Curcumin and Ethanol Effects in Trembler-J Schwann Cell Culture

Abstract

Charcot-Marie-Tooth (CMT) syndrome is the most common progressive human motor and sensory peripheral neuropathy. CMT type 1E is a demyelinating neuropathy affecting Schwann cells due to peripheral-myelin-protein-22 (PMP22) mutations, modelized by Trembler-J mice. Curcumin, a natural polyphenol compound obtained from turmeric (Curcuma longa), exhibits dose- and time-varying antitumor, antioxidant and neuroprotective properties, however, the neurotherapeutic actions of curcumin remain elusive. Here, we propose curcumin as a possible natural treatment capable of enhancing cellular detoxification mechanisms, resulting in an improvement of the neurodegenerative Trembler-J phenotype. Using a refined method for obtaining enriched Schwann cell cultures, we evaluated the neurotherapeutic action of low dose curcumin treatment on the PMP22 expression, and on the chaperones and autophagy/mammalian target of rapamycin (mTOR) pathways in Trembler-J and wild-type genotypes. In wild-type Schwann cells, the action of curcumin resulted in strong stimulation of the chaperone and macroautophagy pathway, whereas the modulation of ribophagy showed a mild effect. However, despite the promising neuroprotective effects for the treatment of neurological diseases, we demonstrate that the action of curcumin in Trembler-J Schwann cells could be impaired due to the irreversible impact of ethanol used as a common curcumin vehicle necessary for administration. These results contribute to expanding our still limited understanding of PMP22 biology in neurobiology and expose the intrinsic lability of the neurodegenerative Trembler-J genotype. Furthermore, they unravel interesting physiological mechanisms of cellular resilience relevant to the pharmacological treatment of the neurodegenerative Tremble J phenotype with curcumin and ethanol. We conclude that the analysis of the effects of the vehicle itself is an essential and inescapable step to comprehensibly assess the effects and full potential of curcumin treatment for therapeutic purposes.

Keywords: CMT1E; Hsps; Trembler-J; autophagy; curcumin; ethanol.

Figure 1

Morphology of different cell types in enriched SC cultures. (A) In both +/+ and TrJ/+ genotypes, fibroblasts (FBs) and Schwann cells (SC) show morphological differences. FBs show a heterogeneous flattened shape with abundant cytoplasm and a large nucleus easily distinguishable under light microscopy. SCs show typically bipolar shape with lateral extensions and a smaller size compared to FBs; (B) Confocal microscopy image showing PMP22 (red) and nuclear labeling counterstain (DAPI, blue). Scale in (A) = 250 µm. Scale in (B) = 20 µm.

Figure 2

Enriched SC–FB cultures obtained by different protocols. Images of mixed primary culture obtained from the “collagenase-trypsin” protocol are visualized in the left panels in the +/+ and TrJ/+ cultures. This protocol allowed the obtaining of a large number of cells in eight days, but the SC/FB ratio is rather low, as observed after 15 days of seeding. Images of the mixed primary culture obtained with the modified protocol of Rosso et al., 2017 [71] in +/+ and TrJ/+ are displayed in the right panels. This method requires more time to obtain a large number of cell volumes. The epineurium removal and enrichment by Cold-Jet (CJ) allowed us to obtain a higher SC/FB ratio, compared to the collagenase-trypsin protocol. Arrowheads point to SCs. Scale = 250 µm for all panels.

Figure 3

Cell viability by MTT assay. Enriched SCs cultures +/+ and TrJ/+ were treated for six days with different concentrations of curcumin (added to the culture medium) in addition to the evaluation of N.ctrl (without curcumin and EtOH) and Et.ctrl (without curcumin, with EtOH, 0.43 mM). (A) Control comparison within each genotype show a significant increase in viability of the Et.ctrl compared to N.ctrl. Comparison +/+ vs. TrJ/+ no significant differences (data not shown); (B) Comparison of the different curcumin concentrations to Et.ctrl in +/+ and TrJ/+. (C) Comparison +/+ vs. TrJ/+ of the lowest concentration (0.25µM) without differences with respect to Et.ctrl. obtained in (B) for both genotypes. The data, expressed as median ± SIR, were analyzed in (A,C) by Mann–Whitney U signed-rank test and in (B) by the Kruskal–Wallis test. * p < 0.05 ** p < 0.002; *** p < 0.0002. SIR: semi-interquartile range.

