Mice deficient in synaptic protease neurotrypsin show impaired spaced long-term potentiation and blunted learning-induced modulation of dendritic spines.

Neurotrypsin (NT) is a neuronal trypsin-like serine protease whose mutations cause severe mental retardation in humans. NT is activated in vitro by Hebbian-like conjunction of pre- and postsynaptic activities, which promotes the formation of dendritic filopodia via proteolytic cleavage of the proteoglycan agrin. Here, we investigated the functional importance of this mechanism for synaptic plasticity, learning, and extinction of memory. We report that juvenile neurotrypsin-deficient (NT-/-) mice exhibit impaired long-term potentiation induced by a spaced stimulation protocol designed to probe the generation of new filopodia and their conversion into functional synapses. Behaviorally, juvenile NT-/- mice show impaired contextual fear memory and have a sociability deficit. The latter persists in aged NT-/- mice, which, unlike juvenile mice, show normal recall but impaired extinction of contextual fear memories. Structurally, juvenile mutants exhibit reduced spine density in the CA1 region, fewer thin spines, and no modulation in the density of dendritic spines following fear conditioning and extinction in contrast to wild-type littermates. The head width of thin spines is reduced in both juvenile and aged NT-/- mice. In vivo delivery of adeno-associated virus expressing an NT-generated fragment of agrin, agrin-22, but not a shorter agrin-15, elevates the spine density in NT-/- mice. Moreover, agrin-22 co-aggregates with pre- and postsynaptic markers and increases the density and size of presynaptic boutons and presynaptic puncta, corroborating the view that agrin-22 supports the synaptic growth.

Activation of the 5-HT7 receptor and MMP-9 signaling module in the hippocampal CA1 region is necessary for the development of depressive-like behavior.

Major depressive disorder is a complex disease resulting from aberrant synaptic plasticity that may be caused by abnormal serotonergic signaling. Using a combination of behavioral, biochemical, and imaging methods, we analyze 5-HT7R/MMP-9 signaling and dendritic spine plasticity in the hippocampus in mice treated with the selective 5-HT7R agonist (LP-211) and in a model of chronic unpredictable stress (CUS)-induced depressive-like behavior. We show that acute 5-HT7R activation induces depressive-like behavior in mice in an MMP-9-dependent manner and that post mortem brain samples from human individuals with depression reveal increased MMP-9 enzymatic activity in the hippocampus. Both pharmacological activation of 5-HT7R and modulation of its downstream effectors as a result of CUS lead to dendritic spine elongation and decreased spine density in this region. Overall, the 5-HT7R/MMP-9 pathway is specifically activated in the CA1 subregion of the hippocampus during chronic stress and is crucial for inducing depressive-like behavior.

The cell adhesion protein dystroglycan affects the structural remodeling of dendritic spines.

Dystroglycan (DG) is a cell membrane protein that binds to the extracellular matrix in various mammalian tissues. The function of DG has been well defined in embryonic development as well as in the proper migration of differentiated neuroblasts in the central nervous system (CNS). Although DG is known to be a target for matrix metalloproteinase-9 (MMP-9), cleaved in response to enhanced synaptic activity, the role of DG in the structural remodeling of dendritic spines is still unknown. Here, we report for the first time that the deletion of DG in rat hippocampal cell cultures causes pronounced changes in the density and morphology of dendritic spines. Furthermore, we noted a decrease in laminin, one of the major extracellular partners of DG. We have also observed that the lack of DG evokes alterations in the morphological complexity of astrocytes accompanied by a decrease in the level of aquaporin 4 (AQP4), a protein located within astrocyte endfeet surrounding neuronal dendrites and synapses. Regardless of all of these changes, we did not observe any effect of DG silencing on either excitatory or inhibitory synaptic transmission. Likewise, the knockdown of DG had no effect on Psd-95 protein expression. Our results indicate that DG is involved in dendritic spine remodeling that is not functionally reflected. This may suggest the existence of unknown mechanisms that maintain proper synaptic signaling despite impaired structure of dendritic spines. Presumably, astrocytes are involved in these processes.

Combination of dasatinib and quercetin improves cognitive abilities in aged male Wistar rats, alleviates inflammation and changes hippocampal synaptic plasticity and histone H3 methylation profile.

