P42 SIGNALING PATHWAYS INVOLVED IN SBMA PATHOGENESIS
SARMA T, BRADY S
University of Illinois at Chicago, Department of Anatomy and Cell Biology, Chicago, IL, United States
E-mail address for correspondence: [email protected]
Keywords: SBMA, flutamide, fast axonal transport
Background: Spinal and bulbar muscular atrophy (SBMA) is an inherited neurodegenerative disorder caused by expansion of a CAG repeat (glutamine) in the gene encoding the androgen receptor (AR). The role of polyglutamine expansion in human AR function and SBMA pathogenesis remains unclear. AR is a transcription factor that translocates to the nucleus in the presence of the AR ligand testosterone. The polyglutamine-expanded AR (polyQ-AR) is proposed to alter transcription after ligand binding and contribute to pathogenesis. However, polyQ-AR also alters cytoplasmic signaling pathways and inhibits fast axonal transport (FAT), leading to reduction or mistargeting of critical components delivered by FAT.
Objectives: In this study, we analyzed the effects of androgen agonists and antagonists in a SBMA cell model.
Methods: We evaluated the relative contribution of androgen binding and polyQ-AR translocation to the nucleus to effects on cell morphology, activation of c-Jun N-terminal kinases and FAT.
Results: Using androgen antagonists we compared the role of ligand-dependent nuclear versus cytoplasmic signaling. Interestingly, we found that the androgen antagonist, flutamide, restores FAT in squid axoplasm and modifies the cellular phenotype of SBMA pathogenesis, while inhibiting translocation of AR to the nucleus. In contrast, a second androgen antagonist, cyproterone acetate alters nuclear translocation, but does not prevent polyQ-AR effects on FAT or cell morphology. Moreover, androgen antagonist flutamide, changed the subcellular localization of phospho-JNK in cells expressing polyQ-AR. This indicates that androgen signaling is not critical for JNK activity.
Discussion and conclusions: These studies suggest that polyQ-AR-induced changes contribute to neuropathology through a cytoplasmic signaling pathway and that nuclear signaling is not required for polyQ-AR-induced pathology.
P43 IDENTIFICATION OF FUS INTERACTING PROTEINS IN NEURONAL CELLS
FUMOTO K1, GROEN E2, VAN DEN BERG LH2, PASTERKAMP J1
1Department of Neuroscience and Pharmacology, 2Department of Neurology; Rudolf Magnus Institute of Neuroscience, Utrecht, Netherlands
E-mail address for correspondence: [email protected]
Keywords: FUS, NSC34 cells, protein-protein interaction
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder, selectively affecting motor neurons and pathologically characterized by the presence of intracellular ubiquitin (Ub)-positive aggregates. TDP-43 has been identified as one of the major constituents of Ub aggregates in ALS and its dysfunction is thought to contribute to the neurotoxicity underlying the ALS neurodegenerative process. More recently, missense mutations in FUS (fused in sarcoma, also known as TLS) were identified and associated with the pathogenesis of familial ALS. FUS is an RNA binding protein and regulates RNA processing, DNA repair, nucleocytoplasmic shuttling, and axonal trafficking in neurons. Like TDP-43, mutations in FUS cause protein mislocalization and induce cytoplasmic aggregation. We recently analyzed mutations in the coding region of the FUS gene in a cohort of Dutch familial ALS patients. Two previously identified mutations (R521C and R521H) and one novel mutation (S462F) were found. Additionally, a Q210H polymorphism was identified in one proband and three healthy controls. However, the functional consequences of these novel mutations await analysis.
To analyze the cellular localization of FUS, we tagged FUS (wild type, R521C, S462, or Q210H) with enhanced green fluorescence protein (GFP) at the N-terminus and expressed the fusion proteins in COS7 and NSC34 cells. Wild type GFP-FUS was confined to the nucleus, resembling the endogenous FUS expression pattern. In contrast, the R521C mutant was mainly distributed in the cytoplasm and protein aggregations were frequently observed. Q210H and S462F were mostly confined to the nucleus although a weak cytoplasmic distribution was observed. To further understand the cellular function of FUS in motor neurons, biotinylation (Bio) peptide-tagged GFP, wild type GFP-FUS or GFP-FUS mutants (S462F, R521C) were transiently co-expressed with BirA enzyme in NSC34 cells so that Bio-tagged proteins were biotinylated in cells. Then, the lysates were pulled down with streptavidin beads and samples were size-separated. Gels were stained with silver to visualize differential bands followed by whole lane sequencing by Mass-spectrometry. The effect of mislocalization of mutated FUS on cell viability and toxicity through the newly discovered interacting proteins will be discussed.
P44 DEVELOPMENT OF A NOVEL NON-RADIOMETRIC ASSAY FOR NUCLEIC ACID BINDING TO TDP-43 SUITABLE FOR HIGH-THROUGHPUT SCREENING USING ALPHASCREEN TECHNOLOGY
PAWLYK A, CASSEL J, BLASS B, REITZ A
ALS Biopharma, LLC, Doylestown, PA, United States
E-mail address for correspondence: [email protected]
Keywords: TDP-43, high-throughput screen, TAR DNA
Background: TAR DNA binding protein 43 (TDP-43) is a nucleic acid binding protein that is associated with the pathology of amyotrophic lateral sclerosis (ALS), the related disorder frontotemporal lobar dementia, certain forms of cystic fibrosis, and HIV infection. Assays to examine nucleic acid binding to TDP-43 typically use qualitative techniques that are not amenable to the high-throughput screening techniques necessary to discover small molecule probes or therapeutics.
Methods: We have developed a robust, quantitative, non-radiometric high-throughput assay measuring oligonucleotide binding to TDP-43 using AlphaScreen technology. AlphaScreen technology works by detecting the proximity of TDP-43 tethered to an acceptor bead and a DNA oligonucleotide tethered to a donor bead. This permits energy transfer via excited singlet oxygen when the two macromolecules are associated. We have established this assay in a 384-well plate format in which beads and macromolecules are pre-incubated in separate reactions and then combined in the presence or absence of a competing molecule. This allows the assay to detect direct binding of biotinylated oligonucleotides or competitive binding of a test oligonucleotide or small molecule. A chemical diversity library of 7,360 predominantly heterocyclic drug-like molecules was screened for their ability to disrupt this interaction.
Results: Biotinylated single-stranded TAR DNA (bt-TAR-32) and six TG repeats (bt-TG6) bound with high affinity to TDP-43, with KD values of 0.75 nM and 0.63 nM, respectively. Both oligonucleotides exhibited slow dissociation rates, with half-lives of 750 min for bt-TAR-32 and 150 min for bt-TG6. The relative affinities of unlabeled DNA and RNA oligonucleotides, as determined by displacement of either bt-TAR-32 or bt-TG6, were consistent with previous reports of nucleic acid interactions with TDP-43, where increasing TG or UG repeats yields greater affinity. We also found that DNA oligonucleotides bound with a greater affinity than RNA oligonucleotides. Screening the library of 7,360 compounds for inhibition of TDP-43 binding to bt-TAR-32 identified a series of compounds with nascent SAR and IC50 values ranging from 100 nM to 10 μM.
Discussion and conclusions: We have established a homogenous, quantitative, high-throughput assay for the binding of nucleic acids to TDP-43 and demonstrated that this assay can be used to assess both direct and competitive binding interactions. We have demonstrated it is capable of identifying small molecule inhibitors of the nucleic acid-TDP-43 interaction from compound libraries. These compounds may prove to be useful biochemical tools to facilitate the elucidation of the function of TDP-43 and may lead to novel therapeutics for indications where the TDP-43-nucleic acid interaction is causal to the associated pathology. Screening of additional compound libraries and the development of downstream assays of TDP-43 function will enhance our understanding of the pathogenic role of TDP-43 in ALS and other diseases.
P45 INHIBITION OF SIRT1 FUNCTIONS PROMOTES NEURAL PROGENITORS TOWARD MOTONEURON DIFFERENTIATION FROM HUMAN EMBRYONIC STEM CELLS
ZHANG Y1, WANG J1, CHEN G2, FAN D1, DENG M1
1Department of Neurology, 2Reproductive Center; Peking University Third Hospital, Beijing, China
E-mail address for correspondence: [email protected]
Keywords: Sirt1, resveratrol, nicotinamide
Background and objectives: A few protocols of directing embryonic stem cells (ESCs) to differentiate into functional motoneurons have been established, but the efficiency of motoneuron generation varied from different human ESC lines. We tried to search a novel protocol to increase the formation of motoneurons from ESCs.
