Murine Cortical Neurons Research Articles

Memantine Improves Safety of Thrombolysis for Stroke

Despite side effects including N-methyl-d-aspartate-mediated neurotoxicity, recombinant tissue-type plasminogen activator (rtPA) remains the only approved acute treatment for ischemic stroke. Memantine, used for treatment of Alzheimer disease, is an antagonist for N-methyl-d-aspartate receptors. We investigated whether memantine could be used as a neuroprotective adjunct therapy for rtPA-induced thrombolysis after stroke. In vitro N-methyl-d-aspartate exposure, oxygen and glucose deprivation, and N-methyl-d-aspartate-mediated calcium videomicroscopy experiments were performed on murine cortical neurons in the presence of rtPA and memantine. The therapeutic safety of rtPA and memantine coadministration was evaluated in mouse models of thrombotic stroke and intracerebral hemorrhage. Ischemic and hemorrhagic volumes were assessed by MRI and neurological evaluation was performed by the string test and automated gait analysis. Our in vitro observations showed that memantine was able to prevent the proneurotoxic effects of rtPA in cultured cortical neurons. Although memantine did not alter the fibrinolytic activity of rtPA, our in vivo observations revealed that it blunted the noxious effects of delayed thrombolysis on lesion volumes and neurological deficits after ischemic stroke. In addition, memantine rescued rtPA-induced decrease in survival rate after intracerebral hemorrhage. Memantine could be used as an adjunct therapy to improve the safety of thrombolysis.

Adenovirus vector‐based in vitro neuronal cell model for Huntington's disease with human disease‐like differential aggregation and degeneration

Neuronal degeneration, in particular in the striatum, and the formation of nuclear and cytoplasmic inclusions are characteristics of Huntington's disease (HD) as a result of the expansion of a polyglutamine tract located close to the N-terminus of huntingtin (htt). Because of the large (10-kb) size of the htt cDNA, expression of full-length htt in primary neurons has proved difficult in the past. We generated a new chronic in vitro model that is based on high-capacity adenovirus vector-mediated transduction of primary murine striatal and cortical neurons. Because the vector has a large capacity for transport of foreign DNA, it was possible to quantitatively express in these primary cells normal and mutant full-length htt (designed as fusion proteins with enhanced green fluorescent protein) in addition to its truncated versions. Pathological changes caused by mutant htt were characterized. The model mimicked several features observed in HD patients: prominent nuclear inclusions in cortical but not in striatal neurons, preferential neuronal degeneration of striatal neurons and neurofilament fragmentation in this cell type. Compared with expressed truncated mutant htt, the expression of full-length mutant htt in neurons resulted in a much slower appearance of pathological changes. Different from cortical neurons, the vast majority of nuclei in striatal cells contained only diffusely distributed N-terminal htt fragments. Cytoplasmic inclusions in both cell types contained full-length mutant htt. This model and the adenovirus vectors used will be valuable for studying the function of htt and the pathogenesis of HD at molecular and cellular levels in different neuronal cell types.

Antagonism of Neuronal Prostaglandin E2 Receptor Subtype 1 Mitigates Amyloid β Neurotoxicity In Vitro

Multiple lines of evidence indicate that regional brain eicosanoid signaling is important in initiation and progression of neurodegenerative conditions that have neuroinflammatory pathologic component, such as AD. We hypothesized that PGE(2) receptor subtype 1 (EP1) signaling (linked to intracellular Ca(2+) release) regulates Aβ peptide neurotoxicity and tested this in two complementary in vitro models: a human neuroblastoma cell line (MC65) producing Aβ(1-40) through conditional expression of the APP C-terminal portion, and murine primary cortical neuron cultures exposed to Aβ(1-42). In MC65 cells, EP1 receptor antagonist SC-51089 reduced Aβ neurotoxicity ~50 % without altering high molecular weight Aβ immunoreactive species formation. Inositol-3-phosphate receptor antagonist 2-aminoethoxy-diphenyl borate offered similar protection. SC-51089 largely protected the neuron cultures from synthetic Aβ(1-42) neurotoxicity. Nimodipine, a Ca(2+) channel blocker, was completely neuroprotective in both models. Based on these data, we conclude that suppressing neuronal EP1 signaling may represent a promising therapeutic approach to ameliorate Aβ peptide neurotoxicity.

ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA.

BackgroundNeurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.Methodology/Principal FindingsHere we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.ConclusionsproBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.

Distinct Dendritic Spine and Nuclear Phases of Calcineurin Activation after Exposure to Amyloid-β Revealed by a Novel Fluorescence Resonance Energy Transfer Assay

Calcineurin (CaN) activation is critically involved in the regulation of spine morphology in response to oligomeric amyloid-β (Aβ) as well as in synaptic plasticity in normal memory, but no existing techniques can monitor the spatiotemporal pattern of CaN activity. Here, we use a spectral fluorescence resonance energy transfer approach to monitor CaN activation dynamics in real time with subcellular resolution. When oligomeric Aβ derived from Tg2576 murine transgenic neurons or human AD brains were applied to wild-type murine primary cortical neurons, we observe a dynamic progression of CaN activation within minutes, first in dendritic spines, and then in the cytoplasm and, in hours, in the nucleus. CaN activation in spines leads to rapid but reversible morphological changes in spines and in postsynaptic proteins; longer exposure leads to NFAT (nuclear factor of activated T-cells) translocation to the nucleus and frank spine loss. These results provide a framework for understanding the role of calcineurin in synaptic alterations associated with AD pathogenesis.

Poster #136 DISRUPTION OF CDC42 AND DUO/PAK1 PATHWAY DRIVES MYOSIN LIGHT CHAIN PHOSPHORYLATION IN SCHIZOPHRENIA

Bone Morphogenetic Proteins (BMPs) are crucial for many aspects of the development and differentiation of the nervous system and are important in controlling cytoskeletal remodeling during neuronal morphogenesis. BMPs are TGFβ superfamily members that signal through a heteromeric complex of type I and type II BMP receptors. The BMPRII receptor is particularly important in mediating remodeling of the neuronal cytoskeleton through the activation of BMPRII-bound cytoskeletal regulators, such as LIM Kinase (LIMK). Here, we show that PAK1, a key regulator of diverse neuronal processes and an upstream activator of LIMK, binds to the BMP type I receptor, ALK2. Although, PAK1 is dispensable for activation of the Smad transcriptional mediators, abrogation of PAK1 expression or inhibition of PAK1 activity prevents BMP-induced neurite outgrowth in cultured neuroblastoma cell lines. Moreover, in primary murine embryonic cortical neurons, inhibition of PAK activity blocks BMP7-induced cofilin phosphorylation, prevents remodeling of the actin cytoskeleton and thereby blocks BMP7-induced dendrite formation. Thus, we propose a model in which BMP7 signaling leads to the recruitment of ALK2-bound PAK1 to BMPRII, which binds a downstream regulator of the actin cytoskeleton, LIMK1, and that the BMP receptor complex thereby acts as a scaffold to localize and coordinate actin cytoskeletal remodeling. We propose that this scaffold plays a key role in mediating BMP7-dependent dendritogenesis in primary cortical neurons.

Lipocalin 2: Novel component of proinflammatory signaling in Alzheimer's disease

Alzheimer's disease (AD) is associated with an altered immune response, resulting in chronic increased inflammatory cytokine production with a prominent role of TNF-α. TNF-α signals are mediated by two receptors: TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Signaling through TNFR2 is associated with neuroprotection, whereas signaling through TNFR1 is generally proinflammatory and proapoptotic. Here, we have identified a TNF-α-induced proinflammatory agent, lipocalin 2 (Lcn2) via gene array in murine primary cortical neurons. Further investigation showed that Lcn2 protein production and secretion were activated solely upon TNFR1 stimulation when primary murine neurons, astrocytes, and microglia were treated with TNFR1 and TNFR2 agonistic antibodies. Lcn2 was found to be significantly decreased in CSF of human patients with mild cognitive impairment and AD and increased in brain regions associated with AD pathology in human postmortem brain tissue. Mechanistic studies in cultures of primary cortical neurons showed that Lcn2 sensitizes nerve cells to β-amyloid toxicity. Moreover, Lcn2 silences a TNFR2-mediated protective neuronal signaling cascade in neurons, pivotal for TNF-α-mediated neuroprotection. The present study introduces Lcn2 as a molecular actor in neuroinflammation in early clinical stages of AD.

