P18 EFFECT OF HUMAN MESENCHYMAL STEM CELLS INTRACARDIAC TRANSPLANTATION ON SOD1-G93A MICE
ZHANG C1, ZHAO C-P1, WANG Y-H1, XIE Y-M2, ZHOU C1, LI W-Y1
1Department of Neurology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China, 2Department of Neurology, University of North Carolina, Chapel Hill, North Carolina, United States
E-mail address for correspondence: [email protected]
Keywords: human mesenchymal stem cells, SOD1-G93A mice, transplantation
Background: Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disease, currently without any effective therapy. Multiple advantages make mesenchymal stromal cells (MSC) a good candidate for cellular therapy of ALS. Usually, the MSCs were transplanted to SOD1-G93A mice by intravenous injection, but the lung arrests a lot of the BMSs. In order to overcome the problem, may we transplant the MSCs to SOD1-G93A mice by artery injection?
Objective: To study the changes of life span and pathology in SOD1-G93A mice after intracardiac transplantation of human mesenchymal stem cells (hMSCs).
Methods: hMSCs were isolated from bone marrow cells obtained from healthy donors and cultured. The purity and morphology were assessed by flow cytometry (FCM). hMSCs (3×106) resuspended in 0.2 ml DMEM was injected into the heart of 8 week-old SOD1-G93A mice. In non-transplantation control SOD1-G93A mice, only DMEM was injected. The mice were evaluated for signs of motor deficit with a 4-point scoring system previously described by Weydt et al (1). The age of onset and life span in mice were assessed. The pathological change including number of motor neurons was investigated by Nissl staining. Immunofluorescence staining with specific human nuclear antibody was used to confirm the transplant of hMSCs in mice.
Results: The onset of symptoms in untreated SOD1-G93A mice appeared at (156.56±3.60) days of age and the average life span was (188.32±3.51) days. hMSCs transplantation delayed the onset of ALS type symptoms about 16 days (χ2=10.888, P=0.001) and prolonged the life span about 14 days compared to the untreated SOD1-G93A littermates ((202.19±4.09) days vs (188.32±3.51) days, χ2=3.917, P=0.04). The loss of motor neurons in untreated mice was earlier and more severe than in hMSCs transplanted mice. At 20 weeks, the number of motor neurons in transplanted mice was significantly higher than those in untreated mice. Human specific nuclear antigen in brain and spinal cord was detected in transplanted SOD1-G93A mice.
Discussion and conclusions: hMSCs can be implanted for a long-term into the central nervous system by intracardiac transplantation and the transplantation can prolong life span, and delay the onset of the disease and motor neuron loss in SOD1-G93A mice.
References:
- Weydt P, Hong SY, Kliot M, et al. Neuroreport 2003;14: 1051–4.
P19 FLUOXETINE TREATMENT HAS MODEST EFFECTS ON DISEASE PROGRESSION IN THE mSOD1G93A MOUSE MODEL OF ALS
SCHUSTER J, FU R, HECKMAN CJ
Northwestern University Feinberg School of Medicine, Chicago, IL, United States
E-mail address for correspondence: [email protected]
Keywords: fluoxetine, serotonin, excitability
Background: Amyotrophic Lateral Sclerosis (ALS) is an adult onset neurodegenerative disease that results in the retraction of spinal motoneuron axon terminals from muscle fibers and motoneuron death. Since the discovery of inherited genetic mutations in some ALS patients and subsequent development of transgenic ALS mouse models, studies have shown that many factors contribute to disease onset and progression. Many of these factors can be aggravated directly or indirectly by increasing motoneuron excitability. Seroton-ergic inputs to spinal motoneurons are known to increase motoneuron excitability by increasing persistent inward currents (PICs) which amplify synaptic inputs. Fluoxetine (trade name Prozac), a selective serotonin reuptake inhibitor (SSRI) and a commonly prescribed antidepressant, may therefore have detrimental effects when given to ALS patients.
Objectives: The objective of this study was to determine if increasing synaptic serotonin levels negatively effects the progression of ALS by increasing motoneuron excitability.
Methods: In this study, fluoxetine was administered to the mutant superoxide dismutase G93A (mSOD1G93A) mouse model of ALS. Fluoxetine was administered in the drinking water with target concentrations of either 5 or 10 μg/g/day. Groups were gender balanced. Treatment was started at postnatal day 70 (P70) near the onset of overt motor symptoms (ie appearance of tremor, mean 83 ±9) and continued until death. Water consumption, weight, rotorod performance, tremor severity (4 point scale), and end stage were monitored. Repeated measure ANOVAs were used for water consumption, weight, and rotorod performance over time with sex as a covariate. Bonferroni adjustments were made for multiple comparisons. Kaplan Meier log rank tests were used for time to tremor onset, the first decrease in rotorod performance, and end stage with the factor (drug concentration) being pooled for each stratum (sex).
Results : Fluoxetine treatment slightly increased tremor severity (F=3.759, P=0.041) and the decline in rotorod performance (3.774, P=0.041); although post hoc tests did not reveal any pair wise differences. The time to the first decrease in rotorod performance was also significantly different in female mice with performance decreasing sooner in the 10 μg/g/day (chi-squared=6.261, P=0.044) but not male mice (chi-squared=3.666, P=0.160). There were no main effects of drug treatment on water consumption, weight, tremor onset, or end stage.
Discussion: Fluoxetine given near symptom onset had modest effects on disease progression. This is encouraging given the frequent administration of SSRIs and other antidepressant medications to ALS patients. Ongoing studies will determine the effects of fluoxetine administered at earlier time points and will test the apparent increase in tremor severity with ventral root recordings in adult mice.
Conclusion: Fluoxetine, given at symptom onset, does not affect end stage in the SOD1G93A ALS mouse model. It may however increase the severity of tremors and, in females, accelerate the onset of motor impairment.
P20 HUMAN SOD1/G93A-EXPRESSING MICE PROVE INCREASED EXOCYTOTIC RELEASE OF GLUTAMATE
BONANNO G1,2, MILANESE M1, ONOFRI F1, MUSAZZI L3, TARDITO D3, BONIFACINO T1, MESSA M4, GIRIBALDI F1, BENFENATI F1,4, POPOLI M3
1Department of Experimental Medicine, 2Center of Excellence for Biomedical Research; University of Genova, Genova, Italy, 3Center of Neuropharmacology - Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milano, Milano, Italy, 4Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, Gevova, Italy
E-mail address for correspondence: [email protected]
Keywords: SOD1/G93A mice, glutamate release, exocytosis
Selective vulnerability of motor-neurons in ALS has been in turn ascribed to protein misfolding, mitochondrial dysfunction, oxidative damage, insufficient growth factor signaling, inflammation and glutamate-mediated excitotoxicity. High glutamate levels have been reported in ALS patients and in animal models of the disease, and reduced glutamate transport, mainly due to astroglial GLT1 defects, was suggested as a cause. Due to the complex interplay of multiple mechanisms in the aetiology of ALS, defects of glutamate transport may not be the only reason for excitotoxicity-based neurodegeneration and other causes should be considered for the increased glutamate availability, including increase of glutamate release. We have previously found that the non-exocytotic glutamate release, due to activation of glycine and GABA transporters, heterologously sited on glutamate-releasing nerve terminals, is enhanced in the spinal cord of mice expressing human SOD1 carrying the G93A mutation (SOD1/G93A(+)), a widely used animal model of ALS. We extend here our investigation to the study of the modifications of the exocytotic release of glutamate, as a potential cause for hyper-glutamatergicity in ALS. The spontaneous outflow of endogenous glutamate and of the glutamate analogue [3H]D-aspartate was more elevated in SOD1/G93A(+) mice, as compared to mice expressing wild type human SOD1 or to non-transgenic controls. Exposure to 15 mM KCl or 0.3 μM ionomycin provoked Ca2+-dependent glutamate release that was dramatically increased in symptomatic transgenic mutated mice. Contrary to glutamate, the stimulus-evoked release of [3H]GABA or [3H]glycine in the spinal cord of SOD1/G93A(+) mice did not differ from controls, and the same was true for [3H]D-aspartate release in the motor-cortex. The augmentation of basal and stimulated glutamate release was already present in pre-symptomatic mutant mice, suggesting it as a cause instead of a consequence of the progression of pathology. Further studies revealed the existence of increased resting and stimulated Ca2+ levels in nerve terminals from the SOD1/G93A(+) mouse spinal cord, accompanied by increased activation of Ca2+/calm-odulin-dependent kinase II and increased phosphorylation of synapsin I, a cascade of events leading to higher availability of synaptic vesicles for exocytosis. In line with these findings, release experiments suggested the involvement of the readily releasable pool of vesicles in the production of the abnormal glutamate release in SOD1/G93A(+) mice, as well as a greater capability of these vesicles to fuse upon stimulation. To conclude, glutamate exocytosis is elevated in symptomatic and pre-symptomatic G93A mutant mice and changes in cyto-solic Ca2+, Ca2+/calmodulin-dependent protein-kinase II auto-activation and synapsin I phosphorylation seem to be major causes of the augmented neurotransmitter release. The discovery of this excessive glutamate exocytosis may have translational perspectives. Presynaptic release inhibition is a common regulatory process of CNS transmission; already available or newly designed efficacious agents can be identified to selectively inhibit abnormal glutamate exocytosis in ALS.
