THEME 3 IN VITRO EXPERIMENTAL MODELS

P44 SIGNALS FROM CNS EXTRACTS OF SOD1-G93A MICE PROMOTE hMSCS NEUROGENIC DIFFERENTIATION IN VITRO

ZHANG C, ZHAO C, ZHOU S, XIE Y, WANG Y

Department of Neurology, First Affilated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China

E-mail address for correspondence: [email protected]

Keywords: SOD1-G93A mice, CNS extracts, neurogenic differentiation

Background: Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disease, currently without any effective therapy. Multiple advantages make mesenchymal stromal cells (MSC) a good candidate for cellular therapy of ALS, but the capabilities of MSCs to differentiate into neurons in ALS mice still need to be proven.

Objectives: To investigate whether signals from pathological CNS extracts of SOD1-G93A (20 weeks of age) ALS mice promote hMSCs neurogenic differentiation in vitro.

Methods: The brain and spinal cord of SOD1-G93A mice at symptomatic stages (20 weeks of age) and normal mice were removed and homogenized by adding DEME, respectively. The supernatant was collected and filtered, then turned into a conditioned medium for the hMSCs. Passage 5 of hMSCs was seeded into a 6-well plate and cultured with the conditioned medium. Proliferaton of hMSCs was observed by phase-contrast light microscope. The cell viability and the phenotype of hMSCs and neural like cells were analyzed by FMC, RT-PCR, immunofluorescence, and Western blots, respectively.

Results: Incubation of hMSCs with the conditioned medium prepared from the CNS extracts of SOD1-G93A mice resulted in a time-dependent morphological change from fibroblast-like into neuron-like. Similarly, the expression of Nestin and subsequent beta-tubulin III, NSE and GAP43 increased over time. Moreover, signals in the pathological CNS extracts of SOD1-G93A mice were more effective in promoting hMSCs neurogenic differentiation than those in physiological extracts of normal adult mice.

Conclusion: These results show that the pathological condition of ALS is endowed with capacity to induce hMSCs neurogenic differentiation and that the hMSCs may be a potential candidate in cellular therapy for ALS.

P45 PREVENTION OF EXCITOTOXIC MOTONEURON DEATH BY LITHIUM IN ORGANOTYPIC SLICE CULTURES OF SPINAL CORD

BRUNET N, TARABAL O, ESQUERDA J E, CALDERÓ J

Facultat de Medicina, Universitat de Lleida, Irblleida, Catalonia, Spain

E-mail address for correspondence: [email protected]

Keywords: lithium, excitotoxicity, motoneuron

Background: Several in vitro and in vivo studies have reported the neuroprotective effects of lithium (Li) and suggest its potential in the treatment of acute and chronic neurological disorders. Recently, it has been demonstrated that Li delays the progression of amyotrophic lateral sclerosis (ALS) in animal models and humans [1]. We have recently developed an organotypic slice culture of chick embryo spinal cord that provides a convenient model for in vitro studies on mature motoneurons (MNs) [2].

Objectives: Since we have found that chick MNs in organotypic spinal cord cultures are vulnerable to excitotoxic insults, the presumptive neuroprotective effects of Li in this paradigm has been investigated.

Methods: Slices of lumbar spinal cord from E16 chick embryos were cultured for 7 days in vitro (DIV) and treated with different regimens of kainate (KA) in the absence or presence of LiCl (4–10 mM) and chronically applied. MN survival and ultrastructural morphology was evaluated and the neuroprotective mechanisms of Li on this system were analyzed. Moreover, LiCl was chronically administered in ovo in order to evaluate its effects on programmed cell death of MNs during development.

Results: LiCl inhibited excitotoxic MN death induced by KA in a dose dependent manner. However, MNs rescued by Li displayed conspicuous structural changes consisting of an accumulation of neurofilaments, increased large dense core vesicles and autophagic vacuoles. In congruence with these changes, an increased immunostaining was found for: phosphorylated neurofilaments, calcitonin gene-related peptide (CGRP) and LC3. Li treatment resulted in an inhibition of GSK-3β and indirubin-3’-monoxamine, a specific GSK-3β inhibitor, mimicked the effects of Li. LY294002, a PI3-K inhibitor, blocked the anti-excitotoxic effects of Li. The involvement of the inositol pathway in the neuroprotective effects of Li was also explored with negative results. Li was not able to prevent programmed (apoptotic) MN death when administered in ovo.

Conclusions: In an in vitro paradigm, Li protects mature MNs against excitotoxic injury induced by KA. These effects appeared to be mediated by the inhibition of GSK-3??and PI3-K/Akt pathways. Li-rescued MNs remains alive but exhibited prominent structural abnormalities indicative of chronic pathology. In addition, Li does not prevent developmentally regulated and target dependent apoptotic MN death in the chick embryo.

