Ethanol sustains phosphorylated tau protein in the cultured neonatal rat hippocampus: Implications for fetal alcohol spectrum disorders
Introduction
Fetal alcohol spectrum disorders (FASDs) result from teratogenic prenatal ethanol exposure and include individuals who are diagnosed with partial or full Fetal Alcohol Syndrome (pFAS; FAS), alcohol-related neurodevelopmental disorder (ARND), alcohol-related birth defects (ARBD), and neurobehavioral disorders associated with prenatal alcohol exposure (ND-PAE). These conditions are on a continuum of severity, with FAS patients presenting a greater magnitude of physical (lower birth weight, thin vermilion, smooth philtrum, flat midface, shortened palpebral fissure) and neurodevelopmental (microcephaly, cerebellar hypoplasia) abnormalities compared to other FASDs (Bertrand et al., 2004; de la Ferreira & Cruz, 2017; Jones & Smith, 1973; Kully-Martens, Denys, Treit, Tamana, & Rasmussen, 2012). Prenatal ethanol exposure is the most common cause of preventable developmental disability: an estimated 2–5% of children are affected by FASDs, annually costing up to $4 billion in the United States (Lupton, Burd, & Harwood, 2004; May et al., 2009). Although early behavioral intervention somewhat improves behavioral and cognitive outcomes for children diagnosed with FASDs (for a comprehensive analysis, see Bertrand & Interventions for Children with Fetal Alcohol Spectrum Disorders Research Consortium, 2009), there are currently no pharmacological treatments specifically for FASDs. In order to develop such treatments and the current standard of care for patients, the neurobiological mechanisms that underpin these disorders need further elucidation.
The rodent CNS is an excellent animal model of human CNS development, and also shows susceptibility to ethanol insult. During perinatal rodent CNS development, ethanol is capable of inducing aberrant neonatal epigenetic modification (for a review, see Basavarajappa & Subbanna, 2016) and disrupting proliferation, axonal growth, white matter production, and synaptic communication (Lindsley, Kerlin, & Rising, 2003; Mathews, Dewees, Diaz, & Favero, 2021; Redila et al., 2006; Sadrian, Subbanna, Wilson, Basavarajappa, & Saito, 2012). Additionally, ethanol can produce deficits observed in paradigms thought to depend upon proper function of the rodent hippocampus, such as performance in Y-maze, radial arm maze, and object recognition tasks (Berman & Hannigan, 2000; Goodlett & Johnson, 1997; Johnson & Goodlett, 2002; Subbanna & Basavarajappa, 2014; Wagner, Zhou, & Goodlett, 2014). The deleterious effects that ethanol has upon the axonal development in the hippocampus may partially be explained by its action on microtubules, microtubule-associated proteins (MAPs) (Smith, Butler, & Prendergast, 2013), and their kinases, which have the capacity to alter the function of MAPs by way of regulatory phosphorylation (Ahluwalia, Ahmad, Adeyiga, Wesley, & Rajguru, 2000). Of the ethanol-sensitive MAPs, the tau protein plays an integral role in ubiquitous axonal development and structural maintenance (Gendron, McCartney, Causevic, Ko, & Yen, 2008). However, the tau protein can become pathological under dysregulated or untimely hyperphosphorylation, resulting in a battery of cognitive deficits and disorders known as tauopathies (for a review, see Orr, Sullivan, & Frost, 2017).
The most carefully studied tauopathies are neurodegenerative disorders. There are likely to be similarities and differences in the mechanisms and dysfunctions of hippocampal hyperphosphorylated tau (p-Tau) dysfunction in neurodegenerative tauopathies compared to FASDs. Specifically, the effects of p-Tau are functionally distinct at different stages in brain development (Yoshida & Ihara, 1993; Yu et al., 2009). For instance, during early CNS development, tau proteins are involved in catalyzing microtubule polymerization, synaptogenesis, neural migration, axonal transport, and cytoskeletal support (Cleveland, Hwo, & Kirschner, 1977; Dayanandan et al., 1999; Gendron et al., 2008; Mandelkow, Stamer, Vogel, Thies, & Mandelkow, 2003; Saito et al., 2010; Wang & Liu, 2008) – functions that are critical for proper development of the fetal and perinatal CNS (Tau & Peterson, 2010).
