Effects of Biotin on survival, ensheathment, and ATP production by oligodendrocyte lineage cells in vitro

Qiao-Ling Cui; Yun Hsuan Lin; Yu Kang T Xu; Milton G F Fernandes; Vijayaraghava T S Rao; Timothy E Kennedy; Jack Antel

doi:10.1371/journal.pone.0233859

. 2020 May 29;15(5):e0233859. doi: 10.1371/journal.pone.0233859

Effects of Biotin on survival, ensheathment, and ATP production by oligodendrocyte lineage cells in vitro

Qiao-Ling Cui ¹, Yun Hsuan Lin ¹, Yu Kang T Xu ¹, Milton G F Fernandes ¹, Vijayaraghava T S Rao ^2,^¤, Timothy E Kennedy ¹, Jack Antel ^1,^*

Editor: Ken Arai³

PMCID: PMC7259710 PMID: 32470040

Abstract

Mechanisms implicated in disease progression in multiple sclerosis include continued oligodendrocyte (OL)/myelin injury and failure of myelin repair. Underlying causes include metabolic stress with resultant energy deficiency. Biotin is a cofactor for carboxylases involved in ATP production that impact myelin production by promoting fatty acid synthesis. Here, we investigate the effects of high dose Biotin (MD1003) on the functional properties of post-natal rat derived oligodendrocyte progenitor cells (OPCs). A2B5 positive OPCs were assessed using an in vitro injury assay, culturing cells in either DFM (DMEM/F12+N1) or “stress media” (no glucose (NG)-DMEM), with Biotin added over a range from 2.5 to 250 μg/ml, and cell viability determined after 24 hrs. Biotin reduced the increase in OPC cell death in the NG condition. In nanofiber myelination assays, biotin increased the percentage of ensheathing cells, the number of ensheathed segments per cell, and length of ensheathed segments. In dispersed cell culture, Biotin also significantly increased ATP production, assessed using a Seahorse bio-analyzer. For most assays, the positive effects of Biotin were observed at the higher end of the dose-response analysis. We conclude that Biotin, in vitro, protects OL lineage cells from metabolic injury, enhances myelin-like ensheathment, and is associated with increased ATP production.

Introduction

Multiple sclerosis (MS) most frequently follows an initial relapsing course that then evolves into a more progressive disorder. Mechanisms implicated in disease progression include continued tissue injury and failure of repair. The initial lesions of MS feature destruction of myelin with variable extent of loss of its cell of origin, the oligodendrocyte (OL). In chronic lesions, there is universal loss of OLs [1, 2]. The degree of recovery from relapses in MS following the acute event is attributed, at least in part, to remyelination dependent on recruitment of OL progenitor cells (OPCs) with differentiation into myelin-sheath forming mature OLs [3]. Recent evidence also implicates participation of existent OLs [4–6]. OPCs are increased in periplaque white matter (PPWM) in early MS lesions, but decreased in chronic MS lesions [7, 8]. We postulated there was a differentiation block of OPCs in chronic multiple sclerosis lesions, which may contribute to failure of remyelination [8].

Causes of OL injury and loss in MS include micro-environmental conditions of metabolic stress such as ischemia/hypoxia and mitochondrial injury [9–12]. OL loss is best recognized in the pattern 3 lesion subtype [1], that features an “oligodendrogliopathy” defined by retraction of OL terminal processes (“dying back”) [2]. We have observed retraction of terminal processes of OLs even in absence of cell death in the majority of early MS lesions and at the rims of chronic active lesions [13]. OL/myelin loss impacts the survival and function of neurons/axons [14]. There are as yet no agents approved for use in MS based on neuroprotective or remyelination effects.

Biotin is a co-factor required for the activity of a family of carboxylases involved in pathways that could potentially serve protective roles for cells exposed to hypoxic/ischemic conditions as implicated in MS, as well as enhance myelination (reviewed in [15]). These carboxylases include three that are central to aerobic energy production and generate intermediates for the tricarboxylic acid (TCA) cycle. Such effects are predicted to enhance ATP production. Biotin is also a co-factor for acetyl-CoA carboxylases involved in fatty acid synthesis, a process underlying myelin production. High dose Biotin is demonstrated to reverse rare genetic CNS disorders involving energy metabolism defects [16]. In an initial double-blind placebo controlled study, high dose Pharmaceutical grade Biotin, MD1003 (100 mg three times daily), achieved sustained reversal of MS-related disability in a subset of patients with progressive MS, not seen in the placebo group [17]. However the second pivotal Phase III trial (SPI2) did not meet its primary and secondary end points (Medday announcement (03/10/2020).

In previous studies, we have used in vitro systems to model the response of human and rodent OL lineage cells to ischemic/hypoxic conditions and to assess myelination capacity [13, 18–21]. For the former, we used dissociated cell cultures of such cells subjected to conditions of glucose and nutrient deprivation [2, 20–22]. We observe initial process retraction followed by cell death [22]. This metabolic insult reduces the rate of oxygen consumption and ATP production by these cells. OL lineage cells derived from postnatal rats are still at an OPC stage and are more susceptible to cell death under these conditions than are adult human brain derived mature OLs [20, 22]. The rodent cells display activated caspase 3 and a high percentage are TUNEL+ [13, 20, 21]; and this cell death can be inhibited by a pan caspase inhibitor [13]. To assess the capacity to myelinate, we used ensheathment of synthetic nanofibers, quantified using a high-throughput program with single-cell resolution [23].

In the current study, we directly examine the in vitro effects of high dose biotin on functional properties of post-natal rat derived OPCs as related to protection from conditions of glucose deprivation and capacity to ensheath nanofibers. We then directly assess the biotin effect on baseline oxidative metabolism and ATP production by the cells using a Seahorse XF Analyzer. In addition, we examined the expression of biotin-dependent carboxylases [15] in adult human oligodendrocytes under physiological or metabolic stress conditions (low glucose), mimicking MS lesion microenvironment.

Materials and methods

Cell isolation and culture

Rodent oligodendrocyte cultures

All procedures involving animals were performed in accordance with the Canadian Council on Animal Care’s guidelines for the use of animals in research and approved by the McGill University Animal Care Committee. OPCs were prepared from the brains of newborn Sprague-Dawley rats (purchased from Charles River, Saint Constant, Canada) as previously described [24]. Microglia were removed by an initial shake-off, total cells in flasks were digested by trypsin, and the cells were selected using magnetic beads conjugated with monoclonal antibody A2B5, which recognizes a cell surface ganglioside [25] to select a progenitor cell pool that comprises ~30% of the total cells. OPCs were initially plated at a density of 2.5 × 10⁵ cells per mL on poly-lysine-coated chamber slides, and cultured in defined medium (DFM) consisting of Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12) supplemented with N1, 0.01% bovine serum albumin (BSA), 1% penicillin-streptomycin, and B27 supplement (Invitrogen, Burlington, ON), platelet derived growth factor (PDGF)-AA (10ng/ml), and basic fibroblast factor (bFGF, 10ng/ml) (Sigma, Oakville, ON). Culture media was changed every 48 hrs under the stated conditions. >80–90% % of cells were O4+ after the initial 4 days in culture following cell isolation.

Proliferation and protection assays

For injury assays, cells were cultured in either DFM (DMEM/F12+N1) or “stress media” (no glucose (NG)-DMEM). OPCs were treated for 24 hrs with the indicated concentrations of high dose Pharmaceutical grade Biotin (MD1003), dissolved in PBS. Cell viability was assessed after 24 hrs by live-staining with propidium iodide (PI) (1:200, 15 min, 37° C, Invitrogen, Burlington, ON). To identify OPCs, cells were incubated with monoclonal O4 antibody (IgM, 1:200, R&D Systems, Oakville, ON) [26] for 30 min at 4°C then fixed in 4% paraformaldehyde for 10 min at 4°C, washed twice with PBS, followed by blocking with HHG (1 mM HEPES, 2% horse serum, 10% goat serum, Hanks’ balanced salt solution) for 10 min. Secondary antibodies were either goat anti-mouse IgM Alexa Fluor 488 (1:500, Thermo Fisher Scientific, Eugene, OR) or goat anti-mouse IgM-Cy3 (1:250, Abcam, Toronto, ON) added for 30 min at 4°C. Proliferating cells were identified by immunostaining with Ki67-FITC antibody (1:200 dilution, 4° C, overnight, Cell Signaling Technology). Monoclonal antibodies against galactocerebroside (GC) (IgG₃ 1:50, 30 min, 4° C, derived from hybridoma, [27]) and myelin basic protein (MBP) (IgG_2b, 1:500 dilution, 4° C, overnight, Sternberger, Lutherville, MD) were used to stain the cells to determine OPC differentiation after 24 hrs and 3 days of biotin treatment, respectively. The corresponding secondary antibodies were Goat anti-mouse IgG₃-FITC (1:100, 1 hour, room temperature, Molecular Probes, Eugene, OR) and Goat anti-mouse IgG_2b-FITC or TxR (1:100, 1 hour, room temperature, Biosource, Camarillo, CA). Cell nuclei were stained with Hoechst 33258 (10 μg/ml, Invitrogen) for 10 min at room temperature. Cells were then imaged via an epifluorescent microscope (Zeiss).

PI+ and Ki67+ cells were analyzed using a MATLAB program. The script first identified all circular Hoechst+ nuclei using the extended local minima of the distance transformed binary image for watershed segmentation. A similar procedure is used to identify PI+ and Ki67+ blebs in their respective channels. All circular blebs were then subjected to a circularity metric to ensure exclusion of non-circular artifacts and only circular nuclear-like structures retained. Finally, Hoechst+ nuclei were co-localized with PI+ and Ki67+ blebs on a per pixel basis, and the number of positive cells counted. PDGFAA/bFGF was used as positive control.

Nanofiber ensheathment assay

OPCs were plated in multi-well aligned Nanofiber plates (The Electrospinning Company Ltd., Didcot, Oxfordshire, OX11 0RL) and treated with biotin for 3 days (earliest time to observe nanofiber ensheathment by OPCs—see Xu et al [23]), followed by immunostaining with O4 primary antibody and corresponding secondary antibodies conjugated with either Alexa Fluor 488 (1:500, Thermo Fisher Scientific, Eugene, OR) or Texas Red (1:100, Biosource, Camarillo, CA). Images were acquired with a Zeiss fluorescence microscope, % ensheathment by O4+ cells was quantified using MATLAB software as described by Xu et al [23]. BDNF and PDGFAA/bFGF were used as positive and negative controls respectively.

