Optogenetic-induced α-synuclein accumulation reveals early synaptic dysfunction in experimental models of Parkinson’s disease

Rodriguez-Aller, Raquel; Romero-Quineche, Beatriz; Morissette, Marc; Sheta, Razan; Di Paolo, Thérèse; Saghatelyan, Armen; Oueslati, Abid

doi:10.1038/s41531-025-01201-x

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Published: 13 December 2025

Optogenetic-induced α-synuclein accumulation reveals early synaptic dysfunction in experimental models of Parkinson’s disease

Raquel Rodriguez-Aller^1,2,3,
Beatriz Romero-Quineche³,
Marc Morissette²,
Razan Sheta²,
Thérèse Di Paolo^2,4,
Armen Saghatelyan³ &
…
Abid Oueslati^1,2

npj Parkinson's Disease volume 11, Article number: 356 (2025) Cite this article

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Abstract

Presynaptic accumulation of misfolded α-synuclein (α-syn) and altered synaptic transmission are considered early events in the pathogenesis of Parkinson’s disease (PD), suggesting a potential causal link between these two events. However, the mechanisms by which α-syn aggregation induces synaptic dysfunction and the subsequent progressive neurodegeneration remain elusive. In the present study we leveraged the high temporal resolution of the Light-Inducible Protein Aggregation (LIPA) system in vivo and in human dopaminergic neurons to explore the early sequence of α-syn-induced pathological events leading to synaptopathy. We observed that nigrostriatal axonal transport and presynaptic accumulation of α-syn aggregates altered the activity of different neuronal populations in the mouse striatum. The results of histological and metabolite analyses show that presynaptic accumulation of α-syn induced a shift in the activation pattern of D1- and D2-expressing striatal medium spiny neurons, caused an increase in the size and density of dopaminergic synapses, and disrupted striatal dopamine signaling. Altogether, our findings reveal that the accumulation of α-syn in dopaminergic terminals triggered early presynaptic impairments, which subsequently altered striatal neuronal activity. Our study provides new insights into the molecular mechanisms underlying early synaptopathy in PD.

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Introduction

α-Synuclein (α-syn) is a ubiquitous protein, particularly enriched at neuronal presynaptic terminals^1,2, where it exists in equilibrium between a soluble monomeric form and a membrane-bound, high-order multimeric state^3,4. Under physiological conditions, α-syn plays key roles in the synaptic vesicle cycle, including promoting SNARE complex formation, regulating synaptic vesicle trafficking, participating in neurotransmitter release and endocytosis processes, and controlling the clustering of synaptic vesicles within distinct vesicle pools^{2,5,6,7,8,9,10}. Pathologically aggregated α-syn is the primary component of intracellular inclusions known as Lewy bodies (LBs) and Lewy neurites, hallmark features of Parkinson’s disease (PD) and other synucleinopathies, including PD with dementia (PDD), dementia with LBs (DLB), and multiple system atrophy (MSA)^11,12,13,14.

Growing evidence suggests that synaptic dysfunction is an early event in the pathogenesis of PD, occurring prior to nigrostriatal axonal and neuronal degeneration^15,16, supporting the “dying-back” hypothesis^15,17. Additionally, postmortem studies of brains from patients with DLB and other synucleinopathies have revealed that α-syn aggregates appear earlier in presynaptic terminals than in cell bodies^18,19, accompanied by a reduction in various presynaptic markers¹⁹. Moreover, several imaging studies in PD patients have also confirmed synaptic dysfunction during the prodromal phase, occurring several years before the onset of motor symptoms^20,21. Taken together, these observations suggest that aberrant α-syn accumulation at the synaptic terminals might represent an early initiating event in the cascade leading to synaptic dysfunction. Over time, the progressive loss of synaptic integrity directly impacts neuronal activity, disrupting synaptic communication and ultimately resulting in circuit-level defects²². This scenario is strongly supported in the nigrostriatal pathway, which is particularly vulnerable to α-syn aggregation^23,24,25. Dopaminergic cells in the substantia nigra pars compacta (SNc) project their long axons to the dorsal striatum where they release dopamine (DA) to modulate the activity of striatal medium spiny neurons (MSNs)^25,26,27. The balanced activity between the two main subtypes of MSNs, D1 receptor-expressing MSNs (D1-MSNs), which regulate the basal ganglia direct pathway (dMSNs), and D2 receptor-expressing MSNs (D2-MSNs), which regulate the indirect pathway (iMSNs), is essential for fine control of movement^28,29,30,31. However, the abnormal accumulation of α-syn in dopaminergic terminals has been shown to be detrimental to nigrostriatal transmission^15,25,32,33. Studies involving the overexpression of α-syn or using α-syn preformed fibrils have shown that there are early changes in the excitability of dopaminergic neurons, leading to alterations in striatal synaptic transmission^34,35,36. In addition, other studies have reported that α-syn oligomers prompt the early impairment of long-term potentiation in MSNs, reflecting a decrease in synaptic strength and an alteration of the synaptic plasticity^22,36,37.

Although previous research has made significant strides toward understanding the relevance of synaptic impairment in the pathogenesis of PD, the precise sequence of events and underlying mechanisms by which α-syn aggregates induce early synaptic dysfunction in the nigrostriatal pathway remain unresolved. This gap in knowledge is mainly due to the limitations of the experimental models of α-syn aggregation, which often lack temporal resolution and only allow studies of the late stages of aggregation^38,39. To overcome this limitation, we developed an optogenetic-based cellular and animal model that allows for the induction and the formation of α-syn aggregates under the control of the blue light, referred to as the light-inducible protein aggregation (LIPA) system^40,41. The LIPA system not only enables the formation of pathological LB-like inclusions in neuronal cultures and in the mouse brain, but also allows for the real-time monitoring α-syn aggregation and inclusions formation with unprecedented spatial and temporal resolution⁴⁰. Interestingly, by combining the LIPA-induced α-syn aggregation system and in vivo Ca²⁺ imaging in the striatum of mice, we recently reported that the induction of LIPA-α-syn inclusions in the midbrain leads to rapid and sustainable changes in striatal neurons activity, thus highlighting the advantage of the LIPA system for studying the early stages of nigrostriatal transmission alteration⁴⁰.

In the present work, we leveraged the advantages of the LIPA system to unravel the early sequence of pathological events leading to synaptic impairment and the dysfunction of the nigrostriatal circuit. Using in vivo Ca²⁺ imaging in the striatum of freely behaving mice following light-induced α-syn accumulation, we found that the formation of LIPA-α-syn inclusions in the midbrain differentially affected the activity patterns of striatal MSNs populations, prior to the onset of motor symptoms. Further analyses using immunolabeling revealed that the activities of D1-MSNs and D2-MSNs were unbalanced. To explore the underlying mechanisms, we next investigated synaptic dysfunction and structural remodeling as potential contributors to the altered neuronal activity. Our results showed that the accumulation of LIPA-α-syn in dopaminergic terminals disrupted presynaptic structures, altered synaptic architecture, and impaired dopamine homeostasis. Notably, these pathological changes occurred before the degeneration of dopaminergic axons. Collectively, our results provide new insights into the initial pathophysiological changes within the nigrostriatal circuit triggered by α-syn accumulation, and highlight synaptic impairment as an early event in the cascade that drives the onset and progression of the disease.

