Novel insights in the pathophysiology of α-synuclein dysregulation on D2 receptor activity contributing to the vulnerability of dopamine neurons

Background

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is characterised by the loss of dopaminergic neurones and proteinous inclusions known as Lewy bodies in the ventral midbrain. These neurones play a vital role in the initiation and control of movement meaning that patients often present with rigidity, tremor, and slowness of movement and inability to initiate movement. It is known that damage or loss of function of these neurones precedes their death¹.

Lewy bodies are proteinous accumulations of primarily alpha-Synuclein. The gene that encodes for alpha-Synuclein, SNCA, is known to be mutated, duplicated or triplicated in autosomal dominant forms of PD. There is also evidence that overexpression of alpha-Synuclein plays a role in idiopathic PD. Overexpression of wild type (WT) alpha-Synuclein is sufficient to alter the pathophysiology of dopaminergic neurones resulting in their vulnerability and eventual loss. The exact mechanism for this is not yet known.

In this preprint, the authors used primary mouse cultures of dopaminergic neurones in conjunction with adeno virus expressing WT human alpha-Synuclein under a dopaminergic promoter (Tyrosine Hydroxylase (TH)). These neurones were compared with control non alpha-Synuclein expressing cells. They saw high specificity of alpha-Synuclein in TH positive/Dopaminergic neurones (Figure 1).

Figure 1: Immunofluorescence images showing expression and co-localisation of TH and -Synuclein in naïve and -Synuclein overexpression dopaminergic neuron model. Adapted from Figure 1C in the preprint under a CC-BY 4.0 licence.

Key findings

The overexpression of alpha-Synuclein disrupts Dopamine (D2) receptor autoinhibition resulting in altered calcium signalling and increased firing rate of mouse dopaminergic neurones.

It is known that loss of function precedes neurone loss in PD¹. The authors wanted to investigate if the overexpression of WT human alpha-Synuclein in mouse dopaminergic neurones altered their electrical activity.

Following overexpression of alpha-Synuclein the width and amplitude of the Ca²⁺ signal increased, which was complemented by an increase in neuronal firing recorded by whole cell current recordings. This suggests that overexpression of alpha-Synuclein can increase firing rate of dopaminergic neurones.

Both firing activity and Ca²⁺ dynamics are tightly regulated by D2 autoinhibitory receptor which suppresses electrical activity of dopaminergic neurones upon dopamine binding^2,3. The D2 receptor agonist, Quinpirole, was utilised to show that those cells expressing alpha-Synuclein maintained their Ca²⁺ signal and firing rate unlike control neurones which saw an inhibition of both Ca²⁺ signals and firing rate when treated with D2 agonist. Furthermore, blocking D2 receptors, in control neurones mirrored the effects of firing rate as seen with alpha-Synuclein overexpression. These data suggests that -Synuclein impacts D2 receptors to alter Ca²⁺ signalling and firing rate of mouse DA neurones.

The overexpression of alpha-Synuclein decreases the complexity and survival of Dopaminergic neurones.

Dopaminergic neurones are neuronally complex, have large branching neurites and projections fields. In PD patients there is a decrease in complexity of dopaminergic neurones⁴. The authors investigated the complexity and survival of mouse dopaminergic neurones overexpressing alpha-Synuclein. To do this they use Sholl analysis, which examines neurite projection area and field, number of intersections (branching) and circularity, alongside Tyrosine Hydroxylase (TH) immunofluorescence which marks dopaminergic neurones (Figure 2a).  In neurones overexpressing alpha-Synuclein there was a decrease in neuronal processes, projection area and circularity accompanied by a decreased in the amount of TH expressing cells (Figure 2 (b,c). This suggests that neurones expressing alpha-Synuclein are less complex and may be informative about the progression of alpha-Synuclein pathology prior to neuronal loss.

Figure 2: (a) Schematic illustrating the measurable parameters of dopaminergic neurone. (b,c,d) schematic representations of naïve, those with overexpressed alpha-Synuclein and D2 agonist pre-treatment. Adapted from Figure 6 in the preprint under a CC-BY 4.0 licence

Pharmacological activation of D2 receptors could be a potential target to alleviate alpha-Synuclein pathology.

The authors had previously found an unexpected effect of prolonged exposure of dopaminergic neurones to the agonist Quinone. They saw that this rescued the firing properties to the alpha-Synuclein expressing neurones to that of the control. They further investigated other key defects seen in these alpha-Synuclein expressing neurones such as: neuron complexity, survival, Calcium dynamics, spontaneous firing and dopamine release and synthesis. They found partial rescue of these factors with prolonged exposure (48h) of neurones to Quinone. This highlights D2 receptor agonists as potential drug targets and an area for further investigation (Figure 2)

Importance of this work/ How it moves to field forwards

This paper highlights D2 receptors as a potential target that could be used to alleviate alpha-Synuclein pathology in a range of diseases including PD and Lewy body dementia. With an aging population it is increasingly important to highlight a diverse range of treatments. What is especially interesting is that any treatments involving D2 dopamine receptor could be given preventatively treating the cause of disease rather than the symptoms. However, early diagnosis of neurodegenerative disease is still difficult. I specifically liked the use of many complementary methods to solidify their finding in addition to examining the functionality of the neurone not just survival.

Open Questions

1. Was this aggregated alpha-Synuclein? Could this have a different effect?
2. What do you think the effects of mutated alpha-Synuclein on these parameters such as firing rate and the impact that D2 agonists could have? Have you thought about investigating this?
3. This is conducted in mouse cells, are there plans for this be conducted using human model? IPSC’s?
4. Are there any drugs which target D2 receptors, that have already been approved for other uses which could be tested in your model and potentially re-purposed?

 

 

Tags: alpha-synuclein, dopaminergic, parkinson's disease

doi: https://doi.org/10.1242/prelights.29164

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