Dopamine-dependent early synaptic and motor dysfunctions induced by α-synuclein in the nigrostriatal circuit

doi:10.1093/brain/awab242

. 2021 Dec 16;144(11):3477-3491.

doi: 10.1093/brain/awab242.

Dopamine-dependent early synaptic and motor dysfunctions induced by α-synuclein in the nigrostriatal circuit

Alessandro Tozzi¹, Miriam Sciaccaluga¹, Vittorio Loffredo^{1

2}, Alfredo Megaro¹, Ada Ledonne³, Antonella Cardinale^{4

5}, Mauro Federici³, Laura Bellingacci¹, Silvia Paciotti¹, Elena Ferrari⁶, Antonino La Rocca², Alessandro Martini³, Nicola B Mercuri^{3

7}, Fabrizio Gardoni⁶, Barbara Picconi^{4

8}, Veronica Ghiglieri⁸, Elvira De Leonibus^{2

9}, Paolo Calabresi¹⁰

Affiliations

¹ Department of Medicine and Surgery, University of Perugia, 06132 Perugia, Italy.
² Institute of Biochemistry and Cell Biology - IBBC, CNR, 00015 Monterotondo scalo, Italy.
³ Laboratory of Experimental Neuroscience, Santa Lucia Foundation IRCCS, 00143 Rome, Italy.
⁴ Department of Experimental Neurophysiology, IRCCS San Raffaele, 00166 Rome, Italy.
⁵ Neurology, Department of Neuroscience, Faculty of Medicine, Università Cattolica del "Sacro Cuore", 00168 Rome, Italy.
⁶ Department of Pharmacological and Biomolecular Sciences, University of Milan, 20133 Milan, Italy.
⁷ Neurology Unit, Department of Systems Medicine, University of Rome "Tor Vergata", 00133 Rome, Italy.
⁸ San Raffaele University, 00166 Rome, Italy.
⁹ Telethon Institute of Genetics and Medicine, 80078 Pozzuoli, Italy.
¹⁰ Neurological Clinic, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, 00168 Rome, Italy.

PMID: 34297092
PMCID: PMC8677552
DOI: 10.1093/brain/awab242

Dopamine-dependent early synaptic and motor dysfunctions induced by α-synuclein in the nigrostriatal circuit

Alessandro Tozzi et al. Brain. 2021.

. 2021 Dec 16;144(11):3477-3491.

doi: 10.1093/brain/awab242.

Authors

Affiliations

¹ Department of Medicine and Surgery, University of Perugia, 06132 Perugia, Italy.
² Institute of Biochemistry and Cell Biology - IBBC, CNR, 00015 Monterotondo scalo, Italy.
³ Laboratory of Experimental Neuroscience, Santa Lucia Foundation IRCCS, 00143 Rome, Italy.
⁴ Department of Experimental Neurophysiology, IRCCS San Raffaele, 00166 Rome, Italy.
⁵ Neurology, Department of Neuroscience, Faculty of Medicine, Università Cattolica del "Sacro Cuore", 00168 Rome, Italy.
⁶ Department of Pharmacological and Biomolecular Sciences, University of Milan, 20133 Milan, Italy.
⁷ Neurology Unit, Department of Systems Medicine, University of Rome "Tor Vergata", 00133 Rome, Italy.
⁸ San Raffaele University, 00166 Rome, Italy.
⁹ Telethon Institute of Genetics and Medicine, 80078 Pozzuoli, Italy.
¹⁰ Neurological Clinic, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, 00168 Rome, Italy.

PMID: 34297092
PMCID: PMC8677552
DOI: 10.1093/brain/awab242

Abstract

Misfolding and aggregation of α-synuclein are specific features of Parkinson's disease and other neurodegenerative diseases defined as synucleinopathies. Parkinson's disease progression has been correlated with the formation and extracellular release of α-synuclein aggregates, as well as with their spread from neuron to neuron. Therapeutic interventions in the initial stages of Parkinson's disease require a clear understanding of the mechanisms by which α-synuclein disrupts the physiological synaptic and plastic activity of the basal ganglia. For this reason, we identified two early time points to clarify how the intrastriatal injection of α-synuclein-preformed fibrils in rodents via retrograde transmission induces time-dependent electrophysiological and behavioural alterations. We found that intrastriatal α-synuclein-preformed fibrils perturb the firing rate of dopaminergic neurons in the substantia nigra pars compacta, while the discharge of putative GABAergic cells of the substantia nigra pars reticulata is unchanged. The α-synuclein-induced dysregulation of nigrostriatal function also impairs, in a time-dependent manner, the two main forms of striatal synaptic plasticity, long-term potentiation and long-term depression. We also observed an increased glutamatergic transmission measured as an augmented frequency of spontaneous excitatory synaptic currents. These changes in neuronal function in the substantia nigra pars compacta and striatum were observed before overt neuronal death occurred. In an additional set of experiments, we were able to rescue α-synuclein-induced alterations of motor function, striatal synaptic plasticity and increased spontaneous excitatory synaptic currents by subchronic treatment with l-DOPA, a precursor of dopamine widely used in the therapy of Parkinson's disease, clearly demonstrating that a dysfunctional dopamine system plays a critical role in the early phases of the disease.

