Dopamine Synthesis in the Nigrostriatal Dopaminergic System in Patients at Risk of Developing Parkinson's Disease at the Prodromal Stage.

Parkinson's disease (PD) is diagnosed by the onset of motor symptoms and treated long after its onset. Therefore, the development of the early diagnosis of PD is a priority for neurology. Advanced methodologies for this include (1) searching for patients at risk of developing prodromal PD based on premotor symptoms; (2) searching for changes in the body fluids in these patients as diagnostic biomarkers; (3) verifying the diagnosis of prodromal PD and diagnostic-value biomarkers using positron emission tomography (PET); (4) anticipating the development of motor symptoms. According to our data, the majority of patients (n = 14) at risk of developing PD selected in our previous study show pronounced interhemispheric asymmetry in the incorporation of 18F-DOPA into dopamine synthesis in the striatum. This was assessed for the caudate nucleus and putamen separately using the specific binding coefficient, asymmetry index, and putamen/caudate nucleus ratio. Interhemispheric asymmetry in the incorporation of 18F-DOPA into the striatum provides strong evidence for its dopaminergic denervation and the diagnostic value of previously identified blood biomarkers. Of the 17 patients at risk of developing prodromal PD studied using PET, 3 patients developed motor symptoms within a year. Thus, our study shows the promise of using the described methodology for the development of early diagnosis of PD.

Application of OpenArray Technology to Assess Changes in the Expression of Functionally Significant Genes in the Substantia Nigra of Mice in a Model of Parkinson's Disease.

Studying the molecular mechanisms of the pathogenesis of Parkinson's disease (PD) is critical to improve PD treatment. We used OpenArray technology to assess gene expression in the substantia nigra (SN) cells of mice in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD and in controls. Among the 11 housekeeping genes tested, Rps27a was taken as the reference gene due to its most stable expression in normal and experimental conditions. From 101 genes encoding functionally significant proteins of nigrostriatal dopaminergic neurons, 57 highly expressed genes were selected to assess their expressions in the PD model and in the controls. The expressions of Th, Ddc, Maoa, Comt, Slc6a3, Slc18a2, Drd2, and Nr4a2 decreased in the experiment compared to the control, indicating decreases in the synthesis, degradation, and transport of dopamine and the impaired autoregulation of dopaminergic neurons. The expressions of Tubb3, Map2, Syn1, Syt1, Rab7, Sod1, Cib1, Gpx1, Psmd4, Ubb, Usp47, and Ctsb genes were also decreased in the MPTP-treated mice, indicating impairments of axonal and vesicular transport and abnormal functioning of the antioxidant and ubiquitin-proteasome systems in the SN. The detected decreases in the expressions of Snca, Nsf, Dnm1l, and Keap1 may serve to reduce pathological protein aggregation, increase dopamine release in the striatum, prevent mitophagy, and restore the redox status of SN cells.

Compensatory Processes in Striatal Neurons Expressing the Tyrosine Hydroxylase Gene in Transgenic Mice in a Model of Parkinson's Disease.

The mammalian striatum is known to contain non-dopaminergic neurons that express dopamine (DA)-synthesizing enzymes and produce DA, responsible for the regulation of motor function. This study assessed the expression of DA-synthesizing enzymes in striatal neurons and their role in DA synthesis in transgenic mice expressing the green fluorescent protein (GFP) gene under the tyrosine hydroxylase (TH) gene promoter in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease (PD). We showed that, in Parkinsonian animals, the number of neurons expressing the TH gene increased by 1.9 times compared with the control (0.9% NaCl), which indicates a compensatory response to the DAergic denervation of the striatum. This assumption is supported by a 2.5-fold increase in the expression of genes for TH and transcription factor Nurr1 and a 1.45-fold increase in the expression of the large amino acid transporter 1 gene. It is noteworthy that, in Parkinsonian mice, in contrast to the controls, DA-synthesizing enzymes were found not only in nerve fibers but also in neuronal cell bodies. Indeed, TH or TH and aromatic L-amino acid decarboxylase (AADC) were detected in GFP-positive neurons, and AADC was detected in GFP-negative neurons. These neurons were shown to synthesize DA, and this synthesis is compensatorily increased in Parkinsonian mice. The above data open the prospect of improving the treatment of PD by maintaining DA homeostasis in the striatum.

Searching for Biomarkers in the Blood of Patients at Risk of Developing Parkinson's Disease at the Prodromal Stage.

