Title: TrkB-mediated neuroprotection in female hippocampal neurons is autonomous, estrogen receptor alpha-dependent, and eliminated by testosterone: a proposed model for sex differences in neonatal hippocampal neuronal injury
Legend: p-TrkB immunoactivity increased following OGD/REOX .ERa+/+ and ERa−/−hippocampal neurons were grown on coverslips and subjected to 4 h OGD followed by VC or 3 μM 7,8-DHF during 24 h REOX. Cells were fixed and stained with anti-p-TrkB and anti-MAP-2 antibodies. A. Representative images of male and female ERa+/+ hippocampal neurons under normoxic conditions or after 4 h OGD followed by 24 h REOX with and without 7,8-DHF treatment (3μM). OGD/REOX resulted in increased p-TrkB staining in male and female hippocampal neurons. However, 7,8-DHF treatment resulted in a further increase in p-TrkB staining in female neurons, but not males. Inset: primary antibody control (p-TrkB and MAP-2). Arrow = co-localized p-TrkB and MAP-2 staining; Arrowhead = lack of p-TrkB staining. B. Summary figure of p-TrkB staining intensity in ERa+/+ and ERa−/− hippocampal neurons grown on coverslips and subjected to 4 h OGD followed by 24 h REOX with and without 7,8-DHF treatment (3μM). Data are mean±SEM, ERa+/+, n=4–11; ERa−/−, n=6. Significance was determined by multi-factorial analysis of variance.
Citation: Chanana V, Zafer D, Kintner DB, Chandrashekhar JH, Eickhoff J, Ferrazzano PA, Levine JE, Cengiz P. TrkB-mediated neuroprotection in female hippocampal neurons is autonomous, estrogen receptor alpha-dependent, and eliminated by testosterone: a proposed model for sex differences in neonatal hippocampal neuronal injury. Biol Sex Differ. 2024 Apr 2;15(1):30. doi: 10.1186/s13293-024-00596-1. PMID: 38566248; PMCID: PMC10988865.
Abstract:
Background – Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of learning disabilities and memory deficits in children. In both human and animal studies, female neonate brains are less susceptible to HI than male brains. Phosphorylation of the nerve growth factor receptor TrkB has been shown to provide sex-specific neuroprotection following in vivo HI in female mice in an estrogen receptor alpha (ERa)-dependent manner. However, the molecular and cellular mechanisms conferring sex-specific neonatal neuroprotection remain incompletely understood. Here, we test whether female neonatal hippocampal neurons express autonomous neuroprotective properties and assess the ability of testosterone (T) to alter this phenotype.
Methods – We cultured sexed hippocampal neurons from ERa+/+ and ERa−/− mice and subjected them to 4 h oxygen glucose deprivation and 24 h reoxygenation (4-OGD/24-REOX). Sexed hippocampal neurons were treated either with vehicle control (VC) or the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following in vitro ischemia. End points at 24 h REOX were TrkB phosphorylation (p-TrkB) and neuronal survival assessed by immunohistochemistry. In addition, in vitro ischemia-mediated ERa gene expression in hippocampal neurons were investigated following testosterone (T) pre-treatment and TrkB antagonist therapy via q-RTPCR. Multifactorial analysis of variance was conducted to test for significant differences between experimental conditions.
Results – Under normoxic conditions, administration of 3 µM 7,8-DHF resulted an ERa-dependent increase in p-TrkB immunoexpression that was higher in female, as compared to male neurons. Following 4-OGD/24-REOX, p-TrkB expression increased 20% in both male and female ERa+/+ neurons. However, with 3 µM 7,8-DHF treatment p-TrkB expression increased further in female neurons by 2.81 ± 0.79-fold and was ERa dependent. 4-OGD/24-REOX resulted in a 56% increase in cell death, but only female cells were rescued with 3 µM 7,8-DHF, again in an ERa dependent manner. Following 4-OGD/3-REOX, ERa mRNA increased ~ 3 fold in female neurons. This increase was blocked with either the TrkB antagonist ANA-12 or pre-treatment with T. Pre-treatment with T also blocked the 7,8-DHF-dependent sex-specific neuronal survival in female neurons following 4-OGD/24-REOX.
Conclusion: OGD/REOX results in sex-dependent TrkB phosphorylation in female neurons that increases further with 7,8-DHF treatment. TrkB phosphorylation by 7,8-DHF increased ERa mRNA expression and promoted cell survival preferentially in female hippocampal neurons. The sex-dependent neuroprotective actions of 7,8-DHF were blocked by either ANA-12 or by T pre-treatment. These results are consistent with a model for a female-specific neuroprotective pathway in hippocampal neurons in response to hypoxia. The pathway is activated by 7,8-DHF, mediated by TrkB phosphorylation, dependent on ERa and blocked by pre-exposure to T.
Keywords: Neurotrophin receptor, Estrogen receptor alpha, Neonatal, Hypoxia Ischemia, 7,8-dihydroxyflavone, Tyrosine kinase B receptor
Investigator: Pelin Cengiz, MD
About the Lab: Pelin Cengiz’s lab focuses on finding a novel therapy for neonatal encephalopathy subsequent to hypoxia ischemia (HI).