Keywords: Parkinson’s disease; long-term depression; long-term potentiation; substantia nigra; synaptic plasticity.

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Figures

**Figure 1**
**Graphical representation of the general experimental plan; procedure for aggregating α-syn and time-dependent immunofluorescence striatal characterization of the α-syn-PFF-injected rat model.** (A) Scheme representing the timeline of the experimental procedures and the organization of the experimental groups. Rats were injected with α-syn-PFF or PBS at 7–8 weeks of age. After injection, the rats were enrolled in behavioural tests and then used for immunofluorescence (IF), electrophysiological or amperometric experiments. (B) Time-course graph of α-syn (1 mg/ml) aggregation *in vitro*, as increase in thioflavin T fluorescent staining. Note that after 160 h incubation, complete stable aggregates of α-syn (preformed fibrils, α-syn-PFF) are formed. (C) Transmission electron microscopy images showing α-syn-PFF at different magnifications before sonication. (D) Representative coronal brain images (i–**iii**) of p-α-syn immunofluorescence in the striatum of (i) sham (PBS-injected) and α-syn-PFF-injected rats at (ii) 6 weeks- and (**iii**) 12 weeks-post-injection, showing the site of α-syn-PFF injections (20×, scale bar = 500 µm). The *insets* show higher magnification (digital zoom) of α-syn-positive aggregates (scale bar = 100 µm). Representative images (i′–**iii′**) of p-α-syn (red)/NeuroTrace^TM 455 (green) double immunolabelling in striatum of (i′) sham and α-syn-PFF-injected rats at (**ii′**) 6 weeks- and (**iii′**) 12 weeks-post-injection (20× objective, scale bar = 50 µm). The insets show examples of higher magnification (40× with 2× digital zoom, scale bar = 20 µm). (E) Representative images of coronal brain sections showing reduced TH immunoreactivity in the dorso-lateral (DL) and dorso-medial (DM) striatum of α-syn-PFF-injected rats (20×, scale bar = 500 µm). The *second* and *third row* show the DL and DM striatal regions at higher magnification (×20, scale bar = 50 µm) of the square areas outlined. The graph shows quantification of the TH immunoreactivity in the DL and DM striatum of sham- and α-syn-PFF-injected rats. Note the significant reduction of the TH staining both in the DL and DM striatum of 6- and 12-week-injected rats with respect to shams. Data are presented as mean ± SEM of the optical density, as a percentage of the control. Sham n = 8; α-syn-PFF 6 weeks n = 6; α-syn-PFF 12 weeks n = 6; **P < 0.01, ***P < 0.001. One-way ANOVA, followed by Bonferroni’s *post hoc* test.

**Figure 2**
**Striatal α-syn-PFF injection leads to a time-dependent increase of p-α-syn in the SNpc and VTA with parallel loss of SNpc TH-positive neurons.** (A) Representative photomicrographs of SNpc (*top*) and VTA sections (*bottom*) of Sham- (*left*) and α-syn-PFF-injected rats, at 6 (*middle*) and 12 weeks after the injection (*right*), showing DAPI, DAT and p-α-syn triple co-immunofluorescence staining. Note that p-α-syn is mainly detected in dopaminergic neurons, as revealed by DAT immunostaining (20× magnification, 2× digital zoom, scale bar = 100 μm). Graphs showing the counting of p-α-syn-positive cells of the SNpc (*top*) and VTA (*bottom*). Note the significant increase in the number of p-α-syn-positive neurons 12 weeks after α-syn-PFF injection compared with the sham (SNpc: sham n = 5; α-syn-PFF 6 weeks n = 9; α-syn-PFF 12 weeks n = 10. VTA: sham n = 5; α-syn-PFF 6 weeks n = 9; α-syn-PFF 12 weeks n = 10). (B) Representative photomicrographs sections, including the SNpc and VTA regions, of sham- (*left*) and α-syn-PFF-injected rats at 6 (*middle*) and 12 weeks (*right*) after injection, showing different degrees of TH immunofluorescence. *Left* graph shows the number of TH-positive neurons counted in the SNpc of α-syn-PFF-injected rats at 6 and 12 weeks post-injection (as a % of counts in shams). Note the significant reduction of TH-positive neurons in the SNpc of α-syn-PFF 12 weeks after the striatal injection (sham n = 5; α-syn-PFF 6 weeks n = 9; α-syn-PFF 12 weeks n = 12). The *right* graph shows the number of TH-positive neurons counted in the VTA of α-syn-PFF-injected rats at 6 and 12 weeks post-injection (as a % of counts in shams). Note that no significant differences between groups were found in the VTA (sham n = 5; α-syn-PFF 6 weeks n = 9; α-syn-PFF 12 weeks n = 12. *P < 0.05. One-way ANOVA followed by Bonferroni’s *post hoc* test).

**Figure 3**
**Intra-striatal α-syn-PFF injections lead to time-dependent onset of motor impairment in the grid-walking task and to early onset of anxiety-like behaviours.** (A and B) Graphs of the performance of α-syn-PFF-injected rats in the grid-walking task showing increasing latency to climb (A) and immobility time (B) at 6 and 12 weeks after α-syn-PFF injection compared with sham rats; note that comparisons were significant 12 weeks after the injection (latency to climb and immobility time sham n = 14, α-syn-PFF 6 weeks n = 11; α-syn-PFF 12 weeks n = 10, *P < 0.05, mean ± SEM. One-way ANOVA followed by Bonferroni’s *post hoc* test). (C) Representative track plots of the route to the top of the grid for sham rats (*left*) and α-syn-PFF-injected rats 6 weeks (*centre*) and 12 weeks (*right*) post-injection. (D–F) Graphs of the distance travelled (D), immobility time (E) and time spent in the centre of the arena (F) of α-syn-PFF-injected and sham rats tested using the open field task. Note the reduced time spent in the centre of the arena for α-syn-PFF-injected rats with respect to Shams in the second part of the test (300–600 s), suggesting anxiety-like behaviour (sham n = 15, α-syn-PFF 6 weeks n = 8, α-syn-PFF 12 weeks n = 16) *P < 0.05, mean ± SEM. One-way ANOVA followed by Bonferroni’s *post hoc* test. (G) Representative track plots of rats performing the open field test. Note the reduced time spent in the centre of the arena of α-syn-PFF-injected rats at 6 and 12 weeks after the injection with respect to shams.

**Figure 4**
**Electrical firing properties of nigral neurons and constant potential amperometry of striatal DA in slices of α-syn-PFF-injected and sham rats.** (A) Representative traces of spontaneous firing recorded in cell-attached patch-clamp in DA neurons of SNpc in sham (black traces) and α-syn-PFF-injected (cyan traces) animals at 6 (*left*) or 12 weeks (*right*) after injection. Scale bars = 20 pA, 0.5 s. (B) Graph of the mean spontaneous firing frequency of active DA neurons in SNpc recorded from sham and α-syn-PFF rats at 6 and 12 weeks post-injection, showing a significative increase in the mean frequency in neurons of 12 weeks α-syn-PFF-injected rats respect to the sham animals (6 weeks: sham n = 17, α-syn-PFF n = 9; 12 weeks: sham n = 17, α-syn-PFF n = 21). Values are expressed as mean ± SEM, *P < 0.05, Student’s t-test. (C) Bar graph showing the number of recorded DA cells and the percentage of silent versus active neurons in the SNpc of sham (grey) and α-syn-PFF-injected (cyan) rats at 6 or 12 weeks post-surgery (active cells: 6 weeks, sham 21/25, α-syn-PFF 9/22; 12 weeks, sham 18/27, α-syn-PFF 21/25, **P = 0.002, χ² test. (D) Representative traces of action potentials discharge in response to +50 and +150 pA current steps (*top*) and graphs of the number of action potentials (*bottom*) of SNpc DA neurons of sham and α-syn-PFF rats at 6 or 12 weeks after injection. Data are expressed as mean ± SEM, *P < 0.05, Student’s t-test. Scale bars = 40 mV, 0.5 s. (E) Representative traces of spontaneous firing recorded in GABAergic neurons of SNpr in sham (black traces) and α-syn-PFF (cyan traces) animals at 6 or 12 weeks post-injection. Scale bars = 25 pA, 250 ms. Graph of the mean spontaneous firing frequency of GABAergic neurons of the SNpr showing no differences between sham and α-syn-PFF rats at both 6 and 12 weeks post-injection (6 weeks: sham n = 15, α-syn-PFF n = 15; 12 weeks: sham n = 13, α-syn-PFF n = 21). (F) Representative traces (*left*) and graph (*right*) of constant potential amperometry of electrically-stimulated DA release performed in slices containing the dorsal striatum of sham and α-syn-PFF animals at 6 or 12 weeks after injection (6 weeks: sham n = 62, α-syn-PFF n = 70; 12 weeks: sham n = 77, α-syn-PFF n = 87). Data are expressed as mean ± SEM, **P < 0.01; ***P < 0.001, Student’s t-test. Scale bars = 50 pA, 0.2 s.