Metabolic analysis

OPCs were analyzed after first being grown in DFM for 4 days and then treated with biotin for 1 day. Metabolic measurements were carried out as described previously [22]. The cells were washed with XF assay medium (pH adjusted to 7.4) and equilibrated for 1 hr in the Seahorse incubator. The XF96 plate was inserted into the Seahorse analyzer (Seahorse Bioscience, Billerica, MA) where 4 basal assay cycles were performed consisting of a 3 min mix followed by 3 min measure. After completion, oligomycin (OLIGO, 0.5 μM), a mitotoxin that inhibits ATP synthase (complex V), decreasing electron flow through the electron transport chain (ETC) with resultant reduction in mitochondrial respiration or Oxygen consumption rate (OCR), was added by automatic pneumatic injection for 3 assay cycles. Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 0.5 μM), an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential, was then added for an additional three assay cycles followed by rotenone (R, 0.1μM), a complex I inhibitor, plus antimycin A (AA, 0.2μM), a complex III inhibitor, for another three assay cycles. This combination (R/AA) shuts down mitochondrial respiration and enables the calculation of nonmitochondrial respiration driven by processes outside the mitochondria (Agilent, manufacturer’s instructions). The coupling efficiency to calculate mitochondrial ATP production was derived from differences in oxygen consumption rate (OCR) upon addition of oligomycin compared to basal rate, converting OCR to ATP production using a phosphate/oxygen ratio of 5.5 [28, 29]. Extracellular acidification rates (ECAR) were calculated by the addition of 2-deoxy-glucose (2DG; 1M). Proton production rate (PPR) was utilized to estimate ATP production from glycolysis in a 1:1 ratio [30]. All reagents were purchased from Sigma (St. Louis, MO).

Gene expression

Human adult brain derived OL expression of biotin-dependent carboxylases–these data were derived from a previously reported microarray analysis performed on human adult brain derived OLs maintained in dissociated culture under control or 48 hrs of glucose/nutrient deprivation conditions [18].

Statistical analysis

All statistical analyses were performed with GraphPad Prism. 1 way-ANOVA was performed followed by Dunnett’s multiple comparison Test for Fig 1A and 1B, 2A, and 3E. For Fig 2A, the % of ensheathed O4+ cells was compared between the DFM control and each concentration of biotin by paired T-test. For Fig 2B, 1 way-ANOVA (Kruskal-Wallis test) was performed followed by Dunnett’s multiple comparison Test. For Fig 2C, unpaired T-test was performed to compare the control and each concentration of biotin. For Fig 3C, we compared the means of OCR for each seahorse treatment (basal, oligomycin (OLIGO), FCCP, R/AA and 2DG) between control vs 250 μg/ml, control vs. 25 μg/ml, using paired t-test. For Fig 3B, 2 way-ANOVA was performed. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig 2 — OPCs growing on nanofibers were treated with indicated concentrations of biotin in DFM for 3 days. The cell cultures were immunostained with O4 (red) and MBP (green) antibodies. Cells were imaged. % of ensheathed O4+ cells (panel A), number of sheaths per cells (panel B) and the proportion of length of sheath segment per cell (panel C) were analyzed using a MATLAB program and illustrated (mean and/or SEM for each condition are listed in the table underneath each graph). Panels D and E are examples of cells under DFM alone and biotin (250 μg/ml) treated conditions, respectively. Panel F is a higher magnification of a cell from panel E. N = 7. Comparison to control DFM (pair t-test): *, p < 0.05; ** p < 0.01, *** p < 0.001. Scale bars:10 μm in panel E, 20 μm in panel F.

Fig 3 — OPCs growing on seahorse plates were treated with indicated concentrations of biotin in DFM for 24 hr, followed by Seahorse analyzer measurement. Panel A presents OCR data normalized to basal values under DFM control conditions that ranged from 2.2 to 4.8 pmol O2/min/μg protein. The time point for applying OLIGO, FCCP, R/AA and 2DG is indicated. Panel B shows the mean relative basal OCR and after addition of mitotoxins. Panel C shows the p values for Biotin compared with DFM control for Panel B. Panel D shows OCR normalized; panel E shows calculated ATP production, **, p < 0.01 for 250 μg/ml Biotin compared to DFM or 25 μg/ml Biotin (1 way-ANOVA followed by Tukey multiple comparison test, n = 4).

Results

Biotin mediated protection from “stress” conditions—Under glucose-free (NG) conditions, the mean % cell death (PI+ OPCs) at 24 hrs was 29 ± 7% (11–68%) vs. 8± 2% (2,5–12%) for control conditions (n = 8, p = 0.02, S1 Fig). As shown in Fig 1A, supplementation with biotin was done over a concentration range of 0.25 ng/ml to 250 μg/ml; significant reductions in the % PI+ cells were noted over the 2.5μg/ml to 250 μg/ml range (mean % reduction 35 ± 5%, n = 8, p < 0.001 at 250 μg/ml). PI staining is illustrated in Fig 1C–1F. Biotin had no significant effects on protection of OPCs at concentrations of 0.25 ng/ml to 1.25 μg/ml. Biotin supplementation did not induce a significant change in cell proliferation as measured by % Ki67 + cells (Fig 1B), at any of concentrations used. The % of Ki67+ cells is 16.1±2.6% under basal DFM conditions. Ki67 staining is illustrated in Fig 1G–1J. Cultures supplemented with PDGFAA and bFGF were used as the positive control in the proliferation assay. There was a trend for Biotin to increase GC+ cells after 1 day treatment (1.4 ± 0.2 fold of control by 250μg/ml biotin, n = 8, p = 0.07 S2 Fig). The % of MBP+ cells was not increased after 3 days treatment.

