Sustained chemogenetic activation of locus coeruleus norepinephrine neurons promotes dopaminergic neuron survival in synucleinopathy

Abstract

Dopaminergic neuron degeneration in the midbrain plays a pivotal role in motor symptoms associated with Parkinson's disease. However, non-motor symptoms of Parkinson's disease and post-mortem histopathology confirm dysfunction in other brain areas, including the locus coeruleus and its associated neurotransmitter norepinephrine. Here, we investigate the role of central norepinephrine-producing neurons in Parkinson's disease by chronically stimulating catecholaminergic neurons in the locus coeruleus using chemogenetic manipulation. We show that norepinephrine neurons send complex axonal projections to the dopaminergic neurons in the substantia nigra, confirming physical communication between these regions. Furthermore, we demonstrate that increased activity of norepinephrine neurons is protective against dopaminergic neuronal depletion in human α-syn A53T missense mutation over-expressing mice and prevents motor dysfunction in these mice. Remarkably, elevated norepinephrine neurons action fails to alleviate α-synuclein aggregation and microgliosis in the substantia nigra suggesting the presence of an alternate neuroprotective mechanism. The beneficial effects of high norepinephrine neuron activity might be attributed to the action of norepinephrine on dopaminergic neurons, as recombinant norepinephrine treatment increased primary dopaminergic neuron cultures survival and neurite sprouting. Collectively, our results suggest a neuroprotective mechanism where noradrenergic neurons activity preserves the integrity of dopaminergic neurons, which prevents synucleinopathy-dependent loss of these cells.

Fig 1. Chronic stimulation of LC-NE neurons using hM3D-DREADD.

A- Experimental approach to chronically activate LC-NE neurons. B- Coronal section showing mCherry labeled neurons (red) at the injection sites (locus coeruleus), scale bar 300μm. C- Representative image showing colocalization of mCherry (red) and TH positive cells (green) in locus coeruleus, scale bar 200 μm. D- Representative image showing LC-NE projections to substantia nigra, mCherry positive fibers (red), TH positive cells (green), scale bar 200 μm. E, F-Activation of TH neurons in LC, transduced with AAV containing double floxed Gq-coupled hM3D DREADD fused with mCherry under the control of human synapsin promoter, upon injection of saline or CNO, respectively, mCherry (red) and c-Fos positive cells (green) in locus coeruleus, scale bar 50 μm and 200 μm.

Fig 2. Improved motor function after 6 weeks of LC-NE stimulation.

A- Representative track (blue) and rearing (red dots) patterns of all groups in open field test. B- Total activity, percentage of center activity, and rearing in open field test (n = 6–9). C- Distance and latency to fall over the period of 3 days in rotarod test (n = 6–9). ***p<0.0001, **p<0.001, *p<0.05, all values denote means ± SEM.

Fig 3. Neuroprotective effect of LC-NE action on SNc dopaminergic neurons.

A-Representative images of TH staining in SNc from all groups, TH (green), scale 200μm. B-TH-positive cells count in SNc (n = 6–9). C- Representative absolute-eigen images of TH staining from all groups in SNc, scale 50μm. D- Quantification of TH⁺ cellular morphology integrity in SNc (n = 6–9). E- Representative images of TH staining in LC from all groups, TH (green), scale 200μm.G- Quantification of TH-positive cells in LC (n = 6–9). ****p<0.00001, ***p<0.0001, **p<0.001, *p<0.05, all values denote means ± SEM.

Fig 4. LC-NE activation does not mitigate α-synuclein aggregation.

A- Immunohistochemistry against α-syn and TH in SNc of SNCA^Control and SNCA^LC mice, TH (purple), α-syn (green), DAPI (blue), scale bar 20μm. B- Quantification of the total area of α-syn positive staining in the SNc (n = 6–9). C- Analysis of α-syn particle size distribution in SNCA^Control and SNCA^LC mice (n = 4–6), right upper panel: representative image used in the analysis, obtained by BX-Z Analyzer software, scale 50μm. D- Immunohistochemistry against pSer129-αSyn from all groups, pSer129-αSyn (green), Nissl (magenta), DAPI (blue), scale bar 20μm. Arrow denotes Lewy neurites. ****p<0.00001, all values denote means ± SEM.

Fig 5. LC-NE chronic stimulation promotes systemic insulin resistance in α-syn overexpressing animals.

A- Changes in body weight over 6 weeks during CNO treatment, (n = 6–9). B- Lean and fat mass at weeks 2 and 6 of chronic LC-NE stimulation (n = 6–9). C-Fasting blood glucose levels on week 6 of LC-NE chronic stimulation (n = 6–9). D- Insulin tolerance test performed at week 6 of CNO treatment, (n = 6–9). E- Insulin tolerance testing area under the curve analysis 6 weeks after beginning of the CNO treatment (n = 6–9). *p<0.05, all values denote means ± SEM.

Fig 6. Neuroprotective role of NE in AAV-α-synuclein transduced primary DA cell culture.

A- Time line for primary mouse ventral midbrain dopaminergic neurons tissue culture and representation of neurite analysis showing examples of roots, branch points, and total extremities. B- Representative images of dopaminergic neurons infected with AAV-hSynA53T α-synuclein or AAV-GFP, supplemented with vehicle, NE, or GDNF and immunostained with TH (red), GFP (green) or α-synuclein (green) with DAPI nuclear stain (blue), scale bar 20 μm. C- TH neurons survival. D-H- Neurite growth analysis quantifying branching, root number, extremities and neurite length. ****p<0.00001, ***p<0.0001, **p<0.001, *p<0.05, all values denote means ± SEM.