Aging is associated with cognitive decline and accumulation of senescent cells in various tissues and organs. Senolytic agents such as dasatinib and quercetin (D+Q) in combination have been shown to target senescent cells and ameliorate symptoms of aging-related disorders in mouse models. However, the mechanisms by which senolytics improve cognitive impairments have not been fully elucidated particularly in species other than mice. To study the effect of senolytics on aging-related multifactorial cognitive dysfunctions we tested the spatial memory of male Wistar rats in an active allothetic place avoidance task. Here we report that 8 weeks treatment with D+Q alleviated learning deficits and memory impairment observed in aged animals. Furthermore, treatment with D+Q resulted in a reduction of the peripheral inflammation measured by the levels of serum inflammatory mediators (including members of senescent cell secretome) in aged rats. Significant improvements in cognitive abilities observed in aged rats upon treatment with D+Q were associated with changes in the dendritic spine morphology of the apical dendritic tree from the hippocampal CA1 neurons and changes in the level of histone H3 trimethylation at lysine 9 and 27 in the hippocampus. The beneficial effects of D+Q on learning and memory in aged rats were long-lasting and persisted at least 5 weeks after the cessation of the drugs administration. Our results expand and provide new insights to the existing knowledge associated with effects of senolytics on alleviating age-related associated cognitive dysfunctions.

S-Palmitoylation of Synaptic Proteins as a Novel Mechanism Underlying Sex-Dependent Differences in Neuronal Plasticity.

Although sex differences in the brain are prevalent, the knowledge about mechanisms underlying sex-related effects on normal and pathological brain functioning is rather poor. It is known that female and male brains differ in size and connectivity. Moreover, those differences are related to neuronal morphology, synaptic plasticity, and molecular signaling pathways. Among different processes assuring proper synapse functions are posttranslational modifications, and among them, S-palmitoylation (S-PALM) emerges as a crucial mechanism regulating synaptic integrity. Protein S-PALM is governed by a family of palmitoyl acyltransferases, also known as DHHC proteins. Here we focused on the sex-related functional importance of DHHC7 acyltransferase because of its S-PALM action over different synaptic proteins as well as sex steroid receptors. Using the mass spectrometry-based PANIMoni method, we identified sex-dependent differences in the S-PALM of synaptic proteins potentially involved in the regulation of membrane excitability and synaptic transmission as well as in the signaling of proteins involved in the structural plasticity of dendritic spines. To determine a mechanistic source for obtained sex-dependent changes in protein S-PALM, we analyzed synaptoneurosomes isolated from DHHC7-/- (DHHC7KO) female and male mice. Our data showed sex-dependent action of DHHC7 acyltransferase. Furthermore, we revealed that different S-PALM proteins control the same biological processes in male and female synapses.

MMP-9 Signaling Pathways That Engage Rho GTPases in Brain Plasticity.

The extracellular matrix (ECM) has been identified as a critical factor affecting synaptic function. It forms a functional scaffold that provides both the structural support and the reservoir of signaling molecules necessary for communication between cellular constituents of the central nervous system (CNS). Among numerous ECM components and modifiers that play a role in the physiological and pathological synaptic plasticity, matrix metalloproteinase 9 (MMP-9) has recently emerged as a key molecule. MMP-9 may contribute to the dynamic remodeling of structural and functional plasticity by cleaving ECM components and cell adhesion molecules. Notably, MMP-9 signaling was shown to be indispensable for long-term memory formation that requires synaptic remodeling. The core regulators of the dynamic reorganization of the actin cytoskeleton and cell adhesion are the Rho family of GTPases. These proteins have been implicated in the control of a wide range of cellular processes occurring in brain physiology and pathology. Here, we discuss the contribution of Rho GTPases to MMP-9-dependent signaling pathways in the brain. We also describe how the regulation of Rho GTPases by post-translational modifications (PTMs) can influence these processes.

Stress-induced Changes in the S-palmitoylation and S-nitrosylation of Synaptic Proteins.

