THEME 10 IN VIVO EXPERIMENTAL MODELS

P230 COMPARISON OF ZEBRAFISH MODELS OF ALS

Lissouba A

Armstrong G

Drapeau P

Research Centre of the University of Montréal Health Centre, Montreal, Quebec, Canada

Email address for correspondence: [email protected]

Keywords: TARDBP, RNA-sequencing, zebrafish

Background: ALS-causing mutations have been discovered in several genes involved in RNA metabolism, such as TARDBP (coding for TDP-43) and FUS, two structurally and functionally related DNA/RNA binding proteins. However, the pathological dysfunction caused by ALS mutations in these genes is unknown. Zebrafish are optimal for the development of genetic models of ALS; the zebrafish embryos develop externally and are optically transparent, allowing for fluorescent labelling of live cells. Additionally, their swimming behaviour is well characterized, electrophysiological recording in whole live embryos is easily obtained and genetic manipulations are highly feasible.

Objectives: Using next-generation RNA-sequencing to study the transcriptome modifications induced by ALS-causative mutations of FUS and TARBDP, using our different genetic models of ALS.

Methods: We have used zebrafish to generate genetic models of ALS. Overexpression models were generated by injecting human ALS-related mutant TARDBP mRNA or FUS mRNA into 1-cell stage embryos. A stable TARDBP transgenic line was also generated, with the human gene placed under the control of the heat shock inducible hsp70l promoter. We extracted high quality mRNA from larvae obtained for each of these models of ALS and performed RNA sequencing using the Illumina Hi-seq 2000 technology.

Results: In all these models, the embryos exhibited an abnormal touch-evoked escape response, with a reduction in the distance swam, the maximum velocity of swim and in the swim duration. The motoneurons of these embryos also had abnormal, overbranched axons. Over 40,000 transcripts were sequenced and some 26,000 had human homologs. Analyses using an adjusted p-value < 0.1 revealed that any single condition altered the level of expression (up or downregulated) of only 32 to 278 genes; 22 genes were upregulated and 14 genes downregulated in at least two of these conditions. As TDP-43 and FUS are implicated in RNA processing, we are currently performing a transcriptome-wide analysis of splicing.

Discussion and conclusion: Our preliminary results indicate that surprisingly few genes are commonly altered between these different models, helping to define potential disease-related targets. We are determining if these commonly modified genes or pathways could be common denominators of the ALS pathology by testing if their overexpression or downregulation in wild-type zebrafish embryos can cause an ALS phenotype similar to the one we observe in our genetic models of ALS. Additionally, we are testing by qRT-PCR if the hits found in our TARDBP and FUS models are also misregulated in the presence of other ALS-causing genes, such as SOD1 and C9ORF72.

Acknowledgements: Financial support for this work from: ALS Society of Canada and the Canadian Institutes of Health Research; Fonds de la Recherche en Santé du Québec; Groupe de Recherche sur le Systeme Nerveux Central.

P231 INTERACTIONS OF ENVIRONMENTAL NEUROTOXINS WITH SOD1 IN AMYOTROPHIC LATERAL SCLEROSIS IN A ZEBRAFISH MODEL

Sher R

Powers S

Lavin T

Kwok S

Lovejoy E

University of Maine, Orono, ME, USA

Email address for correspondence: [email protected]

Keywords: BMAA, zebrafish, SOD1^G93A

Background: Both genetic and environmental causes of amyotrophic lateral sclerosis (ALS) have been identified, and the scientific consensus is that a combination of gene-environment interactions are key for the development of ALS, but how either toxicants or genes lead to a disease mechanism is currently unknown. This represents a major gap in our understanding of the pathogenesis of ALS (1). A suite of environmental neurotoxins has been associated with the development of ALS, with epidemiological, clinical, and experimental evidence indicating that early developmental exposures to neurotoxins can have consequences for neurotoxicity later in life (2). Potentially, impairment of genes crucial to early neuronal differentiation makes neurons more susceptible to additional environmental disruptions, leading to late-onset disease (3). By determining cellular pathways involved in modifying neurological defects, both by toxicants and genetic influences, we hope to gain a better understanding of the root causes of this disorder.

Objectives: The zebrafish, Danio rerio, has been shown to be a robust model organism for modelling human neurodegenerative diseases, including ALS. Our research aims to study the intersection of environmental neurotoxins on motor neuron defects in a zebrafish model of ALS.

Methods: We have determined the impact of exposure to varying doses (0–25μg/L) of BMAA (β-methylamino-L-alanine) on early neurological defects in mutant SOD1-ALS zebrafish.

Results: We have found that: (i) Motor neuron length (30hpf) is decreased in control and SOD1^G93A fish when exposed to BMAA, but not in SOD1-wt overexpressing fish; (ii) SOD1^G93A embryos are more sensitive to lower doses of BMAA than are control fish, and (iii) 72hpf neuromuscular junction architecture is significantly altered in BMAA-exposed SOD1^G93A but not in SOD1-wt overexpressing embryos.

Discussion and conclusion: Our results indicate that genetic and environmental insults combine to facilitate neurological dysfunction in ALS, and that overexpression of wt-SOD1 may have protective effects against neurotoxin damage.

References:

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P232 NEW INSIGHTS ON HEMATOPOIETIC STEM CELLS DIFFERENTIATION IN TRANSGENIC SOD1G93A MICE

Gasco S¹

Calvo AC¹

Rando A¹

Oliván S¹

Toivonen JM¹

Esteban Pérez J²

Zaragoza P¹

Garcia Redondo A²

Osta R¹

^aLaboratorio de Genética Bioquímica (LAGENBIO-I3A), Aragon's Institute of Health Sciences (IACS), Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain

^bUnidad de ELA, Instituto de Investigación Hospital 12 de Octubre de Madrid, SERMAS, Madrid, Spain, and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER U-723), Spain. Edificio C, Madrid, Spain

Email address for correspondence: [email protected]

Keywords: hematopoietic stem cell, flow cytometry, SOD1^G93A

Background: Hematopoietic stem cells (HSC) are multipotent cells with self-renewal capacity that can give rise to their closest oligopotent hematopoietic progenitor cells, best known as Common Lymphoid Progenitors (CLP) and Common Myeloid Progenitors (CMP) (1). In this study we propose to investigate the frequencies of HSC, CLP and CMP in a murine model along disease progression.

Objectives: The main aim of this study was to characterize for the first time the frequency of HSC, CLP and CMP in serial blood extractions from transgenic SOD1^G93A mice.

Methods: Blood samples from transgenic mice were collected and treated to study the frequency of HSC, CLP and CMP by real time PCR and flow cytometry. The blood samples were first collected at the age of 30 days and then serial samples were obtained from each animal along disease progression till the end-point. Two-tailed t-Student tests were used to assess statistical significance between groups. A total of 40 mice were included in this study, 20 control and 20 transgenic mice of both sexes.

Results: The results obtained in serial blood samples suggested at transcriptional level, HSC were almost significantly activated in transgenic mice at terminal stage (p = 0.06). Interestingly in the flow cytometry study, the frequency of HSC was significantly increased since symptomatic stage to the end-point of transgenic mice (p < 0.01). However, the frequencies of CLP and CMP decreased along disease progression, starting at late (p < 0.05) and early symptomatic stage (p < 0.05), respectively.

Discussion and conclusion: The time dependent increase in HSC frequencies together with the time dependent decrease in CLP and CMP frequencies could suggest a time-point dependent differentiation of HSC to CLP and CMP, which could be influenced by the degenerative progression of the disease. This result is in accordance with recent studies that reveal a potential therapeutic effect based on stem/progenitor cell transplantation at presymptomatic or early symptomatic stages in animal models for the disease (2) and in ALS patients (3). The time dependent activation of HSC could shed light on the role of hematopoietic system in the progression of ALS and future therapeutic targets could be defined.

Acknowledgements: We wish to thank Miriam de la Torre for her technical assistance. This work was supported by grants PI10/01787 and PI10/00092 (Fondo Investigación Sanitaria, Spain).

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P233 ANDROGEN RECEPTOR DYSREGULATION IN ALS MOUSE MODELS

Sheean RK

Perera ND

Weston R

Turner BJ

Florey Institute of Neuroscience and Mental Health, Melbourne, Australia

Email address for correspondence: [email protected]

Keywords: SBMA, androgen receptor, SOD1^G93A

Background: Spinal bulbar muscular atrophy (SBMA) is an adult-onset lower motor neuron disease affecting males that results from pathogenic androgen receptor (AR) expansion and inactivation. AR inactivation is linked to neurodegeneration by loss of AR trophic signalling, leading to transcriptional dysregulation of growth factors required for motor neuron survival, and nuclear accumulation of toxic AR-polyglutamine inclusions. Interestingly, AR is abundant on spinal and cranial motor neurons also affected in ALS. The increased incidence of ALS in males also implicates AR signalling in pathogenesis. We therefore sought to determine a potential role for AR dysregulation in ALS.

Objectives: 1) To investigate AR expression and distribution in spinal cords of transgenic SOD1^G93A and TDP-43^A315T mice with disease progression; 2) To determine the impact of androgen ablation on AR expression, localisation and motor neuron loss, in SOD1^G93A mice.

Methods: AR expression level was analysed in spinal cords of SOD1^G93A and TDP-43^A315T mice at presymptomatic and symptomatic ages compared to age-matched wild-type controls by Western blotting. AR localisation in NeuN, GFAP and CD11b-positive cells in spinal cord was established by immunohistochemistry. SOD1^G93A mice were sham operated or surgically castrated and studied for AR expression, localisation and motor neuron loss in spinal cords.

Results: AR protein was significantly and progressively downregulated by 50% in presymptomatic and 80% in symptomatic SOD1^G93A mouse spinal cords (p < 0.05). AR was abnormally depleted from cell bodies and accumulated in proximal axons of motor neurons in SOD1^G93A mice, but not in astrocytes or microglia. Androgen ablation in SOD1^G93A mice significantly enhanced AR depletion in motor neurons (p < 0.01). AR was also depleted in spinal motor neurons of presymptomatic TDP-43^A315T mice.

Discussion and conclusion: We show for the first time that AR expression is diminished in spinal motor neurons early in the disease course of multiple mouse models of ALS, resembling AR inactivation in SBMA. We therefore propose that AR reduction in spinal motor neurons may render them susceptible in ALS and furthermore, ALS and SBMA may share a common disease pathway mediated by disruption of AR expression and trophic signalling.

P234 HFE H63D MODIFIES DISEASE PATHOPHYSIOLOGY IN AN ALS MOUSE MODEL

Nandar W

Neely E

Simmons Z

Connor J

Penn State University, College of Medicine, Hershey, PA, USA

Email address for correspondence: [email protected]

Keywords: HFE H63D, SOD1^G93A, oxidative stress

Background: The HFE H63D gene variant is present in as many as 30% of individuals with amyotrophic lateral sclerosis (ALS). Despite increasing evidence suggesting the association of HFE H63D with ALS, how HFE H63D influences disease processes in ALS remains unclear. HFE H63D is associated with disease processes implicated in ALS such as iron dyshomeostasis and oxidative stress. Thus, HFE H63D is proposed to be a genetic modifier for the risk of ALS.

Objectives: To determine how HFE H63D impacts ALS pathogenesis, we generated a double transgenic mouse line (SOD1/H67D) carrying the HFE H67D (homologue of human H63D) and SOD1^G93A mutations.

Methods: We crossed mice carrying HFE H67D with SOD1^G93A mice to generate double transgenic mice (SOD1/H67D). A gripstrength meter was used to measure forelimb and hindlimb strength as markers for disease progression. End-stage was defined as the inability of the animal to right itself within 30 s after being placed on its side. Disease duration was the mean time from onset to end-stage. To determine mechanisms by which HFE H63D contributes to ALS pathogenesis, we examined a number of parameters in the lumbar spinal cord of double transgenic mice at 90 days (presymptomatic), 110 days (symptomatic) and end-stage by immunoblot and immunohistochemical analyses.

