Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

Oxidative stress has been implied in a wide variety of diseases, such as cancer, myocardial infarction, and neurodegenerative diseases including Parkinson's diseases (PD). PD is characterized by the degeneration of dopaminergic (DA) neurons; genetic studies have identified gene mutations causal to PD. Accumulating studies hypothesize that these genes protect DA neurons against oxidative stress. However, lack of experimental tools to target oxidative stress in specific cells has prevented direct evaluation of the hypothesis. We established a novel method to use KillerRed (KR), a genetically-encoded protein that generates radicals upon light activation. We showed its efficacy in live animals by cell-specific ablation of neurons in C. elegans. We applied KR to degenerate DA neurons. By controlling the level of stress via activation light, the protective role of PD-gene, LRRK2, against oxidative stress was confirmed. Thus, we established a method to address the role of oxidative stress in a cell-specific manner.

Oxidative stress has been implied in a wide variety of diseases, such as cancer, myocardial infarction, and neurodegenerative diseases including Parkinson's diseases (PD). PD is characterized by the degeneration of dopaminergic (DA) neurons; genetic studies have identified gene mutations causal to PD. Accumulating studies hypothesize that these genes protect DA neurons against oxidative stress. However, lack of experimental tools to target oxidative stress in specific cells has prevented direct evaluation of the hypothesis. We established a novel method to use KillerRed (KR), a genetically-encoded protein that generates radicals upon light activation. We showed its efficacy in live animals by cell-specific ablation of neurons in C. elegans. We applied KR to degenerate DA neurons. By controlling the level of stress via activation light, the protective role of PD-gene, LRRK2, against oxidative stress was confirmed. Thus, we established a method to address the role of oxidative stress in a cell-specific manner.

Friedreichs ataxia (FRDA) is a progressive spinocerebellar ataxia (SCA) inherited as an autosomal recessive trait. The neurodegeneration, cardiomyopathy, diabetes mellitus and skeletal deformities characteristic to FRDA result from a deficiency in the mitochondrial protein frataxin. Frataxin chaperones iron to heme and iron-sulfur clusters and its deficiency causes mitochondrial iron accumulation and oxidative stress.<p> To address the effect of frataxin deficiency on mitochondrial iron chemistry, mitochondria were isolated from FRDA and control fibroblasts. X-ray absorption spectroscopy showed that ferrihydrite was the predominant form of iron in both. Near edge analysis showed that the ferrihydrite in the FRDA mitochondria resembled the highly organized ferrihydrite of ferritin. Western blotting confirmed that FRDA mitochondria had 3-fold more holoferritin containing stainable iron. I conclude that mitochondria from FRDA fibroblasts mineralize excess iron as ferrihydrite within mitochondrial ferritin.<p> To address how cellular iron dysregulation affected metal distribution in brain and spinal cord, a new synchrotron imaging technique, rapid-scanning x-ray fluorescence (RS-XRF) was employed and validated. Brain structures were readily identified by their unique metal content and distribution. This showed that RS-XRF could be used to reveal metal pathologies associated with diseases of metal metabolism such as FRDA. Since human FRDA tissues were not available for a detailed study, RS-XRF was employed to study the distribution of metals in normal cerebellum, a major site of FRDA-associated neurodegeneration, and to localize and quantify metals in the brain and spinal cord from a patient with a SCA of unknown aetiology. The motivation for this work is the prospect of future systematic studies on metal pathology in neurodegenerative diseases with direct application to FRDA. Novel findings arising from this work were the metal segmentation of the dentate nucleus, the high copper content of the olivary region and the different metal content of lesions at different stages of neurodegeneration. My results suggest that not only iron, but also copper and zinc may play a role in the physiopathology of neurodegeneration. Therefore, all three metals should be investigated in FRDA and other SCA of both known and unknown aetiologies to identify possible new therapeutic targets.

Expanded polyglutamine proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. Polyglutamine tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyglutamine proteins into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of expanded polyglutamine proteins, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyglutamine protein, inclusion body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death.