Results

The induction of α-syn inclusions in the midbrain results in a rapid spatial reorganization of the striatal neuronal circuitry

To determine how the induction of α-syn accumulation in the SNc dopaminergic neurons affects the functional organization of the nigrostriatal pathway, we analyzed Ca²⁺ activity in the striatal neurons of freely moving mice following midbrain induction of LIPA-α-syn aggregation⁴⁰ (Fig. 1A). To do so, we implanted a double optogenetic/mini-endoscopic cannula into C57Bl6 male mice previously injected with AAV2/6-LIPA-α-syn-mCherry into SNc. The optic fiber was positioned just above SNc while a gradient index imaging lens (GRIN) was positioned in the striatum to perform Ca²⁺ imaging⁴⁰ (Fig. 1A). We first performed Ca²⁺ imaging analyses under baseline conditions every other day for 4 days followed by imaging with blue light stimulation every other day for 7 days (Fig. 1B). After the 7-day blue light stimulation period, we assessed Ca²⁺ activity every other day for 10 more days (Fig. 1B). This experimental design previously revealed that both the synchronization and frequency of striatal neurons are specifically and rapidly impaired following the induction of LIPA-α-syn accumulation in the midbarin⁴⁰. Based on these observations, we next focused on the baseline day 3 and post-stimulation day 2 and 10 time points to investigate the cellular and molecular consequences of LIPA-α-syn accumulation underlying the striatal responses (Fig. 1B–D).

**Fig. 1: Early functional changes in MSNs caused by the formation of LIPA-α-syn inclusions.**

To assess the functional impact of LIPA-α-syn inclusions on the activity of striatal cells, we monitored the frequency and amplitude of Ca²⁺ events and fitted the results to a Gaussian distribution. In line with our previous data⁴⁰, our results show that the induction of LIPA-α-syn inclusions prompted a significant shift in the frequency of Ca²⁺ transients at both post-stimulation days as compared to baseline (Fig. 1E). Interestingly, our results also show that LIPA-α-syn accumulation affected the amplitude of Ca²⁺ events in a heterogenous manner such that there was a decrease in the lower and medium amplitudes of Ca²⁺ transients, while higher amplitude Ca²⁺ events increased even further (Fig. 1E). The same pattern of activity was observed on post-stimulation days 2 and 10 (Fig. 1E), suggesting that LIPA-α-syn-accumulation induced changes appeared as early as post-stimulation day 2 and were largely sustained at post-stimulation day 10. These results also suggest that LIPA-α-syn accumulation might affect different populations of striatal cells in dissimilar ways. We, thus, next assessed the possibility of functionally distinguishing between different populations of striatal neurons by performing a cluster analysis based on the properties of Ca²⁺ activity, such as amplitude and frequency, and to evaluate the effects of LIPA-α-syn accumulation on these distinct striatal neuronal populations. Under baseline conditions, we distinguished three different clusters. Cluster 1, which consisted of 40.2% of the total number of striatal neurons and exhibited a high amplitude of Ca²⁺ transients (ΔF/F = 2.49 and frequency = 0.073 Hz); cluster 2, which consisted of 52.1% of the total number of striatal neurons and exhibited a low amplitude of Ca²⁺ transients (ΔF/F = 1.27 and frequency = 0.069 Hz), and cluster 3, which consisted of the remaining 7.7% of striatal cells and exhibited the highest amplitude of Ca²⁺ transients (ΔF/F = 4.75 and frequency = 0.067 Hz). Interestingly, the distribution of the clusters differed between baseline and after blue light-induced LIPA-α-syn accumulation at post-stimulation days 2 and 10. After inducing the formation of LIPA-α-syn inclusions, there was a significant increase in the percentage of cells in cluster 2 (64.5%, ΔF/F = 1.15 and frequency = 0.064 Hz) and a significant decrease in cluster 1 (22%, ΔF/F = 2.83 and frequency = 0.062 Hz) at post-stimulation day 2 (Fig. 1F). Only mild, non-significant changes were observed in the percentage of striatal neurons in cluster 3 after the formation of LIPA-α-syn inclusions (13.5%, ΔF/F = 5.61 and frequency = 0.066 Hz) (Fig. 1F). Similarly, at post-stimulation day 10, the percentage of cells in cluster 1 decreased (29.1%, ΔF/F = 3.03 and frequency = 0.063 Hz) and increased, albeit not significantly, in cluster 2 (58.2%, ΔF/F = 1.35 and frequency = 0.063 Hz) (Fig. 1F). To better understand the changes observed in our cluster analysis, we mapped the distribution of Ca²⁺ dynamics using the Kernel Density Estimation (KDE). Our results show that, after inducing LIPA-α-syn accumulation at both post-stimulation days, cells in cluster 1 tended to move toward cluster 2 (Fig. 1G). These results suggest that the induction of LIPA-α-syn inclusions promoted a shift in the activity of striatal cells in cluster 1, making their activity resemble the Ca²⁺ dynamics of cells in cluster 2.

Given the role played by striatal neurons, particularly MSNs, in encoding mouse locomotion^42,43, we next determined whether the reorganization of striatal neuronal activity was associated with changes in motor behavior. We recorded the behavior of mice placed in an open field arena while Ca²⁺ activity was being monitored⁴⁰ and determined whether specific neuronal clusters were more tuned to locomotion and, if so, whether α-syn accumulation disrupted the recruitment of striatal neurons during movement. As reported previously^43,44,45, we observed an increase in the activity of striatal cells when the mice were moving (Fig. 1H). No significant changes in the mobile and immobile periods after light-induced accumulation of α-syn at post-stimulation days 2 and 10 were observed (Fig. 1I). This is in line with the timeline of light-induced α-syn accumulation used in our study which was prior to the onset of the characteristic motor symptoms. We next determined whether the activity of striatal cells from a specific cluster was linked to the movement of the mice. As expected, the frequency of Ca²⁺ transients at baseline was significantly higher during mobile periods in all three clusters (Fig. 1J–L). Similarly, the frequency of Ca²⁺ transients was significantly higher during mobile periods in all three clusters after light-induced α-syn accumulation at post-stimulation days 2 and 10 (Fig. 1J–L). Only cells in cluster 2 exhibited a significant increase in the amplitude, in addition to changes in the frequency, of Ca²⁺ transients when the mice were moving before and after inducing LIPA-α-syn accumulation (Fig. 1K). However, the increase in the frequency of Ca²⁺ transients during mobile periods on post-stimulation days 2 and 10 was comparable to that at baseline. Cells in cluster 1 and 3 showed no significant differences in the amplitude of Ca²⁺ transients during immobile and mobile periods at baseline and on post-stimulation days 2 and 10, and the increase in the frequency of Ca²⁺ transients during mobile periods after inducing LIPA-α-syn accumulation was comparable to that at baseline (Fig. 1J–L). These observations suggest that Ca²⁺ transients of striatal neurons increased in all clusters during movement and light-induced LIPA-α-syn accumulation induced a functional reorganization of the neuronal network by triggering a shift of striatal cells from cluster 1 to cluster 2, even prior to the development of motor symptoms mimicking prodromal PD.