Results: The double transgenic mice have shorter survival and accelerated disease progression. Transferrin receptor and L-ferritin expression, both indicators of iron status, were altered in double transgenic and SOD1 mice starting at 90 days, indicating iron dyshomeostasis in these mice. However, double transgenic mice had higher L-ferritin expression than SOD1 mice suggesting higher iron in double transgenic mice. In addition to increased L-ferritin, double transgenic mice exhibited increased Iba-1 immunoreactivity and caspase-3 levels, indicating increased microglial activation. Although both SOD1 and double transgenic mice had increased GFAP expression, the magnitude of the increase was higher in double transgenic mice at 110 days, suggesting increased gliosis in these mice. Increased hemeoxygenase-1 and decreased nuclear factor E2-related factor 2 levels in double transgenic mice strongly suggest the accelerated disease process could be associated with increased oxidative stress. There was no evidence of TDP-3 mislocalization to the cytoplasm in double transgenic mice. However, there was evidence suggesting neurofilament disruption in double transgenic mice that has been reported in ALS.

Discussion and conclusion: Our findings indicate HFE H63D modifies ALS pathophysiology via pathways involving oxidative stress, gliosis and disruption of cellular functions. Thus, we hypothesize that HFE H63D increases the risk of ALS by promoting the convergence of disease processes implicated in ALS.

Acknowledgements: This work is supported by Judith and Jean Pape Adams Charitable Foundation, the Paul and Harriett Campbell Fund for ALS research, Zimmerman Family Love Fund and the Robert Luongo ALS Fund.

P235 NEW MODEL OF UBIQUILIN2-RELATED ALS USING AAV VECTORS

Biferi MG¹

Bos C¹

Tanguy Y¹

Alonso-Martin S¹

Artegiani B²

Roda M¹

Cohen-Tannoudji M¹

Ferry A¹

Camerini S³

Crescenzi M³

Calegari F²

Relaix F¹

Barkats M¹

^cCenter of Research in Myology, Inserm-CNRS-UPMC-UMR 974-Faculté de Médecine, Paris, France

^dDFG-Research Center and Cluster of Excellence for Regenerative Therapies, Dresden, Germany

^eDept. of Cell Biology and Neurosciences Higher Institute of Health (ISS), Rome, Italy

Email address for correspondence: [email protected]

Keywords: mouse model, ubiquilin 2, adeno-associated virus (AAV)

Background: Ubiquilin 2 is a protein involved in degradation pathways via the ubiquitin proteasome system and autophagy. Mutations in Ubiquilin 2 have been identified in inherited forms of ALS or ALS-FTD and represent the first reported mutations in a protein that is directly linked to the protein degradation pathways (1). Ubiquilin 2-positive inclusions have been observed in the spinal cord and brain of both familial and sporadic ALS patients (1), but the pathogenic role of this protein is still unclear. The generation of animal models of the disease is a prerequisite for the understanding of physiopathological mechanisms and identification of new therapeutic strategies for ALS. However, no animal model recapitulating all Ubiquilin 2-related ALS features has been achieved to date using classical transgenesis.

Objectives: According to the evidence suggesting a toxic gain of function of the mutated Ubiquilin 2, we developed a new animal model of Ubiquilin 2-ALS using recombinant adeno-associated-virus (AAV) to overexpress either the wild- type (AAV-UbiWT) or mutant (AAV-UbiPro497His) human Ubiquilin2.

Methods: The vectors were delivered through intracerebroventricular (ICV) injection in newborn FVB mice. An AAV encoding the green fluorescent protein (GFP), injected under the same conditions, was used as control.

Results: The expression of human Ubiquilin2 in brain and spinal cord extracts of infected animals was first evidenced by western blot analysis, one month after injection. The presence of Ubiquilin2-positive inclusions was further detected in the brain and the spinal cord of UbiPro497His overexpressing animals, similarly to ALS-FTD patients. Moreover, the injected animals displayed a reduced brain size compared to controls and a severe astrogliosis in both the spinal cord and the brain. Interestingly, the number of ChAT+ motor neurons was also significantly decreased in the whole spinal cord. Importantly, the AAV-UbiPro497His injected mice had a shortened lifespan (50% of survival at 80 days) and presented a body weight loss phenotype. Mice overexpressing UbiPro497His also developed neurological phenotype with clasping, abnormal spinning and tremors. Muscle weakness, with loss of muscle mass and decrease of muscle strength was observed. The AAV-UbiPro497His-injected mice also displayed enhanced anxiety-related behaviour, one of the first signs of dementia. Overexpression of wild-type Ubiquilin also led to pathological phenotype (albeit to a lesser extent than the one generated by the mutant form), suggesting a crucial role of Ubiquilin 2 pathways also in sporadic ALS forms.

Discussion and conclusion: In conclusion, we have generated the first mouse model of Ubiquilin 2-ALS-FTD displaying most of the clinical and histological features of the human disease. This innovative and successful application of AAV for ALS modelling will be useful to further dissect the molecular mechanisms of this complex pathology and to envision therapeutic strategies.

Reference:

• Deng HX et al. Nature 2011; 477: 211–215.
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P236 HMG-COA REDUCTASE INHIBITORS AND HFE POLYMORPHISM ACCELERATE DISEASE PROGRESSION AND SHORTEN SURVIVAL IN THE SOD1G93A ALS MOUSE MODEL

Su X

Nandar W

Neely E

Simmons Z

Connor J

Penn State College of Medicine, Hershey, PA, USA

Email address for correspondence: [email protected]

Keywords: HMG-CoA reductase inhibitor, HFE polymorphism, coenzyme Q10

Background: Both HMG-CoA reductase inhibitors (statins) and the H63D polymorphism in the HFE iron regulatory gene may impact ALS risk or disease progression. Mitochondrial dysfunction contributes to ALS, and statins perturb mitochondrial enzymes.

Objectives: To determine if statins accelerate disease progression or decrease survival in ALS mouse models; if HFE genotype influences these effects; and if the effects are mediated via statin-induced mitochondrial dysfunction.

Methods: Double transgenic mice harboring SOD1^G93A and HFE H67D (homologous to human HFE H63D); single transgenic SOD1 or HFE H67D mice; or wild type (WT) mice, balanced for gender, were used in accordance with IACUC guidelines. For the survival study, 2 mg/kg simvastatin or vehicle was administered daily from disease onset, as determined by rotarod, animals were allowed to reach endstage, defined as the inability to right themselves within 30 seconds. Disease progression was measured by gripstrength. Plasma cholesterol and ferritin levels were measured by colorimetric assay or ELISA. Western blots of mitochondrial fractions from gastrocnemius muscle and lumbar spine were performed. For the mechanism study, animals were administered simvastatin in a similar fashion, gastrocnemius muscle and lumbar spine were collected at the symptomatic 120-day timepoint, and Western blots were performed as above. For the rescue study, 2 mg/kg simvastatin, 10 mg/kg coenzyme Q10, or both, was administered daily from disease onset, animals were allowed to reach endstage. There were 8–13 animals per group. SAS 9.3 or NCSS 9 was used for statistical analyses.

Results: SOD1 mice had increased plasma ferritin levels compared to WT mice. Simvastatin administration and HFE H67D accelerated disease progression as measured by gripstrength. Cox proportional hazards analysis indicated simvastatin administration adversely impacted survival, whereas HFE H67D had a benefit. Coenzyme Q10 administration did not rescue the statin-induced decrease in survival in SOD1 or double transgenic mice. At 120 days and endstage, SOD1 mutant mice had significantly decreased levels of complexes I and IV of the electron transport chain, cytochrome c, and the VDAC1 mitochondrial anion channel in gastrocnemius muscle and lumbar spine compared to WT mice. However, statins did not alter levels of mitochondrial proteins, at 120 days or endstage, in lumbar spine or gastrocnemius muscle.

Discussion and conclusion: Statins accelerate disease progression and decrease survival in SOD1 mutant mice. HFE H67D worsens the statin effect on disease progression while paradoxically benefiting survival. Mitochondrial dysfunction does not mediate these effects. These results suggest patients with ALS receiving statins, especially those harbouring HFE H67D, should be monitored for changes in disease progression. Studies of the effects of statins on disease trajectory in patients with ALS harbouring H63D versus WT HFE may guide clinician use of statins in patients with ALS.

P237 INVOLVEMENT OF MONOCARBOXYLATE TRANSPORTER 1 IN SOD1G93A DISEASE PATHOGENESIS

Philips T¹

Hughes EG²

Lee Y¹

Morrison BM¹

Sattler R¹

Bergles DE²

Rothstein JD^1,2

^fDepartment of Neurology, Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

^gThe Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Email address for correspondence: [email protected]

Keywords: oligodendrocytes, metabolism, SOD1^G93A

Background: In the CNS, oligodendroglia are well established as being involved in myelination of axons and providing rapid saltatory conduction of action potentials. Besides their role in myelination, new studies have provided strong evidence that oligodendrocytes are involved in metabolic support of neurons through the expression of a particular monocarboxylate transporter (MCT) MCT1, which catalyzes the proton linked transport of lactate, ketone bodies and pyruvate across plasma membranes. In the spinal cord of human amyotrophic lateral sclerosis (ALS) patients and SOD1^G93A ALS mice, we previously found that MCT1 expression was significantly reduced. The loss of MCT1 mediated trophic support has been shown to impair neuron and axon viability this might contribute to the selective death of interneurons and motor neurons. In the SOD1^G93A mouse decreased MCT1 expression is concomitant with the death and degeneration of oligodendrocytes, and with the failure of newly generating oligodendrocytes to fully mature.

Objectives: The current study is aimed to gain further insight into the involvement of MCT1 transporters during the disease pathogenesis of SOD1^G93A mice.

Methods: Using MCT1tdTomato transgenic reporter mice crossbred with SOD1^G93A mice, we will explore the percentage of oligodendrocytes with active MCT1 reporter expression at different disease stages. Using PDGFRaRCreER mice crossbred with RosaYFP mice, MCT1tdTomato mice and SOD1^G93A mice, we will explore the ability of oligodendrocyte progenitor cells to generate MCT1 reporter expressing oligodendrocytes at both early and late stages of disease.

Results: We found that early disease stage SOD1^G93A mice (P60) and WT littermates at all ages analyzed (up to P150) have about 90% of oligodendrocytes, as assessed by CC1 expression, and show MCT1 reporter expression. At symptomatic disease stages in SOD1^G93A mice, around 50% of oligodendrocytes had maintained active reporter expression. Fate-map analysis of oligodendrocyte progenitor cells differentiating into CC1 oligodendrocytes demonstrated that at all disease stages at least some oligodendrocyte progenitor cells were still able to generate MCT1 reporter expressing oligodendrocytes, but MCT1 reporter expression was only initiated late during the differentiation, well after initiation of MBP protein expression. At these later disease stages, many oligodendrocytes were immature and had not initiated MCT1 reporter expression yet, and as such failed to provide trophic support to motor neurons.

Discussion and conclusion: In SOD1^G93A mice, many oligodendrocytes die and are replaced with newly generated oligodendrocytes generated form oligodendrocyte progenitor cells. At later disease stages, despite the ability of newly generated oligodendrocytes to turn on MCT1 reporter expression, many of these cells had not matured fully and failed to provide trophic support to motor neurons. We are currently trying to modulate MCT1 expression in oligodendrocytes and explore whether this affects the oligodendrocyte ability to provide trophic support to neurons.

Acknowledgements: Support from DOD, RO1 and Packard Center and TargetALS

P238 HSP110 AS A MODIFIER OF TOXICITY IN A MOUSE MODEL OF SOD1-LINKED ALS

Fenton WA¹

Nagy M^1,2

Li D^1,2

Furtak K^1,2

Horwich Al^1,2

^hYale University School of Medicine, New Haven, CT, USA

ⁱHoward Hughes Medical Institute, New Haven, CT, USA

Email address for correspondence: [email protected]

Keywords: molecular chaperone, HSP110, SOD1^G85R

Background: A number of recent studies have suggested that Hsp110 chaperones can collaborate with Hsp70s to forestall or reverse protein aggregation. In an earlier study, we observed that all three mouse Hsp110s were recovered in association with affinity-captured SOD1^G85R -YFP from the spinal cord of ALS mice expressing the mutant SOD1. More recently, we showed that human HSPA4L, an Hsp110, could prevent the toxic effect of purified SOD1^G85R -YFP on anterograde fast axonal transport in squid giant axon axoplasm, preventing activation of a MAP kinase cascade by the mutant misfolded protein.

