Characterization of substantia nigra neurogenesis in homeostasis and dopaminergic degeneration: beneficial effects of the microneurotrophin BNN-20.

BACKGROUND: Loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) underlines much of the pathology of Parkinson's disease (PD), but the existence of an endogenous neurogenic system that could be targeted as a therapeutic strategy has been controversial. BNN-20 is a synthetic, BDNF-mimicking, microneurotrophin that we previously showed to exhibit a pleiotropic neuroprotective effect on the dopaminergic neurons of the SNpc in the "weaver" mouse model of PD. Here, we assessed its potential effects on neurogenesis. METHODS: We quantified total numbers of dopaminergic neurons in the SNpc of wild-type and "weaver" mice, with or without administration of BNN-20, and we employed BrdU labelling and intracerebroventricular injections of DiI to evaluate the existence of dopaminergic neurogenesis in the SNpc and to assess the origin of newborn dopaminergic neurons. The in vivo experiments were complemented by in vitro proliferation/differentiation assays of adult neural stem cells (NSCs) isolated from the substantia nigra and the subependymal zone (SEZ) stem cell niche to further characterize the effects of BNN-20. RESULTS: Our analysis revealed the existence of a low-rate turnover of dopaminergic neurons in the normal SNpc and showed, using three independent lines of experiments (stereologic cell counts, BrdU and DiI tracing), that the administration of BNN-20 leads to increased neurogenesis in the SNpc and to partial reversal of dopaminergic cell loss. The newly born dopaminergic neurons, that are partially originated from the SEZ, follow the typical nigral maturation pathway, expressing the transcription factor FoxA2. Importantly, the pro-cytogenic effects of BNN-20 were very strong in the SNpc, but were absent in other brain areas such as the cortex or the stem cell niche of the hippocampus. Moreover, although the in vitro assays showed that BNN-20 enhances the differentiation of NSCs towards glia and neurons, its in vivo administration stimulated only neurogenesis. CONCLUSIONS: Our results demonstrate the existence of a neurogenic system in the SNpc that can be manipulated in order to regenerate the depleted dopaminergic cell population in the "weaver" PD mouse model. Microneurotrophin BNN-20 emerges as an excellent candidate for future PD cell replacement therapies, due to its area-specific, pro-neurogenic effects.

Anti-neuroinflammatory, protective effects of the synthetic microneurotrophin BNN-20 in the advanced dopaminergic neurodegeneration of "weaver" mice.

BNN-20 is a synthetic microneurotrophin, long-term (P1-P21) administration of which exerts potent neuroprotective effect on the "weaver" mouse, a genetic model of progressive, nigrostriatal dopaminergic degeneration. The present study complements and expands our previous work, providing evidence that BNN-20 fully protects the dopaminergic neurons even when administration begins at a late stage of dopaminergic degeneration (>40%). Since neuroinflammation plays a critical role in Parkinson's disease, we investigated the possible anti-neuroinflammatory mechanisms underlying the pharmacological action of BNN-20. The latter was shown to be microglia-mediated, at least in part. Indeed, BNN-20 induced a partial, but significant, reversal of microglia hyperactivation, observed in the untreated "weaver" mouse. Furthermore, it induced a shift in microglia polarization towards the neuroprotective M2 phenotype, suggesting a possible beneficial shifting of microglia activity. This observation was further supported by morphometric measurements. Moreover, BDNF levels, which were severely reduced in the "weaver" mouse midbrain, were restored to normal even after short-term BNN-20 administration. Experiments in "weaver"/NGL (dual GFP/luciferase-NF-κВ reporter) mice using bioluminescence after a short BNN-20 treatment (P60-P74), have shown that the increase of BDNF production was specifically mediated through the TrkB-PI3K-Akt-NF-κB signaling pathway. Interestingly, long-term BNN-20 treatment (P14-P60) significantly increased dopamine levels in the "weaver" striatum, which seems to be associated with the improved motor activity observed in the treated mutant animals. In conclusion, our findings suggest that BNN-20 may serve as a lead molecule for new therapeutic compounds for Parkinson's disease, combining strong anti-neuroinflammatory and neuroprotective properties, leading to elevated dopamine levels and improved motor activity.

BNN-20, a synthetic microneurotrophin, strongly protects dopaminergic neurons in the "weaver" mouse, a genetic model of dopamine-denervation, acting through the TrkB neurotrophin receptor.