P21 OVER-EXPRESSION OF GLUTAMATE CYSTEINE LIGASE CATALYTIC SUBUNIT IN THE LUMBAR SPINAL CORD OF SOD1-G93A TRANSGENIC MICE
GUO Y, LIU Y, LI Z, WANG Q, ZHANG K, HUANG J, LI C
Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang, China
E-mail address for correspondence: [email protected]
Keywords: GCLC, astrocyte, transgenic mice
Background: Glutamate cysteine ligase catalytic subunit (GCLC) regulates GSH synthesis and induction of GCLC is capable of enhancing the antioxidant capability of the cell. GCLC expression in the central nervous system of amyotrophic lateral sclerosis (ALS) SOD1-G93A transgenic mouse model has not been previously examined.
Objectives: To investigate whether GCLC expression is changed in the susceptible spinal cord as well as in the motor cortex.
Methods: Immunohistochemistry, Western blot and confocal microscopy were used to detect GCLC expression and its cellular location.
Results: The results showed that GCLC was up-regulated increasingly in the lumbar spinal cord of SOD1-G93A transgenic mice when the disease progressed. GCLC expression was located in neurons and did not show any co-localization with GFAP-positive astrocytes. Remnant interneurons in the lumbar anterior horn over-expressed GCLC and may contribute to the combat against motor neuron-destroying oxidative stress. In contrast, no alteration of GCLC expression was observed in the motor cortex of SOD1-G93A transgenic mice at any disease stages.
Discussion: The main discovery of this study was that over-expression of GCLC, the catalytic subunit of glutamate-cysteine ligase, was examined in the lumbar spinal cord of SOD1-G93A transgenic mice and increased as the disease progressed. In contrast, GCLC over-expression was not found in the motor cortex of the transgenic mice. With the degeneration of motor neurons, interneurons in the lumbar anterior horn over-expressed GCLC, which might contribute to the antioxidant response. However, the dramatically proliferated astrocytes did not show any GCLC immunoreactivity. To the best of our knowledge, this is the first report about GCLC expression in the CNS tissues of SOD1-G93A transgenic mice.
Conclusions: It is suggested that GCLC up-regulation may represent a protective response against motor neuron degeneration in ALS.
P22 GENETIC BACKGROUND EFFECTS ON LIFESPAN OF SOD1 MOUSE MODELS OF ALS
SHER R, SOUNDARARARJAN P, COX G
The Jackson Laboratory, Bar Harbor, ME, United States
E-mail address for correspondence: [email protected]
Keywords: modifier, genetic background, embryonic stem cells
Background: Dominant SOD1 mutations account for ∼25% of familial forms of ALS (FALS), though heterogeneity in age of onset or symptom severity exist within families carrying the same SOD1 mutation, suggesting that modifier genes significantly impact the disease. Similarly, there is variation in onset and severity in hSOD1Tg mice on genetically heterogeneous backgrounds. For testing genetic modifiers of the disease, it is crucial to eliminate genetic heterogeneity in the animals to reduce phenotypic variability.
Objectives: The goals of our studies were to test the hypothesis that genetic modifiers can significantly affect the onset or progression of ALS symptoms in G93A mutant SOD1 transgenic mice, and to identify major QTL loci associated with longevity.
Methods: We developed a range of inbred strains containing the SOD1-G93A mutation with varying lifespans. One long-lived and one short-lived strain were used in reciprocal back-crosses for QTL analysis of modifier loci.
Results: We identified two inbred strains (ALR/LtJ, NOD/ LtSz-Rag1tm1Mom) that significantly accelerate disease, and three that significantly delay disease (C57BL/6J, DBA/2J and BALB/cByJ). Through reciprocal backcrosses between B6 (∼161d) and ALR (∼114d) lines we have mapped two major QTL on Chr 4 (LOD 4.76) and Chr 17 (LOD 11.99) that significantly modifies the lifespan of G93A SOD1 mice. The Chr 17 locus has also been identified at DUCOM through our collaborative research.
We crossed B6.SOD1-G93A with a B6.NOD-Chr17 congenic. Lifespan for SOD1 NOD/B6 mice (N=14) was 149.8±9.0 d, and for SOD1 NOD/NOD mice (N=14) was 136.4±7.0 d, a statistically significant difference between groups and from the B6.SOD1 line (all P<0.001). The decrease in lifespan is regulated in a dose-specific manner, with one NOD copy resulting in an ∼7.1% lifespan reduction, and two NOD copies resulting in an ∼15.4% reduction.
Discussion and conclusions: We have demonstrated that the genetic background of hSOD1-G93A transgenic mice significantly affects lifespan, and that a region of Chr 17 has a major dose-dependent effect on lifespan.
We are presently crossing the B6.NOD-Chr17 congenic to B6.SOD1-G85R and B6.SOD1-G37R lines to determine if this region has a general effect on SOD1-ALS lifespan. We are also crossing a NOD.B10-Chr17 congenic line to our NOD.SOD1-G93A line to determine if this increases lifespan in a short-lived strain. In addition, crosses of a TDP-43 mutant mouse to the B6.NOD-Chr17 congenic will determine if the Chr 17 QTL affects lifespan in other ALS models. We have resequenced the Chr 17 region for mutations in gene(s) affecting lifespan. We are deriving embryonic stem cells from the G93A, G85R, and G37R SOD1 mice, along with the TDP-43 mouse, and are differentiating them into motor neurons. We will use these motor neuron cultures to test for effects of up- and down-regulation of genes of interest in our region.
P23 DIFFERENTIAL REGULATION OF THE GLUTAMATE TRANSPORTER VARIANTS GLT-1A AND GLT-1B IN THE CORTEX AND SPINAL CORD OF TRANSGENIC RATS EXPRESSING HSOD1G93A
GOURSAUD S, BERGER J, MALOTEAUX J-M, ERMANS E
Group of Neuropharmacology, Institute of Neurosciences, Université catholique de Louvain, Brussels, Belgium
E-mail address for correspondence: [email protected]
Keywords: glutamate transporter 1, splice variants, hSOD1G93A
Background: Loss of activity of the glutamate transporter GLT-1 and increased extracellular concentration of glutamate have been documented in sporadic and familial cases of amyotrophic lateral sclerosis (ALS) and transgenic rodent models expressing mutated human superoxide dismutase 1 (hSOD1). The cellular and molecular mechanisms causing the loss of GLT-1 expression have been largely investigated and identification of mutations in the GLT-1 gene and detection of aberrant GLT-1 transcripts in disease-affected areas have been reported. Interestingly, some experimental data indicate that the expression of the two C-terminus splice variants of GLT-1, namely GLT-1a and GLT-1b could be differentially and independently regulated in the motor cortex of ALS patients.
Objective: We herein characterized the GLT-1a and GLT-1b mRNAs and activity of the transporters at different symptomatic stages of the disease in the fronto-temporal cortex and in the lumbar spinal cord of a transgenic rat model expressing hSOD1G93A.
Methods: Tissues were isolated from 60, 120 and 195 day old wild-type and transgenic animals. Number of copies of GLT-1a or GLT-1b mRNAs were obtained by quantitative real-time PCR. Activities of the transporters were evaluated in tissue synaptosomes by measuring D-[3 H]-aspartate uptake velocity in the presence of DHK, a selective GLT-1 blocker.
Results: GLT-1a transcripts were four-fold more abundant than GLT-1b transcripts in the fronto-temporal cortex of wild-type rats. During development of disease, expression of GLT-1a strongly decreased and expression of GLT-1b gradually increased so that abundance of the two transcripts was identical at the end stage of life (195 days) in the cortex of transgenic rats. Also, the cortical activity of GLT-1 decreased during progression of ALS. The quantity of GLT-1a and GLT-1b mRNAs was identical in the lumbar spinal cord of wild-type rats. In transgenic animals, GLT-1a and GLT-1b transcripts copy number were dramatically reduced in the ventral horn of the lumbar spinal cord of transgenic rats. In correlation with this loss of expression, activity of GLT-1 specifically decreased in this area.
Conclusions: Our results demonstrate that GLT-1a and GLT-1b are differentially expressed in the fronto-temporal cortex and the lumbar spinal cord of wild-type and transgenic animals. During the progression of the symptoms, expression of the mRNAs differentially and independently changed although GLT-1 activity constantly decreased in both structures.
Supported by the FNRS, FMRE, DIANE and ABMM.
