Role of an interdependent Wnt, GSK3-ß/ß-catenin and HB-EGF/EGFR mechanism in arsenic-induced hippocampal neurotoxicity in adult mice.

We previously reported the neurotoxic effects of arsenic in the hippocampus. Here, we explored the involvement of Wnt pathway, which contributes to neuronal functions. Administering environmentally relevant arsenic concentrations to postnatal day-60 (PND60) mice demonstrated a dose-dependent increase in hippocampal Wnt3a and its components, Frizzled, phospho-LRP6, Dishevelled and Axin1 at PND90 and PND120. However, p-GSK3-ß(Ser9) and ß-catenin levels although elevated at PND90, decreased at PND120. Additionally, treatment with Wnt-inhibitor, rDkk1, reduced p-GSK3-ß(Ser9) and ß-catenin at PND90, but failed to affect their levels at PND120, indicating a time-dependent link with Wnt. To explore other underlying factors, we assessed epidermal growth factor receptor (EGFR) pathway, which interacts with GSK3-ß and appears relevant to neuronal functions. We primarily found that arsenic reduced hippocampal phosphorylated-EGFR and its ligand, Heparin-binding EGF-like growth factor (HB-EGF), at both PND90 and PND120. Moreover, treatment with HB-EGF rescued p-GSK3-ß(Ser9) and ß-catenin levels at PND120, suggesting their HB-EGF/EGFR-dependent regulation at this time point. Additionally, rDkk1, LiCl (GSK3-ß-activity inhibitor), or ß-catenin protein treatments induced a time-dependent recovery in HB-EGF, indicating potential inter-dependent mechanism between hippocampal Wnt/ß-catenin and HB-EGF/EGFR following arsenic exposure. Fluorescence immunolabeling then validated these findings in hippocampal neurons. Further exploration of hippocampal neuronal survival and apoptosis demonstrated that treatment with rDkk1, LiCl, ß-catenin and HB-EGF improved Nissl staining and NeuN levels, and reduced cleaved-caspase-3 levels in arsenic-treated mice. Supportively, we detected improved Y-Maze and Passive Avoidance performances for learning-memory functions in these mice. Overall, our study provides novel insights into Wnt/ß-catenin and HB-EGF/EGFR pathway interaction in arsenic-induced hippocampal neurotoxicity.

Hypothyroidism induces motor deficit via altered cerebellar HB-EGF/EGFR and autophagy.

Thyroid hormones (TH) are vital for brain functions, while TH deficiency, i.e. hypothyroidism, induces neurological impairment in children and adults. Cerebellar neuronal apoptosis and motor deficits are crucial events in hypothyroidism; however, the underlying mechanism is less-known. Using a methimazole-treated hypothyroidism rat model, we investigated cerebellar autophagy, growth factor, and apoptotic mechanisms that participate in motor functions. We first identified that methimazole up-regulated cerebellar autophagy, marked by enhanced LC3B-II, Beclin-1, ATG7, ATG5-12, p-AMPKα/AMPKα, and p62 degradation as well as reduced p-AKT/AKT, p-mTOR/mTOR, and p-ULK1/ULK1 in developing and young adult rats. We probed upstream effectors of this abnormal autophagy and detected a methimazole-induced reduction in cerebellar phospho-epidermal growth factor receptor (p-EGFR)/EGFR and heparin-binding EGF-like growth factor (HB-EGF). Here, while a thyroxine-induced TH replenishment alleviated autophagy process and restored HB-EGF/EGFR, HB-EGF treatment regulated AKT-mTOR and autophagy signaling in the cerebellum. Moreover, neurons of the rat cerebellum demonstrated this reduced HB-EGF-dependent increased autophagy in hypothyroidism. We further checked whether the above events were related to cerebellar neuronal apoptosis and motor functions. We detected that comparable to thyroxine, treatment with HB-EGF or autophagy inhibitor, 3-MA, reduced methimazole-induced decrease in Nissl staining and increase in c-Caspase-3 and TUNEL-+ve apoptotic count of cerebellar neurons. Additionally, 3-MA, HB-EGF, and thyroxine attenuated the methimazole-induced diminution in riding time on rota-rod and grip strength for the motor performance of rats. Overall, our study enlightens HB-EGF/EGFR-dependent autophagy mechanism as a key to cerebellar neuronal loss and functional impairments in developmental hypothyroidism, which may be inhibited by HB-EGF and 3-MA treatments, like thyroxine.

Arsenic Induces GSK3ß-Dependent p-Tau, Neuronal Apoptosis, and Cognitive Impairment via an Interdependent Hippocampal ERα and IL-1/IL-1R1 Mechanism in Female Rats.