Methods: In this study, we tested a nuclear protein, histone deacetylase Sirt1 to influence neural precursor cells (NPCs) development during differentiation of hESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide dramatically increased motoneuron formation.
Results: We found that about 60% cells from the total NPCs express HB9 and βIII-tubulin proteins, the typical motoneuron markers derived from ESCs after nicotinamide treatment. A functional marker of mature motoneurons, choline acetyltransferase (ChAT) was found positive from the derived cells. The inhibition of Sirt1 promotes more motoneurons (59.7%) to differentiate from a human embryonic stem cell line PKU1.1 than that of traditional protocol (32.8%) which used a simple sequential application of retinoid acid (RA) and sonic hedgehog (SHH) in a chemically defined suspension culture. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 μM) or inhibitor nicotinamide (100 μM) for 6 days by a quantitative RT-PCR, respectively. The levels of Mash1, Ngn2, and HB9, mRNAs are consistently increased significantly after nicotinamide treatment compared with control groups which used the traditional protocol. On the contrary, the levels of Mash1, Ngn2, and HB9 mRNAs by resveratrol treatment were all significantly lower than that of control group.
Conclusions: Our results suggested that increase of Mash1 and Ngn2 levels by inhibition of Sirt1 could elevate HB9 expression, which promote motoneuron differention. This study provides an alternative method for the production of sufficient amounts of engraftable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.
P46 GRADIENT CENTRIFUGATION ENRICHMENT OF MOTOR NEURONS DERIVED FROM EMBRYONIC STEM CELLS
MILAZI S, PRICE A, BROOKS BR, MOUGEOT J-L
Carolinas Medical Center, Department of Neurology, Neuromuscular/ALS Research Laboratory, Charlotte, NC, United States
E-mail address for correspondence: [email protected]
Keywords: ES Cells, motor neurons, enrichment
Background: The etiology of Amyotrophic Lateral Sclerosis (ALS) is still poorly understood. There is no cure and only one FDA approved drug, riluzole, is known to extend the lifespan of ALS patients by a few months on average. Several groups have produced MNs derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) from ALS patients to study ALS and develop new therapies. One limitation of isolating pure MNs is that, following induced differentiation, undifferentiated cells are still present in embryoid bodies (EBs) that contain MNs. EBs are aggregates of cells that are difficult to dissociate into single cell suspension without affecting MN viability. Undifferentiated cells may interfere with the interpretation of the results in drug screening assays that affect the survival of MNs and therefore render understanding of ALS-specific cell death mechanisms more difficult. We have developed a method based on step-gradient centrifugation to isolate viable MNs from dissociated EBs.
Objective: To isolate and subculture differentiated motor neurons derived from mouse embryonic stem cells within embryoid bodies for the development of drug screening assays and transplantation therapies.
Methods: Mouse ESCs (HBG3:GFP:HB9) were differentiated into MNs using a modified protocol by Wichterle et al. (2008). EBs containing GFP-positive MNs and undifferentiated cells were gently dissociated into single cells by enzymatic and chemical treatments without mechanical dissociation. The single cell suspension was layered on OptiprepTM medium step-gradient ranging from 8-20%. To determine purity, we used FACS to quantify GFP-positive cells corresponding to MNs.
Results: We were able to consistently produce EBs which in total contained over 68%±8 (mean±std dev) of MNs when dissociated and analyzed by FACS. After dissociation, the single cell suspension was co-cultured onto myoblast cells (C2C12) and neuromuscular junction formation was verified by immunohistochemistry using alpha-bungarotoxin conjugate which binds to acetylcholine receptor. In addition, we were able to obtain MN fractions enriched to 88%±6 (mean±std dev) by step-gradient centrifugation.
Discussion and conclusion: The development of standards for MNs addressing physiological relevance such as the phenotype (neurite outgrowth), and the ability of MNs to form neuromuscular junctions with muscles is necessary to design an appropriate assay. Our data supports dissociation without trituration and step-gradient centrifugation can be used to enrich ES cell-derived MNs retaining high viability, without the use of a cell sorter or the extensive use of antibodies for negative or positive selection. This study provides data for developing criteria for using MNs enriched by gradient centrifugation for cell-based assays for preclinical drug screening and cell transplantation therapies in ALS and other motor neuron disorders.
P47 DEVELOPMENT OF IN VITRO HUMAN NEUROMUSCULAR JUNCTION SYSTEMS
GUO X1, GONZALEZ M1,2, DAS M3, RUMSEY J4, STANCESCU M1, JOHE K5, DAVIS H1,2, MOLNAR P1, HICKMAN J1,2
1Nanoscience Technology Center, 2Biomolecular Science Center, Burnett School of Biomedical Sciences; University of Central Florida, Orlando, FL, United States, 3Bioscience and Bioengineering, Indian Institute of Technology, Kanpur (UP), India, 4Sanford-Burnham Medical Research Institute, Lake Nona, FL, United States, 5Neuralstem, Inc., Rockville, Maryland, United States
E-mail address for correspondence: [email protected]
Keywords: human, in vitro, neuromuscular junction
Background: To date, studies concerning motoneuron diseases (MNDs) have been primarily carried out in animal models or postmortem human tissues. However, animal models do not recreate the full process of human diseases, emphasizing a need for human-based systems. Conversely, studies from postmortem human tissues usually only capture late stage pathology rather than the real cause which occur at early stages. In addition, the effectiveness and toxicity of therapeutic drugs on human cells/systems are generally not assessed until preclinical studies, which are under strict regulations and expensive and time consuming. Therefore, human stem cell-based in vitro model systems are necessary for the study of MNDs and their therapies.
Objectives: In this study, we strived to build an in vitro human-based Neuromuscular Junction (NMJ) system to address this need.
Methods: Due to the limited availability of primary human tissue, human stem cell-derived motoneurons and myoblasts were utilized, in addition to rat embryonic skeletal muscle. NMJs were analyzed by phase microscopy, immunocytochemistry with confocal microscopy, and functional assays.
Result: First, motoneurons were differentiated from the human spinal cord stem cell line NSI-566RSC (1). A serum-free media was developed and the cells were cultured on a synthetic non-biological surface. These motoneurons were functionally mature based on the analysis with immunocytochemistry and electrophysiology. Then, the motoneurons were co-cultured with rat embryonic skeletal muscle to test their capability to form chimeric, functional NMJs. Based on the immunocytochemical analysis and the functional assays, successful NMJs were formed between human motoneurons and rat myotubes (2). These motoneurons were also co-cultured with myotubes developed from human skeletal muscle stem cells in a similar defined system. NMJ formation was demonstrated by the co-localization of motoneuron terminals and Acetylcholine (Ach) receptor clusters on the myotubes. Particularly, the observation of muscle contractions that could be ended by the Ach receptor antagonist, curare, confirmed the formation of functional NMJs.
Discussion and conclusions: The cross-species NMJ formation between human motoneurons and rat skeletal muscles not only uncovers the essential elements for NMJ formation shared in both human and rat, but also provides scientific basis for stem cell replacement studies which are typically conducted in rats. Due to their defined nature, these human-based systems, especially the human motoneuron-human skeletal muscle pairing, can be easily dissected and manipulated to study human NMJ formation, maintenance and repair, to investigate the mechanism of MND, and to perform high throughput drug screening. In summary, these human-based in vitro model systems would provide a novel avenue for the study and therapeutic investigation of MND.
References:
- Guo X, Johe K, Molnar P, et al. Journal of Tissue Engineering and Regenerative Medicine 2010;4(3):181–93.
- Guo X, Das M, Rumsey J, et al. Tissue Engineering Part C: Methods. in press
P48 COMPARTMENTALISED EXCITOTOXICITY IN PRIMARY CULTURED NEURONS: A NOVEL IN VITRO MODEL OF ALS PATHOLOGY
HOSIE KA1,2, KING AE1,2, YAP YC1,2, BLIZZARD CA1,2, VICKERS JC1,2, DICKSON TC1,2
1Menzies Research Institute, Hobart, Tasmania, Australia, 2University of Tasmania, Hobart, Tasmania, Australia
E-mail address for correspondence: [email protected]
Keywords: excitotoxicity, neuromuscular junction, axon degeneration
Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterised by distal axon degeneration prior to symptoms. We have linked glutamate excitotoxicity in primary motoneurons to ALS-like distal axon degeneration. It currently remains unclear if toxicity is directed through the distal axon at the neuromuscular junction, or the somatodendritic compartment within the spinal cord.