Methods: We programmed transgenic mice expressing this chaperone from a Thy1.2 (neuronal) promoter and crossed them with ALS mice expressing SOD1^G85R -YFP, a misfolded protein that forms large aggregates in spinal cord motor neurons. The latter animals develop typical signs of lower motor neuron disease and paralysis by 5–6.5 months.

Results: Two independent doubly transgenic lines were evaluated. For one, transgenic Hsp110 mRNA is expressed in brain at 2–2.5 times the level of the corresponding mouse Hsp110. Sixty-five animals of this strain, with Hsp110 copy numbers of ∼80–150, have been followed for the past year. We have observed median survival to be increased by 2 months, compared both with littermates lacking Hsp110 (35 mice), none of which survived beyond 6.5 months, and with our parental ALS line. Strikingly, ∼30% of the double transgenic animals of this line continue to survive beyond 8 months of age, with several now 11 months old. Sections of the spinal cord of one of the Hsp110 animals at 5 months of age has been examined microscopically, revealing a larger number of surviving motor neurons than typically seen in a pre-end-stage G85R mouse and fewer neurons with large aggregates. Animals from the second line are several months younger, but also appear to be exhibiting extended survival. We are further evaluating these lines by qRT-PCR and LC/MS analysis of motor neuron cell bodies captured by LMD.

P239 MUSCLE-SPECIFIC CONTROL OF HSP70 IN POLYGLUTAMINE INDUCED MOTOR NEURON DISEASE

Kondo N

Katsuno M

Adachi H

Sahashi K

Miyazaki Y

Iida M

Tohnai G

Ishigaki S

Fujioka Y

Tanaka F

Sobue G

Nagoya University, Nagoya, Japan

Email address for correspondence: [email protected]

Keywords: SBMA, heat shock proteins, skeletal muscle

Background: Spinal and bulbar muscular atrophy (SBMA), also called Kennedy's disease, is an adult-onset motor neuron disease caused by the expansion of a CAG repeat within the first exon of the androgen receptor gene. Heat shock proteins (Hsps) such as Hsp70 play a defensive role in the pathophysiology of neurodegenerative disorders by solubilising pathogenic abnormal proteins. Although heat shock factor-1 (Hsf-1) controls the expression levels of Hsps, the molecular basis for tissue specific control of Hsps in motor neuron diseases was not clear.

Methods: To reveal the mechanism of Hsps regulation in the skeletal muscle of SBMA, we studied the skeletal muscle from the model mice of SBMA, in which Hsf-1 is heterozygously knocked out. We performed western blotting and Immunohistochemistry of skeletal muscle from wild-type; AR-97Q (SBMA model: 97Q^Tg/-, Hsf-1^+/+); and AR-97QHsf-1^+/− (heterozygous Hsf-1 knockout SBMA model: 97Q^Tg/-, Hsf-1^+/−) mice using anti-Hsf-1, anti-Hsp72, anti-Nfya, anti-Sp1, anti-p53, anti-Tbp and anti-polyglutamine antibodies.

Results: On western blot analysis, the expression level of Hsp72, the inducible form of Hsp70, in the spinal cord of SBMA mice was downregulated by heterozygous knock out of Hsf-1. Conversely, Hsp72 expression level in the skeletal muscle of these mice was maintained despite Hsf-1 depletion. Moreover, the depletion of Hsf-1 did not enhance the pathogenic AR accumulations in the skeletal muscle of SBMA mice. Surprisingly, in the skeletal muscle of SBMA mice, Nfya and Sp1 were upregulated compared with wild-type mice both on immunohistochemistry and western blotting. Furthermore, this reaction was prominent in the Hsf-1 depleted SBMA mice. In contrast to the skeletal muscle, neither Nfya nor Sp1 were upregulated by Hsf-1 depletion in the spinal cord of SBMA and heterozygous Hsf-1 knockout SBMA mice.

Conclusion: These results suggest that the regulatory system of Hsps in skeletal muscle is distinct from that in the central nervous system in SBMA.

P240 ABSENCE OF C5A-C5AR1 SIGNALLING DIMINISHES INFILTRATION OF PERIPHERAL IMMUNE CELLS IN SKELETAL MUSCLE OF HSOD1G93A MICE

Wang H¹

Lee J¹

Woodruff T¹

Noakes P^1,2

^jSchool of Biomedical Sciences

^kQueensland Brain Institute, The University of Queensland, Queensland, Australia

Email address for correspondence: [email protected]

Keywords: complement system, C5aR1, peripheral immune cells

Background: The terminal innate immune complement system has recently been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Our previous studies in the hSOD1^G93A mouse model of ALS demonstrated excessive complement activation, C5a receptor (C5aR1) microglial upregulation in the lumbar spinal cord (1). Importantly, the absence of C5aR1 in these hSOD1^G93A mice reduced disease pathology (2).

Objectives: The present study aimed to determine the expression of complement components (C1qB, C3 and C5aR1) at both mRNA and protein levels in tibialis anterior (fast-twitch) and soleus (slow-twitch) muscles of hSOD1^G93A mice. Furthermore, we investigated the role of C5a-C5aR1 signalling in the infiltration of peripheral immune cells (macrophages and helper T lymphocytes) in tibialis anterior (TA) and soleus (SOL) muscles of hSOD1^G93A mice.

Methods: TA and SOL muscles from hSOD1^G93A, hSOD1^G93A lacking C5aR1 (hSOD1^G93A×C5aR1^−/−) and wild-type (WT) mice were examined at 3 different stages of disease progression. The mRNA level of complement factors C1qB, C3 and C5aR1 were measured in WT and hSOD1^G93A mice using quantitative real-time PCR. Protein expression level of C5aR1 was also examined using western blotting. Cellular localisation of C5aR1 was investigated using immunohistochemistry with combinations of antibodies specific for neuromuscular junctions (α-Bungaratoxin), Schwann cells (S100), macrophages (CD11b) and helper T lymphocytes (CD4). The number of macrophages and helper T lymphocytes were also counted in TA and SOL muscles of WT, hSOD1^G93A and hSOD1^G93A×C5aR1^−/− mice.

Results: We found elevated levels of C1qB, C3 and C5aR1 in TA and SOL muscles of hSOD1^G93A mice during disease progression. Immuno-localisation showed that C5aR1 was expressed predominantly on the macrophages in WT and hSOD1^G93A mice. Furthermore, we demonstrated a significant increase of macrophage and helper T lymphocyte numbers in TA and SOL muscles of hSOD1^G93A mice when compared to WT mice during disease progression. The infiltration of immune cells in TA muscle was far greater when compared to SOL muscle in hSOD1^G93A mice. Interestingly, hSOD1^G93AxC5aR1^-/- mice showed decreased numbers of macrophages and helper T lymphocytes when compared to hSOD1^G93A mice.

Discussion and conclusion: These results indicate that complement activation occurs in the muscle tissue of hSOD1^G93A mice. In addition, increased C5a-C5aR1 signalling may contribute to the recruitment of peripheral immune cells that may accelerate muscle denervation. The amounts of infiltrating immune cells may also reflect the degree of muscle denervation as more immune cells were observed in TA, a fast-twitch muscle that is more vulnerable to degeneration in ALS.

Acknowledgements: We acknowledge the assistance of Mary White and Maryam Shayegh. Haitao Wang is a recipient of UQ scholarship. The work was funded by MNDRI-Australia to PGN and TMW.

References:

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P241 INDUCTION OF IMMUNOPROTEASOME AND MAJOR HISTOCOMPATIBILITY COMPLEX I (MHCI) IN MOTOR NEURONS OF A FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS (ALS) MOUSE MODEL

Nardo G

Trolese MC

Bosani B

Rossi M

Bendotti C

IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy

Email address for correspondence: [email protected]

Keywords: immune system, MHCI, transgenic mouse model

Background: We recently found, in the spinal MN of SOD1^G93A mice a significant upregulation of the immunoproteasome subunit, the large multifunctional peptidase 7 (LMP7), which starts at the presymptomatic stage and progressively increased during the disease progression (1, 2). Immunoproteasome recognizes and degrades polyubiquitinated protein substrates to generate small protein fragments that can be used by major histocompatibility complex (MHC) class I molecules for the display of antigens to CD8+ T cells (3).

Objectives: We aimed to examine the expression of MHCI pathway at the central and peripheral nervous system of a familial ALS mouse model.

Methods: An extensive immunohistochemical and confocal microscopy analysis has been performed in the spinal cord, peripheral nerves and muscles of SOD1^G93A mice during the disease course and the associated β2 microglobulin (β2m), together with LMP7.

Results: We report that MN and surrounding glial cells (microglia, olygodendrocytes but not astrocytes) exhibit the activation of LMP7, MHC-I and β2m at very early stages of the disease. Notably, while the immunostaining of LMP7 and β2m were highly increased in the perikarya of MNs and motor axons, the MHC-I immunoreactivity was increased exclusively in the motor axons and neuromuscular junction (NMJ) of SOD1^G93A mice during the disease course. Consistently, we found CD8+ T lymphocytes infiltrates in the spinal cord, sciatic nerve and muscle of SOD1^G93A as demonstrated by CD3 positive immunoreactivity and real time PCR, suggesting the interaction of MNs with cytotoxic T cells through MHCI.

Discussion and conclusion: These data point out that the activation of the adaptive immune system molecules both at central and peripheral level may take part in the pathogenesis and / or progression of ALS. Studies are ongoing to investigate the beneficial or detrimental effect of this immune response in SOD1^G93A mice.

Acknowledgements: This work is supported by the Thierry Latran Foundation and AriSLA.

References:

• Cheroni C et al. Hum Mol Genet 2009; 18(1): 82–96.
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P242 BENEFICIAL EFFECTS OF A SYNTHETIC PROSTAGLANDIN I2 AGONIST, ONO1301, IN ANIMAL MODELS OF ALS

Tada S^1,2

Okuno T³

Yasui T¹

Nakatsuji Y³

Sakai Y⁴

Miyagawa S⁴

Sawa Y⁴

Hazama T²

Kikutani H¹

Sakoda S⁵

Mochizuki H³

^lDepartment of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

^mDepartment of Neurology, Osaka General Medical Center, Osaka, Japan

ⁿDepartment of Neurology

^oDepartment of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan

^pNational Toneyama Hospital, Osaka, Japan

Email address for correspondence: [email protected]

Keywords: ONO1301, vasodilation therapy, HIF1 alpha

Background: Accumulating studies suggest that there is a link between altered vascular structure, reduced blood flow in the spinal cord and neurodegeneration in animal models of ALS (1, 2). However, the effect of vasodilation therapy with a prostaglandin I2 agonist, which is an effective vasodilator, against ALS in vivo remains totally unknown.

Objectives: Our objective here is to explore the role of blood flow increase in neurodegeneration of ALS by using ONO1301, a novel synthetic prostaglandin I2 agonist with a vasodilator effect.

Methods: We prepared a novel sustained-release prostaglandin I2 agonist polymerized with poly (D,L-lactic-co-glycolic acid) (PLGA) microspheres to realize long-lasting effects (ONO1301MS). We treated mice carrying the human SOD-1 mutation G93A (mSOD1^G93A mice) with ONO1301MS (n = 14) or vehicle (n = 13), and assessed neurodegeneration by measuring body weight, motor function and survival time. We assessed the expression of several genes associated with hypoxia in the spinal cord of ONO1301MS or vehicle treated mSOD1^G93A mice with quantitative RT-PCR. We also conducted immunohistochemistry to show the different expression levels of hypoxia inducible factor 1 (HIF1alpha) in the spinal cords of mSOD1^G93A mice.

Results: ONO1301MS significantly improved motor function of mSOD1^G93A mice at 17, 19 and 20 weeks of age although it did not affect body weight and survival time of them. To confirm the vasodilation effect of ONO1301MS in vivo, we conducted immunohistochemical analysis of the spinal cord of mSOD1^G93A mice. ONO1301MS significantly decreased the expression level of HIF1 alpha, suggesting that it had increased the blood flow and ameliorated the hypoxia the spinal cord of mSOD1^G93A mice.

