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 SNCAControl and SNCALC 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 SNCAControl and SNCALC 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.