Étude des huiles essentielles d'espèces végétales de la flore laurentienne : composition chimique, activités pharmacologiques et hémi-synthèse

Au Québec, le potentiel pharmacologique des huiles essentielles issues de plantes de la flore laurentienne a été très peu étudié. Dans ce contexte et en tenant compte du savoir médicinal des autochtones, les chercheurs du laboratoire LASEVE de l'UQAC étudient leur potentiel pharmacologique ainsi que celui de leurs constituants volatils. Dans cette optique, ce projet de maîtrise visait à évaluer les activités anticancéreuse, antioxydante, anti-inflammatoire, antifongique et antibactérienne de plusieurs huiles essentielles issues de plantes de la forêt boréale. Suite à ce criblage biologique, l'huile essentielle de bourgeons de peuplier baumier s'est remarquablement distinguée des autres espèces de par son activité anticancéreuse (IC50 12-16 Hg/mL). Une étude approfondie de la composition chimique de cette huile essentielle a donc été entreprise à partir de bourgeons récoltés à différentes périodes de l'année. Le composé majoritaire, l'ot-bisabolol, un alcool sesquiterpénique bien connu pour sa non-toxicité, a été identifié et dosé dans chaque huile essentielle. Une activité cytotoxique marquée envers les lignées cancéreuses de poumon (A549) et de côlon (DLD-1) a été observée pour ce composé (IC50 18 et 26 u.g/mL). L'a-bisabolol P-D-fucopyranoside, un sesquiterpène glycosidé naturel, a été synthétisé avec cinq autres glycosides d'a-bisabolo! O-D-glucoside, p-D-galactoside, p-D-xy!oside, a-L-rtiamnoside et a-D-mannoside). Pour la première fois, la synthèse de glycosides d'un sesquiterpène selon la procédure inverse de Schmidt a été réalisée et avec d'excellents rendements (83-95%). Leur activité anticancéreuse a été évaluée contre un large éventail de cellules cancéreuses. Dans la plupart des cas, l'addition d'une section sucre a eu pour effet d'améliorer l'activité cytotoxique de l'a-bisabolol. Parmi les six glycosides synthétisés, l'a-bisabolol a-L-rhamnoyranoside a exercé la plus forte cytotoxicité (!C50 40-64 u.M) surpassant celle de la molécule initiale. Par ailleurs, au cours de ce projet, l'huile essentielle des fleurs de Solidago puberula a également été étudiée car son profil chimique n'avait jamais été établi. Une activité anticancéreuse modérée a été détectée pour cette huile essentielle (IC50 DLD-1: 30 ± 4 u,g/mL). Une autre partie importante de ce projet visait à étudier Sa composition et le potentiel pharmacologique des hydrolats, produits secondaires obtenus !ors de l'extraction des huiles essentielles par entraînement à la vapeur d'eau. Les hydrolats ont en effet été très peu étudiés. Pour la première fois, la composition chimique de plusieurs hydrolats a été analysée ainsi que leur activité biologique. Ainsi, i'hydrolat de bourgeons de Populus balsamifera a démontré une activité antioxydante intéressante à l'aide du test chimique ORÂC (23 ± 10 u.mo! Trolox/mg). Ceux de Solidago puberula et Monarda didyma ont démontré une activité anti-inflammatoire intéressante en inhibant fortement le relâchement d'oxyde nitrique dans les macrophages murins stimulés avec une molécule induisant l'inflammation (LPS). Au final, les résultats de ce projet de maîtrise auront permis de mettre en évidence le potentiel thérapeutique des huiles essentielles et hydrolats de plantes de la flore laurentienne et d'ouvrir la voie à de futures études