Discussion and conclusion: Our study showed that a synthetic prostacyclin agonist, ONO1301, had beneficial effects against neurodegeneration in mSOD1^G93A mice. The favourable effects could be attributable to the increased blood flow and amelioration of hypoxia in the spinal cord of mSOD1^G93A mice. Further study is necessary to fully characterize the neuroprotective action of ONO1301. We might consider other vasodilation agents as therapeutic potentials for the treatment of ALS.

Acknowledgements: This study was financially supported in part by an Inochi-no-Iro ALS Research Grant and a Grant-in-Aid for Young Scientists (B) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

References:

• Miyazaki K et al. Journal of Cerebral Blood Flow & Metabolism 2012; 32:456–467.
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P243 THE ROLE OF EPHRIN-B2 IN AMYOTROPHIC LATERAL SCLEROSIS

Schoonaert L^1,2

Rué L^1,2

Poppe L^1,2

Timmers M^1,2

Van Hoecke A⁴

Van Damme P^2,3

Lemmens R^2,3

Robberecht W^1,2

^qKU Leuven - University of Leuven, Department of Neurosciences, Experimental Neurology and Leuven Research Institute for Neuroscience and Disease (LIND), Leuven, Belgium

^rVIB, Vesalius Research Center, Laboratory of Neurobiology, Leuven, Belgium

^sUniversity Hospitals Leuven, Department of Neurology, Leuven, Belgium

^tMax-Planck-Institute of Neurobiology, München, Germany

Email address for correspondence: [email protected]

Keywords: ephrin-B2, astrocyte, EphA4

Background: Amyotrophic lateral sclerosis (ALS) is characterized by considerable genetic heterogeneity since mutations in more than 10 different genes (eg, SOD1, FUS, TDP, C9ORF72) are known to cause the hereditary form of ALS. Similar heterogeneity is observed in the clinical presentation. This indicates that there are factors that modify the phenotypic expression of the disease. The tyrosine kinase receptor EphA4 was recently shown to be a modifier of ALS. Genetic and pharmacological inhibition of EphA4 rescued the phenotype in a zebrafish model of ALS and increased survival in ALS rodent models. In ALS patients an inverse correlation was found between EphA4 expression and disease onset. However, the mechanism of action has not yet been fully elucidated. EphA4 interacts with ephrin-a and ephrin-b ligands. Several of these EphA4 interaction partners have been shown to be not only expressed on motor neurons, but also on astrocytes, microglia and oligodendrocytes. These cells surrounding the motor neurons play an important role in the pathogenesis of ALS. Here, we aimed to determine the contribution of these various cell types and one specific EphA4 ligand, ephrin-b2, in ALS disease progression.

Methods: First we performed immunofluorescence staining of ephrin-b2 in the spinal cord of an ALS mouse model, overexpressing mutant SOD1 (SOD1^G93A) and compared this pattern to mice overexpressing wild-type SOD1 (SOD1^WT) at different stages of the disease.

Results: In SOD1^WT spinal cord we observed ephrin-b2 to be highly expressed in motor neurons and oligodendrocytes, while only faint expression was detected in astrocytes. In symptomatic SOD1^G93A spinal cord the expression pattern of ephrin-b2 in astrocytes and motor neurons changed. Immunoreactivity was markedly upregulated in astrocytes, but the presence of ephrin-b2 was clearly reduced in the neuronal population. As the expression pattern changed in the different cell types with disease progression, we next explored a possible modifying cell-specific role of ephrin-b2 in ALS, by generating a conditional ephrin-b2 knockout mouse, in which ephrin-b2 is deleted upon GFAP expression. Deleting ephrin-b2 in reactive astrocytes of the SOD1^G93A ALS mouse model resulted in a delay of disease onset and prolonged disease duration. These results suggest astrocytic ephrin-b2 to play a role in modifying ALS.

Discussion and conclusion: In future experiments we intend to further explore the cellular mechanism of ephrin signalling in the pathophysiology of motor neurodegeneration.

P244 INCREASED OREXIN PROMOTES SLEEP/WAKE DISTURBANCES AND INTERACTS WITH SIRT1 IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

Liu R

Cai B

Fan D

Peking University Third Hospital, Beijing, China

Email address for correspondence: [email protected]

Keywords: sleep/wake disturbance, orexin, SIRT1

Objective: Sleep/wake disturbances of amyotrophic lateral sclerosis (ALS) patients are well documented, whereas corresponding animal or mechanistic study on sleep disturbances in ALS are lacking Orexin and SIRT1 are important molecules in sleep/wake regulation.

Methods: In this study, we used SOD1^G93A transgenic mice as ALS mouse model. EEG/EMG recordings, quantitative reverse transcriptase PCR, western blot, ELISA, co- immunoprecipitation and immunofluorescence were performed between SOD1^G93A transgenic mice and their littermate control mice at the age of 90 days and 120 days.

Results: In SOD1^G93A transgenic mice, for the first time, we observed significantly enhanced wakefulness time, reduced sleep time and up-regulated orexins (prepro-orexin, orexin A and B). SIRT1 is also increased in symptomatic SOD1^G93A transgenic mice and interacts with prepro-orexin in hypothalamus of SOD1^G93A transgenic mice.

Discussion and conclusion: Increased orexins promote wakefulness, result in sleep/wake disturbances and interact with SIRT1 in the hypothalamus in ALS mouse model. This interaction could prevent SIRT1 from neuroprotective effects in ALS and contribute to the disease process. Therefore, sleep disturbances are not only symptoms that occur early in ALS, but also factors that promote ALS progression. This could be common mechanisms in all neurodegenerative diseases, and orexins/SIRT1 pathways might be potential target to retard the disease process in the early stages.

P245 GOLGI FRAGMENTATION IN PMN MICE IS DUE TO A DEFECTIVE ARF1/TBCE CROSS TALK THAT COORDINATES COPI VESICLE FORMATION AND TUBULIN POLYMERIZATION

Bellouze S¹

Schaefer M²

Buttigieg D¹

Baillat G¹

Rabouille C³

Haase G¹

^uCNRS UMR7289 and Aix-Marseille University, Marseille, France

^vKlinik für Anaesthesiologie, Universität Mainz, Mainz, Germany

^wHubrecht Institute-KNAW and University Medical Center Utrecht and The Department of Cell Biology, Utrecht, The Netherlands

Email address for correspondence: [email protected]

Keywords: Golgi fragmentation, microtubules, vesicle trafficking

Background: Golgi fragmentation is an early hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases affecting motor neurons. Yet, its pathophysiological relevance and molecular mechanisms are unclear.

Objectives: To better understand the mechanisms of Golgi fragmentation, we investigated a model of human motor neuron disease, progressive motor neuronopathy (pmn) mice mutated in the Golgi-localized tubulin-binding cofactor E (TBCE), together with TBCE-depleted motor neuron cultures.

Results: We demonstrate severe and progressive Golgi fragmentation in motor neurons of pmn mice. Loss of TBCE function in mutant pmn and TBCE-depleted motor neuron cultures causes defects in Golgi-derived microtubules, as expected, but surprisingly also reduced levels of the COPI subunits β-COP and ϵ-COP, decreased recruitment of the Golgi tethering factors p115/GM130 and impaired vesicle fusion mediated by the Golgi SNAREs GS15/GS28. Conversely, the small GTPase ARF1, which stimulates COPI vesicle formation, enhances the recruitment of TBCE to the Golgi, increases polymerization of Golgi-derived microtubules and rescues TBCE-linked Golgi fragmentation. Importantly, transgenic mutant SOD1 mice display a similar Golgi fragmentation and dys-regulation of COPI subunits, Golgi tethering factors and Golgi SNAREs as pmn mice.

Discussion and conclusion: Our data unravel a novel ARF1/TBCE-mediated cross talk that coordinates COPI vesicle formation and tubulin polymerization at the Golgi apparatus. Interruption of the ARF1/TBCE cross talk causes Golgi fragmentation in pmn mice and possibly also in human SOD1-linked ALS.

P246 ROLE OF CYSTEINE RESIDUE OF MUTANT SOD1 IN THE PATHOGENESIS OF ALS

Nagano S¹

Yamamoto K¹

Urushitani M²

Fujiwara N³

Araki T¹

^xNational Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan

^yDepartment of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Japan

^zDepartment of Biochemistry, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan

Email address for correspondence: [email protected]

Keywords: SOD1, cysteine, oxidation

Background: Previously we have reported that the cysteine residue (Cys111) near the dimer contact site is critical to generate monomers and aggregates of mutant Cu Zn-superoxide dismutase (SOD1) protein, which is thought to be toxic to motor neurons in familial ALS. However, it is not ascertained whether the residue contributes to the motor-neuronal toxicity of mutant SOD1 in vivo.

Objectives: To verify the significance of Cys111 in the pathogenesis of ALS, we aimed to analyze the phenotype of mutant SOD1 transgenic mice in relation to the existence of Cys111.

Methods: We generated SOD1 transgenic mice harbouring H46R mutation alone or H46R plus C111S mutations. Motor performance test and pathological/biochemical analyses of the spinal cord were done to compare the toxicity of mutant SOD1 to motor neurons in each mouse line.

Results: The onset of the disease was delayed and the survival time was extended in SOD1^H46R/C111S mice compared with SOD1^H46R mice. Motor neuron loss and astrocyte/microglia activation was trivial in the spinal cord of SOD1^H46R/C111S mice at the time H46R mice reached to the endpoint. Misfolded or insolubly aggregated SOD1 was seen in the spinal cord of H46R mice at the endpoint, whereas such SOD1 species was not detected in age-matched H46R/C111S mice. Cys111-oxidized SOD1 appeared in the spinal cord of H46R mice throughout their life, which was more prominent than that in wild type SOD1 transgenic mice.

Discussion and conclusions: It was suggested that Cys111 of mutant SOD1 have a key role in the appearance of ALS by generating oxidation-mediated monomerization and aggregation of the protein. Cys111 is thought to take part in the first step to change the high-order structure than the aggregation process of mutant SOD1. The blockage of oxidative modification of Cys111 in mutant SOD1 will be expected as a novel treatment strategy of ALS.

P247 GENDER SPECIFIC BENEFICIAL EFFECTS OF DOCOSAHEXAENOIC ACID DIETARY SUPPLEMENTATION IN ALS MICE

Torres P¹

Cacabelos D¹

Ayala V¹

Boada J¹

Cabré R¹

Naudí A¹

Povedano MP²

Pamplona R¹

Portero-Otin M¹

^aaIRBLLEIDA-UdL, Lleida, Catalonia, Spain

^abHospital de Bellvitge, Barcelona, Catalonia, Spain

Email address for correspondence: [email protected]

Keywords: dietary intervention, fatty acid metabolism, SOD1^G93A

Background: Although the underlying causes of motor neuron degeneration in ALS remain largely unknown, docosahexaenoic acid (DHA), a key fatty acid in nervous system homeostasis, is depleted in spinal cord post-mortem samples of ALS patients, confirming the contribution of changes in fatty acid metabolism to the pathogenesis of ALS (1). A gender bias exists in ALS, with higher incidence and prevalence in men than in women, and different clinical phenotypes, which is also observed in animal models of ALS (2).

Objectives: In this study we evaluated the effect of dietary changes (n-3 fatty acid depletion with and without DHA supplementation) aimed to modify tissue fatty acid composition (3) on survival, disease onset and inflammatory and oxidative stress markers in male and female hSOD1^G93A transgenic mice.

Methods: Survival was assessed by Kaplan-Meier analysis. Spinal cord fatty acid profiles were determined after whole lipid extraction, hydrolysis and gas chromatography analysis of their methyl esters.

Results: Male ALS mice survival was extended (p < 0.0001; n = 14) under DHA dietary supplementation and equalled female lifespan, without modifying the age of disease onset, whereas n-3 fatty acid depletion had no effect in both genders. DHA supplementation resulted in an increase in DHA content at p60 (pre-symptomatic stage) and p90 (disease onset), whereas the peroxidability index was diminished at endpoint. The content of arachidonic acid, a precursor of proinflammatory mediators, and the derived anti-inflammatory index decreased at the three time points. These changes in fatty acid profiles and derived indexes were similar in both genders. On the other hand, n-3 PUFA depletion did not provoke marked changes in the fatty acid profiles. In line with DHA content increase, WB analyses showed that DHA supplementation increased levels of syntaxin 3-DHA dependent synaptic protein in spinal cord of male, but not female, ALS mice.