Autophagy regulates endoplasmic reticulum stress in ischemic preconditioning

Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5–20 mM) and bafilomycin A1 (75–150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50–200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.

Calpains Are Downstream Effectors ofbax-Dependent Excitotoxic Apoptosis

Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

Chronic Excitotoxin-Induced Axon Degeneration in a Compartmented Neuronal Culture Model

Glutamate excitotoxicity is a major pathogenic process implicated in many neurodegenerative conditions, including AD (Alzheimer's disease) and following traumatic brain injury. Occurring predominantly from over-stimulation of ionotropic glutamate receptors located along dendrites, excitotoxic axonal degeneration may also occur in white matter tracts. Recent identification of axonal glutamate receptor subunits within axonal nanocomplexes raises the possibility of direct excitotoxic effects on axons. Individual neuronal responses to excitotoxicity are highly dependent on the complement of glutamate receptors expressed by the cell, and the localization of the functional receptors. To enable isolation of distal axons and targeted excitotoxicity, murine cortical neuron cultures were prepared in compartmented microfluidic devices, such that distal axons were isolated from neuronal cell bodies. Within the compartmented culture system, cortical neurons developed to relative maturity at 11 DIV (days in vitro) as demonstrated by the formation of dendritic spines and clustering of the presynaptic protein synaptophysin. The isolated distal axons retained growth cone structures in the absence of synaptic targets, and expressed glutamate receptor subunits. Glutamate treatment (100 μM) to the cell body chamber resulted in widespread degeneration within this chamber and degeneration of distal axons in the other chamber. Glutamate application to the distal axon chamber triggered a lesser degree of axonal degeneration without degenerative changes in the untreated somal chamber. These data indicate that in addition to current mechanisms of indirect axonal excitotoxicity, the distal axon may be a primary target for excitotoxicity in neurodegenerative conditions.

Multifaceted role of nitric oxide in an in vitro mouse neuronal injury model: transcriptomic profiling defines the temporal recruitment of death signalling cascades

Nitric oxide is implicated in the pathogenesis of various neuropathologies characterized by oxidative stress. Although nitric oxide has been reported to be involved in the exacerbation of oxidative stress observed in several neuropathologies, existent data fail to provide a holistic description of how nitrergic pathobiology elicits neuronal injury. Here we provide a comprehensive description of mechanisms contributing to nitric oxide induced neuronal injury by global transcriptomic profiling. Microarray analyses were undertaken on RNA from murine primary cortical neurons treated with the nitric oxide generator DETA-NONOate (NOC-18, 0.5 mM) for 8–24 hrs. Biological pathway analysis focused upon 3672 gene probes which demonstrated at least a ±1.5-fold expression in a minimum of one out of three time-points and passed statistical analysis (one-way anova, P < 0.05). Numerous enriched processes potentially determining nitric oxide mediated neuronal injury were identified from the transcriptomic profile: cell death, developmental growth and survival, cell cycle, calcium ion homeostasis, endoplasmic reticulum stress, oxidative stress, mitochondrial homeostasis, ubiquitin-mediated proteolysis, and GSH and nitric oxide metabolism. Our detailed time-course study of nitric oxide induced neuronal injury allowed us to provide the first time a holistic description of the temporal sequence of cellular events contributing to nitrergic injury. These data form a foundation for the development of screening platforms and define targets for intervention in nitric oxide neuropathologies where nitric oxide mediated injury is causative.