Friedreichs ataxia (FRDA) is a progressive spinocerebellar ataxia (SCA) inherited as an autosomal recessive trait. The neurodegeneration, cardiomyopathy, diabetes mellitus and skeletal deformities characteristic to FRDA result from a deficiency in the mitochondrial protein frataxin. Frataxin chaperones iron to heme and iron-sulfur clusters and its deficiency causes mitochondrial iron accumulation and oxidative stress.<p> To address the effect of frataxin deficiency on mitochondrial iron chemistry, mitochondria were isolated from FRDA and control fibroblasts. X-ray absorption spectroscopy showed that ferrihydrite was the predominant form of iron in both. Near edge analysis showed that the ferrihydrite in the FRDA mitochondria resembled the highly organized ferrihydrite of ferritin. Western blotting confirmed that FRDA mitochondria had 3-fold more holoferritin containing stainable iron. I conclude that mitochondria from FRDA fibroblasts mineralize excess iron as ferrihydrite within mitochondrial ferritin.<p> To address how cellular iron dysregulation affected metal distribution in brain and spinal cord, a new synchrotron imaging technique, rapid-scanning x-ray fluorescence (RS-XRF) was employed and validated. Brain structures were readily identified by their unique metal content and distribution. This showed that RS-XRF could be used to reveal metal pathologies associated with diseases of metal metabolism such as FRDA. Since human FRDA tissues were not available for a detailed study, RS-XRF was employed to study the distribution of metals in normal cerebellum, a major site of FRDA-associated neurodegeneration, and to localize and quantify metals in the brain and spinal cord from a patient with a SCA of unknown aetiology. The motivation for this work is the prospect of future systematic studies on metal pathology in neurodegenerative diseases with direct application to FRDA. Novel findings arising from this work were the metal segmentation of the dentate nucleus, the high copper content of the olivary region and the different metal content of lesions at different stages of neurodegeneration. My results suggest that not only iron, but also copper and zinc may play a role in the physiopathology of neurodegeneration. Therefore, all three metals should be investigated in FRDA and other SCA of both known and unknown aetiologies to identify possible new therapeutic targets.

The peripheral immune system plays a role in delayed neural injury after stroke. This response originates from the spleen as splenectomy prior to middle cerebral artery occlusion (MCAO) in rats significantly reduces infarct volume in the brain. This research is based on the hypothesis that inhibiting the splenic response will reduce neurodegeneration after stroke. Studies in animals have implicated lymphocytes as the immune cell type that is detrimental following MCAO. Interferon gamma (IFNγ) has been identified as a pro-inflammatory cytokine that is also detrimental following stroke. IFNγ is important because it activates microglia and macrophages in a pro-inflammatory nature that increases neural injury following stroke. Therefore IFNγ was examined in the brain and the spleen following MCAO. IFNγ protein was elevated at 24 h in the spleen and at 72 h in the brain post MCAO. Microglia/macrophages become maximally activated at 72 h in the brain after MCAO. Splenectomy decreases the levels of IFNγ in the brain following MCAO. Systemic administration of IFNγ reversed the protective effects of splenectomy. The cellular response to MCAO was examined next because of the difference in time between the spike in IFNγ in the spleen and the delayed increase in the brain. The cellular response from the spleen was studied by labeling splenocytes five days prior to MCAO with a fluorescein dye. Tissues were examined 48 and 96 h post MCAO or sham MCAO for fluorescence. These cells were released from the spleen into circulation at 48 h post MCAO and migrated to the brain where the cells produced IFNγ at 96 h post MCAO. IFNγ appears to play a role in the splenic response to stroke. One protein that is up regulated by cells that have been activated by IFNγ, interferon-inducible protein 10 (IP-10) is part of the inflammatory cycle driven by IFNγ. IP-10 recruits more IFNγ producing T helper (Th) cells to the site of injury. IP-10 has the unique ability to attract Th1 cells, the pro-inflammatory Th cells, and inhibit Th2 cells, the anti-inflammatory Th cells. This leads to more IFNγ production as IFNγ is the signature cytokine of a Th1 response. IP-10 is significantly increased in the brain at 72 h post MCAO, similar to IFNγ expression. In the spleen IP-10 increased at 24 h and remained elevated out to 96 h following MCAO. IFNγ signaling was inhibited by utilizing an IFNγ neutralizing antibody administered beginning 24 h post MCAO. The IFNγ antibody treated group had decreased infarct volumes, IP-10 levels in the brain, and appeared to have decreased T cells in the ipsilateral hemisphere at 96 h post MCAO. Following ischemic stroke splenocytes are released into circulation and migrate to the brain. They release IFNγ to activate microglia/macrophages in a proinflammatory phenotype causing an increase in IP-10 levels. IP-10 then potentiates the Th1 driven inflammation which inhibits the Th2 response. The elevated levels of IFNγ increase neural injury following MCAO. Blocking IFNγ selectively blocks the inflammatory facet of the immune response to reduce stroke induced neurodegeneration. This leaves the other immune responses intact and able to contribute to tissue repair, regeneration, and able to respond to infections. Selectively inhibiting IFNγ signaling is a promising stroke therapeutic.