P24 REDUCING CASPASE-3 ACTIVITY UPREGULATES THE GLUTAMATE TRANSPORTER GLT-1A IN THE CORPUS CALLOSUM AND IN CULTURED CALLOSAL ASTROCYTES FROM A RAT MODEL OF AMYOTROPHIC LATERAL SCLEROSIS (HSOD1G93A)
HERMANS E1, FOCANT M1, NIZET Y2, BERGER J1, MALOTEAUX J-M1, GOURSAUD S1
1Group of Neuropharmacology, Institute of Neurosciences 2Institute of Experimental and Clinical Research; Universit é catholique de Louvain, Brussels, Belgium
E-mail address for correspondence: [email protected]
Keywords: glutamate, transporter, caspase
Background: Upper motor neuron dysfunction in amyotrophic lateral sclerosis (ALS) has been associated with a deficit of transcallosal connections and a reduced volume of the corpus callosum. Besides, impairment of the astroglial glutamate transporter GLT-1 and related excitotoxicity are likely to participate in the progression of the disease. At the molecular level, caspase-3-mediated cleavage of GLT-1 leading to a selective inhibition of the transporter was evidenced in spinal cord homogenates of a transgenic mouse model of fALS. In addition, recent reports have documented alternative splicing mechanisms for GLT-1 and the differential expression of the isoforms GLT-1a and GLT-1b in ALS patients.
Objectives: We herein characterised the expression of GLT-1a and GLT-1b and the activity of the transporters after pharmacological inhibition of caspase-3 in callosal homogenates and in cultured callosal astrocytes isolated from transgenic rats expressing a mutated form of human superoxide dismutase 1 (hSOD1G93A).
Methods: The experiments were performed in callosal homogenates isolated from symptomatic and wild-type rats and in cultured callosal astrocytes obtained from transgenic and wild-type newborn rats. The expression of GLT-1a and GLT-1b was characterized by quantitative RT-PCR and Western-blotting studies and the GLT-1 activity was determined in d-[3H]-aspartate uptake assays in the presence of the inhibitor DHK. Caspase-3 activity was evaluated using the fluorogenic substrate Ac-DEVD-AMC. In some experiments, tissues were collected 48 h after intracallosal injection of 1 μg Peptide Histidine Isoleucine (PHI), a neuroprotective neuropeptide in the white matter.
Results: Differential expression of GLT-1a and GLT-1b was evidenced in the corpus callosum and in callosal astrocytes. Also, in the transgenic animals, GLT-1a was less abundant while robust expression of GLT-1b was observed. Local PHI instillation induced an upregulation of GLT-1a protein only in transgenic rats and specifically in the ipsilateral side. Expression of GLT-1b was unchanged. In these experimental conditions, activity of GLT-1 was also strongly upregulated. Similar results were obtained in cultured astrocytes treated with PHI or with Ac-Asp-Met-Gln-Asp-aldehyde, a selective inhibitor of caspase-3. Moreover, PHI also reduced caspase-3 activity after injection in the corpus callosum and in callosal astrocytes selectively in transgenic animals.
Conclusions: Reducing the activity of caspase-3 could exclusively upregulate GLT-1a in callosal astrocytes and in the corpus callosum of a transgenic rat model of ALS.
Supported by the FNRS, FMRE and ABMM.
P25 ABSENCE OF GFAP DOES NOT AFFECT DISEASE ONSET AND PROGRESSION IN SOD1H46R-EXPRESSING MICE
YOSHII Y1,2, OTOMO A2, LEI P2, IKEDA K1, IKEDA J-E2, IWASAKI Y1, HADANO S2
1Department of Neurology, Toho University Medical Center Omori Hospital, Ota-ku, Tokyo, Japan, 2Department of Molecular Life Sciences, Tokai University School of Medicine, Isehara, Kanagawa, Japan
E-mail address for correspondence: [email protected]
Keywords: GFAP, SOD1, astrocyte
Background: Interactions between glia and neurons have long been investigated in neuroscience fields. Glial fibrillary acidic protein (GFAP), vimentin and nestin are among the intermediate filaments (IFs) to form a group of cytoskeletal proteins. IFs play a role in the mechanical strength and shape of cells and their processes in physiological conditions. By contrast, in pathological conditions such as neurodegenerative disease, some of the IFs are up-regulated in activated astrocytes and are believed to have deteriorating factor for neuronal survival. Astrogliosis is one of the cardinal features for the progression of amyotrophic lateral sclerosis (ALS). Recent studies have demonstrated that glial cells carrying a human SOD1G93A mutation adversely affect motor neuron survival with a non-cell autonomous trait in embryonic stem cell based ALS model, and expression of mutated SOD1 in rodent spinal astrocytes specifically contributes to neurodegeneration. Although GFAP is highly expressed in activated astrocytes and frequently used as a marker of them, it is still unclear whether GFAP by itself plays a role in the pathogenesis for ALS.
Objectives: To explore the effect of GFAP on disease onset and progression of ALS using a mutated SOD1-expressing mouse model.
Methods: We crossed two congenic lines; Gfap-knockout (KO) and SOD1H46R transgenic mice on a C57BL/6 background, and generated mice with 6 different genotypes; Gfap+/+, Gfap+/−, Gfap−/−, Gfap+/+; SOD1H46R, Gfap+/−; SOD1H46R, and Gfap−/−; SOD1H46R. We monitored body weight, disease onset, spontaneous motor activity (cage activity and rearing activity), and lifespan.
Results: No significant differences in the body weight, spontaneous motor activity, disease onset (160.3 ±1.47 days; mean±SEM), and survival (172.4±1.19 days; mean±SEM) were observed among SOD1H46R mice with different Gfap genotypes.
Conclusions: GFAP by itself may play a limited role in the pathogenesis for the mutated SOD1-linked motor neuron disease. It is possible that other IFs, such as vimentin and nestin, are up-regulated for compensation, therefore analysis of these levels of these proteins is currently underway by Western blotting and immunohistochemistry.
P26 DO NEUROTOXIC INSULTS CAUSE MISFOLDING OF SOD1 IN AN ANIMAL MODEL OF SPORADIC ALS?
ZWIEGERS P1, LEE G1, LIU H-N2, ROBERTSON J2, SHAW CA1
1University of British Columbia, Vancouver, British Columbia, Canada, 2University of Toronto, Toronto, Ontario, Canada
E-mail address for correspondence: [email protected]
Keywords: ALS-PDC, stigmasterol β-D-glucoside, SOD1 conformation
Background: Amyotrophic lateral sclerosis with missense mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD1) account for approximately 1-2% of all ALS cases, and is purported to be both clinically and neuropathologically indistinguishable from sporadic forms of the disease. The underlying pathobiological mechanism implicated in SOD1-mediated familial ALS (fALS) involves a toxic gain-of-function mutation with resultant misfolding and aggregation of the aberrant protein. The above observations, coupled with research demonstrating that both wild-type and mutant SOD1 can misfold and aggregate upon in vitro oxidation, has led to a hypothesis that aberrant SOD1 conformational changes underlie all ALS etiology. However, a recent study by Liu et al (2009) using the misfolded SOD1-specific SEDI antibody in sporadic and familial ALS cord samples does not support this interpretation.
Objectives: Previous work conducted by our group has shown that a Guamanian ALS-Parkinsonism dementia complex (ALS-PDC) phenotype can be replicated by exposing mice to stigmasterol β-D-glucoside (SG). Utilizing SOD1 conformation-specific antibodies, our studies attempted to determine if SG induced SOD1 conformational changes, as well as to probe for any synergistic effect on SOD1 misfolding arising from exposure of mSODG37R mice to this toxin.
Methods: The human IMR-32 neuroblastoma cell line was exposed to SG at a concentration of 50 μM for a period of up to 9 days. Wild-type and transgenic mSODG37R mice were treated with 42 μg/kilogram of body weight daily starting at 10 weeks of age. The role of SG regarding induction of aberrant SOD1 protein folding was investigated with the use of novel antibodies selective for epitopes indicative of misfolded SOD1.
Results: The results do not implicate SOD1 misfolding and aggregation as the mechanism of SG mediated neurotoxicity. Prolonged SG treatment of IMR-32 cells exhibited cell loss but failed to induce SOD1 misfolding. Additionally, SG exposure in mice did not demonstrate a synergistic interaction on SOD1 conformational state with the G37R mutation.
Discussion: Although the mechanism of SG toxin neuropathology is uncertain, it has been shown that dietary exposure to SG alone is sufficient to produce a disease phenotype and a more severe phenotype in conjunction with a genetic predisposition to ALS. These results posit that aberrant SOD1 folding is not implicated as a mechanism for SG neuropathology and support the notion that SOD1 conformational changes are unique to some forms of fALS.
