Comparative Analysis of Gelsemine and Gelsemium sempervirens Activity on Neurosteroid Allopregnanolone Formation in the Spinal Cord and Limbic System

Centesimal dilutions (5, 9 and 15 cH) of Gelsemium sempervirens are claimed to be capable of exerting anxiolytic and analgesic effects. However, basic results supporting this assertion are rare, and the mechanism of action of G. sempervirens is completely unknown. To clarify the point, we performed a comparative analysis of the effects of dilutions 5, 9 and 15 cH of G. sempervirens or gelsemine (the major active principle of G. sempervirens) on allopregnanolone (3α,5α-THP) production in the rat limbic system (hippocampus and amygdala or H-A) and spinal cord (SC). Indeed, H-A and SC are two pivotal structures controlling, respectively, anxiety and pain that are also modulated by the neurosteroid 3α,5α-THP. At the dilution 5 cH, both G. sempervirens and gelsemine stimulated [3H]progesterone conversion into [3H]3α,5α-THP by H-A and SC slices, and the stimulatory effect was fully (100%) reproducible in all assays. The dilution 9 cH of G. sempervirens or gelsemine also stimulated 3α,5α-THP formation in H-A and SC but the reproducibility rate decreased to 75%. At 15 cH of G. sempervirens or gelsemine, no effect was observed on 3α,5α-THP neosynthesis in H-A and SC slices. The stimulatory action of G. sempervirens and gelsemine (5 cH) on 3α,5α-THP production was blocked by strychnine, the selective antagonist of glycine receptors. Altogether, these results, which constitute the first basic demonstration of cellular effects of G. sempervirens, also offer interesting possibilities for the improvement of G. sempervirens-based therapeutic strategies

HS3ST2 expression is critical for the abnormal phosphorylation of tau in Alzheimer's disease-related tau pathology

Heparan sulphate (glucosamine) 3-O-sulphotransferase 2 (HS3ST2, also known as 3OST2) is an enzyme predominantly expressed in neurons wherein it generates rare 3-O-sulphated domains of unknown functions in heparan sulphates. in Alzheimer's disease, heparan sulphates accumulate at the intracellular level in disease neurons where they co-localize with the neurofibrillary pathology, while they persist at the neuronal cell membrane in normal brain. However, it is unknown whether HS3ST2 and its 3-O-sulphated heparan sulphate products are involved in the mechanisms leading to the abnormal phosphorylation of tau in Alzheimer's disease and related tauopathies. Here, we first measured the transcript levels of all human heparan sulphate sulphotransferases in hippocampus of Alzheimer's disease (n = 8; 76.8 +/- 3.5 years old) and found increased expression of HS3ST2 (P < 0.001) compared with control brain (n = 8; 67.8 +/- 2.9 years old). Then, to investigate whether the membrane-associated 3-O-sulphated heparan sulphates translocate to the intracellular level under pathological conditions, we used two cell models of tauopathy in neuro-differentiated SH-SY5Y cells: a tau mutation-dependent model in cells expressing human tau carrying the P-301L mutation hTau P-301L, and a tau mutation-independent model in where tau hyperphosphorylation is induced by oxidative stress. Confocal microscopy, fluorescence resonance energy transfer, and western blot analyses showed that 3-O-sulphated heparan sulphates can be internalized into cells where they interact with tau, promoting its abnormal phosphorylation, but not that of p38 or NF-kappa B p65. We showed, in vitro, that the 3-O-sulphated heparan sulphates bind to tau, but not to GSK3B, protein kinase A or protein phosphatase 2, inducing its abnormal phosphorylation. Finally, we demonstrated in a zebrafish model of tauopathy expressing the hTau P-301L, that inhibiting hs3st2 (also known as 3ost2) expression results in a strong inhibition of the abnormally phosphorylated tau epitopes in brain and in spinal cord, leading to a complete recovery of motor neuronal axons length (n = 25; P < 0.005) and of the animal motor response to touching stimuli (n = 150; P < 0.005). Our findings indicate that HS3ST2 centrally participates to the molecular mechanisms leading the abnormal phosphorylation of tau. By interacting with tau at the intracellular level, the 3-O-sulphated heparan sulphates produced by HS3ST2 might act as molecular chaperones allowing the abnormal phosphorylation of tau. We propose HS3ST2 as a novel therapeutic target for Alzheimer's disease.Association France Alzheimer & Maladies ApparenteesSATT Idf InnovCONACyT, MexicoFrench Ministry of Higher Education and ResearchInstitute de Recherche ServierUniv Paris Est, CNRS, Lab Cell Growth Tissue Repair & Regenerat CRRET, UPEC,EA 4397,ERL 9215, F-94000 Creteil, FranceUPMC, Univ Paris 04, Inst Cerveau & Moelle Epiniere, CNRS,UMR 7225,INSERM,U1127,UM75, Paris, FranceHop Robert Debre, INSERM, UMR 1141, F-75019 Paris, FranceSorbonne Paris Cite, Univ Paris Diderot, Paris, FranceUniversidade Federal de São Paulo, Aging & Neurodegenerat Dis Brain Bank Invest Lab, BR-04023062 São Paulo, BrazilGrp Hosp Pitie Salpetriere, Biochim Malad Neurometab, F-75013 Paris, FranceRadboud Univ Nijmegen, Med Ctr, Radboud Inst Mol Life Sci, NL-6525 ED Nijmegen, NetherlandsUniv Strasbourg, INSERM, U1119, FMTS, F-67000 Strasbourg, FranceUniversidade Federal de São Paulo, Aging & Neurodegenerat Dis Brain Bank Invest Lab, BR-04023062 São Paulo, BrazilCONACyT, Mexico: 308978Web of Scienc