Objectives: The aim of this study is to investigate the hypothesis that ‘somatodendritic excitotoxin exposure can result in primary degeneration of the distal axon’. We are investigating this hypothesis in a compartmentalized primary culture model involving growth of primary spinal motor neuron cell bodies on a feeder layer of glial cells and distal axons extending to cultured skeletal muscle, allowing formation of a neuromuscular synaptic structure.
Methods: Primary cortical and spinal motoneurons are derived from E15 and E13 embryonic rodents respectively. Primary glia and skeletal muscle cultures are derived from neonatal rodent pups. Primary cortical neurons are grown as monocultures in compartmentalized microfluidic chambers (Xona) that allow separation of somatodendritic and axonal compartments. Spinal motoneurons are grown on feeder layers of glial cells or skeletal muscle or in microfluidic chambers with glial and muscle cultures as described above. Control and treated cultures are analysed using standard immunocytochemical methods.
Results: Cortical neurons grown in microfluidic chambers demonstrated separation of axonal and somatodendritic compartments as evidenced by MAP2 immunoreactivity (dendrites) confined to the somal chamber, and NFM immunoreactivity (axons) present in the axonal chamber. Skeletal myoblast cultures matured into contractile, multinucleated myotubes, with rudimentary neuromuscular junctions evident when co-cultured with spinal motoneurons. Furthermore spinal motoneurons cultured on muscle exhibited significantly (P<0.05) diminished survival and altered morphology, with fewer dendrites (n=5, 2.6±0.3) and significantly longer axons (n=5, 1363 μm±104) compared to motoneurons co-cultured with astrocytes (dendrite number: n=4, 4.5±0.8, axon length: n=5, 759 μm±105). To investigate the hypothesis that excitotoxicity mediated through the somatodendritic compartment results in distal axon degeneration, cultured cortical neurons (14 days in vitro) in microfluidic chambers were compartmentally exposed to 100 μM glutamate.
Excitotoxin exposure to the somatodendritic compartment resulted in axonal blebbing and fragmentation within both compartments, with increased overall distal axon damage (preliminary data). Somatodendritic excitotoxin exposure resulted in dendritic beading and a significant (P<0.05) increase in apoptotic nuclei (n=4, 64%±4.5) relative to untreated controls (n=4, 40%±4.4). Axonal excitotoxin exposure resulted in increased axonal fragmentation relative to control chambers, decreased relative to somatodendritic treated chambers (preliminary data). Future experiments will investigate mechanisms of site-specific excitotoxicity in the spinal motoneuron model.
Discussion and conclusion: These data indicate that microfluidic chambers can be used to investigate the role of different neuronal compartments axon degeneration and may provide insights in mechanisms of degeneration in ALS.
P49 EPIGALLOCATECHIN-3-GALLATE PROTECTS MOTOR NEURONS AND REGULATES GLUTAMATE LEVEL
LIU Y, YU J, GUO Y, JIA Y, DUAN W, LI Z, LI C
Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang, China
E-mail address for correspondence: [email protected]
Keywords: motor neuron, glutamate excitotoxicity, EGCG
Background: Amyotrophic lateral sclerosis (ALS) is a progressive and lethal neurodegenerative disease, characterized by degeneration of motor neurons from cortex, brainstem and spinal cord. In ALS, glutamate-mediated neurotoxicity was first suggested as a mechanism of motor neuron death. Increased levels of glutamate were detected in the cerebrospinal fluids of 40% sporadic ALS patients. THA-induced glutamate excitotoxicity in organotypic spinal cord cultures has been one of the widely used models of motor neuron degeneration and has also been applied for development of neuroprotective strategies.
Objectives: To investigate the protective effects of EGCG on the glutamate excitotoxicity induced motor neuron injury.
Methods: Organotypic spinal cord cultures were prepared from lumbar spinal cord explants of 7-day-old SD rat pups. The cultures were divided into four groups at random: control, THA, EGCG+THA, NAC+THA (EGCG or NAC pretreated 48 h then treated with EGCG+THA or NAC+THA). The number of motor neurons was assessed by immunohistochemistry, and glutamate concentrations in the culture medium and lipid peroxidation in spinal cord explants were measured using glutamate detection and TBARS assay kits, respectively. EAAT2 expression was measured by Western blot.
Results: Both 5 μM EGCG and 100 μM NAC blocked THA-induced motor neuron death and decreased TBARS levels. Different from NAC, protection of motor neurons by 5 μM EGCG is associated with regulating the glutamate level in the culture medium. However, there was no change of EAAT2 expression after treatment with 5 μM EGCG for 48 h. This property of EGCG may be not due to its intrinsic antioxidative activity, because another antioxidant NAC could not regulate glutamate levels under the same condition.
Discussion: In the present study, we found that motor neuron protection by EGCG was accompanied by regulation of glutamate levels in the synaptic cleft, and there have been few studies investigating this mechanism of EGCG. We speculate that EGCG could increase the activity of EAAT2 and the glutamate uptake of astrocytes, so the medium glutamate levels decreased after treatment with EGCG for 3 weeks. In view of the importance of glutamate excitotoxicity in ALS, EGCG may be a potential candidate for ALS therapy.
Conclusions: EGCG can regulate glutamate levels and inhibit lipid peroxidation and protect motor neurons against THA-induced toxicity.
P50 PROTECTIVE EFFECT OF COMBINATION OF SULFORAPHANE AND RILUZOLE ON GLUTAMATE-MEDIATED EXCITOTOXICITY
LI C, CHANG G, GUO Y, JIA Y, LIU Y, SONG X
Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, China
E-mail address for correspondence: [email protected]
Keywords: excitotoxicity, sulforaphane, riluzole
Background: Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by progressive and selective death of motor neurons. Evidences suggest that glutamate-induced excitotoxicity is an important pathogenic factor.
Objectives: To investigate whether the combination of sulforaphane (SF) and riluzole was more effective than either used alone in the protection against glutamate-mediated excitotoxicity.
Methods: Threohydroxyaspartate (THA) treated organotypic spinal cord cultures was used as a selective motor neuron injury model. Five groups were included: control group, THA group (100 μM), riluzole treatment group (5 μM or 2 μM), SF treatment group (10 μM or 4 μM) and SF-riluzole combined treatment group. Explants from each group were harvested for immunohistochemical staining with anti-neurofilament (SMI-32) antibody. Expression of erythroid 2-related factor 2 (Nrf2), NADPH: quinone oxidoreductase 1 (NQO1) and heme oxygenase 1(HO-1) were measured by Western blot analysis. Levels of LDH, MDA and glutamate in the culture medium were measured.
Results: In our study, SF-riluzole combined treatment not only stimulated the expression of Nrf2, NQO1 and HO-1, but also reduced the extracellular accumulation of glutamate. When used at optimal doses, SF (10 μM) and riluzole (5 μM), either alone or in combination, all exerted significant and similar neuroprotection, as measured by the number of motor neurons, medium MDA and LDH level. When used at lower doses, SF (4 μM) and riluzole (2 μM), the combined treatment group was better than either used alone.
Discussion: The results presented in this study strongly suggest that the combination of SF and riluzole at lower doses was more effective than either used alone in the protection against glutamate-mediated excitotoxicity. Such a combination has never been studied before. It represents a novel approach in the potential cocktail therapy, as riluzole primarily modulates glutamate signaling, whereas sulforaphane modulates antioxidative functions.
Conclusions: SF and riluzole combined treatment may represent a potential therapy against excitotoxicity induced motor neuron injury.
P51 PROTECTIVE EFFECTS OF RESVERATROL ON AN ALS CELL CULTURE MODEL
WANG J, ZHANG Y, FAN D, DENG M
Peking University Third Hospital, Beijing, China
E-mail address for correspondence: [email protected]
Keywords: VSC4.1 cell line, hSOD1G93A, resveratrol
Background and objectives: Resveratrol has recently been widely reported as an age delaying and neuroprotective compound, and it seems to exert benefit by a mimicking calorie restriction effect and activating SIRT1. In in vivo and in vitro studies of amyotrophic lateral sclerosis (ALS), the effect of calorie restriction and SIRT1 activation is controversial.
Methods: In the present study, we constructed an ALS in vitro cell culture model by stably expressing human superoxide dismutase 1(hSOD1)wt and mutant hSOD1G93A in a motor neuron like VSC4.1 cell line, which expressed matured motor neuron specific marker HB9 after differentiation. Then, we investigated the effect of resveratrol on this cell culture model.