**Figure 5**
**Time course of LTD and LTP of striatal spiny projection neurons of α-syn-PFF- and PBS-injected rats at 6 and 12 weeks post-injection.** (A and B) Representative traces of superimposed evoked EPSCs recorded from a SPN before and 20 min after a high-frequency stimulation (HFS) protocol in sham (black traces) and α-syn-PFF-injected (cyan traces) animals at 6 weeks (A, *top*) or 12 weeks (B, *top*) from injection. The time-course graphs show the mean EPSC amplitudes, as a percentage of the baseline, of SPNs recorded before and for 20 min after an HFS (LTD protocol) in both sham and α-syn-PFF rats at 6 weeks (A) and 12 weeks (B) post-surgery. Note the reduced LTD in α-syn-PFF rats at 12 weeks post-injection (6 weeks: sham n = 6, α-syn-PFF n = 7; 12 weeks: sham n = 8, α-syn-PFF n = 9), **P < 0.01, two-way ANOVA. (C and D) Representative traces of superimposed evoked EPSCs recorded from a SPN in a Mg²⁺-free Krebs solution before and 20 min after an HFS protocol, in sham (black traces) and α-syn-PFF (cyan traces) animals at 6 weeks (C, *top*) or 12 weeks (D, *top*) from injection. The time-course graphs show the mean EPSC amplitudes, as a percentage of the baseline, of SPNs recorded before and for 20 min after the HFS (LTP protocol) in both sham and α-syn-PFF rats at 6 weeks (C) and 12 weeks (D) post-surgery. Note the absence of LTP in SPNs of α-syn-PFF rats at both 6 and 12 weeks post-surgery (6 weeks: sham n = 9; α-syn-PFF n = 13; 12 weeks: sham n = 9; α-syn-PFF n = 9), ***P < 0.001, two-way ANOVA. Scale bars = 100 pA, 20 ms.

**Figure 6**
**Effect of subchronic l-DOPA treatment on synaptic spontaneous currents, synaptic plasticity and motor performance of rats at 12 weeks post α-syn-PFF- or PBS-injection.** (A and B) Graphs of the performance of α-syn-PFF- and PBS-injected rats treated with l-DOPA in the grid-walking task show that the latency to climb (A) and the immobility time (B) at 12 weeks after α-syn-PFF injection are not significantly different, unlike the observations of α-syn-PFF and sham rats that were naïve to l-DOPA (naïve to l-DOPA: sham n = 14, α-syn-PFF n = 10, *P < 0.05; l-DOPA: sham n = 7, α-syn-PFF n = 6, one-way ANOVA followed by Bonferroni’s *post hoc* test). (C) Representative superimposed evoked EPSCs recorded from a SPN before and 20 min after a high-frequency stimulation (HFS) protocol in sham (black traces) and α-syn-PFF (cyan traces) animals at 12 weeks from injection. Scale bar 100 pA, 20 ms. Time-course graphs showing the mean EPSC amplitude, as a percentage of the baseline, of SPNs recorded before and for 20 min after an HFS (LTD protocol) in both sham and α-syn-PFF rats at 12 weeks post-surgery. Note the restored LTD in α-syn-PFF rats treated with l-DOPA (naïve to l-DOPA: α-syn-PFF n = 9; l-DOPA: sham n = 8; α-syn-PFF n = 8), **P < 0.01, two-way ANOVA. (D) Representative superimposed evoked EPSCs recorded from an SPN before and 20 min after the HFS protocol in Mg²⁺-free external solution in sham (black traces) and α-syn-PFF (cyan traces) animals at 12 weeks from injection. Scale bar = 100 pA, 20 ms. Time-course graphs show the mean EPSC amplitude, as percentage of the baseline, of SPNs recorded before and for 20 min after the HFS (LTP protocol) in both l-DOPA-treated sham and α-syn-PFF rats at 12 weeks post-surgery. Note that, after the l-DOPA treatment, the impaired LTP observed in SPNs of α-syn-PFF rats at 12 weeks post-surgery was completely restored (naïve to l-DOPA: α-syn-PFF n = 9; l-DOPA: sham n = 7; α-syn-PFF n = 11, ***P < 0.001, two-way ANOVA). (E) Representative traces of sEPSCs of SPNs of sham and α-syn-PFF rats at 12 weeks post-injection in the absence (*left*) or presence (*right*) of l-DOPA treatment. Scale bar = 20 pA, 1 s. Graphs of the sEPSC mean amplitude (*left*) and frequency (*right*) of SPNs recorded from sham and α-syn-PFF rats at 12 weeks post-injection in the presence or in the absence of l-DOPA treatment. Note that the increase of sEPSC frequency in α-syn-PFF rats is completely reverted by l-DOPA treatment (naïve to l-DOPA: sham n = 11; α-syn-PFF n = 28. l-DOPA: sham n = 9; α-syn-PFF n = 10), **P < 0.01, Student’s t-test.