Inhibition of the Mitochondrial Enzyme ABAD Restores the Amyloid-β-Mediated Deregulation of Estradiol

Alzheimer's disease (AD) is a conformational disease that is characterized by amyloid-β (Aβ) deposition in the brain. Aβ exerts its toxicity in part by receptor-mediated interactions that cause down-stream protein misfolding and aggregation, as well as mitochondrial dysfunction. Recent reports indicate that Aβ may also interact directly with intracellular proteins such as the mitochondrial enzyme ABAD (Aβ binding alcohol dehydrogenase) in executing its toxic effects. Mitochondrial dysfunction occurs early in AD, and Aβ's toxicity is in part mediated by inhibition of ABAD as shown previously with an ABAD decoy peptide. Here, we employed AG18051, a novel small ABAD-specific compound inhibitor, to investigate the role of ABAD in Aβ toxicity. Using SH-SY5Y neuroblastoma cells, we found that AG18051 partially blocked the Aβ-ABAD interaction in a pull-down assay while it also prevented the Aβ42-induced down-regulation of ABAD activity, as measured by levels of estradiol, a known hormone and product of ABAD activity. Furthermore, AG18051 is protective against Aβ42 toxicity, as measured by LDH release and MTT absorbance. Specifically, AG18051 reduced Aβ42-induced impairment of mitochondrial respiration and oxidative stress as shown by reduced ROS (reactive oxygen species) levels. Guided by our previous finding of shared aspects of the toxicity of Aβ and human amylin (HA), with the latter forming aggregates in Type 2 diabetes mellitus (T2DM) pancreas, we determined whether AG18051 would also confer protection from HA toxicity. We found that the inhibitor conferred only partial protection from HA toxicity indicating distinct pathomechanisms of the two amyloidogenic agents. Taken together, our results present the inhibition of ABAD by compounds such as AG18051 as a promising therapeutic strategy for the prevention and treatment of AD, and suggest levels of estradiol as a suitable read-out

Analyse cellulaire et intégrée de l'implication du neurostéroïde déhydroépiandrostérone dans la régulation des processus nociceptifs