Discussion and conclusion: Our results demonstrate that DHA supplementation in diets during the pre-symptomatic stage extends survival in male but not female ALS mice, concomitant with an increase in spinal cord DHA content until disease onset, but not at endpoint. These results reinforce the role of gender as a relevant factor in the design of dietary interventions in ALS patients, and suggest a greater beneficial effect of early DHA supplementation in male ALS patients.

Acknowledgements: This study was supported by grants from Instituto de Salud Carlos III to Manuel Portero-Otin (PI11/01532) and FUNDELA to Victoria Ayala and Jordi Boada. Pascual Torres received a fellowship from the Ministerio de Educación, Ciencia y Deporte.

References:

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P248 NEUROPROTECTIVE EFFECTS OF A CELL-FREE EXTRACT DERIVED FROM HUMAN ADIPOSE STEM CELLS IN AMYOTROPHIC LATERAL SCLEROSIS

Park KH¹

Jeon GS¹

Sohn SY¹

Sung J-J¹

Oh J-H²

^acSeoul National University Hospital, Seoul, Republic of Korea

^adJeju National University Hospital

Email address for correspondence: [email protected]

Keywords: SOD1, apoptosis, human adipose stem cells

Background: Amyotrophic lateral sclerosis (ALS) is a devastating human neurodegenerative disease. The aetiology and pathogenic mechanisms of the disease remain unknown, and there is no effective treatment. Human adipose stem cells (hASC) are an easily available source of stem cells. Since hASC can be differentiated into neuronal stem cells, they have clinically feasible potential for neurodegenerative disease.

Objectives: The cytosolic extracts of hASC contain a number of neurotrophic factors. Here, we investigated effects of the hASC extract on SOD1^G93A mouse ALS model and motor neuronal cells (NSC-34).

Results: The hASC extract administration improved motor function and prolonged the time until symptom onset, rotarod failure, and death in transgenic mice with ALS compared to control mice. NSC-34 cells treated with the hASC extract showed decreased mutant SOD1-induced cell toxicity and cell apoptosis.

Conclusion: These results suggest that the hASC is promising as a novel therapeutic strategy for ALS.

P249 REDUCING THE EXPRESSION OF MGLU1 AND MGLU5 RECEPTORS AMELIORATES SURVIVAL AND DISEASE PROGRESSION IN SOD1G93A MICE

Milanese M¹

Bonifacino T¹

Melone M²

Giribaldi F¹

Musante I³

Puliti A^3,4

Conti F²

Bonanno G^1,4

^aeDepartment of Pharmacy, Pharmacology and Toxicology Unit

^afCenter of Excellence for Biomedical Research, University of Genoa, Genoa, Italy

^agDepartment of Experimental and Clinical Medicine, Unit of Neuroscience and Cell Biology, Università Politecnica delle Marche and Center for Neurobiology of Aging, INRCA IRCCS, Ancona, Italy

^ahDepartment of Neuroscience, Rehabilitation, Ophthalmology, Genetics and Maternal and Child Health, Medical Genetics Unit, University of Genoa and Gaslini Institute, Genoa, Italy

Email address for correspondence: [email protected]

Keywords: Group I metabotrophic glutamate receptors, glutamate-mediated excitotoxicity, SOD1^G93A

Background: Glutamate (Glu)-mediated excitotoxicity plays a major role in the degeneration of motor neurons (MNs) in amyotrophic lateral sclerosis (ALS). Besides the impaired glial glutamate transport (1), an excessive Glu release has been also found in the spinal cord of experimental ALS mice (2). Our recent pharmacological studies suggest that the hyper-activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5), expressed at pre-synaptic level in the spinal cord of ALS mice, play a role in this scenario (3).

Objectives: To investigate the effect of the mGluR1 or mGluR5 down-regulation in ALS.

Methods: To provide a genetic tool to evaluate the role of mGluR1 and mGluR5 in ALS, we generated mice carrying half expression of mGluR1 in the SOD1^G93A background, by crossing SOD1^G93A mice with heterozygous mGluR1^+/- mice. In the same line, we also generated mice carrying half expression of mGluR5, by crossing SOD1^G93A mice with mGluR5^+/- mice. Life span, motor abilities, MNs preservation, mitochondrial damage, oxidative stress markers, astrogliosis and microglia activation, receptor expression and glutamate release were investigated to characterize double mutant mice compared to the SOD1^G93A ALS model.

Results: The SOD1^G93AmGluR1^+/- mice showed a delayed pathology onset, improved motor performances and prolonged survival probability, compared to SOD1^G93A mice. These results were associated with reduction of spinal cord motoneuron death, decreased astrocyte and microglia activation, down-regulation of oxidative stress markers and reduced mitochondrial damage. As functional results we also registered a normalization of the abnormal glutamate release induced by the activation of mGluR1 and mGluR5 in SOD1^G93A mGluR1^+/- compared to SOD1^G93A mice. Interestingly, knocking-down mGluR1 also reduced mGluR5 spinal cord expression. SOD1^G93A mGluR5^+/- animals showed a delayed pathology onset and a remarkable prolonged life span, although these data were not accompanied by improved motor performances and phenotype amelioration respect to SOD1^G93A mice. Differently from what we observed in SOD1^G93A mGluR1^+/- mice, reducing mGluR5 does not affect mGluR1 expression.

Discussion and conclusion: Our findings so far demonstrate that mGluR1 or mGluR5 down-regulation has a significant impact in vivo on ALS clinical outcome and provide a rationale for pharmacological approaches based on the selective block of Group I mGluRs.

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P250 SYNAPTIC BOUTONS AND DENDRITIC VACUOLIZATION IN ADULT MOTONEURONS OF MSOD1-G93A MOUSE MODEL OF ALS

Delestrée N

Martinot C

Manuel M

Zytnicki D

University Paris Descartes, UMR CNRS 8119, Paris, France

Email address for correspondence: [email protected]

Keywords: varicosities, synapses, SOD1^G93A

Background: A striking pathological feature observed in mSOD1 mouse models of ALS is the presence of vacuoles in the soma, the axon and also the dendrites of their motor neurons (1). The mechanism leading to the vacuolization is not known. However, vacuoles might originate through dilation of the mitochondrial intermembrane space (2).

Objectives: We investigated where the vacuoles appear along the dendritic tree, how they evolve during the time course of the disease and how synaptic boutons are distributed with respect to the dendritic vacuoles.

Methods: Spinal motor neurons were recorded intracellularly and labelled with neurobiotin in anaesthetized mice. Our preparation also allows us to record motor units EMG in response to intracellular stimulation to asses the state of denervation of motoneurons. After intracardiac perfusion of the mouse, spinal sections were processed for immunolabelling of excitatory (VGLUT1 and VGLUT2) and inhibitory (VGAT) boutons. Z-stacks were analysed using Neurolucida software.

Results: Both WT and SOD1^G93A mice display swellings along their dendrites (same density: WT 67 ± 28 vs. SOD 65 ± 21 swellings/mm), but vacuolization appears in the dendritic swellings of mSOD1 motoneurons only. This vacuolization process has already started by P40 in motoneurons still able to excite their muscle fibers, indicating that the neuromuscular junctions are still intact. Afterward, dendritic vacuoles dramatically grow throughout the disease to reach diameters as large as 15 micrometers at P110. Swellings are also present in mice expressing non-mutated human SOD1 but at P180 they still do not display vacuolization. The average densities of excitatory (VGLUT1 and VGLUT2) and inhibitory (VGAT) boutons that contact the dendritic tree (measured on 350–1500 μm of dendritic length for each cell) are unchanged at P40–50 in mSOD1 (n = 6) mice compared to WT (n = 6) mice (VGAT: WT 0.54 ± 0.09 vs. SOD1 0.45 ± 0.08 boutons/μm; VGLUT1: WT 0.06 ± 0.04 vs. SOD1 0.07 ± 0.02 boutons/μm; VGLUT2: WT 0.42 ± 0.10 vs. SOD1 0.49 ± 0.07 boutons/μm). Unexpectedly, we found that VGLUT2 and VGAT boutons tend to cluster on swellings in both WT and mSOD1 dendrites creating hotspots of extensive ion influx and high metabolic activity.

Discussion and conclusion: Our data suggest that there might be a causal link between synaptic activity and dendritic vacuolization in spinal motor neurons of ALS mice. Our work is reinforcing the hypothesis that excitotoxicity can lead to mitochondrial damages and ultimately to degeneration of motor neurons in ALS.

Acknowledgements: NIH 1R01NS077863-01, ANR-2010-BLAN-1429, Target-ALS, Foundation Thierry Latran “OHEX project”, S. Boilée for the gift of hSOD1-WT mice.

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P251 NEUROPROTECTIVE AND ANTI-NEUROINFLAMMATORY EFFECTS OF MECASIN VIA UP-REGULATION OF HEME OXYGENASE-1 IN MOUSE HIPPOCAMPAL AND MICROGLIAL CELLS

Lee J-C¹

Kim S-C¹

Lee D-S²

Kim Y-C³

^aiCenter of Amyotrophic lateral Sclerosis, Wonkwang University Hospital, Gwangju, Republic of Korea

^ajDepartment of Biochemistry, Inha University School of Medicine, Incheon, Republic of Korea

^akInstitute of Pharmaceutical Research and Development, College of Pharmacy, Wonkwang University, Iksan, Jeollabuk-do, Republic of Korea

Email address for correspondence: [email protected]

Keywords: anti-glutamate toxicity, neuroprotective, anti-neuroinflammatory

Background: One of the various causes of ALS is known as glutamate toxicity. Oxidative stress and neuroinflammation have also been implicated in many neurodegenerative diseases. In previous studies on microglial responses in central nervous system (CNS) inflammation, microglial activation was induced by a variety of agents, including the bacterial product lipopolysaccharide (LPS), and proinflammatory cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Heme oxygenase (HO)-1, an enzyme essential for heme degradation, has been shown to exert antioxidative and anti-inflammatory effects under various conditions.

Objectives: The aim of this study was to evaluate neuroprotective and anti-neuroinflammatory effects of mecasin in mouse hippocampal and microglial cells on glutamate- or LPS-induced neuronal cell injury.

Methods: Mecasin, a combination of natural plant extracts, was obtained from Curcuma longa, Salvia miltiorrhiza, Gastrodia elata, Chaenomeles sinensis, Polygala tenuifolia, Paenia japonica, Glycyrrhiza uralensis, Atractylodes japonica and Aconitum carmichaeli, and was freshly dissolved in dimethyl sulfoxide. The effects of Mecasin on cell viability were studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of prostaglandin E2 (PGE2), TNF-α, and interleukin-1β (IL-1β) were also evaluated. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, HO-1, and the nuclear factor-E2-related factor 2 (Nrf2) were evaluated by Western blot using specific antibodies. In addition, the translocation of Nrf2 was also assessed by immunofluorescence microscopy.

Results: At the non-cytotoxic concentrations, Mecasin (10, 50, 100, 200 μM) increased the cellular resistance of HT22 cells to oxidative injury caused by the glutamate-induced cytotoxicity by nuclear translocation of Nrf2-mediated HO-1 expression in a concentration-dependent manner. Furthermore, mecasin significantly suppressed the LPS-induced expression of pro-inflammatory enzymes and inflammatory mediators, and also inhibited the production of NO, PGE2, COX-2, iNOS through Nrf2-mediated HO-1 expression.

Discussion and conclusion: Mecasin, a combination of natural plant extracts, showed potent cytoprotective effects on glutamate-induced neurotoxicity in the mouse hippocampal HT22 cells, presumably through Nrf2 pathway-dependent HO-1 expression. Also, the mecasin as an anti-neuroinflammatory agent was investigated in microglia activation by LPS. We have demonstrated that Mecasin suppresses pro-inflammatory mediators through Nrf2-dependent expression of anti-inflammatory HO-1 in BV-2 microglia. These results suggest that Mecasin possesses therapeutic potentials against neurodegenerative diseases with oxidative stress and neuroinflammation.