Biotin mediated enhancement of nanofiber ensheathment under DFM condition–Overall biotin supplementation increased the % of nanofiber ensheathing O4+ cells compared to defined media (DFM) control conditions over the 2.5–250 μg/ml dose range (27.58±6.25 for 250 μg/ml biotin vs. 16.31±4.54 DFM, n = 7, p <0.05) (Fig 2A). At the individual cell level, biotin supplementation also increased the number of ensheathed segments per cell (panel 2B), and the proportion of cells with higher log length of segments (panel C). As expected, BDNF (10 ng/ml) enhanced ensheathment whereas PDGFAA/bFGF was inhibitory. Examples of nanofiber ensheathment under control and biotin supplemented conditions are provided in Fig 2D and 2E, respectively. Almost all cells are O4+ but only a small minority (<5%) are MBP+.

Biotin increased ATP production by OPCs under DFM control conditions. As shown in Fig 3A and 3B, OCR under all conditions (basal and after addition of mitotoxins) was higher for cells supplemented with biotin at 250 and 25 μg/ml compared to control conditions. P values are shown in Panel C. ECAR were at the margin of detection and no biotin-related effect could be observed (S3 Fig). In order to determine the contribution of non-mitochondrial, ATP-linked, and proton leak components to overall OCR, we expressed each as a % of total OCR for each condition, as shown in panel D. Biotin did not change non-mitochondria oxygen consumption that accounts for ~50% of the OCR. Biotin did significantly increase the ATP-linked contribution to OCR at the higher concentration (250 μg/ml) as shown in Panel E (the calculation is described in method). The calculated ATP-linked OCR production was 6.0±0.22 pmol/min/μg protein with 250 μg/ml biotin vs. 2.7±0.61 in DFM conditions alone (n = 4, p < 0.01).

Expression of Biotin regulated carboxylase genes in human OLs subjected to glucose/nutrient deprivation–Table 1 shows a suggestive decrease of biotin-regulated carboxylase genes except for MCCC1 in adult human OLs cultured for 2 days in the deprivation conditions. At this time there was minimal cell death and only initial indication of process retraction as shown in our previous study [13].

Table 1. Biotin regulated carboxylase gene expression in human OLs.

Carboxylases	AveExpr (LG)	AveExpr (N1)	geneSymbols	LogFC	P.Value
Acetyl-Coa Carboxylase A	7.5	7.7	ACACA	-0.223	0.24
Acetyl-Coa Carboxylase B	5.9	6.0	ACACB	-0.071	0.66
Methylcrotonyl-CoA carboxylase 1	6.8	6.6	MCCC1	0.097	0.52
Methylcrotonyl-CoA carboxylase 2	5.2	5.5	MCCC2	-0.271	0.16
Pyruvate carboxylase	6.4	6.6	PC	-0.168	0.28
Propionyl-Coa carboxylase A	6.7	7.0	PCCA	-0.494	0.01*
Propionyl-Coa carboxylase B	7.0	7.3	PCCB	-0.285	0.23

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Discussion

The current studies demonstrate the measurable capacity of high dose Biotin to protect OL lineage cell from metabolic stress, enhancing nanofiber ensheathment, and increasing ATP production. Our in vitro studies are based on the use of post-natal rat derived OL linage cells, obtained using a shake off method and then further enrich for the population using A2B5 antibody coated immunomagnetic beads. As expected, the majority of cells obtained, from the outset, are in the OL lineage as assessed by expression of O4; not many are yet MBP positive. Our basal culture media, DMEM F12, contained 0.0035mg/L of biotin https://www.thermofisher.com/ca/en/home/technical-resources/media-formulation.55.html. We did not assess biotin free media as our focus was on effects of high dose biotin modeling the in vivo effects of this approach.

We elected to assess dose-dependent protective effects of biotin using glucose depletion as the insult. These conditions model those that can occur in the MS lesion microenvironment [31–35]. Our previous in vitro studies indicate that these can induce a dying back response of OLs that may be reversible or preventable by protective strategies [13]. We had also shown using human adult brain derived OLs that this metabolic insult down-regulated pathways designated as cell morphology, cell metabolism, and cell signaling, as well as pathways specifically implicated in myelination [13]. We recognize that the OPCs used as the targets in this study may be more susceptible to the insults used compared to more mature cells derived in vitro from these OPCs or directly from the adult CNS [20]. OPCs injury may itself be directly relevant to the evolution of the MS disease process as such damage in MS lesions could contribute to impaired remyelination as considered below [36].