Les douleurs chroniques rebelles constituent un problème majeur de santé publique car elles sont résistantes aux analgésiques actuels et provoquent une souffrance persistante ainsi que des difficultés socio-économiques telles qu une baisse de productivité et des dépenses élevées. La découverte de nouvelles molécules capables de traiter efficacement les douleurs rebelles est donc aujourd hui une nécessité absolue pour la recherche biomédicale. Sont incluses dans la catégorie des douleurs rebelles, les douleurs neuropathiques qui sont liées à des lésions du système nerveux ou à des perturbations de l équilibre fonctionnel des structures spinales et supraspinales contrôlant la nociception. Il est donc évident que les composés à découvrir pour la thérapie des douleurs neuropathiques devraient nécessairement être de puissants modulateurs de l activité des neurones sensoriels spinaux et supraspinaux. Diverses pistes sont explorées parmi lesquelles les neurostéroïdes qui sont des modulateurs des récepteurs GABAA, NMDA et P2X intervenant dans la nociception. Les médias présentent souvent le neurostéroïde déhydroépiandrostérone (DHEA) comme étant une molécule miracle capable de réduire les déficits physiologiques liés au vieillissement. La DHEA est considérée comme un neuromodulateur endogène dont la biosynthèse diminue avec l âge. Paradoxalement, les preuves scientifiques démontrant les effets bénéfiques de la DHEA sont rares. Contrairement aux humains, les concentrations plasmatiques de DHEA sont quasiment indétectables chez les rongeurs adultes puisque l enzyme de synthèse de la DHEA, le cytochrome P450c17 (P450c17), est présente dans les glandes surrénales humaines mais n est pas exprimée dans les surrénales de rongeurs. Par conséquent, même si l administration de DHEA synthétique contrôle plusieurs processus neurophysiologiques chez les rongeurs adultes, le rôle de la DHEA comme neuromodulateur endogène n est possible que si la DHEA est produite dans le système nerveux central. Pour obtenir des renseignements utiles sur la DHEA, notamment sur son rôle régulateur de processus importants tels que la nociception et la douleur, nous avons élaboré un projet permettant d intégrer des données depuis les niveaux moléculaire et cellulaire jusqu à l échelle de l organisme entier. Nous avons étudié chez le rat adulte l éventualité de la biosynthèse de DHEA dans la moelle épinière (ME), une structure cruciale dans la nociception et la douleur. Nous avons ensuite déterminé les modifications intervenant dans l expression du gène et de l activité enzymatique du P450c17 dans la ME au cours du développement de la douleur chronique neuropathique induite par ligature lâche du nerf sciatique. Nos travaux ont été complétés par une évaluation comportementale des effets de la DHEA et du kétoconazole (un inhibiteur pharmacologique du P450c17) sur la sensibilité à la douleur.La PCR quantitative en temps réel après transcription inverse a révélé l existence d ARNm codant le P450c17 dans tous les segments de la ME. La présence de la protéine enzymatique a été mise en évidence dans la ME par Western Blot avec un anticorps dirigé contre le P450c17. Des études immunohistochimiques ont permis de localiser de nombreux neurones et cellules gliales P450c17-positifs dans la corne dorsale de la ME. Des expériences de pulse-chase combinées à l analyse HPLC et à la détection en flux continu ont montré que les tranches de ME sont capables de convertir la [ H]prégnènolone en [ H]DHEA. Lorsque les rats sont soumis à une douleur neuropathique, les analyses moléculaires et biochimiques ont révélé une diminution de la biosynthèse de DHEA provoquée par la répression du gène et de l activité du P450c17 dans la ME. Les études comportementales ont démontré que la DHEA induit une action bi-phasique sur la sensibilité à la douleur à savoir un effet pro-nociceptif à court terme et une action antalgique tardive liée à la conversion de la DHEA en métabolites androgéniques. L effet propre de la DHEA semble effectivement être pro-nociceptif puisque le blocage in vivo de la synthèse de DHEA dans la ME par injection intrathécale de kétoconazole induit une analgésie chez les rats neuropathiques. Contrairement au kétoconazole, l injection intrathécale de DHEA potentialise l hyperalgie thermique et l allodynie mécanique caractérisant la douleur neuropathique.Parallèlement au travail principal de thèse sur la DHEA, j ai participé à un projet satellite sur les interactions entre la neurostéroïdogenèse spinale et les neurotransmetteurs impliqués dans la transmission des messages douloureux. Nos résultats montrent que la substance P, en agissant par les récepteurs neurokinine-1, diminue de façon dose-dépendante la biosynthèse du neurostéroïde alloprégnanolone (un puissant activateur des récepteurs GABAA) dans la corne dorsale de la ME.Compte tenu de la nature endogène des neurostéroïdes et de leur capacité à interagir avec l ensemble des systèmes majeurs de la neurotransmission, nous espérons qu à terme nos travaux ouvriront des perspectives sérieuses pour le développement de médicaments efficaces contre les douleurs rebelles.Stubborn chronic pain constitutes a major public health concern because this category of pain is refractory to the currently available analgesics and provokes persistent suffering in several thousands of patients with socio-economical problems such as substantial costs due to disability and decreased productivity. Therefore, development of novel therapeutic strategies against stubborn pain has become a real challenge for biomedical research. Included in the category of stubborn pain is neuropathic pain generated by lesions or injuries of the nervous system or by disturbances in the activity of spinal and supraspinal neural networks controlling nociception. Thus, it appears that the compounds to be characterized for the treatment of stubborn neuropathic pain must necessarily be capable of modulating the activity of spinal and supraspinal neuronal pathways. Various family of molecules are currently explored among which are neurosteroids that strongly modulates GABAA, NMDA and P2X receptors intervening in nociception and pain control. Since several years, the neurosteroid dehydroepiandrosterone (DHEA) is excessively advertised as a miraculous pill capable of reducing various physiological deficits associated with aging. DHEA is considered as an endogenous neuromodulator the synthesis of which strongly decreases with aging. Paradoxically, scientific proofs supporting beneficial effects of DHEA are rare. Unlike in humans, plasma concentrations of DHEA are undetectable in adult rodents concurrently with the fact that cytochrome P450c17 (P450c17), the key DHEA-synthesizing enzyme, is expressed by the adrenals in humans but not in rodents. Consequently, even though the administration of synthetic DHEA controls several processes in the central nervous system of adult rodents, the role of DHEA as endogenous neuromodulator remains possible only if DHEA is produced by nerve cells. To obtain fundamental and useful results on DHEA, particularly on its possible role in the regulation of important neurobiological processes such as nociception and pain, we developed a multidisciplinary project allowing integration of data from genomic, molecular and cellular levels to behavioral components of the whole individual. In a first step, we investigated the occurrence of DHEA biosynthesis in the adult rat spinal cord (SC), a crucial structure involved in nociceptive transmission and pain sensation. Afterwards, we identified changes occurring in P450c17 gene expression and enzymatic activity in the SC of rats submitted to chronic neuropathic pain provoked by sciatic nerve ligature. Our studies were completed with behavioral analysis allowing in vivo assessments of the effects of DHEA and ketoconazole (a pharmacological inhibitor of P450c17) on pain sensitivity.Quantitative real-time PCR after reverse transcription revealed P450c17 gene expression in all SC segments. Western blot analyses allowed identification of a specific P450c17 protein in the SC and immunohistochemical studies localised P450c17 in neurons and glial cells. Pulse-chase experiments combined with HPLC and continuous radioactive flow detection showed that SC slices converted [3H]pregnenolone into [3H]DHEA, a conversion markedly reduced by ketoconazole. When the animals were submitted to chronic neuropathic pain situation, molecular and biochemical analyses revealed a significant decrease of DHEA synthesis which resulted from the down-regulation of P450c17 gene expression and bioactivity in the SC. Behavioral investigations showed that DHEA induced a biphasic action on pain sensitivity: a short term pro-nociceptive effect followed by a late analgesic action due to DHEA conversion into androgenic metabolites. The proper action of DHEA seems effectively to be pro-nociceptive because in vivo blockade of DHEA biosynthesis in the SC by intrathecal injection of ketoconazole induced analgesia in neuropathic rats. Unlike ketoconazole, intrathecal administration of DHEA potentiated both thermal hyperalgesia and mechanical allodynia characterizing the neuropathic pain.In addition to the main PhD program on DHEA, I have also participated to a side-project aiming to investigate anatomic and functional interactions between spinal neurons secreting neurosteroids and classical neurotransmitters involved in painful message transmission. We demonstrated that substance P, acting through neurokinin-1 receptors, inhibits the production of the neurosteroid allopregnanolone (a potent stimulator of GABAA receptors) in the SC dorsal horn.Owing to the fact that neurosteroids are endogenous compounds capable of interacting with main systems of the central neurotransmission, we hope that our project, at full completion, may open new perspectives for the development of efficient therapeutic drugs against stubborn pain.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Tryptophan metabolites modify brain Aβ peptide degradation: A role in Alzheimer’s disease?