LIPA-α-syn inclusions induce an imbalance in the direct and indirect striatal pathways

The results of the Ca²⁺-based neuronal activity analysis show that the early functional disruption induced by LIPA-α-syn inclusions mostly affected two striatal neuronal populations that were defined based on their functional activity. These two populations might be the MSNs that expressed either the D1 DA receptor or the D2 DA receptor (D1-MSNs and D2-MSNs) that govern the basal direct and indirect ganglia pathways, respectively. These neuronal populations are functionally connected to nigrostriatal afferents²⁹. Given that these two neuronal populations represent a large percentage of mouse striatal cells and are highly susceptible to DA modulation³⁰, it is plausible that LIPA-α-syn-induced neuronal alterations primarily affect these two populations. To test this hypothesis, we injected LIPA-α-syn AAV into the SNc of wild-type (WT) mice and, 15 days later, induced LIPA-α-syn inclusions in the SNc by blue light stimulation (Fig. 2A). We assessed the activation of striatal cells by analyzing c-fos expression 10 days post-stimulation (Fig. 2A). We used LIPA-Empty, a LIPA construct lacking the sequence of α-syn, as a control⁴⁰. The results of c-fos analysis show that there was a significant decrease in the number of activated striatal cells after inducing α-syn accumulation, while no changes were observed in the control groups, LIPA-α-syn non-stimulated and LIPA-Empty stimulated (Fig. 2B, C).

**Fig. 2: Induction of LIPA-α-syn inclusions in the midbrain affects D1 and D2 striatal neuronal activity.**

We then immunolabeled D1- and D2-MSNs and assessed the activation of these cells in the striatum. The accumulation of LIPA-α-syn induced a significant decrease in the number of activated D1+ cells (Fig. 2D, E) and a significant increase in the number of activated D2+ cells (Fig. 2F, G), while no changes were observed in the LIPA-α-syn non-stimulated and LIPA-Empty stimulated groups for either D1- or D2-MSNs (Fig. 2D–G). These results are similar to those of our analysis of Ca²⁺ activity showing that there was a decrease in the number of cells in cluster 1 and an increase in the number of cells in cluster 2. Overall, these results show that the formation of LIPA-α-syn inclusions in the nigrostriatal pathway disrupted the activity of D1- and D2-MSNs, causing an imbalance in the striatal network that mimicked the early stages of PD³⁶. Given that the activity of MSNs in the striatum is tightly controlled by dopaminergic projections from the SNc^27,46, our results suggest that the altered activity might be caused by a pre-synaptic dysfunction induced by LIPA-α-syn accumulation.

Accumulation of LIPA-α-syn inclusions at dopaminergic terminals induces synaptic dysfunction in the striatum of mice

Previous studies have reported that synaptic pathology can arise early in the pathogenesis of PD^15,16. We thus wondered whether the accumulation of LIPA-α-syn accumulation at DA terminals would induce presynaptic damage, leading to the altered activity observed in striatal cells. We first determined whether the early imbalance observed in the activity of direct- and indirect-striatal neurons was caused by dopaminergic axonal degeneration. To do so, we overexpressed LIPA-α-syn or LIPA-Empty in the SNc of WT mice (Fig. 3A). Fifteen days post-surgery, we induced LIPA-α-syn accumulation by optogenetic stimulation with blue light and assessed tyrosine hydroxylase (TH) and DA transporter (DAT) expression 10 days post-stimulation (Fig. 3A). Since in these experiments we performed an immunohistochemical assessment of the expression pattern of DAT and TH, which is unlikely to be changed at post-stimulation day 2, we analyzed samples only at post-stimulation day 10. We did not detect any differences in the densities of TH (Fig. 3B, C) or DAT (Fig. 3D, E) fibers between the LIPA-α-syn stimulated, LIPA-α-syn non-stimulated, and LIPA-Empty stimulated groups at post-stimulation day 10. These results show that the induction of LIPA-α-syn accumulation in the nigrostriatal pathway did not induce dopaminergic fiber degeneration, suggesting that the abnormal activity observed in MSNs was probably due to a synaptic dysfunction and not to dopaminergic degeneration. We next determined whether the presynaptic accumulation of LIPA-α-syn inclusions could lead to synaptic dysfunction and alterations in the activity of MSNs. To do so, we first confirmed the presence of α-syn in the presynaptic terminals by immunolabeling. At 10 days post-stimulation, we observed a considerable accumulation of α-syn in the axons of the blue light-stimulated LIPA-α-syn group and, to a lesser extent, in the non-stimulated LIPA-α-syn group (Fig. 3F). Interestingly, we did not detect the presence of LIPA-Empty inclusions in the axons of the LIPA-Empty group, suggesting that only LIPA-α-syn was transported to the nigral axonal terminals (Fig. 3F). We then assessed the effect of the presence of LIPA-α-syn inclusions at the synaptic terminals. Interestingly, we observed LIPA-α-syn accumulation colocalizing with the presynaptic marker synaptophysin in the LIPA-α-syn group stimulated with blue light (Fig. 3F arrowhead). However, we observed no such level of colocalization in the LIPA-α-syn non-stimulated group (Fig. 3F arrowhead). An assessment of the number and morphology of the synaptic varicosities using the presynaptic marker synaptophysin showed that the number of synaptophysin varicosities and the surfaces of the overall synapses had significantly increased in the blue light-stimulated LIPA-α-syn group compared to the control groups (Fig. 3G, H). In addition, the accumulation of LIPA-α-syn at the dopaminergic terminals resulted in a significant increase in the expression levels of synaptophysin in the striatum (Fig. 3I). These observations suggest that the accumulation of LIPA-α-syn inclusions at the presynaptic structure might lead to synaptic impairment. Altogether, our results show that synaptic pathology occurred because LIPA-α-syn accumulated in the dopaminergic terminals and highlighted the role of α-syn accumulation in inducing the first event of a cascade leading to neuronal dysfunction prior to axonal neurodegeneration.

**Fig. 3: LIPA-α-syn inclusions induce early synaptic dysfunction in dopaminergic axons.**

Nigral induction of LIPA-α-syn inclusions disrupts dopamine homeostasis in the mouse striatum

Given the early impact of LIPA-α-syn inclusions on striatal neuronal activity, the known susceptibility of these striatal neurons to dopaminergic modulation^46,47, and the early synaptic defects observed when LIPA-α-syn accumulation is induced, we determined whether the formation of LIPA-α-syn inclusions would lead to early dysregulation of the neurotransmitter DA in the nigrostriatal pathway^21,48. To address this question, we injected an AAV encoding LIPA-α-syn or the control LIPA-Empty in the SNc of WT mice and implanted an optic fiber in the midbrain. Fifteen days later, we induced the formation of LIPA-α-syn inclusions using blue light stimulation for one week and assessed the levels of DA and its metabolites in the striatum of mice by high-performance liquid chromatography (HPLC) (Fig. 4A). Strikingly, we observed that the nigral induction of LIPA-α-syn accumulation resulted in a significant increase in striatal DA content, while no changes were observed in the control groups (LIPA-α-syn –light and LIPA-Empty +light) (Fig. 4B). Unlike the changes in DA content we observed, we did not detect any significant differences in the content of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) (Fig. 4C) and 3-methoxytyramine (3-MT) across the experimental groups (Fig. 4D). In addition, the striatal DOPAC/DA ratio significantly decreased in mice with LIPA-α-syn inclusions (Fig. 4E), suggesting that while striatal DA levels had increased, the turnover of DA was not affected. The striatal 3-MT/DA ratio was significantly decreased in the blue light-stimulated LIPA-α-syn group compared to mice overexpressing LIPA-Empty and stimulated with blue light, while no differences were observed with the non-stimulated LIPA-α-syn group (Fig. 4F). Altogether these results suggest that the accumulation of LIPA-α-syn induced a deregulation of striatal DA transmission without affecting the catabolism of the neurotransmitter.