P46 HUMAN NMDA RECEPTORS MODULATION BY SERA FROM AMYOTROPHIC LATERAL SCLEROSIS PATIENTS AND MUTATED SUPEROXIDE DISMUTASE TRANSGENIC RATS

TEXIDO L¹, HERNANDEZ S², POVEDANO M³, SOLSONA C¹, ESQUERDA J², MARSAL J¹

¹Ciberned Laboratory of Cellular and Molecular Neurobiology, Department of Pathology and Experimental Therapeutics, Medical School-Bellvitge Campus, Idibell-University of Barcelona, L'Hospitalet de Llobregat, Spain, ²Department of Cellular Neurobiology. Medical School, University of Lleida, Spain, ³Neurology Unit, Bellvitge Hospital, L'Hospitalet de Llobregat, Spain

E-mail address for correspondence: [email protected]

Keywords: excitotoxicity, NMDA, sera

Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease, characterized by the selective degeneration of the superior motor neurons in the motor cortex and of the inferior motor neurons in the brain-stem and spinal cord. So far, no treatment has achieved significant improvement. The familial form of the illness is associated with the mutation of the superoxide dismutase enzyme (SOD-1), but it accounts for fewer than 10% of cases; the rest, more than 90%, correspond to the sporadic form.

In this study we tested the effect of sera from sporadic ALS patients and from mutated human SOD-1 (mSOD1 G93A) transgenic rats on N-methyl-D-aspartate receptors (NMDAR). We hypothesize that an endogenous excitotoxic factor is implicated in neuronal death in ALS, mediated by the activation of NMDAR noncanonical signalling pathways [1].

Sera from ALS patients or healthy subjects were pretreated to inactivate complement pathways and dialysed to remove glutamate. Sera from mSOD1 G93A rats were obtained at different stages of the neurodegenerative progression. Sera from transgenic rats were also pretreated to eliminate complement system and glutamate. Human NMDAR were expressed in Xenopus laevis oocytes, and glutamate-induced currents were recorded using the two electrode voltage clamp technique

We observed that sera from sporadic ALS patients induced transient oscillatory currents in Xenopus oocytes expressing NMDAR with a total electric charge (14594±603 nC, n = 76 oocytes) significantly higher than the electric charge carried by currents induced by sera from healthy subjects (4219±206 nC, n = 76 oocytes) p< 0.001. The currents were inhibited by MK-801, a noncompetitive blocker of NMDAR. Results of sera from mSOD1 G93A transgenic rats were similar to those of sera from ALS patients; samples from patients with another type of neuromuscular disease did not exert this effect.

The oscillatory currents recorded are due to internal calcium mobilization [2], our data therefore agree with the view that ALS patients sera contain some soluble factor/s that activates intracellular calcium concentration.

P47 THE INVOLVEMENT OF THE KYNURENINE PATHWAY AND INFLAMMATION IN AMYOTROPHIC LATERAL SCLEROSIS

CHEN Y¹, STANKOVIC R³, CULLEN K³, MEININGER V⁵, BREW BJ⁴, GUILLEMIN GJ²

¹University of New South Wales, Sydney, ²Centre for Immunology, St. Vincent's Hospital, Sydney, ³University of Sydney, Sydney, ⁴Department of Neurology, St. Vincent's Hospital, Sydney, NSW, Australia, ⁵Hopital Pitie-Salpetriere, Paris, France

E-mail address for correspondence: [email protected]

Keywords: kynurenine pathway, quinolinic acid, NSC-34

Background: Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease that selectively targets motor neurons in the motor cortex, brain stem and spinal cord. The kynurenine pathway (KP), which catabolizes tryptophan and generates neuroactive compounds such as picolinic acid (PIC) and quinolinic acid (QUIN), is emerging as a possible pathogenic component of ALS. The first enzyme in the KP, indoleamine-2,3 dioxygenase (IDO), can be stimulated by cytokines.

Objectives: This study aims to characterize the KP in ALS patients and the NSC-34 rodent motor neuron cell line; and assess the effect of QUIN toxicity on NSC-34 cells.

Methods: GC/MS and HPLC were used to analyze CSF and serum QUIN and PIC levels and IDO activity, respectively, of ALS patients (n = 155) and controls (n = 20). Antibodies to HLA-DR, IDO and QUIN were used on paraffin embedded ALS human spinal cord and motor cortex sections. In NSC34 cells, RT-PCR and immunocytochemistry were used to characterize KP enzymes and catabolites. LDH test assessed the effect of QUIN, with and without antagonist MK801, APV and memantine.

