Dopaminergic Retinal Cell Loss and Visual Dysfunction in Parkinson Disease

Abstract

Objective: Considering the demonstrated implication of the retina in Parkinson disease (PD) pathology and the importance of dopaminergic cells in this tissue, we aimed to analyze the state of the dopaminergic amacrine cells and some of their main postsynaptic neurons in the retina of PD.

Methods: Using immunohistochemistry and confocal microscopy, we evaluated morphology, number, and synaptic connections of dopaminergic cells and their postsynaptic cells, AII amacrine and melanopsin-containing retinal ganglion cells, in control and PD eyes from human donors.

Results: In PD, dopaminergic amacrine cell number was reduced between 58% and 26% in different retinal regions, involving a decline in the number of synaptic contacts with AII amacrine cells (by 60%) and melanopsin cells (by 35%). Despite losing their main synaptic input, AII cells were not reduced in number, but they showed cellular alterations compromising their adequate function: (1) a loss of mitochondria inside their lobular appendages, which may indicate an energetic failure; and (2) a loss of connexin 36, suggesting alterations in the AII coupling and in visual signal transmission from the rod pathway.

Interpretation: The dopaminergic system impairment and the affection of the rod pathway through the AII cells may explain and be partially responsible for the reduced contrast sensitivity or electroretinographic response described in PD. Also, dopamine reduction and the loss of synaptic contacts with melanopsin cells may contribute to the melanopsin retinal ganglion cell loss previously described and to the disturbances in circadian rhythm and sleep reported in PD patients. These data support the idea that the retina reproduces brain neurodegeneration and is highly involved in PD pathology. ANN NEUROL 2020;88:893-906.

Fig. 1

Dopaminergic cells display an impaired morphology in PD. Dopaminergic cells (green) in healthy retinas (A) have long thin dendrites that create a dense plexus with ring structures (arrows) surrounding their post-synaptic cell bodies. In PD (B), dendrites are shorter and display some swollen regions (arrowheads), a sign of degeneration. Their plexus is less continuous, and it has lost the typical dopaminergic rings. In transversal sections, the density of the dopaminergic plexus can be observed in control (C) and PD (D) retinas. Dopaminergic plexus in control subjects (C) is much thicker, denser and more continuous than in PD patients (D). In addition, while a dopaminergic plexus at IPL S3 can be identified in controls, it is almost lost in PD. Dopaminergic projections towards the outer retina (OPL and ONL) can be observed in controls (C) but are strongly reduced in PD (D) (arrows). n=5 control, n=8 PD. Scale bar A-D:10 μm.

Fig. 2

Density of dopaminergic amacrine cells is reduced in PD. Color-coded density maps of dopaminergic cells in the macular region (temporal-central pieces) of control (A) and PD (B) reflect the decreased dopaminergic density in patients. Each dopaminergic cell is represented by a black dot and, in the color gradient, yellow represents the highest cell density (45 cells/mm²) and dark blue the lowest (0 cells/mm²). Insets in (A) and (B) show high magnification images of the parafovea where changes in cell number between groups can be observed. Confocal images of dopaminergic amacrine cells in the parafovea of control (C) and PD (D) also show the decline in cell number observed in the disease. Dopaminergic cell density is highest at the parafovea, concentrically decreases towards higher eccentricities, and it is reduced in PD compared to controls in all the locations tested (E). In the temporal-central quadrant the dopaminergic density decreased from 20.2 cells/mm² in controls to 13.13 cells/mm² in PD, changing from 45.13 in controls to 19.16 cells/mm² in PD in the parafovea, from 29.67 cells/mm² to 14.43 cells/mm² in the perifovea, and from 18.48 cells/mm² to 12.89 cells/mm² in the next 3mm.. In the superonasal quadrant, density maps show the differences in dopaminergic cell density between control (F) and PD (G) and how the density is gradually reduced towards the periphery. This density is significantly reduced in PD, changing from 18.4 cells/mm² in the retina of healthy individuals to10.8 cells/mm² in the retina of PD patients, and differences are maintained along the retina: from 2 to 7 mm to optic nerve dopaminergic cells change from 19.44 cells/mm² in controls to 13.38 cells/mm² in PD, from 7 to 11 mm to optic nerve it is reduced from 17.3 cells/mm² in controls to 12.82 cells/mm² in PD, and in the most peripheral region, from 11 to 16 mm to the optic nerve, dopaminergic density diminishes from 14.40 cells/mm² in controls to 8.79 cells/mm² in PD, being statistically significant in all cases. ON: Optic Nerve; Fv: Fovea. A-E: n=3 control, n=2 PD; F-H: n=5 control, n=8 PD. Scale bar A-B, F-G: 1mm; C-D: 20 μm. *: P<0.05; **: P<0.01.