Prion diseases are fatal and incurable neurodegenerative diseases that share many pathological similarities to other neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease. One of the earliest pathological signs commonly detected in all of these diseases is the dysfunction followed by loss of neuronal synapses, spines and eventually dendrites that collectively contribute to disruption of normal brain function. These pathologies tend to progressively accumulate within the brain tissue such that extensive damage typically precedes clinical symptom manifestation and ultimate death of neurons. Clearly, understanding the molecular processes responsible for these pathologies could uncover critical pathway(s) that are responsible for propagating this brain damage and could therefore be exploited for therapy development. However, molecular mechanisms implicated in this early pathology remain unidentified. To address this gap in knowledge, this thesis describes a transcriptional approach coupled with specific isolation of neuronal-enriched tissue which was used to help delineate cellular pathways involved in prion-induced neurodegeneration. Profiling cell bodies of CA1 hippocampal neurons known to be affected during early prion disease revealed temporal alteration in both gene and microRNA (gene regulators) expression throughout disease. On a gene expression level, changes in transcript expression during preclinical disease were reminiscent of an activity-dependent neuroprotective gene signature previously described in the literature. These neuroprotective genes were induced during preclinical disease, diminished as disease progressed and were abolished at clinical disease. In support of this process, upregulation of the phosphorylated form of the neuroprotective transcription factor CREB was detected during preclinical disease in these neurons. Furthermore, several genes known to be induced by CREB were also upregulated at preclinical disease in prion-infected mice. Interestingly, expression of numerous deregulated microRNAs paralleled the neuroprotective gene signature of which several are known to remodel neuronal spines and dendrites. To determine whether other preclinically induced microRNAs were also capable of remodeling neuronal structures, gain-of-function studies were performed in primary mouse hippocampal neurons for the uncharacterized miR-26a-5p. Neurons over-expressing miR-26a-5p had enhanced spine density and dendrite arborization, similar to other preclinically-induced microRNAs. Together, these data suggests that CA1 hippocampal neurons induce a neuroprotective transcriptional signature that is evident early in the course of disease within CA1 hippocampal neurons and is abolished by clinical disease. Reestablishment of key molecules that can induce this neuroprotective signature at a time when these genes begin to dissipate could prolong prion disease onset and delay clinical symptom manifestation. / October 2015

<p>Neurons, with their enormous membrane contents, depend heavily on regulated membrane trafficking processes to maintain their morphology and function. The phosphatidylinositol 3-kinase class III, or PIK3C3, plays a critical role in various membrane trafficking processes including both the endocytic and autophagic pathways. The functions of PIK3C3 in the nervous system in vivo are un-characterized. We reasoned that studying PIK3C3 in neurons would provide us an entry point into understanding the regulations and functions of the neuronal membrane trafficking processes and their