Results: IDO activity was significantly increased in ALS patients and ALS serum and CSF PIC levels were significantly lower but serum QUIN levels were significantly higher. ALS motor cortex and corticospinal tracts showed extensive microglial activation. IDO immunoreactivity was evident in the ventral horn motor neurons and in the motor cortex neurons, including remaining Betz cells. QUIN immunoreactivity was seen mostly in glial cells and in motor cortex and spinal cord neurons. NSC34 cells stained positive for KP enzymes and catabolites and RT-PCR showed the presence of most of the KP enzymes. LDH production displayed a dose dependant increase with QUIN which was partially inhibited by MK801 and completely inhibited by MK801, APV and memantine combined.

Conclusion: Our results provide in vivo and in vitro support for the involvement of KP in ALS.

P48 A NOVEL MOTONEURON DEATH PATHWAY MEDIATED BY THE TNF SUPERFAMILY LIGAND, LIGHT

AEBISCHER J, MOUMEN A, PETTMANN B, RAOUL C

Inserm, The Mediterranean Institute of Neurobiology, Marseille, France

E-mail address for correspondence: [email protected]

Keywords: motoneuron death signalling, LIGHT, caspase

Death pathways restricted to specific neuronal classes could explain the selectivity of neuronal loss in neurodegenerative diseases, such as the loss of motoneurons in amyotrophic lateral sclerosis (ALS). We previously showed that Fas-induced death of motoneurons involves a motoneuron-specific cell death pathway, which is exacerbated in motoneurons expressing ALS-linked mutated SOD1 [1],[2]. Importantly, involvement of the Fas death pathway in the pathogenesis has been documented [3]. However, the Fas death pathway may not be responsible for the loss of all motoneurons, suggesting that other death pathways might be implicated.

LIGHT is a member of the tumor necrosis factor receptor (TNFR) superfamily, which upon binding to the herpes virus entry mediator (HVEM) and/or the lymphotoxin-β receptor (LT-βR) can trigger the death program. In this study we show that cultured motoneurons express both LT-βR and HVEM, and that soluble recombinant LIGHT induces death of approximately 50% of motoneurons in a dose-dependent manner. LIGHT-mediated death might be motoneuron-specific since striatal, cortical, hippocampal and sensory neurons are resistant to LIGHT effect. We show that LIGHT is additive to FasL since both ligands together induce death of about 70% of cultured motoneuron. To our surprise, LIGHT-induced motoneuron death is triggered in a Caspase-9 and -6 dependent but Caspase-8 and -3 independent manner.

This study indicates that LIGHT acts as a selective trigger of motoneuron death in vitro, which involves an unconventional caspase pathway. We are currently investigating the potential involvement of LIGHT death signalling in the pathogenesis of motoneuron disease.

P49 DO ALS ASSOCIATED SOD1 MUTANTS INTERACT WITH LIPID MEMBRANES?

BYSTRÖM R, AISENBREY C, GRÖBNER G

Dept of Chemistry, University of Umea, Sweden

E-mail address for correspondence: [email protected]

Keywords: SOD1, membranes, stability

More than 130 mutations in the human gene encoding the cytosolic homodimeric enzyme Cu/Zn-Superoxide Dismutase (SOD1) have been linked to the familial form of ALS (fALS). The key event in SOD1 associated fALS seems to be the pathological formation of toxic protein aggregates formed by initially unfolded or partly structured SOD1 protein mutants.

We have compared the folding behavior for a set of SOD1 mutants. The common denominator for all of them is the move of the folding equilibrium towards a denatured monomer. It is believed that these denatured monomers gain properties which might be toxic to the cell. The apo form of the protein is partly folded under reduced conditions and therefore exposes both a hydrophobic dimer-interface but also charged patches. This exposure is ideal for interactions with cellular membranes to occur.

Our results show electrostatic interactions between the reduced apo form of ALS associated SOD1-mutants and charged membrane surfaces. The interaction changes the secondary structures of these mutants in a way that is quite different from the situation found in the membrane free aqueous environment. Mutants of SOD1 seem to interact differently compared to wildtype SOD1.

P50 CU,ZN SUPEROXIDE DISMUTASE IS SECRETED VIA EXOSOMES IN A CELL MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

GOMES C¹, KELLER S², ALTEVOGT P², COSTA J¹

¹Instituto de Tecnologia Química e Biológica, Oeiras, Portugal, ²German Cancer Research Center (DKFZ), Heidelberg, Germany

E-mail address for correspondence: [email protected]

Keywords: SOD1, exosomes, NSC-34 cells

Background: A familial form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS), is caused by dominant mutations in the cytosolic Cu,Zn superoxide dismutase (SOD1). There has been evidence for secretion of SOD1, by an unknown mechanism that might involve a vesicle dependent pathway. SOD1 is a cytoplasmic protein that lacks a signal peptide, so there must be an alternative mechanism for the extracellular export of this protein.

Exosomes are small membrane vesicles (30–100 nm diameter) secreted by various cell types, including neurons as a consequence of fusion of multivesicular late endosomes/lysosomes with the plasma membrane.