International audienceAmong several processes, a decrease in amyloid-beta (Aβ) peptide elimination is thought to be one of the major pathophysiological factors in Alzheimer’s disease (AD). Neprilysin (NEP) is a key metalloproteinase controlling the degradation and clearance of Aβ peptides in the brain. NEP is induced by several pharmacological substances, amyloid deposits and somatostatin, but the physiological regulation of its expression remains unclear. This situation hampers the exploitation of NEP regulatory factors/mechanisms to develop effective strategies against Aβ peptide accumulation-induced brain toxicity. Based on recent data aimed at elucidating this major question, the present paper addresses and critically discusses the role of 5-hydroxyindole-acetic acid (5-HIAA) and kynurenic acid (KYNA) in the regulation of NEP activity/expression in the brain. Both 5-HIAA and KYNA are endogenous metabolites of tryptophan, an essential amino-acid obtained through diet and gut microbiome. By interacting with the aryl hydrocarbon receptor, various tryptophan metabolites modulate several metalloproteinases regulating brain Aβ peptide levels under normal and pathological conditions such as AD. In particular, interesting data reviewed here show that 5-HIAA and KYNA stimulate NEP activity/expression to prevent Aβ peptide-induced neurotoxicity. These data open promising perspectives for the development of tryptophan metabolite-based therapies against AD