**Fig. 4: Accumulation of LIPA-α-syn inclusions affects dopamine levels and its metabolites in the striatum of mice.**

LIPA-α-syn inclusions induce early synaptic dysfunction in human dopaminergic neurons

Human-derived induced pluripotent stem cells (hiPSCs) are an excellent model for studying the pathogenesis of PD in vitro⁴⁹. Combined with our LIPA system, hiPSCs provide an exceptional opportunity to study the early alterations that occur in human cells after the induction of α-syn accumulation. We previously established a rapid, efficient differentiation protocol for turning hiPSCs into mature dopaminergic neurons (iDA) by combining neurogenin-2 programming and midbrain differentiation and maturation media kits^50,51,52 (Fig. 5A). To determine whether α-syn inclusions induce an early synaptic dysfunction in human dopaminergic neurons as observed in our in vivo model, at day 28 we overexpressed the LIPA-α-syn construct and the two control constructs, LIPA-Empty and a non-aggregatable form of α-syn LIPA-α-syn^ΔNAC⁵³. in the iDA (Fig. 5A, Supplementary Fig. S1). Two days later, the cells were optogenetically stimulated with blue light to induce α-syn accumulation (Fig. 5A). As previously reported⁴⁰, only iDA neurons expressing LIPA-α-syn and LIPA-Empty stimulated with blue light form inclusions (Fig. 5B, Supplementary Fig. S1). To evaluate the impact of LIPA-α-syn inclusions on synaptic integrity, we assessed the number and morphology of synaptic varicosities in the neurites of dopaminergic neurons by immunolabelling with the presynaptic marker synaptophysin (Fig. 5B). We observed a colocalization of LIPA-α-syn inclusions with a synaptophysin signal (Fig. 5B yellow arrowhead). In line with the results obtained with our in vivo model, the formation of LIPA-α-syn inclusions induced a significant increase in the number of synaptophysin varicosities as well as in their surface while no changes were observed in the control groups (Fig. 5C, D). Moreover, the accumulation of LIPA-α-syn significantly increased the expression levels of synaptophysin (Fig. 5E). These results suggest that the accumulation of LIPA-α-syn inclusions in the dopaminergic neurites led to early synaptic dysfunction.

**Fig. 5: The accumulation of LIPA-α-syn in the neurites of human dopaminergic neurons leads to early synaptic disruption.**

As shown in Fig. 5B, not all the synaptophysin varicosities in the neurites of dopaminergic neurons were positive for LIPA-α-syn or LIPA-Empty aggregates (Fig. 5B yellow and white arrowheads). Indeed, 50% of the total synaptophysin varicosities colocalized with LIPA-α-syn inclusions, while only 11% colocalized with LIPA-Empty inclusions (Fig. 5F). We thus determined whether the increase in the surface of synaptophysin-positive synaptic varicosities was due to the presence of LIPA-α-syn inclusions. Interestingly, the surface of synaptophysin varicosities colocalizing with LIPA-α-syn inclusions was significantly higher than that of non-colocalizing varicosities (Fig. 5G). In addition, the area of synaptophysin varicosities non-colocalizing with LIPA-α-syn inclusions was similar to that of the control groups (Fig. 5G). Moreover, synaptophysin expression levels significantly increased only when synaptophysin varicosities colocalized with LIPA-α-syn inclusions, while no differences of synaptophysin varicosities non-colocalizing with LIPA-α-syn inclusions with the other control groups were observed (Fig. 5H). Of note, the surface of synaptophysin varicosities colocalizing and non-colocalizing with LIPA-Empty inclusions was not significantly different from the other control groups (Fig. 5G). In addition, no differences were observed in the synaptophysin expression of varicosities colocalizing or non-colocalizing with LIPA-Empty inclusions (Fig. 5H). These observations suggest that the presence of LIPA-α-syn inclusions might rapidly trigger synaptic disruption. Overall, these results indicate that, in human dopaminergic neurons, a synaptic dysfunction caused by LIPA-α-syn inclusions occurs early in the progression of the disease and may lead to the progressive neurodegeneration observed in PD. Overall, our results indicated that a dysfunction in nigrostriatal transmission occurs early in the progression of the disease, highlighting the presynaptic structure as one of the first elements affected following α-syn accumulation.

Discussion

Several lines of evidence support the “dying-back” hypothesis in PD, where the pathology starts by affecting presynaptic structures, inducing axonal degeneration, and gradually progressing toward the cell bodies of neurons, eventually culminating in their death^15,17. In the present study, we leveraged the temporal resolution of the LIPA system to gain new insights into the early pathological events that occur in the nigrostriatal pathway of mice and in human dopaminergic neurons and that are associated with α-syn aggregation. We provide evidence that LIPA-α-syn inclusions induced in the SNc rapidly accumulated in the dopaminergic terminals in the striatum leading to structural changes in the presynaptic boutons and altering DA levels. We also show that LIPA-α-syn aggregates differentially induced changes in the activity of different striatal populations, suggesting that there were early underlying defects in the nigrostriatal circuit. Additionally, a histological analysis revealed that there was an opposite effect on the activation of D1- and D2-MSNs, as assessed by c-fos labeling, after the induction of the accumulation of LIPA-α-syn. In parallel, our in vitro findings using human dopaminergic neurons also brought to light changes in the architecture of presynaptic structures.

Dopaminergic neurons in the SNc are particularly vulnerable to α-syn accumulation. Research over the last few decades has focused on developing models to understand the effects of α-syn inclusions in this specific population^{23,54,55,56,57}. Previous work by our group has shown that the LIPA system prompts the induction of α-syn inclusions in the dopaminergic neurons of the SNc in mice, reproducing several key features of LBs observed in PD patients⁴⁰. In addition, an important pathological characteristic of α-syn aggregates is their ability to be transported and spread to different areas of the brain, exacerbating the progression of the disease^56,58,59. Our results show that, as early as 10 days following the induction of LIPA-α-syn aggregation in the SNc, there was a substantial accumulation of LIPA-α-syn in the presynaptic terminals in the striatum, suggesting that α-syn aggregates can be transported to the striatum via anterograde transport^56,60. Indeed, the accumulation of α-syn aggregates in the presynaptic terminals has also been commonly observed in PD animal models and human tissue^19,61,62,63. Given this, the deposition of α-syn in dopaminergic projections might trigger structural and functional synaptic alterations².

Cumulative evidence suggests that synaptic dysfunction arises early in the progression of PD^15,16. However, the mechanisms driving the synaptic pathology remain unclear. In the present study, we uncovered early presynaptic alterations caused by LIPA-α-syn inclusions before the onset of dopaminergic axonal degeneration. Interestingly, our results show that the accumulation of LIPA-α-syn caused a significant increase in the number of synaptic boutons in the striatum. This observation contrasts with previous studies that have reported either no change or a significant decrease in the number of synaptic varicosities associated with α-syn accumulation^64,65,66. This discrepancy may stem from the fact that these studies were performed at more advanced stages of the disease where axonal degeneration may have already occurred^55,65. Moreover, the increase in presynaptic boutons reported in our study may have uncovered early compensatory synaptic changes aimed at counteracting the malfunction in DA transmission⁶⁷ or underly early synaptic dysfunction. In addition, previous studies overexpressing α-syn in mice or using brain tissue from patients suffering from DLB have reported an increase in synapse size^63,66,68. We also observed an enlargement of the presynaptic structures, which is consistent with the increase in synapse size. In addition to synaptic structural alterations, our findings also show that there was an increase in the expression of synaptophysin resulting from LIPA-α-syn inclusions. Given that synaptophysin is a presynaptic protein localized to synaptic vesicles^69,70, it is conceivable that LIPA-α-syn inclusions might cause an abnormal accumulation of synaptic vesicles at the synapses. Although previous ultrastructural analyses have determined that the overall number of synaptic vesicles is not affected and that pathological α-syn induces an accumulation of enlarged synaptic vesicles and changes in the distribution of synaptic vesicles in the synaptic pools, pointing to a malfunction in exocytosis/endocytosis processes or the trafficking of synaptic vesicles^{7,33,71,72,73,74}.