The precise regulation of synaptic integrity is critical for neuronal network connectivity and proper brain function. Essential aspects of the activity and localization of synaptic proteins are regulated by posttranslational modifications. S-palmitoylation is a reversible covalent modification of the cysteine with palmitate. It modulates affinity of the protein for cell membranes and membranous compartments. Intracellular palmitoylation dynamics are regulated by crosstalk with other posttranslational modifications, such as S-nitrosylation. S-nitrosylation is a covalent modification of cysteine thiol by nitric oxide and can modulate protein functions. Therefore, simultaneous identification of endogenous site-specific proteomes of both cysteine modifications under certain biological conditions offers new insights into the regulation of functional pathways. Still unclear, however, are the ways in which this crosstalk is affected in brain pathology, such as stress-related disorders. Using a newly developed mass spectrometry-based approach Palmitoylation And Nitrosylation Interplay Monitoring (PANIMoni), we analyzed the endogenous S-palmitoylation and S-nitrosylation of postsynaptic density proteins at the level of specific single cysteine in a mouse model of chronic stress. Among a total of 813 S-PALM and 620 S-NO cysteine sites that were characterized on 465 and 360 proteins, respectively, we sought to identify those that were differentially affected by stress. Our data show involvement of S-palmitoylation and S-nitrosylation crosstalk in the regulation of 122 proteins including receptors, scaffolding proteins, regulatory proteins and cytoskeletal components. Our results suggest that atypical crosstalk between the S-palmitoylation and S-nitrosylation interplay of proteins involved in synaptic transmission, protein localization and regulation of synaptic plasticity might be one of the main events associated with chronic stress disorder, leading to destabilization in synaptic networks.

Insights Into Protein S-Palmitoylation in Synaptic Plasticity and Neurological Disorders: Potential and Limitations of Methods for Detection and Analysis.

S-palmitoylation (S-PALM) is a lipid modification that involves the linkage of a fatty acid chain to cysteine residues of the substrate protein. This common posttranslational modification (PTM) is unique among other lipid modifications because of its reversibility. Hence, like phosphorylation or ubiquitination, it can act as a switch that modulates various important physiological pathways within the cell. Numerous studies revealed that S-PALM plays a crucial role in protein trafficking and function throughout the nervous system. Notably, the dynamic turnover of palmitate on proteins at the synapse may provide a key mechanism for rapidly changing synaptic strength. Indeed, palmitate cycling on postsynaptic density-95 (PSD-95), the major postsynaptic density protein at excitatory synapses, regulates the number of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and thus affects synaptic transmission. Accumulating evidence suggests a relationship between impairments in S-PALM and severe neurological disorders. Therefore, determining the precise levels of S-PALM may be essential for understanding the ways in which this PTM is regulated in the brain and controls synaptic dynamics. Protein S-PALM can be characterized using metabolic labeling methods and biochemical tools. Both approaches are discussed herein in the context of specific methods and their advantages and disadvantages. This review clearly shows progress in the field, which has led to the development of new, more sensitive techniques that enable the detection of palmitoylated proteins and allow predictions of potential palmitate binding sites. Unfortunately, one significant limitation of these approaches continues to be the inability to use them in living cells.

Dystroglycan controls dendritic morphogenesis of hippocampal neurons in vitro.

Dendritic outgrowth and arborization are important for establishing neural circuit formation. To date, little information exists about the involvement of the extracellular matrix (ECM) and its cellular receptors in these processes. In our studies, we focus on the role of dystroglycan (DG), a cell adhesion molecule that links ECM components to the actin cytoskeleton, in dendritic development and branching. Using a lentiviral vector to deliver short-hairpin RNA (shRNA) that specifically silences DG in cultured hippocampal neurons, we found that DG knockdown exerted an inhibitory effect on dendritic tree growth and arborization. The structural changes were associated with activation of the guanosine triphosphatase Cdc42. The overexpression of DG promoted dendritic length and branching. Furthermore, exposure of the cultures to autoactivating matrix metalloproteinase-9 (aaMMP-9), a ß-DG-cleaving protease, decreased the complexity of dendritic arbors. This effect was abolished in neurons that overexpressed a ß-DG mutant that was defective in MMP-9-mediated cleavage. Altogether, our results indicate that DG controls dendritic arborization in vitro in MMP-9-dependent manner.

CD44 regulates dendrite morphogenesis through Src tyrosine kinase-dependent positioning of the Golgi.

