part of a mammalian cerebellum

Most scientists attend conferences armed with posters and presentations. But in addition to those, protein chemist Jonathan Elegheert came to the 2018 Molecular Neurobiology Workshop with a protein and a pitch.

Unveiled in a 2020 study in Science, the protein CPTX was capable of restoring eroded synapses in diseased mice. It was born from an idea that turned the traditional therapeutic approach to degenerative brain diseases on its head. Instead of targeting the mechanisms that cause synapses to erode, the researchers wanted to instead add a synthetic protein molecule to bring them back together.

“It was a wild idea at the beginning,” said Elegheert, now a team leader at the Interdisciplinary Institute for Neuroscience of the French National Center for Scientific Research. “And the results actually surprised us.”

His pitch for the meeting? He wanted to engage expert cellular neurobiologists to scrutinize and help him strengthen his team’s preliminary data on how the protein functioned in neuronal cells and mice.

A ‘chimera,’ thanks to carefully orchestrated experiments

The preliminary data Elegheert brought to the workshop came from a complex series of experiments — and a question that began five years earlier at the University of Oxford in the laboratory of Radu Aricescu.

Man with glasses standing in office.
Jonathan Elegheert of the Interdisciplinary Institute for Neuroscience. Photo courtesy of Jonathan Elegheert.

Elegheert and Aricescu knew that an imbalance between excitatory and inhibitory neurotransmission underlies many conditions such as autism spectrum disorder and Alzheimer’s disease. A previous study led by the pair had shown that the formation of precise and balanced neuronal circuits requires structural proteins, called synaptic organizers, that bridge synaptic clefts and mediate bi-directional signaling.

With that knowledge, they hatched the idea of engineering a synthetic synaptic organizer protein to bridge and restore broken synapses.

They began by combining structural elements from two proteins key to neuronal development: cerebellin-1 and neuronal pentraxin-1.

Cerebellin is an extracellular scaffolding protein that facilitates synapse formation. Previous research showed that a single dose of cerebellin-1 rapidly and lastingly restored roughly 75% of synapses as well as motor coordination in genetically engineered mice where cerebellin-1 was knocked out. But the protein is limited by its capacity to bind only to one specific receptor that’s largely confined to the cerebellum.

By contrast, neuronal pentraxins induce clustering of postsynaptic AMPA-type ionotropic glutamate receptors. AMPA receptors, which help usher electrical impulses across synapses, are enriched at excitatory synapses throughout the brain. In short, pentraxin-1 could act as a gateway into other brain regions.

“And so we came up with CPTX, which is basically a chimera,” Elegheert said. “It’s taking parts of cerebellin and fusing them to parts of pentraxin.”

Partnering with Michisuke Yuzaki, an expert on the biology of cerebellins, and his team, Elegheert and Aricescu showed that the protein worked structurally in cultured neuronal cells and brain tissue slices. CPTX successfully connected with the signaling molecule neurexin, located at the front end of the synapse, and AMPA-type ionotropic glutamate receptors (AMPARs), at the back end.

The researchers also demonstrated that CPTX worked functionally. When injected into the cerebellum of mice, it induced excitatory synapses in the hippocampus and caused clustering of AMPARs. It also increased the frequency and amplitude of excitatory postsynaptic currents.

Based on those results, the researchers proposed a mechanism for how CPTX achieved success: By bridging presynaptic neurexins and postsynaptic AMPARs, the molecule would nucleate the initial formation of immature synapses, which would then evolve into functional, mature ones.

Backed by promising results, they turned to three experts to test CPTX in mice.

Putting data to the test

The first was Yuzaki, who had a murine model of ataxia that mimicked the deteriorated synapses in the cerebellum and impaired motor skills in humans. When his team injected the animals with CPTX, they showed improvement in mobility, as measured in part by tracking mice footfalls on a plexiglass path.

With the help of Alexander Dityatev, they were able to test CPTX in mice modeling Alzheimer’s, which heavily impacts the hippocampus. In addition to slightly improving spatial memory, CPTX enabled the mice to better distinguish dangerous situations from neutral ones, suggesting that the protein rescued their ability to discern context.

Finally, joined by Kosei Takeuchi, the researchers tested CPTX in mice modeling spinal cord injuries, where the protein restored spinal motor circuits and motor coordination. Biochemical analysis revealed that CPTX was undetectable in the brain and spinal cord just days after injection, evidence that one dose of CPTX, like cerebellin-1, had lasting effects.

With structural elements from the cerebellin-1 and pentraxin-1, CPTX rapidly induced the formation of excitatory synapses, restoring and reorganizing neuronal circuits in mouse modeling ataxia, Alzheimer’s disease and spinal cord injury. Preprint figure courtesy of Jonathan Elegheert.

Eager to bolster his team’s data on how the protein functioned, Elegheert took his protein on the road, engaging with neurobiologists to get ideas and suggestions on worthwhile experiments.

Among other meetings, he found the inaugural 2018 Molecular Neurobiology Workshop, hosted by the European Molecular Biology Organization. Drawing neurobiologists, structural biologists and biophysicists together with top journal editors, the workshop was the perfect place to present his ideas and get feedback.

“It’s already pretty rare that you have a lot of structural biologists meeting cell biologists,” Elegheert said. “But we were actively encouraged to show prelim, unpublished data.”

“That proved to be very beneficial for the work,” he added. “After sharing data and taking up many suggestions, our work became so comprehensive that the actual manuscript reviewing and correction process was very smooth.”

The results established CPTX as the first synthetic synaptic organizer and a potential template for future therapies. Elegheert says interdisciplinary networking at the workshop was pivotal to the success.

“I think it’s a nice illustration of the philosophy behind the meeting,” said Elegheert, who will present at the 2022 workshop. “It’s exactly to foster this kind of collaboration and exchange of ideas, where structure meets function.”

Writer: Halle Marchese,
Source: Jonathan Elegheert,

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