Enhancing remyelination remains a central challenge for MS with most efforts beginning with screening for agents that promote ensheathment as we have done in this study [37, 38]. Most implicate this process as being dependent on OPCs that need to survive, proliferate, migrate, differentiate, and then contact and ensheath their targets, an energy-consuming process [39]. The use of artificial nanofibers provides a convenient platform to assess ensheathment although lacking complete features of myelination such as formation of specialized para-nodal loops [40, 41]. Artificial fiber assays have been used to screen for agents that promote myelination [42, 43]. The changes in the number of ensheathing cells we observed with biotin are unlikely to be related to cell proliferation/differentiation as we did not observe differences in cell numbers in our dissociated cell cultures and no differences in % O4 (or MBP) + cells. Myelination and remyelination depend on fatty acid synthesis by oligodendrocytes. Biotin is required to synthesize fatty acids and membrane formation [44], therefore we did not use biotin-free medium in our experiments. Although we did not specifically measure the expression or activity of the acetyl-coA carboxylase which could be involved in myelination [15, 45, 46], the increase in number of ensheathed segments per cell and changes in sheath length would be consistent with enhanced lipid synthesis.

To evaluate whether the increase in the number of ensheathed segments was associated with changes in energy metabolism, we assessed the effects of biotin on the metabolic status of cells under DFM basal culture conditions using a Seahorse XF bio-analyzer. As confirmed in the current study, post-natal rat OPCs mainly utilize oxidative phosphorylation to produce ATP [20, 22]. The OCR levels recorded in the current study are characteristic and comparable to our previously published respiration profiles of post-natal rat brain derived oligodendrocytes [20, 22]. The mechanisms whereby Biotin supplementation increases the resting OCR could include biotin being a co-factor of carboxylases. Three out of five biotin dependent mammalian carboxylases lie in mitochondria (Pyruvate carboxylase, Propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase) [15] and have a role in providing intermediates to the TCA cycle. Increased processing of intermediates through the TCA cycle generates more NADH and FADH2. Increased NADH and FADH2 levels necessitate processing through oxidative phosphorylation accounting for OCR increment. Oxygen consumption has been shown to reflect NADH turnover in brain tissue [47]. Changes in biotin availability in yeast have been shown to impact OCR levels, at concentration of 200 μg/L [48]. As shown in rat oligodendroglia, carboxylase activities are sensitive to levels of biotin availability [49]. Further, biotin can attenuate oxidative stress and mitochondrial dysfunction in murine oligodendrocytes [50]. Our findings that biotin-dependent carboxylase gene expression level is reduced in human OLs exposed to metabolic stress conditions that have been used to model what is encountered in MS lesions [22, 51–53] suggest a basis whereby biotin may target the MS disease process within the CNS.

Conclusion

Our in vitro based results indicate that high dose biotin supplementation protects OL lineage cells from metabolic injury and enhances their ensheathment capacity; such effects are associated with enhanced ATP production.

Supporting information

S1 Fig. Glucose-free (NG) conditions trigger cell death in OPCs.

OPCs were cultured in DFM control or NG conditions for 24 hrs. OPCs were immunostained with monoclonal antibody O4 and PI. PI+ cells were analyzed using a MATLAB program described in the methods. Comparison between DFM and NG was performed by paired t-test, p = 0.02, N = 8.

(TIF)

Click here for additional data file.^{(1.3MB, tif)}

S2 Fig. Effects of biotin on OPC differentiation.

Panel A, OPCs were treated with indicated concentrations of biotin in DFM for 24 hrs. OPCs were immunostained with monoclonal antibodies O4 and GC. Cells were imaged and GC+ cells were analyzed using a MATLAB program described in the methods. Panel B, OPCs were treated with indicated concentrations of biotin in DFM for 3 days. OPCs were immunostained with monoclonal antibodies O4 and MBP. 1-way ANOVA was performed followed by Dunnett’s multiple comparison test.

(TIF)

Click here for additional data file.^{(4.8MB, tif)}

S3 Fig. Effects of biotin on OPC metabolism.

ECAR data normalized to basal values under control conditions that ranged from 0.03 to 0.3 pmol O2/min/μg protein. The time point for applying OLIGO and 2DG is indicated.

(TIF)

Click here for additional data file.^{(2.9MB, tif)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was funded by a research grant to McGill University (Jack Antel, PI) from Medday Pharmaceuticals The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0233859.r001

Decision Letter 0

Ken Arai

5 Feb 2020

PONE-D-19-34914

Effects of Biotin on survival, ensheathment, and ATP production by oligodendrocyte lineage cells in vitro

PLOS ONE

Dear Dr. Cui,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PLOS ONE

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2. Please check that the reference to previous work that described biotin-dependent carboxylase expression in human-adult-brain-derived OLs is correct. This work seems to be cited as both reference 13 and reference 18 in the text.

3. Thank you for providing the following Funding Statement:

"Yes, this study was funded by Medday Pharmaceuticals SA, who also provided the Biotin (MD1003), the same product that is being used in their clinical trial program. The data were generated independently of Medday although by agreement was reviewed by the company."

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4. Please provide copyright information for table 1, as the data presented therein seem to be derived from prev publ from this group (https://doi.org/10.1002/ana.24944).

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the manuscript entitled “Effects of Biotin on survival, ensheathment, and ATP production by oligodendrocyte lineage cells in vitro” by Qiao-Ling Cui, Yun Hsuan Lin, Yu Kang T. Xu, Milton Fernandes, Vijayaraghava TS Rao, Timothy E Kennedy, and Jack Antel, the authors assessed the effect of biotin on oligodendrocyte lineage cell (OPC), and concluded that biotin can be decrease the ratio of cell death of OPC, and promote ensheathment of myelin, and ATP synthesis in vitro assay. In addition, the authors examined gene expression analysis using data from human adult brain oligodendrocyte, and presented the list of carboxylase genes which biotin regulated in human OL.