Interestingly, the tau protein exists in a highly phosphorylated state during fetal and perinatal development in both humans and rodents (Brion, Smith, Couck, Gallo, & Anderton, 1993; Kenessey & Yen, 1993), and this phosphorylation state dynamically changes across different amino acid residues on the tau protein and across different developmental periods and subregions in the rat hippocampus (Yu et al., 2009). While it has been shown that the tau protein is sensitive to ethanol exposure in humans and rats (Ahluwalia et al., 2000; Gendron et al., 2008) and that tau phosphorylation changes across different residues at different periods of neonatal development in the rat hippocampus (Yu et al., 2009), the effects that ethanol exerts on the constitutive changes to tau phosphorylation in the developing neonatal rat hippocampus have yet to be established.
In this study, we investigate the distinct changes to the phosphorylation state of the tau Threonine 231 (Thr231) residue. In some cases, the developmental changes in the tau Thr231 phosphorylation state are inconclusive (Yu et al., 2009); however, assuming there are mechanistic similarities between neurodegenerative tauopathies and the neurodevelopmental tauopathy we aim to establish here (FASDs), we selected the Thr231 residue for three reasons: 1) Thr231 phosphorylation disrupts microtubule polymerization by tau (Lin et al., 2007); 2) Thr231 phosphorylation has been established as a pathological residue in neurodegenerative diseases involving the hippocampus (Chen, 2005); and 3) Thr231 has a distinct relationship with and is often hyperphosphorylated by the kinase glycogen synthase kinase-3β (GSK-3β) (Lin et al., 2007), the activity of which is disinhibited by ethanol exposure (Chen et al., 2009; Liu et al., 2009; Luo, 2009). Taken together, we hypothesize that perinatal ethanol exposure may sustain Thr231 tau phosphorylation across time.
This is the first study to use the organotypic hippocampal slice culture (OHSC) model to document the effect of ethanol in the phosphorylated state of the tau Thr231 residue across time. In this report, we show the effects of ethanol exposure on the phosphorylation state of the tau Thr231 residue out to 24 days in vitro (DIV) in the cultured rat hippocampus.
Section snippets
Organotypic hippocampal slice culture
All experiments complied with the National Research Council's Guide for the Care and Use of Laboratory Animals and were approved by the University of Kentucky Institutional Animal Care and Use Committee. Thirty-one male (n = 21) and female (n = 10) Sprague Dawley pups (Harlan Laboratories; Indianapolis, Indiana, United States) were humanely euthanized at postnatal day 10 (PND10). The rodent CNS at PND10 is roughly equivalent to gestational termination in the third trimester human (for a review,
Preliminary analyses for western blot p-Tau measures
As shown in Table 1, the observations for p-Tau signal are evenly distributed across sex and experimental conditions. Assignment to ethanol condition was unrelated to sex or p-Tau signal. Greater days in vitro was associated with lower p-Tau signal, r = −.50, p < .05.
Results for western blot p-Tau signal
As shown in Table 2, there was a significant interaction between ethanol and days in vitro, β = .63, p < .05, accounting for 13% of the variance in p-Tau signal beyond Male and the main effect of days in vitro. For the control
Discussion
To our knowledge, this is the first study to investigate the impact of ethanol exposure on tau protein Thr231 phosphorylation across neonatal hippocampal culture. In Experiment 1, we demonstrated that ethanol exposure had no effect on Total Tau expression between 12 and 24DIV. The phosphorylated state of the tau Thr231 residue, however, was sensitive to ethanol exposure. We demonstrated that under control conditions, increased DIV was associated with significantly lower phosphorylation state of
Author statement
Caleb Bailey: Conceptualization, Methodology, Writing – Original Draft, Visualization, Investigation, Project administration, Funding acquisition. Julia Jagielo-Miller: Investigation, Writing – Review & Editing. Peggy Keller: Formal analysis. Ethan Glaser: Investigation, Resources. Abigail Wilcox: Investigation. Mark Prendergast: Supervision, Funding acquisition.
Declaration of competing interest
The authors declare no conflicts of interest.
Acknowledgments
This study was supported by a University of Kentucky Substance Use Priority Research Area (SUPRA) Super Student Grant awarded to the first and fifth authors. Partial support comes from the NIAAA Interdisciplinary Training in Alcohol Research (T32 AA027488) awarded to the first, second, and fourth authors. Partial support comes from the SUPRA Graduate Student Pilot Grant awarded to the first author. SUPRA awards are supported by the Vice President for Research at the University of Kentucky.
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