Our results show that LIPA-α-syn accumulation induced early alterations in DA homeostasis. Surprisingly, our findings also show that there was an increase in striatal DA contents without particularly affecting its metabolites. Following its synthesis at synapses, DA is rapidly internalized into synaptic vesicles via VMAT2 to avoid DA oxidation^75,76,77. After neurotransmitter release, DA can either be degraded by Monoamine oxidase and Catecholamine-O-methyltransferase enzymes or recycled into synaptic vesicles after reuptake^78,79. It is thus conceivable that LIPA-α-syn inclusions induce the trapping of DA in synaptic vesicles, making it inaccessible for enzymatic degradation. Taken together with the alterations observed at the structural level, our results may suggest that LIPA-α-syn inclusions induce an early impairment in DA signaling³³. However, the synaptic vesicle cycle is a complex process, and which stages are impacted by LIPA-α-syn aggregates remain to be determined. Given the physiological role of α-syn in synaptic vesicle clustering and neurotransmitter release through the stabilization of the SNARE complex^3,4 and observations of changes in synaptic structure and DA homeostasis, LIPA-α-syn inclusions may impact the normal clustering of synaptic vesicles, interfere with their trafficking, or alter DA release by preventing SNARE complex formation^7,74,80. Importantly, while our current study provides a steady-state view and primarily focuses on dopamine (DA) content rather than DA release, future experiments combining LIPA with temporally resolved DA release assays, such as genetically encoded DA sensors (e.g., dLight)^81,82,83, will be essential to determine how DA release is altered during the early stages of α-syn accumulation at striatal dopaminergic terminals.

As a result of these LIPA-α-syn-induced presynaptic changes, striatal neurons may also undergo early alterations. Indeed, previous work by our team and others has shown that a disruption of the nigrostriatal pathway leads to dysfunctional striatal cell activity^36,40,44 and that this can occur early in the progression of the disease^36,40. We monitored neuronal activity with single-cell resolution using mini-endoscopes and found that the induction of LIPA-α-syn inclusions in the midbrain not only caused a decrease in the frequency of Ca²⁺ transients in striatal cells but also distinctly affected the amplitude of their Ca²⁺ transients. Although Ca²⁺ transients with low and medium amplitude values decreased, transients with high amplitude values increased. These results suggest that LIPA-α-syn accumulation might affect different populations of striatal cells in distinct ways. The cytoarchitecture of the striatum encompasses various types of neuronal populations, including a large population of GABAergic MSNs (95%) and a small population of interneurons (5%)^84,85,86. MSNs, in turn, can be divided into three populations depending on the expression of the DA receptor, i.e., D1-MSNs (or dMSNs), D2-MSNs (or iMSNs), and a smaller proportion of D1/D2-coexpressing MSNs^28,87. Our cluster analysis based on the properties of Ca²⁺ dynamics shows that, under baseline conditions, striatal cells were divided into three different clusters. Cluster 1 and cluster 2 comprised the largest percentage of neurons, making up 40.2% and 52.1% of the total number of striatal neurons recorded, respectively. Given that MSNs constitute the largest population of neurons in the striatum^84,85,86, we propose that cells from cluster 1 and cluster 2 could comprise the neuronal populations of D1- and D2-MSNs, respectively. In addition, while some studies that performed Ca²⁺ imaging in freely behaving mice showed Ca²⁺ transients with similar frequencies and amplitudes in D1- and D2-MSNs^43,44, it is worth noting that the work by Varin et al.⁸⁸, who recorded the activities of D1- and D2-MSNs using endoscopic imaging, revealed significantly higher activity in D1-MSNs than in D2-MSNs⁸⁸. Given that our cluster analysis shows that neurons in cluster 1 exhibited a higher frequency of Ca²⁺ events than neurons in cluster 2, it is conceivable that cluster 1 might consist of D1-MSNs while cluster 2 might consist of D2-MSNs. This hypothesis is supported by the results of our assessment of c-fos+ cells showing that LIPA-α-syn inclusions induced a decrease in the number of activated D1+ cells and an increase in the number of activated D2+ cells, providing support for the changes observed in cluster organization following LIPA-α-syn accumulation. In the classical view, D1 DA receptors are coupled to a Gα_s/olf protein, and their activation triggers a downstream signaling cascade that enhancemes the neuronal excitability of D1-MSNs^27,89. In contrast, D2 DA receptors are coupled to G_i/o protein, and their activation initiates a downstream signaling cascade that reduces the excitability of D2-MSNs²⁷. The observed reduction in D1 activity concomitant with an increase in D2 activity supports the hypothesis that DA release is diminished, despite elevated tissue DA contents^29,90.

Converging lines of evidence have shown that enhanced striatal activity is associated with mouse movement for both D1- and D2-MSNs, suggesting that the coactivation of these two populations of striatal neurons encodes mouse locomotion^42,43,44,45. Consistent with this, we found that under both baseline and after light-induced accumulation of α-syn on post-stimulation days 2 and 10 conditions, all three clusters exhibited increased activity during mobile periods. However, the accumulation of LIPA-α-syn did not affect the activity of striatal cells during locomotion at this early stage.

In line with our observations in the mouse brain, we found, in human iDA neurons, that the early accumulation of LIPA-α-syn in dopaminergic axons led to a significant increase in the number of synaptic varicosities, an enlargement of synaptic structures, and an accumulation of the presynaptic marker synaptophysin. Interestingly, changes in synaptic size and synaptophysin expression were specifically associated with the presence of LIPA-α-syn inclusions. This observation contrasts with previous studies that used human dopaminergic neurons derived from patients harboring the A53T mutation to show that there is a decrease in the number of synaptic varicosities^65,91. These discrepancies may be attributed to differences in the time points chosen to assess synaptic pathology. Our observations highlight the early synaptic changes caused by LIPA-α-syn accumulation, which may trigger synaptic dysfunction and alter the activity of human dopaminergic neurons, potentially compromising cell viability⁹².

In conclusion, our study shows that the accumulation of LIPA-α-syn inclusions in the dopaminergic terminals at early stages of the disease triggered synaptic dysfunction and dysregulation in DA homeostasis. In addition, we speculate that the presynaptic impairment observed in mouse brains following α-syn accumulation led to alterations in striatal neuronal activity that occur prior to the onset of dopaminergic fiber neurodegeneration in the striatum and the emergence of the characteristic motor symptoms of PD. Ultimately, these events may progress and result in the development of dopaminergic neurodegeneration.

Methods

Animals

Adult 2- to 3-month-old C57Bl/6 WT male mice (n = 47) (Charles Rivers, Wilmington, MA, USA) were kept on a 12-h light/dark cycle at 22 °C with ad libitum access to food and water. All experiments were approved by the institutional Animal Welfare Committee of Université Laval, protocol 2020–641 to AO, in accordance with the Canadian Council on Animal Care guidelines. All mini-endoscopic imaging coupled with optogenetic stimulation experiments were approved by the Animal Welfare Committee of Université Laval, protocol 2019–020 to AS.

Mice were anesthetized with 2% isoflurane-O₂ and were placed in a stereotactic frame (David Kopf Instruments, CA, USA). An incision was made in the skin, and the skull was cleared to visualize the bregma. After drilling a hole above the SNc, the mice received a unilateral 2-μL injection of either AAV2/6-LIPA-α-syn-mCherry or AAV2/6-LIPA-Empty at a rate of 0.2 μL/min. The injections were performed using the following coordinates with respect to the bregma: AP − 3.08 mm, ML − 1.5 mm, and DV − 4.25 mm. Following the viral injection, a wireless optogenetic device (Eicom/Amuza, San Diego, CA, USA) was slowly implanted in the SNc using the following coordinates with respect to the bregma: AP − 3.08 mm, ML − 1.5 mm, and DV − 4.20 mm. The optogenetic device was fixed to the skull using cement.

Fifteen days after the surgery, the mice received 1.76-mW/mm² 10-ms 20-Hz pulses over a 1-h period for 7 days using wireless optogenetic devices (Eicom/Amuza, San Diego, CA, USA). Just before each optogenetic stimulation session, a battery (Eicom/Amuza, San Diego, CA, USA) was directly connected to the implant.

Ca²⁺ dynamics analysis

All the Ca²⁺ results described in the present paper have been reanalyzed from previous experiments we conducted⁴⁰. As previously described⁴⁰, 2-month-old C57Bl/6 WT male mice (n = 5) (Charles Rivers, Wilmington, MA, USA) received an injection of AAV2/6-LIPA-α-syn-mCherry in the SNc (AP − 3.5, ML + / − 1.2, DV − 4) and AAV2/5 CAG-GCaMP6s in the striatum (AP + 1.5, ML + / − 1.2, DV − 2.5). Two to three weeks later, a double optogenetic/mini-endoscopic cannula was implanted. The GRIN lens was placed above the dorsal striatum and the optic fiber just on top of the SNc. Three weeks after surgery, Ca²⁺ activity was recorded at a rate of 10 fps in the striatum of freely behaving mice twice for 10 min every other day. Four days after beginning the Ca²⁺ recordings, the SNc of the mice were subjected to a 1-h stimulation with blue light before the Ca²⁺ imaging using the following pattern 1.76-mW/mm² 10-ms 20-Hz pulses. After the 7-day stimulation protocol, the mice continued to be imaged every second day for up to 10 days. Ca²⁺ recorded images were processed as previously described⁴⁰. Briefly, the raw images were time-averaged in 200-ms bins using ImageJ software. Afterwards, Doric Neuroscience Studio software (Doric Lenses, Quebec City, QC, Canada) was used to correct the images in XY for brain motion, apply ΔF/F0 processing, and subtract the background. ROIs were manually traced around the cell bodies, and the mean fluorescence values were obtained. Ca²⁺ traces were analyzed using a custom-script created in MATLAB (The MathWorks, Natick, MA, USA) to obtain the mean amplitude and frequency of the Ca²⁺ transients. Of note, while we cannot formally rule out the possibility that GCaMP6 excitation induced by blue light in the striatum may also contribute to α-syn accumulation at dopaminergic terminals in the striatum, our previous experiments argue against this possibility⁴⁰. We have previously shown Ca²⁺ imaging in the striatum without delivering a blue light in the SNc does not induce α-syn accumulation at dopaminergic terminals and changes in Ca²⁺ activity in the striatum⁴⁰.

Mouse behavior tests

All the behavior experiments were reported in a previously published paper⁴⁰ and were reanalyzed for the present study. Briefly, mice (n = 5) implanted with a mini-endoscope were placed in an open-field arena and their behavior was monitored twice for 10 min every other day while Ca²⁺ activity was being recorded. To record behavior, we used a video camera (30 fps) (Doric Lenses, Quebec City, QC, Canada) positioned above the arena and controlled by Doric Neuroscience Studio software (Doric Lenses, Quebec City, QC, Canada). We used DeepLabCut software^93,94 to track the nose; head; left and right ears; upper, center, and lower parts of the body; and base of the tail of the mice. To train the DeepLabCut algorithm, we extracted 20 frames from each of the recorded videos and manually labeled the abovementioned regions of the mice. The network was trained for up to 500,000 iterations and was used to obtain the coordinates of all ROIs in the complete recorded footage. The tracked region of the center part of the body was used to calculate the speed and to measure the time the mice spent immobile and mobile throughout the videos. The percentage of time the mice spent mobile and immobile, along with their speed, was determined using a custom-written script created in python.

Cluster analysis of Ca²⁺ transients

A clustering analysis was performed based on the frequencies and amplitudes of the Ca²⁺ transients. To determine the optimal number of clusters for each condition, we computed three different scores: Calinski-Harabasz, Elbow, and Silhouette. Given the variability in the Calinski-Harabasz and Elbow scores, we used the recommendations resulting from the Silhouette score. The clustering was performed using the K-Means algorithm included in scikit-learn 1.5.0. The Python package seaborn 0.13.2 was used to generate the KDE maps.

To investigate potential trends in cluster activity changes between the mobile and immobile periods, we merged the DeepLabCut results with the raw results, maintaining the labels from the cluster analysis results for each ROI/Animal ID. We first processed the DeepLabCut results to be able to categorize the results between the mobile and immobile periods using a binary scoring system (1 for mobile, 0 for immobile). We then computed the mean values for the Ca²⁺ result columns of interest, frequency and amplitude, before merging the mobility and calcium data. Once combined, we separated the results between mobile and immobile periods for each cluster to obtain movement-specific activity data.

Immunostaining

The mice (n = 17) were deeply anesthetized with an intraperitoneal injection (0.1 mL/30 g) of a mixture of ketamine (100 mg/mL) and xylazine (10 mg/mL). They were then perfused intracardially with PBS followed by 4% paraformaldehyde (PFA). The brains were collected and were post-fixed overnight in 4% PFA at 4 °C. They were then incubated in 20% sucrose-PBS for 24 h at 4 °C. Coronal slices (30-μm thick) of the brains were cut using a microtome (Leica SM2000R), and the slices were stored in cryoprotection solution (0.05 M sodium phosphate (pH 7.3), 30% ethylene glycol, and 20% glycerol) at −20 °C.