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References

1. Spillantini MG, Goedert M.. Synucleinopathies: Past, present and future. Neuropathol Appl Neurobiol. 2016;42(1):3–5. - PubMed
1. Luk KC, Song C, O’Brien P, et al.Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc Natl Acad Sci U S A. 2009;106(47):20051–20056. - PMC - PubMed
1. Hijaz BA, Volpicelli-Daley LA.. Initiation and propagation of alpha-synuclein aggregation in the nervous system. Mol Neurodegener. 2020;15(1):19. - PMC - PubMed
1. Cremades N, Cohen SI, Deas E, et al.Direct observation of the interconversion of normal and toxic forms of alpha-synuclein. Cell. 2012;149(5):1048–1059. - PMC - PubMed
1. Ghiglieri V, Calabrese V, Calabresi P.. Alpha-synuclein: From early synaptic dysfunction to neurodegeneration. Front Neurol. 2018;9:295. - PMC - PubMed

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[1] Spillantini MG, Goedert M.. Synucleinopathies: Past, present and future. Neuropathol Appl Neurobiol. 2016;42(1):3–5. - PubMed

[2] Spillantini MG, Goedert M.. Synucleinopathies: Past, present and future. Neuropathol Appl Neurobiol. 2016;42(1):3–5. - PubMed

[3] Luk KC, Song C, O’Brien P, et al.Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc Natl Acad Sci U S A. 2009;106(47):20051–20056. - PMC - PubMed

[4] Luk KC, Song C, O’Brien P, et al.Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc Natl Acad Sci U S A. 2009;106(47):20051–20056. - PMC - PubMed

[5] Hijaz BA, Volpicelli-Daley LA.. Initiation and propagation of alpha-synuclein aggregation in the nervous system. Mol Neurodegener. 2020;15(1):19. - PMC - PubMed

[6] Hijaz BA, Volpicelli-Daley LA.. Initiation and propagation of alpha-synuclein aggregation in the nervous system. Mol Neurodegener. 2020;15(1):19. - PMC - PubMed

[7] Cremades N, Cohen SI, Deas E, et al.Direct observation of the interconversion of normal and toxic forms of alpha-synuclein. Cell. 2012;149(5):1048–1059. - PMC - PubMed

[8] Cremades N, Cohen SI, Deas E, et al.Direct observation of the interconversion of normal and toxic forms of alpha-synuclein. Cell. 2012;149(5):1048–1059. - PMC - PubMed

[9] Ghiglieri V, Calabrese V, Calabresi P.. Alpha-synuclein: From early synaptic dysfunction to neurodegeneration. Front Neurol. 2018;9:295. - PMC - PubMed

[10] Ghiglieri V, Calabrese V, Calabresi P.. Alpha-synuclein: From early synaptic dysfunction to neurodegeneration. Front Neurol. 2018;9:295. - PMC - PubMed

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Dopamine-dependent early synaptic and motor dysfunctions induced by α-synuclein in the nigrostriatal circuit

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Dopamine-dependent early synaptic and motor dysfunctions induced by α-synuclein in the nigrostriatal circuit

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