roles in neuronal morphogenesis and homeostasis. </p><p>We generated a conditional allele of Pik3c3 and first deleted it specifically in the peripheral sensory neurons. Mutant large-diameter myelinated sensory neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated slower than large-diameter neurons. These differential degenerative phenotypes are unlikely caused by a disruption of the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct subcellular phenotypes and very slow degenerations of all sensory neurons. More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. This work uncovered unexpected differences of the endo-lysosomal systems in different types of neurons and discovered a novel autophagy initiation pathway in vivo in neurons. </p><p>To examine the role of PIK3C3 in the central nervous system (CNS), we next deleted Pik3c3 in CNS neural progenitor cells using the Nestin-Cre transgenic line. The resulting conditional knockout mice displayed a severe cortical lamination abnormality caused by defective cortical neuron migration. This finding uncovered a previously under-appreciated role of endocytic trafficking in neural migration, which was further confirmed by electron microscopic analyses of the developing cortex. Moreover, overexpressing the dominant negative forms of Dynamin2 or Rab5, two regulators of endocytosis, caused similar migration defects as Pik3c3-deletion. Mechanistically, Pik3c3-deficient cortical neurons drastically reduced surface Reelin binding sites, and showed significantly decreased levels of Dab1 phosphorylation, despite expressing normal total amount of Reelin receptor ApoER2. This work suggests endocytosis and recycling of Reelin receptors are likely to play an important role in cortical migration regulated by the Reelin signaling pathway. </p><p>These studies represent the first in vivo characterization of PIK3C3 functions in mammals, and provide insight into the complexity and functional importance of neuronal endo-lysosomal and autophagic pathways.</p> / Dissertation

Η Νόσος του Πάρκινσον αποτελεί τη δεύτερη συχνότερη νευροεκφυλιστική ασθένεια μετά τη νόσο του Αλτσχάιμερ, η οποία εμφανίζεται στο 2% των ανθρώπων άνω των 65 ετών. Η μέχρι στιγμής αντιμετώπισή της περιορίζεται σχεδόν αποκλειστικά στη χορήγηση του προδρόμου μορίου της ντοπαμίνης L-DOPA, με σκοπό την αντιμετώπιση των κινητικών προβλημάτων της ασθένειας, η οποία επιφέρει όμως ισχυρές παρενέργειες. Για το λόγο αυτό κρίνεται σκόπιμη η εύρεση νευροπροστατευτικών ουσιών οι οποίες θα καθυστερούν ή θα αναστέλλουν την εξέλιξη της νόσου, με τις ελάχιστες δυνατές παρενέργειες. Ο μυς weaver, φέρει μία αυτοσωμική υπολειπόμενη μετάλλαξη στο γονίδιο Girk2 και αποτελεί το μοναδικό γενετικό μοντέλο της νόσου του Πάρκινσον, το οποίο εμφανίζει προοδευτική απώλεια των ντοπαμινεργικών νευρώνων της μέλαινας ουσίας, η οποία συμβαίνει ενδογενώς. Για το λόγο αυτό θεωρείται ιδανικό για μελέτες νευροπροστασίας. Τόσο in vitro, όσο και in vivo μελέτες στο μοντέλο weaver αλλά και σε άλλα παρκινσονικά μοντέλα (συμπεριλαμβανομένων προηγούμενων αποτελεσμάτων της ομάδας μας, (Διδακτορική Διατριβή Κωνσταντίνου Μποτσάκη, Σεπτέμβριος 2013) αναδεικνύουν ότι το ενδογενές νευροστεροειδές δεϋδροεπιανδροστερόνη (DHEA), καθώς και ο θειικός της εστέρας (DHEA-S) ασκούν ισχυρότατη νευροπροστατευτική δράση στους ντοπαμινεργικούς νευρώνες της μέλαινας ουσίας των παρκινσονικών αυτών ζώων. Επιπλέον, το συνθετικό ανάλογο του DHEA 17β-spiro[5-androstene-17,20-oxiran]-3β-ol (ΒΝΝ-50), το οποίο δεν μεταβολίζεται ενδογενώς σε οιστρογόνα (και θα μπορούσε κατά συνέπεια να είναι κατάλληλο για κλινική χρήση), φαίνεται να ασκεί την ίδια νευροπροστατευτική επίδραση στους ντοπαμινεργικούς νευρώνες της μέλαινας ουσίας των ομοζυγωτικών μυών weaver (Δ.