Neurotrophic factors are among the most promising treatments aiming at slowing or stopping and even reversing Parkinson's disease (PD). However, in most cases, they cannot readily cross the human blood-brain-barrier (BBB). Herein, we propose as a therapeutic for PD the small molecule 17-beta-spiro-[5-androsten-17,2'-oxiran]-3beta-ol (BNN-20), a synthetic analogue of DHEA, which crosses the BBB and is deprived of endocrine side-effects. Using the "weaver" mouse, a genetic model of PD, which exhibits progressive dopaminergic neurodegeneration in the Substantia Nigra (SN), we have shown that long-term administration (P1-P21) of BNN-20 almost fully protected the dopaminergic neurons and their terminals, via i) a strong anti-apoptotic effect, probably mediated through the Tropomyosin receptor kinase B (TrkB) neurotrophin receptor's PI3K-Akt-NF-κB signaling pathway, ii) by exerting an efficient antioxidant effect, iii) by inducing significant anti-inflammatory activity and iv) by restoring Brain-Derived Neurotrophic Factor (BDNF) levels. By intercrossing "weaver" with NGL mice (dual GFP/luciferase-NF-κΒ reporter mice, NF-κΒ.GFP.Luc), we obtained Weaver/NGL mice that express the NF-κB reporter in all somatic cells. Acute BNN-20 administration to Weaver/NGL mice induced a strong NF-κB-dependent transcriptional response in the brain as detected by bioluminescence imaging, which was abolished by co-administration of the TrkB inhibitor ANA-12. This indicates that BNN-20 exerts its beneficial action (at least in part) through the TrkB-PI3K-Akt-NF-κB signaling pathway. These results could be of clinical relevance, as they suggest BNN-20 as an important neuroprotective agent acting through the TrkB neurotrophin receptor pathway, mimicking the action of the endogenous neurotrophin BDNF. Thus BNN-20 could be proposed for treatment of PD.

Exposure to novel environment is characterized by an interaction of D1/NMDA receptors underlined by phosphorylation of the NMDA and AMPA receptor subunits and activation of ERK1/2 signaling, leading to epigenetic changes and gene expression in rat hippocampus.

Interactions between dopamine and glutamate receptors are essential for prefrontal cortical (PFC) and hippocampal cognitive functions. The hippocampus has been identified as a detector of a novel stimulus, where an association between incoming information and stored memories takes place. Further to our previous results which showed a strong synergistic interaction of dopamine D1 and glutamate NMDA receptors, the present study is going to investigate the functional status of that interaction in rats, following their exposure to a novel environment. Our results showed that the "spatial" novelty induced in rat hippocampus and PFC (a) a significant increase in phosphorylation of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, which are both dependent on the concomitant stimulation of D1/NMDA receptors and are both abolished by habituation procedure, (b) chromatin remodeling events (phosphorylation-acetylation of histone H3) and (c) an increase in the immediate early genes (IEGs) c-Fos and zif-268 expression in the CA1 region of hippocampus, which is dependent on the co-activation of D1/NMDA and acetylcholine muscarinic receptors. In conclusion, our results clearly show that a strong synergistic interaction of D1/NMDA receptor is required for the novelty-induced phosphorylation of NMDA and AMPA receptor subunits and for the robust activation of ERK1/2 signaling, leading to chromatin remodeling events and the expression of the IEGs c-Fos and zif-268, which are involved in the regulation of synaptic plasticity and memory consolidation.

Blockade of adenosine A2A receptors downregulates DARPP-32 but increases ERK1/2 activity in striatum of dopamine deficient "weaver" mouse.

In the present study we investigated the signal transduction cascade modulated by adenosine A(2A) receptors under chronic dopamine deficiency in the "weaver" mouse. We determined the phosphorylation state of cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at Thr34 and of Extracellular Signal-regulated Protein Kinases 1/2 (ERK1/2), under basal conditions and after in vivo stimulation of A(2A) receptors by administration of the agonist CGS21680. Our results revealed that the endogenous levels of phospho-DARPPP-32 and phospho-ERK1/2 are elevated in "weaver" striatum probably as an adaptation phenomenon to gradual dopaminergic neurodegeneration appearing in this animal model, characterized as phenocopy of Parkinson's disease. Stimulation of A(2A) receptors by CGS21680 further increases phospho-DARPP-32 but downregulates significantly the elevated phospho-ERK1/2 levels bringing them close to those observed in wild type animals. Consistently, blockade of A(2A) receptors by MSX-3 (A(2A) receptor antagonist) downregulates phospho-DARPP-32 but significantly increases even more the phosphorylation/activation of ERK1/2. These results indicate that under chronic dopamine deficiency (a) the A(2A)/cAMP/PKA/DARPP-32 cascade is overactive due to the elevated endogenous phospho-DARPP-32 levels and (b) the A(2A) receptor modulatory effect on ERK1/2 signaling is dysregulated exerting opposing action compared to that observed in normal animals (Quiroz et al., 2006), i.e. in "weaver" animals A(2A) receptor blockade increases the activity of ERK1/2 cascade. This could be of clinical relevance since A(2A) antagonists are already used in clinical trials for ameliorating Parkinson's disease (PD) symptoms.

Differential pharmacological properties of GABAA/benzodiazepine receptor complex in dorsal compared to ventral rat hippocampus.

Several studies have indicated a functional differentiation across the septotemporal axis of rat hippocampus. Our previous results have shown that the alpha 1 beta 2 gamma 2-GABAA receptor subtype dominates in dorsal hippocampus (DH), while the alpha 2 beta 1 gamma 2-subtype prevails in ventral hippocampus (VH). We therefore studied possible differences in the pharmacological properties and receptor binding parameters of the GABAA receptor subtypes between DH and VH, by examining: (1)(a) the specific binding of [3H]-flunitrazepam (Benzodiazepine sites agonist) by using quantitative autoradiography, (b) the kinetic parameters of [3H]-flunitrazepam specific binding, by using the "wipe off" technique and (2) the competitive displacement of [3H]-flunitrazepam binding by using zolpidem (selective agonist of the alpha 1-subtype) and L-655,708 (selective inverse agonist of the alpha 5-subtype) and the enhancement of [3H]-flunitrazepam binding by using etomidate (selective positive modulator of the beta 2-subunit), in an autoradiographical saturation kinetic study. Our results showed in VH compared to DH: (A) lower level of [3H]-flunitrazepam binding, apparently due to weaker binding affinity (higher KD value), since no differences in the Bmax value could be detected, (B) higher IC50 values for zolpidem and lower IC50 values for L-655,708 and (C) higher EC50 values for etomidate. In conclusion, the lower binding for zolpidem and etomidate and the higher binding for L-655,708 observed in VH support the evidence that the alpha 1 beta 2 gamma 2-GABAA receptor subtype dominates in DH and the alpha 5-subtype prevails in VH. Further, our results suggest differential pharmacological effects of the benzodiazepines in DH compared to VH, with the sedative effects being more potent in the dorsal hippocampus.

Thiol redox state and lipid and protein oxidation in the mouse striatum after pentylenetetrazol-induced epileptic seizure.

PURPOSE: In the present study, we examined the effects of pentylenetetrazol (PTZ) administration on the thiol redox state (TRS), lipid peroxidation, and protein oxidation in the mouse striatum to (a) quantitate the major components of TRS and relate them to oxidative stress, and (b) investigate whether neuronal activation without synchronization, induced by subconvulsive doses of PTZ, can cause similar qualitative effects on TRS in this brain area. Specifically, we examined the TRS components glutathione (GSH), glutathione disulfide (GSSG), cysteine (CSH), protein thiols (PSH), and the protein (P) and nonprotein (NP/R) disulfides PSSR, NPSSR, NPSSC, and PSSP. METHODS: TRS components were measured photometrically (GSSG enzymatically) as were lipid peroxidation and protein oxidation. RESULTS: GSH, GSSG, and NPSSC levels are decreased by 45%, 38% and 26%, respectively, at 15 min after seizure; PSSP and PSSR levels and lipid peroxidation are increased by 47%, 200% and 22%, respectively, whereas CSH, NPSSR, PSH, PSSC, and protein carbonyl levels do not change. At 30 min after seizure, GSH, GSSG, CSH, NPSSC, and protein carbonyl levels are decreased by 26%, 62%, 25%, 40%, and 13%, respectively. PSSP and NPSSR levels are increased by 30% and 42%, respectively, whereas PSH, PSSC, PSSR, and lipid peroxidation remain unchanged. At 24 h after seizure, GSH, NPSSR, PSSR, and lipid-peroxidation levels return to normal; GSSG, CSH, NPSSC, and protein carbonyl levels are decreased by 44%, 22%, 30%, and 27%, respectively. CONCLUSIONS: The significant decrease in GSH, GSSG, CSH, and NPSSC and the increase in PSSP, NPSSR, PSSR, and lipid peroxidation after PTZ-induced seizure strongly suggest increased oxidative stress in the mouse striatum.

