Endogenous versus exogenous cell replacement for Parkinson’s disease: where are we at and where are we going?

Parkinson’s disease is the second most common neurodegenerative disease and has currently no effective treatment, one that would be able to stop or reverse the loss of dopaminergic neurons in the substantia nigra pars compacta. In addition, Parkinson’s disease diagnosis is typically done when a significant percentage of the dopaminergic neurons is already lost. In neurodegenerative disorders, some therapeutic strategies could be effective only at inhibiting further degeneration; on the other hand, cell replacement therapies aim at replacing lost neurons, an approach that would be ideal for the treatment of Parkinson’s disease. Many cell replacement therapies have been tested since the 1970s in the field of Parkinson’s disease; however, there are still significant limitations prohibiting a successful clinical application. From the first fetal midbrain intrastriatal graft to the most recent conversion of astrocytes into dopaminergic neurons, we have gained equally, significant insights and questions still looking for an answer. This review aims to summarize the main milestones in cell replacement approaches against Parkinson’s disease. By focusing on achievements and failures, as well as on the additional research steps needed, we aim to provide perspective on how future cell replacement therapies treats Parkinson’s disease

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

Enhancement of endogenous midbrain neurogenesis by microneurotrophin BNN-20 after neural progenitor grafting in a mouse model of nigral degeneration

We have previously shown the neuroprotective and pro-neurogenic activity of microneurotrophin BNN-20 in the substantia nigra of the “weaver” mouse, a model of progressive nigrostriatal degeneration. Here, we extended our investigation in two clinically-relevant ways. First, we assessed the effects of BNN-20 on human induced pluripotent stem cell-derived neural progenitor cells and neurons derived from healthy and parkinsonian donors. Second, we assessed if BNN-20 can boost the outcome of mouse neural progenitor cell intranigral transplantations in weaver mice, at late stages of degeneration. We found that BNN-20 has limited direct effects on cultured human induced pluripotent stem cell-derived neural progenitor cells, marginally enhancing their differentiation towards neurons and partially reversing the pathological phenotype of dopaminergic neurons generated from parkinsonian donors. In agreement, we found no effects of BNN-20 on the mouse neural progenitor cells grafted in the substantia nigra of weaver mice. However, the graft strongly induced an endogenous neurogenic response throughout the midbrain, which was significantly enhanced by the administration of microneurotrophin BNN-20. Our results provide straightforward evidence of the existence of an endogenous midbrain neurogenic system that can be specifically strengthened by BNN-20. Interestingly, the lack of major similar activity on cultured human induced pluripotent stem cell-derived neural progenitors and their progeny reveals the in vivo specificity of the aforementioned pro-neurogenic effect

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

Endogenous versus exogenous cell replacement for Parkinson’s disease: where are we at and where are we going?

Parkinson’s disease is the second most common neurodegenerative disease and has currently no effective treatment, one that would be able to stop or reverse the loss of dopaminergic neurons in the substantia nigra pars compacta. In addition, Parkinson’s disease diagnosis is typically done when a significant percentage of the dopaminergic neurons is already lost. In neurodegenerative disorders, some therapeutic strategies could be effective only at inhibiting further degeneration; on the other hand, cell replacement therapies aim at replacing lost neurons, an approach that would be ideal for the treatment of Parkinson’s disease. Many cell replacement therapies have been tested since the 1970s in the field of Parkinson’s disease; however, there are still significant limitations prohibiting a successful clinical application. From the first fetal midbrain intrastriatal graft to the most recent conversion of astrocytes into dopaminergic neurons, we have gained equally, significant insights and questions still looking for an answer. This review aims to summarize the main milestones in cell replacement approaches against Parkinson’s disease. By focusing on achievements and failures, as well as on the additional research steps needed, we aim to provide perspective on how future cell replacement therapies treats Parkinson’s disease

Induction of adult dopaminergic neurogenesis in the substantia nigra of the "weaver" mouse (preclinical model of parkinson's disease), under long-term administration of the synthetic microneurotrophin BNN-20