The acquisition of proper dendrite morphology is a crucial aspect of neuronal development towards the formation of a functional network. The role of the extracellular matrix and its cellular receptors in this process has remained enigmatic. We report that the CD44 adhesion molecule, the main hyaluronan receptor, is localized in dendrites and plays a crucial inhibitory role in dendritic tree arborization in vitro and in vivo. This novel function is exerted by the activation of Src tyrosine kinase, leading to the alteration of Golgi morphology. The mechanism operates during normal brain development, but its inhibition might have a protective influence on dendritic trees under toxic conditions, during which the silencing of CD44 expression prevents dendritic shortening induced by glutamate exposure. Overall, our results indicate a novel role for CD44 as an essential regulator of dendritic arbor complexity in both health and disease.

DP-b99 modulates matrix metalloproteinase activity and neuronal plasticity.

DP-b99 is a membrane-activated chelator of zinc and calcium ions, recently proposed as a therapeutic agent. Matrix metalloproteinases (MMPs) are zinc-dependent extracellularly operating proteases that might contribute to synaptic plasticity, learning and memory under physiological conditions. In excessive amounts these enzymes contribute to a number of neuronal pathologies ranging from the stroke to neurodegeneration and epileptogenesis. In the present study, we report that DP-b99 delays onset and severity of PTZ-induced seizures in mice, as well as displays neuroprotective effect on kainate excitotoxicity in hippocampal organotypic slices and furthermore blocks morphological reorganization of the dendritic spines evoked by a major neuronal MMP, MMP-9. Taken together, our findings suggest that DP-b99 may inhibit neuronal plasticity driven by MMPs, in particular MMP-9, and thus may be considered as a therapeutic agent under conditions of aberrant plasticity, such as those subserving epileptogenesis.

Neuroprotection from tissue inhibitor of metalloproteinase-1 and its nanoparticles.

Matrix metalloproteinases (MMPs) are family of zinc dependent endopeptidases, which cleave extracellular matrix proteins, and play an important role in tissue remodelling in physiological and pathological processes. There is enhanced expression of MMPs, in particular MMP-9, during numerous pathological conditions, including epilepsy and ischemic stroke. Therefore, inhibition of MMP-9 is considered as a potential therapeutic target. Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) is a 28kDa endogenous inhibitor of MMP-9. In this study we examined recombinant mouse TIMP-1 for its in-vitro neuroprotective effects, against Kainic Acid (KA) induced excitotoxicity in organotypic hippocampal slice culture (OHC) model. We also studied, sustained release effects of TIMP-1 in OHC by using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). TIMP-1 and TIMP-1 PLGA NPs were added to the slice cultures at different time points, i.e., 30min before treatment with KA and 6h after KA treatment. Propidium iodide staining was used to reveal cell toxicity in the cultures. In addition, neurotoxicity was assessed using standard lactate dehydrogenase (LDH) release assay. Gelatinolytic activity in conditioned cultured medium of OHC was accessed by a fluorescent substrate assay. Briefly, our result show that TIMP-1 provided significant level of neuroprotection, especially when given before 30min of KA and released from the NPs. Since gelatinolytic activity assay showed a decrease in MMP-9 activity, it can be suggested that this neuroprotection might be mediated by the gelatinase inhibition.

Silencing of ICERs (Inducible cAMP Early Repressors) results in partial protection of neurons from programmed cell death.

ICERs proteins (Inducible cAMP Early Repressors) are the most effective endogenous repressors of CREB/CREM/ATF transcription factors family (CREB-cAMP Responsive Element Binding protein, CREM-cAMP Responsive Element Modulator, ATF-Activating Transcription Factor) that have repeatedly been shown to have a prosurvival function. It has been reported previously that neuronal death is accompanied by increased expression of ICERs and, furthermore, their overexpression provokes neuronal cell death in culture. However, it was not explained whether endogenously activated by proapoptotic stimuli ICERs contribute to the neuronal cell death. Herein, we have examined the involvement of endogenous ICERs in the apoptosis by checking whether it is possible to protect neurons from cell death by blocking the ICER gene. We applied two different in vitro models of neuronal death of primary neuronal cultures: excitotoxic death of neurons derived from dentate gyrus, and cortical cell loss provoked by trophic deprivation. Using the lentiviral vector (LV) to deliver shRNA, specifically silencing ICERs, but not other CREM proteins, we have found that silencing of ICERs enhances the CRE-driven transcription and exerts a mild, although significant, neuroprotective effect in both models. Since we demonstrated that silencing of endogenous ICERs have protective effect on neurons exposed to apoptosis-provoking conditions, targeting ICERs might be a novel strategy to prevent neuronal loss during degenerative processes.

