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Mechanisms of abnormal spinal cord development and biomarkers of open neural tube defects: insights from a fetal rat model
Janik, Karolina Joanna
Janik, Karolina Joanna
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2025-08
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Biomedical Sciences
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https://doi.org/10.34944/2tte-7n80
Abstract
Neural tube defects (NTDs) are major birth defects affecting the central nervous system (CNS). They arise as a result of incomplete closure of the neural tube during early embryonic development, leading to defects in the brain and/or the spinal cord. The pattern of occurrence suggests the multifactorial etiology of NTDs, involving complex interactions between genetic predisposition and environmental factors. However, the precise mechanisms underlying their etiology and pathogenesis remain poorly understood. Myelomeningocele (MMC), the most common and severe form of spina bifida, is an open NTD that affects the developing spinal cord. Afflicted fetuses usually survive birth; however, MMC leaves most individuals with substantial and life-long deficits, including reduced or absent motor and sensory function, bowel and bladder dysfunction and cognitive disabilities. MMC defect, which is typically found in the lumbar region, is characterized by herniation of neural tissue and meninges through a pathological opening in the overlying structures, leading to spinal cord exposure to the surrounding amniotic fluid (AF). The progressive nature of MMC during gestation along with its life-long clinical impact, points to the importance of early prenatal diagnosis. Currently, the measurement of alpha fetoprotein (AFP) levels and/or fetal imaging techniques such as ultrasound are typically used for prenatal diagnosis of open NTDs. However, due to limited sensitivity and specificity, detection of open NTDs through elevated levels of AFP is not definitive, making the early diagnosis of these defects more challenging. Thus, there is a need for the identification of novel biomarkers enabling early and accurate prenatal diagnosis of open NTDs.
The chondroitin sulfate proteoglycans (CSPGs), neurocan and phosphacan, are extracellular matrix (ECM) components of neural origin that are abundant in the developing CNS. With the occurrence of open NTD, these highly soluble forms of ECM proteins could be released from the site of the spinal cord defect into the surrounding AF, resulting in increased levels of neurocan and phosphacan in the AF of affected fetuses. Therefore, using the well-accepted retinoic acid (RA)-induced fetal rat model of MMC, the objective of this part of the project was to determine whether neurocan and/or phosphacan levels are elevated in the AF of affected fetuses compared to age-matched controls at various points of gestation, particularly in early development, and whether the elevated AF levels of neurocan and phosphacan constitute potential biomarkers for open NTDs.
We identified significantly elevated levels of both neurocan and phosphacan in the AF of fetal rats with MMC, when compared to age-matched normal controls. These CSPGs were detected in MMC fetuses starting early in gestation, with their levels increasing with the progression of MMC. We demonstrated that significant differences in the AF levels of neurocan and phosphacan allow for distinction between MMC and normal controls at all examined gestational ages. Therefore, these studies provided two prospective biomarkers with potential applications in early prenatal diagnosis of open NTDs.
In MMC, the failure in neural tube closure results in spinal cord malformation, leading to the disruption in the normal development of the spinal cord at the affected site. The pathological development of MMC spinal cord involves enhanced generation of glial fibrillary acid protein (GFAP)-expressing astrocytes in the spinal cord at the site of MMC defect. However, the mechanisms underlying the abnormal generation of astrocytes in MMC spinal cord are poorly understood. Therefore, investigating the dysregulation in cellular and molecular mechanisms underlying the process of astrogenesis in the MMC spinal cord is essential for better understanding of its impaired development.
The Janus kinase - signal transducer and activator of transcription (JAK-STAT) signaling is the most prominent pathway that regulates GFAP expression and, therefore, the differentiation of neural progenitor cells (NPCs) into astrocytes. However, its effect on GFAP expression is influenced by epigenetic changes, including DNA methylation and histone modifications. In line with the importance of epigenetic changes in the process of astrogenesis, studies of cortical progenitors demonstrated that STAT3 induces GFAP expression and differentiation of NPCs into astrocytes following demethylation of STAT3 binding site in the Gfap promoter. While the relationship between the activation of STAT3 signaling and the epigenetic modifications was investigated in the context of normal astrogenesis using cultures of cortical progenitors, the dysregulation of these mechanisms in the developing MMC spinal cord have not been explored. Therefore, using the fetal rat model of MMC and NPC cultures obtained from MMC spinal cords, the objective of the second part of this project was to determine whether the abnormal activation of STAT3 in NPCs and the epigenetic changes within the Gfap promoter at the STAT3 binding site are involved in the abnormal generation of astrocytes in the MMC spinal cord, and to determine the role of AF exposure in this pathological process.
We found that NPCs in the ventricular zone (VZ) of developing MMC spinal cord, exhibit abnormal STAT3 activation, a feature that is not observed in normal controls. These changes, along with the accelerated epigenetic modifications characterized by reduced DNA methylation at the STAT3 consensus binding site within the Gfap promoter and enrichment of dimethylated histone 3 lysine 4 (H3K4me2), induced early GFAP expression in NPCs, promoting premature astrocyte generation in MMC spinal cords. Moreover, we demonstrated that AF exposure serves as a stimulus for STAT3 activation in NPCs of MMC spinal cord, thereby promoting GFAP expression and driving their differentiation into astrocytes. To validate this mechanism, we showed that exposure of NPCs to MMC AF following CRISPR/Cas9-mediated elimination of STAT3 expression, abolished GFAP expression and inhibited their differentiation into astrocytes. These findings from the fetal rat model of MMC and NPCs isolated from MMC spinal cords reveal the cellular origin and novel mechanistic basis of the abnormal astrocyte development in MMC spinal cords.
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