Arsenic is an environmental contaminant with potential neurotoxicity. We previously reported that arsenic promoted hippocampal neuronal apoptosis, inducing cognitive loss. Here, we correlated it with tau pathology. We observed that environmentally relevant arsenic exposure increased tau phosphorylation and the principal tau kinase, glycogen synthase kinase-3 beta (GSK3ß), in the female rat hippocampal neurons. We detected the same in primary hippocampal neurons. Because a regulated estrogen receptor (ER) level and inflammation contributed to normal hippocampal functions, we examined their levels following arsenic exposure. Our ER screening data revealed that arsenic down-regulated hippocampal neuronal ERα. We also detected an up-regulated hippocampal interleukin-1 (IL-1) and its receptor, IL-1R1. Further, co-treating arsenic with the ERα agonist, 4,4',4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), or IL-1R antagonist (IL-1Ra) resulted in reduced GSK3ß and p-tau, indicating involvement of decreased ERα and increased IL-1/IL-1R1 in tau hyperphosphorylation. We then checked whether ERα and IL-1/IL-1R1 had linkage, and detected that although PPT reduced IL-1 and IL-1R1, the IL-1Ra restored ERα, suggesting their arsenic-induced interdependence. We finally correlated this pathway with apoptosis and cognition. We observed that PPT, IL-1Ra and the GSK3ß inhibitor, LiCl, reduced hippocampal neuronal cleaved caspase-3 and TUNEL+ve apoptotic count, and decreased the number of errors during learning and increased the saving memory for Y-Maze test and retention performance for Passive avoidance test in arsenic-treated rats. Thus, our study reveals a novel mechanism of arsenic-induced GSK3ß-dependent tau pathology via interdependent ERα and IL-1/IL-1R1 signaling. It also envisages the protective role of ERα agonist and IL-1 inhibitor against arsenic-induced neurotoxicity.

Arsenic Induces Differential Neurotoxicity in Male, Female, and E2-Deficient Females: Comparative Effects on Hippocampal Neurons and Cognition in Adult Rats.

We earlier reported that arsenic induced hippocampal neuronal loss, causing cognitive dysfunctions in male rats. This neuronal damage mechanism involved an altered bone morphogenetic protein (BMP2)/Smad and brain-derived neurotrophic factor (BDNF)/TrkB signaling. Susceptibility to toxicants is often sex-dependent, and hence we studied the comparative effects of arsenic in adult male and female rats. We observed that a lower dose of arsenic reduced learning-memory ability, examined through passive avoidance and Y-maze tests, in male but not female rats. Again, male rats exhibited greater learning-memory loss at a higher dose of arsenic. Supporting this, arsenic-treated male rats demonstrated larger reduction in the hippocampal NeuN and %-surviving neurons, together with increased apoptosis and altered BMP2/Smad and BDNF/TrkB pathways compared to their female counterparts. Since the primary female hormone, estrogen (E2), regulates normal brain functions, we next probed whether endogenous E2 levels in females offered resistance against arsenic-induced neurotoxicity. We used ovariectomized (OVX) rat as the model for E2 deficiency. We primarily identified that OVX itself induced hippocampal neuronal damage and cognitive decline, involving an increased BMP2/Smad and reduced BDNF/TrkB. Further, these effects appeared greater in arsenic + OVX compared to arsenic + sham (ovary intact) or OVX rats alone. The OVX-induced adverse effects were significantly reduced by E2 treatment. Overall, our study suggests that adult males could be more susceptible than females to arsenic-induced neurotoxicity. It also indicates that endogenous E2 regulates hippocampal BMP and BDNF signaling and restrains arsenic-induced neuronal dysfunctions in females, which may be inhibited in E2-deficient conditions, such as menopause or ovarian failure.

Hypothyroidism Induces Interleukin-1-Dependent Autophagy Mechanism as a Key Mediator of Hippocampal Neuronal Apoptosis and Cognitive Decline in Postnatal Rats.

Thyroid hormone (TH) is essential for brain development, and hypothyroidism induces cognitive deficits in children and young adults. However, the participating mechanisms remain less explored. Here, we examined the molecular mechanism, hypothesizing the involvement of a deregulated autophagy and apoptosis pathway in hippocampal neurons that regulate cognitive functions. Therefore, we used a rat model of developmental hypothyroidism, generated through methimazole treatment from gestation until young adulthood. We detected that methimazole stimulated the autophagy mechanism, characterized by increased LC3B-II, Beclin-1, ATG7, and ATG5-12 conjugate and decreased p-mTOR/mTOR and p-ULK1/ULK1 autophagy regulators in the hippocampus of developing and young adult rats. This methimazole-induced hippocampal autophagy could be inhibited by thyroxine treatment. Subsequently, probing the upstream mediators of autophagy revealed an increased hippocampal neuroinflammation, marked by upregulated interleukin (IL)-1alpha and beta and activated microglial marker, Iba1, promoting neuronal IL-1 receptor-1 expression. Hence, IL-1R-antagonist (IL-1Ra), which reduced hippocampal neuronal IL-1R1, also inhibited the enhanced autophagy in hypothyroid rats. We then linked these events with hypothyroidism-induced apoptosis and loss of hippocampal neurons, where we observed that like thyroxine, IL-1Ra and autophagy inhibitor, 3-methyladenine, reduced the cleaved caspase-3 and TUNEL-stained apoptotic neurons and enhanced Nissl-stained neuronal count in methimazole-treated rats. We further related these molecular results with cognition through Y-maze and passive avoidance tests, demonstrating an IL-1Ra and 3-methyladenine-mediated improvement in learning-memory performances of the hypothyroid rats. Taken together, our study enlightens the critical role of neuroinflammation-dependent autophagy mechanism in TH-regulated hippocampal functions, disrupted in developmental hypothyroidism.