Lidia Strużyńska

Astroglial reaction during the early phase of acute lead toxicity in the adult rat brain

The developing nervous system is susceptible to lead (Pb) exposure but less is known about the effect of this toxic agent in adult rat brain. Since astrocytes serve as a cellular Pb deposition site, it is of importance to investigate the response of astroglial cells in the adult rat brain in a model of acute lead exposure (25 mg/kg b.w. of lead acetate, i.p. for 3 days). An increased immunoreactivity of glial fibrillary acidic protein (GFAP) on Western blots was noticeable in fractions of astroglial origin-glial plasmalemmal vesicles (GPV) and in homogenates from the hippocampus and cerebral cortex but not in the cerebellum. The features of enhanced astrocytic reactivity (i.e. large accumulation of mitochondria, activated Golgi apparatus and increment of gliofilaments) were observed in electron microscopy studies in the same tissues. Total glutathione levels increased both in GPV fractions and in brain homogenates-in the cerebellum (120% above control) and in hippocampus (30% above control). The results of current studies indicate that acute lead exposure is accompanied by astrocyte activation connected with the presence of the enhanced expression of GFAP. It may indicate lead-induced neuronal injury. At the same time, a regional enhancement of detoxicative mechanisms (GSH) was noticed, suggesting activation of astrocyte-mediated neuroprotection against toxic Pb action.

Synaptic degeneration in rat brain after prolonged oral exposure to silver nanoparticles.

Neurotoxicity of silver nanoparticles has been confirmed in both in vitro and in vivo studies. However, the mechanisms of the toxic action have not been fully clarified. Since nanoparticles are likely to have the ability to enter the brain and significantly accumulate in this organ, it is important to investigate their neurotoxic mechanisms. Here we examine the effect of prolonged exposure of rats to small (10nm) citrate-stabilized silver nanoparticles (as opposed to the ionic silver) on synapse ultrastructure and specific proteins. Administration of both nanosilver and ionic silver over a two-week period resulted in ultrastructural changes including blurred synapse structure and strongly enhanced density of synaptic vesicles clustering in the center of the presynaptic part. Disturbed synaptic membrane leading to liberation of synaptic vesicles into neuropil, which testifies for strong synaptic degeneration, was characteristic feature observed under AgNPs exposure. Also a noteworthy finding was the presence of myelin-like structures derived from fragmented membranes and organelles which are associated with neurodegenerative processes. Additionally, we observed significantly decreased levels of the presynaptic proteins synapsin I and synaptophysin, as well as PSD-95 protein which is an indicator of postsynaptic densities. The present study demonstrates that exposure of adult rats to both forms of silver leads to ultrastructural changes in synapses. However, it seems that small AgNPs lead to more severe synaptic degeneration, mainly in the hippocampal region of brain. The observations may indicate impairment of nerve function and, in the case of hippocampus, may predict impairment of cognitive processes.

A glutamatergic component of lead toxicity in adult brain: the role of astrocytic glutamate transporters.

Astroglial cells have a variety of roles in the central nervous system (CNS), providing a homeostasis for the proper functioning of neuronal cells. The classical view concerning the supportive role of astroglia towards associated neurons has to be extended. A great number of new evidences suggest that astrocytes interact closely with neurons being involved in the active control of neuronal activity and metabolism, forming with pre- and postsynaptic nerve terminals a tripartite synapse. Astrocytes control many aspects of brain function. Regulation of extracellular glutamate concentration, potentially neurotoxic neurotransmitter, is fundamental. Glial glutamate transporters system is of importance in protection against glutamate excitotoxicity and antioxidant defence system which is glutathione. When astrocytes fail to function properly, they influence the degree of neuronal damage. Thus, astrocytes are involved to a very great extent into numerous brain pathologies, including toxicity of heavy metals, like lead (Pb). Under pathological conditions they appear to express two opposite features: they are neuroprotective (until they can) or deleterious for neurons and may participate in neuronal damage. The very well known affinity of Pb to astroglia and the changes in glutamatergic transmission upon Pb toxicity, led us to discuss the role of astroglia and astrocytic glutamate transporters in the neurotoxicity of this metal. Our observations are viewed against a background of other results.

Perinatal exposure to lead induces morphological, ultrastructural and molecular alterations in the hippocampus.