Objectives: To investigate the capacity of NSC-34 cells expressing wild-type or mutant hSOD1 to secrete SOD1 protein via exosomes.

Methods: Stable mouse motor neuron-like NSC-34 cells overexpressing human SOD1 wild-type hSOD1^wt (NSC-34/hSOD1^wt) and mutant hSOD1^G93A (NSC-34/hSOD1^G93A) have been used as an ALS cellular model.

Results: Wild-type and mutant SOD1 were present in the supernatant medium from NSC-34 cells, and they were found to be secreted in association with a membrane fraction, vesicle tethering containing a mixture of exosomes and apoptotic membrane blebs, that pelleted at 100,000×g. Sucrose density gradient separation of this fraction showed that wild-type and mutant SOD1 were found between 0.5 and 1.16M sucrose and co-localized with the exosomal marker CD9. Therefore, SOD1 secretion occurred via exosomes. p 115, a cytosolic and Golgi apparatus protein involved in vesicle tethering was also found in exosomes, contrary to the endoplasmic reticulum protein calnexin.

Discussion and Conclusions: SOD1 secretion mediated by exosomes might constitute a new cellular mechanism of cell-to-cell communication and dissemination of mutant SOD1 toxicity in ALS pathogenesis.

P51 AAV-MEDIATED EXPRESSION OF WILDTYPE OR MUTANT VAP-B TRIGGERS DEATH OF EMBRYONIC MOTONEURONS THROUGH IMPAIRMENT OF ENDOPLASMIC RETICULUM FUNCTIONS

LANGOU K¹, MOUMEN A¹, AEBISCHER J¹, PETTMANN B¹, AEBISCHER P², RAOUL C¹

¹Inserm, The Mediterranean Institute of Neurobiology, Marseille, France, ²Ecole Polytechnique Fédérale de Lausanne, EPFL, Lausanne, Switzerland

E-mail address for correspondence: [email protected]

Keywords: ALS, VAP-B, ER stress

Amyotrophic Lateral Sclerosis (ALS) is a late-onset motoneuron disorder characterized by selective loss of motoneurons in the spinal cord, brainstem and motor cortex. Disturbed homeostasis of the early secretory pathway is a pathological hallmark of the disease. However, molecular mechanisms leading to motoneuron death following impairment of the endoplasmic reticulum (ER) function remain unclear.

Recently, an autonomic dominant mutation (P56S) in VAMP-associated protein subtype B (VAP-B) has been associated with familial forms of ALS. VAP-B, an endoplasmic reticulum (ER) resident protein and ER-Golgi intermediate vesicle, has been proposed to play a role in ER-Golgi transport and in the unfolded protein response (UPR). A P56S mutation leads VAP-B to form cytosolic aggregates that might impair the early secretory pathway. It remains unknown how VAP-B^P56S leads to motoneuron degeneration.

Adeno-Associated Viruses (AAV), which can transduce up to 90% of purified motoneurons, were used to express either wild type (hVAPB^wt) or mutant forms (hVAP-B^P56S) of human VAP-B. We show that hVAP-B^WT localizes to the ER and ER-Golgi intermediates, whereas hVAP-B^P56S forms cytosolic aggregates in motoneurons. Interestingly, over-expression of both hVAPB^wt and hVAP-B^P56S induces death of motoneurons but has no effect on cortical and striatal neurons survival. This selective death of motoneurons correlates with an increased susceptibility of motoneurons to ER stress as elicited by thapsigargin. We show that VAP-B-induced motoneuron death requires activation of p38 kinase, the ER-associated caspase-12 and the effector caspase, caspase-3. Additionally, we observe that the VAP-B killing effect involves a Ca^2 +-dependent pathway acting through calpain activation. We also observed that expression of hVAP-B^P56S or hVAPB^wt in motoneurons leads to fragmentation of the Golgi apparatus. These data suggest that overexpression of either VAP-B^WT or VAP-B^P56S leads to death of motoneurons by disturbing the ER homeostasis. We are currently investigating molecular mechanisms of motoneuron susceptibility to ER stress elicited by VAP-B.

P52 EFFECT OF MITOCHONDRIAL SUBSTRATES ON CELL SURVIVAL AND ROS GENERATION IN AN IN VITRO MODEL OF PROPOSED MECHANISMS OF ALS PATHOGENESIS

HEMENDINGER R, ARMSTRONG E, PANOV A, BROOKS BR

Carolinas Medical Center, Charlotte, NC, United States

E-mail address for correspondence: [email protected]

Keywords: neurotoxicity, neurorescue, cell death

Background: Metabolic changes and mitochondrial dysfunction have been implicated in the pathogenesis of ALS and other motor neuron diseases. We have shown that there are intrinsic differences between brain (BM) and spinal cord mitochondria (SCM) in relation to respiratory substrates, which become even greater in the presence of mutant SOD1. These observations suggest that metabolic differences between BM and SCM may be both of pathogenic and therapeutic significance for ALS.

