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Parkinson’s disease (PD) affects an estimated 8.5 million people worldwide and is the second most common neurodegenerative disorder (1–3). A cure for PD is currently unavailable, but other clinical challenges are obstacles to improving the lives of those affected by this disease. A definite diagnosis only happens when symptoms emerge, and by this stage, neurodegeneration has advanced beyond the point of preventing progression (4–6). But the development of early-detection tests can also have a major impact on the management of PD (7). As a result, patients can have early access to current and emerging treatments for PD and experience better overall clinical outcomes (7).
Using preclinical models, Zhou et al. developed the Dopamine Neuron Challenge (DNC), a test capable of detecting early-stage PD in mice (8). Keep reading to learn more about Zhou et al.’s study and the translational relevance of the data they acquired.
Progressive loss of dopamine (DA) neurons in the substantia nigra pars compact (SNc) drives the pathophysiology of PD (9). These neurons control movement and muscle tone, and their loss contributes to muscle stiffness, slow movement, tremors, and impaired balance (9). However, these symptoms only appear after a 60% loss of DA neurons in the SNc (4–6). From then on, symptoms worsen as the disease progresses (4–6).
Zhou et al. hypothesized that during the early stages of PD, increased release of DA by viable neurons compensates for the ongoing neuronal loss. And for this reason, patients don’t show any functional impairment. At symptoms onset, homeostatic increase in DA release can no longer compensate for massive neurodegeneration, leading to motor deficits. If Zhou et al.’s hypothesis is correct, disruption to the compensatory release of DA can help detect neurodegeneration during the early stages of PD.
Researchers tested their hypothesis by quantifying DA release in the striatum before and after using drugs that impact DA synaptic transmission to exhaust the compensatory release of this neurotransmitter. The initial drug candidates and their respective mechanisms of action were:
Zhao et al. used fiber photometry to quantify DA release in vivo in response to the drug challenge. This technique uses an optical fiber probe to stimulate neurons and measures changes in fluorescence signal corresponding to variations in dopamine concentration. C57BL/6J mice received microinjections of viral vectors expressing DA receptors conjugated to dLight1.1 and tdTomato fluorescent probes. Once released DA binds to its receptors, fluorescence levels increase. The combination of methylphenidate and haloperidol led to the most abundant release of DA and was used for all subsequent experiments.
After selecting the ideal drug combination, researchers used MitoPark mice, a preclinical genetic model of PD, to answer the study’s central research question: Can the DNC test detect early-stage PD?
First, Zhao et al. used immunohistochemistry (IHC) followed by stereology to quantify DA neurons in the striatum. They followed a standard IHC protocol and started by blocking brain sections with Normal Goat Serum Blocking Solution (S-1000-20) to reduce the chances of unspecific staining. After incubation with polyclonal primary antibody against tyrosine hydroxylase (TH; DA neuronal marker), researchers incubated the sections with biotinylated secondary antibody followed by avidin-biotin peroxidase complex and impregnation with DAB. Stereological quantification revealed that MitoPark mice lost 28% of DA neurons at 20 weeks.
Next, Zhao et al. quantified the levels of DA metabolites in cerebral spinal fluid (CSF) and plasma samples from control and MitoPark mice. No differences in DA metabolites in CSF and plasma were observed at baseline. But after the DNC test, the concentration of DA metabolites in CSF and plasma samples was lower in MitoPark mice than in control mice.
Researchers validated the initial results using a different mouse model of PD generated with 6-hydroxidopamine (6-OHDA)-induced selective loss of DA neurons in C57BL/6J mice. Unilateral injection of 6-OHDA in the striatum resulted in a 57% loss of DA neurons on the injected side and an overall 29% loss considering both sides. Results with this second model of PD confirmed the data acquired using MitoPark mice. At baseline, control and lesioned mice had similar levels of DA metabolites in plasma. After the drug challenge, plasma levels of DA metabolites were significantly lower in lesioned mice than in control mice.
With this set of experiments, Zhao et al. confirmed their hypothesis that a homeostatic increase in DA release compensates for ongoing neurodegeneration. In addition, data demonstrate that disruption to this compensatory mechanism can help detect early-stage PD in mice.
Researchers continuined on to answer one final question: How early can the DNC test detect PD in MitoPark mice? This preclinical model of PD shows age-dependent progressive neurodegeneration, and at 15 weeks of age, they don’t display any loss of DA neurons. The only observable pathological change is a 44% loss of TH+ axonal terminals in the dorsal striatum, which can be considered an ultra-early stage of PD.
The new set of experiments with 15-week-old MitoPark mice yielded results similar to those acquired with 20-week-old animals. At baseline, control and 15-week-old MitoPark mice had similar levels of DA metabolites in the CSF and plasma. However, after the drug challenge, 15-week-old MitoPark mice showed lower levels of DA metabolites in CSF and plasma than control mice.
The long-term goal of preclinical research is to translate findings into interventions that advance the clinical management of a disease. And data from Zhao et al.’s study show that the DNC test has the potential to meet this requirement. First, the test had a good safety profile when administered to mice. Although haloperidol inhibited mice locomotor activity, its co-administration with methylphenidate restored locomotor activity to normal levels. Overall, the DNC test was safe, and changes in motor function were mild and reversible.
The DNC test has the potential to be used in a clinical setting as both drugs used in the test are approved by the FDA. The combination of haloperidol and methylphenidate can also help improve the safety profile of the DNC test application in humans, as haloperidol alone can cause extrapyramidal symptoms in elderly adults. In addition, the DNC is relatively easy to be performed in humans as plasma collection is minimally invasive. If needed, CSF collection can also be performed, despite being more invasive.
Clinical diagnostic interventions need to be sensitive and specific. Sensitivity refers to a test’s ability to correctly identify patients with a disease. Specificity quantifies how precisely a test can identify people without a disease. When using CSF samples from 20-week-old MitoPark mice with 28% loss of DA neurons, the DNC Test showed a sensitivity and specificity of 100%. In those same animals, plasma sample analyses resulted in a sensitivity of 100% and specificity of 82%.
Preclinical studies are the first step in developing new diagnostic and therapeutic strategies. Data from Zhou et al.’s study serve as a proof of concept that the DNC Test can detect dopaminergic dysfunction in early and ultra-early PD in mice. Future studies are needed to confirm DNC test safety and feasibility for implementation in a clinical setting.
Check out other pieces on the blog to keep learning about relevant scientific advances and tips and tricks to optimize your experiments.