Efficient syntheses of a series of trehalose dimycolate (TDM)/trehalose dicorynomycolate (TDCM) analogues and their interleukin-6 level enhancement activity in mice sera.

We found an IL-6 level-enhancing compound during our synthetic study of trehalose-6,6'-dimycolate (1, TDM, formerly called cord factor) analogues. TDM is a glycolipid distributed in the cell wall of Mycobacterium tuberculosis and shows significant antitumor activity based on an immunoadjuvant activity. However, due to its significant toxicity, TDM is not yet applicable for practical use. In 1993, Datta and Takayama reported the purification of trehalose-6,6'-dicorynomycolate (2c, TDCM) from Corynebacterium spp. We have previously reported the synthesis of four diastereomeric TDCMs and showed that the synthetic (2R,3R,2'R,3'R)-TDCM (2c, hereafter abbreviated RRRR-TDCM-C14) is identical to natural TDCM; we also demonstrated that 2c and SSSS-TDCM-C14 (3c) showed significant antitumor activity as well as inhibitory activity in experimental lung metastasis based on the immunoadjuvant activity. Furthermore, we found that the significant lethal toxicity in mice by TDM (1) was no longer observed with the shorter-chain analogues of TDCMs. Therefore, we have elucidated that the 2,3-antistereochemistry (RR or SS) of the fatty acid residue is promising for biological activities. The chain length of the fatty acid residue should also be important for the biological activity, and thus, we designed a general synthetic procedure for trehalose diesters with 2,3-antistereochemistry and a series of chain lengths by using Noyori's asymmetric reduction of beta,beta-ketoesters followed by antiselective alkylation according to Frater to give beta,beta-hydroxy alcohols as the key steps. Thus, we prepared trehalose diesters (TDCM) 2a-d, 3a-d, and 4a-d as well as monoesters (TMCM) 5a-d and 6a-d. Immunological activities of TDCMs and TMCMs were evaluated by determining IL-6 level enhancement in mouse serum, and we found that RRRR-TDCM-C14 (2c) and RRSS-TDCM-C14 (4c) showed significant IL-6 level enhancement activities

New Roles of Glycosaminoglycans in α-Synuclein Aggregation in a Cellular Model of Parkinson Disease

International audienceThe causes of Parkinson disease (PD) remain mysterious, although some evidence supports mitochondrial dysfunctions and α-synuclein accumulation in Lewy bodies as major events. The abnormal accumulation of α-synuclein has been associated with a deficiency in the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Cathepsin D (cathD), the major lysosomal protease responsible of α-synuclein degradation was described to be up-regulated in PD model. As glycosaminoglycans (GAGs) regulate cathD activity, and have been recently suggested to participate in PD physiopathology, we investigated their role in α-synuclein accumulation by their intracellular regulation of cathD activity. In a classical neu-roblastoma cell model of PD induced by MPP + , the genetic expression of GAGs-biosynthetic enzymes was modified, leading to an increase of GAGs amounts whereas intracellular level of α-synuclein increased. The absence of sulfated GAGs increased intracellular cathD activity and limited α-synuclein accumulation. GAGs effects on cathD further suggested that specific sequences or sulfation patterns could be responsible for this regulation. The present study identifies, for the first time, GAGs as new regulators of the lysosome degradation pathway , regulating cathD activity and affecting two main biological processes, α-synuclein ag-gregation and apoptosis. Finally, this opens new insights into intracellular GAGs functions and new fields of investigation for glycobiological approaches in PD and neurobiology

Insights on a new path of pre-mitochondrial apoptosis regulation by a glycosaminoglycan mimetic

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Characterization of MPP⁺-induced apoptosis.

<p>Total, mitochondrial and cytosolic extracts were obtained from normal and MPP⁺-stressed SHSY5Y cells (0.5 mM, 6 h).a) CathD activity in cytosolic extracts. CathD activity was normalized by cell number in each sample and expressed as percentage of the activity in normal cells. Results are expressed in percentage of normal cell extract levels and presented as mean ± S.E.M. of four independent experiments in triplicate. *** p<0.01 compared to control cells. b) Immunofluorescence detection of total cathD in cells. Left: unstressed control cells (Ctrl), right: MPP⁺-stressed cells (6 h). Cells were observed with a confocal microscope Zeiss Axio Observer Z.1. Scale bar represents 10 μm. Negative control omitting the first antibody but in the presence of the second one (anti-goat Fluo 546) did not show any signal. c) Analysis by western blot of Bax relocation in mitochondrial membranes at 6 h, just after the end of stress. The amount of protein was normalized by the number of cells to avoid taking into account the increase of protein amount induced by the MPP⁺ stress. Succinate dehydrogenase-A (SDHA) was used as the loading control. This immunoblot is representative of three independent experiments in duplicate. d) Assessment of respiratory chain function 24 h after MPP⁺ treatment. The respiratory control index (RCI) i.e. ratio [state 3 rate] / [state 4 rate] was calculated. Results are expressed as percentage of unstressed control group and represent three independent experiments in triplicate. Results are presented as the mean ± S.E.M. *** p<0.01 compared to control cells.</p

Glycosaminoglycans modulation of α-synuclein aggregation and degradation by cathepsin D <i>in vitro</i>.

<p>a) <i>In vitro</i> α-synuclein aggregation in the presence of different GAGs. Commercial α-synuclein (25 μg/mL) was incubated 24 h at 37°C in the presence of 1, 10 or 100 μg/mL of commercial Hep, CS or HS. Monomeric, dimeric and oligomeric forms of α-synuclein were detected by western blot and quantified with ImageJ software. b)Inhibition of cathD degradation of α-synuclein by different commercial GAGs. α-synuclein was incubated in the presence of cathD (50 mU/mL) and pepstatin A, Hep, HS or CS (100 μg/mL) during 30 min at 37°C. Residual α-synuclein was detected by western blot and quantified with ImageJ software. Results are presented as the mean ± S.E.M. *** p<0.01 compared to control.</p

Regulation of cathepsin D activity by glycosaminoglycans.

<p>a) SH-SY5Y cells were treated with sodium chlorate (75 mM) during 24 h and sample were extracted at different times after sodium chlorate removal. GAGs amounts were measured in cells after 0, 1, 3 and 6 h after the end of the chlorate treatment. Results are presented as mean ± S.E.M. of three independent experiments in triplicate. *** p<0.001 compared to control. b) Total cathD activity was measured at different times after sodium chlorate removal (75 mM, 24h). Results are expressed in percentage of cathD in normal cell extract and presented as mean ± S.E.M. of three independent experiments in triplicate. *** p<0.001 compared to control cells. c) GAGs were extracted from control, MPP⁺-stressed cells, chlorate treated cells 3 and 6 h after chlorate removalas described in Material and Methods. GAG amounts were measured by DMMB assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116641#pone.0116641.ref027" target="_blank">27</a>]. The activity of cathD was measured in the presence of various concentrations of these extracted GAGs (white bars: 1μg/mL; light grey2μg/mL, medium grey:5 μg/mL and dark grey: 7 μg/mL) and expressed as the percentage of the specific CathD activity. Results are mean ± S.E.M. of three independent experiments in duplicate. ** p<0.005; *** p<0.001 compared to control cathD activity, ψ p<0.01; ψψ p<0.005; ψψψ p<0.001 compared to results obtained with 1 μg/mL of GAGs, ΦΦ p<0.005 comparison between 3 and 6 h extracted GAGs. d) Total cathD activity was measured after different times (0, 1, 3 and 6 h) of treatment with MPP⁺ (0.5 mM) with or without pre-treatement of cell with chlorate (75 mM, 24h). CathD activity was normalized by cell number in each sample and expressed as percentage of the activity in normal cells. ** p<0.005; *** p<0.001 compared to control. e) CathD activity in cells subjected or not to MPP⁺ stress (6 h) and followed by commercial GAGs treatment (Hep, HS and CS, 1 μg/mL). After 24 h, GAGs effects were compared to the complete inhibition of cathepsin activity by pepstatin A (peps), a specific inhibitor of this enzyme and expressed as percentage of cathD activity in control cells.</p

Interplay between CathD, glycosaminoglycans (heparan sulfate (HS), chondroitin sulfate (CS)) and α-synuclein on MPP⁺-stressed cells.

<p>See details in the text.</p