Results: During a 24∼48 hour course, we found that 0.5∼50 μM resveratrol showed dose-dependent protective effects on the hSOD1G93A bearing ALS cell culture model, by increasing cell viability, promoting neurite outgrowth, preventing cell apoptosis course and elevating cellular ATP level. We also showed in our study that this effect was at least partly achieved by accelerating mitochondrial biosynthesis, as resveratrol remarkably increased the mRNA level of PGC1-α and mitofusin 2 in the hSOD1G93A bearing ALS cell model 24∼48 hours after treatment, which was prevented by the SIRT1 inhibitor nicotinamide. Meanwhile, the ability of resveratrol to promote neurite outgrowth could not be blocked by SIRT1 inhibition, which indicated that resveratol might affect another SIRT1 independent pathway to exert benefit on the hSOD1G93A bearing ALS cell model.
Conclusions: Our results suggest that resveratrol protects hSOD1G93A bearing ALS cell culture model from mutant SOD1-mediated motor neuron cell toxicity partially by activation of SIRT1, which may be a potent therapeutic target for preventing the motor neuron degeneration of ALS.
P52 MUTANT TDP-43 INDUCED OXIDATIVE INJURY IN A MOTOR NEURON-LIKE CELL LINE
DUAN W, GUO Y, YU X, JIANG H, LU J, LI C
Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang, China
E-mail address for correspondence: [email protected]
Keywords: oxidative stress, HO-1, Nrf2
Background: Since the transactive response DNA-binding protein 43 (TDP-43) positive inclusions were detected in patients with frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-Us) and ALS, various missense mutations have been identified in this protein. TDP-43 is a nuclear protein which regulates transcription and RNA splicing, and participates in neurofilament mRNA transport and stability. To date, oxidative stress is regarded as an important pathway for the selective degeneration of motor neurons. In the present study, we hypothesized that mutant TDP-43 could mediate oxidative stress.
Objectives: To explore whether mutant TDP-43 could induce mitochondrial dysfunction and oxidative damage in the NSC34 cell line and whether sulforaphane could protect the cell line from the toxicity of mutant TDP-43.
Methods: Stable wild type and mutant TDP-43 (Q331K and M337V) transfected NSC34 cell lines were established; Western blot, quantitative real time PCR, confocal microscopy; and immunocytochemistry analysis were used. Cell proliferation was measured by Cell Counting Kit-8. Mitochondria transmembrane potential was measured by flow cytometry. TBARS in the cells and LDH content in the medium were tested.
Results: Mutant TDP-43 induced mitochondrial dysfunction, oxidative damage and nuclear accumulation of nuclear factor E2-related factor 2 (Nrf2). HO-1 was down-regulated in cells expressing the mutant TDP-43, and could not be restored by sulforaphane. Nevertheless, sulforaphane reduced the level of lactate dehydrogenase and lipoperoxidation products in cells expressing mutant TDP-43. However, sulforaphane could up-regulate the expression of HO-1 and NAD(P)H/quinone oxidoreductase-1 (NQO-1) in cells transfected with empty vector and the wild-type TDP-43.
Discussion: We found that mutant TDP-43 induced mitochondrial injury and oxidative damage, especially TDP-43 Q331K. The similar changes of mitochondrial dysfunction and oxidative damage in motor neurons have been reported in patients who suffered from sporadic or familiar ALS. Therefore, motor neuron-like cell line expressing mutant TDP-43 mimics the pathological changes of motor neurons in vivo. Activating Nrf2 is a new and effective therapeutic strategy for ALS. Antioxidant defense elevated by activating Nrf2 can protect motor neurons from oxidative damage and apoptosis. Subsequently, sulforaphane protected cells against mutant TDP-43 independent of Nrf2-ARE pathway, nevertheless it had an action on the empty and wild-type TDP-43 cell lines by activating Nrf2 and up-regulating the expression of HO-1 and NQO-1 in a dose-dependent manner.
Conclusions: Mutant TDP-43 results in mitochondrial dysfunction, oxidative injury and reduced HO-1 expression in NSC34 cell lines.
P53 MG132 PROMOTES NEURITE OUTGROWTH INHIBITED BY MUTANT TAR DNA-BINDING PROTEIN-43 (TDP-43) VIA ACTIVATING HEME OXYGENASE-1(HO-1)
DUAN W, GUO Y, JIANG H, YU X, LU J, LI Z, LI C
Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang, China
E-mail address for correspondence: [email protected]
Keywords: TDP-43, HO-1, Nrf2
Background: TDP-43 is a nuclear protein which regulates transcription and RNA splicing, and participates in neurofilament mRNA transport and stability. Recently, we established wild type and mutant TDP-43 (Q331K and M337V) cell lines. Furthermore, we observed that mutant TDP-43 had an adverse effect on neurite outgrowth in stable cell lines.
Objectives: To explore the effect of MG132 on the mutant TDP-43 cell lines.
Methods: Stable wild type and mutant TDP-43 (Q331K and M337V) transfected NSC34 cell lines were established, and the length of neurites were measured by FV10-SVW 1.7 viewer. Western blot, lipoperoxidation and LDH analysis were used.
Results: The neurites of NSC34 cell line expressing mutant TDP-43, especially TDP-43 Q331K, were significantly shorter than that expressing WT TDP-43. Lower doses (0.1 and 1 μM) of MG132 didn't increase mutant TDP-43 expression or LDH level in the cells transfected mutant TDP-43. However, 5 μM MG132 significantly induced cellular damage. Lower dose inhibition of the activity of proteasome promoted the neurite outgrowth in the mutant TDP-43 expressing cells. In our previous studies, mutant TDP-43 down-regulated the expression of HO-1. So, we speculate whether lower dose MG132 could restore the expression of HO-1. Indeed, HO-1 was restored by MG132. The transcription factor Nrf2 regulates the endogenous antioxidative capacity through transactivating phase-II detoxification genes, known as HO-1. MG132 induced the accumulation of Nrf2 into the nucleus in cells stably transfected WT TDP-43 in a dose-dependent manner, however reduced the accumulation of Nrf2 in the nucleus in mutant TDP-43 expressing cells.
Discussion: We found that mutant TDP-43 inhibited the neurite outgrowth. The shorter neurites in the cells stably transfected with mutant TDP-43 could suggest that mutant TDP-43 lost its normal function and gained toxicity. It is well known that inhibiting UPS by both pharmacological and genetic means delays axon degeneration and promotes neurite outgrowth. Indeed, we found that MG132 promoted the neurite outgrowth in the cells stably transfected with mutant TDP-43 as well as wild type TDP-43. In ALS, spinal motor neurons demonstrate ubiquitin immunopositive cytoplasmic inclusions which are also immunopositive for TDP-43. Its presence in ubiquitin positive inclusions strongly indicated TDP-43 was at least regulated by the UPS. We also found that non-toxic MG132 up-regulated HO1 expression in a dose-dependent manner and increased the antioxidative capacity accompanied with stimulating neurite outgrowth.
Conclusions: Mutant TDP-43 inhibits neurite outgrowth and lower dose MG132 stimulates neurite outgrowth. This action of MG132 could be associated with increase of HO1 expression.
P54 ENDOPLASMIC RETICULUM STRESS IS LINKED WITH TDP-43 REDISTRIBUTION IN ALS
WALKER A1,2, MA Y2, TURNER B2, PARISH C2, SOO K Y1, FARG M1, HORNE M2,3, ATKIN J1,2
1Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia, 2Howard Florey Institute, Florey Neuroscience Institutes, Parkville, Victoria, Australia, 3Department of Neurology, St Vincent's Hospital, Fitzroy, Victoria, Australia
E-mail address for correspondence: [email protected]
Keywords: ER stress, TDP-43, redistribution
Background: Endoplasmic reticulum (ER) stress results from a variety of cellular insults, and leads to activation of signalling pathways known as the unfolded protein response (UPR). The UPR can alleviate ER stress by increasing chaperone production, inhibiting general protein translation and enhancing protein degradation. However if ER stress is prolonged, as in ALS, then cell death is triggered. ER stress occurs prior to symptom onset in SOD1G93A mice, and genetic ablation of UPR proteins delays disease onset and increases survival of these mice, suggesting an important role for ER stress in pathogenesis. Recently, ER stress was also identified in sporadic ALS patients. TDP-43 is the major constituent of inclusions in most ALS patients, and altered TDP-43 protein processing and distribution is a key feature of disease. Mutations to the gene encoding TDP-43 are also linked with familial and sporadic forms of ALS.
Objectives: The aims of this study were to investigate whether ER stress leads to altered cellular distribution or aggregation of TDP-43, and whether TDP-43 expression causes UPR induction in cell culture.
Methods: Plasmids encoding full-length or C-terminal fragment TDP-43 were constructed. Neuroblastoma cells and primary rodent cortical neurons were transfected, and TDP-43 expression, sub-cellular location and solubility were monitored using immunocytochemistry, confocal microscopy and immunoblotting. ER stress was monitored using immunocytochemistry and immunoblotting for UPR markers.
Results: The majority of cells expressing either full-length wildtype or six different ALS-linked mutants had primarily nuclear localised TDP-43 while the C-terminal fragment proteins were located throughout the cytoplasm. Proteasome inhibition caused a dramatic increase in the proportion of cells with cytoplasmic localisation of TDP-43 and resulted in the formation of inclusion-like structures, particularly when the C-terminal TDP-43 fragments were expressed. Pharmacologically-induced ER stress also caused TDP-43 redistribution, and cells expressing mutant TDP-43 showed increased UPR induction compared to controls.
Discussion and conclusions: These findings indicate that ER stress is a feature of mutant TDP-43 linked ALS, and that UPR induction results in redistribution of TDP-43 from the nucleus to the cytoplasm in a manner reminiscent of disease pathology. ER stress could therefore be an early triggering event in ALS, and therapeutic targeting of the UPR could be beneficial in treatment of both familial and sporadic forms of disease.
P55 METALS AND OXIDATIVE STRESS INDUCE MISLOCALIZATION AND AGGREGATION OF ENDOGENOUS TDP-43
MEYEROWITZ J1,2, PARKER S1,2, CARAGOUNIS A1,2, PRICE K1,2, SOON C1,2, FILIZ G1,2, MASTERS C3, LI Q-X1, CROUCH P1,2, WHITE A1,2
1Department of Pathology, 2Centre for Neuroscience; The University of Melbourne, Victoria, Australia, 3Mental Health Research Institute, Victoria, Australia
E-mail address for correspondence: [email protected]
Keywords: TDP-43, oxidative stress, metals
Background: Neuronal aggregates containing ubiquitinated and phosphorylated TDP-43 are pathological hallmarks in the spectrum of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Changes to TDP-43 metabolism have also been reported in other neurodegenerative diseases. In affected neurons, TDP-43 undergoes C-terminal fragmentation, phosphorylation and ubiquitination and forms aggregates in the cytoplasm or nucleus. Loss of nuclear TDP-43 expression is also prominent. While in vitro studies have been able to re-capitulate many of these features using transfected cell culture models, little is known about the biochemical mechanisms that underlie pathological changes to endogenous TDP-43.
Objective: As altered metal ion homeostasis and oxidative stress are central features of neurodegeneration, including FTD and ALS, we sought to determine the effects of these factors on endogenous TDP-43 metabolism in mammalian cells.
Methods: Cultures of neuronal-like SY5Y cells were treated with biometals or subjected to mild oxidative insults and changes to TDP-43 metabolism were examined by immunoblot and immunofluorescence.
Results: Treatment of SY5Y cells expressing endogenous TDP-43 with zinc (Zn) induced depletion of nuclear TDP-43 expression and formation of inclusions in the nucleus and cytoplasm that were TDP-43 positive. No evidence of C-terminal fragmentation, phosphorylation or ubiquitination was observed. The depletion and aggregation of TDP-43 was associated with the specific action of Zn and was not seen with copper or iron. Exposure of cells to specific modulators of oxidative stress induced depletion of nuclear TDP-43, increased diffuse cytoplasmic TDP-43 localization, elevated levels of a 35 kDa C-terminal fragment and led to formation of cytoplasmic ubiquitin-positive inclusions containing TDP-43. Inhibition of caspase activation partially abrogated cytoplasmic inclusion formation concomitant with reduced expression of the 35 kDa C-terminal fragment.
Conclusions: These findings suggest that altered metal homeostasis or oxidative stress, which are common features of neurodegeneration, may affect metabolism of endogenous neuronal TDP-43 resulting in accumulation in cytoplasmic inclusions.
P56 MODE OF DEATH AND ROLE OF ROS IN HOMOCYSTEINE (HCY) AND AAPH (2,2'-AZOBIS-2-METHYL-PROPANIMIDAMIDE)-INDUCED CELL DEATH IN DIFFERENTIATED MOTOR NEURON - NEUROBLASTOMA HYBRID NSC-34D CELLS
HEMENDINGER R, ARMSTRONG III E, BROOKS BR
Carolinas Medical Center, Charlotte, NC, United States
E-mail address for correspondence: [email protected]
Keywords: oxidative stress, cell death mechanisms, NSC-34 cells
Background: Oxidative stress has been implicated as an early step in the pathogenesis of ALS and animal models of ALS. Hydrogen peroxide is commonly used in vitro to study oxidative stress. Our previous work with the antioxidants, luteolin and huperzine A, demonstrated both neuroprotection and neurorescue against hydrogen peroxide (H2O2)-induced cell death in an in vitro model of sporadic ALS (1,2). As H2O2 is a general oxidative stressor, we studied a free radical generator capable of generating peroxyl radicals and molecular nitrogen, AAPH.
Objective: The current study examines the toxicity of AAPH in our in vitro model of sporadic ALS in comparison to H2O2 and Hcy (a compound shown to induce oxidative stress in other cell types).
Methods: The toxicity of AAPH was examined in differentiated motor neuron-neuroblastoma hybrid NSC-34 (NSC-34D) cells. Cell death was assessed after a 24 h exposure to AAPH doses ranging from 0 mM to 5 mM. Cell death was determined using nuclear staining with Hoechst 33342 and propidium iodide and a CellQuanti assay for metabolic activity at 24 h of exposure. Additional assays investigating the dose-response induction of ROS production during a 1 h exposure to AAPH and homocysteine were performed. Analysis of variance and Fisher's LSD test were used to anlyze the data at a P value of 0.05.
Results: Cell death increased in a dose-dependent manner with AAPH, reaching 46% at 3.0 mM and increasing to 84% at 5.0 mM (P<0.05). The mode of cell death was primarily via necrosis with AAPH. In contrast, Hcy induced primarily apoptotic cell death (3). Metabolic activity assays demonstrated a significant dose-dependent reduction in metabolic activity at 24 h with AAPH (P<0.05) while Hcy had no effect on metabolic activity. Preliminary studies examining the role of ROS in the cell death induced by these agents were performed. A dose-response induction of ROS during a 1 h exposure to AAPH was observed in NSC-34D cells and in the control heptocarcinoma cell line, HepG2. In contrast, homocysteine failed to induce ROS up to 500 mM dose in this assay with either cell line.
Conclusions: AAPH induces necrotic cell death and ROS generation in a dose-dependent fashion in the differentiated motor neuron-neuroblastoma hybrid NSC-34D cells. In contrast, Hcy, known to play a role in the pathogenesis of ALS and cause oxidative stress in endothelial cells, induces apoptotic cell death in these NSC-34D cells without significant ROS generation following a short-term exposure. Understanding how AAPH and Hcy modulate molecular targets will allow us to understand the processes involved in neuronal cell death in our in vitro model and in patients with ALS.
References:
- Hemendinger R, et al. Neurotox Res. 2008;13:49–61.
- Hemendinger R, et al. ALS. 2007;9(Suppl 1):83.
- Hemendinger, et al. Neurology. 2010;74(Suppl):A208.
P57 PROTEIN DISULPHIDE ISOMERASE REGULATES SOD1 ACTIVITY AND CONTROLS CYTOCHROME C-CATALYZED PEROXIDATION – IMPLICATIONS FOR MITOCHONDRIAL ROS PRODUCTION IN ALS MODELS
JARONEN M1, AHTONIEMI T2, KOISTINAHO J1, GOLDSTEINS G1
1A. I. Virtanen Institute for Molecular Sciences University of Eastern Finland, Kuopio, Finland, 2Medeia Therapeutics Ltd, Kuopio, Finland
E-mail address for correspondence: [email protected]
Keywords: SOD1, protein disulfide isomerase, ROS
Background: Increased mitochondrial production of reactive oxygen species (ROS) has been implicated in a number of neurodegenerative diseases, including ALS. Some familial forms of ALS are associated with point mutations in the superoxide dismutase 1 (SOD1) gene. Nevertheless, the mechanism of the gain of toxic function remains unknown.
