Establishment of new experimental models of progressive prodromal Parkinson’s disease using viral-mediated overexpression of wild-type human α-synuclein in the substantia nigra compacta and the locus coeruleus in mice
Part 1. Summary Alpha-7-nicotine-acetylcholine-receptor (α7-nAChR) agonists modulate the cholinergic anti-inflammatory pathway to attenuate proinflammatory signals and reduce dopaminergic neuronal cell loss in toxin-induced experimental murine models of Parkinson’s disease (PD). However, no researc...
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|Summary:||Part 1. Summary
Alpha-7-nicotine-acetylcholine-receptor (α7-nAChR) agonists modulate the cholinergic anti-inflammatory pathway to attenuate proinflammatory signals and reduce dopaminergic neuronal cell loss in toxin-induced experimental murine models of Parkinson’s disease (PD). However, no research has been performed to evaluate the effect of α7-nAChR agonists in human α-synuclein (hαSyn) mediated models of PD. We, therefore, investigated the effect of the compound JN403, an α7-nAChR specific agonist, in αSyn treated in-vitro microglia culture and human αSyn overexpression in-vivo mouse model of PD. In primary mouse microglia cells, αSyn fragment 61-140 treatment increased the release of nitric oxide (NO), TNF-α and IL-6, and decreased cell viability. 100 nM of JN403 pre- and co-incubation significantly reduced the level of NO and TNF-α release in the microglial cells. However, cell viability and IL-6 cytokine release did not revert to control level. For in-vivo testing of JN403, unilateral intranigral overexpression of WT-αSyn or of the control protein luciferase (luc) was induced in C57/BL6N mice via stereotaxic delivery of recombinant adeno-associated virus (rAAV) vectors. Targeted WT-αSyn overexpression reduced 20% of the number of tyrosine hydroxylase (TH) immunoreactive (ir) nigral neurons after 10 weeks. Subcutaneous daily treatment of 30 mg/kg JN403 over 9 weeks starting at postoperative week 1 did not reverse the decline of the number of TH-ir nigral neurons (nor Iba1-ir density) in WT-αSyn overexpression mouse model. The reduced density of TH-ir striatal terminals in the WT-αSyn groups was also not recovered by the JN403 treatment. In summary, JN403, an α7-nAChR specific agonist shows a beneficial effect on ameliorating proinflammatory signals in αSyn exposed microglia cells. However, no significant treatment effect is found in an intranigral WT-αSyn overexpression in-vivo mouse model with JN403 therapy employed.
Part 2. Summary
The locus coeruleus (LC) is a small nucleus among the catecholaminergic cell groups, but it has extensively branched axons, and it innervates broad areas in the brain. According to the widely accepted Parkinson’s disease (PD) staging model of Braak, the LC is affected by alpha-synuclein (αSyn) pathology at the second stage, but the possible neurochemical changes induced by axonal αSyn-transport from the LC have never been investigated. Therefore, we overexpressed human wild-type αSyn (WT-αSyn) or the control protein Luciferase (Luc) in the mouse LC via unilateral stereotaxic delivery of recombinant adeno-associated viral vectors (rAAV) and subsequently investigated pathological alterations in a time-dependent manner. Unbiased stereology demonstrated that the number of LC neurons did not significantly decrease upon WT-αSyn overexpression, yet rigorous microgliosis and astrogliosis occurred post 3 weeks post-injection. Intriguingly, the number of choline acetyltransferase (ChAT) immunoreactive (ir) neurons in the dorsal motor nucleus of the vagus (DMnX) and in the nucleus ambiguus (nAmb) significantly decreased in the αSyn group as early as 3 weeks. The putative axonal connectivity between the LC and the cholinergic motor nuclei as well as the loss of ChAT-ir were further elucidated by microinjection of Tyrosine Hydroxylase (TH) promoter-specific viral vectors (rAAV2/5-TH-EGFP or WT-αSyn) in the LC. We found robust αSyn-ir axonal varicosities around the DMnX neurons, and remarkably, intraneuronal αSyn was present in the ambiguus neurons in both hemispheres 3 weeks post-injection. As the compact formation of the nAmb controls the esophagus, we investigated the presence of αSyn in the cervical, thoracic and abdominal parts of the esophagus tissues of injected mice. As a result of aSyn overexpression in the LC, we observed αSyn-ir cells and varicosities in the muscle layers, and notably, some of these αSyn-ir neurons co-localized with myenteric plexus ganglia post 3 and 9 weeks. Furthermore, we found that, unlike the majority of the Vesicular Acetylcholine Transporter (VAChT) signals, some nerve endings were weak in signal and dispersed from the motor receptors. Our data reveal that LC neurons induced the transsynaptic and transneuronal spreading of αSyn in the vagal motor nuclei and suppressed cholinergic efferents terminating on motor endplates in the esophagus. It appears that αSyn can travel through anatomically connected brain regions as proposed in the early staging model of PD; implying α-synucleinopathy in noradrenergic neurons may potentially disturb the cholinergic modulation of the upper gastrointestinal tract rostral-caudally.|
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