Acknowledgements: This work was supported by R & D(B110076) of Korea Institute, Ministry of Health and Welfare in 2013.

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P252 SPECIFIC LOSS AND DYSFUNCTION OF CALRETININ INTERNEURONS IN THE SOD1G93A AND HUMAN ALS CORTEX: A PRIMARY MECHANISM OF ALS?

Clark R¹

Fielder T¹

King A²

Dickson T¹

^alMenzies Research Institute

^amSchool of Medicine, University of Tasmania, Hobart, Tasmania, Australia

Email address for correspondence: [email protected]

Keywords: pathology, interneurons, excitotoxicity

Background: In amyotrophic lateral sclerosis (ALS), increased excitability of circuitry precedes motor neuron degeneration, suggesting that ALS results from disturbances in regulation of cell excitability. However, the mechanism of presymptomatic excitability remains unknown. There is strong clinical evidence, in both cortical and spinal regions, of reduced inhibition implicating this as a potential primary mechanism. We examined the motor and somatosensory cortex of the SOD1^G93A and non-transgenic mice and human ALS and control tissue for expression of interneuron-specific calcium binding and neuropeptide protein markers.

Objectives: To characterise the pathological alterations to cortical interneuron subpopulations in ALS mouse models over the time course of symptom development compared to that present in ALS tissue.

Methods: Cortical tissue from presymptomatic (8 week) and end-stage (20 week) SOD1^G93A and ALS human cortex were serially sectioned (40μm), alongside age-matched controls, and immunohistochemically labelled with antibodies against calretinin (CR), parvalbumin (PV), somatostatin (SOM), Neuropeptide Y (NPY) and Vasoactive Intestinal protein (VIP). Morphometric analysis was also performed using Neurolucida to investigate changes in neurite length and branching patterns.

Results: In SOD1 mice we find that in late symptomatic stages, specific interneuron subsets of the motor cortex display contrasting (and potentially complimentary) pathology; the density of calretinin (CR) populations is significantly reduced by 37% in supragranular lamina (WT, n = 4, 55.3 ± 6.9mm²; SOD1, n = 4, 35.3 ± 6.0mm²), whilst neuropeptide Y (NPY) populations are increased by 40% in the infragranular lamina (WT, n = 4, 18.6 ± 2.4mm²; SOD1, n = 4, 31.0 ± 4.2mm²) (p < 0.05, two-way ANOVA with Bonferroni post-hoc test). Moreover, using morphometric approaches, we show that remaining CR populations have undergone early and continuing alteration to neurite labelling patterns, with progressive reductions in neurite complexity from presymptomatic- to late- symptomatic stages.

Discussion and conclusion: These findings indicate that inhibitory regulation of cortical circuitry may be impaired in a motor- and lamina-specific manner, prior to motor neuron loss, in ALS. Differential involvement of CR- and NPY-positive interneurons suggests interplay of these specific populations may drive altered regulation, as the majority of remaining cortical interneuron populations are not affected. Furthermore, analysis of human ALS post-mortem brain tissue revealed a cluster of ALS cases with reduced CR density in lamina II/III compared with controls. This may be suggestive of unique motor system vulnerabilities involving the early susceptibility of interneurons in the pathogenesis of ALS.

P253 THE ELECTROPHYSIOLOGICAL PROPERTIES OF THE DIFFERENT MOTOR UNIT SUBTYPES ARE NOT EQUALLY AFFECTED IN ADULT SOD1-G93A MICE

Manuel M^1,2

Heckman CJ²

Zytnicki D¹

^anUniv Paris Descartes, UMR CNRS 8119, Paris, France

^aoDept of Physiology, Northwestern University, Chicago, IL, USA

Email address for correspondence: [email protected]

Keywords: excitotoxicity, spinal motoneuron, intracellular recording

Background: One of the proposed mechanisms to explain the death of motoneurons (MNs) in ALS is an excitotoxic process, ie, an excess electrical activity leading to an overload of intracellular calcium triggering apoptotic death. However, we have recently shown (1) that spinal MNs of adult mice are not intrinsically hyperexcitable in SOD1^G93A mice. Instead, we found that a subpopulation of MNs lost their ability to fire repetitively in response to a stationary input. Yet, at the time, we could not determine whether this hypo-excitability was restricted to a specific physiological type of motor unit.

Objectives: The goal of this work is to correlate changes in excitability with the physiological type of the recorded motor units.

Methods: We have developed an in vivo adult mouse preparation allowing simultaneously recording intracellularly spinal MNs and recording the force developed by their motor unit. We characterized the properties of motor units (MUs) from the Triceps Surae muscles of SOD1 mice and their non-transgenic controls (WT) at the stage preceding their denervation (P35-P65). We classified the motor units in physiological types (“Slow” - S, “Fast fatigue-Resistant” - FR and “Fast Fatigable” - FF) on the basis of their contractile properties (contraction time, twitch force, fatigability, sag in unfused tetanus).

Results: During the time frame studied, the contractile properties of the MUs are not affected by the disease, and we could use the same criteria to classify MUs as in WT mice. We found that many electrical properties of SOD1 MNs are unchanged. For example, there were no differences between SOD1 and WT MNs in term of input conductance or rheobase in each of the physiological types taken separately. However, in keeping with our earlier results, we found that a large proportion of MNs (33% vs. 9% in WT mice) lost the ability to produce a sustained firing in response to a stationary input. Interestingly, all of these MUs had a fast contraction time (mean ± SD 13 ± 3 ms, N = 13), and produced twitch forces ranging from 0.4 to 34 mN. Overall 4 out of the 6 MUs that we classified as FF units, and 9 out of the 18 classified as FR units had lost the ability to produce a sustained firing.

Discussion and conclusion: We were therefore able to demonstrate that FF and FR MUs (that are vulnerable in ALS), but not the S type MUs (that are resistant in ALS), become progressively hypoexcitable before they lose their connections to their muscle fibers. We are now investigating if an excitotoxic process could nevertheless arise from changes in excitatory and inhibitory inputs to MNs.

Acknowledgements: NIH 1R01NS077863-01, ANR-2010-BLAN-1429, TARGET ALS, Fondation Thierry Latran “OHEX project”

Reference:

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P254 VISUALIZATION OF CSMN IN THE DISEASE MODELS OF ALS USING NOVEL UCHL1-EGFP REPORTER MICE REVEALS DETAILS OF CELLULAR VULNERABILITY

Gautam M¹

Sekerkova G²

Ravi V³

Yasvoina M¹

Jara J¹

Martina M²

Ozdinler H¹

^apDepartment of Neurology

^aqDepartment of Physiology, Northwestern University, Chicago, IL, USA

^arIllinois Math and Science Academy, Chicago, IL, USA

Email address for correspondence: [email protected]

Keywords: CSMN, upper motor neurons, reporter line

Background: Corticospinal motor neurons (CSMN) are unique in their ability to collect, integrate, translate and transmit cerebral cortex input towards spinal cord targets. Their location in cerebral cortex and their complex network with neighbouring cells make it extremely arduous to study CSMN specifically. Progressive degeneration of CSMN is prominent feature in many neurodegenerative diseases such as ALS. Despite their immensely important functions, lack of appropriate methods to study CSMN hinders our knowledge about the impact of their health and diseases. TDP-43 is a DNA/RNA binding protein showing pathological signatures in ALS. There are several mutations in the TDP-43 gene involved in ALS disease (1). In addition, mutations in the Alsin 2 (ALS2) gene are responsible for juvenile primary lateral sclerosis, infantile onset ascending hereditary spastic paraplegia, and are the most common cause for autosomal recessive juvenile ALS (2). Upper motor neuron signs and bulbar symptoms are often prevalent in patients with juvenile ALS. Therefore, it is important to investigate the health of CSMN in regard to these diseases.

Objective: Here, we investigate the health, stability and cellular vulnerability of CSMN in hTDP-43^A315T and Alsin KO mice. We crossed UCHL1-^eGFP reporter mice (3) with Alsin KO and hTDP-43^A315T mice which overexpresses human TDP-43^A315T gene (4), to generate hTDP-43^A315T-UeGFP and AlsinKO-^UeGFP mice respectively. In the reporter disease mice, the CSMN are genetically labelled with stable eGFP expression allowing visualization and cellular analysis of CSMN through adulthood and late ages.

Results: Our ongoing studies suggest very subtle, yet important cellular changes that occur in CSMN. There is axonal degeneration of the subcerebral projection neurons, including CSMN, increased autophagy with age and changes in the localization of autophagic vesicles toward apical dendrites. Even though the neurons are not completely cleared from the motor cortex, detailed cellular visualization and analysis using immunocytochemistry coupled with electron microscopy (EM) reveal very precise aspects of cellular vulnerability and ongoing degeneration.

Discussion and conclusion: Investigation of pure upper motor neuron defects in mouse is challenging, but here we demonstrate that using UCHL1-eGFP mice as a reporter for CSMN, their health and potential pathways that contribute to their vulnerability can be studied at a cellular level with high precision.

Acknowledgements: We thank Dr. T. Siddique for providing Alsin^-/- mice. This work was supported by the grants from Les Turner ALS Foundation and Herbert C Wenske Foundation (PHO), and NIH-R21 NS085750-01 (PHO).

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P255 IMPORTANCE OF UCHL1 FUNCTION FOR THE MOTOR NEURON CIRCUITRY AND THE HEALTH OF THE CORTICOSPINAL MOTOR NEURONS

Genc B¹

Jara J¹

Ulupinar E²

Manuel M³

Cox G⁴

Bohn M¹

Heckman CJ³

Roos R⁵

Macklis JD⁶

Didonato CJ⁷

Ozdinler H¹

^asDepartment of Neurology, Northwestern University, Chicago, IL, USA

^atOsmangazi University Medical School, Eskisehir, Turkey

^auDepartment of Physiology, Northwestern University, Chicago, IL, USA

^avThe Jackson Laboratories, Bar Harbor, ME, USA

^awDepartment of Neurology, University of Chicago, Chicago, IL, USA

^axDepartment of Stem Cell and Regenerative Biology, Harvard Medical School, Boston, MA, USA

^ayDepartment of Pediatrics, Northwestern University, Chicago, IL, USA

Email address for correspondence: [email protected]

Keywords: corticospinal motor neurons CSMN, upper motor neurons, UCHL1

Background: Corticospinal motor neurons (CSMN) receive, integrate and relay cerebral cortex input towards spinal targets to initiate and modulate voluntary movement. CSMN degeneration is central for numerous motor neuron disorders and neurodegenerative diseases, but the cellular and molecular basis of CSMN vulnerability and progressive degeneration remains unknown. Mutations in the ubiquitin C-terminal hydrolase-L1 (UCHL1) gene have been detected in patients with neurodegenerative disease that affect motor function; recently three siblings displayed early neurodegeneration, including upper motor neuron dysfunction. In the absence of UCHL1 function, CSMN show profound degeneration, it is important to understand the importance of CSMN for the health of the motor neuron circuitry, and how UCHL1 function affects CSMN health and stability.

Objectives: This study aims to understand the possible function of UCHL1 for the health and stability of CSMN, as well as the proper function of the motor neuron circuitry.

Methods: Using the UCHL1^nm3419 mice, which lack all UCHL1 function, we have analyzed motor neuron function at multiple levels: 1) behavioural tests to evaluate overall motor function (rotarod, Digigait, and grip test); 2) in vivo analysis of spinal motor neuron function; 3) immunocytochemical analysis of muscle, spinal cord and cerebral cortex in combination with retrograde labelling of CSMN approaches by Fluoro-Gold and retrograde transduction via AAV2-2; 4) generated novel conditional mutant mice in which UCHL1 function is removed either from the corticospinal or the spinal motor neurons, respectively.

