What's in a neuron?

Why are certain cells and brain regions more affected in neurodegenerative diseases than others? This question - referred to as selective vulnerability - has been baffling scientists for over 100 years.

Selective vulnerability is a fundamental feature of these neurodegenerative diseases, in which different neurons (brain cells) lie on a spectrum of susceptibility to decay.

Selective vulnerability at the network level (i.e. in brain regions) has been extensively explored in both Alzheimer's and Parkinson's.

However, little is known about the mechanisms underlying selective vulnerability at the cellular level.

If scientists could figure out why and how α-synuclein and tau affect different types of neurons, they could start to understand the mechanisms underlying why some cells are more resilient to these toxic proteins.

This could, in turn, provide insight into disease mechanisms and lead to therapeutic strategies to tackle neurodegenerative disorders.

Scientists at the VIB-KU Leuven Center for Brain & Disease Research have been actively contributing to the single-cell revolution for the past few years. In particular, the Stein Aerts laboratory was a pioneer for the use of single-cell technologies.

In a interview for VIB News in 2018, Aerts explained how his fascination with single-cell technology all began: "We got really interested when two specific papers were published in the same issue of Cell in 2015, which described the use of droplet microfluidics to sequence the RNA of tens of thousands of individual cells in parallel - and more cheaply than ever. We were so excited that we called Jeroen Lammertyn, from the KU Leuven bioengineering faculty, who taught our PhD student Kristofer Davie how to design and fabricate our own microfluidic devices. Our first successful experiment came a few months later - it was a great moment to see cells cluster together as anticipated."

Thanks to single-cell technologies, Stein and his team accomplished a world first: a gene expression map of every cell in the brain of an ageing fly. The atlas is a major step towards a better understanding of human disease development.

This is where Roman's research comes in.

I caught up with Roman in a lab at the VIB-KU Leuven Center for Brain & Disease Research, where I asked him to describe his latest work, published in Neuron. "In neurodegenerative diseases such as Parkinson's and Alzheimer's, some nerve cells are highly vulnerable and degenerate, while others are resilient and survive." Roman explains. "In this project, we explored the idea that these resilient neurons might be able to tell us some of nature’s own secrets. After all, these resilient neurons seem to be doing something right - so why not learn from them?" His aim is that in the future, this knowledge might be harnessed to treat the vulnerable neurons, whose loss causes debilitating neurological symptoms.

They focused specifically on the two proteins already known to be critical drivers of neurodegeneration: α-synuclein and tau. "Remarkably, even though α-synuclein and tau can be toxic to neurons in general, we can find multiple resilient neuron types that express high levels of α-synuclein and tau in human brain datasets and fruit-fly models," Roman continues. "This suggests that they instead use strategies to better deal with potentially toxic proteins."

To find out what these resilience strategies are, Roman and his colleagues compared gene expression differences between vulnerable and resilient neurons with a technique called single-cell RNA sequencing. "We found that α-synuclein and tau resilient neurons differentially regulate mitochondrial energy metabolism, Ca2+ homeostasis and synapses (i.e. contact sites between neurons). Remarkably, when we experimentally modulated genes in these pathways we found several of them to potently reduce the toxicity of abnormal tau in our fly models, confirming that resilient neurons contain very valuable insights for potential new treatments."

I sit with Suresh - head of Single-Cell Microfluidics at VIB Technologies, to find out more about what specific single-cell technology was used in this project, and why.

VIB was one of the earliest adopters of single-cell technologies. In 2016, VIB Tech Watch team invested in one of the first 10x Genomics Chromium platforms in Europe, a ground-breaking technology that enables large scale single-cell RNA sequencing. However, applying these commercial setups to large scale single cell profiling has been far too expensive.

To solve this problem, Suresh worked with other researchers in the center (including Stein Aerts, who we introduced earlier!) to develop HyDrop, a powerful yet affordable droplet microfluidics platform.

"In projects like Roman's - where we're dealing with small fly cells that have low amounts of RNA transcripts - it's important to have really high encapsulation rates. The more cells you can efficiently capture, the higher chance of getting more diverse dataset," Suresh explains, before diving into the technical details of the project.

"We used multiple sequencing techniques for single cells, and bulk sequencing workflows were used to understand the underlying cellular diversity of fly brain. For high-throughput single-cell profiling, 10X Genomics’ 3’ Whole Transcriptome Analysis and for bulk sequencing of limited FACS sorted cell population, modified version of SMART seq 3 workflow was used."

"What we did with Roman's project was a novel adaptation of using the HyDrops’ droplet generation chip with the 10X Genomics reagents to encapsulate single cells. We have seen that the encapsulation efficiency with the HyDrop chips on ONYX platform are far superior and reliable compared to the 10X Genomics chromium controller.”