Free-floating brain slices were permeabilized with 0.3% Triton X-100 for 30 min at RT and were then blocked with a mixture of 0.3% Triton X-100, 1% bovine serum albumin (BSA), and 5% normal goat serum (NGS) for 2 h at RT. The brain slices were then incubated with the following primary antibodies diluted in a mixture of 0.1% Triton X-100, 1% BSA, and 1% NGS overnight at 4 °C: rat anti-mCherry (16D7) (1:500, Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-human α-syn (LB509) (1:400, Thermo Fisher Scientific, Waltham, MA, USA), guinea pig anti-cfos (1:1000, Synaptic Systems, Göttingen, Germany), mouse anti-TH (1:1000, EMD Millipore, Temecula, CA, USA), rabbit anti-TH (1:1000, Pel-Freez, Rogers, AR, USA), rat anti-DAT (1:500, Millipore Sigma, Burlington, MA, USA), rabbit anti-dopamine receptor 1 (1:250, Novus Biologicals, Centennial, CO, USA), rabbit anti-dopamine receptor 2 (1:250, Proteintech, Rosemont, IL, USA), and rabbit anti-synaptophysin (1:200, Thermo Fisher Scientific, Invitrogen, Waltham, MA, USA) (Table 1). The slices were then incubated with the appropriate secondary antibodies diluted in 0.1% Triton X-100 in PBS for 2 h at RT: Alexa Fluor 488 anti-rabbit (1;1000, Invitrogen, Waltham, MA, USA), Alexa Fluor 488 anti-mouse (1;1000, Invitrogen, Waltham, MA, USA), Alexa Fluor 488 anti-guinea pig (1:250, Jackson ImmunoResearch Laboratories Inc, West Grove, PA, USA), Alexa Fluor 568 anti-rat (1:1000, ThermoFisher, Waltham, MA, USA), Alexa Fluor 633 anti-rabbit (1:250 for DRD1 and DRD2 staining and 1:400 for synaptophysin staining, Invitrogen, Waltham, MA, USA), Mouse IgG (680RD) (1:1000, LI-COR Biosciences, Lincoln, NE, USA), Cyanine5 anti-rat (1:500, Jackson ImmunoResearch Labs, West Grove, PA, USA) (Table 1). Lastly, the brain slices were counterstained with DAPI (1:5000) diluted in PBS and were mounted on Superfrost Plus microscope slides with Fluoromount mounting medium (Thermo Fisher Scientific, Waltham, MA, USA).

Table 1 List of antibodies used in the present study

Full size table

We used an antigen retrieval protocol for the immunostaining to assess c-fos activation in D2 positive cells. Briefly, the brain slices were mounted on Superfrost Plus microscope slides and were left to dry overnight. The brain slices were then subjected to an antigen retrieval protocol by incubating them in Tris-EDTA (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) for 10 min at 95 °C. The subsequent steps of the protocol were the same as described above.

For the immunohistological analyses of human dopaminergic neurons, the cells were permeabilized with 0.3% Triton X-100 for 10 min at RT and were blocked with a mixture of 0.1% Triton X-100, 1% BSA, and 5% NGS for 1 h at RT. The cells were then incubated with primary antibodies diluted in blocking solution overnight at 4 °C. The following primary antibodies were used: mouse anti-TH (1:1000, EMD Millipore, Temecula, CA, USA) and rabbit anti-synaptophysin (1:1000, Thermo Fisher Scientific, Invitrogen, Waltham, MA, USA). The cells were then incubated with the appropriate secondary antibodies diluted in blocking solution (1:800) for 2 h at RT. Lastly, the cells were counterstained with DAPI (1:5,000), and the coverslips were mounted on Superfrost Plus microscope slides with Fluoromount mounting medium (Thermo Fisher Scientific, Waltham, MA, USA).

Quantification

To evaluate the activation of D1- and D2-MSNs in the striatum of the mice, five random images were acquired from the dorsal striatum of each brain slice using a Zeiss LSM800 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 40x oil immersion objective (Plan-Apochromat 40x/NA 1.4; Zeiss). Zen software was used to blindly quantify the number of c-fos + /D1-MSNs+ and c-fos + /D2-MSNs + . A cell was considered to be a c-fos + /D1-MSNs+ or a c-fos + /D2-MSNs+ when the c-fos staining was surrounded by D1 or D2 staining, respectively.

To assess the density of TH⁺ and DAT⁺ fibers in the striatum, images were acquired using a Zeiss Axio Imager M2 equipped with an ApoTome epifluorescent microscope (Carl Zeiss, Oberkochen, Germany) with a 20x air objective (EC Plan-Neofluar 20x/0.50 M27; Zeiss). Tiling was performed to obtain an image of the entire striatum. ImageJ software was used to manually delimit the area of the dorsal striatum and quantify the mean pixel intensity in the dorsal part of the striatum of the injected hemisphere.

To evaluate synaptic density in the striatum of the mice, six random images were acquired from the dorsal striatum of each brain slice using a Zeiss LSM800 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 63x oil immersion objective (Plan-Apochromat 63x/NA 1.4; Zeiss). The number of synaptophysin varicosities, surface, and integrated density were blindly analyzed using the 3D Objects Counter plugin from ImageJ software. An intensity threshold of 7842 was determined for all the groups analyzed.

To evaluate the synaptic density in human dopaminergic cells, we acquired images using a Zeiss LSM800 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 63x oil immersion objective (Plan-Apochromat 63x/NA 1.4; Zeiss) and performed tiling. The synaptophysin varicosities were manually quantified along the neurites of mCherry-expressing neurons using ImageJ software. The number of synaptophysin varicosities was corrected by the length of the neurite, which was also quantified using ImageJ software. We also extracted the values for the mean surface and integrated density of the synaptophysin varicosities.

Striatal biogenic amine assay

Mice (n = 25) were deeply anesthetized with an intraperitoneal injection (0.1 mL/30 g) of ketamine (100 mg/mL), and xylazine (10 mg/mL). They were then intracardially perfused with PBS containing a cocktail of phosphatases (50 mM sodium fluoride and 1 mM sodium pyrophosphate) and protease inhibitors (Sigma-Aldrich, Burlington, MA, USA), and the regions containing the SNc and striatum were separated. The brain area containing the striatum was rapidly frozen in liquid nitrogen and was stored at −80 °C until it was processed for high performance liquid chromatography (HPLC). The brain region including the SNc was immediately fixed in 4% PFA overnight at 4 °C, was incubated in 4% PFA and 20% sucrose for 24 h at 4 °C, and was then processed for immunohistochemistry using the same protocol described in the Immunostaining section.

For HPLC analysis, the right anterior part of the striatum (bregma ~1.18 at ~0.14 mm, Paxinos and Franklin, 2001⁹⁵) was dissected, homogenized in 250 μl of 0.1 N HClO4 (Sigma-Aldrich, Burlington, MA, USA) at 4 °C and then centrifuged at 10,000 × g for 30 min (4 °C) to precipitate proteins. The resulting pellet was homogenized in 250 μl of 0.1 M NaOH, and the protein concentrations were then assayed using a Micro BCA Protein Assay kit (Thermo Scientific, Rockford, IL). The supernatant containing the striatal contents of dopamine and its metabolites DOPAC and 3-MT were measured by HPLC with electrochemical detection. Supernatants of striatal tissue were directly injected into the chromatograph consisting of a Waters 717 plus autosampler automatic injector equipped with a sample heater/cooler set at 4 °C (Waters, Mississauga, ON, Canada), a Waters 515 pump equipped with a C-18 column (Waters Nova-Pak C18, 3 μm, 3.9 mm × 150 cm), and a BAS LC-4C amperometric detector connected to a dual glassy carbon electrode (BASi research Products, West Lafayette, IN, USA). The electrochemical potential was set at 0.8 V with respect to an Ag/AgCl reference electrode. The mobile phase consisted of 0.025 M citric acid, 1.7 mM 1-heptane-sulfonic acid (Sigma-Aldrich, MilliporeSigma Canada Ltd, Oakville, ON, Canada), and 10% methanol (VWR International, Mississauga, ON, Canada), in filtered distilled water, delivered at a flow rate of 0.8 ml/min. The final pH of 3.99 was obtained by addition of NaOH. To prevent bubble formation in the liquid chromatography system, the mobile phase is degassed by bubbling helium through it for a few minutes just before use. Throughout the chromatography process, the amperometric signal from the detector was transferred directly to the Kipp-Zonen BD41 2-channel chart recorder. Peak heights corresponding to the different amines were measured directly on the chromatogram plotted on the chart recorder using a digital scale ruler. Standard curves containing a mixture of different concentrations of dopamine, DOPAC, and 3-MT, dissolved in HClO₄, were obtained to measure the concentrations of each amine present in the samples. The amine levels in the samples, expressed in nanogram, were transferred to a Microsoft Excel spreadsheet and converted to nanograms of amine per milligram of protein based on the injection volume in the liquid chromatography system and the protein content for each sample.