Neurotrophic factors are among the most promising therapeutic candidates against Parkinson's disease (PD), but their clinical use is limited due to their inability to cross the BBB. Previous results of our research team in the preclinical PD model, the “weaver” mouse, suggest that the synthetic micro-neurotrophin BNN-20 exhibits pleiotropic neuroprotective activity on the dopaminergic neurons of the “weaver” SNpc, significantly inhibiting neurodegeneration when its administration starts before the onset of neurodegeneration (P0-P20). The aforementioned neuroprotective effect is mainly exerted due to BNN-20's ability to selectively activate the TrkB receptor of BDNF, and its downstream signaling pathways, TrkB-PI3K-Akt-NFκB and TrkB-ERK1/2-NFκB, acting as a mimetic molecule of BDNF, while crossing the BBB. The primary purpose of the present PhD thesis was to investigate the neuroprotective effect of BNN-20 when administration starts in P14, where neurodegeneration has already reached 40%, similar to the degeneration observed at the onset of motor symptoms in human PD. In the first part of the present study, we have observed that BNN-20 administration (P14-P40 and P14-P60) not only inhibited further neurodegeneration but also increased the total number of dopaminergic neurons in SNpc, indicating the presence of adult dopaminergic neurogenesis.Consequently, the second objective of this thesis was a) to confirm the existence of adult dopaminergic neurogenesis in the SNpc (a fact that remained controversial to the moment); b) to evaluate the effect of chronic BNN-20 administration (P14-P60) on neurogenesis; as well as c) to investigate the origin of the newly born dopaminergic neurons of the SNpc. The presence of adult dopaminergic neurogenesis was assessed in vivo by double immunofluorescence against the proliferation marker5-bromo-2'-deoxyuridine (BrdU) and the marker of the dopaminergic neurons tyrosine hydroxylase (TH), in wild-type and the “weaver” SNpc following daily BNN-20 (P14-P60) and BrdU (P40-P60 or P20-P40) administration.In addition, the neurogenic ability of the adult Substantia Nigra (SN), as well as the neurogenic activity of BNN-20, was further evaluated by isolation and culture of adult neural stem cells (NSCs) from the SN and sub-ependymal zone of the lateral ventricles (SEZ).The results suggest that the wild-type SNpc (P60) exhibits a low basal rate of adult dopaminergic neurogenesis, which is significantly increased in the “weaver” mouse, probably as a compensatory mechanism. Chronic BNN-20 administration led to a further, dramatic increase in the rate of dopaminergic neurogenesis in SNpc, indicating its neurogenic effect. The aforementioned are consistent with the in vitro results, where few primary neurospheres were grown from the wild-type SN, while significantly more were developed from the "weaver" SN. Both the chronic in vivo administration of BNN-20 as well as the direct addition of BNN-20 in the cell culture medium affected neurogenesis primarily by promoting the differentiation of NSCs into neurons and glia, and, to a much lesser extent, by affecting their proliferation.In addition, the neurogenic effect of BNN-20 appeared to be tissue-specific, since no change in the rate of neurogenesis was observed in the major neurogenic areas of the adult brain (the subgranular layer of the dentate gyrus - SGZ and SEZ) after BNN-20 administration. Finally, by labeling the NSCs of the SEZ using stereotaxic infusion of the lipophilic dye DiI into the wild-type and “weaver” lateral ventricles, we have concluded that the newly-born dopaminergic neurons of SNpc originate, at least partially, from the NSCs of the SEZ.Taken together, the results of the present PhD thesis highlight BNN-20 as a promising micromolecular agent that may not only inhibit, but also potentially reverse the progression of PD.