Sanghamitra Bandyopadhyay

Characterization of Developmental Neurotoxicity of As, Cd, and Pb Mixture: Synergistic Action of Metal Mixture in Glial and Neuronal Functions

Neurotoxicity of individual metals is well investigated but that of metal mixture (MM), an environmental reality, in the developing brain is relatively obscure. We investigated the combinatorial effect of arsenic (As), cadmium (Cd), and lead (Pb) on rat brain development, spanning in utero to postnatal development. MM was administered by gavage to pregnant and lactating rats, and to postweaning pups till 2 months. The pups exhibited behavioral disturbances characterized by hyperlocomotion, increased grip strength, and learning-memory deficit. Disruption of the blood-brain barrier (BBB) was associated with dose-dependent increase in deposition of the metals in developing brain. Astrocytes were affected by MM treatment as evident from their reduced density, area, perimeter, compactness, and number of processes, and increased apoptosis in cerebral cortex and cerebellum. The metals induced synergistic reduction in glial fibrillary acidic protein (GFAP) expression during brain development; however, postweaning withdrawal of MM partially restored the levels of GFAP in adults. To characterize the toxic mechanism, we treated rat primary astrocytes with MM at concentrations ranging from lethal concentration (LC)(10) to LC(75) of the metals. We observed synergistic downregulation in viability and increase in apoptosis of the astrocytes, which were induced by proximal activation of extra cellular signal-regulated kinase (ERK) signaling and downstream activation of Jun N-terminal kinase (JNK) pathway. Furthermore, rise in intracellular calcium ion ([Ca(2+)](i)) and reactive oxygen species generation promoted apoptosis in the astrocytes. Taken together, these observations are the first to show that mixture of As, Cd, and Pb has the capacity to induce synergistic toxicity in astrocytes that may compromise the BBB and may cause behavioral dysfunction in developing rats.

Interleukin-1α stimulates non-amyloidogenic pathway by α-secretase (ADAM-10 and ADAM-17) cleavage of APP in human astrocytic cells involving p38 MAP kinase

Interleukin‐1α (IL‐1α) stimulates a disintegrin and metalloproteinase, ADAM‐17 synthesis, consistent with activation of the soluble fragment of Amyloid Precursor Protein, APP, (sAPPα) in human primary astrocytes. To characterize the mechanism by which IL‐1α promotes the non‐amyloidogenic pathway of APP metabolism, we used U373 MG astrocytoma cells. IL‐1α significantly increased levels of ADAM‐10 and ADAM‐17 mRNA in 16 hr. Upregulation of ADAM‐17 mRNA by IL‐1α was more pronounced despite higher basal levels of ADAM‐10 mRNA. This pattern was also observed at the protein level with the upregulation of α‐secretase. RNA interference (RNAi) of ADAM‐10 and ADAM‐17 inhibited IL‐1α‐stimulated sAPPα release and the effect was more pronounced with ADAM‐17 RNAi. Concomitantly, the level of sAPPα was significantly increased by IL‐1α in 48 hr; however, IL‐1α stimulated cell‐associated APP levels maximally at 6 h but the induction declined at 48 hr. IL‐1α treatment of cells for 48 h reduced both intracellular and secreted levels of amyloid‐β, Aβ‐40, and Aβ‐42 peptides. Multiple MAP kinases (MAPK), including MEK/ERK, p38 kinase, PI3 kinase (PI3K) but not JNK were involved in the regulation of IL‐1α‐stimulated α‐secretase activity and sAPPα release. p38 MAPK seems to be the most proximal of these MAPKs, as it was the earliest to be activated by IL‐1α and blocking this pathway attenuated activation of IL‐1α‐induced MEK and PI3K pathways. Our data show a complex mechanism of sAPPα regulation by IL‐1α that involves ADAM‐10, ADAM‐17 and p38 MAPK upstream of MEK and PI3K.

Anti-apoptotic role of omega-3-fatty acids in developing brain: perinatal hypothyroid rat cerebellum as apoptotic model.