Objective: The ability of mitochondrial substrates to rescue a motor neuron-like cell line from cell death in our cell-based model of ALS pathogenesis utilizes neurotoxic agents with mechanisms of action similar to the pathogenic mechanisms seen in ALS. Based on our in vivo work with SOD1 rats, we studied the effects of glutamate, pyruvate and malate, alone or in combination, on cell viability and reactive oxygen species (ROS) generation in this sporadic ALS model system.

Methods: The toxicity of glutamate, pyruvate or malate was examined in differentiated NSC-34 cells. Cell death was assessed after a 24 hour (hr) exposure to doses ranging from 0 mM to 7.5 mM for each substrate. We examined whether glutamate, pyruvate and malate could improve cell survival of NSC-34 cells following (1) staurosporine-induced caspase-3 activation, (2) thapsigargin-induced endoplasmic reticulum Ca^2 +-ATPase inhibition, (3) carbonyl cyanide chlorophenyl hydrazone (CCCP)-induced mitochondrial uncoupling and (4) hydrogen peroxide-induced free radical production. NSC-34 cells were exposed to the mitochondrial substrates (singly, in pairs or as a triple combination) simultaneously with each of the neurotoxic agents listed and then assayed at 24hr for viability. Cell death was determined using nuclear staining and morphology with Hoechst 33342 and propidium iodide.

Results: Mitochondrial substrates were not toxic to NSC-34 cells using a nuclear morphology staining assay. NSC-34 cells were then simultaneously exposed to the substrates (alone or in combination) and either thapsigargin or hydrogen peroxide for 24hrs. The triple combination of glutamate, pyruvate and malate improved viability when given simultaneously with hydrogen peroxide as compared to the inducer alone (increased by 58.8%; p ≤0.05). Only the combinations containing pyruvate or pyruvate alone showed improved survival (increased by 59–63%). No significant improvement in cell survival was observed with thapsigargin, although there was a trend towards improvement with addition of the triple combination (increased by 11.6–13.6%). Additional studies examining the other neurotoxic agents and effects on ROS generation are currently underway.

Conclusions: Mitochondrial substrates, glutamate, pyruvate and malate, particularly pyruvate, improve differentiated NSC-34 cell survival following hydrogen peroxide-induced cytotoxicty compared with the effects of these substrates on other toxicological mechanisms and warrant further study as potential therapeutic agents.

P53 ACTIVATION OF THE HEAT SHOCK RESPONSE IN A CELLULAR MODEL OF ALS

KALMAR B, GREENSMITH L

UCL Institute of Neurology, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: Heat shock response, arimoclomol, celastrol

Background: Heat shock proteins (Hsps) are chaperones that aid protein folding during protein synthesis and under conditions of stress Hsps protect cells from stress. Although up-regulation of Hsps rescues motoneurons in a mouse model of ALS, the relationship between increased Hsp expression and neuronal survival is not straightforward ([4],[5]). In SOD1 mice, over-expression of Hsp70 has no beneficial effects [6] whereas overexpression of Hsp27 offers marginal benefits [8]. However, induction of multiple Hsps is more effective than individual family members [1],[7]. Some inducers of Hsps, such as arimoclomol and celastrol act on the transcription factor of Hsps, HSF-1, thereby achieving induction of members the whole heat shock response (HSR) machinery [3],[9]

Objectives: We compared the neuroprotective and hsp inducing effects of two inducers of hsp expression, celastrol and arimoclomol following exposure of primary motoneurons to apoptotic stimuli in-vitro.

Methods: Primary rat motoneuron cultures were obtained [2]. Cells were exposed to either 200 nMS taurosporin or 100 µM H₂O₂ and upregulation of Hsps was achieved by co-treating some cultures with either arimoclomol (0.1–100 µM) or celastrol (0.01–3 µM). The effects on motoneurons were assessed by immunostaining for the inducible Hsp70 and activated caspase-3. Motoneuron survival was assessed by counting surviving MAP-2 cells.

Results: H₂O₂ or Staurosporin treatment leads to the activation of apoptosis, and treated cells show increased expression of activated caspase-3. Treatment of stressed cells with hsp inducers induces expression of Hsp70. However, although both arimoclomol and celasterol induce Hsp70, their neuroprotective effects are very different. Thus, whereas arimoclomol protects motoneurons from apoptotic death at a concentration of 1 µM, celastrol showed no neuroprotective effects at any dose applied.

Discussion: Results indicate that not all agents that activate the HSR will necessarily have neuroprotective effects. Furthermore, the mechanism of action of arimoclomol may involve activation of other coupled mechanisms that interfere with the apoptotic pathway, a mechanism that is not activated in celastrol treated cells.

