NEUROPLASTICITY IN THE NIGROSTRIATAL SYSTEM OF MPTP-TREATED MICE AT THE PRESYMPTOMATIC AND EARLY SYMPTOMATIC STAGES OF PARKINSONISM

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1 NEUROPLASTICITY IN E NIGROSTRIATAL SYSTEM OF MPTP-TREATED MICE AT E PRESYMPTOMATIC AND EARLY SYMPTOMATIC STAGES OF PARKINSONISM Michael V. Ugrumov Institute of Developmental Biology RAS, Moscow, Russia Russian Academy of Sciences

2 Brain plasticity Ernesto Lugaro ( ) V.M. Bekhterev ( ) The Italian psychiatrist E. Lugaro had introduced the term plasticity into the neurosciences considering that throughout life the anatomo-functional relations between neurons can be changed in an adaptive fashion to enable psychic maturation, learning, and even functional recovery after brain damage. the function of the damaged brain areas is restored due to the undamaged areas. This is accounted for by the reorganization of the interneuronal relations under the information, coming from the target organs. Berlucchi G. 22. J. History Neurosciences, 11,

3 Neuroplasticity in the nigrostriatal system of MPTP-treated mice at the presymptomatic and symptomatic s of parkinsonism Introduction. New insight into the pathogenesis of Parkinson s disease; Dopamine turnover in the nigrostriatal system of MPTP-treated mice at modeling Parkinson s disease at the preclinical and early clinical s with focus on neuroplasticity; Dopamine production by non-dopaminergic expressing individual enzymes of dopamine synthesis as a mechanism of neuroplasticity under a failure of dopaminergic neurons; Conclusions.

4 Bezard and Gross, Prog. Neurobiol. 55, ; Trends Neurosci. 26, ;. Ugrumov, M.V., 28. In: Handbook of Neurochemistry and Molecular Neurobiology. Neurotransmitter Systems, Springer, Heidelberg, pp New insight into the pathogenesis of Parkinson s disease %, % Dopamine in the striatum Dopamine loss Compensatory processes Threshold loss of dopamine Onset of the disease First symptoms Age (years) Pathogenesis: Neurodegeneration; Prion-like propagation; Neuroplasticity; Inflammation Unspecific mechanisms providing neuroprotection: Stimulation of secretion of the growth factors; Activation of the antioxidant systems. Specific mechanisms serving to maintain neurotransmission: Stimulation of neurotransmitter secretion; A reduction of the enzymatic degradation of neurotransmitters; Increase of the sensitivity of the target neurons.

5 Neuroplasticity in the nigrostriatal system of MPTP-treated mice at the presymptomatic and symptomatic s of parkinsonism Introduction. New insight into the pathogenesis of Parkinson s disease; Dopamine turnover in the nigrostriatal system of MPTP-treated mice at modeling Parkinson s disease at the preclinical and early clinical s with focus on neuroplasticity; Dopamine production by non-dopaminergic expressing individual enzymes of dopamine synthesis as a mechanism of neuroplasticity under a failure of dopaminergic neurons; Conclusions.

6 Dopamine content / number of neurons (% of control) Dopamine concentration / number of axons (% of control) Neurotoxic (MPTP) models of Parkinson s disease in mice: Preclinical and early clinical s % 1 Dopamine Striatum Axon 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, pretoxin) (glial cells) 1-methyl-4-pyridine (MPP +, toxin) Axons Clinical Preclinical Injections of MPTP: 1, 2 or 4 times at the individual dose of 12 mg/kg with 2-hours intervals between the injections. Decapitation: 2 weeks after the treatment Striatum Dopaminergic neurons Dopamine-sensitive neurons Substantia nigra Ugrumov M. et al. (211) Neuroscience 181, ; Kozina et al. (214) J Neurol. Sci. 34: % х12 2х12 4х12 N injections x 12 mg/kg MPTP Substantia nigra Cell bodies Dopamine Clinical Preclinical 1х12 2х12 4х12 N injections x 12 mg/kg MPTP Cell body