First of all, while I appreciate the effort of this experiment, I have no idea whether there is a relationship between the results of in vitro assay from rat OPC and that of genes from human OL in this study. I agree that the authors research points are important to resolve how oligodendrocyte or OPC regulate the mechanism(s) to recover from injuries like MS and other white matter injuries. But in this paper, the authors did not show in detailed explanation in the “Results” and “Discussion” about human, so I could not imagine the relationship between both. If the authors want to include this argument, please add more detailed explanations in “Introduction”, “Results”, and “Discussion”. Or, the authors should analyze gene expression using rat OPC after biotin treatment, and you can compare the results from rat and human, and discuss this point.

In the second point, the authors mentioned that high concentration of biotin was effective on cell viability, ensheathment, and metabolic activity. But in the Fig.2B and C, it seemed that the concentration of 2.5 µg/ ml was more effective than the highest concentration (250 µg/ ml). What is the most effective concentration of biotin? And also, the authors examined the effect of 2 concentrations (25 µg/ ml and 250 µg/ ml) of biotin in Fig.3. Why did the authors choose these 2 concentrations to analyze metabolic activities? In Fig.3, it seemed that concentration of 25 µg/ ml of biotin was more effective than that of 250 µg/ml after oligomysin treatment. In Fig.3E, however, the concentration of 250 µg/ ml was effective than that of 25 µg/ ml on ATP production. I was so confused because I have no idea what the best concentration was to discuss what the authors wanted to say. Please explain more clearly in this point, and it is helpful for readers to understand this study. Also there seem some more points that need to be addressed by the authors. Please see below.

Comments;

1. In the “Materials and Methods”, the authors used some media in this study. Are there some differences in defined medium (DFM) and optimal media? If so, please unify the notation. If not, please write differences more clearly.

2. Please describe procedure of ICC; especially, dilution ratio, incubation time, washing conditions, and so on.

3. As discussed above, please describe more detailed explanation in “Gene expression”.

4. P.9, 1st paragraph, the authors said the cell death ratio under the glucose-free conditions. Please show us the data (e.g. graph). And also, the authors mentioned that biotin did not increase the MBP-positive cells, though GC-positive cells were increased after biotin treatment (P.9). Generally speaking, we know that the expression of GC is earlier than that of MBP in myelination processes. The authors assessed ICC at 24 h after biotin treatment, so it was easy to imagine that results. I have no idea why the authors mentioned.

5. P.10, 1st paragraph, the authors used the word “mitotoxins”. The authors should write all reagents name, or you should define what mitotoxins are. It is helpful for all readers to understand. Similarly, the authors wrote the word “ECAR rates”. This expression is not correct, because ECAR is an abbreviation for “ExtraCellular Acidification Rates”. In P.8, 1st paragraph, the authors wrote “extracellular acidification rate”, so please define here.

6. Please check your manuscript carefully. I can’t understand in some sentences in your manuscript.

Minor points;

- In Fig.1, 2, and supplementary Fig.1, use correct unit in your graph. Change “ug/ml” to “¨µg/ml”.

- In Fig. 3, R/AA is not common word for all readers. Please define an abbreviation in “Materials and Methods”(P.7).

- There are some typos. So please check the words carefully, e.g. KI-67, DMF.

Reviewer #2: In the context of multiple sclerosis, the authors study the influence of a high dose of biotin on oligodendrocyte precursor cells (OPCs) under metabolic stress (low-glucose concentration in the medium). The OPC cell death is increased in this condition, but the increase can be reduced in presence of a high concentration of biotin. The authors also used nanofiber myelination assays to show that biotin increases the percentage of ensheathing cells, the number of ensheathed segments per cell, and the length of ensheathed segments. They also show that in cell culture, biotin increases oxygen consumption rate and ATP production.

The article is well written and interesting.

Here are my remarks:

1- There are several names that should be explained, so that non-specialists could fully understand the paper:

- A2B5

- what are mitotoxins and what are their effects?

- same question with oligomycin.

- In the Materials and Methods section, please give the concentration of antibodies in the Proliferation and Protection assays and the Nanofiber ensheathment assay paragraphs, so that the experiments could be reproduced.

2. Figures

In Figure 1 and 2, the conditions significantly different from the control do not apple clearly. For Example, in Figure 1A: it seems that only the concentrations of 250 and 2.5 ug/mL are significantly different from the control (the only two points with an asterisk). However, at the beginning of the Result Section, it is written: "As shown in Figure 1A, supplementation with biotin over a concentration range of 2.5 to 250 μg/ml significantly reduced the % PI+ cells (mean % reduction 35+-5 %, n = 8, p < 0.001 at 250 μg/ml).

An asterisk should be put above all the data points, or the sentence should be changed.

- Same remark for Figure 2A: is only the 250 ug/mL condition statistically significant? If this is the case, it should be mentioned in the text.

- Figure 2C: I do not see the experimental data points, it seems that it is only a fit that is showed here. Please add the experimental data points, so that I could see the error bars. How are estimated the error bars on the proportion of length of sheath segment per cell? are the differences statistically significant?

- Figure 2 D and E: the figures of cells on nanofibers are not very clear, the contrast is not the same and it is really difficult to see a difference. Would it be possible to show a magnified image so that we could clearly see a few ensheathed nanofibers?

