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.

Role Portrayed by Serum NGAL and Vitamin D in Patients with Bone Tumors.

BACKGROUND: Bone tumors are responsible for considerable morbidity and mortality at an early age. Malignant bone tumors are quite aggressive in nature. Thus, an accurate and timely diagnosis is essential for bone tumors. Neutrophil gelatinase associated lipocalin (NGAL) and vitamin D have been found to be associated with cancer and may have potential to act as biomarkers for bone tumors also. METHODS: Serum levels of NGAL and 25-OH vitamin D were estimated in 14 patients with benign and 14 with malignant bone tumors and compared with 14 apparently healthy controls. The data collected was compared among different groups using appropriate statistical analysis. NGAL was estimated by enzyme linked immunosorbent as-say (ELISA) and 25-OH vitamin D by radioimmunoassay (RIA) in the serum samples. RESULTS: Serum NGAL levels were found to be increased significantly and 25-OH vitamin D levels decreased significantly in patients with malignant bone tumors as compared to healthy controls (p < 0.001) while this difference was not statistically significant in patients with benign bone tumors (p = 0.05). The difference in serum levels of NGAL and 25-OH vitamin D in patients with malignant bone tumors was found to be statistically significant as compared to patients with benign bone tumors (p < 0.05). The correlation was not statistically significant between the levels of 25-OH vitamin D and NGAL in group I (r = 0.067, p = 0.819), group II (r = 0.204, p = 0.483), and group III (r = -0.086, p = 0.772). CONCLUSIONS: Serum NGAL and 25-OH vitamin D may be used as important serological biomarkers in patients with bone tumors along with other standard investigative modalities.

Chemoradiation in locally advanced Ca Cx: Effect on NGAL levels.

INTRODUCTION: Neutrophil gelatinase-associated lipocalin (NGAL) has been reported to be unregulated in many cancers and to suppress tumor suppressor genes like p53 leading to cell proliferation. Studies to report its relationship with carcinoma cervix (Ca Cx) are still scant. MATERIALS AND METHODS: Serum NGAL levels were analyzed in 30 patients of histopathologically proven locally advanced Ca Cx at the time of diagnosis and 3 weeks after standard chemoradiation by enzyme-linked immunosorbent assay. These patients underwent either brachytherapy or supplementary external beam radiotherapy (EBRT) depending on the response of treatment. The results were analyzed statistically by applying Student's paired t-test. RESULTS: No statistically significant difference (P > 0.05) was observed in patients of Ca Cx before and after treatment or when compared stage wise, histopathological grade wise, or response wise. But the levels were found to increase when duration of treatment was ≥8 weeks (P = 0.040) and to decrease significantly when duration of treatment was <8 weeks (P = 0.0052). The NGAL levels also increased significantly after treatment in patients who received EBRT and supplementary radiotherapy (P = 0.019) while the pre- and post-treatment difference in NGAL levels was not statistically significant in patients who received EBRT + intracavitary brachytherapy (P > 0.05). CONCLUSION: As the duration as well as modality of treatment is quite important in Ca Cx, shorter duration associated with better results and lower NGAL levels, NGAL might prove to be a useful biomarker although further studies are needed to support the claim.

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.

Chemoradiation in Lung Cancer: Effect on Levels of NGAL and Vitamin D in Serum.

BACKGROUND: Mortality due to lung cancer is one of the growing concerns worldwide. Accurate and timely diagnosis is the key to treatment of this disease. Neutrophil gelatinase associated lipocalin (NGAL) and vitamin D have been found to be associated with cancer and may have potential to act as biomarkers for lung cancer. METHODS: Serum levels of NGAL and 25 hydroxy vitamin D (25OH vitamin D) were estimated in 25 patients with lung cancer before (Group I) and 4 weeks after standard treatment (Group II) by chemoradiation. The levels were also analyzed in 25 apparently healthy controls and data was compared among different groups using appropriate statistical analysis. NGAL was estimated by enzyme linked immunosorbent assay (ELISA) and 25OH vitamin D by radioimmunoassay (RIA) in the serum samples. RESULTS: Serum NGAL levels were found to be increased significantly in patients before treatment as compared to healthy controls (p < 0.001) while the levels decreased significantly after treatment (p < 0.01). The levels of vitamin D were found to be decreased in lung cancer patients as compared to healthy controls (p < 0.05) while after treatment the levels of vitamin D were found to be significantly increased (p < 0.001). The correlation was not statistically significant between the levels of vitamin D and NGAL in Group I (r = 0.12, p = 0.57), Group IIa (r = 0.037, p = 0.86), and Group IIb (r = 0.091, p = 0.66). CONCLUSIONS: Serum NGAL and vitamin D bear the potential to act as biomarkers in patients with lung cancer.

