Could Overcrowded Cells Kickstart Neurodegeneration?

2020 has been a year where distancing is paramount. Whilst we have been (for the most part) able to adjust our lives to a more spacious setup, one place where social distancing isn’t possible is inside our cells. An ambitious new research project powered by hitherto inaccessible techniques is investigating whether a crowded cellular environment might have a role to play in the origin of neurodegeneration.

“One of the characteristic features in diseases like amyotrophic lateral sclerosis (ALS) is the aggregation of proteins,” says Hemali Phatnani, director of the Center for Genomics of Neurodegenerative Disease (CGND) at the New York Genome Center. This is a process where certain proteins, molecules that control virtually every function of our cells, change from their usually ordered molecular formation into tangled masses. This messy process is a hallmark of the process of neurodegeneration, the death of nerve cells that occurs in brain diseases such as ALS and dementia.

Virtually all efforts to treat neurodegenerative disease have revolved around stopping or slowing cell death by targeting the process of protein disruption and aggregation. These efforts have long been hamstrung by the lack of knowledge about the origin of aggregation. “We don't know what drives this kind of aggregation, because sometimes you can have mutations in proteins that cause them to become more aggregation prone, but in about 95% of ALS cases, there's no identifying cause that's driving why these proteins decide to aggregate,” Phatnani says.

Cell-cial distancing?

Phatnani has recently received a grant from the Chan Zuckerberg Initiative (CZI) to investigate this process, armed with new techniques that could revolutionize how we study the degenerating neuron. Those techniques, as it happens, have not come from neuroscience, but from biophysics. They were dreamed up in the lab of Liam J Holt, assistant professor in the Department of Biochemistry and Molecular Pharmacology at New York University.

By combining microscopy, live cell imaging and innovative nanoparticle technology, Holt and Phatnani hope to answer a long-standing question in neuroscience – what happens in the cell prior to protein aggregation?

Speaking to Technology Networks, Holt says that to understand why this question has proved so difficult to answer, one must get a sense of what the cellular environment looks like. Remember when clubbing was a thing?

“Imagine stuffing people into a dance club,” says Holt. “At a certain point, everyone's fine. They can all dance around – everyone's comfortable. This cell is way beyond that. It is at the point that there's barely any room to move.” Huge macromolecules, like nucleic acids, cramp out the cell, which means their movement in the cellular morass is dramatically slowed down. Cells are often drawn in textbook as a sphere with a few structures merrily floating about like a Windows screensaver. The reality is more like a micro-sized mosh pit.

But generally, cells function normally in this crowded configuration. It is when the delicate balance of the cell is disrupted that things start to go wrong, explains Phatnani. “When you make the inside more crowded, certain sized assemblies tend to crash out of solution, so there's a size regime that's particularly vulnerable to changes in the extent of intracellular crowding.”

An even more crowded cell means that structures that wouldn’t normally interact are forced together, with unpredictable consequences. Holt goes back to our clubbing scenario, “This effect can also be seen in a nightclub occasionally. People that might have only had weak affinity for one another are maybe driven together into more permanent unions, and so the same thing happens with molecules.”

Filling in the blanks in neurodegeneration

Holt is using nanoparticles as a tool to determine whether changes in cellular crowdedness precede the formation of protein aggregates. If the answer is yes, it would fill in a huge blank for neurodegeneration theorists and could even provide researchers with a mechanism to therapeutically target. But how does this nanoparticle approach work?

“The issue with trying to understand how a cell is controlling its interior environment,” says Holt, “is that looking at a cell, you can't really see much. It's pretty much transparent.”

The nanoparticles are telltales, he explains. The neurons that Holt studies have been genetically modified to produce these nanoparticles naturally. Attached to a fluorescent molecule, their movement can be tracked easily. “Imagine you know you're looking at a landscape and you’re tracking, with GPS, a dog running around in the landscape and you don't know anything about where your dog is,” says Holt. If the GPS shows long, straight lines, perhaps the dog is in a field, but if the tracks are ever-changing and hard to follow, perhaps your pooch is in a tangled forest. The nanoparticles will inform Holt and Phatnani of what their cells’ interior looks like, a process that was previously done with a particularly agonizing process called microinjection. This, as it might sound, literally involved taking a tiny needle and trying to inject particles into cells. With Holt’s genetically-expressed molecules, experimental timelines are massively condensed. “We’ve gone from hours, to five seconds for an experiment,” says Holt.

Holt and Phatnani are careful to say that their new approach is still far from being a guaranteed path to new therapies or game-changing discoveries. But what it will enable them to do is answer long-standing questions about neurodegenerative disease that have puzzled researchers for decades. “When you look at pathology images, sometimes you see these groups of proteins in the nucleus, sometimes in the cytoplasm. Sometimes you see these protein groups in neurons, or glial cells, sometimes they can be all over the neuropil and we don't know the significance of any of them. And we don't know how it correlates with disease either. So those are some of the things that we want to investigate,” says Phatnani.

A canary in the coal mine?

The studies also hope to answer why neurodegenerative diseases have shared mechanisms, such as protein aggregation, but such different courses of progression. People with Parkinson’s disease can expect to live 14.6 years after onset, whilst ALS patients have a median expectancy of just two or three years.

Furthermore, Holt and Phatnani hope to answer one very long-standing question about the behavior of proteins in ALS and other conditions – are they a causal factor, or just a canary in a coal mine of cellular dysfunction?

With their technique, Phatnani explains, they have the basis for a “screen”, an experiment in which thousands of cells are each slightly altered in different ways, and their resulting cellular environment monitored. Would reducing a crowded cell’s busyness give it a longer lifespan? Does restoring the balance of proteins in the cell, which is called proteostasis, reverse protein aggregation and cellular dysfunction. Now, researchers have the tools they need to find an answer. 

“We're not claiming that we've solved the neurodegeneration problem,” concludes Phatnani. “We've just found a way to investigate a facet of it that researchers have wanted to examine for a long time and we're very excited to be able to have the chance to do this together.”