Results: Our results demonstrate a unique function of UCHL1 in maintaining CSMN viability and cellular integrity. Uchl1^nm3419 (UCHL1 ^−/−) mice, which lack all UCHL1 function, display motor neuron circuitry defects. Even though spinal motor neurons remain intact with subtle dysfunction, CSMN show early, selective, progressive and profound cell loss. CSMN degeneration is mediated via increased ER stress and becomes evident at pre-symptomatic stages by cytoarchitectural defects primarily involving the apical dendrites. The novel transgenic mice in which UCHL1 function is selectively blocked in CSMN began to reveal the impact of the CSMN health on motor neuron circuitry.

Conclusion: We report that UCHL1 is essential for motor circuitry and is especially important for CSMN health. We now characterize a novel tool especially for motor neuron diseases with prominent CSMN involvement.

Acknowledgments: The Milton Safenowitz Post-Doctoral Fellowship from the ALS (JHJ). NUCATS Translational Innovation award, Les Turner ALS Foundation, and Wenske Foundation (PHO). NIH M.A.D. Training Grant 5T32AG020506-09 (BG) and NIH- RO1NS085161-01 (PHO)

P256 DEVELOPMENT AND CHARACTERIZATION OF MUTANT HUMAN PROFILIN1 TRANSGENIC MICE AS NEW MODEL FOR ALS

Yadav S

Kiaei M

University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA

Email address for correspondence: [email protected]

Keywords: profilin1, actin binding protein, transgenic mouse model

Background: The mechanism of neuronal degeneration and muscle atrophy in ALS is poorly understood. So far, ALS genes identified account for approximately only 50% for familial ALS patients. Recently, five mutations in profilin1 (PFN1) gene (ALS18) were linked to a subpopulation of fALS patients which had none of the previously known mutated genes in fALS (1). PFN1 is an Actin-binding protein essential for regulation of filamentous F-actin formation from monomeric G-actin. Whether PFN1 mutations in this group of ALS patients is a cause of ALS, remain unknown. Identification of PFN1 mutation in human ALS patients with approximately 10 years earlier average age of onset than other ALS patients, and common clinical limb onset makes a strong case for its involvement.

Objectives: To develop animal model overexpressing profilin1 with one of the mutations found in ALS patients and study whether expression of mutant profilin1 cause ALS-like phenotypes.

Methods: Standard transgenic methodology was employed and transgenic mice were monitored for general beings, behavior, weights, motor performance and survival length with standard techniques.

Results: We have successfully created three lines of transgenic mice overexpressing mutant human PFN1, High (H), Medium (M) and low (L) expressing lines. While these mice are healthy enough to breed and generate viable offspring, the H line mice develop ALS-like phenotypes; ie, hindlimb tremor, clasping, gait abnormality leading to low body profile, reduced stride length, gradual weakness and atrophy in muscle of limbs, hunched back posture (kyphosis) toward later part of the disease, and reduced life-span. Staining with the astrocyte marker, glial fibrillary acidic protein (GFAP), also indicate astrocytosis. The average survival is 177 ± 5 days for H line (n = 9), so far. The other two lines have subtle phenotypes that progress slowly and are still alive at 430 days, thus far.

Discussion and conclusion: We have generated three lines of transgenic mice overexpressing mutant human profilin1. Two low expressing lines have subtle phenotypes, slow progression and no change in survival length observed. The high expressing line exhibit subtle phenotype as early as weaning age and progressed slowly. They become progressively worse from 140 days, began rapid weight loss and motor weakness which resulted in premature death. To our knowledge this model with profilin1 mutation is the first to be produced and develop symptoms and signs that resembles ALS.

Acknowledgements: Authors acknowledge support by grants from UAMS startup funds and the College of Medicine Research Council. Also, this research is funded by a pilot study award from the Center for Translational Neuroscience, NIGMS IDeA Program award P20 GM103425-10.

Reference:

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P257 GENERATION OF A NOVEL MOUSE MODEL OF FUS-ALS USING BACTERIAL ARTIFICIAL CHROMOSOME (BAC) TECHNOLOGY

Vizard T¹

Kent L¹

Thomas M²

Alegre-Abarrategui J²

Davies B³

Ansorge O⁴

Wade-Martins R²

Talbot K¹

^azNuffield Department of Clinical Neurosciences

^baDepartment of Physiology, Anatomy and Genetics

^bbWellcome Trust Centre for Human Genetics, University of Oxford, UK, Oxford, UK

^bcDepartment of Neuropathology, John Radcliffe Hospital, Oxford, UK

Email address for correspondence: [email protected]

Keywords: FUS, mouse, BAC

Background: Mutations in the gene encoding the RNA-binding protein FUS (Fused in sarcoma) cause a subtype of ALS characterized by cytoplasmic mislocalisation of FUS, the extent of which correlates with disease onset and severity. A number of rodent models have been created in an attempt to recapitulate key features of FUS-ALS. The majority of these have been generated using cDNA-based expression strategies, where a FUS cDNA transgene is often expressed at levels higher than that of endogenous FUS. Expression of a wild-type FUS transgene using this approach can result in a neurodegenerative phenotype in rodents, albeit with later onset than that of an ALS-associated FUS mutant. This observation highlights whether these models truly reflect the human disease or instead demonstrate toxicity from FUS overexpression. Here we sought to generate a novel FUS mouse model relevant to human disease using the entire human FUS genomic locus together with BAC technology to express the human FUS transgene in a physiological manner.

Objectives: To create a mouse model of FUS-ALS by expressing FUS from the entire human FUS gene using BAC transgenesis.

Methods: BAC constructs containing the entire human FUS locus were generated harbouring either the human wild-type (WT) FUS sequence or the ALS-derived P525L mutation. The FUS gene including its introns, downstream of its own promoter and regulatory sequences, was cloned into a BAC. An N-terminal mCherry fluorescent tag was fused to Exon 1 of human FUS and LoxP sites were introduced flanking the FUS promoter. Founder mice were generated from pronuclear injection of fertilized oocytes with WT FUS-BAC and P525L FUS-BAC. Independent mouse lines were established from each founder.

Results: We have successively generated independent mouse lines from pronuclear injections of WT and P525L FUS-BAC constructs. Presence of the intact BAC was determined using PCR from Founder and F1 mouse genomic DNA. FUS-BAC copy number in each of the mouse lines has been determined using a qPCR assay and has shown to be present in the mouse genome from ∼9 to 90 copies. Successful transgene expression has been established by Western blot and correlates with FUS-BAC copy number. Motor neurons cultured from the spinal cord of WT FUS-BAC mice reveal a predominantly nuclear localisation of human WT FUS. P525L FUS-BAC motor neurons, however, demonstrate mislocalisation of mutant FUS to the cytoplasm, consistent with the established cellular phenotype associated with this mutation.

Discussion and conclusion: Here we describe the generation of a novel FUS mouse model using BAC transgenesis. Early experimental evidence reveal that mouse lines from each of the WT and P525L FUS-BAC constructs show successful expression of the transgene.

Acknowledgements: We thank the MND Association and the Patrick Berthoud Trust for funding.

P258 AN INVESTIGATION OF THE ROLES OF TDP-43 AND C9ORF72 IN REGULATION OF THE NEURONAL CYTOSKELETON IN AMYOTROPHIC LATERAL SCLEROSIS

Baskaran P

Shaw CE

Guthrie S

Kings College London, London, UK

Email address for correspondence: [email protected]

Keywords: TDP-43, C9ORF72, toxicity

Background: The presence of ubiquitinated misfolded protein inclusions in the cytoplasm/nucleus of neurons is the key feature of most neurodegenerative diseases. This project will directly investigate the molecular mechanisms underlying amyotrophic lateral sclerosis (ALS). Mutations in the TDP-43 and C9ORF72 genes have been shown to cause ALS. TDP-43 has DNA and RNA binding properties, and is involved in RNA splicing, transport and stability (1, 2).

Local translation of mRNA plays a key role in axonal guidance to targets, synapse formation and maintenance and neuronal survival processes which may mis-function in ALS. Genetic studies have also identified expansion repeat number as a key factor in several degenerative diseases and currently the GGGGCC (G₄C₂) intronic repeat expansion within C9ORF72 has been identified as the most common genetic cause of ALS and frontotemporal dementia (FTD) (3,4) .

Objectives: Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G₄C₂ RNA may be toxic. We are using rat primary cortical neurons in vitro and the chick embryonic system in vivo to model the acute effects of TDP-43 and C9ORF72 hexanucleotide repeats. We aim to test the role of TDP-43 in subcellular localisation of a key range of mRNA species, whose mis- localization can cause defective cytoskeletal dynamics, synaptogenesis/synapse maintenance and neuronal death.

Results: Our experiments show that that TDP-43 mis-localizes to the cytoplasm over time and that it directly or indirectly affects mRNA localization and axonal transport thereby affecting the cytoskeletal organization. We also demonstrate that the G4C2 repeats implicated in C9ORF72 pathogenesis form intranuclear RNA foci that initiate apoptotic cell death in chick spinal motor neurons in vivo.

Discussion and conclusion: The phenotypic changes observed in the motor axon projections in embryos transfected with the G₄C₂ repeats are similar to those measured in embryos transfected with TDP-43 mutations, which suggests that there may be some shared mechanism, possibly involving the sequestering of the TDP-43 protein. We propose that RNA toxicity and protein sequestration may disrupt RNA processing, along with the disruption of cytoskeletal integrity, may contribute to neurodegeneration.

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P259 TDP-43 AND THE AXONAL CYTOSKELETON

King A

Liu Y

Atkinson R

Vickers J

Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, Tasmania, Australia

Email address for correspondence: [email protected]

Keywords: TDP-43, axon, cytoskeleton

Background: TDP-43 is an RNA/DNA binding protein with a primary pathogenic role in ALS. Although a ubiquitously expressed protein, studies have recently shed light on how TDP-43 may have specialised functions in neurons. TDP-43, although predominantly a nuclear protein, is transported into axons and dendrites where it is involved in the transport of mRNA. Axonal and dendritic protein synthesis enables rapid alterations in local structure and directly results in adaptive responses ‘on site’. TDP-43 is a known regulator of neurofilament light (NFL) mRNA thus we propose that TDP-43 plays a role in plasticity and remodelling through modulation of the cytoskeleton.

Objectives: We aim to investigate the role of TDP-43 in cytoskeletal alterations, axonal function and plasticity. In preliminary investigations, expression of TDP-43 decreased during mouse development as expression of neurofilament increased. We hypothesised that TDP-43 may be a plasticity-related protein and that increased demand for neurofilament or other cytoskeletal proteins due to neurite outgrowth or remodelling could drive increased expression of TDP-43. Therefore we investigated whether decreased expression of neurofilament protein results in alterations to TDP-43 expression, localization or phosphorylation.

Methods: We investigated TDP-43 expression in the cortex, hippocampus and spinal cord of adult (10 week) and aged (12 month) mice lacking NF-L protein. Western blotting and immunohistochemistry was performed using antibodies against phosphorylation independent TDP-43 and phosphorylated forms of TDP-43 implicated in ALS (n = 5 animals per group)

Results: There was a significant (p < 0.05) increase in TDP-43 in the spinal cord of adult mice (49 ± 0.08%) and in cortex and lumbar spinal cord of aged NFL-KO mice (20 ± 0.05%, 41% ± 0.09% respectively). Increased TDP-43 expression was not associated with increased cytoplasmic localization of TDP-43 or with alterations in TDP-43 phosphorylation. However, altered phosphorylation of TDP-43 was associated with ageing with a significant (p < 0.05, 241 ± 0.07%) increase in phospho-TDP-43 (pS409/410) and a significant decrease (p < 0.05, 22.82 ± 0.03%) in phospho TDP-43 (pS403/404) in aged mice. Our current studies continue to investigate the role of altered TDP-43 (increased/decreased expression or mutation) on axon dynamics and plasticity.

Discussion and conclusion: Developmental expression of TDP-43 declines in association with the increased expression of neurofilament proteins within the axon, with the latter linked with reduced neurite plasticity and the stabilization of networks. Our study suggests that decreased expression of neurofilament can drive alterations in TDP-43 expression in vulnerable regions of the nervous system. However, in our study, increased expression of TDP-43 alone did not r esult in pathological alterations including TDP-43 phosphorylation.

Investigating the role of TDP-43 in plasticity dependent remodelling could lead to new avenues for investigating the cause of neuronal dysfunction in ALS.