Climbing Fiber Signaling and Cerebellar Gain Control

The physiology of climbing fiber signals in cerebellar Purkinje cells has been studied since the early days of electrophysiology. Both the climbing fiber-evoked complex spike and the role of climbing fiber activity in the induction of long-term depression (LTD) at parallel fiber-Purkinje cell synapses have become hallmark features of cerebellar physiology. However, the key role of climbing fiber signaling in cerebellar motor learning has been challenged by recent reports of forms of synaptic and non-synaptic plasticity in the cerebellar cortex that do not involve climbing fiber activity, but might well play a role in cerebellar learning. Moreover, cerebellar LTD does not seem to strictly require climbing fiber activity. These observations make it necessary to re-evaluate the role of climbing fiber signaling in cerebellar function. Here, we argue that climbing fiber signaling is about adjusting relative probabilities for the induction of LTD and long-term potentiation (LTP) at parallel fiber synapses. Complex spike-associated, dendritic calcium transients control postsynaptic LTD and LTP induction. High calcium transients, provided by complex spike activity, do not only favor postsynaptic LTD induction, but simultaneously trigger retrograde cannabinoid signaling, which blocks the induction of presynaptic LTP. Plasticity of the climbing fiber input itself provides additional means to fine-tune complex spike associated calcium signaling and thus to adjust the gain of heterosynaptic climbing fiber control. In addition to dendritic calcium transients, climbing fiber activity leads to the release of the neuropeptide corticotropin-releasing factor (CRF), which facilitates LTD induction at both parallel fiber and climbing fiber synapses

Intrinsic plasticity complements long-term potentiation in parallel fiber input gain control in cerebellar Purkinje cells

Synaptic gain control and information storage in neural networks are mediated by alterations in synaptic transmission, such as in long-term potentiation (LTP). Here,weshowusingboth in vitroandin vivo recordingsfromthe rat cerebellum that tetanization protocols for the induction of LTP at parallel fiber (PF)-to-Purkinje cell synapsescanalsoevokeincreases in intrinsic excitability. Thisformof intrinsic plasticity shares with LTP a requirement for the activation of protein phosphatases 1, 2A, and 2B for induction. Purkinje cell intrinsic plasticity resembles CA1 hippocampal pyramidal cell intrinsic plasticity in that it requires activity of protein kinase A(PKA) and casein kinase 2 (CK2) and is mediated by a downregulation of SK-type calcium-sensitive K conductances. In addition, Purkinje cell intrinsic plasticity similarly results in enhanced spine calcium signaling. However, there are fundamental differences: first, while in the hippocampus increases in excitability result in a higher probability for LTP induction, intrinsic plasticity in Purkinj

Pinceau Organization in the Cerebellum Requires Distinct Functions of Neurofascin in Purkinje and Basket Neurons during Postnatal Development

Basket axon collaterals synapse onto the Purkinje soma/axon initial segment (AIS) area to form specialized structures, the pinceau, which are critical for normal cerebellar function. Mechanistic details of how the pinceau become organized during cerebellar development are poorly understood. Loss of cytoskeletal adaptor protein Ankyrin G (AnkG) results in mislocalization of the cell adhesion molecule Neurofascin (Nfasc) at the Purkinje AIS and abnormal organization of the pinceau. Loss of Nfasc in adult Purkinje neurons leads to slow disorganization of the Purkinje AIS and pinceau morphology. Here we utilized mouse conditional knockout techniques to show that selective loss of Nfasc specifically in Purkinje neurons during early development prevented maturation of the AIS and resulted in loss of Purkinje neuron spontaneous activity and pinceau disorganization. Loss of Nfasc in both Purkinje and basket neurons caused abnormal basket axon collateral branching and targeting to Purkinje soma/AIS, leading to extensive pinceau disorganization, Purkinje neuron degeneration and severe ataxia. Our studies reveal that the Purkinje Nfasc is required for AIS maturation and for maintaining stable contacts between basket axon terminals and the Purkinje AIS during pinceau organization, while the basket neuron Nfasc in combination with Purkinje Nfasc is required for proper basket axon collateral outgrowth and targeting to Purkinje soma/AIS. Thus, cerebellar pinceau organization requires coordinated mechanisms involving specific Nfasc functions in both Purkinje and basket neurons

Pre and Post Synaptic NMDA Effects Targeting Purkinje Cells in the Mouse Cerebellar Cortex