Parkinson's disease (PD) is diagnosed many years after its onset, under a significant degradation of the nigrostriatal dopaminergic system, responsible for the regulation of motor function. This explains the low effectiveness of the treatment of patients. Therefore, one of the highest priorities in neurology is the development of the early (preclinical) diagnosis of PD. The aim of this study was to search for changes in the blood of patients at risk of developing PD, which are considered potential diagnostic biomarkers. Out of 1835 patients, 26 patients were included in the risk group and 20 patients in the control group. The primary criteria for inclusion in a risk group were the impairment of sleep behavior disorder and sense of smell, and the secondary criteria were neurological and mental disorders. In patients at risk and in controls, the composition of plasma and the expression of genes of interest in lymphocytes were assessed by 27 indicators. The main changes that we found in plasma include a decrease in the concentrations of l-3,4-dihydroxyphenylalanine (L-DOPA) and urates, as well as the expressions of some types of microRNA, and an increase in the total oxidative status. In turn, in the lymphocytes of patients at risk, an increase in the expression of the DA D3 receptor gene and the lymphocyte activation gene 3 (LAG3), as well as a decrease in the expression of the Protein deglycase DJ-1 gene (PARK7), were observed. The blood changes we found in patients at risk are considered candidates for diagnostic biomarkers at the prodromal stage of PD.

LUHMES Cells: Phenotype Refinement and Development of an MPP+-Based Test System for Screening Antiparkinsonian Drugs.

The low effectiveness of symptomatic pharmacotherapy for Parkinson's disease (PD), which compensates for dopamine (DA) deficiency under degeneration of nigrostriatal dopaminergic (DAergic) neurons, could apparently be improved with neuroprotective therapy, which slows down neurodegeneration and PD progression. For this, it is necessary to have a DAergic cell line for the development of a PD model to screen neuroprotectors. We used immortalized human embryonic mesencephalon LUHMES cells (LCs) differentiated into DAergic neurons. The aim of this study was to characterize the phenotype of differentiated LCs and develop an 1-methyl-4-phenylpyridinium iodide (MPP+)-based test system for screening neuroprotectors. Using polymerase chain reaction (PCR) and immunocytochemistry, it has been shown that all differentiated LCs express genes and synthesize proteins characteristic of all neurons (microtubule-associated protein 2, bIII-tubulin, synaptotagmin 1) and specifically of DAergic neurons (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, DA transporter, vesicular monoamine transporter 2). Furthermore, LCs are able to produce a small amount of DA, but under special conditions. To assess the mechanisms of neurodegeneration and neuroplasticity under the influence of toxins and antiparkinsonian drugs, including neuroprotectors, we have developed an LCs-based MPP+ PD model and proposed an original panel of markers for testing functional and structural cell disorders.

Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson's disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.

Striatal Neurons Partially Expressing a Dopaminergic Phenotype: Functional Significance and Regulation.

Since the discovery of striatal neurons expressing dopamine-synthesizing enzymes, researchers have attempted to identify their phenotype and functional significance. In this study, it was shown that in transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase (TH) gene promoter, (i) there are striatal neurons expressing only TH, only aromatic L-amino acid decarboxylase (AADC), or both enzymes of dopamine synthesis; (ii) striatal neurons expressing dopamine-synthesizing enzymes are not dopaminergic since they lack a dopamine transporter; (iii) monoenzymatic neurons expressing individual complementary dopamine-synthesizing enzymes produce this neurotransmitter in cooperation; (iv) striatal nerve fibers containing only TH, only AADC, or both enzymes project into the lateral ventricles, providing delivery pathways for L-3,4-dihydroxyphenylalanine and dopamine to the cerebrospinal fluid; and (v) striatal GFP neurons express receptor genes for various signaling molecules, i.e., classical neurotransmitters, neuropeptides, and steroids, indicating fine regulation of these neurons. Based on our data, it is assumed that the synthesis of dopamine by striatal neurons is a compensatory response to the death of nigral dopaminergic neurons in Parkinson's disease, which opens broad prospects for the development of a fundamentally novel antiparkinsonian therapy.

The Periventricular Nucleus as a Brain Center Containing Dopaminergic Neurons and Neurons Expressing Individual Enzymes of Dopamine Synthesis.