Complex Regulation of p73 Isoforms after Alteration of Amyloid Precursor Polypeptide (APP) Function and DNA Damage in Neurons

Genetic ablations of p73 have shown its implication in the development of the nervous system. However, the relative contribution of ΔNp73 and TAp73 isoforms in neuronal functions is still unclear. In this study, we have analyzed the expression of these isoforms during neuronal death induced by alteration of the amyloid-β precursor protein function or cisplatin. We observed a concomitant up-regulation of a TAp73 isoform and a down-regulation of a ΔNp73 isoform. The shift in favor of the pro-apoptotic isoform correlated with an induction of the p53/p73 target genes such as Noxa. At a functional level, we showed that TAp73 induced neuronal death and that ΔNp73 has a neuroprotective role toward amyloid-β precursor protein alteration or cisplatin. We investigated the mechanisms of p73 expression and found that the TAp73 expression was regulated at the promoter level. In contrast, regulation of ΔNp73 protein levels was regulated by phosphorylation at residue 86 and multiple proteases. Thus, this study indicates that tight transcriptional and post-translational mechanisms regulate the p73 isoform ratios that play an important role in neuronal survival.

Dysregulated LRRK2 Signaling in Response to Endoplasmic Reticulum Stress Leads to Dopaminergic Neuron Degeneration in C. elegans

Mutation of leucine-rich repeat kinase 2 (LRRK2) is the leading genetic cause of Parkinson's Disease (PD), manifested as age-dependent dopaminergic neurodegeneration, but the underlying molecular mechanisms remain unclear. Multiple roles of LRRK2 may contribute to dopaminergic neurodegeneration. Endoplasmic reticulum (ER) stress has also been linked to PD pathogenesis, but its interactive mechanism with PD genetic factors is largely unknown. Here, we used C. elegans, human neuroblastoma cells and murine cortical neurons to determine the role of LRRK2 in maintaining dopaminergic neuron viability. We found that LRRK2 acts to protect neuroblastoma cells and C. elegans dopaminergic neurons from the toxicity of 6-hydroxydopamine and/or human α-synuclein, possibly through the p38 pathway, by supporting upregulation of GRP78, a key cell survival molecule during ER stress. A pathogenic LRRK2 mutant (G2019S), however, caused chronic p38 activation that led to death of murine neurons and age-related dopaminergic-specific neurodegeneration in nematodes. These observations establish a critical functional link between LRRK2 and ER stress.

RIG-I Mediates Innate Immune Response in Mouse Neurons Following Japanese Encephalitis Virus Infection

BackgroundNeuroinflammation associated with Japanese encephalitis (JE) is mainly due to the activation of glial cells with subsequent release of proinflammatory mediators from them. The recognition of viral RNA, in part, by the pattern recognition receptor retinoic acid-inducible gene I (RIG-I) has been indicated to have a role in such processes. Even though neurons are also known to express this receptor, its role after JE virus (JEV) infections is yet to be elucidated.Methodology/Principal FindingsUpon infecting murine neuroblastoma cells and primary cortical neurons with JEV the expression profile of key proinflammatory cyto/chemokines were analyzed by qRT-PCR and bead array, both before and after ablation of RIG-I. Immunoblotting was performed to evaluate the levels of key molecules downstream to RIG-I leading to production of proinflammatory mediators. Changes in the intracellular viral antigen expression were confirmed by intracellular staining and immunoblotting. JEV infection induced neuronal expression of IL-6, IL-12p70, MCP-1, IP-10 and TNF-α in a time-dependent manner, which showed significant reduction upon RIG-I ablation. Molecules downstream to RIG-I showed significant changes upon JEV-infection, that were modulated following RIG-I ablation. Ablation of RIG-I in neurons also increased their susceptibility to JEV.Conclusions/SignificanceIn this study we propose that neurons are one of the potential sources of proinflammatory cyto/chemokines in JEV-infected brain that are produced via RIG-I dependent pathways. Ablation of RIG-I in neurons leads to increased viral load and reduced release of the cyto/chemokines.