The aim of this paper is to examine if pre- and neonatal exposure to lead (Pb) may intensify or inhibit apoptosis or necroptosis in the developing rat brain. Pregnant experimental females received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring; the control group received distilled water. During the feeding of pups, mothers from the experimental group were still receiving PbAc. Pups were weaned at postnatal day 21 and the young rats of both groups then received only distilled water until postnatal day 28. This treatment protocol resulted in a concentration of Pb in rat offspring whole blood (Pb-B) below the threshold of 10 μg/dL, considered safe for humans.We studied Casp-3 activity and expression, AIF nuclear translocation, DNA fragmentation, as well as Bax, Bcl-2 mRNA and protein expression as well as BDNF concentration in selected structures of the rat brain: forebrain cortex (FC), cerebellum (C) and hippocampus (H). The microscopic examinations showed alterations in hippocampal neurons.Our data shows that pre- and neonatal exposure of rats to Pb, leading to Pb-B below 10 μg/dL, can decrease the number of hippocampus neurons, occurring concomitantly with ultrastructural alterations in this region. We observed no morphological or molecular features of severe apoptosis or necrosis (no active Casp-3 and AIF translocation to nucleus) in young brains, despite the reduced levels of BDNF. The potential protective factor against apoptosis was probably the decreased Bax/Bcl-2 ratio, which requires further investigation. Our findings contribute to further understanding of the mechanisms underlying Pb neurotoxicity and cognition impairment in a Pb-exposed developing brain.

The role of the glutamatergic NMDA receptor in nanosilver-evoked neurotoxicity in primary cultures of cerebellar granule cells.

Nanoparticles are known to enter the vertebrate brain, but little is known about their neurotoxicity. The aim of this study is to investigate mechanisms of the contribution of AgNPs to neuronal cell death using primary cultures of rat cerebellar granule cells (CGCs). We tested the role of glutamatergic N-methyl-d-aspartate receptors (NMDA) in AgNP-evoked neurotoxicity using MK-801, a noncompetitive inhibitor of NMDAR. We used commercially available 0.2% PVP-coated AgNPs <100 nm in a concentration range of 2.5-75 μg/ml sonicated with fetal calf serum. After a 10 min incubation period, a dose-dependent increase in the uptake of (45)Ca(2+) into neurons was observed in the presence of 25-75 μg/mL AgNPs which was completely abolished by addition of MK-801. Using the fluorescent dye fluo3 AM we observed an increase in the intracellular calcium level by 87% compared to control. ROS production was found to increase by about 30% over control after a 30-min incubation with 75 μg/mL AgNPs. Further, we observed a significant decrease in the mitochondrial potential during a 30-min incubation with AgNPs. Administration of MK-801 was found to provide a protective effect. Our results show that excitotoxicity via activation of NMDA receptor, followed by calcium imbalance, destabilization of mitochondrial function and ROS production, indicate an important mechanism involved in neurotoxicity evoked by AgNPs in cultured neurons.

Effects of antagonists of glutamate receptors on pro-inflammatory cytokines in the brain cortex of rats subjected to experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Inflammatory cytokines and glutamate neurotoxicity have been proposed as major determinants accompanying the demyelination and axonal degeneration observed during the course of MS. The present study using the animal model of MS known as experimental autoimmune encephalomyelitis (EAE) demonstrates that pharmacological inhibition of ionotropic NMDA glutamate receptors by their antagonists (amantadine and memantine) suppresses neurological symptoms of disease in EAE rats and reduces expression of pro-inflammatory cytokines in the brain. Conversely, antagonists of group I metabotropic glutamate receptors, mGluRs (LY 367385 and MPEP), do not affect the inflammatory process and the neurological condition of EAE rats.

Changes in expression of neuronal and glial glutamate transporters in lead-exposed adult rat brain.

Excitatory amino acid transporters (EAATs) are membrane-bound proteins localized in glial and neuronal cells which transport glutamate (Glu) in a process essential for terminating its action and protecting neurons from excitotoxic damage. Since Pb-induced neurotoxicity has a glutamatergic component and astrocytes serve as a cellular Pb deposition site, it was of interest to investigate the response of main glutamate transporters to short-term lead exposure in the adult rat brain (25mg/kg b.w. of lead acetate, i.p. for 3 days). We examined the expression of mRNA and protein of GLAST, GLT-1 and EAAC1 in homogenates obtained from cerebellum, hippocampus and forebrain. Molecular evidence is provided which indicates that, of the two glial transporters, GLT-1 is more susceptible than GLAST to the neurotoxic effect arising from Pb. RT-PCR analysis revealed highly decreased expression of GLT-1 mRNA in forebrain and hippocampus. In contrast, GLAST was overexpressed in forebrain and in cerebellum. In the case of EAAC1, the enhanced expression of mRNA and protein of transporter was observed only in forebrain. The results demonstrate regional differences in the expression of glutamate transporters after short-term exposure to Pb. In forebrain, downregulation of GLT-1 is compensated by enhanced expression of GLAST, while in hippocampus, the expression of both is lowered. This observation suggests that under conditions of Pb toxicity in adult rat brain, the hippocampus is most vulnerable to the excitotoxic cell damage arising from impaired clearance of the released glutamate.