P54 INTERACTIONS BETWEEN SOD1 AND SMALL HEAT SHOCK PROTEIN 22

KWOK A¹, TURNER B¹, AGASHE V², TALBOT K¹

¹MRC Functional Genetics Unit, University of Oxford, ²Neurosciences Group, Weatherall Insitute of Molecular Medicine, Oxford, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: heat shock protein, SOD1, chaperone

Background: Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive neuromuscular disease characterized by progressive loss of motor neurons in the brain and spinal cord. A proportion of familial cases are due to mutations in the gene for SOD1. Whilst the mechanisms underlying mutant SOD1-mediated cytotoxicity are currently unknown, a possible mechanism may be an impairment in the protein quality control machinery of motor neurons. Heat shock protein 22 (Hsp22/HSPB8) is a molecular chaperone belonging to the small heat shock protein family which is selectively expressed in motor neurons and mutations affecting residue 141 of the Hsp22 protein lead to hereditary motor neuropathy.

Objectives: To determine whether Hsp22 levels are altered by mutant SOD1 and whether any interaction may be relevant to SOD1 mediated neuronal cell death

Methods: NSC-34 cells stably overexpressing SOD1 mutants G37R, and G93A, were created and Hsp22 transcript and protein levels were measured by rtPCR and Western blotting respectively. The effect of transfection of wild type and mutant Hsp22 on SOD1 aggregate formation was assessed by fluorescence microscopy.

Results: Western blots showed a decrease in Hsp22 protein expression compared to cells expressing wild type SOD1. In contrast to Hsp22 protein levels, Hsp22 mRNA, as determined by RT-PCR, remains unchanged suggesting that the decrease in Hsp22 protein levels occurs post-transcriptionally. The use of proteasome inhibitor, MG132, was found to restore Hsp22 levels in mutant SOD1 expressing NSC-34 cells to levels comparable to wild type (WT) SOD1 expressing cells, suggesting that mutant SOD1 may specifically modulate proteasome activity. Over expression of wild type Hsp22 suppressed the formation of SOD1 protein aggregates while expression of K141N mutant Hsp22 led to enhanced aggregate formation.

Conclusions: Hsp22 displays a pattern of expression which is consistent with a potential role as a motor neuron specific chaperone. Expression studies in neuronal cells stably expressing mutant and wild type SOD1 indicate that mutant SOD1 may exert a specific effect in down regulating Hsp22 levels via the proteasome. Conversely, mutant Hsp22 may be deficient in chaperoning SOD1 and preventing the formation of insoluble protein aggregates.

P55 MANIPULATION OF THE HEAT SHOCK RESPONSE IN MSOD1 ASTROCYTES

YIP J, KALMAR B, GREENSMITH L

Institute of Neurology, UCL, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: astroglia, heat shock proteins, heat shock response

Background: Although the precise mechanism by which the mutant SOD1 (mSOD1) results in selective motoneuron degeneration remains unclear, it has recently become clear that mSOD1 toxicity is not restricted to expression within motoneurons alone. Several lines of evidence have shown that the expression of mSOD1 in non-neuronal cells such as astrocytes, microglia and oligodendrocytes plays a critical role in motoneuron degeneration in mSOD1-ALS ([17–21]). In view of the role that non-neuronal cells play in ALS pathogenesis, it is possible that targeting these cells may be a successful approach in the development of a therapeutic strategy for ALS. In particular, exploitation of endogenous defense mechanisms that exists within these different cell types, particularly in astrocytes, may be effective. The heat shock response (HSR) is one such ubiquitious cellular defense mechanism that involves the expression of a family of proteins called heat shock proteins (Hsps). These proteins are molecular chaperones that carry out a range of house-keeping functions and protect cells from a variety of cell stress. Therefore, a better understanding of the HSR in different cell populations may help to establish whether this mechanism may a targeted as a therapeutic approach in ALS.

Objectives: To establish the effect of expression of mSOD1 in astroglia on various components of the HSR in astroglia from different regions of the CNS and to evaluate the effect of upregulation of the HSR in astrocytes on motoneuron survival.

Methods: In established in vitro cultures of isolated primary astrocytes cultures from cortex and spinal cord of SOD1^G93A transgenic mice, elements of the HSR will be analysed. These assessments will involve immunocytochemistry, and Western blot analysis. Hsp expression in WT and mSOD1 astrocytes will be upregulated by treatment with agents that are known to upregulate Hsp expression, and the effect on the survival of co-cultured primary WT and mSOD1 motoneurons will be assessed.

Results, Discussion and Conclusions: We found that astrocytes from SOD1-G93A mice have regional specificity in which cortical mSOD1 astrocytes are less activated than spinal astrocytes, as shown by reduced level of GFAP expression, NO level production and iNOS expression. This indicates that a regional difference in mSOD1 toxicity in astrocytes exists. This heterogeneity among astrocytes suggests that their role in motoneuron survival is more complex than is commonly recognized. In addition, under basal conditions, mSOD1 expressing astrocytes have a much reduced stress response compared to WT astrocytes indicated by reduced levels of expression of a number of Hsps. This reduced stress response in spinal astrocytes of SOD1 mice may play a role in ALS pathogenesis.