SOD1, an abundant cytosolic enzyme, is present in an inactive form also in the mitochondrial intermembrane space (IMS). SOD1 activity in this compartment is controlled by the redox state of an intramolecular disulphide bond. The formation of this bond and activation of SOD1 is executed by protein disulfide isomerase (PDI), which has been found also in mitochondria.
We have previously demonstrated that in G93A-SOD1 rats the mutant SOD1 is up-regulated in the IMS and possesses increased ability to bind the inner membrane of isolated mitoplasts. In parallel, we have shown that SOD1 activity in the IMS increases mitochondrial ROS production by enhancing hydroperoxide production, eventually resulting in augmented cytochrome c-catalyzed peroxidation. Finally, we have been able to demonstrate that PDI expression peaks in the spinal cord of a G93A-SOD1 rat model as well as in a mouse model at presymptomatic stage of the disease.
Objectives: Our previous findings allowed us to hypothesize that increased PDI expression leads to the aberrant control of SOD1 activity in the mitochondrial IMS and causes increased hydroperoxide production in this compartment, eventually resulting in neuronal vulnerability. In the current study our aim was to investigate whether PDI can exercise redox control of SOD1 activity leading to increased hydroperoxide production and cytochrome c-catalysed peroxidation in vitro and HEK-293 cell culture.
Methods: In vitro superoxide production was generated by xanthine/xanthine oxidase. Cytochrome c-catalyzed peroxidation was measured with dichlorodihydrofluorescein (DCF), a specific substrate for hydroperoxide, both in vitro and in HEK-293 cell culture. In cell culture model superoxide production was induced with paraquat.
Results: Our results show that PDI catalyzes reactivation of SOD1 after inactivation by disulphide bond reduction. This reactivation resulted in increased hydroperoxide production and cytochrome c-catalysed peroxidation. Both reactivation and increased peroxidation were inhibited by bacitracin, a PDI inhibitor. Inhibition of PDI by bacitracin suppressed also paraquat-induced hydroperoxide production in HEK-293 cells.
Discussion and conclusions: These results elucidate the possible role of PDI in controlling SOD1 activity within the IMS and its impact on mitochondrial ROS production in ALS models.
This work has been kindly funded by Sigrid Juselius foundation.
P58 EFFECTS OF PALMITOYL CARNITINE ON BRAIN, SPINAL CORD AND HEART MITOCHONDRIA FROM WILD TYPE AND TRANSGENIC SOD1 RATS
PANOV A, KUBALIK N, BROOKS BR
Carolinas Medical Center, Charlotte, North Carolina, United States
E-mail address for correspondence: [email protected]
Keywords: brain, spinal cord heart mitochondria, ROS
Background: Mitochondrial dysfunctions contribute to the loss of motor neurons in ALS. Several metabolites are oxidized simultaneously by mitochondria in vivo (1). The higher vulnerability of motor neuron mitochondria may be associated with some metabolic features unique for central nervous system tissue. Brain and spinal cord mitochondria from SOD1 transgenic rats have substrate-specific increases in ROS production (2). Unlike heart, brain and spinal cord mitochondria do not oxidize fatty acids presumably due to low activity of 3-ketoacyl-coenzyme A thiolase.
Objectives: Do the tissue-specific metabolic preferences affect mitochondrial dysfunctions in ALS?
Methods: Heart, brain and spinal cord mitochondria were isolated from wild type and G93A SOD1 transgenic rats. Pyruvate, glutamate, malate, succinate, palmitoyl-carnitine and their mixtures were used as substrates. Respiratory activity was determined with a Clark electrode, and ROS generation was measured by the Amplex Red method.
Results: Heart mitochondria oxidized palmitoyl-carnitine or succinate alone very poorly in State 3, and respiration was inhibited upon uncoupling. However, when palmitoyl-carnitine was combined with any other mitochondrial substrate (pyruvate, glutamate, or succinate), the rates of oxidative phosphorylation and uncoupled respiration increased by 40 to 70%. Importantly, resting respiration increased 64% with glutamate and succinate. As a result, production of ROS increased 3-4 fold with these combinations of substrates. This trend was more pronounced in SOD1 transgenic rat heart mitochondria. Brain and spinal cord mitochondria did not oxidize palmitoyl-carnitine. However, when palmitoyl-carnitine was added to glutamate, pyruvate or succinate, the rates of ROS generation increased 4-fold as compared with glutamate and malonate. Malonate, an inhibitor of succinate oxidation, completely abolished increased ROS with Succinate and palmitoyl-carnitine, but not glutamate and palmitoyl-carnitine. The latter was inhibited by addition of uncoupler CCCP. This suggests that with glutamate and palmitoyl-carnitine the reverse electron transport was also involved in increased ROS production, but the electrons were fed to the membrane's pool of coenzyme Q by the FAD enzyme acyl CoA dehydrogenase. However, the exact mechanisms by which substrates and palmitoyl-carnitine facilitate ROS production remain obscure.
Conclusions: Rat heart mitochondria, brain and spinal cord mitochondria show strong substrate preferences for maximal rates of oxidative phosphorylation and ROS production. Palmitoyl carnitine significantly increased ROS production in all types of mitochondria. Although normally brain mitochondria do not oxidize palmitoyl-carnitine, its presence may facilitate oxidative stress in spinal cord of SOD1 transgenic rats.
References:
- Panov A P, Schonfeld S, Dikalov R, et al. J Biol Chem. 2009;284(21):14448–56.
- Panov A, Kubalik N, Hemendinger R, et al. ALS;9:S2–S3.
P59 GLIAL DYSFUNCTION IN ALS: REDUCED ACTIVITY OF THE MONOCARBOXYLATE TRANSPORTER, MCT1, PRODUCES CELL DEATH OF OVERACTIVE MOTOR NEURONS
MORRISON B, LI Y, TSINGALIA A, COCCIA C, ROTHSTEIN J
The Johns Hopkins University, Baltimore, Maryland, United States
E-mail address for correspondence: [email protected]
Keywords: metabolism, glutamate, astrocyte
Background: Several lines of evidence have suggested a role for astrocytes in the degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Astrocytes support the function of motor neurons through several mechanisms, including secretion of trophic factors, removal of glutamate from the synapse, and supplying the energy substrate lactate. This latter function is termed the astrocyte-neuron lactate shuttle. In this proposed shuttle, lactate produced by astrocytes through glycolysis is transported into the extracellular space through monocarboxylate transporters (MCTs) where it can be taken up by neurons using distinct MCTs, converted to pyruvate, and used within mitochondria to produce ATP. One of the primary astrocytic MCTs, MCT1, has been shown to be reduced in patients with ALS, and we propose that this contributes to motor neuron vulnerability by interrupting supply of lactate. Of course, motor neurons can also generate ATP from glucose; and therefore, we hypothesize that lactate is particularly critical for motor neurons when glucose supply is not sufficient to maintain neuronal metabolic activity.
Objectives: To investigate whether motor neurons in vitro and in vivo are more dependent on astrocyte-derived lactate when metabolically stressed by depolarization, overactivity, or injury.
Methods: To investigate this hypothesis in vitro, we exposed organotypic spinal cord cultures to three treatments expected to produce motor neuron depolarization and increased metabolic activity (ie, high potassium, the GABA antagonist bicuculline, and glutamate) in the presence or absence of a pharmacologic inhibitor to MCT1. In vivo, we used sciatic nerve crush as an injury paradigm, which is expected to increase the energy requirements of recovering motor neurons, in rats treated with intrathecal MCT1 inhibitor or vehicle alone.
Results: There was no cell death from the MCT1 inhibitor or any of the treatments that increase metabolic activity when added to the media alone. However, when the pharmacologic MCT1 inhibitor was combined with high potassium, bicuculline, or glutamate, there was a significant increase in cell death. Similarly in vivo, motor neurons in the lumbar spinal cord that had undergone nerve crush degenerated in rats treated with intrathecal MCT1 inhibitor, but not in those treated with vehicle alone.
Discussion: Elevated glutamate secondary to reduced activity of the astrocytic glutamate transporter, GLT1, has been shown to occur in ALS. In addition to potentially causing excitotoxic cell death, this elevated glutamate would lead to depolarization and hyperactivity of motor neurons. In combination with reduced expression of MCT1, our results from cell culture and animal models suggest that significant motor neuron cell loss would occur due to reduced lactate production from astrocytes and insufficient production of ATP to maintain critical neuronal functions.
Conclusion: Reduced levels of MCT1 in patients with ALS may contribute to motor neuron degeneration by increasing the vulnerability of overactive neurons.