Rôle des neurostéroïdes endrogènes dans la régulation des processus neurodégénératifs impliqués dans la maladie d'Alzheimer et dans l'étiologie des douleurs neuropathiques

La transfection des cellules humaines SH-SY5Y par des protéines clés de la maladie d Alzheimer telles que la protéine native hTau40, Tau mutée P301L ou APP provoque une perturbation de la biosynthèse des neurostéroïdes. hTau40 stimule fortement la neurostéroïdogenèse alors que Tau mutée P301L est dépourvue d activité. Les peptides b-amyloïdes exercent une action dose- et séquence peptidique-dépendante sur la neurostéroïdogenèse. L H2O2 induit une mort cellulaire en réprimant l expression d aromatase et la synthèse d oestradiol (E2). Le prétraitement des SH-SY5Y avec l E2 les protège contre la mort liée à l H2O2.Dans le modèle de douleur neuropathique chez le rat, nous avons détecté une apoptose de cellules satellites gliales dans les ganglions rachidiens et une surproduction d E2 dans les neurones voisins des cellules apoptotiques.Des perspectives intéressantes sont ouvertes pour le développement de stratégies neuroprotectrices basées sur l exploitation des neurostéroïdes.Transfection of human neuroblastoma SH-SY5Y cells with Alzheimer s disease key proteins including native hTau40, mutant Tau P301L or APP resulted in neurosteroid biosynthesis dysregulation. hTau40 strongly stimulated neurosteroidogenesis while Tau P301L was devoid of action. Amyloid-b peptides exerted a dose-and sequence-dependent effect on neurosteroidogenesis. H2O2 killed SH-SY5Y cells through inhibition of aromatase gene expression and estradiol (E2) synthesis. Pretreatment with E2 rescued SH-SY5Y cells from H2O2-induced death.Apoptotic satellite glial cells were evidenced in dorsal root ganglia of neuropathic pain rats. E2 production increased in sensory neurons surrounded by apoptotic satellite cells, suggesting that endogenous neurosteroids may control neurodegenerative events in dorsal root ganglia.Together, the data open interesting possibilities for the development of neurosteroid-based neuroprotective strategies.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceSwitzerlandFRC

Neurosteroids and neuropathic pain management: Basic evidence and therapeutic perspectives

International audienc

Neurosteroid 3α-androstanediol efficiently counteracts paclitaxel-induced peripheral neuropathy and painful symptoms.

Painful peripheral neuropathy belongs to major side-effects limiting cancer chemotherapy. Paclitaxel, widely used to treat several cancers, induces neurological symptoms including burning pain, allodynia, hyperalgesia and numbness. Therefore, identification of drugs that may effectively counteract paclitaxel-induced neuropathic symptoms is crucial. Here, we combined histopathological, neurochemical, behavioral and electrophysiological methods to investigate the natural neurosteroid 3α-androstanediol (3α-DIOL) ability to counteract paclitaxel-evoked peripheral nerve tissue damages and neurological symptoms. Prophylactic or corrective 3α-DIOL treatment (4 mg/kg/2 days) prevented or suppressed PAC-evoked heat-thermal hyperalgesia, cold-allodynia and mechanical allodynia/hyperalgesia, by reversing to normal, decreased thermal and mechanical pain thresholds of PAC-treated rats. Electrophysiological studies demonstrated that 3α-DIOL restored control values of nerve conduction velocity and action potential peak amplitude significantly altered by PAC-treatment. 3α-DIOL also repaired PAC-induced nerve damages by restoring normal neurofilament-200 level in peripheral axons and control amount of 2',3'-cyclic-nucleotide-3'-phosphodiesterase in myelin sheaths. Decreased density of intraepidermal nerve fibers evoked by PAC-therapy was also counteracted by 3α-DIOL treatment. More importantly, 3α-DIOL beneficial effects were not sedation-dependent but resulted from its neuroprotective ability, nerve tissue repairing capacity and long-term analgesic action. Altogether, our results showing that 3α-DIOL efficiently counteracted PAC-evoked painful symptoms, also offer interesting possibilities to develop neurosteroid-based strategies against chemotherapy-induced peripheral neuropathy. This article shows that the prophylactic or corrective treatment with 3α-androstanediol prevents or suppresses PAC-evoked painful symptoms and peripheral nerve dysfunctions in rats. The data suggest that 3α-androstanediol-based therapy may constitute an efficient strategy to explore in humans for the eradication of chemotherapy-induced peripheral neuropathy

Xanthurenic acid is localized in neurons in the central nervous system

Kynurenine pathway metabolites (KPM) are thought to be synthesized mainly by non-neuronal cells in the mammalian brain. KPM are of particular interest because several studies demonstrated their implication in various disorders of the nervous system. Among KPM is xanthurenic acid (XA) deriving from the catabolism of 3-hydroxykynurenine. Based on its chemical structure, XA appears as a close analog of kynurenic acid which has been extensively investigated and is considered as a potent neuroprotective compound. Contrary to kynurenic acid (KYNA), XA has received little attention and its role in the brain remains not elucidated. We have previously described several characteristics of XA, suggesting its possible involvement in neurotransmission. XA is also proposed as a potential modulator at glutamatergic synapses. Here, we used a selective antibody against XA and various neuronal, glial and synaptic markers to show that XA is essentially localized in the soma and dendrites of brain neurons, but is absent from axonal compartments and terminal endings. Our results also reveal that XA-like immunoreactivity is not expressed by glial cells. To double-check our findings, we have also used another XA antibody obtained from a commercial source to confirm the neuronal expression of XA. Together, our results suggest that, differently to several other KPM produced by glial cells, XA exhibits a neuronal distribution in the mouse brain