Conclusion: In conclusion, the environmental agent studied here is not sufficient to induce or exacerbate SOD1 protein conformational changes. Through the use of novel conformation-specific antibodies we have demonstrated that the mechanism of SG neurotoxicity is similar to sALS in that misfolded SOD1 was undetectable. The environmental mouse model may be used to further understand the mechanism of disease progression in degenerative conditions exacerbated by environmental agents.
P27 OXIDATIVE MODIFICATION OF CYSTEINE 111 PROMOTES FORMATION OF DISULFIDE BOND-INDEPENDENT AGGREGATION OF SOD1
CHEN X1, CUI L2, QI X2, SHANG H3, KONG J1
1University of Manitoba, Winnipeg, Canada, 2Union Hospital, Beijing, China, 3West-China Hospital, Chengdu, China
E-mail address for correspondence: [email protected]
Keywords: oxidation, SOD1 aggregation, disulfide bond
Aggregation of mutant human SOD1 has been implicated in the ‘gain of toxic property’ that plays a role in the pathogenesis of ALS. To study the role of oxidative modification in SOD1 aggregation, we examined the redox state of SOD1 in the G37R transgenic mice at different stages of the disease. Our data show that the cysteine residues in SOD1 from the spinal cord of G37R transgenic mice exhibited decreased accessibility to modification by malPEG with disease progression. This was at least partially caused by oxidative modification of the cysteine thiol groups because treatment of human SOD1 with H2O2 led to similar decreased accessibility to modification by malPEG as well. Using an antibody (anti-C111ox-SOD1) specific for oxidized SOD1, C111-peroxidized SOD1 was found to be increased in abundance with the progression of the disease. Levels of oxidation correlated with the formation of SOD1 aggregation. Oxidation of both WT and Mutant forms of SOD1 with H2O2 induced formation of high molecular weight aggregates even in the presence of 5% β-mercaptoethanol, suggesting that SOD1 aggregation was not mediated by the disulfide bond between C111 and C6. Cysteine 111 existed in sulfhydryl state under normal circumstance compared with other cysteine residues. Endogenous mouse SOD1, which lacks C111, was not modifiable by malPEG. When cysteine 111 in human SOD1 was mutated to serine, it became unmodifiable by malPEG, while several other cysteine-residue-mutated SOD1s behaved just like wt SOD1 and were easily modifiable by malPEG.
The results suggest that cysteine 111 is a primary site of malPEG modification. Since malPEG can only react with the sulfhydryl group (-SH) of reduced cysteine residues, our data suggest that the sulfhydryl group (-SH) of cysteine 111 in human SOD1 is the target of thiolate anion (S-) for the oxidative modification, while other cysteine residues exist in disulfide (-S-S-) state.
In conclusion, cysteine 111 provides thiolate anion (S-) for oxidative modification. Oxidative modification of cysteine 111 promotes the formation of disulfide bond-independent aggregation of SOD1
Supported by MDA (USA), ALS Canada, CIHR and NSFC (China).
P28 THE CARBOXY-TERMINUS OF TDP-43 IS THE PRIMARY DETERMINANT OF ITS TOXICITY IN THE NERVOUS SYSTEM
HAZELETT D, STEWART J, MORTON D
Oregon Health & Science University, Portland, OR, United States
E-mail address for correspondence: [email protected]
Keywords: TDP-43, Drosophila, cell biology
Background: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease resulting from the loss of upper and lower motor neurons in affected individuals. Recently TDP-43 was identified as the major constituent of cytoplasmic aggregates in motor neurons. TDP-43 is an highly conserved RNA-binding protein normally expressed in the nucleus. In disease neurons, TDP-43 is excluded from the nucleus.
Objectives: We made overexpression transgenic lines and knockouts to test competing hypotheses that TDP-43 gain-of-function or loss-of-function might contribute to a motor-deficient phenotype.
Methods: We employed the yeast GAL4-UAS expression system to overexpress different isoforms of TDP-43 in larval and adult flies.
Results: TDP-43 knockouts do not eclose, but larvae have severely impaired locomotion. RNAi against TDP-43, which reduces expression about 2-fold, exhibits a significantly milder phenotype of age-dependent neurode generation. Overexpression of three naturally occurring c-terminal variant splice-forms of TDP-43 in the nervous system resulted in early lethality, depending on the c-terminus. Expression of the c-terminus alone had no effect. All three isoforms caused motor deficits when misexpressed with a motor-neuron driver, but the isoform lacking a c-terminus showed the mildest effect. All three isoforms also caused degeneration when expressed in photoreceptors, again with the c-terminal truncation having the mildest phenotype. In both cases overexpression of c-terminus alone had little or no effect. To compliment previously published analyses of neuromuscular junction (NMJ) anatomy in TDP-43 mutants, we examined NMJ in TDP-43 misexpressing larvae with motor neuron driver. Opposite to what was previously observed for loss of function, overexpression of all three isoforms promoted branching, outgrowth, and supernumerary bouton formation in Type IS sub-class of boutons but not Type IB.
Discussion: These data are not consistent with the model that TDP-43 overexpression causes a TDP-43 loss of function. However it remains possible that TDP-43 overexpression has pleiotropic effects that include its own loss-of-function in the nucleus. Together with the effects on motor neurons and photoreceptors, our findings indicate that the c-terminus is necessary, but not sufficient, for the toxic effects of TDP-43 in neuronal cells.
P29 DIFFERENTIAL EXPRESSION AND ALTERNATIVE SPLICING OF GENES IN THE LUMBAR SPINAL CORD OF SOD1-G93A TRANSGENIC MICE
GUO Y, CHEN H, HU M, ZHANG K, WANG Q, LI Z, LI C
Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang, China
E-mail address for correspondence: [email protected]
Keywords: exon array, gene expression, alternative splicing
Background: Amyotrophic lateral sclerosis (ALS) is one of the most common adult-onset neurodegenerative diseases, for which the exact pathogenic mechanism remains unknown. Recent evidence suggests that differential gene expression and alternative gene splicing may play a significant role.
Objectives: To identify differentially expressed genes and alternatively spliced exons in the lumbar spinal cord of SOD1-G93A transgenic mice at both presymptomatic and symptomatic stages.
Methods: Affymetrix GeneChip® Mouse Exon 1.0 ST Array was used to investigate the expression profiling and alternative splicing of genes in the lumbar spinal cord from both presymptomatic (30 days of age, 30A) and symptomatic (disease onset, about 120 days of age, 120A) SOD1-G93A mice and their non-transgenic littermates (30C and 120C, respectively). We selected transcripts that showed an increase/decrease of at least two-fold or Splice Index (SI) ≥ 2 or ≤ 0.5 with a statistical significance (P < 0.05). Cluster analysis 3.0 software was used for hierarchical cluster (HC) analysis and gene ontology (GO) hierarchy analysis was then carried out. CapitalBio® MAS V4.0 software was used to identify known pathways. RT-PCR was performed to validate.
Results: The gene level analyses identified 263 (9.17%) up-regulated and 71 (2.47%) down-regulated genes in 120A vs 30A group and only one 2.8-fold up-regulated gene (with no annotation) in 30A vs 30C group. 322 (2.2%) genes were differentially expressed in 120A vs 120C group, of which 309 (95.96%) were up-regulated and 13 (4.04%) were down-regulated. The exon level analysis identified 63 alternatively spliced exons derived from 27 transcripts in 120A vs 120C group, 630 alternatively spliced exons derived from 563 transcripts in 120A vs 30A group, and 85 alternatiively spliced exons derived from 85 transcripts in 30A vs 30C group. Several differentially expressed genes (CD68, LPL, NOX 2, PIK3CG) and candidate splice variants (PDCD1, FYB) were validated with RT-PCR and consistency with exon array was found. Our findings indicate that cell adhesion, immune-inflammation response, lipid metabolism, and oxidative stress play important roles in the onset of mutant SOD1-related motor neuron degeneration.
Discussion: At 30 days of age, no significant change was found at both gene and exon levels between transgenic and non-transgenic mice, but 120-day transgenic mice differ significantly from their non-transgenic littermates and 30-day transgenic mice. The results suggest that a number of differentially expressed genes and alternatively spliced exons are involved in mutant SOD1-related disease onset of ALS. Functional and pathway analysis demonstrated the importance of transcription and splicing regulation in physiological and pathological processes.
Conclusions: We speculate that it is a multi-factor interactive mechanism that participates in the onset and progression of ALS, although much more remains to be understood.
P30 DIFFERENTIAL NEUREGULIN 1 ISOFORM EXPRESSION CORRELATES WITH MOTOR NEURON DEGENERATION AND GLIAL CELL ACTIVATION IN THE ALS-SOD1 MOUSE MODEL
SONG F, CHIANG P, LOEB J
Hiller ALS Clinic and Research Center, Department of Neurology, Center of Molecular Medicine and Genetics, Wayne State University, Detroit, MI 48201, United States
E-mail address for correspondence: [email protected]
Keywords: neuregulin, neurodegeneration, glial activation
Background: Neuregulin 1 (NRG1) is a neuronally-expressed factor that supports axoglial and neuromuscular development. It can be regulated by neurotrophic factors derived from surrounding cells in both secreted (type I) and membrane-bound (type III) forms (1). To date, this important reciprocal signaling pathway has not been explored in mouse models of ALS. Since both NRG1 and neurotrophic factors are potent survival factors, understanding their normal function as well as their altered function in models of ALS could lead to biologically-driven therapeutics to combat the progressive neuromuscular degeneration in ALS and other motor neuron diseases.