7 MPTP models in mice of PD at the preclinical and early clinical s: Prospect of the use Evaluation of the molecular mechanisms of neurodegeneration in the central and peripheral nervous system; Evaluation of the mechanisms of neuroplasticity in the central and peripheral nervous system; Search for mechanisms triggering a transition from preclinical to clinical ; Search for peripheral biomarkers for the development of preclinical diagnostics of Parkinson s disease; Development of the provocation test for the diagnostics of Parkinson s disease at the preclinical ; Development of preventive therapy in Parkinson s disease: to slow down neurodegeneration; to control and improve neuroplasticity; to deactivate triggers of motor disorders. Ugrumov (29) J Chem Neuroanat. 38, ; Ugrumov.(211) Neuroscience 181, ; Kozina et al. (214) J Neurol Sci. 34,198-27; Ugrumov (213) Adv Pharmacol. 68:37-91.

8 Nigrostriatal system in MPTP-treated mice: Evaluation of functional activity Substantia nigra Indicators of functional activity: Gene expression, synthesis and activity of tyrosine hydroxylase; Spontaneous and stimulated release of dopamine; Dopamine uptake; Content and turnover of dopamine; Dopamine synthesis by non-dopaminergic neurons; Gene expression and activity of MAO A and B; Gene expression of D2 receptors. The data obtained for the striatum and substantia nigra as a whole were adjusted to individual nigral cell bodies and striatal axons Cell body Axon Striatum

9 Functional activity of the nigrostriatal system in MPTP-treated mice at the presymptomatic and symptomatic s of parkinsonism Conclusions Compensatory processes: An increase of gene expression and activity of tyrosine hydroxylase (), An increase of dopamine (DA) release and turnover, A decrease of MAO B activity. Non-expected results: Regulations of transcription, translation and enzymatic activity of are different; An increase of activity despite and increase of D2 gene expression. Triggers of motor disorders: A decrease of DA stimulated release, An increase of DA uptake, An increase of MAO A activity Object Parameter Tyrosine hydroxylase mrna Tyrosine hydroxylase protein Tyrosine hydroxylase activity Dopamine content Dopamine release spontaneus / stimulated Dopamine uptake Dopamine turnover Neuron Cell body Presympt vs. control Axon Presympt vs. control _ / / Neuron Cell body Presympt vs. sympt. Axon Presympt vs. sympt. D2 receptor mrna / _ / Increase; Decrease; No change; _ No data Ugrumov et al. (211) Neuroscience 181, ; Kozina Ugrumov (214) J Neurol Sci, 34:

10 Neuroplasticity in the nigrostriatal system of MPTP-treated mice at the presymptomatic and symptomatic s of parkinsonism Introduction. New insight into the pathogenesis of Parkinson s disease; Dopamine turnover in the nigrostriatal system of MPTP-treated mice at modeling Parkinson s disease at the preclinical and early clinical s with focus on neuroplasticity; Dopamine production by non-dopaminergic expressing individual enzymes of dopamine synthesis as a mechanism of neuroplasticity under a failure of dopaminergic neurons; Conclusions.

11 Neurons partly expressing dopaminergic phenotype Dopaminergic neurons Striatum Hypothalamus Substantia nigra Pituitary Principal characteristics Non-dopaminergic neurons partly expressing dopaminergic phenotype Two enzymes of dopamine synthesis Dopamine membrane transporter Vesicular membrane transporter, type 2 Individual enzymes of dopamine synthesis No dopamine membrane transporter No vesicular membrane transporter, type 2 Meister B., Hökfelt T. (Sweden), Steinbusch H.W.M. (the Netherlands), Skagerberg G., Lindvall O., Geffard M. (France), Joh T., Cuello A.C., Goldstein M. (1988) Do tyrosine hydroxylase-immunoreactive neurons in the ventro-lateral arcuate nucleus produce dopamine or only L-DOPA? J. Chemical Neuroanat. 1, Hoffman, B.J., Hansson, S.R., Mezey, E., Palkovits, M. (1998). Localization and dynamic regulation of biogenic amine transporters in the mammalian central nervous system. Front. Neuroendocrinol. 19,