Generation of human dopaminergic neurons

We used a previously published protocol⁵¹ to generate Ngn2 iNeurons from hiPSCs and to differentiate them into dopaminergic neurons. Prior to the initiation of the differentiation protocol, hiPSCs expressing the doxycycline-inducible Ngn2 cassette were seeded on Matrigel-coated plates (Matrigel Matrix hESC-qualified) (Thermo Fisher Scientific, Waltham, MA, USA) and were kept in mTesr™ Plus medium (Stemcell Technologies, Vancouver, BC, Canada), as previously described⁵⁰. To differentiate the Ngn2 hiPSC line into iNeurons, one day after seeding the cells were cultured in Day 0/1 medium containing DMEM/F12 (Thermo Fisher Scientific/Gibco, Waltham, MA, USA), N2 (Thermo Fisher Scientific/Gibco, Waltham, MA, USA), B27 (Thermo Fisher Scientific, Waltham, MA, USA), NEAA (Wisent Life Sciences, Saint-Jean-Baptiste, QC, Canada), BDNF (Peprotech, Cranbury, NJ, USA), GDNF (Peprotech, Cranbury, NJ, USA), mouse laminin (Thermo Fisher Scientific, Waltham, MA, USA), and 2 µg/mL doxycycline (Takara Bio Company, Kusatsu, Japan) to induce Ngn2 expression. On day 2 (DIV 2), the cells were dissociated with Accutase (Thermo Fisher Scientific, Waltham, MA, USA), were seeded on coverslips coated with 50 µg/mL of poly-L-ornithine (Sigma-Aldrich, St. Louis, MO, USA) and 10 µg/mL of laminin, and were maintained in Day 2 neurobasal medium (Thermo Fisher Scientific/Life Technologies, Waltham, MA, USA), N2, B27, GlutaMax (Thermo Fisher Scientific, Waltham, MA, USA), NEAA, BDNF, GDNF, mouse laminin, and 2 µg/mL of doxycycline. From DIV 2, iNeurons were induced to differentiate into dopaminergic neurons using a STEMdiff™ Midbrain Neuron Differentiation Kit (Stemcell Technologies, Vancouver, BC, Canada). From DIV 2 to DIV 4, the medium, which was supplemented with 2 µM Ara-C, was replaced every day. From DIV 5 to DIV 8, only half the medium was changed every day. At Day 9 (DIV 9), the medium was completely replaced with midbrain maturation medium from a STEMdiff™ Midbrain Neuron Maturation Kit (Stemcell Technologies, Vancouver, BC, Canada) supplemented with 2 µg/mL of doxycycline. From day 10 (DIV 10) onward, and every two days, half the medium was replaced with fresh maturation medium. At Day 28 (DIV28), the cells were transfected with the LIPA constructs⁴⁰: LIPA-α-syn (0.5 μg), LIPA-Empty (0.4 μg), and LIPA-α-syn^ΔNAC (0.6 μg) using Lipofectamine® 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The maturation medium was replaced with Opti-MEM medium (Thermo Fisher Scientific, Waltham, MA, USA) 30 min before transfecting the cells. The DNA was combined with the liposome mix at a ratio of 1:3 (DNA:Reagent) and was added to the Opti-MEM medium. Six hours later, the Opti-MEM medium was replaced with maturation medium. Two days post-transfection, the cells were stimulated with blue light (0.3 mW/mm²) for 6 h and were then fixed using 4% PFA and 3% sucrose for 15 min at RT. After three 5-min washes in PBS, the cells were stored in PBS at 4 °C for immunohistological analyses.

Statistical analysis

In vitro assays were performed in three independent experiments for the LIPA-α-syn group, and two independent experiments were carried out for the control groups.

For the in vivo analyses, a one-way ANOVA followed by Tukey’s multiple comparisons test was used to assess differences between the three experimental groups used. In addition, an unpaired two-sided Student’s t test was used to analyze the differences in the frequencies and amplitudes of Ca²⁺ transients between the mobile and immobile periods. For the in vitro analyses, a one-way ANOVA followed by Tukey’s multiple comparisons test was used to determine the differences between the experimental conditions used. Outliers (values greater than the 1.5 interquartile intervals below the first quartile or 1.5 interquartile intervals above the third quartile) were excluded from the analysis. To test statistical significance for the frequency and amplitude of Ca²⁺ events, two-sided Kolmogorov–Smirnov test was used. All values are expressed as means ± SEM. The statistical analyses were performed using Prism 9 (GraphPad). p < 0.05 was required for the rejection of the null hypothesis.

Data availability

Data are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the Canadian Institutes of Health Research (CIHR: PJT-162400) to A.O., (CIHR; PJT-159680) to T.D.P., (CIHR; PJT-186027) to A.S., the Natural Sciences and Engineering Research Council (NSERC; RGPIN-2023-05581) to A.O. A.O. was supported by Junior 2 salary Awards from the Fonds de Recherche du Québec-Santé (FRQS) and la Société Parkinson du Québec. A.S. holds a Canada Research Chair Tier 1 position.

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Authors and Affiliations

Department of Molecular Medicine, Faculty of Medicine, Université Laval, Quebec City, QC, Canada
Raquel Rodriguez-Aller & Abid Oueslati
CHU de Québec Research Center, Axe Neurosciences, Quebec City, QC, Canada
Raquel Rodriguez-Aller, Marc Morissette, Razan Sheta, Thérèse Di Paolo & Abid Oueslati
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada
Raquel Rodriguez-Aller, Beatriz Romero-Quineche & Armen Saghatelyan
Faculty of Pharmacy, Université Laval, Quebec City, QC, Canada
Thérèse Di Paolo

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Raquel Rodriguez-Aller
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Beatriz Romero-Quineche
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Marc Morissette
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Razan Sheta
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Thérèse Di Paolo
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Armen Saghatelyan
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Abid Oueslati
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Contributions

A.O. and A.S. conceived the study, designed the experiments, and supervised the project. R.R.A. collected and analyzed the in vivo data and data from iPSC-derived neurons. B.R.Q. contributed to data analysis. R.S. conducted the experiments involving iPSC-derived neuron generation. M.M. and T.D.P. collected and analyzed dopamine and dopamine metabolite data. R.R.A., A.O., and A.S. wrote the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Armen Saghatelyan or Abid Oueslati.

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Rodriguez-Aller, R., Romero-Quineche, B., Morissette, M. et al. Optogenetic-induced α-synuclein accumulation reveals early synaptic dysfunction in experimental models of Parkinson’s disease. npj Parkinsons Dis. 11, 356 (2025). https://doi.org/10.1038/s41531-025-01201-x

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Received: 23 July 2025
Accepted: 02 November 2025
Published: 13 December 2025
Version of record: 24 December 2025
DOI: https://doi.org/10.1038/s41531-025-01201-x