Objectives: To determine the relationship of NRG1 gene and protein expression with motor neuron loss and glial cell activation in spinal cords of SOD1 (G93A) mice compared to wild type (wt) littermate controls at serial life stages in order to develop novel therapeutic strategies in ALS.
Methods: Pathological changes were measured histologically for motor neuron number, astrocytosis, and microgliosis in both SOD1 mice and wt littermates at days 35, 56, 90 and end-stage (>day 117 but before death). Gene expression was determined by quantitative PCR for brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) and the NRG1 gene isoforms (Type I and Type III) that have important roles in nervous system development and compared to protein levels.
Results: Significant pathological changes including motor neuron loss, demyelination, astrocytosis and microgliosis were observed in spinal cords in SOD1 mice starting around or before 90 days. The development of these changes paralleled reciprocal changes in type I and type III NRG1 gene expression. While type I NRG1 was significantly upregulated, type III NRG1 was down-regulated. GDNF and BDNF, known to induce both transcriptional as well as post-translational expression of NRG1 were mostly upregulated, even before periods of increased motor neuron loss. Studies defining NRG1 protein expression and distribution with respect to glial activation are currently underway. Through the regulation of NRG1 isofrom expression, neurotrophic factors may therefore play a regulatory role in glial activation and disease development in SOD1 mice.
Conclusions: Reciprocal changes in NRG1 isoform type expression and glial activation in the SOD1 model of ALS suggests a potential casual role for NRG1 in glial changes that occur together with motor neuron loss in this model. At the same time points, we observed changes in neurotrophic factor expression, known to regulate NRG1.
Discussion: The differential expression of NRG1 isoforms (membrane bound versus soluble) could be important either in the degeneration or repair process in ALS and therefore represent a potential therapeutic target by either up- or down-regulating NRG1 in ALS.
Reference:
- Esper RM, Pankonin MS, Loeb JA, Brain Res Rev 2006; 51:161–75.
P31 MICROARRAY ANALYSIS OF THE TRANSCRIPTOME FOLLOWING GLIA- AND MOTONEURON-SPECIFIC EXPRESSION OF MUTANT SOD1 IN DROSOPHILA
QUEEN E1, GRISSOM J2, CONWAY T2, ZHANG B1
1Department of Zoology, University of Oklahoma, Norman, OK, United States, 2Department of Botany and Microbiology, Norman, OK, United States
E-mail address for correspondence: [email protected]
Keywords: SOD1, Drosophila, microarray
Background: Mutations in the Cu/Zn superoxide dismutase (SOD1) gene account for about 20% of familial ALS cases. The normal function of SOD1 is to neutralize superoxide free radicals. Mutant SOD1 are thought to confer a toxic gain of function to the protein rather than loss of dismutase activity. However, the cellular and molecular mechanisms by which mutant SOD1 induces neurodegeneration remain unclear. Transgenic animals expressing mutant SOD1 are useful models for investigating ALS and have greatly enhanced our understanding the disease. Similar to the mouse model, transgenic Drosophila selectively expressing mutant SOD1 in motoneurons shows an age-dependent decline in motor behavior, defects in synaptic transmission, and the appearance of SOD1 protein aggregates.
Objectives: Although motoneurons are the affected tissue in ALS, research indicates that SOD1 linked ALS involves multiple cell types. Astrocytes and microglial cells, in particular, seem to contribute to the pathology attributed to mutant SOD1. The objective of this project is to investigate the contributions of glia and motoneurons in SOD1-linked defects in flies.
Methods: To fully understand the role of glia and motoneurons in ALS, we performed a large-scale screen to identify genes that are differentially expressed in mutant SOD1 flies. Using the UAS/GAL4 system, we generated flies expressing wild type Drosophila dSOD1 or mutant human SOD1 (G85R) in motoneurons, glia, and concurrently in motoneurons and glia. An Affymetrix GeneChip microarray was conducted on 5 and 45 day old flies expressing a mutant human SOD1 (G85R) and wild type Drosophila SOD1 in each of the above cell types.
Results: With the help of the bioinformatics team at the University of Oklahoma, we are analyzing genes that show a significant change in expression across the multiple groups. Preliminary analysis reveals detectable changes in many potentially interesting genes. For example, a 3 fold increase was observed in a neurotransmitter transport gene (CG33296) and a 4 fold decrease was detected in an oxidation reduction gene (CG18233), suggesting potential direct functional links with SOD1. Fully analyzed transcriptome data from these experiments will be presented in the meeting.
Discussion and conclusions: Drosophila is relevant to understanding human diseases because flies and humans share highly conserved genes and have similar cellular organization and function of the nervous system. Further, flies offer the advantage of a large and highly developed genetic toolkit that allows manipulations of specific genes in specific cell types. Several neurological diseases, such as Parkinson's and Alzheimer's, have been successfully modelled in flies and have provided important insights into human neurological disorders. A preliminary analysis of our microarray data has revealed similarities to microarrays conducted in mutant SOD1 mice and cell culture, illustrating the usefulness of a fly model of ALS.
P32 INVESTIGATION OF VESICLE TRAFFIC DEFECTS ASSOCIATED WITH MOTOR NEURON DEGENERATION IN WOBBLER MICE
SCHMITT-JOHN T, DROCE A, KARLSSON PR, ORTIZ-PADILLA C, MOSER JM
Molecular Biology Department, Aarhus University, Aarhus, Denmark
E-mail address for correspondence: [email protected]
Keywords: vesicle traffic, wobbler mouse model, motor neuron degeneration
Background: The wobbler mouse serve as an animal model for human ALS/MND since the wobbler phenotype has been shown to closely resemble the human disease. We have identified the gene affected by the wobbler mutation to be the ubiquitously expressed vesicle traffic factor Vps54. Vps54 is a component of the GARP (Golgi associated retrograde protein) complex, a vesicle tethering factor involved in the retrograde vesicle traffic from endosomes to the Golgi apparatus and thus suggests a role for retrograde vesicle traffic in motor neuron degeneration.
Objectives: We investigated if vesicle traffic is affected in wobbler mice and analyzed the cellular effects of the Vps54 wobbler point mutation as well as Vps54 null mutations. Furthermore, we tried to find ways to investigate the retrograde vesicle traffic in human ALS/MND patients.
Methods: For this purpose, we utilized embryonic fibroblasts and embryonic stem cells from wobbler and Vps54 null mutant mice as well as cultured wobbler skin fibroblasts for the functional analysis of the retrograde vesicle traffic. Finally, we used cultured skin fibroblasts of human ALS patients in order to evaluate the option of screening ALS patients for wobbler-like defects in the retrograde vesicle traffic.
Results: In murine Vps54 mutant cells we observed an affected retrograde vesicle transport and mis-distributed mannose-6-phosphate receptors, while endocytosis was not affected. Similar experiments were performed with human skin fibrob-lasts but the variation from cell to cell was much higher in human skin fibroblast as compared to the murine cells.
Discussion and conclusions: Our results indicate that Vps54 and thereby the GARP complex is affected in wobbler mice and leads to a disturbance of retrograde vesicle traffic and thereby to mis-distribution of mannose-6-phosphate receptors in all cells tested so far. The vesicle traffic assays used for murine cells can be applied to human skin fibroblasts, even though the variability is much higher in human cells, probably due to the higher genetic variability as compared to mouse inbred strains. However, we think that a higher number of individual cells analyzed per patient will allow screening for vesicle transport defects among sporadic ALS patients.
P33 H63D VARIANT OF HFE ALTERS CHOLESTEROL METABOLISM: A POTENTIAL MECHANISM FOR DISRUPTION OF CELL SIGNALING IN AMYOTROPHIC LATERAL SCLEROSIS
ALI-RAHMANI F, LEE S, NANDAR W, NEELY B, SIMMONS Z, CONNOR J, SCHENGRUND C-L
Pennsylvania State University, Hershey, PA, United States
E-mail address for correspondence: [email protected]
Keywords: cholesterol, HFE, lipid rafts
Background: The HFE gene variant, H63D, has been under investigation as a risk factor for amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Previous studies indicated that possession of at least one H63D allele is associated with a four-fold increased risk of ALS. HFE protein and glutamate transporters are localized in lipid rafts. Lipid rafts are microdomains in the plasma membrane and enriched in cholesterol and sphingolipids. Alterations in raft components can affect cell signaling functions of these proteins and can also affect cellular activities such as endo- and exocytosis. Reduction in membrane cholesterol has been shown to increase dissociation of the glutamate transporter from lipid rafts and causes loss of glutamate uptake. Recently, alterations in lipid metabolism have been observed in ALS patients and decreased glutamate uptake is reported in a cell model expressing H63D. The role of cholesterol metabolism in the pathogenesis of ALS is unclear but important to clarify as cholesterol-lowering agents are commonly prescribed.