Inadequate maternal intake of omega‐3‐fatty acids (ω3 FAs) causes adverse neurodevelopmental outcome in the progeny; however, their molecular mechanism of action is obscure. Since ω3 FAs are known to inhibit neuronal apoptosis during neuro‐degeneration, we investigated their possible contribution in regulating neuronal apoptosis during brain development. Using rat model of hypothyroidism‐induced neuronal apoptosis, we provide evidence for anti‐apoptotic role of ω3 FAs during cerebellar development. ω3 FAs were supplemented as a mixture of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to pregnant and lactating rats, and primary hypothyroidism was induced by administering methimazole. The cerebella from postnatal day 16 (d16) pups were isolated, and studies on apoptosis were conducted. We observed that ω3 FA‐supplementation significantly reduced DNA fragmentation and caspase‐3 activation in developing cerebellum of hypothyroid pups. The protection provided by ω3 FAs was associated with their ability to prevent increases in the level of pro‐apoptotic basal cell lymphoma protein‐2 (Bcl‐2)‐associated X protein (Bax) in the cerebellum during thyroid hormone (TH) deficiency. ω3 FAs increased the levels of anti‐apoptotic proteins like Bcl‐2 and Bcl‐extra large (Bcl‐xL), known to be repressed in hypothyroidism. ω3 FAs also restored levels of cerebellar phospho (p)‐AKT, phospho‐extracellular regulated kinase (p‐ERK) and phospho‐c‐Jun N‐terminal kinase (p‐JNK), which were altered by hypothyroid insults, without interfering with the expression of TH responsive gene, myelin basic protein (mbp). Taken together, these results supplement an insight into the molecular mechanism of action of ω3 FAs in developing brain that involves regulation of apoptotic signaling pathways under stress.

Novel drug targets based on metallobiology of Alzheimer's disease.

Importance of the field: Increased localization of Zn, Fe, Cu and Al within the senile plaques (SP) exacerbates amyloid beta (Aβ)-mediated oxidative damage, and acts as catalyst for Aβ aggregation in Alzheimers disease (AD). Thus, disruption of aberrant metal–peptide interactions via chelation therapy holds considerable promise as a rational therapeutic strategy against Alzheimers amyloid pathogenesis. Areas covered in this review: The complexities of metal-induced genesis of SP are reviewed. The recent advances in the molecular mechanism of action of metal chelating agents are discussed with critical assessment of their potential to become drugs. What the reader will gain: Taking into consideration the interaction of metals with the metal-responsive elements on the Alzheimers amyloid precursor protein (APP), readers will gain understanding of several points to bear in mind when developing a screening campaign for AD-therapeutics. Take home message: A functional iron-responsive element (IRE) RNA stem loop in the 5′ untranslated region (UTR) of the APP transcript regulates neural APP translation. Desferrioxamine, clioquinol, tetrathiolmolybdate, dimercaptopropanol, VK-28, and natural antioxidants, such as curcumin and ginko biloba need critical evaluation as AD therapeutics. There is a necessity for novel screens (related to metallobiology) to identify therapeutics effective in AD.

Novel 5′ Untranslated Region Directed Blockers of Iron-Regulatory Protein-1 Dependent Amyloid Precursor Protein Translation: Implications for Down Syndrome and Alzheimer's Disease

We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP), which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1) whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE) RNA stem loop in the 5′ untranslated region (UTR) of APP mRNA. These agents were 10-fold less inhibitory of 5′UTR sequences of the related prion protein (PrP) mRNA. Western blotting confirmed that the ‘ninth’ small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009), a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5′UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimers disease (AD). RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5′UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5′UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down syndrome (DS) that can also cause familial Alzheimers disease.

Diverse roles of extracellular calcium-sensing receptor in the central nervous system.