Fig. 3

AII amacrine cells maintain their cell density in PD although their dopaminergic inputs are reduced. Double immunostaining using tyrosine hydroxylase (TH, green) and calretinin (CR, red) shows the interactions between dopaminergic (green) and AII (red) amacrine cells. In control retinas (A), dopaminergic cells make several synaptic contacts around AII soma, which are severely lost in PD (B). High magnification images show the contacts and the ring structures in controls (C, D) and PD (E, F). Their quantification revealed a significant loss of dopaminergic contacts per AII cell, from 9.3±0.2 in controls to 3.7±0.8 in PD (G) but no differences in the number of AII amacrine cells was observed (H). n=4 control, n=7 PD, H: 20 different frames per retina. Scale bar A-B: 20 μm; C-F: 5 μm; **: P<0.01; n.s.: non-significant

Fig. 4

AII amacrine cells loss mitochondria in their lobular appendages in PD. Calretinin (CR) shows AII amacrine cells (red) and cytochrome C (CytC) shows mitochondria in control (A) and PD (B) retinas. AII amacrine cells (red) present small dendrites that stratify close to the cell body in both sublamina a and b of the IPL (A). In PD, their typical morphology has been partially disrupted, showing an apparent loss of lobular appendages and stratification. Also, healthy subjects have big and abundant mitochondria within the AII lobular appendages (C, arrows) while fewer and smaller mitochondria are found in the retina of PD (D, arrows). Higher magnification images of the areas indicated by doted lines show differences in mitochondria size and number between controls, where lobular appendages are full of big mitocondria (E, arrows) and PD, where mitochondria are small, sparse (F, arrows) and some lobular appendages seem empty. n=3 control, n=5 PD. Scale bar A-D: 10 μm; E-F: 5 μm

Fig. 5

AII amacrine cells in PD loss connexin 36 at the ON sublamina of the IPL. In healthy controls (A), AII (calretinin, CR, red) gap junctions are mediated by connexin 36 (Cx36, green), which is abundant at the IPL ON sublamina. In PD (B), connexin 36 is reduced, probably altering gap junctions. (C) and (D) are the same images as (A) and (B) respectively but showing the green channel alone, to properly observe the connexin 36 reduction in PD (D). (E-F) are high magnification images of (A-B) dotted areas. Notice the altered morphology of AII cells (less lobular appendages and impaired stratification) and the reduction in connexin 36 in PD (F) compared to controls (E). (G-H) images display the green channel from (E-F) images, and are high magnification of (C-D); they show the strong decline in connexin 36 in the sublamina ON of the IPL in PD (H) compared to controls (G). n=3 control, n=5 PD Scale bar: A-D: 10 μm; E-F: 5 μm

Fig. 6

Dopaminergic synaptic contacts to melanopsin retinal ganglion cells are reduced in PD. In controls, melanopsin cells (red) display their normal morphology (A), while in PD cell dendrites are less complex, having lower number of branches and shorter dendrites (B). Also, controls display a dense dopaminergic plexus (green) (C), that is reduced in PD (D). In high-magnification images, details of synaptic contacts of dopaminergic cells in melanopsin dendrites can be observed (E-H), showing that controls (E, F) have a higher number of contacts than PD (G, H). Its quantification revealed a loss of dopaminergic synaptic contacts in melanopsin dendrites: from 70±14 contacts per 100 μm in controls to 43±9 contacts per 100 μm in PD (P<0.0001) (i). n=3 control, n=3 PD, 15 different frames per retina. Scale bar: A-D: 20 μm; E-H: 1 μm