Objectives: The aim of this study was to investigate the effect(s) of the H63D-HFE mutation on cholesterol metabolism and its association with ALS pathology.
Methods: We used SH-SY5Y human neuroblastoma cells transfected to stably express either wild type (WT) or the H63D variant of HFE. Total cholesterol content, targeted gene array analysis and protein expression was determined. In addition, brains were isolated from six and twelve month old mice expressing WT HFE and H67D (equivalent to H63D) and used for analysis of total cholesterol content and total protein expression.
Results: Analysis of cholesterol content indicated that cells expressing the H63D variant had 50% less cholesterol than cells expressing WT HFE. Targeted gene array analysis showed a six fold increase in the expression of cholesterol 24 hydroxylase (CYP46A1), an enzyme that converts cholesterol to 24S-hydroxycholesterol. Because sphingolipids are also key components of lipid rafts, we measured expression of mRNA for 13 genes involved in their metabolism. Transcription of genes encoding sphingosine kinase and GD3 synthase was reduced, suggesting alterations in sphingolipid metabolism. Analyses of 6 and 12 month old mice revealed that compared to WT-HFE expressing mice, homozygous H67D mice showed significant increase in the expression of cholesterol synthesis protein (DHCR24) and proteins regulating cholesterol efflux (CYP46A1, APOE, and ABCA1).
Discussion and conclusions: The H63D-HFE variant induces alterations in key lipid raft components including cholesterol and sphingolipids. This supports the hypothesis that disruption of lipid rafts induced by the HFE mutant may contribute to development of ALS. These data also have implications for the use of cholesterol-lowering agents to treat individuals with the H63D mutation.
P34 INCREASED HUMAN MUTANT SUPEROXIDE DISMUTASE 1 IN SPINAL CORD MITOCHONDRIA COMPARED WITH BRAIN MITOCHONDRIA AND SKELETAL MUSCLE MITOCHONDRIA IN THE RAT MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
KUBALIK N, HEMENDINGER R, BROOKS BR
Carolinas Medical Center, Charlotte, NC, United States
E-mail address for correspondence: [email protected]
Keywords: superoxide dismutase, mitochondria, neurodegneration
Background: Amyotrophic lateral sclerosis (ALS) is a destructive fulminate neurodegenerative disease with multifactorial pathogenesis including: mitochondrial dysfunctions, oxidative stress, SOD1 mutations, and protein misfolding. In animal models of ALS, the mutated human SOD1 (mtSOD1) protein may be associated with neuronal mitochondria (1). Spinal cord mitochondria from tgSOD1 rodents are more dysfunctional as compared with the brain mitochondria (2). Our laboratory has reported differences in metabolic activity and ROS generation between brain mitochondria and spinal cord mitochondria from wild-type and tgSOD1 (3,4). The reason for this dysfunction is unknown but may depend upon the amount of mtSOD1 associated with between brain mitochondria or spinal cord mitochondria.
Objective: To examine the distribution of the human SOD1 expression in isolated mitochondria from different tissues from G93A-SOD1 transgenic rats in presymptomatic and symptomatic stages of ALS.
Methods: Brain, spinal cord, heart and skeletal muscle mitochondria were isolated from tgG93A SOD1 rats at three time points in the disease process: pre-symptomatic, at disease onset and at endstage. Mitochondria were isolated using the Mitosciences Mitochondria Isolation Kit for Tissue. Human SOD1 was quantified in rodent mitochondrial samples by immnoblotting and analyzed with General-Purpose Analysis Software Multi-Gauge V3.0. All rats were genotyped and copy number was controlled for these studies.
Results: Human SOD1 was detected in mitochondria from brain, spinal cord and muscle tissues, but within the central nervous system, the content of human SOD1 protein was 24.9% higher in spinal cord mitochondria in comparison with the brain mitochondria in pre-symptomatic stage. Human SOD1 was not increased in the mitochondria from skeletal muscle.
Conclusion: Our study supports the hypothesis that mitochondrial dysfunction may depend on the amount of mtSOD1 protein associated with mitochondria. The increased level of mtSOD1 in spinal cord mitochondria pre-symptomatically compared with brain mitochondria may result from SOD1 aggregation in spinal cord mitochondria leading to neurode-generation in motor neurons.
References:
- Higgins C, et al. J Neurosci 22:RC215.
- Sullivan P, et al. J. Comparative Neurol 2004;474,524–34.
- Panov A, et al. ALS 2007;8(Suppl 1):179.
- Panov A, et al. ALS 2008;9(Suppl 1):2.
P35 THE ROLE OF ACTIVITY IN NEUROMUSCULAR SYNAPTIC DEGENERATION: INSIGHTS FROM WLDS MICE
BROWN R, THOMSON D, SHEWARD J, HARTLEY R, HARMAR A, RIBCHESTER R
University of Edinburgh, Edinburgh, United Kingdom
E-mail address for correspondence: [email protected]
Keywords: neuromuscular junction, degeneration, activity
Background: Evidence for the role of activity in the onset or progression of Motor Neuron Disease (MND/ALS) is controversial. Some patients perceive benefits from taking exercise but conflicting evidence has raised the alternative possibility that some forms of activity may constitute a risk factor for onset of ALS or accelerate disease progression, perhaps through its potentially excitotoxic effects. The neuromuscular junction (NMJ) is the first component of the motor neuron to degenerate in some forms of MND, including the SOD1G93A mouse model and sporadic ALS in humans.
Objectives: Since exercise directly regulates neuromuscular activity, we asked whether blocking or enhancing activity either suppresses or accelerates synaptic degeneration at neuromuscular junctions induced by interruption of axonal transport. To test this, we used mutant WldS mice with slow-Wallerian degeneration in which axons are preserved but synapses degenerate progressively over several days after nerve section (1).
Methods: To study effects of paralysis, chronic sciatic nerve block was initiated in WldS mice and sustained for one week using microcapsules filled with tetrodotoxin (TTX; 10 mM). Following tibial nerve axotomy, neuromuscular synaptic preservation was measured physiologically 3-5 days later by intracellular recording from muscle fibres in isolated flexor digitorum brevis nerve-muscle preparations. The prevalence of fibres showing spontaneous (MEPPs) and/or evoked responses (EPPs) were scored in each muscle. To facilitate activity instead, WldS mice were individually housed and given voluntary access to running wheels for 2-4 weeks, before cutting the sciatic nerve. WldS mice showed a normal circadian rhythm under open-field conditions and typically ran about 13 km per night when provided with running wheels. Functional innervation was then assayed 3-5 days after axotomy in these mice as well. In some mice (transgenic thy1.2YFP16/WldS), isolated lumbrical muscles were stained with rhodamine-conjugated a-bungarotoxin and fluorescence microscopy was used to score the numbers of innervated and denervated motor endplates.
Results: The results showed that priming motor nerve terminals in WldS mice by chronic TTX-induced nerve conduction block significantly accelerated synaptic degeneration compared to controls (68% unresponsive fibres in the TTX pre-treated group compared to 21% in axotomised control groups; P<0.05; ANOVA). Wheel-running liminally increased muscle fibre diameter and EPP amplitude in WldS mice. However, the number of denervated fibres observed 3-5 days after axotomy was neither increased nor decreased following up to a month of voluntary wheel running.
Discussion and conclusions: The data suggest that neuromuscular disuse suppresses the neuroprotective effect of the WldS gene and enhances neuromuscular synaptic degeneration following challenges that disrupt axonal integrity. However, environmental enrichment by facilitated aerobic exercise neither enhanced nor suppressed the protective effect of WldS on synaptic degeneration. We are presently extending this evaluation to SOD1G93A transgenic mice.
Reference:
- Gillingwater TH, Thomson D, Mack TG, et al. J Physiol; 543:739–55.
P36 MUSCLE SATELLITE CELL BEHAVIOUR IN A MOUSE MODEL OF ALS
MANZANO R1, TOIVONEN J1, FERNANDES F1, CALVO A1, OLIVÁN S1, MONTARRÁS D2, UCKINGHAM M2, MUÑOZ JM1, ZARAGOZA P1, RODELLAR C1, OSTA R1
1LAGENBIO-I3A, Facultad de Veterinaria, Instituto Aragonés de Ciencias de la Salud, Universidad de Zaragoza, Zaragoza, Spain, 2Unité de Génétique Moléculaire du Développement, Institut Pasteur, Paris, France
E-mail address for correspondence: [email protected]
Keywords: muscle, satellite cells, culture
Background: Skeletal muscle satellite cells are the main cells implicated in regenerative response following muscle injury. Although ALS is described as a disease related to upper and lower motor neuron degeneration, the first events described in ALS are related to neuromuscular junction perturbation suggesting that an impairment of the muscle could produce toxic signals that would destroy neuromuscular junctions leading to a progressive retrograde axonophathy or ‘dying-back’. If this is the case it is of interest to study the skeletal muscle satellite cell status and regenerating response throughout the disease.