Figure 4

Modulation of PMP22 expression in +/+ and TrJ/+ SCs enriched cultures. Confocal microscopy images of +/+ and TrJ/+ SCs N.Ctrl, Et.ctrl (0.43 mM EtOH) and Et+Cu (0.25 µM curcumin + 0.43 mM EtOH) were quantified at the nuclear and cytoplasmic level. Et.ctrl vs. N.ctrl, had a nuclear decrease in PMP22 expression in +/+ SC, while in TrJ/+ SC it was observed in both compartments. On the other hand, Et+Cu treatment vs. Et.ctrl, showed an increase compared to both cell compartments, only for the +/+ genotype. In turn, the comparison +/+ vs. TrJ/+ SCs of the same condition revealed differences in both compartments, in N.ctrl and Et+Cu treatment. Within each genotype: * p < 0.033; ** p < 0.002; *** p < 0.0002; **** p < 0.0001. Same treatment between +/+ and TrJ/+: ## p < 0.002; #### p < 0.0001. The mean is shown as “+”. Scale = 10 µm for all panels. n = 100 cells on average per condition for each genotype.

Figure 5

Modulation of HSF1 and Hsp27 expression levels in +/+ and TrJ/+ SCs enriched cultures. Images obtained by confocal microscopy of +/+ and TrJ/+ SC N.ctrl, E.ctrl, and Et+Cu were quantified in the nucleus and cytoplasm. (A) HSF1 expression. Et.ctrl vs. N.ctrl had a decrease only in TrJ/+ SC in both compartments. Et+Cu treatment vs. Et.ctrl showed a nuclear increase and a cytoplasmic decrease only in +/+ SCs. In turn, the comparison +/+ vs. TrJ/+ SCs of the same condition revealed only cytoplasmic differences in Et.ctrl and Et+Cu; (B) Hsp27 expression. No differences were observed between Et.ctrl vs. N.ctrl, in both genotypes or both compartments. A comparison +/+ vs. TrJ/+ SCs of the same condition revealed cytoplasmic differences in N.ctrl and Et.ctrl. Within each genotype: * p < 0.033, ** p < 0.002 and **** p < 0.0001. Same treatment between +/+ and TrJ/+: # p < 0.033, ## p < 0.002 and ### p < 0.0001. The mean is shown as “+”. Scale = 10 µm for all panels. n = 100 cells in average per condition for each genotype.

Figure 6

Modulation of HDAC6 and ribosome expression in +/+ and TrJ/+ SC enriched cultures. Images obtained by confocal microscopy of +/+ and TrJ/+ SC in N.ctrl, E.ctrl, and Et+Cu were quantified in the nucleus and cytoplasm. (A) HDAC6 expression. Et.ctrl vs. N.ctrl showed a decrease only in TrJ/+ SCs in both compartments. Et+Cu vs. Et.ctrl showed an increase in +/+ SCs in both compartments. Conversely, the same condition between +/+ and TrJ/+ SCs revealed nuclear differences only with Et+Cu. At the cytoplasmic level, differences were observed with the N.ctrl and Et+Cu; (B) Ribosome’s expression. Et.ctrl vs. N.ctrl, showed a nuclear decrease only in TrJ/+, while the cytoplasmic decrease occurred in both genotypes. Et+Cu vs. Et.ctrl showed an increased expression in +/+ SCs in both compartments. +/+ vs. TrJ/+ SCs revealed nuclear differences with the N.ctrl and the Et.ctrl, and cytoplasmic differences with Et.ctrl and Et+Cu treatment. Within each genotype: * p < 0.033; ** p < 0.002; *** p < 0.0002; **** p < 0.0001. Same treatment, +/+ vs. TrJ/+; # p < 0.033; ## p < 0.002; ### p < 0.0002; #### p < 0.0001. The mean is shown as “+”. Scale = 10 µm for all panels. n = 100 cells in average per condition for each genotype.