P60 INSIGHTS INTO THE REGULATION OF ASTROCYTIC EAAT ACTIVITY FOR MOTOR NEURON PROTECTION
SHEEAN R1,2, LAU C L1,2, O'SHEA RD1,2, BEART PM1,2
1Florey Neuroscience Institute, Parkville, VIC, Australia, 2University of Melbourne, Parkville, VIC, Australia
E-mail address for correspondence: [email protected]
Keywords: glutamate, transporters, pharmacology
Background: In Motor Neuron Disease (MND), glial cells strongly influence the demise of the motor neurons by contributing to inflammatory and excitotoxic mechanisms. Changes in astrocytic phenotype and cytoskeletal arrangement are key indicators of astrocytic reactivity. Astrocytes are responsible for the removal of the neurotransmitter L-glutamate (Glu) from the extracellular space to prevent neuronal injury and maintain synaptic signalling. Glu uptake is performed by excitatory amino acid transporters (EAATs), with the astrocytic EAATs, EAAT1 and EAAT2, responsible for the bulk of Glu uptake in brain. Loss of EAAT function is damaging to MNs, and increased EAAT2 expression is neuroprotective in MND models. The relationship between astrocytic morphology and EAAT function remains poorly understood, and understanding this interface may reveal how EAAT modulation can be exploited as a potential treatment in MND.
Objectives: To investigate the regulation of EAAT activity in astrocytes by altering astrocytic phenotype and EAAT distribution through pharmacological manipulation.
Methods: Primary cultures of mouse astrocytes were treated with rottlerin (a PKCδ inhibitor (100 mM, 6 h)) to reduce EAAT activity. Changes in astrocytic morphology and EAAT distribution were determined using immunocytochemistry, biotinylation and Western blotting. Glu uptake was investigated during and following rottlerin exposure. The rate of recovery of EAAT activity was examined during exposure to various drugs including monensin (a Na+ ionophore (100 mM, 2 h) to explore the involvement of cellular mechanisms.
Results: Rottlerin treatment rapidly decreased [3H]-d-aspartate uptake and caused cytoskeletal rearrangement. Biotinylation revealed increases in EAAT expression in all cellular fractions, including the cell surface with rottlerin treatment. After rottlerin removal, EAAT activity returned to control levels within 2-4 h. Monensin enhanced the recovery of [3H]-d-aspartate uptake, suggesting that Na/K-ATPase may be attenuated by rottlerin treatment. This possibility is being investigated by measuring Rb+ uptake to determine effects of rottlerin and monensin on coupling to EAAT activity.
Discussion: Decreases in [3H]-d-aspartate uptake are often associated with EAAT internalization or degradation. Biotinylation revealed that both total and cell-surface expression of EAATs increased with rottlerin treatment, suggesting a homeostatic response. These results, plus the rapid rate of EAAT recovery following rottlerin removal, revealed this inhibition of EAAT function was not due to changes in protein expression or localization. The ability of monensin to increase the rate of recovery suggests the involvement of Na+ in this EAAT inhibition. These data support the concept that EAATs are part of a molecular transport-complex, including the Na/K-ATPase.
Conclusion: EAAT activity and consequent homeostasis are essential for maintaining neuronal viability during injury. This study reveals that astrocytic EAAT expression is enhanced when EAAT activity is impaired, in an attempt to prevent injury. Additionally, our data suggest that maintenance of Na/K-ATPase activity is an integral component of this homeostatic response.
P61 USING LIVE CELL IMAGING IN A PRIMARY CULTURE MODEL OF ALS AS A METHOD TO DISCOVER THERAPEUTIC TARGETS
TRADEWELL M, COOPER L, DURHAM H
Montreal Neurological Institute, McGill University, Montreal, Canada
E-mail address for correspondence: [email protected]
Keywords: calcium, mitochondria, aggregates
Background: Alterations in calcium homeostasis, mitochondrial function and protein aggregation have been identified as pathogenic mechanisms in models of ALS; however, how each of these mechanisms interact and fit together in a timeline has not been well-established. It is important to understand the primary and most important pathogenic events in order to properly target ALS therapies. Using genetically encoded and chemical fluorescent indicators, we have established a central role for the dysregulation of calcium homeostasis in motor neurons expressing mutant SOD1.
Methods: Mouse dissociated spinal cord cultures were prepared from embryonic mice as described previously (Durham et al, 1997), and plasmids encoding wild-type and mutant SOD1, TDP-43, and TLS-FUS were introduced by intranuclear microinjection. To measure mitochondria and ER calcium levels, we co-injected plasmids encoding mitochondrial ratiometric pericam or DER1 cameleon, respectively. Cytosolic calcium levels and mitochondrial membrane potential were measured using fura-2 and TMRM, respectively. Motor neurons were then imaged at several time points following microinjection.
Results: Measurement of calcium levels within different intracellular compartments indicated an early (day 1) elevation of mitochondrial calcium, which was accompanied by a loss in mitochondrial membrane potential and a rounding of their shape. This was followed by an elevation in ER calcium (day 3) and finally a rise in cytosolic calcium (day 5). Motor neurons with mutant SOD1 inclusions had even higher levels of cytosolic calcium, demonstrating a relationship between elevated calcium levels and inclusion formation. Expression of the calcium binding protein, calbindin D-28K, caused a reduction of cytosolic calcium and reduced mutant SOD1 inclusion formation whereas reducing inclusion formation by treatment with geldanamycin had no effect on calcium levels. Using the same imaging techniques in motor neurons, we have demonstrated that neither TDP-43 nor TLS-FUS mutants have a significant effect on mitochondrial shape; other parameters remain to be investigated.
Discussion and conclusions: Using live cell imaging techniques in primary motor neurons, we have established a timeline for calcium dysregulation in motor neurons expressing mutant SOD1. Calcium plays an early and central role in the demise of motor neurons. Using this model, we plan to examine the pathogenic events caused by the expression of other ALS-linked genes such as TDP-43 and TLS-FUS in order to find common disease mechanisms and potential therapeutic drug targets.
P62 CHRONOLOGICAL SEQUENCE OF EVENTS IN THE PATHOGENIC CASCADE IN ALS
FARG M1, SOO K1, WALKER A2, TURNER B2, HORNE M2, ATKIN J1,2
1Department of Biochemistry, La Trobe University, Bundoora, Australia, 2Howard Florey Institute, University of Melbourne, Melbourne, Australia
E-mail address for correspondence: [email protected]
Keywords: disease mechanisms, protein misfolding, ER stress
Background: In ALS/MND, the pathology centres on central and peripheral motor neurons, and the severely damaged motor neurons observed at post mortem are the end stage of a pathophysiological cascade. Many mechanisms have been implicated in pathology, including ER stress, axonal transport, intracellular inclusion formation, and apoptosis, but their exact role in disease and the sequence in which they occur is unclear.
Objective: In this study, we aimed to identify the earliest events occurring in disease because these events are most likely to be involved in triggering the pathological cascade.
Methods: We used the motor neuron cell line, NSC-34, and neuronal Neuro2a cells transiently transfected with human wild-type and mutant SOD1 constructs tagged with EGFP. We also examined lumbar spinal cords obtained from transgenic SOD1G93A mice at pre-symptomatic (p10, p30, p60), symptom onset (p90) and disease end stage (p120).
Results and discussion: We have established that in motor neuron cell lines expression of SOD1 begins at 10 h. At t 14 hours we detected increased levels of ubiquitinated proteins, at 16 h ER stress is triggered and binding of mutant SOD1 to dynein and cellular transport proteins occurs. However the formation of mutant SOD1 oligomers, intracellular inclusions does not occur until 24 h post transfection. Interestingly, Bax recruitment occurred at 18 hours but apoptotic cell death was not triggered until after 24 h. In SOD1 mice, a physical interaction between mutant (but not wildtype SOD1) with proteins involved in intracellular transport was detected as early as postnatal age 10 days, 20 days prior to the onset of ER stress.
Conclusion: This study suggests that the first upstream events in the pathogenic cascasde triggered by mutant SOD1 are protein misfolding, perturbation of cellular transport and ER stress. The formation of mutant SOD1 intracellular inclusions and oligomers occurs relatively late in pathology, after the triggering of apoptosis by recruitment of Bax to mitochondria.