Acknowledgements: JY is in receipt of a MND Association Prize Studentship

P56 NEURON-GLIA INTERACTIONS UNDERLYING AXONAL HEALTH IN AN IN VITRO SPINAL CULTURE MODEL OF RELEVANCE TO ALS

KING A, DICKSON T, CHUNG R, FOSTER S, BLIZZARD C, WOODHOUSE A, VICKERS J

Menzies Research Institute, University of Tasmania, Hobart, Tasmania, Australia

E-mail address for correspondence: [email protected]

Keywords: spheroid, glia, neurofilament

There is increasing evidence that axonal degeneration underlies initial motor dysfunction prior to motor neuron loss in ALS, and that axonal protection may be an important target for future therapeutic intervention. Several studies have demonstrated the importance of non-neuronal cells in the pathogenesis of ALS and the progression of the disease is accompanied by changes in the glial cell populations including astrogliosis and alterations in progenitor cells. However, there is little information on how these changes affect the health of the axon. Using co-cultures of primary spinal motor neurons and mixed glial cell populations we have investigated how the environment provided by non-neuronal cells affects axonal health. Spinal motor neurons were derived from E13 mice embryos, purified on an optiprep™ density gradient and yielding a subset of motor neurons immunopositive for SMI32, an antibody to de-phosphorylated neurofilaments. Motor neurons were plated onto confluent mixed glial feeder layers derived from P3 mice. In some instances feeder layer cells were derived from G93A mSOD1 mice or were pre-aged for 9 weeks prior to plating of motor neurons. Spinal motor neurons were also plated onto purified astrocytes and meningeal cells. Co-cultures were maintained for up to 21 days, followed by paraformaldehyde-fixation and immunolabelling for cytoskeletal proteins, glial markers, cytochrome c and ubiquitin.

Mixed glial feeder layers consisted predominantly of astrocytes, fibroblasts, microglia, and progenitor cells (GFAP, fibronectin, ferritin and nestin immunoreactivity, respectively). Spinal motor neurons grown on mixed glial feeder layers developed swellings in the proximal portion of the axon, which were up to 40 µ m in diameter. These axonal swellings in vitro were morphologically similar to axonal spheroids in spinal cord tissue from 20 week G93A mSOD1 mice. Swellings in both mSOD1 mice and cultured motor neurons were immunolabelled for neuronal intermediate filament proteins and a subset in both models were immunoreactive for cytochrome c and ubiquitin. The size of the axonal swellings in vitro was increased with the age of the neuron. The density of swellings was influenced by the phenotype of glial feeder layer, with a significant (p < 0.05) increase being induced by a glial feeder layer that was pre-aged prior to plating the motor neurons, or expressing mSOD1. However, 84% swellings were present in areas of the coverslip that were devoid of GFAP –positive astrocytes. These data indicate that the formation of proximal axonal swellings in cultured spinal motor neurons is influenced by non-neuronal cells, and the phenotype and health of these cells affects the severity of this pathology. This model of proximal neurofilament-rich swellings in cultured neurons supports a role for disrupted neuron-glia interaction in the pathogenesis of ALS.

P57 MUSCLE FIBRES EXPRESSING MSOD1 DISPLAY ELEVATED NOS ACTIVITY IN VIVO AND IN VITRO

EDET-AMANA E, KALMAR B, GREENSMITH L

Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: Nitric oxide synthase, fast-twitch, skeletal muscle

Background: Non-neuronal cells in the CNS are known to play a critical role in ALS pathogenesis. It is possible that cells such as muscle, which interact with motoneurons in the periphery, may also influence disease progression. Muscle atrophy is a characteristic feature of ALS, and is associated with increased mitochondrial reactive oxygen species (ROS) and nitric oxide (NO) production, resulting from denervation [1]. NO may have deleterious effects on the muscle function and can act to inhibit force production [2]. In the Wobbler mouse model of motoneuron degeneration, inhibition of NO synthesis improves motor function and delays motoneuron death [3]. Neuronal NOS (nNOS) is expressed at high levels in fast twitch myofibres in normal adult mice (2). However, the role of muscle nNOS in the progression of ALS is not clear.

Objectives: To investigate whether the expression of mutant SOD1 induces changes in NO production in primary myotubes cultured from neonatal wild type (WT) and SOD1 hindlimb muscles, under basal conditions as well as in a model of exercise-induced stress. We also compared nNOS expression in fast and slow muscles of WT and symptomatic SOD1-G93A mice and examined the relationship between nNOS expression and NO production in slow and fast muscle cells cultured from adult mice.