N-methyl-D-aspartate (NMDA) receptors are associated with many forms of synaptic plasticity. Their expression level and subunit composition undergo developmental changes in several brain regions. In the mouse cerebellum, beside a developmental switch between NR2B and NR2A/C subunits in granule cells, functional postsynaptic NMDA receptors are seen in Purkinje cells of neonate and adult but not juvenile rat and mice. A presynaptic effect of NMDA on GABA release by cerebellar interneurons was identified recently. Nevertheless whereas NMDA receptor subunits are detected on parallel fiber terminals, a presynaptic effect of NMDA on spontaneous release of glutamate has not been demonstrated. Using mouse cerebellar cultures and patch-clamp recordings we show that NMDA facilitates glutamate release onto Purkinje cells in young cultures via a presynaptic mechanism, whereas NMDA activates extrasynaptic receptors in Purkinje cells recorded in old cultures. The presynaptic effect of NMDA on glutamate release is also observed in Purkinje cells recorded in acute slices prepared from juvenile but not from adult mice and requires a specific protocol of NMDA application

Total synthesis, isolation, surfactant properties, and biological evaluation of ananatosides and related macrodilactone-containing rhamnolipids

Rhamnolipids are a specific class of microbial surfactants, which hold great biotechnological and therapeutic potential. However, their exploitation at the industrial level is hampered because they are mainly produced by the opportunistic pathogenPseudomonas aeruginosa. The non-human pathogenic bacteriumPantoea ananatisis an alternative producer of rhamnolipid-like metabolites containing glucose instead of rhamnose residues. Herein, we present the isolation, structural characterization, and total synthesis of ananatoside A, a 15-membered macrodilactone-containing glucolipid, and ananatoside B, its open-chain congener, from organic extracts ofP. ananatis. Ananatoside A was synthesized through three alternative pathways involving either an intramolecular glycosylation, a chemical macrolactonization or a direct enzymatic transformation from ananatoside B. A series of diasteroisomerically pure (1→2), (1→3), and (1→4)-macrolactonized rhamnolipids were also synthesized through intramolecular glycosylation and their anomeric configurations as well as ring conformations were solved using molecular modeling in tandem with NMR studies. We show that ananatoside B is a more potent surfactant than its macrolide counterpart. We present evidence that macrolactonization of rhamnolipids enhances their cytotoxic and hemolytic potential, pointing towards a mechanism involving the formation of pores into the lipidic cell membrane. Lastly, we demonstrate that ananatoside A and ananatoside B as well as synthetic macrolactonized rhamnolipids can be perceived by the plant immune system, and that this sensing is more pronounced for a macrolide featuring a rhamnose moiety in its native1C4conformation. Altogether our results suggest that macrolactonization of glycolipids can dramatically interfere with their surfactant properties and biological activity

Molecular interactions of the plasma membrane calcium ATPase 2 at pre- and post-synaptic sites in rat cerebellum.

The plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events [Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35 (Pt 5):919-922], its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre- and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull-down approaches confirmed that PMCA2 interacts with the post-synaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its C-terminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well-known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull-down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95-PMCA2 and syntaxin-1A-PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre- and post-synaptic signalling complexes

Sensory Stimulation-Dependent Plasticity in the Cerebellar Cortex of Alert Mice

In vitro studies have supported the occurrence of cerebellar long-term depression (LTD), an interaction between the parallel fibers and Purkinje cells (PCs) that requires the combined activation of the parallel and climbing fibers. To demonstrate the existence of LTD in alert animals, we investigated the plasticity of local field potentials (LFPs) evoked by electrical stimulation of the whisker pad. The recorded LFP showed two major negative waves corresponding to trigeminal (broken into the N2 and N3 components) and cortical responses. PC unitary extracellular recording showed that N2 and N3 occurred concurrently with PC evoked simple spikes, followed by an evoked complex spike. Polarity inversion of the N3 component at the PC level and N3 amplitude reduction after electrical stimulation of the parallel fiber volley applied on the surface of the cerebellum 2 ms earlier strongly suggest that N3 was related to the parallel fiber–PC synapse activity. LFP measurements elicited by single whisker pad stimulus were performed before and after trains of electrical stimuli given at a frequency of 8 Hz for 10 min. We demonstrated that during this later situation, the stimulation of the PC by parallel and climbing fibers was reinforced. After 8-Hz stimulation, we observed long-term modifications (lasting at least 30 min) characterized by a specific decrease of the N3 amplitude accompanied by an increase of the N2 and N3 latency peaks. These plastic modifications indicated the existence of cerebellar LTD in alert animals involving both timing and synaptic modulations. These results corroborate the idea that LTD may underlie basic physiological functions related to calcium-dependent synaptic plasticity in the cerebellum