Acknowledgements: Motor Neuron Disease Research Institute of Australia, Alzheimer's Australia Dementia Research Foundation, Wicking Dementia Centre.

P260 SYNAPTIC ALTERATIONS IN THE TDP-43A315T MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

Blizzard C

Handley E

Clark R

Fielder T

Dickson T

University of Tasmania, Hobart, Tasmania, Australia

Email address for correspondence: [email protected]

Keywords: TDP-43 mutation, synapse, YFP

Background: TDP-43 is the major component of the inclusions that define ALS pathologically and has been identified as a genetic cause for ALS, highlighting the importance of this protein in this disease (1). Whilst previous research has focused upon the role of TDP-43 in the nucleus under normal conditions and cytoplasmic aggregates pathologically, recent research has indicated that TDP-43 misprocessing, as a consequence of either TDP-43 mislocalisation or TDP-43 mutant gain/loss of function may have an underappreciated pathological role at the synapse

Objectives: To characterise the pre- and post-synaptic pathology occurring in the TDP-43^A315T mouse model of ALS with regional immunohistochemistry (IHC) and spine density analysis. Spine analysis was investigated in TDP-43^A315T YFP-H fluorescent mice. YFP-H mice ubiquitously express yellow fluorescent protein (YFP) in a subset of neocortical pyramidal neurons (2), making it an ideal model to study spine morphology.

Methods: Mice were perfused with 4% paraformaldehyde over a time-course of disease (postnatal day 30 to day 90). Pre- and post-synaptic pathology was investigated using IHC on 60μm coronal sections at day 90 with antibodies directed again VGlut (pre-synaptic excitatory) and GAT1 (pre-synaptic inhibitory) and PSD-95 (post-synaptic). Dendrite spines in the TDP-43^A315T YFP-H mice were investigated in 20μm coronal sections on the Zeiss LSM 510 Meta confocal microscope and Neurolucida software.

Results: Our investigations have identified a significant (p < 0.05) reduction in Glutamatergic (excitatory) and GABAergic (inhibitory) pre-synaptic vesicle transporters by day 90 (symptom onset) in the TDP-43^A315T mice compared to wild-type controls. Of note, these changes were specific to the motor cortex and not present in the somatosensory cortex. Additional IHC investigations revealed that PSD-95, which activity dependently co-localises with TDP-43 (3), was mislocalised to cytoplasmic granules in cortical neurons of the symptom onset TDP-43^A315T mice. Preliminary investigations in TDP-43^A315T x YFP-H mice found dendrite spine alterations during symptom onset, suggesting that early post-synaptic disturbances may be occurring in this model. We are currently using this novel mouse model to determine the precise time-course of spine alterations using multi photon in vivo live imaging.

Conclusion: Synaptic dysfunction is an early pathogenic event in ALS. Our investigations highlight a potential pathogenic role for TDP-43 at the synapse. Understanding the role that TDP-43 plays in this synaptic pathogenesis represents a critical first step towards revealing a new therapeutic window for intervention - targeted at synaptic function - to improve outcomes for people suffering from ALS.

Acknowledgements: This work was supported by the MNDRIA, Alzheimer's Australia and Brain Foundation Australia.

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P261 TDP-43-MEDIATED HDAC6 EXPRESSION PROMOTES THE FORMATION OF LARGER PRE-SYNAPTIC DENSITIES, FACILITATING NEUROTRANSMITTER RELEASE

Miskiewicz K^1,2

Jose L^1,2

Yeshaw W^1,2

Valadas J^1,2

Swerts J^1,2

Munck S^2,3

Feiguin F⁴

Dermaut B^5,6

Verstreken P^1,2

^bdVIB, Center for the Biology of Disease, Leuven, Belgium

^beKULeuven, Center for Human Genetics, Leuven, Belgium

^bfVIB Bio Imaging Core, Leuven, Belgium

^bgInternational Center for Genetic Engineering and Biotechnology, Trieste, Italy

^bhInstitut Pasteur de Lille, Université de Lille Nord de France, Lille, France

^biCenter for Medical Genetics, Ghent University Hospital, Gent, Belgium

Email address for correspondence: [email protected]

Keywords: TDP-43, active zone, neurotransmission

Background: TAR DNA-binding protein TDP-43 acts in different neurodegenerative diseases and mutations in TDP-43 are causative for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. Furthermore the early phases of ALS and other neurodegenerative disorders are characterized by hyper-excitability but the mechanisms resulting in synaptic defects remain enigmatic.

Objectives: The aim of this work is to identify early synaptic defects caused by deregulation of TDP-43/TBPH function and to characterize the mechanisms of synaptic dysfunction.

Methods: We used the neuromuscular junction of Drosophila 3^rd instar larvae expressing pathogenic TDP-43 mutants and tbph/tdp-43 null mutants. We applied super-resolution microscopy and TEM to identify structural changes at synapses as well as synaptopHluorin imaging and electrophysiology to characterize pre-synaptic function.

Results: The expression of pathogenic mutants TDP-43 results in increased synaptic vesicle fusion and a larger readily releasable pool. These defects correlate with enlargement of synaptic vesicle-tethering sites. In contrast tbph loss-of-function mutations cause opposite defects, suggesting that pathogenic TDP-43 expression causes synaptic defects by a gain-of-function mechanism.

The expression of pathogenic TDP-43 mimics phenotypes that were previously linked to the reduction in the acetylation status of the protein bruchpilot (BRP), which is a component of the vesicle tethering site at synapses (1). Using biochemical assays we show that flies expressing pathogenic TDP-43 show much lower BRP acetylation levels in vivo. TDP-43 is known to bind HDAC6 mRNA and regulates its expression such that tbph loss-of-function results in low levels of HDAC6, while over-expression of pathogenic and wild type TDP43 causes up-regulation of HDAC6 mRNA. Using in vivo and in vitro assays we show that HDAC6 is necessary and sufficient to de-acetylate BRP, providing an explanation as to why BRP acetylation is reduced in animals expressing TDP-43. Furthermore, our data indicate that the synaptic defects upon expression of TDP-43 pathogenic mutants correlate with HDAC6 up-regulation, as over expression of HDAC6 also causes larger tethering sites and more synaptic transmission while HDAC6 knock out causes opposite phenotypes, similar to tbph loss-of-function. Finally, genetically correcting the BRP acetylation defects in TDP-43 mutant expressing animals using partial loss of hdac6 or over-expression of ELP3, a BRP acetyltransferase also associated with ALS, rescues the synaptic and adult motor defects.

Discussion and conclusion: We show that de-regulation of TDP-43/TBPH cause defects in neuronal transmission by controlling HDAC6 expression. We identify HDAC6 as a de-acetylase of the active zone protein BRP. Furthermore, our work using fruit flies points to the convergence of two ALS-relevant players, TDP-43 and ELP3, suggesting that HDAC6-dependent de-acetylation and ELP3-dependent acetylation of active zone material is a mechanism by which synaptic vesicle tethering and fusion are regulated.

Reference:

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P262 STRUCTURE-TOXICITY STUDY OF FUS USING DROSOPHILA AS A MODEL SYSTEM

Wilmans N^1,2

Bogaert E^1,2

Scheveneels W^1,2

Van Damme P^1,3

Robberecht W^1,3

Callaerts P⁴

Van Den Bosch L^1,2

^bjVesalius Research Center, Leuven, Belgium

^bkExperimental Neurology (Department of Neurosciences) and Leuven Institute for Neuroscience and Disease (LIND), University of Leuven (KULeuven), Leuven, Belgium

^blNeurology, University Hospitals Leuven, Leuven, Belgium

^bmLaboratory of Behavioural and Developmental genetics VIB, Leuven, Belgium

Email address for correspondence: [email protected]

Keywords: FUS, Drosophila

Background: A subgroup within the ALS/FTD disease spectrum, the FUSopathy, is characterized by FUS inclusions in neurons and glial cells. The observation that mutations in FUS cause ALS emphasizes the involvement of FUS in the disease pathogenesis.

Objectives: We aimed to identify toxicity domains in FUS protein.

Methods: In order to examine the pathogenic role of FUS, we generated four different transgenic fly lines, allowing expression of wild type human FUS (WT hFUS) and three disease-associated mutant human FUS proteins (hFUS^R521G, hFUS^R521H and hFUS^P525L). Selective expression of hFUS transgenes in adult motor neurons via the UAS-GAL4 expression system resulted in a progressive decline of motor performance ultimately leading to early death of the flies.

Results: Overexpression of human FUS in flies leads to severely reduced life span and motor performance defects. However, no differences were observed between wild type and mutant FUS overexpressing flies. To address the toxicity arising from human wild type FUS, we deleted all functional domains from the wild type protein and addressed toxicity in both animal models. We identified important domains present in the N terminal and C terminal part of the full-length protein. In a next set of experiments, we identified the minimal region required for FUS toxicity by evaluating the toxicity arising from overexpression of these domains.

Discussion and conclusion: We used Drosophila as a model system to gain insight into the pathogenic mechanism of FUS induced motor performance defects. Functional regions in the N terminal and C terminal part of the protein are pivotal for toxicity in fly motor neurons.

P263 IDENTIFYING GENETIC SUPPRESSORS OF TDP-43 TOXICITY USING DROSOPHILA

Sreedharan J^1,2

Neukomm L¹

Coleman M²

Brown R¹

Freeman M¹

^bnUMass Medical School, Massachusetts, USA

^boBabraham Institute, Cambridge, UK

Email address for correspondence: [email protected]

Keywords: TDP-43, axon, Drosophila

Background: Pathological and genetic studies suggest that TDP-43 is a final common mediator in sporadic and familial ALS. Understanding TDP-43 pathobiology is a central aim of ALS research but is complicated by the diverse roles of this protein in nuclear, cytoplasmic and axonal compartments of neurons. Determining realistic therapeutic targets will require in vivo approaches that model the earliest changes seen in ALS, such as synapse and axon degeneration. Drosophila offers unrivalled genetic tools to conduct these studies and, importantly, TDP-43 is structurally and functionally conserved in flies. However, while existing fly models recapitulate TDP-43 toxicity there has been limited progress in identifying suppressors of toxicity. This may be because these models focus on eye and larval neuronal phenotypes. A better approach would be to study adult motor neurons, the principle cell type afflicted in ALS.

Objectives: i) Develop a strategy to examine adult Drosophila motor neurons in vivo; ii) Use this approach to model progressive TDP-43-mediated motor neuron degeneration; iii) Perform a forward screen for novel genetic suppressors of TDP-43 toxicity.

Methods and results: We used mosaic analysis with a repressible cell marker (MARCM) to visualize motor neurons with single cell resolution in the adult fly using the glutamatergic OK371-Gal4 driver(1). MARCM clones were induced using flippase under the control of a proneural gene promoter (2). Confocal microscopy was used to resolve axons, neuromuscular junctions (NMJs) and active zones without the need for complex dissection or immunostaining. Overexpression of mutant TDP-43 caused progressive ‘dying back’ neurodegeneration, which preferentially affected motor rather than sensory neurons and could be monitored over two months (the lifetime of the fly). We then screened for novel suppressors of this phenotype using ethyl methanesulphonate (EMS) mutagenesis. After examining over 3000 EMS lines three recessive suppressors of TDP-43 toxicity were isolated and identified through duplication mapping and whole genome resequencing. These loss-of-function hits implicate chromatin, RNA and microtubule biology and are undergoing further validation in mammalian neurons expressing mutant TDP-43.

Discussion and conclusion: Our mosaic approach to studying neurodegeneration permits, for the first time, a simple method of studying individual motor neurons, axons and NMJs in vivo. MARCM permits the long-term study in adult flies of toxic proteins such as TDP-43, which are otherwise lethal at prepupal stages if expressed ubiquitously. Similarly, MARCM makes it possible to isolate recessive suppressors of TDP-43 toxicity, which would be missed using other screening methods. Only 20% of the fly genome was evaluated in this study and many other suppressors of TDP-43 toxicity await identification.

Acknowledgements: Medical Research Council, Motor Neurone Disease Association, Howard Hughes Medical Institute, Max Rosenfeld Fund, UMMS

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