Temporal expression of P2X7 purinergic receptor during the course of experimental autoimmune encephalomyelitis.

Purinergic P2X(7) receptors are nucleotide-gated ion channels widely distributed in brain. Strong evidence suggests that they are involved in cross-talk between glial and neuronal cells. These receptors activated under pathological conditions may participate in regulation of inflammatory response and cell death. In this study we show the expression of P2X(7) protein and mRNA during the course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), in different stages of the disease (4, 6, 8, 10 post-immunization). The enhanced expression of the receptor at the level of both mRNA and protein was observed in the peak of neurological symptoms and was connected mostly with neurons. However, early overexpression of receptor protein was observed also in an asymptomatic phase of EAE and was tightly related to astrocytic pool of cells. This suggests the early involvement of this kind of receptor into pathological mechanisms leading for symptoms characteristic for EAE.

DOES LEAD PROVOKE THE PEROXIDATION PROCESS IN RAT BRAIN SYNAPTOSOMES

Up to now there has been no information concerning the effect of lead on the peroxidation process in brain nerve endings. We have examined whether lead acetate (in chronic and acute models of toxicity in vivo and in vitro) affected the level of free radicals in synaptosomes obtained from rat brain. Simultaneously, we have checked the effect of peroxidation of Pb2+ on brain homogenates and microsomal fraction. Our results indicated that the lead level in synaptosomal fraction obtained from lead-treated rats was much higher than in controls. We did not observe induction of spontaneous and Fe(3+)-dependent peroxidation either in synaptosomes or in homogenates and brain microsomes after chronic and acute lead administration to the rats. Lead itself also did not enhance both processes when added in vitro to the control brain synaptosomes in micromolar concentrations. The lack of the lead effect on the peroxidation process in subcellular fractions of brain was rather surprising, because lead is known to be the accelerator of Fe(3+)-dependent peroxidation processes in liver. Additionally, livers from rats under the same toxicity conditions were examined. We have found that lead did not provoke spontaneous peroxidation in liver, but contrary to brain fractions, it drastically increased iron-dependent peroxidation in liver homogenates and microsomes. The lack of the effect of lead on inducing peroxidation processes in brain is probably the consequence of the brain having stronger protective mechanisms against its toxicity than the liver.

Perinatal exposure to lead (Pb) promotes Tau phosphorylation in the rat brain in a GSK-3β and CDK5 dependent manner: Relevance to neurological disorders

Hyperphosphorylation of Tau is involved in the pathomechanism of neurological disorders such as Alzheimers, Parkinsons diseases as well as Autism. Epidemiological data suggest the significance of early life exposure to lead (Pb) in etiology of disorders affecting brain function. However, the precise mechanisms by which Pb exerts neurotoxic effects are not fully elucidated. The purpose of this study was to evaluate the effect of perinatal exposure to low dose of Pb on the Tau pathology in the developing rat brain. Furthermore, the involvement of two major Tau-kinases: glycogen synthase kinase-3 beta (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in Pb-induced Tau modification was evaluated. Pregnant female rats were divided into control and Pb-treated group. The control animals were maintained on drinking water while females from the Pb-treated group received 0.1% lead acetate (PbAc) in drinking water, starting from the first day of gestation until weaning of the offspring. During the feeding of pups, mothers from the Pb-treated group were still receiving PbAc. Pups of both groups were weaned at postnatal day 21 and then until postnatal day 28 received only drinking water. 28-day old pups were sacrificed and Tau mRNA and protein level as well as Tau phosphorylation were analyzed in forebrain cortex (FC), cerebellum (C) and hippocampus (H). Concomitantly, we examined the effect of Pb exposure on GSK-3β and CDK5 activation. Our data revealed that pre- and neonatal exposure to Pb (concentration of Pb in whole blood below 10μg/dL, considered safe for humans) caused significant increase in the phosphorylation of Tau at Ser396 and Ser199/202 with parallel rise in the level of total Tau protein in FC and C. Tau hyperphosphorylation in Pb-treated animals was accompanied by elevated activity of GSK-3β and CDK5. Western blot analysis revealed activation of GSK-3β in FC and C as well as CDK5 in C, via increased phosphorylation of Tyr-216 and calpain-dependent p25 formation, respectively. In conclusion, perinatal exposure to Pb up-regulates Tau protein level and induces Tau hyperphosphorylation in the rat brain cortex and cerebellum. We suggest that neurotoxic effect of Pb might be mediated, at least in part, by GSK-3β and CDK5-dependent Tau hyperphosphorylation, which may lead to the impairment of cytoskeleton stability and neuronal dysfunction.