Accumulating evidences suggest a strong correlation between metabolic changes and neurodegeneration in CNS demyelinating diseases such as multiple sclerosis (MS). Biotin, an essential cofactor for five carboxylases, is expressed by oligodendrocytes and involved in fatty acid synthesis and energy production. The metabolic effect of biotin or high-dose-biotin (MD1003) has been reported on rodent oligodendrocytes in vitro, and in neurodegenerative or demyelinating animal models. However, clinical studies, showed mild or no beneficial effect of MD1003 in amyotrophic lateral sclerosis (ALS) or MS. Here, we took advantage of a mouse model of myelin deficiency to study the effects of MD1003 on the behavior of murine and grafted human oligodendrocytes in vivo. We show that MD1003 increases the number and the differentiation potential of endogenous murine oligodendroglia over time. Moreover, the levels of MD1003 are increased in the plasma and brain of pups born to treated mothers, indicating that MD1003 can pass through the mother’s milk. The histological analysis of the grafted animals shows that MD1003 increased proliferation and accelerates differentiation of human oligodendroglia, but without enhancing their myelination potential. These findings provide important insights into the role of MD1003 on murine and human oligodendrocyte maturation/myelination that may explain the mitigated outcome of ALS/MS clinical trials.
Biotin is a water-soluble vitamin B-complex and an essential cofactor for five carboxylases involved in various cellular metabolic pathways including gluconeogenesis, fatty acid synthesis, metabolism of amino acids and fatty acids. Studies have reported that nutritional biotin deficiency or genetic defects in biotinidase (an enzyme that helps recycle biotin) can induce immunological disorders, metabolic abnormalities, seizures, demyelination and neurodegeneration.
During central nervous system (CNS) development, oligodendrocyte progenitor cells (OPCs) migrate into—and colonize—the developing white matter, where they undergo differentiation into mature oligodendrocytes by expressing a subset of myelin-associated proteins. In addition to their contributions to neuronal signaling, oligodendrocytes maintain axonal integrity and provide trophic and metabolic support to neurons.
Myelin disorders, as a group, are among the most prevalent and disabling neurological conditions with no cure. In CNS demyelinating diseases, such as multiple sclerosis (MS), although remyelination occurs, it often fails due to (i) a primary deficiency in OPC numbers; (ii) a failure of their recruitment, (iii) differentiation or (iv) full maturation into myelin-forming cells. Two main approaches have been proposed to enhance remyelination. The first approach involves stimulation of endogenous OPCs to form mature myelinating oligodendrocytes and the second one, provides a new pool of myelin-competent cells via transplantation.
Different agents have been studied in vitro or in animal models aiming to enhance endogenous repair. Metabolic pathways have been suggested as possible therapeutic targets for progressive MS. At the preclinical level, biotin enrichment in rodent oligodendrocytes was first described by LeVine and Macklin. It was also found that biotin attenuates oxidative stress, mitochondrial dysfunction, lipid metabolism alteration and 7β-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes, and might promote myelin synthesis by enhancing fatty acid production and increasing energy production by acting as a coenzyme in oligodendrocytes and neurons. High-dose pharmaceutical grade biotin (MD1003) restores redox balance, energy and lipid homeostasis, and axonal health in a model of adrenoleukodystrophy. Finally, biotin promotes survival, ensheathment and ATP production by rat oligodendrocyte lineage cells in vitro. At the clinical level, safety and efficacy of MD1003 were evaluated in ALS patients. Initial clinical data showed that daily doses of MD1003 up to 300 mg over 12 months, can improve objective measures of MS-related disability. Collongues and collaborators reported that MD1003 treatment was associated with clinical as well as brain and cervical spinal cord volume improvements after one year of treatment. However, the more comprehensive SPI2 study showed that MD1003 did not significantly ameliorate disability or walking speed in patients with progressive MS. In spite of such paradoxical reports, a direct effect of MD1003 on the competence of oligodendroglial cells in vivo remains unclear. Here, we took advantage of a mouse model of myelin deficiency to study the effects of MD1003 on the behavior of both murine and grafted human oligodendrocytes in vivo. We show that although MD1003 can accelerate the differentiation potential of both murine and human oligodendrocytes in vivo, it does not increase the myelination potential of mature oligodendrocytes.