3. Metabolism

In Rao et al, PlosOne 2017 (from the same group), it is said that "adult rat oligodendrocytes preferentially use glycolysis whereas [..] oligodendrocyte progenitor cells from which they are derived, mainly use oxidative phosphorylation to produce ATP". In this article, OPCs from the brains of newborn Sprague-Dawley rats were used, as well as human adult brain derived OLs. These two types of cells should have a different metabolism. How does metabolism (glycolysis or oxidative phosphorylation) influence the effect of biotin?

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 May 29;15(5):e0233859. doi: 10.1371/journal.pone.0233859.r002

Author response to Decision Letter 0

14 Apr 2020

Ken Arai

Academic Editor

PLOS ONE

Dear Dr Arai

We are submitting our revised manuscript which we hope you will find addresses the issues and critiques raised by the reviewers. We also address the journal requirements that you outline .Below are our specific responses to all of the above.

Sincerely,

Jack Antel for all the authors

PS As we were preparing to submit the revised manuscript, Medday announced that their current phase 3 high dose Biotin trial did not meet its end points. We added as sentence to the manuscript (there is as yet no scientific presentation of the data).

PLOS ONE related requirements:

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf

We have implemented the above recommended format.

and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

We have implemented the above recommended format.

References 13 or 18 (regarding adult human brain derived oligodendrocytes) do not pertain to biotin dependent carboxylases. Reference 15 has the information regarding carboxylases.

3. Thank you for providing the following Funding Statement:

We have modified our statement in the manuscript regarding support received from Medday for the study. Revised funding statement – this study was funded by a research grant to McGill University (Jack Antel, PI) from Medday Pharmaceuticals.

Author VR’s main contributions to this study occurred while he was a Research Associate at McGill. He joined Medday as the manuscript was being prepared. Medday employees played a consultative role during the under-taking of this project.

If the funding organization did have an additional role, please state and explain that role within your Funding Statement. No other role.

As recommended, we have included the competing interests statement as per PLOSone stipulation "This does not alter our adherence to PLOS ONE policies on sharing data and materials.”

No competing interests to declare. Author contribution section has been updated to address potential questions regarding this.

4. Please provide copyright information for table 1, as the data presented therein seem to be derived from prev publ from this group (https://doi.org/10.1002/ana.24944).

The data depicted in Table 1 has not been published as part of https://doi.org/10.1002/ana.24944.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

5. Review Comments to the Author

Please use the space provided to ex \\plain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Our specific responses are included below and changes to the manuscript are highlighted.

We agree with the reviewer’s comments that in the original manuscript we did not attempt to explain the “relationship between the results of in vitro assay from rat OPC and that of genes from human OL in this study”. We now present our rationale for the human oligodendrocyte studies in the introduction and provide a statement in the discussion to show how our in vitro rat based functional studies link with the human oligodendrocyte molecular studies.

The following sentences have been added in the introduction and discussion sections,

Introduction: “We examined the expression of biotin-dependent carboxylases in adult human oligodendrocytes under physiological or metabolic stress conditions (low glucose), mimicking MS pathology.”

Discussion – new last sentence in discussion: As shown in rat oligodendroglia, carboxylase activities are sensitive to levels of biotin availability [49]. Further, biotin can attenuate oxidative stress and mitochondrial dysfunction in murine oligodendrocytes [50]. Our findings that biotin-dependent carboxylase gene expression levels are reduced in human OLs exposed to metabolic stress conditions that have been used to model what is encountered in MS lesions [22, 51-53]suggest a basis whereby biotin may target the MS disease process within the CNS.

Fig 2 in the results section has been modified. In figure 1 we showed that the protective effect of biotin is observed over the 250-2.5 ug/ml range. We now show more clearly in the revised fig 2, panel A that the ensheathment effect is also seen over this dose range )p values now included). We are less sure about distinguishing between effects within this range. Panel B already showed the significance of the Biotin effect. Panel C – we now include SEMs and p values in the legend. We have added Panel F(a higher magnification of the insert in panel E) so as to better appreciate ensheathment.

And also, the authors examined the effect of 2 concentrations (25 µg/ ml and 250 µg/ ml) of biotin in Fig.3. Why did the authors choose these 2 concentrations to analyze metabolic activities?

We chose these because they were at the extremes of the dose range where we observed effects in figures 1 and 2.

In Fig.3, it seemed that concentration of 25 µg/ ml of biotin was more effective than that of 250 µg/ml after oligomysin treatment. In Fig.3E, however, the concentration of 250 µg/ ml was effective than that of 25 µg/ ml on ATP production. I was so confused because I have no idea what the best concentration was to discuss what the authors wanted to say. Please explain more clearly in this point, and it is helpful for readers to understand this study. Also there seem some more points that need to be addressed by the authors. Please see below.

In panels A and B there are marginal difference between the 2 dosages when compared to each other under all conditions. However when mitochondrial ATP-linked OCR and correlated ATP production is calculated (ie differences between oxygen consumption rate (OCR) upon addition of oligomycin and the basal OCR), there is a significant difference between the 2 dosages (higher with the higher dose) as now indicated in panel E (p values for the comparison are now given)..

Comments;

2. Please describe procedure of ICC; especially, dilution ratio, incubation time, washing conditions, and so on. We added the detailed information of ICC to the methods section.

3. As discussed above, please describe more detailed explanation in “Gene expression”.

Our methods are given in our previously published paper as cited (ref 13)– we can provide these again if requested.