12 Striatum Arcuate nucleus Arcuate nucleus III ventricle Median eminence & (Ikemoto et al., 1997; Cossette et al., 25), aromatic L-amino acid decarboxylase; DAT, dopamine transporter;, tyrosine hydroxylase. Bienzymatic neuron Monoenzymatic neuron Monoenzymatic neuron

13 Dopamine (ng/sample) Dopamine (ng/sample) Dopamine in tissue (pg/mg) Dopamine release (pg/ml) Dopamine in cells (pg/dish) Dopamine release (pg/ml) Cooperative synthesis of dopamine by non-dopaminergic neurons in the arcuate nucleus in rat fetuses ТH 8% 19% ТH+ 1% Arcuate n. 21st embryonic day, ex vivo 3 mm KCl 6 mm KCl th embryonic day, culture 3 mm KCl 6 mm KCl L-Tyrosine Tyrosine hydroxylase Dopaminergic neurons < 1% Tyrosine hydroxylase & aromatic L-amino acid decarboxylase Non-dopaminergic neurons > 99% Dopamine Dopamine synthesis Aromatic L-amino acid decarboxylase.6 Arcuate n..5 STATIC INCUBATION Тyr (-) Тyr (+) Substantia nigra L-DOPA L-Tyrosine Melnikova, Ugrumov (1999) Neuroscience 89, ; Ugrumov et al. (24) Neuroscience 124,

14 Prolactin in plasma (ng/ml) Dopamine (ng / mg tiss) Number of neurons / section Dopamine synthesis by non-dopaminergic neurons as a compensatory mechanism under a failure of tuberoinfundibular dopaminergic neurons Arcuate nucleus ТH & 6-OHDA ТH& ТH& Control 6-OHDA III ventricle & 1 Median eminence Control 6-OHDA 5 6-OHDA.2.1 Norm 14 Ershov, Ugrumov et al., 25, J. Сhem. Neuroanat. 3, 27-33; Ugrumov, 28. In: Handbook of Neurochemistry and Molecular Neurobiology. Neurotransmitter Systems, Springer, Heidelberg, pp Days 14th day 45th day, aromatic L-amino acid decarboxylase; 6-OHDA, 6-hydroxydopamine;, tyrosine hydroxylase

15 Ugrumov, 28. In: Handbook of Neurochemistry and Molecular Neurobiology. Neurotransmitter Systems, Springer, Heidelberg, pp ; Ugrumov M., 213. Adv.Pharmacol. 68, Neurons expressing enzymes of dopamine synthesis in the striatum of rats at 6-OHDA-induced degeneration of nigral dopaminergic neurons 6-hydroxydopamine (6-OHDA) Striatum Substantia nigra Tyrosine hydroxylase Striatum, 6-OHDA Tyrosine hydroxylase Substantia nigra Striatum Tyrosine hydroxylase () Tyrosine hydroxylase Aromatic L-amino acid decarboxylase () Aromatic L-amino acid decarboxylase Control Striatum 6-OHDA 6-OHDA 6-OHDA (Lopez-Real et al., 23)

16 mrna content / striatum, % of control Nigrostriatal system in MPTP-treated mice: Neurons expressing individual enzymes of dopamine synthesis in the striatum Control Preclinical Clinical Fractions of mono- and bienzymatic nerve fibers In the striatum Tyrosine hydroxylase () + 24% 22% 34% 24% 29% 52% 24% 43% 47% Aromatic L-amino acid decarboxylase () & Control gene expression Conclusion: Degeneration of dopaminergic neurons is followed by an increase of the number of monoenzymatic fibers and an appearance of the neurons expressing tyrosine hydroxylase in the striatum Preclinical Preclinical Clinical Clinical