Thiol redox state and oxidative stress in midbrain and striatum of weaver mutant mice, a genetic model of nigrostriatal dopamine deficiency.

In this study we measured thiol redox state (TRS) and the oxidative stress indicator lipid peroxidation in midbrain and striatum of adult (4 months old) male control (+/+) and weaver (wv/wv) mice in order to relate them with oxidative stress in conditions of progressive and severe (approximately 70%) nigrostriatal dopaminergic neurodegeneration. Specifically, we measured the specific TRS components glutathione (GSH), glutathione disulfide (GSSG), cysteine (CSH), and the general classes of TRS components. The latter are the protein thiols (PSH) and the disulfides between (a) protein (P) and protein thiols (PSSP), (b) protein and non-protein (NP/R) thiols (PSSR, PSSC) and (c) non-protein and non-protein thiols (NPSSR, NPSSC). In addition, the main product of lipid peroxidation malonyl dialdehyde (MDA) was estimated. In the midbrain of wv/wv, GSH and NPSSC levels are decreased (44% and 64%, respectively) and GSSG, NPSSR, CSH, PSH, PSSP, PSSR and MDA levels are increased (23%, 660%, 110%, 51%, 68%, 18% and 44%, respectively). In the striatum of male wv/wv, protein and non-protein thiol/disulfide and MDA levels do not change, possibly due to the high decrease in striatal dopamine level versus midbrain. Our data show that the high degeneration of the dopaminergic nigrostriatal neurons in male adult wv/wv mice is accompanied by significant changes in TRS and an increase in lipid peroxidation in the midbrain, suggesting involvement of oxidative stress in the degeneration of dopaminergic neurons. They also strengthen the possible use of thiol antioxidants for the development of new neuroprotective therapeutic strategies for neurodegenerative diseases, such as Parkinson's disease.

Effect of pentylenetetrazol-induced epileptic seizure on thiol redox state in the mouse cerebral cortex.

In the present study we examined the effects of pentylenetetrazol (PTZ) administration on the thiol redox state (TRS), lipid peroxidation and protein oxidation in left and right mouse cerebral cortex in order (a) to quantitate the major components of the thiol redox state and relate them with oxidative stress and cortical laterality, and (b) to investigate whether neuronal activation without synchronization, induced by subconvulsive doses of PTZ, can cause similar qualitative effects on the thiol redox state. Specifically, we examined the TRS components [glutathione (GSH), glutathione disulfide (GSSG), cysteine (CSH), protein (P) thiols (PSH) and protein and non-protein (NP) mixed/symmetric disulfides (PSSR, NPSSR, NPSSC, PSSP)]. At 15 min after seizure, GSH, GSSG, CSH, NPSSC, PSSR and PSSC levels are decreased in left (14-50%) and right (11-53%) cortex while PSSP levels are increased in both left (1400%) and right (1600%) cortex. At 30 min after seizure, GSSG, CSH, NPSSC, PSSR and PSSC levels are decreased in left (14-51%) and right (18-56%) cortex while PSSP and protein carbonyl levels are increased in left (2300% and 20%, respectively) and right (2800% and 21%, respectively) cortex. At 24 h after seizure, the TRS components return to normal and protein carbonyl levels are decreased in left (16%) and right (20%) cortex. The significant decrease in GSH, GSSG, CSH, NPSSC, PSSR and PSSC, as well as the increase in protein carbonyl and the high increase in PSSP levels after PTZ-induced seizure indicate increased oxidative stress in cerebral cortex of mice, and of similar magnitude and TRS-component profiles between left and right cerebral cortex.

Thiol redox state (TRS) and oxidative stress in the mouse hippocampus after pentylenetetrazol-induced epileptic seizure.

In this study we evaluated oxidative stress (lipid peroxidation and protein oxidation) and thiol redox state [TRS: glutathione (GSH), glutathione disulfide (GSSG), cysteine (CSH), protein (P) thiols (PSH) and protein and non-protein (NP) mixed/symmetric disulfides (PSSR, NPSSR, NPSSC, PSSP)] in hippocampus after pentylenetetrazol (PTZ) administration at convulsive and subconvulsive dose. The significant decrease in PSH, CSH and NPSSC, as well as the increase in PSSP, NPSSR, lipid peroxidation and protein oxidation levels after PTZ-induced seizure indicate increased oxidative damage in hippocampus, although the levels of GSH and GSSG do not change significantly.