P63 MODELING ALS AXONAL PATHOLOGY IN VITRO
KING A, HOSIE K, BLIZZARD C, VICKERS J, DICKSON T
Menzies Research Institute, Hobart, Tasmania, Australia
E-mail address for correspondence: [email protected]
Keywords: axon pathology, excitotoxicity, glia
Background: Examination of ALS post-mortem tissue and mouse models of ALS reveals the presence of several types of pathological lesion and provides insight into the pathogenic mechanisms that underlie this devastating disease. Particularly prominent in mouse models of ALS from early time points is evidence of axon degeneration and dysfunction, including distal axon die back from the neuromuscular junction and more proximally within the axon, the presence of large swollen axon segments or spheroids.
Objectives: We have used primary cell culture techniques to investigate how pathological mechanisms implicated in ALS result in the types of pathology described. We have investigated how axonal pathology affects the function of the neuron, and in particular we are focused on determining the role of different neuronal compartments (somatodendritic or axonal) in the development of pathology. These data will be important for provision of therapeutic intervention in ALS.
Methods: Spinal motor neurons or cortical neurons were derived from embryonic rodents and grown for up to 21 days in vitro on glial feeder layers derived from neonatal rodents or Poly-L-lysine/laminin. To determine the role of neuronal compartments, cultures were grown in compartmentalized microfluidic chambers (Xona), which allow physical separation of the somatodendritic compartments from the axonal compartment for manipulation and analysis.
Results: In investigations to date we have been able to model two distinct types of axonal pathology that may have relevance to ALS. Spinal rat or mouse motor neurons, chronically exposed to kainic acid for up to 24 hours, developed bulbous swellings in the distal portion of their axons, which was associated with mislocalization of non-phosphorylated neurofilament protein to this region. This pathology typically progressed to cell death within 48 hours. Preliminary investigations using cortical neurons in compartmentalized chambers have supported the hypothesis that exposure of the somatodendritic compartment to excitotoxins can result in degeneration of the untreated axon segment. In other experiments we have demonstrated that spinal mouse motor neurons grown on a mixed glial feeder layer spontaneously developed swollen axon segments or spheroids filled with neurofilament proteins and organelles. These structures were not present when cells were grown in the absence of glial cells or in glia conditioned media. Strikingly, these spheroids were not associated with apoptosis or rapid cell death, but did involve abnormalities in axonal transport. The connection between distal and proximal axon pathology is the focus of ongoing investigation.
Discussion and conclusion: These data indicate that excitotoxicity can result in distal axon degeneration and that proximal axon spheroids can be the result of disturbed neuron-glia interaction. These primary cell culture models of axon pathology are useful tools for investigating mechanisms of axon degeneration and dysfunction in ALS.
P64 INVOLVEMENT OF THE mTOR SIGNALING IN AMYOTROPHIC LATERAL SCLEROSIS
ZONA C1,2, PIERI M1,2, CANU N1,3, CARUNCHIO I1,2, CAIOLI S1,2
1University of Rome Tor Vergata, Department of Neuroscience, Rome, Italy, 2Fondazione S. Lucia, Rome, Italy, 3Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy
E-mail address for correspondence: [email protected]
Keywords: rapamycin, cortical neurons, electrophysiology
Background: Many papers have reported cortical hyperexcitability both in ALS patients and in the transgenic mouse model G93A of ALS. The involvement of ionic channels in the ALS pathology has been shown in G93A cortical neurons. Since hyperexcitability is an ALS feature, compounds able to reduce the neuronal excitability could be potential drugs useful in ALS clinical treatments. In this context, the mammalian target of rapamycin (mTOR) pathway has been shown to regulate ion channel synthesis and localization, thereby controlling neuronal excitability. In addition, mTOR is a key regulator of cell growth and proliferation and regulates autophagy. Interestingly, it has been reported that autophagy reduces mutant SOD1-mediated toxicity and that the induction of autophagy decreases mutant SOD1 protein levels.
Objectives: The aim of this work is to verify whether the excitability of G93A neurons is modified by rapamycin and whether the mTOR pathway is altered in G93A cortical neurons compared to controls.
Methods: G93A cortical neurons were used for patch clamp and Western blotting experiments. Current clamp cortical neuron recordings were performed and analysed, as previously reported. Proteins were visualized using antibodies to mTOR, PmTOR, Akt, Pakt, p70S6 and Pp70S6. Data are presented as mean + standard deviation (SD). Values of P<0.05 were considered statistically significant.
Results: To study the excitability of control non transgenic neurons and of G93A cortical neurons in control conditions and following rapamycin exposure (50 nM, 24 h), current steps from +40 pA to +200 pA were injected to elicit action potentials. In all tested cells, the inverse of the first interspike interval was taken as an estimate of the cell firing frequency. The treatment of G93A neurons with rapamycin significantly decreased the hyperexcitability (P<0.02) as far as control values. To verify whether in G93A neurons the decreased excitability induced by rapamycin was mediated by the mTOR pathway, we performed Western blotting analysis. We found that rapamycin was able to significantly reduce the level of mTOR phosphorylation both in G93A and in Control neurons. In addition, G93A neurons presented significantly higher levels of Pp70S6 compared to Control neurons, indicating the involvement of the mTOR signalling in ALS pathology because this level was reversed by rapamycin.
Discussion and conclusion: These results indicate the involvement of mTOR pathway in ALS pathology and that mTOR may regulate the electrical activity of the single neuron and mediate mechanisms of neuronal excitability. In conclusion, although our findings do not directly support a pathogenic mechanism of ALS, they point to the importance of the mTOR pathway in ALS pathology, potentially offering novel avenues for developing therapeutic strategies.
Acknowledgement: This work is supported by Wyeth Lederle S.p.A. Italy to C.Z.
P65 EFFECT OF ACYLATED STERYL GLUCOSIDES ON ALPHA-SYNUCLEIN AGGREGATION AND TOXICITY
USUKI S, MATSUO Y, KAMITANI T, YU RK
Medical College of Georgia, Augusta, Georgia, United States
E-mail address for correspondence: [email protected]
Keywords: ALS-PDC, acylated steryl glucoside, alfa-synuclein
Background: The causal factors for sporadic or age-related neurological diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Alzheimer's disease (AD) are still unknown, and disease progression shows involvement of genetic or environmental factors (1). The Guamanian variant of ALS-parkinsonism dementia complex (ALS-PDC) has received much attention, particularly with respect to its potential causes. These causes include its genetic origin, involvement of certain metals in drinking water, eg, low levels of calcium/magnesium and high levels of aluminum; the infectious agents cyanobacteria and Helicobacter pylori, traditional foods made from flying foxes, and consumption of flour made from cycad seed (Cycas micronesica) (2). It has been suggested that cycad seed may be an important factor in the pathogenesis of ALS-PDC as evidenced by a positive correlation between consumption of cycad seed and prevalence of the disease. Several compounds have been isolated from cycad seeds and are suspected to contribute to the motor neuron toxicity. Recently, β-N-methylamino-L-alanine (BMAA) and acylated steryl glucoside (ASG) have been reported to be principal components of cycad neurotoxin by a feeding experiment in mice.
Objectives: Acylated steryl glucosides (ASGs) are ubiquitously distributed in edible plant sources. To know whether edible plant-derived ASGs are a casual factor in ALS-PDC, soybean ASG (S-ASG), pre-germinated brown rice ASG (P-ASG), and Helicobacter pylori-derived ASG (H-ASG) were tested for an in vitro aggregation of α-synuclein and α-synuclein toxicity for yeast cells.
Methods: For α-synuclein aggregation reactions, human normal α-synuclein protein (80 μM) was incubated in a ATP regeneration system with 20 μg/mL of S-ASG, P-ASG and H-ASG, respectively. The aggregate formation was determined by a fluorescence of Thioflavin-T.
For the yeast-cell toxicity experiment, S-ASG, P-ASG and H-ASG were tested for cell growth on the plate of α-synuclein transformant and a control vector transformant of Saccharomycces cerevisiae.
Results: Compared with controls, S-ASG and P-ASG increased α-synuclein aggregation (1.4- and 1.3-fold higher, respectively), but H-ASG was not effective. For the yeast cell toxicity, α-synuclein transformants exhibited a growth defect on control plates as compared to a vector transformant. S-ASG and P-ASG had no effect on the vector transformant, but exhibited inhibition of cell growth on α-synuclein transformants.
Discussion and conclusions: The finding that S-ASG and P-ASG enhanced α-synuclein aggregation and toxicity suggests that ASGs represent a potential agent for disease development and progression, although these lipids are ubiquitously distributed in edible plant sources. S-ASG and P-ASG include a β-glucoside, but H-ASG includes an α-glucoside. The β-glucoside in the ASGs may be responsible for α-synuclein aggregation and toxicity.
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