4. P.9, 1st paragraph, the authors said the cell death ratio under the glucose-free conditions. Please show us the data (e.g. graph).

We show the data in the figure below – we have added it to the manuscript as supplementary figure 1. The original manuscript shows only the mean values in the results section.

And also, the authors mentioned that biotin did not increase the MBP-positive cells, though GC-positive cells were increased after biotin treatment (P.9). Generally speaking, we know that the expression of GC is earlier than that of MBP in myelination processes. The authors assessed ICC at 24 h after biotin treatment, so it was easy to imagine that results. I have no idea why the authors mentioned MBP.

We measured MBP expression after 3 days treatment with Biotin but we did not see expression. (clarified this in the method and results sections).

6. Please check your manuscript carefully. I can’t understand in some sentences in your manuscript.

We hope our revisions have clarified this.

Minor points;

- In Fig.1, 2, and supplementary Fig.1, use correct unit in your graph. Change “ug/ml” to “¨µg/ml”. Done.

- In Fig. 3, R/AA is not common word for all readers. Please define an abbreviation in “Materials and Methods”(P.7). Done.

- There are some typos. So please check the words carefully, e.g. KI-67, DMF. Done.

The article is well written and interesting.

Here are my remarks:

1- There are several names that should be explained, so that non-specialists could fully understand the paper:- A2B5.

We now specify in the methods section that A2B5 cells are recognized based on reactivity with this antibody.

- what are mitotoxins and what are their effects?

- same question with oligomycin.

Below is a detailed description from the literature. We summarize this in our revised manuscript (methods section)

Mitotoxins used in our Seahorse assay are: Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and Rotenone with Antimycin A

Effects of mitotoxins:

Oligomycin inhibits ATP synthase (complex V), and is injected first in the assay following basal measurements. It impacts or decreases electron flow through the electron transport chain (ETC), resulting a reduction in mitochondrial respiration or Oxygen consumption rate (OCR). This decrease in OCR is linked to cellular ATP production.

FCCP is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential. It is the 2nd injection following Oligomycin. As a result, electron flow through the ETC is uninhibited, and oxygen consumption by complex IV reaches the maximum.

The third injection is a mixture of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor. This combination shuts down mitochondrial respiration and enables the calculation of nonmitochondrial respiration driven by processes outside the mitochondria.

Referenced from https://www.agilent.com/cs/library/usermanuals/public/XF_Cell_Mito_Stress_Test_Kit_User_Guide.pdf

2. Figures

An asterisk should be put above all the data points, or the sentence should be changed.

We have done as requested (please see detailed response to reviewer 1.

- Same remark for Figure 2A: is only the 250 ug/mL condition statistically significant? If this is the case, it should be mentioned in the text.

. We have done as requested (please see detailed response to reviewer 1).

We now provide a legend that contains mean values + SEMs + p values. Fitting error bars to the actual curves on the graphs would be difficult.

We added a magnified image of an individual cell in the revised figure.

3. Metabolism

It is correct that postnatal rat OPCs use oxidative phosphorylation to a greater extent as compared to adult rat brain derived oligodendrocytes. It is possible that such cells benefit from increased biotin availability by potentially improving the mitochondrial respiration efficiency. In the manuscript we state that “Three out of five biotin dependent mammalian carboxylases lie in mitochondria (Pyruvate carboxylase, Propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase).”

In the current study we only used the adult human OLs for the studies of gene expression under stress conditions as discussed in our response to reviewer 1.

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PLoS One. doi: 10.1371/journal.pone.0233859.r003

Decision Letter 1

Ken Arai

14 May 2020

Effects of Biotin on survival, ensheathment, and ATP production by oligodendrocyte lineage cells in vitro

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PLoS One. doi: 10.1371/journal.pone.0233859.r004

Acceptance letter

Ken Arai

20 May 2020

PONE-D-19-34914R1

Effects of Biotin on survival, ensheathment, and ATP production by oligodendrocyte lineage cells in vitro

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Supplementary Materials

S1 Fig. Glucose-free (NG) conditions trigger cell death in OPCs.

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S2 Fig. Effects of biotin on OPC differentiation.

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S3 Fig. Effects of biotin on OPC metabolism.

ECAR data normalized to basal values under control conditions that ranged from 0.03 to 0.3 pmol O2/min/μg protein. The time point for applying OLIGO and 2DG is indicated.

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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Effects of Biotin on survival, ensheathment, and ATP production by oligodendrocyte lineage cells in vitro

Qiao-Ling Cui

Yun Hsuan Lin

Yu Kang T Xu

Milton G F Fernandes

Vijayaraghava T S Rao

Timothy E Kennedy

Jack Antel

Roles

Abstract

Introduction

Materials and methods

Cell isolation and culture

Rodent oligodendrocyte cultures

Proliferation and protection assays

Nanofiber ensheathment assay

Metabolic analysis

Gene expression

Statistical analysis

Fig 1. Effects of biotin on OPC survival and proliferation.

Fig 2. Effects of biotin on OL ensheathment.

Fig 3. Effects of biotin on OPC metabolism.

Results

Table 1. Biotin regulated carboxylase gene expression in human OLs.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Ken Arai

Roles

Author response to Decision Letter 0

Decision Letter 1

Ken Arai

Roles

Acceptance letter

Ken Arai

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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