Since the 1980s, the concept of dopamine-rich brain centers as clusters of only dopaminergic neurons has been fundamentally revised. It has been shown that, in addition to dopaminergic neurons, most of these centers contain neurons expressing one of the enzymes of dopamine synthesis: tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC). We have obtained convincing evidence that in rats, the hypothalamic periventricular nucleus (PeVN) is one of the largest dopamine-rich centers, containing dopaminergic and monoenzymatic neurons. Indeed, using double immunostaining for TH and AADC, the PeVN was shown to contain almost three thousand dopaminergic and monoenzymatic neurons. According to high-performance liquid chromatography, PeVN contains L-DOPA and dopamine, which, apparently, are synthesized in monoenzymatic TH neurons and bienzymatic neurons, respectively. According to confocal microscopy, neurons (cell bodies, fibers), which were immunopositive only to TH, only to AADC, or both, are in close topographic relationships with each other and with the 3rd ventricle. These data suggest the mutual regulation of the neurons, as well as the delivery of dopamine and L-DOPA to the third ventricle, which is confirmed by their detection in the cerebrospinal fluid. Thus, evidence has been obtained that PeVN is one of the largest dopamine-rich centers of the brain, containing dopaminergic and monoenzymatic neurons.

A New Method for the Visualization of Living Dopaminergic Neurons and Prospects for Using It to Develop Targeted Drug Delivery to These Cells.

This is the first study aiming to develop a method for the long-term visualization of living nigrostriatal dopaminergic neurons using 1-(2-(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine-BODIPY (GBR-BP), the original fluorescent substance, which is a derivative of GBR-12909, a dopamine uptake inhibitor. This method is based on the authors' hypothesis about the possibility of specifically internalizing into dopaminergic neurons substances with a high affinity for the dopamine transporter (DAT). Using a culture of mouse embryonic mesencephalic and LUHMES cells (human embryonic mesencephalic cells), as well as slices of the substantia nigra of adult mice, we have obtained evidence that GBR-BP is internalized specifically into dopaminergic neurons in association with DAT via a clathrin-dependent mechanism. Moreover, GBR-BP has been proven to be nontoxic. As we have shown in a primary culture of mouse metencephalon, GBR-BP is also specifically internalized into some noradrenergic and serotonergic neurons, but is not delivered to nonmonoaminergic neurons. Our data hold great promise for visualization of dopaminergic neurons in a mixed cell population to study their functioning, and can also be considered a new approach for the development of targeted drug delivery to dopaminergic neurons in pathology, including Parkinson's disease.

The Sphingolipid Asset Is Altered in the Nigrostriatal System of Mice Models of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disease incurable due to late diagnosis and treatment. Therefore, one of the priorities of neurology is to study the mechanisms of PD pathogenesis at the preclinical and early clinical stages. Given the important role of sphingolipids in the pathogenesis of neurodegenerative diseases, we aimed to analyze the gene expression of key sphingolipid metabolism enzymes (ASAH1, ASAH2, CERS1, CERS3, CERS5, GBA1, SMPD1, SMPD2, UGCG) and the content of 32 sphingolipids (subspecies of ceramides, sphingomyelins, monohexosylceramides and sphinganine, sphingosine, and sphingosine-1-phosphate) in the nigrostriatal system in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models of the preclinical and clinical stages of PD. It has been shown that in PD models, the expression of five of the nine studied genes (CERS1, CERS5, ASAH1, ASAH2, and GBA1) increases but only in the substantia nigra (SN) containing dopaminergic cell bodies. Changes in the expression of enzyme genes were accompanied by an increase in the content of 7 of the 32 studied sphingolipids. Such findings suggest these genes as attractive candidates for diagnostic purposes for preclinical and clinical stages of PD.

A Pilot Study of Changes in the Level of Catecholamines and the Activity of α-2-Macroglobulin in the Tear Fluid of Patients with Parkinson's Disease and Parkinsonian Mice.

Development of differential and early (preclinical) diagnostics of Parkinson's disease (PD) is among the priorities in neuroscience. We searched for changes in the level of catecholamines and α-2-macroglobulin activity in the tear fluid (TF) in PD patients at an early clinical stage. It was shown that TF in patients is characterized by an increased level of noradrenaline mainly on the ipsilateral side of pronounced motor symptoms (72%, p = 0.049), a decreased level of adrenaline on both sides (ipsilateral-53%, p = 0.004; contralateral-42%, p = 0.02), and an increased α-2-macroglobulin activity on both sides (ipsilateral-53%, p = 0.03; contralateral-56%, p = 0.037) compared to controls. These changes are considered as potential biomarkers for differential diagnosis. Similar changes in the TF were found in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice when modeling clinical and preclinical stages of PD. These data show the adequacy of models to the pathogenesis of PD along the selected metabolic pathways, and also suggest that the found TF changes can be considered as potential biomarkers for preclinical diagnosis of PD. In Parkinsonian mice, the level of catecholamines also changes in the lacrimal glands, which makes it possible to consider them as one of the sources of catecholamines in the TF.