Δ Κωνσταντίνου Μποτσάκη, Σεπτέμβριος 2013), αλλά και σε κυτταροκαλλιέργειες PC12 κυττάρων. Σκοπός της παρούσας εργασίας ήταν η διερεύνηση του μηχανισμού δράσης των νευροπροστατευτικών αυτών ουσιών. Για το λόγο αυτό διερευνήθηκε η πιθανή αντιαποπτωτική δράση των DHEA-S και ΒΝΝ-50, μέσω προσδιορισμού του λόγου των επιπέδων της αντιαποπτωτικής πρωτεΐνης Bcl-2 προς τα επίπεδα της προαποπτωτικής πρωτεΐνης Bax. Ο προσδιορισμός αυτός πραγματοποιήθηκε στο μεσεγκέφαλο φυσιολογικών μυών και μυών weaver ηλικίας Ρ21 ημερών, μετά από χρόνια χορήγηση των νευροστεροειδών (από την Ρ1 έως την Ρ21). Επιπλέον, διερευνήθηκε η πιθανή αντιοξειδωτική δράση του νευροστεροειδούς ΒΝΝ-50, μέσω του προσδιορισμού της ελεύθερης (frMDA) και προσδεδεμένης σε πρωτεΐνες (prMDA) μαλονικής διαλδεΰδης, στο μεσεγκέφαλο φυσιολογικών μυών και μυών weaver ηλικίας Ρ21 ημερών, μετά από χρόνια χορήγηση του αναλόγου (από την Ρ1 έως την Ρ21). Τα αποτελέσματα ανέδειξαν ότι τόσο το DHEA-S, όσο και το BNN-50 ασκούν ισχυρή αντιαποπτωτική δράση στο μεσεγκέφαλο των μυών weaver, αυξάνοντας τον λόγο Bcl-2/Bax (o οποίος εμφανίζεται μειωμένος στo μεσεγκέφαλο των wv/wv ζώων, σε σχέση με τα φυσιολογικά ζώα) κατά 74% και 83% αντίστοιχα, σε σχέση με τα weaver μύες που έλαβαν φυσιολογικό ορό. Η δράση αυτή επιτυχάνεται με διαφορετικό τρόπο, ανάμεσα στα δύο νευροστεροειδή. Πιο συγκεκριμένα, το DHEA-S επαναφέρει πλήρως τα μειωμένα επίπεδα της αντιαποπτωτικής πρωτεΐνης Bcl-2 στo μεσεγκέφαλο των wv/wv ζώων χωρίς να επηρεάζει τα επίπεδα της προαποπτωτικής πρωτεΐνης Bax, ενώ το ΒΝΝ-50 επαναφέρει πλήρως τα αυξημένα επίπεδα της προαποπτωτικής πρωτεΐνης Βax στo μεσεγκέφαλο των wv/wv ζώων, χωρίς να επηρεάζει τα επίπεδα της αντιαποπτωτικής πρωτεΐνης Bcl-2. Επιπλέον, κατέστει σαφές ότι το στεροειδές ΒΝΝ-50 κατέχει και αντιοξειδωτική δράση, αφού επάγει τη δραματική μείωση των επιπέδων της ολικής μαλονικής διαλδεΰδης, τελικού προϊόντος της υπεροξείδωσης των λιπιδίων που εμφανίζεται αυξημένη στo μεσεγκέφαλο των wv/wv ζώων, φέρνοντάς τα κοντά στα φυσιολογικά επίπεδα. Η μείωση αυτή οφείλεται αποκλειστικά στη μείωση των επιπέδων της προσδεδεμένης σε πρωτεΐνες και όχι της ελεύθερης ΜDA, πιθανότατα λόγω της πολύ μεγαλύτερης αύξησης της πρώτης σε σχέση με τη δεύτερη, στο μεσεγκέφαλο των ομοζυγωτικών weaver μυών. Τα παραπάνω αποτελέσματα οδηγούν στο συμπέρασμα ότι τα νευροστεροειδή DHEA-S και ΒΝΝ-50 ασκούν τη νευροπροστατευτική τους επίδραση δρώντας πλειοτροπικά, τουλάχιστον μέσω δύο γραμμών δράσης, της αντιαποπτωτικής και αντιοξειδωτικής. Το γεγονός αυτό είναι ιδιαίτερα σημαντικό καθώς για πρώτη φορά, γίνεται μελέτη του μηχανισμού της δράσης του στεροειδούς ΒΝΝ-50 in vivo. Καθώς το BNN-50 δεν μεταβολίζεται ενδογενώς σε φυλετικές ορμόνες, και θα μπορούσε συνεπώς να είναι κατάλληλο για κλινική χρήση, κρίνεται απαραίτητη η περαιτέρω διερεύνηση του μηχανισμού δράσης του, ώστε να διευκρινιστεί η πιθανή ικανότητα χρήσης του για αντιμετώπιση της σοβαρής αυτής νόσου, μελλοντικά. / Parkinson’s Disease is the second most common neurodegenerative disease, affecting about 2% of the population aged over 65 years old. The most common treatment of the disease until now, is the administration of L-DOPA, a dopamine precursor, in order to reduce the locomotive defects caused by the disease, a drug that causes severe side effects. Hence, the discovery of neuroprotective compounds that can prohibit or at least prolong