Kainate Promotes Alterations in Neuronal RNA Splicing Machinery

Kainate, a glutamate analogue, activates kainate and AMPA receptors inducing strong synaptic activation. Systemic kainate application to rodents results in seizures, neurodegeneration, and neuronal remodeling in the brain. It is therefore used to investigate molecular mechanisms responsible for these conditions. We analyzed proteome alterations in murine primary cortical neurons after 24 h of kainate treatment. Our 2-D gel based proteomics approach revealed 91 protein alterations, some already associated with kainate-induced pathology. In addition, we found a large number of proteins which have not previously been reported to be associated with kainate-induced pathology. Functional classification of altered proteins revealed that they predominantly participate in mRNA splicing and cytoskeleton remodeling.

Assessment of general anaesthetic cytotoxicity in murine cortical neurones in dissociated culture

General anaesthetics are proposed to cause unconsciousness by modulating neuronal excitability in the mammalian brain through mechanisms that include enhancement of inhibitory GABA A receptor currents and suppression of excitatory glutamate receptor responses. Both intravenous and volatile agents may produce neurotoxic effects during early postnatal rodent brain development through similar mechanisms. In the following study, we investigated anaesthetic cytotoxicity in primary cortical neurones and glia from postnatal day 2–8 mice. Cultures at 4–20 days in vitro were exposed to combinations of ketamine (100 μM to 3 mM), nitrous oxide (75%, v/v) and/or isoflurane (1.5–5%, v/v) for 6–12 h. Neuronal survival and cell death were measured via microtubule associated protein 2 immunoassay and lactate dehydrogenase release assays, respectively. Clinically relevant anaesthetic concentrations of ketamine, nitrous oxide and isoflurane had no significant neurotoxic effects individually or when given as anaesthetic cocktails, even with up to 12 h exposure. This lack of neurotoxicity was observed regardless of whether cultures were prepared from postnatal day 0–2 or day 8 mice, and was also unaffected by number of days in vitro (DIV 4–20). Significant neurotoxic effects were only observed at supraclinical concentrations ( e.g. 1–3 mM ketamine). Our study suggests that neurotoxicity previously reported in vivo is not due to direct cytotoxicity of anaesthetic agents, but results from other impacts of the anaesthetised state during early brain development.

Annexin A3 is associated with cell death in lactacystin-mediated neuronal injury

Massive neuronal apoptosis and accumulation of protein aggregates in the cortex and hippocampus of the brain are hallmarks of several neurodegenerative disorders, indicating ubiquitin proteasome system (UPS) dysfunction. Lactacystin, a classical proteasome inhibitor, is used to simulate ubiquitin proteasome system dysfunction in neurons to mimic pathological features of neurodegenerative disorders. Based on Western blot analyses, we reported for the first time that annexin A3 (AnxA3) is not only endogenously expressed in mouse cortical neurons but also more importantly, by gene expression microarray and real-time RT-PCR that it is greatly transcriptional up-regulated to approximately 11- and 15-fold, respectively in murine primary cortical neurons with 1 μM lactacystin for 24 h. Up-regulation of AnxA3 expression occurred after 12–15 h post-lactacystin treatment, which corresponded with the onset of neuronal injury, with approximately 25% of the neurons being non-viable by that time interval. Western blot analysis with anti-AnxA3 antibodies further validated that up-regulation of AnxA3 only occurs with onset of neuronal death, and not with the onset of proteasome inhibition, which occurs at 4.5 h post-lactacystin treatment. Over-expression studies suggested AnxA3 might be involved in death promotion during lactacystin-mediated neuronal death, since caspase-3 activation was significantly stronger upon neuronal AnxA3 over-expression. We propose AnxA3 up-regulation may have significant relevance in the elucidation of neurodegenerative pathophysiology.

The thiol-modifying agent N-ethylmaleimide elevates the cytosolic concentration of free Zn2+ but not of Ca2+ in murine cortical neurons