The G‐protein‐coupled calcium‐sensing receptor (CaSR), upon activation by Ca2+ or other physiologically relevant polycationic molecules, performs diverse functions in the brain. The CaSR is widely expressed in the central nervous system (CNS) and is characterized by a robust increase in its expression during postnatal brain development over adult levels throughout the CNS. Developmental increases in CaSR levels in brain correlate with myelinogenesis. Indeed, neural stem cells differentiating to the oligodendrocyte lineage exhibit the highest CaSR expression compared with those differentiating to astrocytic or neuronal lineages. In adult CNS, CaSR has broad relevance in maintaining local ionic homeostasis. CaSR shares an evolutionary relationship with the metabotropic glutamate receptor and forms heteromeric complexes with the type B‐aminobutyric acid receptor subunits that affects its cell surface expression, activation, signaling, and functions. In normal physiology as well as in pathologic conditions, CaSR is activated by signals arising from mineral ions, amino acids, polyamines, glutathione, and amyloid‐β in conjunction with Ca2+ and other divalent cationic ligands. CaSR activation regulates membrane excitability of neurons and glia and affects myelination, olfactory and gustatory signal integration, axonal and dendritic growth, and gonadotrophin‐releasing hormonal‐neuronal migration. Insofar as the CaSR is a clinically important therapeutic target for parathyroid disorders, development of its agonists or antagonists as therapeutics for CNS disorder could be a major breakthrough.

Calcium receptor expression and function in oligodendrocyte commitment and lineage progression: Potential impact on reduced myelin basic protein in CaR-null mice

Oligodendrocytes develop from oligodendrocyte progenitor cells (OPCs), which in turn arise from a subset of neuroepithelial precursor cells during midneurogenesis. Development of the oligodendrocyte lineage involves a plethora of cell‐intrinsic and ‐extrinsic signals. A cell surface calcium‐sensing receptor (CaR) has been shown to be functionally expressed in immature oligodendrocytes. Here, we investigated the expression and function of the CaR during oligodendrocyte development. We show that the order of CaR mRNA expression as assessed by quantitative polymerase chain reaction is mature oligodendrocyte > neuron > astrocyte. We next determined the rank order of CaR expression on inducing specification of neural stem cells to the neuronal, oligodendroglial, or astrocytic lineages and found that the relative levels of CaR mRNA expression are OPC > neuron > astrocytes. CaR mRNA expression in cells at various stages of development along the oligodendrocyte lineage revealed that its expression is robustly up‐regulated during the OPC stage and remains high until the premyelinating stage, decreasing thereafter by severalfold in the mature oligodendrocyte. In OPCs, high Ca2+ acting via the CaR promotes cellular proliferation. We further observed that high Ca2+ stimulates the mRNA levels of myelin basic protein in preoligodendrocytes, which is also CaR mediated. Finally, myelin basic protein levels were significantly reduced in the cerebellum of CaR‐null mice during development. Our results show that CaR expression is up‐regulated when neural stem cells are specified to the oligodendrocyte lineage and that activation of the receptor results in OPC expansion and differentiation. We conclude that the CaR may be a novel regulator of oligodendroglial development and function.

Kaempferol and quercetin stimulate granulocyte-macrophage colony-stimulating factor secretion in human prostate cancer cells

Granulocyte-macrophage colony-stimulating factor (GM-CSF) holds immunotherapeutic promise in prostate cancer as it activates the host immune system. Increased production of GM-CSF by cancer cells may facilitate host immunosurveillence by the dendritic cells (DC). Here, we studied the effects of kaempferol (K) and quercetin (Q) on the production of GM-CSF in PC-3 cells. Human cytokine antibody array revealed that treatment with K or Q increased GM-CSF release by PC-3 cells. We further observed by ELISA that K and Q in a concentration-dependent manner increased GM-CSF production without affecting its mRNA levels. Inhibitors of vesicular traffic through the endoplasmic reticulum and Golgi-blocked GM-CSF secretory stimulation. A microtubule-stabilizing agent stimulated GM-CSF release, whereas tubulin and actin depolymerizers suppressed K- or Q-stimulated secretion of GM-CSF. Depletion of extracellular or intracellular calcium ion inhibited the GM-CSF secretion upregulated by both K and Q. Furthermore, we showed that K- and Q-stimulated GM-CSF production involves PLC, PKC, and MEK1/2 activation. Treating human DC with the conditioned medium of K- or Q-incubated PC-3 cells increased chemotaxis of DC, which was significantly attenuated when the conditioned medium was incubated with the neutralizing antibody against GM-CSF. Taken together, our results demonstrate that K and Q activate an immune response in the prostate cancer cells by stimulating GM-CSF production, which in turn could result in the recruitment of DCs to the tumor site.