Methods: Satellite cells of neonatal and adult hindlimb muscles were cultured. Exercise related stress was modelled by glucose deprivation combined with caffeine (1–20 mM). NO levels were measured from cell supernatants using the modified Greiss Reagent assay.

Expression of nNOS was examined in Soleus and EDL muscles of WT and endstage SOD1-G93A mice by immunohistochemistry.

Results: Following exposure to exercise-related stress, in WT muscle cultures there was a dose-dependant increase in NO production in response to glucose deprivation and caffeine treatment, with a 3 fold increase in NO levels after 24 hours. However, in satellite cells of neonatal SOD1 mice, NO production was elevated, even in the absence of stress and these cells responded to stress at a faster rate and to a greater extent than WT cells. In muscle sections from WT and SOD1-G93A mice, there was a disease-specific increase in the expression of nNOS in fast muscles and to a lesser extent in slow muscles. Similarly, in culture, adult fast muscles of SOD1-G93A mice released more NO than either WT muscles or slow muscles of SOD1-G93A mice.

Discussion: In vitro, SOD1 muscle cells intrinsically produce more NO than WT muscle cells. SOD1 muscle cells are also particularly sensitive to exercise-related stress and have an enhanced NO response, which is disease-stage and muscle-type dependant. These characteristics of fast muscles of SOD1 mice, may contribute to their differential vulnerability to disease in ALS.

P58 DIFFERENTIATION OF A FAMILIAL ALS CELLULAR MODEL ALLOWS DETECTION OF ALTERED PROTEIN EXPRESSION IN THE SOD1 TRANSFECTED CELL LINES

COVA E¹, BIANCHI M¹, GHIROLDI A¹, GUARESCHI S¹, CEREDA C¹, MAZZINI G², CERONI M³

¹Foundation Neurological Institute “C. Mondino” IRCCS, Pavia, ²IGM-CNR, Histochemistry and Cytometry, Department of Animal Biology, University of Pavia, ³Foundation Neurological Institute “C. Mondino” IRCCS, Department of Neurological Sciences, University of Pavia, Italy

E-mail address for correspondence: [email protected]

Keywords: SH-SY5Y, differentiation, cdk5/p35

Background: The cdk5/p35 pathway has been suggested to be involved in ALS pathogenesis. Deregulation of the Ser/Thr kinase cdk5 activity has been demonstrated in a transgenic mice model of ALS [1] as its involvement would be associated with hyperphosphorylation of NF-H and tau observed in ALS patients and mice. Deregulation of cdk5 correlates with the increased generation of a truncation product of the cdk5 regulatory unit p35, termed p25. Preliminary experiments performed using an undifferentiated SOD1-mutated transfected neuroblastoma cell line proved that there were no modifications in the cdk5/p35 pathway and in phosphorylated tau. In our laboratory, the treatment of the SH-SY5Y human neuroblastoma cell line with the differentiating agents, retinoic acid and brain derived neurotrophic factor (BDNF), provided morphological and expression evidences of neuron-like phenotype.

Objectives: To evaluate the presence of modified expression of the cdk5/p35 proteins and its substrate, tau, after inducing differentiation of the SOD1-mutated transfected neuroblastoma cell line.

Methods: The human neuroblastoma SH-SY5Y cell line was transfected with the wild-type (WT-SOD1) and the G93A-mutated (G93A-SOD1) SOD1 gene. The un-transfected and transfected cell lines were cultured and treated with All-trans-retinoic acid at a final concentration of 10 µM in DMEM with 15% foetal bovine serum for 5 days and afterwards with 3.75 µM BDNF in serum free DMEM for 7 days. Western blot was performed with anti—actin, -p35, -cdk5, -tau, -tau phosphorylated at ser 202 and thr 205. The expression of neuron-specific markers, tau and tubulin beta 3, was assayed by florescence microscopy, in differentiated compared with undifferentiated cells.

Results: The treatment with the differentiation agents induced a morphological change consisting of a more neuron-like phenotype. Almost all treated cells had long bipolar or multipolar processes and neurite extension that occasionally connected the cells. The results showed an increased expression of tau and tubilin beta 3. The western blotting experiments demonstrated that, respectively from undifferentiated cell lines, the WT- and G93A-SOD1 mutated cell lines had a significantly higher p25/p35 protein ratio compared to untransfected cell line (p < 0.05). Moreover, the G93A SOD1-mutated cell line was significantly hyperphosphorylated in the ser 202 site of tau compared to base cell line (p < 0.05).

Discussion: Our results prove that the undifferentiated neuroblastoma cell line is not a suitable model to study the mechanisms of ALS pathogenesis. The modified pattern of protein expression in the SOD1-transfected cell lines observed after differentiation treatment demonstrates that this model, nearer to the neuronal phenotype, may be useful to studying ALS pathogenic mechanisms.