17 Cooperative synthesis of dopamine in the striatum of normal and parkinsonian mice Synthesis of dopamine (DA) in DA-ergic (bienzymatic) neurons L-tyrosine L-DOPA Cooperative synthesis of DA in nonda-ergic (monoenzymatic) neurons dopamine Design of the experiment Flow incubation of the striatal slices for 2 hours: Incubation with L-leucine Striatum Incubation without L-leucine L-DOPA LAT1 tyrosine hydroxylase, aromatic L-amino acid decarboxylase, LAT1 large neutral amino acid transporter. Collection of incubation medium and perfused slices High-performance liquid chromatography with electrochemical detection (HPLC-ED) L-leucine is a competitive inhibitor of LAT1 transporter, preventing L-DOPA uptake. Slices of substantia nigra, containing only bienzymatic neurons served as an additional control. pmol Total dopamine content in the striatum (slices) and medium following flow incubation without L-leucine with L-leucine % 2 1 A decrease of total dopamine content in the striatum (slices) and medium after the incubation with L-leucine 21% 3% 51% pmol 1 5 Total content of dopamine in substantia nigra (slices) and medium after flow incubation without L-leucine with L-leucine Control Preclinical Clinical NaCl 1x12 4x12 Control Preclinical Clinical NaCl 1x12 4x12 NaCl Control Clinical 4x12

18 Pathways for cooperative synthesis of dopamine Dopamine is produced by non-dopaminergic neurons expressing individual complementary enzymes of its synthesis in cooperation; L-Tyrosine Cooperative synthesis of dopamine is a compensatory reaction under a failure of dopaminergic neurons, mostly in neurodegenerative diseases Dopaminergic neuron L-DOPA Dopamine L-Tyrosine L-DOPA Dopamine L-DOPA transporter L-DOPA & TryH& L-tyrosine Non-dopaminergic neurons L-DOPA Dopamine Dopamine Dopamine, aromatic L-amino acid decarboxylase;, tyrosine hydroxylase; TryH, tryptophan hydroxylase Ugrumov, (28) Handbook of Neurochemistry and Molecular Neurobiology (A. Lajtha, ed.) Neurotransmitter Systems (E.S. Vizi, ed.), Springer, pp ; Ugrumov (29) J Chem Neuroanat. 38, ; Ugrumov (213) Adv.Pharmacol. 68,

19 Neuroplasticity in the nigrostriatal system of MPTP-treated mice at the presymptomatic and symptomatic s of parkinsonism Introduction. New insight into the pathogenesis of Parkinson s disease; Dopamine turnover in the nigrostriatal system of MPTP-treated mice at modeling Parkinson s disease at the preclinical and early clinical s with focus on neuroplasticity; Dopamine production by non-dopaminergic expressing individual enzymes of dopamine synthesis as a mechanism of neuroplasticity under a failure of dopaminergic neurons; Conclusions.

20 Conclusions Experimental models of Parkinson s disease at the preclinical and early clinical (PD) were developed using MPTP-treated mice; A model of PD at the early clinical strongly reproduces the pathogenesis in patients regarding gene expression of α-synuclein and major proteins involved in neurotransmission; Despite a certain degradation of dopaminergic system at the model of PD at the preclinical, motor disorders are prevented due to compensatory processes an increase of the gene expression and activity of tyrosine hydroxylase, an increase of dopamine (DA) release and a decrease of MAO B activity; Dopamine in the striatum is produced besides dopaminergic neurons by non-dopaminergic neurons expressing individual enzymes that is among most efficient mechanisms of neuroplasticity at PD; A transition of the presymptomatic to the symptomatic, manifested by the onset of motor disorders is provoked by a decrease of DA stimulated release and an increase of DA uptake and MAO A activity.

21 ACKNOWLEDGMENTS Laboratory of Neural and Neuroendocrine Regulations, Institute of Developmental Biology RAS, Moscow, Russia Thanks for your attention A. Sapronova (PhD, senior researcher), E. Volina (PhD, senior researcher) T. Pronina (PhD, senior researcher) E. Kozina (PhD, researcher) N. Bondarenko (PhD, researcher) G. Khakimova (PhD, researcher) L. Dilmuchametova (PhD, researcher) A. Kolacheva (PhD student) Y. Zubova (PhD student) A. Kim (PhD student) A. Kurina (PhD student) E. Mingazov (PhD student) V. Safandeev (PhD student) A. Murtazina (PhD student) Michael V. UGRUMOV Institute of Developmental Biology RAS, Moscow, Russia S. Surkov (Researcher) V. Kirillova (technician)