Low density solvent based dispersive liquid–liquid microextraction with gas chromatography–electron capture detection for the determination of cypermethrin in tissues and blood of cypermethrin treated rats

A simple and rapid method to determine the cypermethrin (CYP) insecticide in rat tissues (kidney, liver and brain) and blood has been developed for the first time using low density solvent-dispersive liquid-liquid microextraction (LDS-DLLME) followed by gas chromatography-electron capture detector (GC-ECD) analysis. Initially, tissue samples containing CYP were homoginized in acetone. Subsequently, homogenate was mixed with n-hexane (extraction solvent) and the mixture was rapidly injected into water. The upper n-hexane layer was collected in a separate microtube and injected into GC-ECD for analysis. Blood samples were diluted with ultrapure water and subjected to DLLME through similar procedure. Parameters such as type and volume of disperser and extraction solvent, salting out effect and extraction time, which can affect the extraction efficiency of DLLME, were optimized. Method was validated by investigating linearity, precision, recovery, limit of detection (LOD) and quantification (LOQ). LODs in tissue were in the range of 0.043-0.314 ng mg(-1) and for blood it was 8.6 ng mL(-1) with a signal to noise ratio of 3:1. LOQs in tissue were in the range of 0.143-1.03 ng mg(-1) and for blood it was 28.3 ng mL(-1) with a signal to noise ratio of 10:1. Mean recoveries of CYP at three different concentation levels in all the matrices were found to be in the range of 81.6-103.67%. The results show that, LDS-DLLME coupled with GC-ECD offers a simple, rapid and efficient technique for extraction and determination of CYP in rat tissues and blood samples, which in turn would be useful for toxicological studies of CYP.

Maternal Thyroid Hormone: A Strong Repressor of Neuronal Nitric Oxide Synthase in Rat Embryonic Neocortex

Understanding of how maternal thyroid inadequacy during early gestation poses a risk for developmental outcomes is still a challenge for the neuroendocrine community. Early neocortical neurogenesis is accompanied by maternal thyroid hormone (TH) transfer to fetal brain, appearance of TH receptors, and absence of antineurogenesis signals, followed by optimization of neuronal numbers through apoptosis. However, the effects of TH deprivation on neurogenesis and neuronal cell death before the onset of fetal thyroid are still not clear. We show that maternal TH deficiency during early gestational period causes massive premature elevation in the expression of neuronal nitric oxide synthase (nNOS) with an associated neuronal death in embryonic rat neocortex. Maternal hypothyroidism was induced by feeding methimazole (0.025% wt/vol) in the drinking water to pregnant Sprague Dawley rats from embryonic d 6. Cerebral cortices from fetuses were harvested at different embryonic stages (embryonic d 14, 16, and 18) of hypothyroid and euthyroid groups. Immunoblotting and real-time PCR results showed that both protein and RNA levels of nNOS were prematurely increased under maternal hypothyroidism, and showed reversibility upon T4 administration. Immunohistochemistry revealed an increased nNOS immunoreactivity in both the cortical plate and proliferative zone of neocortex along with a corroborative decrease in the microtubule associated protein-2 positive neurons under maternal TH insufficiency. Results combined, put forth nNOS as a novel target of maternal TH action in embryonic neocortex, and underscore the importance of prenatal screening and timely rectification of maternal TH insufficiency, even of a moderate degree.