Οι νευροτροφικοί παράγοντες αποτελούν μερικούς από τους πλέον υποσχόμενους θεραπευτικούς υποψήφιους κατά της νόσου του Πάρκινσον (PD), αλλά η κλινική τους χρήση είναι περιορισμένη εξαιτίας της ανικανότητάς τους να διασχίσουν τον αιματοεγκεφαλικό φραγμό (BBB). Προηγούμενα αποτελέσματα της ομάδας μας, στο προκλινικό μοντέλο της PD μυ "weaver", υποδηλώνουν ότι η μικρομοριακή συνθετική νευροτροφίνη BNN-20 εμφανίζει πλειοτροπική νευροπροστατευτική δράση επί των ντοπαμινεργικών νευρώνων της SNpc του μυός «weaver», αναστέλλοντας σημαντικά τη νευροεκφύλιση, όταν η χορήγηση ξεκινά πριν την έναρξη της νευροεκφύλισης (Ρ0-Ρ20). Η νευροπροστατευτική του δράση ασκείται, χάρη στην ικανότητα του BNN-20 να ενεργοποιεί εκλεκτικά τον ειδικό υποδοχέα του BDNF ΤrkB, και τις καθοδικές σηματοδοτικές οδούς, TrkB-PI3K-Akt-NFκB και TrkB-ERK1/2-NFκB, δρώντας ως μιμητικό μόριο του BDNF, ενώ παράλληλα διαπερνά το BBB. Αρχικός σκοπός της παρούσας διατριβής ήταν να διερευνηθεί η νευροπροστατευτική επίδραση του ΒΝΝ-20 όταν η χορήγηση ξεκινά την Ρ14, όπου η νευροεκφύλιση έχει φτάσει το 40%, ομοιάζοντας την εξέλιξη της νόσου κατά την εκδήλωση των κινητικών συμπτωμάτων στον άνθρωπο. Στο πρώτο αυτό μέρος της παρούσας διατριβής παρατηρήθηκε ότι η χορήγηση BNN-20 τα διαστήματα Ρ14-Ρ40 και Ρ14-Ρ60, όχι μόνο αναστέλλει την περαιτέρω νευροεκφύλιση, αλλά και αυξάνει το συνολικό αριθμό των ντοπαμινεργικών νευρώνων στην SNpc, υποδεικνύοντας την παρουσία ενήλικης ντοπαμινεργικής νευρογένεσης. Συνεπώς, δεύτερο στόχο της παρούσας διατριβής αποτέλεσε α) η επιβεβαίωση της ύπαρξης ενήλικης ντοπαμινεργικής νευρογένεσης στην SNpc, (η οποία ήταν μέχρι στιγμής αμφιλεγόμενη), β) η αξιολόγηση της επίδρασης της χρόνιας χορήγησης BNN-20 (P14-P60) στη νευρογένεση αυτή και γ) η προέλευση των νεογεννηθέντων νευρώνων της SNpc. Η παρουσία ενήλικης ντοπαμινεργικής νευρογένεσης αξιολογήθηκε in vivo με διπλή ανοσοφθορισμική χρώση έναντι του δείκτη πολλαπλασιαζόμενων κυττάρων BrdU και του δείκτη ντοπαμινεργικών νευρώνων υδροξυλάση της τυροσίνης (ΤΗ), στην SNpc άγριου-τύπου και «weaver» μυών την Ρ60, μετά από καθημερινή χορήγηση BNN-20 (Ρ14-Ρ60) και 5-βρωμο-2'-δεοξυουριδίνης (BrdU) (Ρ40-Ρ60 ή Ρ20-Ρ40). Επιπρόσθετα, η νευρογενετική ικανότητα της μέλαινας ουσίας (SN), καθώς και η νευρογενετική δράση του ΒΝΝ-20 μελετήθηκε περαιτέρω, μέσω απομόνωσης και καλλιέργειας ενήλικών νευρικών βλαστικών κυττάρων (NSCs) από την SN και την υποεπενδυματική ζώνη των πλάγιων κοιλιών (SEZ) in vitro.ΤΤα αποτελέσματα ανέδειξαν ότι στην SNpc άγριου τύπου (Ρ60) υπάρχει χαμηλός βασικός ρυθμός ενήλικης ντοπαμινεργικής νευρογένεσης, ο οποίος αυξάνεται σημαντικά στους μύες «weaver», πιθανώς ως αντισταθμιστικός μηχανισμός. Η χρόνια χορήγηση BNN-20 οδήγησε σε περαιτέρω, δραματική αύξηση του ρυθμού ντοπαμινεργικής νευρογένεσης στην SNpc, γεγονός που υποδηλώνει τη νευρογενετική του δράση. Τα παραπάνω είναι συνεπή με τα in vitro αποτελέσματα, όπου λίγες πρωτογενείς νευρόσφαιρες αναπτύχθηκαν από την SN μυών αγρίου-τύπου, ενώ σημαντικά περισσότερες αναπτύχθηκαν από την SN «weaver» μυών. Τόσο η χρόνια χορήγηση BNN-20, όσο και η απευθείας προσθήκη ΒΝΝ-20 στο θρεπτικό υλικό, φαίνεται ότι δρα κυρίως προάγοντας τη διαφοροποίησή των NSCs σε νευρώνες και νευρογλοία, και, λιγότερο, επηρεάζοντας τον πολλαπλασιασμό τους.Επιπρόσθετα, η νευρογενετική δράση του ΒΝΝ-20 εμφανίζεται ιστοειδική, αφού δεν παρατηρήθηκε καμία μεταβολή στη νευρογένεση στις κύριες νευρογενετικές ζώνες του εγκεφάλου (υποκοκκιώδης στιβάδα της οδοντωτής έλικας του ιπποκάμπου - SGZ και SEZ) μετά τη χορήγηση του BNN-20. Τέλος, πειράματα σήμανσης των NSCs της SEZ, με τη στερεοταξική έγχυση της λιπόφιλης χρωστικής DiI στις πλάγιες κοιλίες αγρίου-τύπου και «weaver» μυών, υποδεικνύουν ότι οι νεογεννηθέντες νευρώνες της SNpc, προέρχονται, τουλάχιστον εν μέρει από τα NSCs της SEZ. Τα παραπάνω αναδεικνύουν το ΒΝΝ-20 ως ένα πολλά υποσχόμενο μικρομοριακό παράγοντα που θα μπορεί δυνητικά, όχι μόνο να αναστείλει, αλλά και να αναστρέψει την εξέλιξη της PD

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

Abstract 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