Objectives: The aim of this study was to analyze behavior of satellite cells in the ALS mouse model hSOD1-G93A in the course of the disease.
Methods: Skeletal muscle satellite cells were isolated from fast and slow type muscles of hSOD1G93A and wildtype male and female mice of ages corresponding to 7, 40, 60, 90 and 120 days. Cell cultures were established and proliferation was measured by fixing the cultures every 24 hours, staining the nuclei of cells with Hoechst 33342 and recording the fluorescence intensity with a plate reader. Myogenic regulatory factors Pax7, Myod1 and Myogenin expression was obtained by Real Time PCR at the same timepoints.
Results: Proliferation ratio differences were found between transgenic and wildtype mice as well as between both sexes and fiber type muscles. Myogenic regulatory factor RNA levels showed that the expression of those factors were also affected; presenting sex, age and fiber-type dependent differences.
Conclusions: Muscle satellite cell proliferation and myogenin regulatory factor expression pattern are affected in the hSOD1G93A ALS mouse model. Further experiments are needed to confirm and determine the cause and pathogenic effects of these findings.
Acknowledgements: This work was supported by the grants: Fondo de Investigaci ó n Sanitaria-Instituto de Salud Carlos III (PI071133) and PAMER from Aragon Health Sciences Institut (PIPAMER 08/08 and 09/09).
P37 CGRP IMMUNOREACTIVITY LEVELS DIFFERENTIATE ALS-VULNERABLE FROM ALS-RESISTANT MOTONEURONS IN SOD1-G93A MICE
RINGER C, WEIHE E, SCHÜTZ B
Institute of Anatomy and Cell Biology, Faculty of Medicine, Philipps-University, Marburg, Germany
E-mail address for correspondence: [email protected]
Keywords: CGRP, vulnerability, immunohistochemistry
Background: In ALS, motor nuclei in brain stem and spinal cord are affected unequally severe by the disease. While some nuclei (eg Ncll. III, IV and VI) are resistant and largely spared from neuropathology, others (e.g. Ncll. V, VII, XII and spinal cord) are vulnerable but show different levels of motoneuron loss. The reasons for this selective resistance and vulnerability are still poorly understood. Calcitonin gene-related peptide (CGRP) is a neuropeptide co-expressed by some but not all motoneurons (1). Recently, we described disease-related changes in the sub-cellular distribution pattern of the ß-iso-form of CGRP in the lumbar spinal cord of SOD1-G93A mice (2). The atypical appearance of ß CGRP in motoneuron dendrites and their close association with reactive astrocytes suggested a role for ß CGRP in the pathology of ALS.
Objective: In the present report we questioned if there is a connection between CGRP expression levels and the vulnerability of motoneurons during ALS pathology.
Methods: Lumbar spinal cord and brain from SOD1-G93A and wild type littermates at age P140-160 (n=3 each) were analyzed by double-immunoflurescence for CGRP and the motoneuron marker choline acteyltransferase (ChAT). For each area of interest all ChAT-positive neurons with clearly cut nucleus were counted on 10 sections and then divided in three groups: non CGRP (ChAT-staining only), low CGRP (weak, diffuse CGRP-staining) and high CGRP (strong, reticular network staining) expressing motoneurons.
Results: In wild type mice, the motor nuclei could be grouped into three clusters that differed in their amount of CGRP co-expression: The first cluster consisted of Ncll. III, IV and VI and contained only very few (<1%) high CGRP, 10-20% low CGRP and 80-90% non CGRP motoneurons. The second cluster (Ncll. V, VII and XII) contained about 30% high CGRP, 40% low CGRP and 30% non CGRP expressing neurons. Finally, Ncl. ambiguus and the lumbar spinal cord contained about 80% high CGRP expressing neurons, 10% low CGRP and 10% non CGRP expressing motoneurons each. While in the first cluster no (VI) or only 20% (III, IV) of all motoneurons had died at end stage in SOD1-G93A mice, the second type lost nearly 50% and the third type about 70% of motoneurons. Motoneuron loss was largely attributable to the level of CGRP expression. On average, high CGRP expressing motoneurons were found reduced by 80%, low CGRP expressing motoneurons by 50%, whereas the number of non CGRP motoneurons was not significantly affected.
Conclusions: Our analysis revealed CGRP immunoreactivity levels as a marker and/or criterion for the selective vulnerability of subsets of motoneurons within distinct motor nuclei during neuropathology in the SOD1-G93A mouse model of ALS.
References:
- Arvidsson, et al. Neuroreport 1993;4:849–56.
- Ringer, et al. Neurobiol. Dis 2009;35:286–95.
P38 UNDERSTANDING THE ROLE OF MACROPHAGE AND SCHWANN CELLS IN PERIPHERAL NERVES OF THE SOD1 MOUSE MODEL OF ALS
WANG M, MOY P, SANCHEZ R, CARRION I, PERRIN S, LINCECUM J
ALS TDI, Cambridge, MA, United States
E-mail address for correspondence: [email protected]
Keywords: macrophage, Schwann cells, SOD1
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the upper and lower motor neurons. The disease mechanism has yet to be fully defined. A hallmark of ALS pathology, in the peripheral nerves, is neuromuscular junction orphaning and demyelination suggesting a chronic injury state. It is well understood that, in response to trauma, peripheral nerves secrete cytokines and chemokines. This potent class of proteins has immunoregulatory and chemoattractive functions that promote a balanced inflammatory and anti-inflammatory response. We have previously established that macrophages accumulate in the peripheral nerves of gastrocnemius tissue as a function of disease progression. This was demonstrated by increased CD68(+) macrophage accumulation in the distal nerves of SOD-G93A transgenic mice relative to age-matched wild-type peripheral nerves.
Here we report on our efforts to further characterize the individual cell population in the peripheral nerves of SOD1-G93A mice. Focusing on macrophage and Schwann cells, we surveyed a panel of antibodies. This survey showed the cell surface protein, galectin-3/Mac2, was expressed on macrophage and Schwann cells in a disease specific pattern. Galectin-3 is a beta-galactosidase binding protein that has been shown to be involved in the inflammatory pathway, specifically in the transition from acute to chronic inflammation. Using immunoblot analysis and quantitative ELISA we show that galectin-3 protein levels are increased throughout disease progression whereas no significant levels are seen in non-transgenic control mice. Furthermore, high resolution, 3-D imaging in muscle tissue revealed extensive galectin-3 expression in distal nerves, specific to CD68+ macrophage and a subset of Schwann cells. To characterize these two cell populations we developed a paramagnetic bead based purification strategy to isolate galectin-3 postive macrophage and Schwann cells. Subsequently, these purified cells were analyzed by transcriptional profiling which established the activation of several inflammatory pathways, such as the TLR pathway, associated with chronic injury. These studies support a role for demyelination and presympotomic peripheral nerve damage in ALS.
P39 NEUROPATHOLOGY OF MICE EXPRESSING A VERY LOW COPY NUMBER OF THE MUTANT HUMAN G93A SOD1 GENE ASSOCIATED WITH ALS
DEITCH J1, MASEL R2, KOCHER K3, HEIMAN-PATTERSON T1
1Drexel University College of Medicine, Philadelphia, PA, United States, 2Muhlenberg College, Allentown, PA, United States, 3Dickenson College, Carlisle, PA, United States
E-mail address for correspondence: [email protected]
Keywords: SOD1, copy number, pathology
Background: Transgenic mice expressing multiple copies of the human mutant SOD1 gene develop motor neuron (MN) pathology and clinical symptoms that are similar to patients with ALS/MND. In mice with 24 copies of the transgene, MN disease is evident as early as 30 d old and degeneration proceeds rapidly. This time course makes it difficult to discern cellular events at onset of pathology. We have developed a line of transgenic mice, expressing a very low number (4-5) of copies of the G93ASOD1 transgene that do not show clinical signs until 650 d old, if at all.
Objective: To identify MN pathology occurring sub-clinically in very low G93ASOD1 expressing (VLE) mice and determine whether onset of pathology begins early and progresses very slowly or if MNs are healthy until onset later in life.
Methods: Four male and four female mice each at approximately 250, 500 and 750 d old were selected. At each age and gender, two were VLE mice and two were non-transgenic littermates. The number of G93ASOD1 gene copies was confirmed by qPCR. MNs in the ventral horns of lumbosacral segments were counted. MNs were easily identifiable by their position and large (>30 μm) cell bodies. The total numbers of MNs in transgenic vs control mice were compared using a paired Student's t-test.