Transient brain ischemia due to cardiac arrest causes irreversible long-lasting cognitive injury.

Herein, we used a clinically-relevant model of 10 min cardiac arrest (CA) in Wistar rats. Histological analyses of the ischemic brains of old rats showed significant atrophy of CA(1) sector of hippocampus (Nissl and NeuN stainings) corresponding with increase of glial fibrillary acidic protein expression. The long-term behavioral consequences of above manipulation producing global brain ischemia were assessed in young, middle-aged and old rats, i.e., 3-, 6- and 18-months post-treatment, respectively. In young animals no differences were found in the context-dependent memory in Fear Conditioning test. The most striking behavioral abnormalities were found in middle-aged rats (6 months post-ischemia). Ischemic rats showed hyperactivity and decreased level of anxiety in Open Field and problems with spatial learning and memory in a Novel Object Location test, T-maze and Morris Water Maze. In old animals, a decline of motor and cognitive functions was found not only in ischemic but also in sham/control ones. This study describes consequences of global brain ischemia in aging animals.

New hippocampal neurons are not obligatory for memory formation; cyclin D2 knockout mice with no adult brain neurogenesis show learning.

The role of adult brain neurogenesis (generating new neurons) in learning and memory appears to be quite firmly established in spite of some criticism and lack of understanding of what the new neurons serve the brain for. Also, the few experiments showing that blocking adult neurogenesis causes learning deficits used irradiation and various drugs known for their side effects and the results obtained vary greatly. We used a novel approach, cyclin D2 knockout mice (D2 KO mice), specifically lacking adult brain neurogenesis to verify its importance in learning and memory. D2 KO mice and their wild-type siblings were tested in several behavioral paradigms, including those in which the role of adult neurogenesis has been postulated. D2 KO mice showed no impairment in sensorimotor tests, with only sensory impairment in an olfaction-dependent task. However, D2 KO mice showed proper procedural learning as well as learning in context (including remote memory), cue, and trace fear conditioning, Morris water maze, novel object recognition test, and in a multifunctional behavioral system-IntelliCages. D2 KO mice also demonstrated correct reversal learning. Our results suggest that adult brain neurogenesis is not obligatory in learning, including the kinds of learning where the role of adult neurogenesis has previously been strongly suggested.

Pro-inflammatory cytokine TNF-alpha as a neuroprotective agent in the brain.

Despite the numerous reports on the role of tumor necrosis factor-alpha (TNF-alpha) in the brain neuropathology, very little is known about the mechanisms by which TNF-alpha may mediate neuroprotection. Different hypotheses pertain to the molecular and cellular effectors triggered by the activation of TNF receptors (TNFRI and TNFR2). They are focused on diminishing the production of nitric oxide and free radicals, alteration of excitatory amino acids neurotransmission, maintenance of neuronal calcium homeostasis and induction of neurotrophic factors synthesis. In this review all these data are summarized. Moreover, possible explanations for the inconsistent data concerning the TNF-alpha effect on neuron are discussed.

Trimethyltin-evoked apoptosis of murine hippocampal granule neurons is accompanied by the expression of interleukin-1beta and interleukin-1 receptor antagonist in cells of ameboid phenotype, the majority of which are NG2-positive.

Interleukin-1beta (IL-1beta) has been implicated in various neuropathologies, while IL-1 receptor antagonist (IL-1ra) has been shown to reduce neuronal injury. We investigated the pattern of expression of both cytokines in murine hippocampus after trimethyltin (TMT) intoxication. Using a ribonuclease protection assay, we demonstrated induction of transcription of IL-1beta and IL-1ra 3 days following TMT treatment which correlated with the peak of neuronal apoptosis. At this time, immunocytochemical staining revealed enhanced expression of both cytokines in NG2 proteoglycan expressing ameboid cells located at the site of neurotoxic insult, some of which bound also the microglial marker, lectin. There was some overlap between NG2 and lectin staining. Our results suggest that the two cytokines are involved in apoptotic processes in dentate granule cells and indicate that the pro-apoptotic effect of IL-1beta prevails over the presumed protective action of IL-1ra. The novel finding of expression of both cytokines in NG2(+) cells of ameboid phenotype indicates that these cells, through the regulatory roles of pro- and anti-inflammatory cytokines, may be involved in control of neuronal death or survival after injury.