the progression of the disease is highly required. The weaver mouse carries an autosomic recessive mutation at the Girk-2 gene and consists the only non-invasive genetic model of Parkinson’s Disease that exhibits progressive neurodegeneration of the nigrostriatal dopaminergic neurons. Thus, it is ideal for neuroprotection studies. The endogenous neurosteroif dehydroepiandrosterone (DHEA) as well as its sulphated ester (DHEA-S) exhibit a significant neuroprotective effect on the dopaminergic neurons of the weaver mouse (K. Botsakis phD thesis, 2013), as well as in other in vitro and in vivo studies. Additionally, the synthetic analogue of DHEA 17β-spiro[5-androstene-17,20-oxiran]-3β-ol (BNN-50), that is not metabolized to estrogens in vivo, exhibits the same neuroprotective effect on the dopaminergic neurons of the weaver mouse as DHEA-S (K. Botsakis phD thesis, 2013) as well as in PC12 cell cultures. The aim of this study was the investigation of the mechanism of action of DHEA-S and BNN-50 in vivo. To that extend, we investigated the possible antiapoptotic action of DHEA-S and BNN -50 by determining the ratio of the levels of the antiapoptotic protein Bcl-2 to the levels of pro-apoptotic protein Bax. The assay was performed in the midbrain of control mice and omozygous weaver mice at P21, after chronic administration of neurosteroids ( from P1 to P21 ) . Furthermore , we investigated the potential antioxidant properties of the neurosteroid BNN -50 , through the determination of free (frMDA) and protein-bound (prMDA) malonic dialdeyde in the midbrain of control mice and omozygous weaver mice at P21 after chronic administration of analogue ( from P1 to P21 ) . The results revealed that DHEA-S as well as the BNN-50 exert a very important antiapoptotic action in the weaver mouse midbrain, increasing the ratio of Bcl-2/Bax (that is reduced in the midbrain of the wv / wv animals, compared to control) by 74 % and 83 % respectively, compared with the weaver mice that received saline. This antiapoptotic action is achieved in different ways for the two neurosteroids . More specifically, DHEA-S fully restore the reduced levels of the antiapoptotic protein Bcl-2 in the wv/wv midbrain, without affecting the levels of the proapoptotic protein Bax, while the BNN -50 fully restored the elevated levels of proapoptotic protein Bax in the wv/wv midbrain, without affecting the levels of the antiapoptotic protein Bcl-2. Moreover , it became clear that the steroid BNN -50 possesses significant antioxidant activity, inducing a dramatic reduction in the levels of total MDA , the end product of lipid peroxidation that is displayed increased in the wv/wv midbrain, bringing the MDA level almost to control. The decrease is due to the reduction of the levels of protein-bound and not free MDA , probably due to the much higher increase of the first than the second , in the midbrain of homozygous weaver mice . The aforementioned results suggest that thee neurosteroids DHEA-S and BNN -50 exert their neuroprotective effects by acting pleiotropically , at least through two lines of action, one antiapoptotic and one antioxidant . This is particularly important because for the first time , there is a study of the mechanism of action of steroid BNN -50 in vivo. As BNN-50 is not metabolized endogenously in sex hormones , it could therefore be suitable for clinical use. Hence, it is necessary to further investigate the mechanism of action of the steroid, in order to clarify its possible usability for treating this serious disease in the future .