Results: There was no statistically significant difference in mean number of MNs in lumbosacral spinal cord at 250 days old (P> 0.2, one-tailed t-test with similar variances) between transgenic (2390±411, mean±std. dev.) and non-transgenic (2149±641) mice. Similar MN counts were found in non-transgenic (2212±492) and transgenic (1908±28) mice at 500 days old (P>0.2). At 750 days old, the number of lumbosacral MNs in non-transgenic mice (2296±233) is comparable to that seen at younger ages, however, the number is significantly reduced by 38% in transgenic mice (1431±63; P<0.02). This reduction is comparable to that seen at 70 d old in the transgenic mice expressing a high copy number of G93ASOD1 genes. The loss of MNs was most noticeable in the lateral motor column.
Discussion and conclusions: The mechanisms by which mutant SOD1 is toxic to MNs is still unclear. In the transgenic mouse model, 24 copies of the mutant gene result in rapid MN loss, making fine distinction between early and late phenomena difficult. In this study we demonstrated that MNs in VLE mice do not degenerate in significant numbers until late in life, but that neurotoxic events occurring much earlier may be studied distinct from cell loss. These data also support the concept that accumulation of mutant SOD1 protein results in cell death when it reaches a critical level, which occurs more slowly with fewer copies of the gene.
P40 AUTONOMIC IMPAIRMENT IN A TRANSGENIC MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
KANDINOV B1, KORCZYN A1, RABINOWITZ R1, NEFUSSY B2, DRORY V2
1Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel, 2Department of Neurology, Tel-Aviv Medical Center, Tel-Aviv, Israel
E-mail address for correspondence: [email protected]
Keywords: autonomic impairment, SOD1 transgenic mice, sympathetic activity
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss of motor neurons, but it is increasingly recognized that non-motor manifestations may occur. The autonomic nervous system may also be affected. To better understand the autonomic involvement in ALS we measured autonomic functions in transgenic (TG) mice carrying a SOD1 (G93A) mutation and wild-type (WT) control mice. TG mice had a higher heart rate at rest and following stress than WT mice at all ages except for the advanced stages of the disease (19-20 weeks of age). The mean pupil diameter at rest was similar in WT and TG mice; however, TG mice had decreased mydriasis following administration of morphine. The rectal temperature did not differ between TG and WT mice at rest, during exposure to cold stress and following administration of morphine (30 mg/ kg) except for the advanced stages of the disease in which TG mice had significantly lower temperatures than WT mice during cold stress and following morphine administration. The results suggest mainly sympathetic cardiac hyperactivity, but also parasympathetic nervous system impairment in this ALS model, consistent with clinical data in humans.
P41 CONTINUOUS NON-INVASIVE INFRARED MOTION SENSING AS A MEASURE OF NEUROLOGICAL DISEASE PROGRESSION IN THE SOD1-G93A TRANSGENIC MOUSE MODEL OF ALS
DHILLON H, REED W, THOMPSON K, KIDD J, AL-NAKHALA B, VIEIRA F, GILL A
ALS TDI, Cambridge, MA, United States
E-mail address for correspondence: [email protected]
Keywords: SOD1-G93A, activity, automation
Background: The SOD1-G93A transgenic mouse manifests all clinical symptoms of sporadic and familial ALS. These animals display an ALS-like phenotype and pathology, including muscle atrophy and motor neuron degeneration. Understanding disease progression in this model is a requirement for drug discovery leading to ALS therapies. ALS-TDI has developed a four-point neurological scoring system (1-4, 0= no neurological signs) that tracks the progression of ALS in these mice. While neurologic scoring (NS) is robust and reproducible, it involves a single observation point per day. Less discreet, gradual changes in neurological disease progression are unlikely to be captured using this method. There is a clear need for a more sensitive method for monitoring disease progression in SOD1-G93A mice in order to allow better assessment of new therapeutics.
Objectives: The aim of the current study was to design, build, develop and test a home-cage continuous monitoring system, and relate mouse activity over time to neurologic disease progression in this ALS model. The longer-term objective is to refine the system for a large-scale use to enhance sensitivity of neurological disease progression tracking during survival.
Methods: A novel Animal Movement Sensing System (AMSS) was built to continuously capture activity levels in each mouse's home cage. Continuous activity data (counts/min) were captured for 32 animals from 50 days to death. Experimental groups were age-matched SOD1-G93A mice (male and female, n=11 each) and WT mice (male and female, n=5 each). Mice were individually housed in cages fitted with Passive Infra Red (PIR) sensors. The cage sensor nodes consist of two PIR sensors connected to Delta Sigma data converters, which connect to a microcontroller. The firmware read the output of the data converters and processed it to detect motion events from the raw sensor data. Motion events were counted by the microcontroller and stored in memory until requested by the computer.
Results and conclusions: Our data showed a significant difference in activity patterns from SOD1-G93A vs WT mice. This activity difference was consistent across genders and correlates well with neurologic disease progression as assessed by our manual scoring system. We observed a decline in activity counts in day 100-120 SOD1-G93A mice vs day 80-100 SOD1-G93A mice (59% males, 35% females). In addition to detecting gradual loss in overall mobility during disease progression, our AMSS accurately captured the diurnal periodicity of mouse activity. These data are the first documentation of continuous activity monitoring of SOD1-G93A transgenic mouse in their home cage. In conclusion, we have generated a simple and robust motion monitoring system that can be scaled up to thousands of sensors for large scale testing. This system will prove highly valuable in testing new drugs in pre-clinical mouse models of ALS.
P41A DISRUPTED TGF-BETA SIGNALING IN SPINAL AND BULBAR MUSCULAR ATROPHY
KATSUNO M1,2, ADACHI H1, MINAMIYAMA M1, DOI H1, KONDO N1, BANNO H1, SUZUKI K1, TANAKA F1, SOBUE G1
1Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan, 2Institute for Advanced Research, Nagoya University, Nagoya, Japan
E-mail address for correspondence: [email protected]
Keywords: spinal and bulbar muscular atrophy, TGF-beta, androgen receptor
Background: Spinal and bulbar muscular atrophy (SBMA) is a late-onset lower motor neuron disease caused by the expansion of a trinucleotide CAG repeat, which encodes a polyglutamine tract in the androgen receptor (AR). Although it is commonly held that the pathogenic polyglutamine proteins accumulate in neurons and thereby induce transcriptional dysregulation through inhibition of histone acetylation, the down-stream molecular events have remained elusive.
Objectives: The aim of this study is to elucidate the molecular events that induce neurodegeneration in SBMA. Since a cDNA microarray study of cultured cells suggests that the expression of genes involved in transforming growth factorbeta (TGF-beta?) pathway is specifically regulated by histone acetylation, we examined whether TGF-beta signaling is dysregulated in SBMA.
Methods: We used a transgenic mouse carrying human AR with 97 CAGs and SH-SY5Y cells expressing truncated AR fragment with 97 CAGs, as mouse and cellular models of SBMA. Histopathological analysis was performed on the autopsy specimens of the spinal cord from SBMA patients and that from the model mice. TGF-beta signal transduction in cells and mice was analyzed using immunoblotting, immunoprecipitation, filter trap assay, RT-PCR and immunohistochemistry. Cellular toxicity analysis was performed using propidium iodide staining and WST-1 viability assay. Promoter activity was measured using luciferase reporter assay.
Results: Nuclear translocation of phosphorylated Smad2/3, a key step in TGF-beta signaling, is suppressed in the spinal motor neurons of male transgenic mice carrying the mutant human AR. A similar finding was also observed in the motor neurons, but not in Purkinje cells, of SBMA patients. The pathogenic AR, the causative protein of SBMA, inhibits the transcription of TGF-beta receptor type II (TbetaRII), via abnormal interactions with NF-Y and p300/CBP-associated factor. Furthermore, overexpression of TbetaRII dampens polyglutamine-induced cytotoxicity in a neuroblastoma cell line expressing the pathogenic AR.
Discussion: It was postulated that TGF-beta signaling plays a fundamental role in neural activity through the regulation of synaptic function. TGF-beta was also shown to protect neurons from glutamate-mediated excitotoxicity, a putative molecular mechanism underlying the pathogenesis of motor neuron diseases. Our findings indicate that the decreased expression of TbetaRII and the resulting perturbation of TGF-beta signaling appear to underlie polyglutamine-dependent neurodegeneration in SBMA. Decrease in TbetaRII expression was also reported in AD patients, suggesting that this molecule plays an important role in various pathogeneses of neurodegeneration.
Conclusion: The present study showed that polyglutamine-dependent neuron damage in SBMA is associated with the disruption of TGF-beta signaling due to transcriptional dysregulation of TbetaRII. Our findings further suggest that restoration of the brain TGF-beta-Smad2/3 pathway might be a potential therapeutic approach to polyglutamine-induced neurodegenerative diseases.