Estrogen deficiency induces memory loss via altered hippocampal HB-EGF and autophagy.

Estrogen deficiency reduces estrogen receptor-alpha (ERα) and promotes apoptosis in the hippocampus, inducing learning-memory deficits; however, underlying mechanisms remain less understood. Here, we explored the molecular mechanism in an ovariectomized (OVX) rat model, hypothesizing participation of autophagy and growth factor signaling that relate with apoptosis. We observed enhanced hippocampal autophagy in OVX rats, characterized by increased levels of autophagy proteins, presence of autophagosomes and inhibition of AKT-mTOR signaling. Investigating upstream effectors of reduced AKT-mTOR signaling revealed a decrease in hippocampal heparin-binding epidermal growth factor (HB-EGF) and p-EGFR. Moreover, 17ß-estradiol and HB-EGF treatments restored hippocampal EGFR activation and alleviated downstream autophagy process and neuronal loss in OVX rats. In vitro studies using estrogen receptor (ERα)-silenced primary hippocampal neurons further corroborated the in vivo observations. Additionally, in vivo and in vitro studies suggested the participation of an attenuated hippocampal neuronal HB-EGF and enhanced autophagy in apoptosis of hippocampal neurons in estrogen- and ERα-deficient conditions. Subsequently, we found evidence of mitochondrial loss and mitophagy in hippocampal neurons of OVX rats and ERα-silenced cells. The ERα-silenced cells also showed a reduction in ATP production and an HB-EGF-mediated restoration. Finally in concordance with molecular studies, inhibition of autophagy and treatment with HB-EGF in OVX rats restored cognitive performances, assessed through Y-Maze and passive avoidance tasks. Overall, our study, for the first time, links neuronal HB-EGF/EGFR signaling and autophagy with ERα and memory performance, disrupted in estrogen-deficient condition.

Rosiglitazone up-regulates glial fibrillary acidic protein via HB-EGF secreted from astrocytes and neurons through PPARγ pathway and reduces apoptosis in high-fat diet-fed mice.

The anti-diabetic drug and peroxisome proliferator-activated receptor-gamma (PPARγ) agonist, rosiglitazone, alters astrocyte activation; however, its mechanism remains less-known. We hypothesized participation of epidermal growth factor receptor (EGFR), known to control astrocyte reactivity. We first detected that rosiglitazone promoted glial fibrillary acidic protein (GFAP) expression in primary astrocytes as well as the mouse cerebral cortex, associated with increased EGFR activation. Screening for EGFR ligands revealed a rosiglitazone-mediated increase of heparin-binding epidermal growth factor (HB-EGF) in astrocytes, resulting in HB-EGF release into culture medium and mouse cerebrospinal fluid too. Treatment with HB-EGF-siRNA and EGFR inhibitors showed that the rosiglitazone-induced HB-EGF and p-EFGR were interdependent, which participated in GFAP increase. Interestingly, we observed that rosiglitazone could induce cellular and secreted-HB-EGF in neurons also, contributing toward the activated EGFR-induced GFAP in astrocytes. Probing whether these effects of rosiglitazone were PPARγ-linked, revealed potential PPARγ-responsive elements within HB-EGF gene. Moreover, gel-shift, site-directed mutagenesis, chromatin-immunoprecipitation and luciferase-reporter assays demonstrated a PPARγ-dependent HB-EGF transactivation. Subsequently, we examined effects of rosiglitazone in a high-fat diet-fed diabetes mouse model, and supporting observations in the normal cortical cells, identified a rosiglitazone-induced GFAP, astrocyte and neuronal HB-EGF and secreted-HB-EGF in the cerebral cortex of diabetic mice. Moreover, assessing relevance of increased HB-EGF and GFAP revealed an anti-apoptotic role of rosiglitazone in the cerebral cortex, supported by a GFAP-siRNA as well as HB-EGF-siRNA-mediated increase in cleaved-caspase 3 and 9 levels in the rosiglitazone-treated astrocyte-neuron coculture. Overall, our study indicates that rosiglitazone may protect the brain, via a PPARγ-dependent HB-EGF/EGFR signaling and increased GFAP.