To study the effect of MD1003 on the behavior of murine oligodendrocytes, myelin-deficient Shi/Shi:Rag2−/− mice (hereafter named shiverer) were fed with either MD1003 or vehicle pellets and analyzed over periods of 12, 16 and 20 weeks post treatment (wpt). While food consumption was regular and similar for all groups of mice, they did not show any sign of morbidity, mortality or any clinical sign of worsening of their “shiverer” phenotype (shivering and convulsion).
The effect of treatment was first analyzed by immunohistochemistry using OLIG2, a general oligodendroglial marker, and CC1, a marker of mature oligodendrocytes (OLs). Results showed a significant increase over time in the percentage of OLIG2+ cells in both vehicle and MD1003 groups (vehicle: 40.28 ± 2.19 and 67.86 ± 1.89 at 12 wpt and 20 wpt, respectively (p value = 0.0001); MD1003: 50.97 ± 2.56% and 71.16 ± 3.87% at 12 wpt and 20 wpt, respectively (p value = 0.0001)). In addition, MD1003 treatment significantly promoted the percentage of murine OPCs (OLIG2+/CC1-) at 12 wpt (vehicle: 7.35 ± 1.29% vs. MD1003: 15.88 ± 1.48%, p value = 0.031), as well as the percentage of murine OLs (OLIG2+/CC1+) at the later time-point of 20 wpt (vehicle: 49.14 ± 2.20 vs. MD1003: 59.62 ± 3.4, p value = 0.027). Thus, the increase in OLIG2+ cells resulted from an increase in OPC at 12 wpt, while that at 20 wpt resulted from an increase in murine OLs, suggesting an effect of MD1003 on two different stages of the lineage. To gain insight into the mechanism by which MD1003 acts on OLIG2+ cell numbers, we examined the effect of the drug on murine OLIG2+ cell proliferation. Immunolabeling for OLIG2 and the proliferation marker KI67 showed no difference in the percentage of OLIG2+KI67+ cells over the total number of cells (vehicle: 1.37 ± 0.16% vs. MD1003: 1.66 ± 0.35% at 12 wpt and vehicle: 1.06 ± 0.35% vs. MD1003: 1 ± 0.14% at 20 wpt), nor in the percentage of OLIG2+KI67+ cells over OLIG2+ cells (vehicle: 12.32 ± 2.58% vs. MD1003: 16.31 ± 2.8% at 12 wpt and vehicle: 10.31 ± 6.67% vs. MD1003: 7.29 ± 2.32% at 20 wpt).
The myelination potential of murine OLs was evaluated by electron microscopy and the percentage of myelinated axons in shiverer mice treated with MD1003 or vehicle for 16 weeks was quantified. Shiverer mice, due to the absence of MBP, are characterized by fewer myelinated axons, and uncompacted myelin at the ultrastructural level, compared to wild-type mice. Our data showed no significant difference in the percentage of myelinated axons between the treatment (70.13%) and vehicle (67.61%, p value = 0.7824) or intact (no treatment) groups (73.98%, p value = 0.5804), suggesting that MD1003 did not increase the percentage of myelinated axons by murine OLs at this time-point.
Next, we investigated the effect of MD1003 on the behavior of human oligodendrocytes in vivo. For this purpose, we used fetal human neural precursor cells (hNPCs). The hNPCs were characterized previously as Nestin+ cells, not expressing mature markers of somatic neural cells. Three fetal hNPC lines derived from ganglionic eminence (GE), were selected for OPC differentiation based on their marker expression, speed of growth and previous validation as lines capable of myelinating axons after engraftment in the developing murine brain. After cell expansion in vitro