Age-dependent changes in neuronal distribution of CacyBP/SIP: comparison to tubulin and the tau protein.

CacyBP/SIP was originally identified as an S100A6 (calcyclin) target and later on as a Siah-1 interacting protein. Recently, we have shown that CacyBP/SIP interacts with tubulin, which suggests its involvement in the reorganization of microtubules. In this work we examined the localization of CacyBP/SIP in cultured neurons and in brain neurons of young and aged rats, and compared this localization with that of tubulin and the tau protein. We have found that in neurons of young rats CacyBP/SIP, tubulin and tau are present in the cytoplasm and in the neuronal processes, whereas in aged animals CacyBP/SIP and tau are mainly seen in the cytoplasm of the neuronal somata. In aged rats, these changes are also accompanied by a different localization pattern of tubulin. Thus, our results show that localization of CacyBP/SIP in brain neurons is similar to that observed for tau and tubulin, which points to the involvement of CacyBP/SIP in cytoskeletal physiology.

The ameboid phenotype of NG2 (+) cells in the region of apoptotic dentate granule neurons in trimethyltin intoxicated mice shares antigen properties with microglia/macrophages.

NG2+, stellate cells present in the adult central nervous system (CNS) have been recently recognized as a distinct glial class, identified as multipotent progenitor cells. Antigenically, they are indistinguishable from oligodendroglia progenitor cells. In response to a variety of CNS insults, these cells become rapidly activated and undergo morphological changes accompanied by increased cellular proliferation. The role they play with respect to injured neurons is not clear. In our studies, we performed immunocytochemical investigations and identified a response of NG2-expressing cells in the model of selective neurodegeneration of murine dentate gyrus granule cells induced by systemic administration of trimethyltin. Dying neurons exhibited features of apoptotic cells. Around the region of neurodegeneration, we observed activation of NG2+ stellate cells and microglia. During the peak of apoptosis, we detected the appearance of NG2+ cells of the ameboid phenotype, intermingled with granule neurons. These cells also expressed markers of microglia/macrophages, OX42- and ED1-recognized antigens, an antigen recognized by O4 antibody-a marker of more differentiated cells of the oligodendroglia lineage and, in some cases, also a protein of mature oligodendroglia adenomatus polyposis coli. They also expressed nestin. Our results suggest that the injury induces a parallel transformation of both the activated glial classes: NG2+ stellate cells and resident microglia, into ameboid cells, sharing properties of both oligodendrocyte and monocyte lineages. These cells may play a role in the phagocytosis. If this assumption is verified by electron microscopy, it would indicate a novel function of NG2 transformed cells under CNS injury conditions.

TNFalpha and TNF receptor 1 expression in the mixed neuronal-glial cultures of hippocampal dentate gyrus exposed to glutamate or trimethyltin.

We examined the expression and cellular localization of tumor necrosis factor alpha (TNFalpha) and its type 1 receptor (TNFR1) in mixed neuronal-glial cultures of rat hippocampal dentate gyrus exposed to glutamate (GLU) or trimethyltin (TMT). Our previous studies demonstrated that both pathogenic factors evoked neuronal apoptosis, however, TMT was more potent and caused cell death in almost 90% of neurons. Observed neurodegeneration was accompanied by morphological changes of microglia. In the current study, using RT-PCR and Western blotting analysis, we found that GLU and TMT induced increase in TNFalpha mRNA and protein levels. The induction of transcription was stronger following GLU treatment, however the protein production was much more intensive after TMT exposure. Double fluorescent labeling for TNFalpha, TNFR1 and cellular markers revealed cytokine expression in microglia and some neurons. On the other hand, majority of neuronal cells displayed TNFR1 immunoreactivity, in control and in treated cultures. Moreover, TMT led to a strong increase in TNFR1 expression in astrocytes, which displayed remarkable, granular staining for the cytokine receptor. Western blotting for TNFR1 revealed enhanced protein expression only in cultures treated with TMT. This is the first report demonstrating the changes of expression of TNFalpha and TNFR1 in hippocampal dentate gyrus cultures treated with GLU or TMT. Our results indicate that TNFalpha may be involved in the mechanism of neurotoxic effects evoked by both pathogenic factors and suggest that astrocytes via TNFR1 may enhance TMT-induced injury.