Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Parkinson's disease is a progressive neurodegenerative disorder caused by a loss of dopamine-producing (dopaminergic) neurons in the substantia nigra of the brain.
That region of the brain “is responsible for coordination of movement,” says Ben Newton, director of neurology at GE Healthcare Life Sciences Business. As the dopaminergic neurons die off, symptoms progress from the relatively minor (muscle stiffness and slowing of muscle movement, for instance) to the severe (uncontrolled tremors and the inability to walk, talk or control facial expressions). Associated cognitive defects, such as dementia, also may occur.
Parkinson's disease affects at least a million Americans and five million people worldwide, according to the Michael J. Fox Foundation. Yet there is no cure, nor even a definitive diagnostic test.
Researchers, of course, are keen to close that gap, and various neuroscientific tools are available to make that happen.
Measuring gene expression
As a graduate student at the University of Ulm, Germany, Falk Schlaudraff was interested in the underlying molecular mechanisms that cause dopaminergic neurons to die in Parkinson's disease. To find out, he and his colleagues compared the gene-expression patterns in samples from post-mortem human individuals with Parkinson's to matching individuals without the disease.
It wouldn’t be enough, however, to simply collect brain tissue, grind it up and measure RNA expression levels. The brain is full of different neurons and other cell types, and clearly identifying differences in a specific cell type, such as dopaminergic neurons, required pure starting material free of surrounding cells.
So, the team turned to laser microdissection (LMD), a method in which a laser is used like a scalpel to excise and collect individual cells from a sample. The researchers harvested dopaminergic neurons from affected and unaffected human tissue, extracted the RNA from individual cells and measured the expression of different dopamine-associated and dopamine-regulated genes via quantitative RT-PCR [1].
“We discovered a kind of compensation mechanism,” says Schlaudraff, now LMD product manager at Leica Microsystems. “All mRNAs of dopamine-associated genes were increased in Parkinson's disease,” suggesting the remaining cells were attempting to cope with the loss of dopaminergic cells by ramping up dopamine availability in the striatum. At the same time, ion channels that tend to dampen neural activity were upregulated, Schlaudraff says—a seemingly contradictory observation that may indicate the cells are trying to protect themselves from hyperexcitability.
Today, Schlaudraff notes, researchers can use laser-microdissected material to obtain a genome-wide view using gene microarrays or next-generation DNA sequencing.
Leica Microsystems sells two dedicated LMD systems based on an upright microscope body, the Leica LMD6 and LMD7, Schlaudraff says. Both allow users to “fine-tune” laser settings, for instance to microdissect subcellular objects, including individual chromosomes.
Going clear
Another option for those studying post-mortem samples is CLARITY. Developed in 2013 in the lab of Karl Deisseroth , D.H. Chen Professor of Bioengineering and of Psychiatry and Behavioral Sciences at Stanford University, CLARITY literally clarifies the brain by removing optically opaque lipids while retaining the overall structure of the organ by encasing it in a gel. As described in a recent Biocompare Bench Tip, “CLARITY transforms intact tissue into a hydrogel-tissue hybrid construct that is perfectly intact and mechanically stable as well as permeable to small molecules and photons.” As a result, researchers can peer deep within the tissue, for instance to track the expression of dopaminergic neuron-specific markers.
Logos Biosystems has commercialized an all-in-one CLARITY sample-preparation system, the X-CLARITY™ Tissue Clearing System, which according to company literature is capable of clarifying a rodent brain in just six hours. In a paper published earlier this year, researchers in South Korea, including at Logos, described a variant of CLARITY and related methods, called ACT-PRESTO (Active CLARITY Technique-Pressure Related Efficient and Stable Transfer of Macromolecules into Organs), which can clear whole organs and even entire animal bodies in less than 20 hours [2].
Researchers also study the diseased brains of live animals using multiphoton imaging, a method for relatively deep-tissue fluorescence microscopy. Among other things, multiphoton microscopy can be used to record cellular activity beneath the surface of a live, active animal through a cranial window, says Joseph Huff, product marketing manager for laser-scanning and superresolution microscopy at Carl Zeiss Microscopy.
Zeiss recently upgraded its multiphoton-capable LSM 880 laser-scanning confocal microscope with an Airyscan detector. According to Huff, instead of using a pinhole to reject out-of-focus light (as in a typical confocal microscope), the Airyscan configuration places a 32-channel detector, like a compound eye, where the pinhole typically is positioned. By projecting an image onto that array, the configuration promises up to a 1.7-fold increase in resolution and an eight-fold improvement in signal to noise, he says—enough to reduce excitation energy, and thus phototoxicity and photobleaching, during live-animal studies. “Because of the signal-to-noise increase, we have a big advantage in reading weak signals as well as picking out finer details,” he says.
Getting clinical
Also available are tools for more translatable preclinical and clinical studies.
Revvity’s G8 PET/CT preclinical imaging system, for instance, is a benchtop whole-body imager capable of providing both high-resolution anatomic computed tomography (CT) and functional/molecular positron emission tomography (PET) data on small-animal models in a single scan, says Vivek Shinde Patil, senior manager for technical applications in the company’s Preclinical Imaging group.
According to Shinde Patil, the advantage of a technique like PET is its immediate applicability to the clinic: Biomarkers and tracers that show promise in preclinical testing can theoretically be adapted for use in clinical trials, as PET is used on humans, too. “There’s a direct translational path … with these probes,” Patil says.
Parkinson's researchers can, for instance, use F18-labeled fluorodeoxyglucose (FDG) to monitor metabolic activity throughout the brain, or F18-Fallypride, which binds D2/3 dopamine receptors, to image dopaminergic neurons specifically. Alternatively, researchers can couple their own candidate drugs or antibodies to PET-compatible isotopes such as Zr89 and I124, both of which are available from Revvity.
Another option is GE Healthcare’s FDA-approved DaTscan™ (ioflupane-I123 injection), a single-photon emission computed tomography (SPECT) tracer that binds to dopamine transporters in the brain.
According to Newton, DaTscan imaging produces a characteristic signature in individuals with Parkinson's disease. “The typical image that is obtained [in normal individuals] after uptake of DaTscan reveals a bright pair of ‘commas’—so there’s a ‘head’ and a pair of ‘tails.’” But in Parkinson's disease, the comma tails are lost. “In that case, you get two period shapes, two full stops.”
Such tools, he notes, cannot cure individuals with Parkinson's disease. But these tools are enabling physicians to make more informed diagnoses and ultimately helping drug developers design better trials.
“If you can identify that symptoms actually are caused by a particular pathology, then it can be transformational in the way a patient is managed,” Newton says.
References
[1] Gründemann, J, et al., “Elevated -synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson’s disease,” Nucleic Acids Research, 36:e38, 2008. [PMID: 18332041]
[2] Lee, E, et al., “ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging,” Scientific Reports, 6:18631, 2016. [PMID: 267505588]
Image: ShutterStock