Fluorescence-Quenched Substrates Measure Parkinson’s Disease-Related Enzymes
By Brittany Wade
August 24, 2022 | More than 10 million people worldwide have been diagnosed with Parkinson’s disease (PD), a brain disorder characterized by involuntary tremors, muscle stiffness, and cognitive decline. Though it ranks as the second most common neurodegenerative disease, scientists know little about its etiology. Hoping to discover more, a research team at Simon Fraser University (SFU) in Burnaby, British Columbia developed a new blood testing method to examine PD development and progression in neurons.
Though scientists struggle to cobble together PD mechanisms, there have been a few critical findings. For example, mutations in the glucocerebrosidase gene, GBA, are thought to be a genetic risk factor for PD. In a Molecular Neurodegeneration study (DOI: 10.1186/s13024-019-0336-2), researchers found at least 495 possible GBA mutations across 11 exons contributing to glucocerebrosidase malfunction and PD symptoms.
Glucocerebrosidase, also called lysosomal glucocerebrosidase or GCase, is an enzyme produced by the lysosome, an organelle that recycles waste products and other components after cell death. Specifically, GCase breaks down a large glycolipid found in neuronal cell membranes called glucocerebroside.
When GCase activity is low, glucocerebroside accumulates in neurons as fat deposits. These deposits are thought to have a solid connection to the development of PD and Gaucher’s Disease (GD). GD is a potentially fatal genetic condition that causes fat-enlarged organs and weakened bones.
Still, the specifics of how GCase contributes to Parkinson’s disease are unknown. Scientists suspect enzyme impairment may be linked to lysosomal downregulation, a buildup of neurotoxins, increased endoplasmic reticulum stress, and mitochondrial dysfunction.
Fluorescence-Based Enzyme Detection
With multiple connections to PD and other neurodegenerative diseases, it is no surprise that GCase has emerged as a potential diagnostic marker and therapeutic target. The SFU team postulated that studying GCase lysosomal activity could disclose the enzyme’s effects on PD disease progression.
“This is the first approach that has been shown to reliably and accurately report on the activity of this enzyme directly within lysosomes of living cells,” says David Vocadlo, co-author of the paper and professor of Chemistry and Molecular Biology and Biochemistry, in a press release. “Being able to measure the lysosomal activity of this enzyme in an accurate way could be very helpful in understanding the root causes of Parkinson’s as well as potentially helping to diagnose or track its progression.”
Measuring GCase activity usually requires complicated quantitative measures and various clinical assays to access lysosomal pathways. Unfortunately, some of these methods disturb the internal environment and dilute enzyme concentrations, thereby losing valuable information.
Looking to provide a more straightforward and accessible method, the team developed a fluorescent biological tool to examine GCase activity in monocytes or white blood cells. Since blood samples are easily accessible, this method could accurately measure Parkinson’s progression at a low cost and without invasive procedures.
Published in Proceedings of the National Academy of Science (DOI: 10.1073/pnas.2200553119), the team successfully measured GCase activity in monocytes, neural progenitor cells–cells that give rise to various specialized cells of the central nervous system–and neurons.
The key to measuring blood GCase is using a fluorescence-quenched substrate: a molecule that emits light in response to target enzymatic processing and whose fluorescence can be measured via microscopy or flow cytometry. Historically, these substrates were used as a qualitative tool; however, the SFU team used them quantitatively to prove their versatility in biological measurement.
The team synthesized a novel substrate, LysoFQ-GBA, with a lysosomotropic dML (lysine derivative) residue to improve substrate solubility, protect surrounding enzymes, enhance selectivity for GCase, and increase substrate retention within the lysosome. Adding this residue was critical, as substrates without it repeatedly diffused out of the cell and could not produce a fluorescent signal.
GCase blood activity matched those found in pluripotent stem cell-derived neurons and confirmed that monocytes provide a valuable pathway to determining enzymatic activity within lysosomes. Furthermore, monocytes are suitable surrogate cells for neurons, which are less accessible and of which exploration poses a tremendous risk to patients. Most importantly, given the GCase connection to PD and GD, enzyme blood examination could become a promising method to depict disease progression in the brain.
The team hopes their findings will lead to diagnostic capabilities and new drug development. “Ultimately, these tools could be used in clinical trials to assess the efficacy of strategies aiming to increase the activity of GCase in patients,” says Vocadlo. The pharmaceutical company Roche is currently using these findings to develop potential PD treatments.