Μηχανισμοί νευροεκφύλισης και νευροπροστασίας στο γενετικό μοντέλο ντοπαμινεργικής απονεύρωσης μυός weaver

Abstract

Parkinson’s disease (PD) is characterized by the progressive degeneration of the nigrostriatal dopaminergic innervation that leads to motor disturbances. It is considered to be a multifactor disease, the etiology of which still remains unknown. Since currently available treatments are only symptomatic, having severe side-effects, neuroprotection from the early stages of the disease has been given much attention as a promising approach to PD management. Indeed, a broad range of agents has been investigated for their neuroprotective role in neurotoxical models of acute dopaminergic degeneration. “Weaver” mutant mouse represents a unique genetic model, in which the nigrostriatal neurodegeneration occurs endogenously and progressively, starting after postnatal day 7 (P7) and reaching 50% at P21. In the present study, aiming to identify neuroprotective mechanisms in the early progression of the “weaver” degenerative process and to achieve a potentially pleiotropic therapeutic action, we applied three pharmaceutical agents with different mechanisms of action, as well as a scheme combining them. Specifically, “weaver” mice were treated, individually and in combination, with N-acetylcysteine (NAC) (antioxidant), aspirine (anti-inflammatory) and 17b-estradiol [E2] (antioxidant, antiapoptotic, neurotrophic) daily, from P1 to P21. The neuroprotective effect was evaluated by immunohistochemical detection of dopaminergic (DA) neurons in the substantia nigra (SNpc) of treated animals. The administration of ΝΑC and aspirine did not influence the survival of (DA) neurons of weaver mice. On the contrary, the administration of 17b estradiol led to significant survival of DA neurons of SNpc, approximately 48%, in weaver mice that received E2, comparatively with weaver mice that received saline. Moreover the administration of the combination of the three drugs (cocktail) promoted the survival of DA neurons of SNpc, approximately 86% to a higher degree. Weaver mice that received cocktail had 26% more DA neurons compared to weaver mice that received individually 17b estradiol, proposing a possible synergistic action between 17b estradiol and NAC. The investigation of mechanism of neurodegeneration in SNpc and provided neuroprotection by 17b estradiol and cocktail, was realised in two levels. Initiall, by determination of oxidative stress markers, like lipid peroxidation and markers of cellular thiol redox (GSH, GSSG, CSH NPSSC, PSH, PSSP, NPSH, NSPSSR). The determination of lipid peroxidation was realised in the midbrain and striatum of normal and weaver mice that received saline (saline +/+ and saline wv/wv), 17 b estradiol (17b +/+ and 17b wv/wv) cocktail (cocktail +/+ and cocktail wv/wv). Lipid peroxidation levels in the midbrain were increased about 98% in saline wv/wv mice comparatively with the saline +/+, showing the presence of intense oxidative stress in the weaver mutant mouse. It was interesting, however, the fact that lipid peroxidation was inhibited approximately 27% in 17b wv/wv mice, while it was reverted at the normal levels in cocktail wv/wv mice. Regarding to the other oxidative markers that were examined, only NPSSC showed differences between saline +/+ and saline wv/wv, while the GSSG, PSSP and PSH followed similar increasement in both cocktail +/+ and cocktail wv/w animals. This observation indicates that these markers alone cannot give a clear figure of oxidative situation, as they constitute rapidly altered components of antioxidant cycles. Afterwards, we investigated the expression of genes Lasp1, Supt14h, Nr4a2 (nurr1), Dlg4 and serotonin transporter’s gene (SERT), which appear to be involved in neurodegeneration pathways, in the midbrain ant striatum of normal and weaver mice. There were not observed differences in their expression levels (using the RT-PCR technique) in both regions investigated. The results of the present study, lead to the conclusion that 17b-estradiol provided important neuroprotection in the DA neurons, for the first time, in a model of in vivo, endogenous, progressive dopaminergic degeneration, the weaver model. The mechanism of E2’s neuroprotective effect appears to be antioxidant as the administration of E2 suspends lipid peroxidation. Moreover the E2’s neuroprotective effect was strengthened significantly by the co-treatment of NAC, proposing the existence of synergy between E2 and GSH, for the first time in an in vivo model of neurodegeneration. The reinforced cocktail’s result gives an additional argument in the hypothesis of antioxidant mechanism of E2’s action, as cocktail, at the same time, restores lipid peroxidation in normal levels. These observations propose E2 as a future candidate pharmaceutic treatment for neurodegenerative situations, like PD, of course for female individuals. Moreover they propose that the combined treatment of E2 and NAC, can lead to the application of lower and, in consequence, less aggravating doses, concerning the side effects, that will lead to same or even higher neuroprotective result with the individual administration of 17b-estradiol