The membrane permeant alkylating agent N-ethylmaleimide (NEM) regulates numerous biological processes by reacting with thiol groups. Among other actions, NEM influences the cytosolic concentration of free Ca2+ ([Ca2+]i). Depending on the cell type and the concentration used, NEM can promote the release of Ca2+, affect its extrusion, stimulate or block its entry. However, most of these findings were obtained in experiments that employed fluorescent Ca2+ probes and one major disadvantage of such experimental setting derives from the lack of specificity of the probes as all the so-called “Ca2+-sensitive” indicators also bind metals like Zn2+ or Mn2+ with higher affinities than Ca2+. In this study, we examined the effects of NEM on the [Ca2+]i homeostasis of murine cortical neurons. We performed live-cell Ca2+ and Zn2+ imaging experiments using the fluorescent probes Fluo-4, FluoZin-3 and RhodZin-3 and found that NEM does not affect the neuronal [Ca2+]i homeostasis but specifically increases the cytosolic and mitochondrial concentration of free Zn2+([Zn2+]i). In addition, NEM triggers some neuronal loss that is prevented by anti-oxidants such as N-acetylcysteine or glutathione. NEM-induced toxicity is dependent on changes in [Zn2+]i levels as chelation of the cation with the cell-permeable heavy metal chelator, N,N,N′N′-tetrakis(−)[2-pyridylmethyl]-ethylenediamine (TPEN), promotes neuroprotection of cortical neurons exposed to NEM. Our data indicate that NEM affects [Zn2+]i but not [Ca2+]i homeostasis and shed new light on the physiological actions of this alkylating agent on central nervous system neurons.

Induction of Autophagy by Cystatin C: A Mechanism That Protects Murine Primary Cortical Neurons and Neuronal Cell Lines

Cystatin C (CysC) expression in the brain is elevated in human patients with epilepsy, in animal models of neurodegenerative conditions, and in response to injury, but whether up-regulated CysC expression is a manifestation of neurodegeneration or a cellular repair response is not understood. This study demonstrates that human CysC is neuroprotective in cultures exposed to cytotoxic challenges, including nutritional-deprivation, colchicine, staurosporine, and oxidative stress. While CysC is a cysteine protease inhibitor, cathepsin B inhibition was not required for the neuroprotective action of CysC. Cells responded to CysC by inducing fully functional autophagy via the mTOR pathway, leading to enhanced proteolytic clearance of autophagy substrates by lysosomes. Neuroprotective effects of CysC were prevented by inhibiting autophagy with beclin 1 siRNA or 3-methyladenine. Our findings show that CysC plays a protective role under conditions of neuronal challenge by inducing autophagy via mTOR inhibition and are consistent with CysC being neuroprotective in neurodegenerative diseases. Thus, modulation of CysC expression has therapeutic implications for stroke, Alzheimer's disease, and other neurodegenerative disorders.

Amyloid seeds formed by cellular uptake, concentration, and aggregation of the amyloid-beta peptide

One of the neuropathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque, primarily composed of aggregated amyloid-beta (Abeta) peptide. In vitro, Abeta(1-42), the major alloform of Abeta found in plaques, self-assembles into fibrils at micromolar concentrations and acidic pH. Such conditions do not exist in the extracellular fluid of the brain where the pH is neutral and Abeta concentrations are in the nanomolar range. Here, we show that extracellular soluble Abeta (sAbeta) at concentrations as low as 1 nM was taken up by murine cortical neurons and neuroblastoma (SHSY5Y) cells but not by human embryonic kidney (HEK293) cells. Following uptake, Abeta accumulated in Lysotracker-positive acidic vesicles (likely late endosomes or lysosomes) where effective concentrations (>2.5 microM) were greater than two orders of magnitude higher than that in the extracellular fluid (25 nM), as quantified by fluorescence intensity using laser scanning confocal microscopy. Furthermore, SHSY5Y cells incubated with 1 muM Abeta(1-42) for several days demonstrated a time-dependent increase in intracellular high molecular weight (HMW) (>200 kDa) aggregates, which were absent in cells grown in the presence of Abeta(1-40). Homogenates from these Abeta(1-42)-loaded cells were capable of seeding amyloid fibril growth. These results demonstrate that Abeta can be taken up by certain cells at low physiologically relevant concentrations of extracellular Abeta, and then concentrated into endosomes/lysosomes. At high concentrations, vesicular Abeta aggregates to form HMW species which are capable of seeding amyloid fibril growth. We speculate that extrusion of these aggregates may seed extracellular amyloid plaque formation during AD pathogenesis.