Contrary to popular belief, there are regions of the human brain where not a single brain cell can be found. These regions consist of a sugar and protein goop known as the brain’s extracellular matrix.

There may be no cells in this space, but there is a plethora of valuable molecular clues that hold the key to unravelling mechanisms behind diseases such as Alzheimer’s or Parkinson’s. Until recently, proteins in the extracellular matrix have been incredibly challenging to measure and monitor in vivo, or in living organisms.

Now, a U-M group led by Chemistry Professor Robert T. Kennedy has developed an expedient protein-detection platform for human and mice α-synuclein, a protein released into the extracellular matrix by pathological cells of Parkinson’s disease.

This exciting discovery opens the door to understanding the disease mechanism on a molecular level. That could ultimately inform treatment and drug design. This work is published in ACS Chemical Neuroscience. First author is research scientist Youngsoo Kim.

“About 20 percent of the brain is extracellular space. Cells can talk to each other in this space using molecular messengers,” explains Kennedy. There is a lot to be learned from such a significant portion of the brain. Measuring the signals of these molecular messengers would be the first step in unravelling the messages they encode.

“In the past, we have been able to study molecular messengers like neurotransmitters and neuropeptides but there really has been very little work studying the bigger molecules. The bigger picture is proteins,” he says

The best available Parkinson’s biomarker poses an analytical conundrum

The current state-of-the-art biomarker for Parkinson’s is a protein called α-synuclein. It is hypothesized to be the main constituent of toxic clumps known as “Lewy bodies” in the brains of patients. Lewy bodies spread through the extracellular matrix of the brain, causing malfunction in each region where they appear. However, measuring α-synuclein in living organisms is hindered by technical challenges.

Proteins have eluded scientists because they are hard to quantify and monitor in minute quantities and complex mixtures. Though a technology known as microdialysis has been used for the last twenty years to collect amino acids, neurotransmitters, and other metabolites, proteins pose a unique challenge because their concentration is much lower and their large size makes them difficult to sample through a membrane.

Proteins that can be detected are also hard to recover and tend to adsorb onto the surfaces of the analytical instrument, reducing the amount of  material to be analyzed. Assays that bind to proteins to give a measurable signal, known commonly as enzyme-linked immunosorbent assay (ELISA), require a large sample size and take as much as an hour to complete.

A new technology to measure protein dynamics in vivo emerges

The Kennedy lab developed a novel microdialysis method that gives the same resolution as state-of-the-art techniques but requires 50 times less sample and is 60 times faster than before. This allows researchers to monitor minute changes in α-synuclein in live mice with higher spatial and temporal resolution.

The Kennedy method establishes a better sampling technique by screening probes that are specific for α-synuclein and uses a washing procedure to prevent loss of sample. Then, they couple this specific sampling probe with a more sensitive detection assay known as amplified luminescent proximity homogenous assay (AlphaLISA) that could screen both human and mouse α-synuclein. The probe and assay together helped researchers develop a useful in vivo sample detection platform that can be used under normal physiological conditions.

Using this innovative platform, researchers established a basal α-synuclein level of 254 pM in the striatum of freely moving mice. Then, they observed that adding known stimulants like potassium ion and nicotine increased α-synuclein production in dialysates.

With this new approach, α-synuclein not only can be measured at smaller concentrations, and therefore on smaller timescales than before, it can also be monitored in vivo to see the effect of potential inhibitors or stimulants on α-synuclein production. This data would aid in understanding Parkinson’s disease progression and treatment.

Future directions in brain chemistry

Measuring protein dynamics on a small timescale and high sensitivity would allow neuroscientists to garner insight into the when and why of the development of these neurodegenerative diseases..

Professor Kennedy envisions that this detection platform will enable researchers to study a whole host of proteins other than α-synuclein. Learning the language of cells through proteins may uncover the deeper mysteries of brain chemistry.

This work has been part of a National Institutes of Health BRAIN Initiative grant. Kennedy is Hobart H Willard Distinguished University Professor of Chemistry and Pharmacology


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