Student Theses and Dissertations
Date of Award
2022
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Timothy Vartanian
Keywords
neurosteroids, oligodendrocyte development, progesterone, myelin basic protein, steroid hormone biosynthesis, multiple sclerosis (MS)
Abstract
The field of neuroendocrinology grew immensely with the realization that steroid hormone production is not confined to the adrenal and reproductive glands but also occurs in the central nervous system (CNS). Steroids synthesized de novo in the brain and spinal cord are referred to as neurosteroid or neuroactive hormones and encompass estrogens, androgens, glucocorticoids, and mineralocorticoids. Though all substrates and enzymes required for neurosteroid biosynthesis exhibit CNS expression, a thorough comprehension of their functionality is lacking. In addition to mediating stress responses, neurosteroids influence CNS-specific processes known to regulate neural development and pathology. Multiple sclerosis (MS), a debilitating neurodegenerative disorder classified by rampant demyelination, exemplifies the neuroendocrine crosstalk facilitated by CNS-resident steroids. Local steroid production from cholesterol, which also happens to be the primary lipid component of myelin sheaths, is critical to myelin repair. Progesterone in particular is implicated in expediting remyelination following demyelinating insults in animal models via an unknown mechanism. Despite the established effect of progesterone on myelin regeneration, its impact on early myelinogenesis remains unclear. This observation inspired the work presented here, in which I investigated a potential role for progesterone in embryonic oligodendrocyte development. Applying the synthetic progestin Nestorone to mouse cerebellar slice cultures, I found that progesterone stimulates the expression of the mature myelin protein, myelin basic protein. Curious as to whether this phenomenon mirrors progesterone-induced remyelination at the molecular level, I implemented the same experimental system in mice genetically altered to delete expression of the nuclear progesterone receptor. Unexpectedly, removal of this receptor from cerebellar slices led not only to an increase in myelin basic protein expression but more robust oligodendrocyte maturation into myelinating cells actively extending processes to axons and participating in fiber formation. The fact that this surprising effect could not be mediated by the nuclear progesterone receptor prompted me to examine potential caveats to studying individual hormones like progesterone in isolation. Due to the existence of multiple non-canonical progesterone receptors expressed in the CNS, the rapid metabolism of progesterone under physiological conditions, and endogenous astrocytic progesterone production, I opted to transition to a mouse devoid of all neurosteroid hormones. To this end, I obtained a transgenic strain with a mutation inhibiting activity of the enzyme that initiates steroid hormone biosynthesis from cholesterol. Histological analyses of E18.5 embryos revealed no differences between wild-type and knockout general anatomy, CNS architecture, or oligodendrocyte quantity and distribution in the brain and spinal cord. I therefore concluded that access to maternal steroid hormones is sufficient to sustain the knockout mice until inevitable perinatal lethality. Unconvinced that the apparent histological normalcy of the knockout CNS represented the full extent of oligodendrocyte functionality, I performed spinal cord explant experiments in which uniform segments of spinal cords dissected from E12.5 embryos were cultured for four days with no exposure to steroid hormones. In this protocol, fluorescent staining with oligodendrocyte transcription factor Nkx2.2 and myelin basic protein revealed a novel CNS phenotype attributed to neurosteroid deficiency. Diffuse Nkx2.2 and myelin basic protein staining was evident across genotypes. However, the explants from knockout embryos also contained a significantly elevated concentration of myelin basic protein-positive cells along the entirety of the midline in the tissue. While further studies are necessary to determine the nature of this abnormality, the results described here suggest excess oligodendrocyte production and/or proliferation defects in spinal cord tissue deprived of neurosteroid hormones may be at play. With our limited understanding of the intricacies governing oligodendrocyte development from neural precursors, relevant neurosteroid activity undoubtedly warrants ongoing pursuit. Taken together, the data holds diverse implications for neurosteroid involvement in oligodendrocyte development, distinct from the myelin repair so urgently needed in MS patients. Spontaneous remyelination proceeds relatively normally in early acute lesions, but progressively declines over time as lesions become chronic and prone to glial scarring and accumulation of inflammatory cellular debris. My finding that unlike in myelin regeneration, progesterone-induced effects on developmental myelin formation do not rely on signaling through the nuclear progesterone receptor, reaffirms the notion that simply recapitulating the process of early oligodendrocyte maturation is inadequate to treat degenerative disorders like MS. The capacity of a sex hormone like progesterone to exert such remarkable effects on oligodendrocyte behavior is in accordance with the striking gender differences characterizing MS, which is nearly three times more common in women but typically more severe in men. Gender-based variations in steroid hormone levels over the course of a lifetime certainly must be considered. Conceivably, men and women may respond uniquely to various pharmacological interventions; thus, MS treatment modalities cannot be approached with a "one size fits all" mentality. Future therapeutic improvements are incumbent on accounting for the explicit sexual dimorphism of MS, to which neurosteroid hormones assuredly contribute.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Winokur, Paige, "Endogenous Neurosteroid Hormone Production and Early Oligodendricyte Development" (2022). Student Theses and Dissertations. 634.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/634
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy