Initial Molecular Mechanisms of the Pathogenesis of Parkinson's Disease in a Mouse Neurotoxic Model of the Earliest Preclinical Stage of This Disease.

Studying the initial molecular mechanisms of the pathogenesis of Parkinson's disease (PD), primarily in the nigrostriatal dopaminergic system, is one of the priorities in neurology. Of particular interest is elucidating these mechanisms in the preclinical stage of PD, which lasts decades before diagnosis and is therefore not available for study in patients. Therefore, our main goal was to study the initial molecular mechanisms of the pathogenesis of PD in the striatum, the key center for dopamine regulation in motor function, in a mouse model of the earliest preclinical stage of PD, from 1 to 24 h after the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). It was shown that the content of tyrosine hydroxylase (TH), the first enzyme in dopamine synthesis, does not change within 6 h after the administration of MPTP, but decreases after 24 h. In turn, TH activity increases after 1 h, decreases after 3 h, remains at the control level after 6 h, and decreases 24 h after the administration of MPTP. The concentration of dopamine in the striatum gradually decreases after MPTP administration, despite a decrease in its degradation. The identified initial molecular mechanisms of PD pathogenesis are considered as potential targets for the development of preventive neuroprotective treatment.

The Gene Expression of Proteins Involved in Intercellular Signaling and Neurodegeneration in the Substantia Nigra in a Mouse Subchronic Model of Parkinson's Disease.

Given the limited access to clinical material for studying the pathogenesis of Parkinson's disease (PD), these studies should be carried out on experimental models. We have recently developed a subchronic model of the progressive development of PD with a gradual transition from the preclinical (asymptomatic) stage to the clinical (symptomatic) one. The aim of this study was to evaluate changes in the expression of a wide range of genes in the substantia nigra (SN), the central link in the regulation of motor function, in mice in our subchronic model of PD. We have found changes in the expression of a number of genes encoding enzymes involved in the synthesis and degradation of dopamine as well as proteins involved in the vesicular cycle, axonal transport, protein degradation in the proteasome system, neuroinflammation, and cell death in the SN of our mouse model of the clinical stage of PD. Similar changes in gene expression were previously demonstrated in patients (postmortem), indicating good reproducibility of PD in our model. Further analysis of the gene expression in the SN of mice has shown that the expression of some genes also changes in the model of the preclinical stage, when dopaminergic neurons have not yet died. Thus, this study opens up broad prospects for further evaluation of the molecular mechanisms of PD pathogenesis and the development of a test system for drug screening.

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.

Development of early diagnosis of Parkinson's disease on animal models based on the intranasal administration of α-methyl-p-tyrosine methyl ester in a gel system.

The fight against neurodegenerative diseases, including Parkinson's disease (PD), is a global challenge of this century. The effectiveness of current PD therapy is limited, since it is diagnosed many years after the onset, following the death of most nigrostriatal dopaminergic neurons regulating motor function. PD treatment could be greatly improved if it was started at an early (preclinical) stage. For this purpose, it is necessary to develop an early diagnosis of PD, which is the goal of our study. We have developed an early diagnosis of PD on animal models using a provocative test by intranasal administration of α-methyl-p-tyrosine methyl ester (αMPTME), a reversible inhibitor of dopamine synthesis. First, we produced the provocative agent, αMPTME in gel, and showed its safety and penetration into the brain bypassing the blood-brain barrier. Then, the optimal dose of αMPTME and time after administration were selected, at which the level of dopamine in the striatum of intact animals decreases, but does not reach the 30% threshold for the appearance of motor disorders in PD patients. Finally, we proved on animal models that intranasal administration of αMPTME can serve as a diagnostic test for preclinical PD. Indeed, intranasal administration of αMPTME to mice in a model of PD at the preclinical stage reversibly reduced the dopamine level in the striatum to the 30% threshold causing short-term motor disorders. Thus, using animal models of PD, we have developed a provocative test for the preclinical diagnosis of PD, a fundamentally new technology in neurology.

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.

Modeling of the Progressive Degradation of the Nigrostriatal Dopaminergic System in Mice to Study the Mechanisms of Neurodegeneration and Neuroplasticity in Parkinson's Disease.

The fight against neurodegenerative diseases, including Parkinson's disease (PD), is among the global challenges of the 21st century. The low efficiency of therapy is due to the late diagnosis and treatment of PD, which take place when there is already significant degradation of the nigrostriatal dopaminergic system, a key link in the regulation of motor function. We have developed a subchronic mouse model of PD by repeatedly administering 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at gradually increasing doses with a 24 h interval between injections, a period comparable to the time of MPTP metabolism and elimination from the body. This model reproduces the main hallmarks of PD: progressive degeneration of dopaminergic neurons; the appearance of motor disorders with a 70-80% decrease in the level of dopamine in the striatum; an increase in dopamine turnover in the striatum to compensate for dopamine deficiency. When comparing the degradation of the nigrostriatal dopaminergic system and motor disorders in mice in the acute and subchronic models of PD, it has turned out that the resistance of dopaminergic neurons to MPTP increases with its repeated administration. Our subchronic model of PD opens up broad prospects for studying the molecular mechanisms of PD pathogenesis and developing technologies for early diagnosis and preventive treatment.

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.